[International Review of Cell and Molecular Biology] Volume 305 || Thy-1-Interacting Molecules and Cellular Signaling in Cis and Trans

CHAPTER FOUR

Thy-1-Interacting Moleculesand Cellular Signaling inCis and TransRodrigo Herrera-Molina*, Alejandra Valdivia†,{, Milene Kong†,{,},Alvaro Alvarez†,{,}, Areli Cárdenas†,}, Andrew F.G. Quest†,{,Lisette Leyton†,{,},1*Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology,Magdeburg, Germany†Programa de Biologıa Celular y Molecular, Facultad de Medicina, Universidad de Chile, Santiago, Chile{Centro de Estudios Moleculares de la Celula, Facultad de Medicina, Universidad de Chile, Santiago, Chile}Biomedical Neuroscience Institute, Instituto de Ciencias Biomedicas (ICBM), Facultad de Medicina,Universidad de Chile, Santiago, Chile1Corresponding author: e-mail address: [email protected]

Contents

1.

InteISShttp

Introduction

rnational Review of Cell and Molecular Biology, Volume 305 # 2013 Elsevier Inc.N 1937-6448 All rights reserved.://dx.doi.org/10.1016/B978-0-12-407695-2.00004-4

165
2. Thy-1 Molecule 167
2.1
Discovery 167 2.2 Expression 168 2.3 Properties 172
3.
Thy-1 Cis-Interacting Molecules and Signaling 174 3.1 Thy-1 cis interactions occur in rafts 174 3.2 Cis-interacting Thy-1 molecules 175 3.3 Thy-1-triggered cell signaling in cis 182
4.
Trans-Interacting Thy-1 Molecules and Signaling 189 4.1 In astrocytes 189 4.2 In melanoma cells 192 4.3 In blood cells 194
5.
Function of Thy-1 Molecule 195 5.1 In fibroblasts 195 5.2 In brain cells 198 5.3 In endothelial cells of vascular and lymphatic endothelium 201 5.4 As a cell biomarker 202
6.
Concluding Remarks 203 Acknowledgments 204 References 204
163

http://dx.doi.org/10.1016/B978-0-12-407695-2.00004-4

164 Rodrigo Herrera-Molina et al.

Abstract

Thy-1, discovered almost 50 years ago, was for many years a subject of great scientificinterest. The putative functions attributed to this molecule could not be confirmed due,at least in part, to a ligand that took a long time to be identified. This chapter describesthe properties of Thy-1 and the regulation of its expression. Also, the interactionsthat have been described for Thy-1 in both cis and trans, and the signaling mechanismsreported to emanate from such interactions are discussed. The consequences ofThy-1-activated signaling pathways for different cell types and organisms are alsoreviewed. Since the discovery of aVb3 integrin as a receptor for Thy-1 in astrocytes, manymore functions have been attributed to Thy-1 interactions in trans toward other celltypes. Recently, a ligand for neuronal Thy-1 was unveiled and shown to elicit signalingin cis. The ligand and the receptor for Thy-1 turned out to be the same molecule, aVb3integrin, which upon interaction with Thy-1 yields bidirectional astrocyte-to-neuroncommunication. Thus, Thy-1 biology is again beginning to make progress in answeringmain questions surrounding this enigmatic molecule. Some of these remainingquestions are highlighted in this chapter.

ABBREVIATIONSATP adenosine triphosphate

CAM cell adhesion molecule

cAMP cyclic adenosyl monophosphate

CBP Csk-binding protein

CREB cAMP response element-binding protein

Csk C-terminal Src kinase

CSPG chondroitin sulfate proteoglycan

DRG dorsal root ganglion

ECM extracellular matrix

FAK focal adhesion kinase

GABA gamma-amino butyric acid

GAP GTPase-activating protein

GFP green fluorescent protein

GPI glycosyl-phosphatidylinositol

HBD heparin-binding domain

HDMEC human dermal microvascular endothelial cell

ICAM-1 intercellular adhesion molecule 1

IL-1 interleukin-1

LAP latency-associated peptide

LAT linker for activation of T-cells

MEK mitogen-activated protein kinase kinase

MHC major histocompatibility complex

MMP matrix metalloproteinase

NF-kB nuclear factor-kappaB

NGF nerve growth factor

PDGF platelet-derived growth factor

165Thy-1 and Its Partners

PI3-K phosphatidylinositol 3-kinase

PI-PLC phosphatidylinositol phospholipase C

PKA cAMP-dependent protein kinase A

PKCa protein kinase C alpha

PTEN phosphatase and tensin homologue

RG retinal ganglion

SFK Src family kinases

SH2 Src-homology 2

STED stimulated emission depletion

TGF-b transforming growth factor beta

TNFa tumor necrosis factor alpha

TRAPs transmembrane adaptor proteins

TSP-1 thrombospondin-1

1. INTRODUCTION

Thy-1 is a small glycoprotein that faces the extracellular matrix

(ECM) and is anchored to the outer leaflet of the plasma membrane

through a glycosyl-phosphatidylinositol (GPI) moiety. Thy-1 was one

of the earliest GPI-anchored proteins to be sequenced and purified.

Insights gained from the study of this protein permitted characterization

of the molecular sequence of events by which the GPI moiety is added

to proteins and how this posttranslational modification anchors proteins

to the exoplasmic leaflet of the cell membrane (Beghdadi-Rais et al.,

1993; Conzelmann et al., 1987; Ferguson and Williams, 1988; Low and

Kincade, 1985; Tse et al., 1985).

Pioneering studies also established that theThy-1 protein core is composed

of 110 amino acids and that carbohydrate modifications contribute to an addi-

tional 30% of Thy-1 mass (Williams et al., 1977). Subsequent technical and

conceptual progress helped elucidate Thy-1 carbohydrate composition,

thereby contributing to our current understanding of posttranscriptional pro-

tein modifications via glycosylation (Luescher and Bron, 1985;Williams et al.,

1993).Moreover, thecharacterizationofThy-1 expressionhashelped improve

our understanding of fundamental immunological mechanisms, many aspects

of central nervous system development, possible mechanisms explaining how

cancer and blood cells reach distant sites, the differences between fibroblast

subpopulations with distinct morphology and capacity to proliferate and

differentiate, as well as, of the formation and function of cell membrane

nano/microdomains in multiple cell types (Hagood et al., 2001; Koumas


et al., 2002; Lang et al., 1998; Morris, 1985; Morris and Grosveld, 1989;

Tiveron et al., 1994; Wandel et al., 2012; Wetzel et al., 2004; Williams,

1982). For all these reasons, Thy-1 surely deserves a privileged position in

the history of modern cell biology.

As pointed out by the famous Chilean writer and poet Pablo Neruda

“Love is so short and oblivion so long.” Likewise for Thy-1, “fame and

glory” of the initial years was followed by many years of subdued interest,

largely due to the absence of a “binding partner” that permitted critically

evaluating functions ascribed to the molecule. Accordingly, during the past

20 years, Thy-1 has been employed as a marker of biochemically isolated

lipid-enriched subcellular fractions, a model to evaluate single-molecule

dynamics at the cell plasma membrane or, alternatively, the Thy-1 pro-

moter was employed with the sole purpose of overexpressing a protein

of interest in a cell-specific manner (Feng et al., 2000; Haeryfar and

Hoskin, 2004; Morris et al., 2011).

On the other hand, a reduced community continued the search to iden-

tify its function and finally in 2001, Thy-1 was shown tomediate adhesion of

neurons to astrocytes through a direct interaction in trans with the receptor

aVb3 integrin, which triggered cell-signaling events and profound morpho-

logical changes in astrocytes (Avalos et al., 2002, 2004, 2009; Henriquez

et al., 2011; Hermosilla et al., 2008; Leyton and Quest, 2002, 2004;

Leyton et al., 2001). Later on, Thy-1–aVb3 integrin interaction became

the starting point for a series of reports showing interactions in trans of

Thy-1 with a selective group of integrins as receptors (Choi et al., 2005;

Saalbach et al., 2005, 2007; Wetzel et al., 2004, 2006).

Recently also, we reported that the glial receptor aVb3 integrin acts as a

Thy-1 ligand, triggering signaling events andmorphological changes in neu-

rons (Herrera-Molina et al., 2012). These findings not only support the par-

adigm of a bidirectional communication between neurons and astrocytes but

have also spawned research efforts to define Thy-1 function in cells other

than those of the nervous system. In this chapter, we summarize the

Thy-1 literature with the objective of highlighting its unique combination

of molecular features and expression patterns. Then, cis-interacting partners

are analyzed, which might be part of a Thy-1-related signaling complex in

particular lipid rafts. Finally, after mentioning some examples of in trans-

interacting counterparts, we review proposed functions of Thy-1, many

of which still await experimental confirmation. Due to space constraints

and the vast body of existing literature on this topic, we apologize for not


mentioning many excellent contributions to the story by colleagues that

during many years have been loving and forgetting Thy-1.

2. Thy-1 MOLECULE

2.1. Discovery
2.1.1 Theta antigenThy-1 was first mentioned by Reif and Allen (1964). The authors were
looking for specific antibodies that would recognize thymoma cells from

AKRmice, but the antibodies produced in C3HeB/Femice also recognized

normal thymus cells (Reif and Allen, 1964). They named it theta antigen

after a suggestion from Amos group to assign Greek letters to the mouse

antigens (Amos et al., 1963). Soon, this AKR antigen was found in

thymus-derived lymphocytes, bone marrow cells, brain cells, and certain

types of fibroblasts (Barclay et al., 1976; Douglas and Dowsett, 1975;

Morris, 1985; Morris et al., 1983; Phipps et al., 1989; Williams, 1976).

But it was only at the beginning of the 1980s when the human version of

Thy-1 was described. This homologue was isolated as a 25 kDa protein from

the human T-lymphoblastoid cell line MOLT-3 and characterized bio-

chemically, as well as genetically (Ades et al., 1980; Bonewald et al.,

1984a,b; Seki et al., 1985d).

2.1.2 Thy-1.1 and Thy-1.2: Genetic descriptionThy-1 was described as Thy-1.1 for AKR mice and as Thy-1.2 for Balb/c

mice. The difference between these isoforms is only one amino acid;

Thy-1.1 contains an arginine in position 89, while Thy-1.2 has a glutamine

in that position. Despite these variations, the genetic characteristics of

Thy-1 from rat, mouse, and human, are very similar (Seki et al., 1985d).

The human Thy-1 gene is located on chromosome 11q22.3, while mouse

Thy-1 is on chromosome 9 (Giguere et al., 1985; Seki et al., 1985a,c,d;

Van Rijs et al., 1985). The gene contains four exons, with two exons 1

(1a and 1b), which upon transcription generate two mRNA splice variants.

There are two transcription initiation sites, one in each version of the first

exon. The second exon contains the translation start site, while the exon 3

encodes the amino acids 7 to 106, and exon 4, the C-terminal ending and

poly-A site. Thus, exons 3 and 4 contain the sequence coding for almost the

complete Thy-1 protein (Fig. 4.1; Giguere et al., 1985).

1a 31b 42

Intron 1 Intron 2 Intron 3

Figure 4.1 Thy-1 gen elements. Thy-1 possesses two alternate weak promoters thatlack cell-type specificity (black hexagons). The 30 region of intron 1 controls brain-specific transcription of the Thy-1 gene inmouse and human, whereas intron 3 regulatesexpression of Thy-1 inmouse thymocytes and human kidney. Exons 1a and 1b codify fortwo alternative spliced mRNA, respectively, and are preceded by a transcription initia-tion site. The 30-end of exon 2 codifies for the translation start site and the signalsequence of Thy-1, exon 3 for the mature protein and the 50-end of exon 4, for the trans-membrane sequence. Portions of the gene encoding for the mature Thy-1 protein areindicated as light gray rectangles. Dark gray rectangles complete the exons.


The Thy-1 gene presents regulatory elements of particular characteristics

that makes it different from other classic genes. It has an inverted CCAAT

box and lacks a TATA box. These regulatory elements are located within a

methylation-free sequence rich in CpG islands. Additionally, using trans-

genic mice, a number of tissue-specific enhancer elements located down-

stream of the Thy-1 promoter have been described, which are reportedly

essential for transcriptional activation (Spanopoulou et al., 1991; Vidal

et al., 1990). Moreover, tissue-specificity relies entirely on these enhancer

sequences, because the promoter itself is not sufficient in this respect and

only initiates transcription in the presence of these downstream enhancer

elements (Spanopoulou et al., 1991; Vidal et al., 1990). Based on these find-

ings, a Thy-1.2 expression cassette containing the second half of intron 1

(Fig. 4.1), defined as necessary to activate Thy-1 expression in the nervous

system (Spanopoulou et al., 1991; Vidal et al., 1990), is widely used to target

Thy-1 expression to the brain of transgenic mice (Aigner et al., 1995; Feng

et al., 2000; Kahle et al., 2000; Moechars et al., 1996).

2.2. ExpressionThy-1 expression is highly regulated; different expression patterns are found

during development and tissue-distribution depends on the species and the

age of the individual. Also, Thy-1 promoters are suggested to possess differ-

ential binding sites depending on the type of tissue in which Thy-1 is being

expressed. The particular characteristics of Thy-1 expression in different cell

types are discussed below.


2.2.1 Regulation of expression in thymocytesIn mouse thymocytes, Thy-1 accounts for 10–20% of the surface protein

reaching almost 1 million molecules per cell (Killeen, 1997). Due to this

trait, Thy-1 has been used as a T-cell marker, which in combination with

other T-cell markers has allowed the identification of each subset of T-cells

(Haeryfar and Hoskin, 2004). Notably, while human thymocytes express

Thy-1, mature human T-cells lack this surface antigen (Haeryfar and

Hoskin, 2004). Similar results have been reported in rat, which, in addi-

tion, express Thy-1 also in a large number of bone marrow cells

(Williams, 1976).

Regulation of Thy-1 expression in thymocytes has been described to

occur both transcriptionally and posttranscriptionally. Evidence indicates

that the third intron possesses enhancer elements required for expression

of Thy-1 in thymocytes (Fig. 4.1; Spanopoulou et al., 1991; Vidal et al.,

1990). Studies performed using a thymoma cell line (EL-4) also indicate that

epigenetic control in the regulatory elements of the Thy-1 gene exists. In

this model, it was shown for the first time that DNA methylation in the

50 region might regulate the expression of Thy-1 (Sneller and Gunter,

1987). At the posttranscriptional level, norepinephrine-induced G protein-

coupled b-adrenergic receptor activation decreases Thy-1 mRNA stability

in a cAMP-dependent protein kinase A (PKA)-dependent manner in mouse

thymocytes and S49 thymoma cells (Wajeman-Chao et al., 1998). Murine

Thy-1 mRNA has a 1146 nucleotide-long 30-untranslated region that pos-

sesses two copies of the AUUUA regulatory element. By using a green fluo-

rescent protein (GFP) reporter gene containing these regulatory Thy-1

regions, it was recently shown that the Thy-1 regulatory element acts as a

cyclic adenosyl monophosphate (cAMP) responsive element that regulates

mRNA stability (Lajevic et al., 2010). Therefore, this mechanism, which

includes a second messenger produced following b-adrenergic receptor

stimulation, might be responsible for norepinephrine-induced decreases

in Thy-1 expression. Importantly, since these same AUUUA regulatory ele-

ments reportedly control cytokine and chemokine expression under inflam-

matory conditions (Wilusz et al., 2001; Zhang et al., 2002), Thy-1

expression regulation in the immune system might also represent part of a

cell stress response.

2.2.2 Regulation of expression in neuronsIn neurons, Thy-1 is also highly expressed, representing up to 7.5% of the

protein present at the plasma membrane. It has been estimated that there are


�500 molecules of Thy-1 per square micron on the surface of retinal gan-

glion (RG) axons (Beech et al., 1983). However, Thy-1 is differentially

expressed in the different types of neurons. For example, neurons with long

processes, such as the RG cells, express Thy-1 at higher density than those

with short processes, like the interneurons (Barnstable and Drager, 1984;

Beale and Osborne, 1982; Morris et al., 1985; Weber et al., 1988).

Thy-1 expression is also regulated during pre- and postnatal development.

Whereas, some neurons like rat RG cells express the Thy-1 glycoprotein on

embryonic day 19 (Schmid et al., 1995), others only start expressing it a few

days after birth (Xue et al., 1991). Apparently, Purkinje neurons regulate

Thy-1 expression posttranscriptionally, since Thy-1 mRNA appears to

accumulate with respect to protein levels (Xue and Morris, 1992). In addi-

tion, Chen and colleagues showed differential expression of Thy-1

depending on the state of neuronal maturation; in the early development

of dorsal root ganglion (DRG) neurons, low levels of Thy-1 are detected.

At the day 2 postnatal, Thy-1 levels are still low, but then increase later as

these neurons mature (Chen et al., 2005). As previously stated

(Section 2.1.2), brain-specific expression of Thy-1 is controlled by enhancer

sequences present in the first intron of the Thy-1 gene, downstream of the

Thy-1 genomic regulatory elements (Gordon et al., 1987; Spanopoulou

et al., 1991; Vidal et al., 1990). Therefore, as for thymocytes, regulation

of Thy-1 expression in the nervous system occurs at both the transcriptional

and the posttranscriptional level. In addition, evidence for the existence of

secreted suppressor factors controlling Thy-1 expression also exists (Saleh

and Bartlett, 1989).

There are also pathological conditions that affect Thy-1 levels, which

include acute and chronic damage in RG cells, in an event that precedes cell

death (Schlamp et al., 2001), in RG cells from a model of experimentally

induced glaucoma (Huang et al., 2006) and, in neurons from the substantia

nigra of individuals with Restless-leg syndrome, where lower levels of

Thy-1 expression have been also observed (Wang et al., 2004). Intriguingly,

the development of many pathological states is associated with down-

regulation of Thy-1 protein levels.

2.2.3 Regulation of expression in fibroblastsIn fibroblasts, Thy-1 has been thoroughly studied, since presence of the pro-

tein appears to be associated with different pathological conditions. Over-

expression of Thy-1 in stromal fibroblasts, for instance, has been


associated with the appearance of prostate cancer (Liu et al., 2004; True

et al., 2010; Zhao and Peehl, 2009). Also, Thy-1-expressing fibroblasts

appear to be predetermined to differentiate to a contractile phenotype in

the human myometrium and orbit (Koumas et al., 2003). This is particularly

relevant, because it correlates the presence of inflammatory stimuli with

higher Thy-1 expression in cells. However, the exact mechanisms

explaining how Thy-1 expression is regulated in these cells is unknown.

Hagood and collaborators have provided some insight to this open question

by showing that hypermethylation of CpG islands in the Thy-1 gene pro-

moter lead to silencing of Thy-1 expression in the lung fibroblast of patients

with idiopathic pulmonary fibrosis and in primary rat lung fibroblasts

(Sanders et al., 2008). Recently, this same group showed that also histone

modifications contribute to defining Thy-1 gene expression and some

changes in methylation have been observed when histone modifications

are altered (Sanders et al., 2011); however, how these epigenetic modifica-

tions are controlled, remains to be defined. A recent report has related these

changes to hypoxia, since increased hypoxia-inducible factor 1 alpha (HIF-

1a) expression has been observed in fibrotic lungs (Tzouvelekis et al., 2007),and also because hypoxia is known to affect DNAmethylation in cancer cells

(Shahrzad et al., 2007; Watson et al., 2009). The authors observed that nor-

mal lung fibroblasts decreased expression of Thy-1 under hypoxia and

suggested that this might be due to increased methylation of the Thy-1 gene

(Robinson et al., 2012). Different studies have also correlated STAT3 phos-

phorylation with reduced Thy-1 expression in lung fibroblasts, but further

studies are required to understand better howThy-1 expression is controlled

in health and disease (Pechkovsky et al., 2012). In any case, up- or down-

regulation of Thy-1 levels in fibroblasts seems to correlate with the appear-

ance of certain diseases (Bradley et al., 2009). Interestingly, the behavior of

Thy-1(þ) and Thy-1(�) cells is reportedly different in lung fibrosis and in

Graves’ disease (Khoo et al., 2008; Koumas et al., 2002, 2003).

2.2.4 Regulation of expression in other cell typesAlthough Thy-1 expression has been reported in various cell types including

activated endothelial and epithelial cells, particular relevance has been attrib-

uted to Thy-1 expression in both stem cells and differentiated cells of hema-

topoietic origin (Craig et al., 1993). Here, other intracellular factors together

with DNAmethylation in the 50 region ofThy-1 gene were held responsiblefor regulation of Thy-1 expression in immature B cells (Shimizu et al.,


1992). These factors remained unresolved for some years, until Murray and

coworkers described that inhibition of histone deacetylases promoted Thy-1

expression in human stem cells (Young et al., 2004). Therefore, epigenetic

changes are observed here again to control Thy-1 expression. Stem cells pre-

sent in various other tissues also express Thy-1 protein (Masson et al., 2006;

Reding et al., 2010), and it is currently used as a marker for adult stem cells

(Jiang et al., 2012; Konishi et al., 2011; Notta et al., 2011; Patel et al., 2010).

In endothelial cells, Thy-1 mRNA and protein levels have been shown

to increase after stimulation with proinflammatory cytokines such as

interleukin-1b (IL-1b) and tumor necrosis factor alpha (TNFa) (Ishizu

et al., 1995; Lee et al., 1998; Saalbach et al., 1999; Takeuchi et al., 1997).

Interestingly, Thy-1 expression is reportedly diminished in lung fibroblasts

exposed to hypoxia or upon treatment with IL-1b or TNFa (Nicola et al.,

2009). Thus, the regulation of Thy-1 expression varies for different cells

exposed to the same stimulus.

2.3. Properties2.3.1 Physical–chemical properties and structureMany of the physical–chemical properties of Thy-1 were deciphered early

on (Barclay et al., 1976; Bonewald et al., 1984a; Williams et al., 1977).

This knowledge has contributed to explaining Thy-1 characteristics in

the cell membrane and also has permitted the generation of experimental

tools required to advance in the study of antigens on the lymphocyte

surface.

Due to the lipid nature of its GPI tail, purification of Thy-1 was com-

plicated by the fact that the correct detergent combination was needed to be

identified to solubilize the protein. The first purification protocols included

lentil lectin and gel filtration columns, but these techniques were quickly

replaced by affinity chromatography using monoclonal antibodies (Feng

and Wang, 1988; Williams et al., 1988). However, the use of deoxycholate

was always an essential requisite to obtain adequate solubilization of Thy-1

(Cotmore et al., 1981).

Along with the purification procedure, information about Thy-1 struc-

ture and composition became available. A molecular weight of 17.5 and

18.7, for brain and thymus forms, respectively, was determined. Currently,

the mature version of Thy-1 is known to contain 110 amino acids, although

when first described, Thy-1 was thought to contain 111 residues in the rat

and human proteins and 112 residues in the mouse version. The immature


protein possesses roughly 31 additional amino acids, which are cleaved prior

to adding the GPI anchor (Seki et al., 1985a,b,c,d).

Three N-glycosylation sites were identified in positions 23, 75, and

99 of the primary sequence for mouse Thy-1. Instead, human Thy-1 con-

tains only two sites for glycosylation. No residues were described for

O-linked glycosylation (Luescher and Bron, 1985; Williams et al., 1993).

Other relevant structural features are two potential disulfide bonds between

cysteines 9 and 112, and between cysteines 19 and 86 (Bonewald et al.,

1984a; Devasahayam et al., 1999).

The presence of the GPI anchor was deduced after the study of Thy-1

properties. Thy-1 exists as an oligomer in the absence of detergents, which

suggested the presence of a hydrophobic domain; however, no such domain

was found in the amino acid sequence, indicating the presence of a non-

proteinaceous domain in the molecule (Seki et al., 1985a). After the analysis

in numerous studies, the domain was identified as a GPI anchor. A GPI

transamidase replaces the Thy-1 C-terminal peptide at the o site in the

N-terminally processed proprotein by a GPI anchor (Beghdadi-Rais

et al., 1993). This occurs rapidly after translation in the endoplasmic retic-

ulum (Conzelmann et al., 1987; Tse et al., 1985). Then, Thy-1 is trans-

ported to the plasma membrane. Thus, this glycolipid molecule is

inserted through its GPI moiety in the cell membrane phospholipids of

the outer leaflet of the bilayer (Fig. 4.2).

The carbohydrates are essential in determining the structure of mature

Thy-1. The first data were obtained performing a profile of brain and thy-

mus Thy-1 after the cleavage of the carbohydrate portion, followed by elec-

trophoretic separation, and gel filtration on a Biogel P4 column. These

experiments revealed that the carbohydrate composition is not the same

in different cell types; however, there is a recognizable pattern in the type

of carbohydrates that bind to a particular residue (Carlsson, 1985).

Thy-1 has been described as an antibody-like molecule, due to its sim-

ilarity to immunoglobulins. The first information pointing in this direction

was obtained by comparison of their sequences. Structurally, the positioning

of disulfide bonds as well as similarities in b-sheet organization suggested theexistence of evolutionarily conserved roots between the two types of

molecules. Particularly, a more in-depth analysis, pointed toward a close

structural relationship with select immunoglobulin V or V-related domains

(Bonewald et al., 1984b; Williams and Gagnon, 1982).

Thus, much is known about the composition and structure of Thy-1

glycoprotein, whereas its function has remained elusive for many years.

Figure 4.2 Three-dimensional modeling of the Thy-1 protein. The Thy-1immunoglobulin-like proteinaceous region appears inserted in the outer leaflet ofthe plasma membrane. The amino acids of Thy-1 that interact with astrocyte integrins,the conserved RLD motif, and the heparin-binding domain of mouse Thy-1, REKRK,which binds to Syndecan-4, are indicated. The model was obtained from the primarysequence of mouse Thy-1 (amino acids 27–161, Accession number: AAA61180.1). Thesequence of Thy-1 was used to generate a 3D model using Expasy (http://expasy.org/). The final PDB file was generated by using a low-energy model, visualized withthe Deepview 4.0 software (http://spdbv.vital-it.ch/). The Thy-1 molecule was then builtwith an Autodesk Maya mMaya v.1 Molecular Maya toolkit. The plasma membrane wascreated using the same software with polygonal modeling and structure amplificationwith Nparticles. Graphics were performed in Adobe Illustrator and the final image inAdobe Photoshop.


3. Thy-1 CIS-INTERACTING MOLECULESAND SIGNALING

3.1. Thy-1 cis interactions occur in rafts
Thy-1 localizes in a subclass of cholesterol-enriched, noncaveolae domains
called “rafts” by inserting the GPI group into the outer leaflet of the cell

membrane. In general, rafts are formed by cholesterol, phosphatidylcholine,

and sphingomyelin (Neumann et al., 2010; Quest et al., 2004). The rigid and

bulky tetracyclic cholesterol structure interacts with other lipids to form

5–200 nm patches of limited stability in the ms-to-min time range

(Kusumi et al., 2004, 2010). These specialized lipid domains create molec-

ular platforms for signal transduction within the disordered, more fluid phase

of the cell membrane. In particular, Thy-1 is present in domains

http://expasy.org/

http://expasy.org/

http://spdbv.vital-it.ch/


distinguishable by their particular composition, as they are enriched in fully

saturated lipids, whereas other raft proteins, like the prion protein PrP, are

present in microdomains with significantly more unsaturated and longer

chain lipids than Thy-1 microdomains (Brugger et al., 2004). Notably,

the existence of Thy-1-containing rafts, depleted of PrP, has been confirmed

by a new biochemical procedure able to stabilize and maintain the correct

inside-out orientation of the cell membrane during extraction with deter-

gent and subcellular fractionation at 37 �C. Importantly, Thy-1-containing

lipid nanodomains have been associated with actin, suggesting a tight inter-

action between Thy-1 rafts and cytoskeletal/cytoplasmatic components

(Chen et al., 2009b; Morris et al., 2011).

This scenario ascribes a number of properties to the Thy-1-containing

domains that must be considered when thinking of Thy-1-interacting mol-

ecules and the signaling pathways triggered in cis. Both composition and

intrinsic dynamics of rafts may not only play a role in Thy-1 mobility but

also in establishing interactions with other molecules within and below

the cell membrane. How do lipids influence protein properties and their

organization in cell membrane? This question has been addressed elsewhere

and the interested reader is referred to ( Jacobson et al., 2007).

3.2. Cis-interacting Thy-1 moleculesIn the next lines, we will discuss evidence suggesting that Thy-1 interacts

with itself, with adaptors, scaffolds, or signaling molecules, such as

reggies-1/2, Src family of protein tyrosine kinases (SFK), and C-terminal

Src kinase (Csk)-binding protein (CBP), in the cell membrane of several cell

types to convey signals to the cell interior. Thus, based on accumulated

evidence in the literature and our own unpublished observations, we pro-

pose that Thy-1 may be an important component of protein complexes,

which initiate cell signaling from rafts (Fig. 4.3). In addition, Thy-1 interacts

with other receptors at the plasma membrane such as the aVb5 integrin in

fibroblasts.

3.2.1 Thy-1–Thy-1 interactionClassical chromatographic and biochemical experimentation revealed that

Thy-1 immunoreactivity could be observed as multimers of 25, 45, and

150 kDa (Mahanthappa and Patterson, 1992b). Themultimeric species were

detected predominantly in primary neurons or differentiated PC12 cells, but

not in proliferating PC12 cells, suggesting that the oligomerization state of

Figure 4.3 Signaling triggered by Thy-1 in cis. Thy-1 binds to its ligand (L) andundergoes molecular clustering at the plasma membrane. Aggregation of Thy-1 mole-cules recruits proteins such as reggies and Src family kinases (SFKs). A transducer hasbeen proposed to connect the intracellular proteins with aggregated Thy-1, transducingsignals that lead to regulation of the cytoskeleton. Rafts in the plasma membrane areindicated as darker areas of the membrane.


Thy-1 might contribute to inhibition of process extension by stabilizing the

surface-membrane complexes formed by Thy-1 with the underlying

cytoskeleton.

Available evidence indicates that 2–20 molecules of Thy-1, spontane-

ously form highly compact nanoclusters, which are as small as 20 nm

according to the results obtained using electron microscopy-associated

immunogold particles (Brugger et al., 2004). Indeed, GFP-GPI molecules

have been observed as close as 4 nm apart using homo-FRET (fluorescence

resonance energy transfer) (Sharma et al., 2004). Considering that two

Thy-1 molecules occupy a surface area of 8�12 nm (Perkins et al.,

1988), direct Thy-1–Thy-1 interaction is plausible and may depend on their

immunoglobulin-like protein structure. Indeed, it is known that the Thy-1

fragment TREKKKHVLCmay be involved in Thy-1 clustering, as incuba-

tion with a Thy-1-derived short peptide promoted Thy-1 cluster


disassembly (Mahanthappa and Patterson, 1992b). Topologically, this puta-

tive Thy-1-aggregating domain is conveniently located at a lateral surface

of the Thy-1 molecule (Fig. 4.2), supporting the idea that it may represent

a sequence element involved in Thy-1-to-Thy-1 docking. Nevertheless,

whether this motive is indeed responsible for Thy-1–Thy-1 interaction has

not been tested and further studies are required.

3.2.2 Thy-1 functionally associates to reggies/flotillinsExisting experimental evidence indicates that Thy-1 may functionally inter-

act with reggie-1 and reggie-2 in specific cell membrane domains to mod-

ulate signaling (Lang et al., 1998). Reggies, also known as flotillins, associate

with the inner layer of the plasma membrane and intracellular membranes

via posttranslational myristoyl and palmitoyl lipid modifications (Morrow

et al., 2002; Neumann-Giesen et al., 2004). It has been proposed that reggies

are scaffolding proteins necessary for the trafficking of specific molecules to

specialized membrane domains as they have been observed decorating post-

Golgi-derived vesicles in transit toward the cell surface (Langhorst et al.,

2008). Interestingly, the presence of reggies has been used to define raft-

related, noncaveolar plasma membrane microdomains, where clustered

Thy-1 is tightly associated (Stuermer et al., 2001). Thy-1 coprecipitates with

reggie-1/2 in extracts from rat brain, DRG neurons, and PC12 cells

(Stuermer et al., 2001). Anti-Thy-1 antibody induces Thy-1 association

with reggie proteins in well-defined nanoclusters in growth cones of

DRG neurons (Lang et al., 1998), as well as in growing neuronal processes

during regeneration in zebrafish (Deininger et al., 2003; Munderloh et al.,

2009). In addition, due to their ability to activate Fyn, mitogen-activated

protein kinase (MAPK), and RhoA GTPases, reggies have been considered

key players in the control of the cytoskeleton and axonal growth

(Stuermer, 2011).

Although direct interaction between reggies and Thy-1 is not possible

due to differential association with the inner and outer leaflet of the plasma

membrane, respectively, reports have indicated that cross-linking of GPI-

anchored molecules, stabilizes raft-like microdomains of the membrane

inner leaflet, causing changes in cell signaling (Eisenberg et al., 2006).

Thus, it is tempting to propose that cross-linking of Thy-1 might recruit

proteins localized in the inner leaflet of the cell membrane (i.e., reggies),

as a necessary step to initiate signaling. Supporting this idea, accumulating

evidence indicates that Thy-1 cross-linking promotes association with


SFK and downstream signaling elements (Chen et al., 2005, 2006, 2009b;

Herrera-Molina et al., 2012).

3.2.3 Src family kinases and Thy-1Despite the fact that Thy-1molecules only span half the cell membrane, they

are able to transmit a signal from the outside to the inside of the cell. It is well

documented that signaling molecules in the inner leaflet of the cell mem-

brane such as members of the SFK (Src, Fyn, Lck, and Csk) can be co-

immunoprecipitated with Thy-1 from homogenates of leukocytes,

thymocytes, murine T-cells, fibroblasts, neurons, as well as from total

extracts of numerous cell lines (Draberova and Draber, 1993; Draberova

et al., 1996; Rege and Hagood, 2006; Rege et al., 2006). SFK are non-

receptor tyrosine kinases that insert their posttranslational lipid modifications

into the cytoplasmic leaflet of the cell membrane to initiate cell signaling via

tyrosine phosphorylation in response to different extracellular stimuli

(Bradshaw, 2010). Thus, it has been proposed that SFKmight be the starting

point for signaling associated with rafts downstream of Thy-1 (Herrera-

Molina et al., 2012; Rege and Hagood, 2006). Accordingly, Thy-1 cluster-

ing induced by Thy-1-specific antibodies modulates SFK in a number of

settings (Barker et al., 2004a; Chen et al., 2006; Stefanova et al., 1991;

Yang et al., 2008).

In addition, Thy-1 clustering requires SFK activity and cholesterol to

become transiently immobile in lipid rafts, as demonstrated using quantum

dot-associated single-molecule tracking in living 3T3 fibroblasts (Chen

et al., 2009a). Moreover, antibody-induced Thy-1 clustering leads to

recruitment of SFK to the membrane and modulates the activity of these

kinases (Chen et al., 2005, 2006, 2009b). Thus, clustered Thy-1, cholesterol,

and SFK seem to represent the elements required for Thy-1 signaling.

How do lipid-anchored proteins attached to opposite sides of the cell

membrane interact? Based on the miscibility of lipid components of the

cell membrane, Kusumi proposed a mechanism to explain cell signaling

by coupling the outer leaflet with the inner leaflet of the bilayer

(Kusumi et al., 2004). When conglomeration of a critical number of

GPI-anchored proteins coincides with interdigitation of miscible unsatu-

rated lipids due to common miscibility in the external layer, then inner-

leaflet rafts and associated proteins are recruited and concentrated beneath

the outer-leaflet domain of the cell membrane. In this manner, the bilayer

could facilitate communication of two proteins on opposite sides of the cell

membrane.


3.2.4 Thy-1 transmembrane transducersIn addition to the evidence pointing toward a mechanism whereby cluster-

ing of GPI-anchored receptors triggers intracellular signals and even con-

nects to the underlying cytoskeleton, a complementary and nonexcluding

alternative hypothesis has been proposed. A transmembrane transducer mol-

ecule might interact with the receptor in the outer leaflet of the bilayer, as

well as with signaling molecules and/or the cytoskeleton via its intracellular

domain. This type of connection has been reported to exist for Thy-1-

induced T-cell activation via LAT (linker for activation of T-cells) phos-

phorylation (Leyton et al., 1999), Thy-1-induced signaling in fibroblasts

via the transmembrane protein PAG1/CBP (protein associated with

glycosphingolipid-enriched microdomains/CBP) (Chen et al., 2009b),

and Thy-1-induced thymocyte activation via the pp85–90 kDa protein,

which was described as being identical to PAG1 (Durrheim et al., 2001).

Altogether, these proteins belong to a group of transmembrane adaptors,

referred to as transmembrane adaptor proteins (TRAPs), possessing a short

extracellular domain, a single-pass transmembrane domain, and a long intra-

cellular tail with the potential to become highly tyrosine phosphorylated

(Leo et al., 2002).

These phosphotyrosine residues might recruit Src-homology 2 (SH2)-

containing proteins, such as SFKs to convey the signaling processes required

to trigger cellular responses. In thymocytes and the T-cell hybridoma 2B4,

LAT is phosphorylated on tyrosine upon Thy-1/CD3 cross-linking with

antibodies, but not upon addition of antibodies to cells lacking GPI-

anchored proteins, suggesting that the LAT adaptor protein plays a role as

a Thy-1-transducer (Leyton et al., 1999). The cytoplasmic domain of LAT

has been found to associate with proteins such as growth factor receptor-

bound protein 2 (Grb-2), phospholipase C gamma 1 (PLC-g1), Vav, casitasB-lineage lymphoma (Cbl), and phosphatidylinositol 3-kinase (PI3-K)

(Cantrell, 1998; Kennedy et al., 1999). CBP has a longer cytoplasmic tail

than LAT and contains multiple tyrosine phosphorylation sites that also serve

as docking sites for SH2-containing proteins. SFKs phosphorylate CBP and

interestingly, one of the proteins recruited via SH2-domains to this tyrosine

phosphorylated protein is the Csk, which inactivates SFKs by phosphorylat-

ing a tyrosine residue that leads to inhibition of their kinase activity (Ingley,

2008; Okada, 2012). Interestingly, LAT and CBP are palmitoylated and this

posttranslational modification is required for partitioning of these molecules

into rafts (Brdicka et al., 1998; Zhang et al., 1998a,b). Thus, evidence

obtained in thymocytes, T-cells, and fibroblasts support a role for TRAPs


as Thy-1-interacting molecules, which could represent the elements

required to initiate Thy-1 signaling in cis; however, whether this is a com-

mon mechanism utilized by other cells, such as neurons or endothelial cells,

remains to be defined.

Another potential transducer has been recently described. The

hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels

were reported to associate with Thy-1 in rat RG cells as shown by

colocalization, coimmunoprecipitation, and electrophysiological experi-

ments. Upon Thy-1 cross-linking with anti-Thy-1 antibodies, the authors

found an activated cation current, which they attributed to these HCN

channels (Partida et al., 2012). Whether different Thy-1 expression levels

in these cells affect the HCN currents or whether the endogenous Thy-1

ligand, the aVb3 integrin (Section 3.3.2.3), triggers changes in the electro-

physiological measurements are issues that have not been approached yet.

3.2.5 Thy-1–aVb5 integrin interactionThy-1 expressed in fibroblasts prevents lung fibrosis (Kis et al., 2011).

Available evidence indicates that Thy-1 interaction with aVb5 integrin pre-

vents transforming growth factor beta 1 (TGF-b1)-induced lung fibroblast

differentiation (Zhou et al., 2010).

TGF-b is produced as an inactive cytokine and forms a latent complex,

which requires activation to bind its cell-surface receptor (Worthington

et al., 2011). Reportedly, integrins aVb3 and aVb5 have been shown to

activate TGF-b by binding to the RGD tripeptide present in the latency-

associated peptide (LAP) of the TGF-b complex (Asano et al., 2005a,b).

Indeed, aVb5 integrin overexpression induces dermal fibroblast differentia-

tion to myofibroblasts, likely through integrin-induced latent TGF-b1activation and subsequent cellular differentiation (Asano et al., 2006). Such a

cascade of events could also be triggered by mechanical stimulation of the

aVb5 integrin by ECM deposition or stiffness (Klingberg et al., 2013; Wipff

et al., 2007). The integrin then interacts with the latent form of TGF-b1through theRGD integrin-binding domain of LAP and activates this cytokine.

Thy-1 possesses an RGD-like domain (Leyton et al., 2001). Via this

RLD tripeptide, it has been demonstrated that, by interacting directly

with aVb5 integrin, Thy-1 prevents the binding of the N-terminal LAP

to the integrin and, in this manner, the activation of TGF-b1, inhibitingalso lung fibroblast differentiation (Fig. 4.4; Zhou et al., 2010). The possi-

bility that a Thy-1-mediated interaction in cis with an integrin could induce

such effects is interesting, particularly in view of the possible therapeutically

Figure 4.4 Thy-1—integrin and Syndecan-4 interactions in trans and cis trigger cellularresponses. In trans, Thy-1 interacts with several integrins to mediate cell–cell bindingand various cellular responses. Thymic Thy-1 mediates binding to thymic epithelia;however, the binding partner(?) and the cellular outcomes are unknown. NeuronalThy-1 binds to aVb3 and Syndecan-4 in astrocytes to induce changes in astrocyte shapeand increased cell adhesion. Thy-1 present in activated endothelial cells interacts withaMb2 and aXb2 integrin, or with CD97 in blood cells to induce extravasation and tissuetransmigration, and with aVb3 integrin in melanoma cells leading to tissue transmigra-tion. Thy-1 in activated fibroblasts associates with a b2-containing integrin to induceadhesion and maturation of dendritic cells. In cis, Thy-1 signaling in neurons is triggeredupon binding with aVb3 in astrocytes, leading to inhibition of neurite outgrowth. Thy-1from endothelial cells induces cell adhesion upon binding to b2- or b3-containingintegrins. In fibroblasts, Thy-1 interaction with a b2-containing integrin triggers cellulardifferentiation; whereas, interaction of Thy-1 with aVb5 integrin inhibits contraction-induced TGF-b1 activation and fibroblast differentiation. Thy-1 is a molecule presentin lipid rafts (represented as darker areas of the lipid bilayer).


beneficial effects that Thy-1 or its peptides could provide in the treatment of

profibrotic disorders of lung fibroblasts.

3.3. Thy-1-triggered cell signaling in cis3.3.1 In fibroblastsFibroblasts are characterized by differential Thy-1 expression, which

accounts, in part, for their heterogeneous phenotypes. Primary Thy-1(þ)

fibroblasts possess well-organized bundles of actin microfilaments, called

stress fibers, as well as elongated points of adhesion, known as focal adhe-

sions, on their ventral surface, as was determined by staining for F-actin

and for the adaptor protein vinculin, respectively. On the other hand,

Thy-1(�) fibroblasts display a rounder cell shape with thinner stress fibers

and smaller adhesion complexes (Barker et al., 2004a; Penney et al., 1992).

Thy-1(�) fibroblasts move faster than Thy-1(þ) ones, as they migrate

more efficiently in in vitro wound-healing assays (Barker et al., 2004a).

A mechanism reported to regulate fibroblast migration involves SFK

and Rho GTPase activation. In embryonic rat lung fibroblasts lacking

Thy-1, heterologous Thy-1 surface expression results in diminished Src

kinase activity, as was shown by decreased activity-associated tyrosine

phosphorylation (Y416). Moreover, decreased activation in the focal

adhesion kinase FAK (Y397) and in the Rho GTPase-activating protein,

p190RhoGAP, provide an explanation for Thy-1-dependent increases in

the RhoA activation state, as assessed in affinity precipitation assays using

Rhotekin-derived Rho-binding domain (RBD)–glutathione sepharose

beads. Thus, because RhoA is known to control functions of the actin

cytoskeleton, it is proposed that Thy-1 expression regulates Src and

FAK kinase activation, as well as phosphorylation of p190RhoGAP,

thereby increasing RhoA-GTP levels, and stress fiber and focal adhesion

formation (Barker et al., 2004a). Thus, decreased migration of Thy-1

(þ) fibroblast subpopulations may occur as the consequence of a complex

Thy-1-triggered signaling process, and not only due to passive Thy-1-

to-matrix adhesion. These results are indicative of Thy-1-dependent

cell-matrix adhesion and migration in fibroblasts.

The participation of Thy-1 in migration of fibroblasts has also been tes-

ted by stimulating themwith the ECM protein thrombospondin-1 (TSP-1),

as well as with a short peptide containing the heparin-binding site, hep I.

TSP-1 expression is upregulated during the initial inflammatory phase of

wound healing and induces a low level of adhesion required for early migra-

tion of fibroblasts in response to injury. Treatment with TSP-1 promoted


disassembly of focal adhesions, surprisingly in Thy-1(þ) fibroblasts only,

suggesting that Thy-1 expression is required for this event (Barker et al.,

2004b). As no differences in expression have been found for components

of the TSP-1 receptor complex, and Thy-1 is not reportedly associated with

this receptor complex, attention has been focused on the aforementioned

Thy-1-controlled Src–FAK–RhoA axis (Rege et al., 2006). Stimulation

with TSP-1 or hep I induces activation of Src within 5 min and FAK after

10 min in Thy-1(þ) fibroblasts. Moreover, Thy-1 coprecipitated with

phospho-Src, phospho-FAK, and FAK, and levels of these components in

the precipitated complex increased upon incubation with hep I. In addition,

fibroblasts transfected with the FAK-related nonkinase, which lacks the

kinase domain, are resistant to hep I-induced FAK phosphorylation and

focal adhesion disassembly.

Thus, Thy-1 is necessary for TSP-1/hep I-induced fibroblast disassem-

bly of focal adhesions, which leads to an intermediate stage of adhesion that

favors cell migration (Rege et al., 2006). Further studies must be performed

in order to establish the mechanism by which Thy-1 modulates TSP-1/hep

I-induced cell migration. Nevertheless, an interesting possibility is that

oligomerized Thy-1 may recruit and inhibit signaling molecules, such as

Src and FAK. As mentioned previously, Thy-1 multimerization might

involve the TREKKKHVLC sequence (Mahanthappa and Patterson,

1992b), which contains the heparin-binding domain (HBD) of Thy-1

(Avalos et al., 2009); therefore, the hep I peptide may promote Thy-1 cluster

disassembly and, as a consequence, release the constraints imposed by such

Thy-1 clusters on Src in these microdomains. This would explain the

requirement for Thy-1 in cells that respond to TSP-1 and, in addition,

why the response appears to be independent of TSP-1 receptor complex

formation.

Subpopulations of Thy-1(þ) and (�) fibroblasts also behave differently

in response to inflammatory processes. Thy-1(þ) fibroblasts are unre-

sponsive to stimulation with different inflammatory factors secreted after tis-

sue injury, such as IL-4, IL-1b, or platelet-derived growth factor (PDGF)

(Zhou et al., 2004). On the other hand, the Thy-1(�) subset of fibroblasts

increase PDGF receptor expression and increase proliferation following

PDGF stimulation (Hagood et al., 1999, 2001, 2002; Shan et al., 2010). This

subpopulation of fibroblasts expresses immune region-associated antigen

(Ia), the rodent major histocompatibility complex (MHC) II antigen, pro-

duces IL-1a, matrix metalloproteinase 9 (MMP-9), and intercellular adhe-

sion molecule 1 (ICAM-1) when exposed to the potent proinflammatory


cytokine TNFa (Phipps et al., 1990). Moreover, upon Thy-1 over-

expression in Thy-1(�) fibroblasts, TNFa-induced expression of MMP-9

and ICAM-1 are attenuated, as their mRNA levels decrease when assessed

by quantitative RT-PCR (Shan et al., 2010). Additionally, MMP-9 lytic

activity is lower in Thy-1(þ) fibroblasts than in their Thy-1(�) counterparts

after treatment with TNFa and TGF-b (Ramirez et al., 2011; Shan et al.,

2010). Thus, the absence of Thy-1 renders fibroblasts highly reactive during

inflammatory processes, whereas the presence of Thy-1 in fibroblasts helps

to modulate their inflammatory response.

Thy-1-dependent molecular mechanisms underlying differential

susceptibility of fibroblasts to inflammatory stimulation also include nuclear

factors. The widely expressed transcription factor NF-kB (nuclear factor-

kappaB) is a well-known mediator of TNFa-induced cellular responses

(Schneider and Tschopp, 2000; Silke, 2011). In nonstimulated cells,

NF-kB remains inactive in the cytoplasm as long as it is bound to its cellular

inhibitor, the I-kB protein. However, upon stress-induced degradation of

I-kB, NF-kB translocates into the nucleus and activates gene transcription

(Li and Lin, 2008). Hagood and colleagues studied the potential role of

NF-kB in Thy-1-mediated TNFa-induced gene expression in fibroblasts.

Notably, in mouse embryonic Thy-1(�) fibroblasts, TNFa stimulation

triggers NF-kB-LUC, but not AP-1-LUC reporter activity, another

transcription factor that mediates TNFa responses (Shan et al., 2010). Thus,

NF-kB seems to be a new actor on the Thy-1 stage that extends the scope of

Thy-1-regulated events into the cell nucleus.

All the above studies characterizing fibroblast subpopulations provide

strong evidence that Thy-1 can modulate the cell phenotype in vitro.

Whereas, Thy-1(þ) fibroblasts are cells that provide support to other cells,

Thy-1(�) fibroblasts secrete inflammatory mediators, as well as proteins

that modify the ECM. Moreover, Thy-1(�) fibroblasts are highly respon-

sive to inflammatory molecules and display morphology characteristics of

migratory/invasive cells under pathological conditions.

Reportedly, loss of Thy-1 is an important event that is observed during

lung fibrogenesis. Thy-1(�) fibroblasts are abundant in lung tissue from

patients suffering from fibrotic lung diseases. In contrast, Thy-1(þ) fibro-

blasts are more prominent in normal lung tissue (Hagood et al., 2005). Strik-

ingly, incubation with TGF-b1, or other molecules related to inflammatory

processes, produces an increase in Thy-1 presence in conditionedmedia, but

this does not correlate with an increase in Thy-1 mRNA in human fibro-

blasts in culture. Also, in a bleomycin-induced lung fibrosis model in Thy-1

knockout mice, the presence of bleomycin-induced proliferative fibroblast


loci correlated with increased amounts of active TGF-b1, as well as phos-phorylated SMA and MAD protein2/3 (SMAD2/3), which are part of

the TGF-b signaling pathway (Hagood et al., 2005). Thus, these results

not only confirm data obtained in vitro but also highlight the relevance of

Thy-1 in the pathogenesis of fibrotic diseases.

Fine-tuning of signals required for survival and apoptosis also determine

the outcome in fibrotic processes. Integrin binding to collagen type-I under

normal conditions results in increased FAK phosphorylation and activation

of the PI3-K/Akt signaling pathway, which leads to either increased cell sur-

vival (Nho et al., 2005; Xia et al., 2004) or cell proliferation (Nho et al.,

2011). On the contrary, when b1 integrin acts as a mechanoreceptor in a

fibrotic process and senses collagen-matrix contraction, the PI3-K/Akt sig-

naling pathway is inhibited and increased apoptosis is observed (Tian et al.,

2002). Such negative regulation is controlled by activation of the phospha-

tase for PI3 lipids, phosphatase and tensin homolog (PTEN), at the plasma

membrane. This phosphatase is present at low levels in fibroblasts from

patients with idiopathic pulmonary fibrosis (Xia et al., 2008). Thus, the

PI3-K/Akt signaling pathway is aberrantly activated leading to pathological

cell proliferation (Xia et al., 2008).

Src negatively regulates PTEN by an unknown mechanism (Liang et al.,

2010), and Thy-1 expression has been reported to inhibit Src in fibroblasts

(Barker et al., 2004a). Thus, in Thy-1(þ) fibroblasts, Src inactivation would

be expected to favor PTEN activity that should diminish PI3-K-mediated

Akt phosphorylation. However, in fibrotic tissues, this regulation would

be lacking, given the absence of Thy-1 and the low levels of PTEN in

fibroblasts. Thus, it would be interesting to test whether overexpression of

Thy-1 leads to PTEN activation and inhibition of the PI3-K/Akt signaling.

3.3.2 In neuronsThy-1-dependent cellular and molecular events in cis in neurons have been

assessed in several ways. Thy-1 levels have been related to neuronal function

or they have been modified to evaluate their impact on cell signaling and

cellular responses. Thy-1-dependent events have also been triggered by

inducing cluster formation with anti-Thy-1 antibodies. More recently,

we have identified the ligand for Thy-1 that, upon binding, assembles

Thy-1 clusters and inactivates Src.

3.3.2.1 Thy-1 levels in cell signaling and cellular responsesStudies performed byRogerMorris described that Thy-1 appearance during

neuronal process establishment coincides with the cessation of neurite


growth in vivo (Morris and Grosveld, 1989). In fact, Thy-1 mRNA is

detected only after new neurons finish migrating into their final niche

and start to form dendrites. Thy-1 protein appears later on in dendrites,

but it is absent from axons until they stop growing (Xue et al., 1990,

1991). In addition, studies reveal that removal of Thy-1 from the neuronal

cell membrane by phosphatidylinositol-specific phospholipase C (PI-PLC)

or silencing of Thy-1 expression facilitates neurite outgrowth in vitro,

leading to the idea that Thy-1 per semight act by inhibiting neurite outgrowth

and/or stabilizing processes that have ceased to grow (Morris, 1992).

Results obtained by Morris indicate that Thy-1 might have little to do

with promotion of neuritogenesis or growth of axonal tracts in the adult

brain (Morris, 1985). Other authors have tested the idea that neuronal

regeneration should be increased in Thy-1 knockout mice; however, the

results were negative (Barlow et al., 2002). Other recent studies have shown

a role for Thy-1 in neuronal regeneration, as Thy-1 levels are drastically

decreased during peripheral regeneration of the sciatic nerve and large

DRG neurons in vivo (Chen et al., 2005). Thus, Thy-1 function in the ner-

vous system remains to be unveiled, but the levels of expression seem to be

related to the capacity of certain neurons to regrow processes.

3.3.2.2 Thy-1-dependent events triggered by anti-Thy-1 antibodiesThy-1 is viewed as a neurite outgrowth inhibitor that stabilizes neuronal

processes. Thus, it was hypothesized that antibodies to Thy-1 should hinder

the inhibitory effects of Thy-1 and thereby increase growth of neurites

(Mahanthappa and Patterson, 1992a). It was later reported that this property

was only detectable with some antibodies (Lipton et al., 1992). In addition,

bivalent, but not monovalent antibodies increased neurite outgrowth of

PC12 cells stimulated with NGF. The signaling events included the influx

of extracellular Ca2þ through the N- and L-type channels (Doherty et al.,

1993). In agreement with these results, abundant andmore complex neurites

were observed in cultured DRG neurons after incubation with a block-of-

function anti-Thy-1 antibody for 6 h. Here, signaling was reported to

involve PKA activation and the activation of the neurogenic mitogen-

activated protein kinase kinase (MEK)–ERK–cAMP response element-

binding protein (CREB) pathway.

The latter pathway coupled PKA activation to Thy-1 function in DRG

neurons (Chen et al., 2007). Presence of the SFK inhibitor PP2 abrogates

the activation of MEK1/2 and CREB, as well as the induction of neurite

outgrowth. Moreover, phosphorylation of Src kinases is induced by anti-


Thy-1 antibodies in DRG neurons (Yang et al., 2008). Since, in these stud-

ies, PKA and Src activation were not shown to occur as a direct consequence

of Thy-1 engagement, the possibility also exists that activation of this signal-

ing cascade is a compensatory response to loss of Thy-1-mediated adhesion.

Moreover, as reported after treatment with block-of-function anti-Thy-1

antibodies, Thy-1 was observed in vesicle-like structures in the cytoplasm

of DRG neurons (Yang et al., 2008). These results were unexpected, since

other authors had reported that Thy-1 is not abundantly endocytosed

(Lemansky et al., 1990). In addition, it has also been described that antibody

treatment induces Thy-1 shedding from the cell membrane (Mahanthappa

and Patterson, 1992a). In any case, the data support a role for Thy-1 in neg-

ative regulation of neurite outgrowth. Thus, unveiling the signaling path-

ways involved in such inhibition will potentially facilitate overcoming the

inhibitory effect of Thy-1 on neurite regeneration. Bearing this in mind,

it would be interesting to know whether compensatory PKA- or Src-

dependent activation of the MEK–CREB cascade would suffice to restore

normal neuronal wiring in Thy-1 knockout mice.

3.3.2.3 Thy-1 responses induced by an endogenous ligandIn 1992, a selective ligand for neuronal Thy-1 was detected in astrocytes cul-

tured for 2–5 months. These astrocytes were able to inhibit neurite out-

growth in Thy-1(þ) but not in Thy-1(�) neurons (Tiveron et al., 1992).

Surprisingly, such inhibition was not observed for astrocytes obtained from

rat embryos and cultured for 2 days. Importantly, the preincubation with

purified Thy-1 abolished astrocyte-mediated inhibition of neurite out-

growth. These results were indicative of the presence of either a ligand or

a strongly attached ECM protein on the astrocyte surface.

In 2001, we described that a b3-containing integrin, present on the surfaceof astrocytes, interacted with Thy-1 present in thymoma cells (Leyton et al.,

2001). These cell–cell interactions were also shown to occur between neuro-

nal Thy-1 and aVb3 integrin in astrocytes, and further described to trigger sig-nals in trans to the astrocytes (Section 4.1). In addition, recombinant aVb3-Fcand Thy-1-Fc fusion proteins were shown to directly bind to one another in a

dose- and bivalent cation-dependent manner, as evaluated by surface plasmon

resonance (Hermosilla et al., 2008). With this in mind, we proposed that

this integrin might also act as a ligand for Thy-1 in neurons and be responsible

for inhibition of neurite outgrowth. Subsequently, we evaluated Thy-

1-mediated effects of aVb3 integrin on growth and retraction of neuronal pro-cesses in astrocyte–neuron cocultures. There, we found that aVb3 integrin,


present on astrocytes or added as an aVb3-Fc fusion protein to primary neu-

rons maintained for 4–7 days in culture, inhibits neurite outgrowth.

Additionally, the integrin binds Thy-1 at the plasma membrane, and this

effect is blocked by pre-treatment of the neurons with PI-PLC. Likewise,

aVb3-Fc is sufficient to inhibit neurite extension in Thy-1(þ), but not in

Thy-1(�) neurons, whereas b3 integrin(�) astrocytes were permissive to

neurite outgrowth. Moreover, aVb3-Fc-induced retraction of Thy-1(þ)-

Figure 4.5 Nanoresolution microscopy to reveal Thy-1 clustering. The panels show live-cell staining against Thy-1 using anti-Thy-1 clone OX7 in hippocampal neurons culturedfor 14 days in vitro following published protocols (Herrera-Molina et al., 2012). An ATTO647N-conjugated secondary antibody was used to label Thy-1 clusters and perform con-focal (upper photograph) and stimulated emission depletion microscopy (STED, middleand bottom photographs). Moreover, STED nanoresolution can be further increased byapplying an image deconvolution procedure (Deconv, bottom). Note that Thy-1 clustersseem to be compact when analyzed by confocal microscopy; however, STED resolutionreveals the presence of several individual nanoclusters of Thy-1 (arrow). Scalebar¼2 mm. Section included in the small rectangle is shown at the upper right of eachpicture with a digital zoom of 3�.


neurites in differentiated neurons. Furthermore, we described that aVb3integrin induces Thy-1 clustering on live neurons, leading to Thy-1–Src

kinase codistribution, and increased inhibition-associated phosphorylation

on Tyrosine-527 of Src kinase (Herrera-Molina et al., 2012).

Currently, we are evaluating how aVb3 integrin modifies Thy-1 lateral

mobility and produces large Thy-1 clusters on the neuronal cell membrane

by using quantum dots and stimulated emission depletion (STED) micros-

copy (Fig. 4.5). Therefore, after years of speculation, the identity of an

endogenous ligand for Thy-1 present in astrocytes has been uncovered.

However, although the enigma of the orphan Thy-1 receptor has been

unveiled, the actual in vivo role of this aVb3 integrin–Thy-1 interaction

remains an important issue to be resolved.

4. TRANS-INTERACTING Thy-1 MOLECULESAND SIGNALING

Available evidence indicates that Thy-1 interacts with specific

molecules on the surface of target cells and induces a variety of physiological

processes, such as adhesion of thymocytes to thymic epithelium, leukocyte/

monocyte extravasation, and tissue transmigration (Fig. 4.4). In addition,

Thy-1 has been linked to pathological conditions, such as atherosclerosis,

glial scar formation, and cancer cell metastasis. In this section, we will focus

on the physiological relevance of cell–cell interactions mediated by Thy-1,

with special emphasis on the cell-signaling events downstream of receptors

present on the surface of astrocytes, melanoma, and blood cells.

4.1. In astrocytesNeuronal Thy-1, as well as recombinant Thy-1-Fc, induces morpholog-

ical changes in astrocytes (Fig. 4.6). Our laboratory has shown that Thy-1

induces focal adhesion and stress fiber formation in astrocytes by the

engagement of aVb3 integrin and Syndecan-4. Astrocytes attached to tissueculture plates increase their adhesion to the underlying substrate when

incubated with the fusion protein Thy-1-Fc or with neuron-like cells,

which contain Thy-1 (Fig. 4.6; Avalos et al., 2004; Avalos et al., 2002,

2009; Hermosilla et al., 2008). Upon Thy-1 binding, the points of contact

of the astrocytes with the matrix increase in size and complexity, forming

focal adhesions. These points of contact between the plasma membrane

and the ECM tether bundles of intracellular microfilaments (stress fibers)

Figure 4.6 Signaling triggered by Thy-1–integrin and Syndecan-4 interactions in trans.Signaling in trans triggered by Thy-1–integrin or Syndecan-4 interactions has beendescribed in astrocytes. Upon integrin ligation, FAK is recruited and phosphorylatedon tyrosine. A complex with paxillin, vinculin, and p130Cas is formed. These eventsare followed by ATP release, which binds to purinergic P2X7 receptors, thereby leadingto influx of Ca2þ to the cytosol. A conventional PKC, PKCa, is thus activated, whichmightform a complex with Thy-1-engaged Syndecan-4 and lead to the activation of the smallGTPase, RhoA, and its effector protein ROCK. RhoA could also be activated downstreamof integrin signaling. These signaling cascades trigger morphological changes andincrease cell adhesion.


to the membrane and increase cellular tension upon integrin engagement

(Dubash et al., 2009). Thus, Thy-1 interaction with aVb3 integrin and

Syndecan-4 promotes adhesion of astrocytes to the underlying substrate,

which in these experiments were presumably ECM proteins secreted by

the astrocytes themselves.

Thy-1 interaction with astrocyte receptors involves different amino acid

sequences that are conserved in rat, mouse, and human. Thy-1 binds to

integrins via a conserved RLD amino acid sequence (Fig. 4.2; Avalos

et al., 2009; Hermosilla et al., 2008; Leyton et al., 2001). Mutation of this

RLD sequence (RLD mutated to RLE) inhibits Thy-1-Fc binding to the

aVb3 heterodimer and Thy-1-induced astrocyte adhesion and spreading,

suggesting that Thy-1–integrin interaction is required to induce the astro-

cyte responses (Hermosilla et al., 2008; Leyton et al., 2001). Alternatively,

Thy-1 interaction with Syndecan-4 occurs via a HBD. The presence of this

putative HBD in the Thy-1 sequence was described in the early 1990s

(Hueber et al., 1992); however, its identity remained unknown. This


sequence was only revealed in 2009, when our group described that the use

of heparin or pretreatment of astrocytes with heparitinase inhibited the focal

adhesion and stress fiber formation triggered by Thy-1 (Avalos et al., 2009).

In addition, substitution of positively charged amino acids in the putative

mouse HBD domain (corresponding to sequence R38EKRK42, Fig. 4.2) by

mainly hydrophobic amino acids (AEAAA) abrogates the interaction of

Thy-1-Fc with heparin beads and inhibits Thy-1-induced focal adhesion

and stress fiber formation in astrocytes (Avalos et al., 2009). These results

revealed the identity of the HBD in the Thy-1 molecule and underscored

the importance of this domain in Thy-1 function. In addition, silencing the

expression of Syndecan-4 with siRNA or using a mutated form of

Syndecan-4 lacking the cytoplasmic domain inhibits Thy-1-induced astro-

cyte adhesion, corroborating the requirement of Syndecan-4 for Thy-1-

induced effects on astrocyte morphology (Avalos et al., 2009).

Stimulation of astrocytes with Thy-1-Fc triggers a signaling cascade

typically described to elicit increased cell adhesion. Thy-1 binding to

the astrocyte receptors recruits FAK, Paxillin, and Vinculin to the focal

contacts. Moreover, p130Cas and FAK are tyrosine phosphorylated after

Thy-1-Fc stimulation (Leyton et al., 2001). In addition, Thy-1-triggered

focal adhesion formation is dependent on the activation of protein kinase

C alpha (PKCa), the small G protein RhoA and its effector p160ROCK

(Avalos et al., 2009). Because PKCa is a conventional PKC, which

requires Ca2þ and diacylglycerol for its activation, we determined the ori-

gin of the intracellular Ca2þ increments required. Our reported data

indicate that a rise in intracellular calcium occurs in astrocytes upon

Thy-1 stimulation via release of adenosine triphosphate (ATP) and subse-

quent activation of the ionotropic P2X7 receptor. This receptor forms a

large, nonselective cation pore upon activation that permits influx of extra-

cellular Ca2þ (Fig. 4.6). Importantly, this signaling mechanism requires

Thy-1–integrin binding, since upon mutation of the Thy-1 integrin-

binding site (RLD to RLE mutation), Thy-1 neither triggers ATP release

nor calcium influx (Henriquez et al., 2011). At present, the complete

sequence of events triggered downstream of integrin or Syndecan-4 in

astrocytes stimulated by Thy-1 is still unclear, but several molecules known

to participate in fibroblast adhesion to the ECM protein fibronectin are

likely to be involved.

Most signaling pathways triggered by Thy-1 in astrocytes have been elu-

cidated by stimulating astrocytes with Thy-1-Fc fusion protein. However,

we have also shown that Thy-1 present on the surface of the neuron-like cell


line CAD induces focal adhesion and stress fiber formation in astrocytes.

These morphological changes occur by activating RhoA/ROCK (Avalos

et al., 2002, 2004, 2009; Hermosilla et al., 2008). Moreover, focal adhesion

formation occurs in the proximity of neuron–astrocyte contact sites and is

inhibited by treating CAD cells with anti-Thy-1 antibodies (Hermosilla

et al., 2008).

Taken together, these results suggest that the engagement of neuronal

Thy-1 with its receptors aVb3 integrin and Syndecan-4 on the surface of

astrocytes, triggers specific signaling pathways that induce actin reorganiza-

tion and changes in astrocyte morphology (Figs. 4.4 and 4.6). This phenom-

enon described in vitro might account for the dramatic morphological

changes produced in astrocytes in response to wounding in the brain, where

an inflammatory environment is created. All three molecules, Thy-1,

integrin, and Syndecan-4, reportedly change their expression in inflamma-

tory conditions (Blain et al., 2004; Ellison et al., 1998, 1999; Lee et al.,

1998). Thus, under these circumstances, where astrocytes become reactive,

adhere, spread, and then migrate to the site of injury to form the glial scar

(Silver and Miller, 2004), Thy-1 interaction with its receptors on astrocytes

might be responsible for some of the changes these cells undergo; however,

this remains a working hypothesis that needs to be tested.

4.2. In melanoma cellsMetastasis depends on the levels and interactions of cell adhesion molecules

(CAMs) expressed on endothelial cells, as well as on tumor cells. Cadherins,

integrins, selectins, and immunoglobulin family molecules are CAMs that

have been implicated in this process. Some of these molecules are expressed

constitutively in a tissue-specific pattern, whereas others are induced by

inflammatory responses, free radicals, bioactive lipids, and growth factors.

Subsequently, cell signaling triggered downstream of these adhesion mole-

cules leads to endothelial cell retraction and cancer cell transmigration

through the blood vessel wall to establish secondary tumors in other organs

(Aplin et al., 1998; Bendas and Borsig, 2012; Howe et al., 1998).

In particular, Thy-1 expression has been demonstrated in situ in endothe-

lial cells activated by inflammation or in adult angiogenesis, but not in resting

endothelial cells (Lee et al., 1998; Saalbach et al., 1999, 2002; Schubert et al.,

2011). In addition, endothelial cells, contiguous to melanoma cells or mela-

noma metastasis, express Thy-1 on their surface. In vitro, Thy-1 expression is

induced by phorbol 12-myristate 13-acetate (PMA), TNFa, or vascular endo-thelial growth factor (VEGF) on human dermal microvascular endothelial


cells (HDMECs) at low passage number (up to 3 passages) suggesting that

Thy-1 expression is due to a specific inflammatory response (Schubert

et al., 2013). Moreover, Thy-1 expression in endothelial cells is also induced

by melanoma cell-conditioned media (Saalbach et al., 1999, 2002); however,

the melanoma cell-derived soluble factors responsible for Thy-1 upregulated

expression remain unknown.

Interestingly, melanoma cells expressing aVb3 integrin can adhere

specifically to Thy-1 expressed on the surface of activated endothelial cells

(Saalbach et al., 2005). The role of aVb3 integrin as a ligand of Thy-1 was

demonstrated by adhesion assays of melanoma cells to an activated

HDMEC monolayer under static, as well as under constant shear flow

conditions. The use of blocking antibodies against Thy-1 (B-C9) and

aVb3 integrin (LM609) results in a significant decrease (about 50–60%)

in melanoma cell adhesion to Thy-1-expressing HDMECs. Additionally,

Thy-1/aVb3 interaction is important for transmigration of melanoma cells

across HDMEC monolayers (Fig. 4.4), as shown using transwell assays.

Even though the decrease in cell adhesion and transmigration in these

assays was significant, neither were completely abolished by blocking

the Thy-1/aVb3 interaction, suggesting that other adhesion molecules

may be implicated. One possible candidate is Syndecan-4, since it also

participates in Thy-1/integrin-mediated cell adhesion in astrocytes

(Avalos et al., 2009). Importantly, Syndecan-4 has been described as an

important player in tumor cell adhesion (Beauvais and Rapraeger, 2004)

and in melanoma invasion (O’connell et al., 2009). Further studies are nec-

essary to establish the potential role of Syndecan-4 in the interaction of

melanoma cells with activated endothelial cells.

Expression of aVb3 integrin in melanoma cells is a marker for poor clin-

ical prognosis in patients, since presence of the integrin is associated with

increased invasive and metastatic potential of human melanoma cells

injected into nude mice (Johnson, 1999). Furthermore, activation of

aVb3 integrin on melanoma cells decreases apoptosis and stimulates tumor

growth, as well as matrix invasion. Indeed, because of the key role of

aVb3 in melanoma progression, antibodies to this integrin have been used

in preclinical and clinical trials (O’day et al., 2011; Trikha et al., 2004).

A different study indicates that the increased expression of aVb3 integrin

is not sufficient to make melanoma cells more invasive. Instead, these cells

are suggested to additionally require constitutive Src activation and/or ele-

vated PKCa or PKCd expression to become highly metastatic cells (Putnam

et al., 2009). All these signaling events could be triggered by Thy-1 upon


interaction with aVb3 integrin (Avalos et al., 2009). Thus, since an active

endothelium is required for increased invasion, and activated endothelial

cells expressed Thy-1, one might speculate that Thy-1–integrin interaction

enhances melanoma cell metastasis. However, whether aVb3 integrin func-

tion in melanoma progression is linked to interaction with Thy-1 is a topic

that requires further investigation.

4.3. In blood cellsInteraction of inflammatory cells with endothelial cells is significant during

immune and inflammatory responses, including infection, artherosclerosis,

psoriasis, rheumatoid arthritis, and asthma. Leukocytes in the blood stream

interact with adhesion molecules on the endothelial cells in a multistep pro-

cess, which includes capture of circulating leukocytes, subsequent rolling of

the leukocytes, arrest and firm adhesion to the endothelium, and transmigra-

tion via diapedesis across the endothelium into perivascular tissues during

inflammation (Ley et al., 2007; Worthylake and Burridge, 2001).

Thy-1 expressed in activated endothelial cells has been involved in the

control of inflammatory cell recruitment and the modulation of the inflam-

matory microenvironment (Schubert et al., 2011;Wetzel et al., 2004). Both,

integrins aXb2 (CD11c/CD18; P150, 95) and aMb2 (CD11b/CD18;

MAC-1) have been described as receptors for Thy-1 in blood cells (Choi

et al., 2005; Wetzel et al., 2004). Thus, Thy-1-containing endothelial cells

may selectively recruit inflammatory cells via binding to these integrins

(Fig. 4.4). The importance of the expression of Thy-1 in activated endothe-

lial cells for adhesion of leukocytes has been studied using various models of

induced inflammation in Thy-1�/� mice and their wild-type littermates.

In the thioglycollate-induced peritonitis model, the recruitment of mono-

cytes and neutrophils was reduced in Thy-1-deficient mice compared with

wild-type mice, where infiltration of CD11b (MAC-1) cells, F4/801 mac-

rophages, and Gr-I1 neutrophil granulocytes was apparent in the peritoneal

tissue. Additionally, levels of eotaxin-2 and MMP-9 were enhanced in the

peritoneal fluid of wild-type mice compared with Thy-1�/� mice

(Schubert et al., 2011). Moreover, the recruitment of eosinophils and mac-

rophages was reduced in Thy-1�/� mice compared with their wild-type

littermates during acute and chronic lung inflammation induced by

ovoalbumin. As a consequence, the broncheo-alveolar lavage of wild-type

mice contained increased levels of IL-4, IL-5, MIP-1a (CCL3), TARC

(CCL17), eotaxin-2 (CCL24), and MMP-9 compared to Thy-1�/� mice


(Schubert et al., 2011). Therefore, Thy-1 appears to modulate the inflam-

matory response by selectively recruiting specific cells to the lesion site.

A nonintegrin-interacting partner for Thy-1 has recently been described

in leukocytes. Thy-1 expressed by activated endothelial cells was shown to

bind leukocytes via interaction with the seven-transmembrane G protein-

coupled receptor CD97 and mediate strong adhesion of these blood cells

to endothelial Thy-1 (Wandel et al., 2012). Thus, reduced recruitment of

leukocytes in Thy-1-deficient mice undergoing inflammatory processes

might be due to the absence of Thy-1 interactions with not only b2integrin-containing cells but also with other counteracting molecules pre-

sent in blood cells (Fig. 4.4).

5. FUNCTION OF Thy-1 MOLECULE

5.1. In fibroblasts
Fibroblasts constitute a multifunctional cell population with an established
role in wound healing, tumor formation, and regulation of the immune

response (Kalluri and Zeisberg, 2006; Tomasek et al., 2002). ECM proteins

are deposited mainly by fibroblasts; in this manner, fibroblasts control many

of their own cellular functions and also those of neighboring cells. Such cel-

lular functions include, cell shape, adhesion, motility, and differentiation

(Laurent et al., 2007; Midwood et al., 2004). Additionally, in stress situa-

tions, fibroblasts secrete a number of inflammatory molecules, which are

indicative of their relevance in pathological conditions (Kisseleva and

Brenner, 2008; Smith, 2005).

In fibroblasts, Thy-1 is expressed constitutively. However, the large

degree of heterogeneity observed among functionally distinct fibroblast sub-

populations may be attributed to differential expression of Thy-1. Accord-

ingly, differences in cell morphology, in the production of and response to

cytokines and growth factors, in proliferation and in differentiation,

between fibroblasts that express Thy-1 and fibroblasts that do not express

this protein have been described (Barker et al., 2004b; Hagood et al.,

1999, 2002; Khoo et al., 2008; Koumas and Phipps, 2002; Koumas et al.,

2002; Lehmann et al., 2010).

5.1.1 In cell morphology, adhesion, and migrationThe structural similarity of this glycoprotein to proteins of the immunoglob-

ulin superfamily suggested early on a role for Thy-1 in cell adhesion and

morphology as previously described (Sections 1 and 2.3.1). Thy-1


expression levels also affect fibroblast migratory properties, with the conse-

quent changes in the arrangement of the cytoskeleton. As stated, fibroblasts

that express surface Thy-1 have stronger focal adhesions and stress fibers

(Barker et al., 2004b). Although some cell adhesion and contraction is

required for cell migration, cells with more static adhesions are reportedly

less motile (Truong and Danen, 2009). However, upon stimulation with

TSP-1 or the short-TSP-1-derived peptide, hep I, only Thy-1(þ) fibro-

blasts activate signaling pathways that lead to disassembly of focal adhesions

and increased migration, possibly by interfering with Thy-1-induced

RhoA activation (Barker et al., 2004b) (see Section 3.3.1 for a detailed

discussion).

5.1.2 In inflammation and the immune responseNumerous molecules, such as growth factors, chemokines, and ECM-

degrading proteases produced by injured tissue and the infiltrating cells,

induce the activation of fibroblasts, which is important in regulation of

the immune response. Upon activation, fibroblasts produce paracrine

immune modulators that include growth factors, cytokines, and inflamma-

tory mediators (Flavell et al., 2008; Smith et al., 1997), key products for tis-

sue remodeling and repair. Interestingly, fibroblasts have been reported to

increase expression of Thy-1 during the early phase of healing, suggesting

that fibroblast activation results in an upregulation of Thy-1 (Bradley

et al., 2009; Saalbach et al., 1998). These findings are in agreement with

the detection of high soluble Thy-1 levels at sites with massive inflammation

in the synovial fluid of patients with rheumatoid arthritis, psoriasis, or other

inflammatory diseases (Saalbach et al., 1999).

Moreover, it has been reported that mouse lung fibroblasts lacking

Thy-1 on their surface express MHC class II molecules and produce

IL-1, when stimulated with interferon-g (IFN-g) or TNFa, respectively(Phipps et al., 1989, 1990). Since IL-1 plays an important role in the initi-

ation of immune responses, and the expression of MHC class-II occurs in

accessory cells for presentation of antigens to T-cells, these observations sup-

port the view that Thy-1(�) fibroblasts may function as antigen-presenting

cells under inflammatory conditions.

A different study demonstrated that fibroblasts actively participate in reg-

ulating the function of dendritic cells, specialized cells that play a key role in

initiating immune responses (Saalbach et al., 2007). The authors suggested

that dendritic cells, during their transit from peripheral to lymphoid tissues,

might adhere to fibroblasts through ICAM-1 and Thy-1. Thy-1 expressed


on activated fibroblasts can interact with b2 integrin on dendritic cells induc-ing cell adhesion and maturation of these cells (Fig. 4.4), as evidenced by the

presence of CD83, C86, CD80, and HLA-DR surface antigens. Cell–cell

contact seems to be necessary to induce the secretion of TNFa by dendritic

cells and their consequent maturation, suggesting that engagement of b2integrin by Thy-1 may activate signaling pathways that lead to the synthesis

and/or secretion of TNFa (Saalbach et al., 2007).

Another model associated with lung inflammation describes the bio-

logical significance of the expression of Thy-1 in fibroblasts during

fibrogenesis. Results obtained in mouse embryonic fibroblast (MEF) cells,

corroborated in the rat fetal lung fibroblast line (RLE6), suggest that the

expression of Thy-1 attenuates TNFa effects on activation of genes,

including MMP9, ICAM-1, and a reporter controlled by the promoter

of IL-8 (Shan et al., 2010). Similar results were obtained in primary human

Thy-1(�) fibroblast lines from patients with idiopathic pulmonary fibrosis,

which express MMP9 upon stimulation with TGF-b, an effect that was not

observed when using an antagonist of TGF-b. These results are in agree-

ment with the elevated expression of MMP9 found in Thy-1(�) fibroblasts

in vivo, in tissue samples obtained from patients with the same disease

(Ramirez et al., 2011).

5.1.3 In cell proliferation and differentiationIt has been reported that the loss of Thy-1 expression in lung fibroblasts

correlates with many aspects of the fibrogenic phenotype including

proliferation. Thy-1(�) cells are able to proliferate in the presence of the

PDGF-AA, a potent fibroblast mitogen, due to the expression of higher

PDGF receptor-a levels (Hagood et al., 1999). Indeed, Imatinib mesylate

(1 mM), a small compound known also as Gleevec, which competitively

inhibits tyrosine kinases, completely precludes proliferation stimulated by

PDGF (Sandler et al., 2006). This feature has been exploited to treat rheu-

matoid arthritis, because PDGF is a potent mitogen for synovial fibroblasts

isolated from these patients (Pereira et al., 2010).

Additional studies indicate that for rat lung fibroblasts, which lack Thy-1

on their surface, expression of myogenic genes, as well as protein levels of

a-SMA, sarcomeric myosin, and MyoD, among others, are elevated in

response to promyofibroblastic stimuli including TGF-b (Sanders et al.,

2007). In addition, Thy-1(�) fibroblasts are more resistant to serum

starvation-induced apoptosis in contracting collagen gels for 48 h and

develop more contractile activity than the Thy-1(þ) fibroblast after


stimulation with fibrogenic agents. These features indicate that Thy-1(�)

fibroblasts are more responsive to profibrotic cytokines than Thy-1(þ) cells

and that absence of Thy-1 facilitates their differentiation to myofibroblasts

and survival (Sanders et al., 2007).

The balance between Thy-1(�) and Thy-1(þ) fibroblast populations

may be critical in the development and progression of diseases. Diff-

erentiation of fibroblasts to adipocytes is also relevant to the Graves’

ophthalmopathy. This is a condition associated with Graves’ hyperthyroid-

ism, in which an inflammatory autoimmune disorder results in remodeling

and expansion of the connective and fat tissue, including proliferation and

differentiation of fibroblasts to adipocytes (Smith, 2005). Interestingly, this

differentiation process correlates inversely with the expression of Thy-1 on

the surface of these cells (Koumas et al., 2002, 2003). However, recent

data strongly support the hypothesis that Thy-1(þ) orbital fibroblasts

may also differentiate to adipocytes, but that they secrete a paracrine factor

which inhibits adipogenesis induced by 15-deoxy-D12,14-prostaglandin J2(15d-PGJ2), a peroxisome proliferator-activated receptor gamma (PPAR-g)ligand (Khoo et al., 2008; Lehmann et al., 2010). These data suggest that a

soluble factor produced by Thy-1(þ) cells inhibits fibroblast differentiation.

Thus, it would be interesting to identify this factor and assess whether

it is a specific inhibitor of adipogenesis or whether it may also inhibit

differentiation of fibroblasts to other cell types, such as myofibroblasts.

Intriguingly in this case, and in contrast to what has been reported for pri-

mary lung fibroblasts, profibrotic cytokines induce differentiation of orbital

Thy-1(þ) fibroblasts into myofibroblasts, while Thy-1(�) cells differentiate

into lipofibroblasts, likely indicating a tissue-specific effect (Koumas et al.,

2002, 2003).

5.2. In brain cells5.2.1 In neuronal process extension and regenerationA number of studies show that Thy-1 is expressed in different neurons and at

different levels in the developing and adult brain (Section 2.2.2). These dif-

ferences in Thy-1 protein levels correlate with decreased axonal elongation

and increased synapse formation (Liu et al., 1996; Schmid et al., 1995),

suggesting that Thy-1 functions by inhibiting the growth of processes to sta-

bilize connectivity once contacts have been formed (Mahanthappa and

Patterson, 1992a; Morris, 1985; Tiveron et al., 1992; Xue and Morris,

1990; Xue et al., 1991).

In peripheral sensory neurons, Thy-1 also has a negative regulatory role

in axonal growth (Chen et al., 2005). Accordingly, levels of Thy-1 mRNA


and protein are reduced following damage to the nervous system (Chen

et al., 2005; Huang et al., 2006). In sciatic nerve crush experiments,

Thy-1 levels are low in DRG cells for the first 2 days, but increase with time

as the animals recover their sensory functions (Chen et al., 2005). Thus, in

agreement with its function as an inhibitor of neurite outgrowth, Thy-1 dis-

appears when axons need to grow, but increases as functional maturation is

reached. Thus, the same function attributed to Thy-1 during development is

observed upon injury, when neurons regenerate their processes.

Interestingly, our unpublished data indicate that both neuron-like cell

lines, PC12 and CAD cells decrease Thy-1 levels when induced to differ-

entiate in culture. Both PC12 and CAD cells recover the initial expression

levels after 24 h in serum-free medium with or without NGF, respectively

(Fig. 4.7 for PC12 cells). These results support the described notion that

Thy-1 presence is a subcellular restriction to process extension.

Huntley and coworkers used Thy-1 null mice to obtain evidence indi-

cating that, after injury in vivo, axonal maintenance or functional synaptic

connections in early stages of development or regeneration and plasticity

in the central nervous system of mature mice occur even in the absence

of Thy-1. Taken together, these observations indicate that Thy-1 is not fun-

damental in these processes (Barlow et al., 2002).While this might indicate a

more restricted role for Thy-1 than anticipated, similar modest effects have

PC12 cells

0 8 2412 72 144 h

Thy-1

β-Actin

Figure 4.7 Thy-1 expression levels initially decreased upon induction of differentiation.For PC12 cells, plates were coated with poly-L-lysine and the cells were added to theplates in RPMI medium supplemented with 10% FBS to allow cell proliferation andadhesion to the plate for 16 h. Then, the medium was changed to starve the cells inRPMI/1% FBS for 15 h. Finally, differentiation was induced by addition of 100 ng/mlnerve growth factor (NGF). Cells were harvested at the time points indicated, then sol-ubilized in a 50 mM Tris–HCl (pH 7.4) lysis buffer containing 0.15 N NaCl, 1% Nadeoxycholate, 1% NP-40, 0.1% SDS, 5 mM EDTA, protease and phosphatase inhibitors,precipitated to yield equivalent amounts of protein per lane and resuspended in samplebuffer. Proteins were separated by gel electrophoresis and transferred to nitrocellulosemembrane. Antigens immobilized on the membrane were detected using polyclonalanti-Thy-1 antibodies followed by an anti-rabbit IgG-horseradish peroxidase andenhanced chemiluminescence to reveal bound peroxidase activity. Actin was used asa control for protein loading. In PC12 cell extracts, a decrease in Thy-1 levels wasobserved during the first 8–12 h.


been reported for other molecules involved in neural adhesion, such as L1

andN-CAM (Hynes, 1996). Moreover, adult neurons are intrinsically capa-

ble of growing long processes when seeded in supportive surroundings.

However, in mammals, neuronal regeneration upon brain lesion or dam-

age is very poor in the central nervous system, suggesting that lack of axo-

nal regeneration is due to the nonpermissive environment that exists in the

brain. Long-term residency of these axonal-growth inhibitory compo-

nents accounts in part for the poor repair capacity of the brain

following injury.

The best-characterized inhibitory molecules to date are those present in

degenerating myelin (Mukhopadhyay et al., 1994; Ng et al., 1996) and in the

ECM (Mckeon et al., 1991, 1995). Myelin-derived proteins include NogoA,

myelin-associated glycoprotein (MAG), and oligodendrocyte-myelin glyco-

protein (OMgp). All of these recognize a single receptor identified as the

GPI-anchored protein NogoR in neurons (Domeniconi et al., 2002;

Fournier et al., 2001; Wang et al., 2002). Another well-known axonal-

growth inhibitory molecule, greatly upregulated upon lesion, is the chondroi-

tin sulfate proteoglycan (CSPG), a component of the ECM in the brain

(Mckeon et al., 1991, 1995) whose receptor was identified as the phosphatase

PTPs present in neurons (Shen et al., 2009). Blocking myelin-associated pro-

teins or CSPG has proven useful to achieve some degree of neuronal repair;

however, none of these strategies restores completely the damaged tissue.

Similarly, deletion of the Thy-1 gene and protein might not be sufficient to

counteract the overall nonpermissive environment that exists in the brain.

Additional reports using Thy-1 null mice indicate that Thy-1 may also

play a role regulating channels or other important constituents of membrane

signaling, since changes in gamma-amino butyric acid (GABA)ergic inhib-

itory function have been described in these knockout mice (Nosten-

Bertrand et al., 1996). Despite a clear impairment in LTP formation due

to exacerbated GABAergic inhibitory activity in the dentate gyrus, Thy-1

knockout mice develop almost normally without other apparent defects

(Mayeux-Portas et al., 2000). However, Thy-1 null mice do display alter-

ations in their social behavior, indicating that Thy-1 may play a role in syn-

aptic activity in the brain. Accordingly, although the location of Thy-1 is

mainly confined to the surface of neurons (Dotti et al., 1991), it has also

been described as an integral component of secretory vesicles, including

synaptic-like vesicles and dense-core vesicles, capable of fusing with the

plasma membrane in a response regulated by increased intracellular calcium

levels in PC12 cells and brain tissue ( Jeng et al., 1998).


Results obtained by analyzing the neuronal phenotype of Thy-1 knock-

out mice have raised more questions than answers. To the best of our under-

standing, there are at least three possible explanations for such a moderate

phenotype. First, Thy-1 deficiency might be compensated by the presence

of other CAMs (quite abundant in neurons), which restore potentially

impaired cell signaling. However, Thy-1 is a highly abundant, and conserved

molecule in neurons; thus, it would be expected to play a more significant

role. Second, Thy-1 function may involve low affinity or low-specificity

interactions. Third, the context in which Thy-1 deficiency was evaluated

might have been inadequate.

5.2.2 In astrocyte cell adhesion and morphologyThy-1 was described to inhibit neurite outgrowth in vitro when induced to

differentiate over a monolayer of mature astrocytes (Tiveron et al., 1992).

These results lead to the hypothesis that a ligand for Thy-1 existed on the

surface of these mature astrocytes. Later on, another report showed that

in the presence of neuronal Thy-1, immature astrocytes promoted neurite

outgrowth, which was blocked by anti-idiotype antibodies against Thy-1

antibodies, arguing that Thy-1 binding to a putative receptor on astrocytes

might induce neurite outgrowth or block an inhibitory effect of Thy-1.

Because the latter experiments were performed using immature astrocytes,

a role for Thy-1 and its ligand or receptor in astrocytes was suggested in brain

development (Dreyer et al., 1995). Interestingly in both cases, a biological

role for Thy-1 was attributed to a molecular partner present on astrocytes.

Using an astrocyte cell line, we described that astrocytes indeed possess

two receptors, aVb3 integrin and Syndecan-4, that interact with different

domains of Thy-1 (Fig. 4.2) and trigger morphological changes in astrocytes

(Avalos et al., 2002, 2009; Hermosilla et al., 2008; Leyton et al., 2001).

These involve dramatic changes in cytoskeletal activity to initiate the forma-

tion of stress fibers and numerous points of adhesion of the cells to the under-

lying substrate. Cell adhesion and spreading, as well as subsequently

increased cell contraction were facilitated by the focal adhesions and stress

fibers that formed in the presence of Thy-1 (see Section 4.1 for a detailed

discussion on this topic).

5.3. In endothelial cells of vascular and lymphatic endotheliumThy-1 is expressed in the endothelium of small blood vessels specifically

those exposed to inflammatory conditions or involved in angiogenesis

(Lee et al., 1998), and thus represents a marker of activated endothelial cells


(Ishizu et al., 1995; Takeuchi et al., 1997). Moreover, its expression is even

higher in the endothelium of lymphatic vessels, especially when these vessels

are associated with tumors (Jurisic et al., 2010).

The activation of endothelial cells at sites of inflammation, injury, or

infection facilitates the adhesion of leukocytes to these activated cells in a

manner that is reportedly Thy-1 dependent (Saalbach et al., 2000). Thy-1

has been shown to specifically interact with Mac-1 (CD11b/CD18;

aMb2) and CD97 from human leukocytes, and thereby permit initial strong

adhesion of these cells to activated endothelium followed by subsequent

trans-endothelial migration (Wandel et al., 2012; Wetzel et al., 2004).

These results point toward a role for cell–cell interactions via a CAM of

the immunoglobulin superfamily and an integrin and/or other molecule

such as CD97, as an additional signaling route that promotes the adhesion

process of leukocytes and potentially promotes invasion of leukocytes into

the inflamed tissue (Wandel et al., 2012; Wetzel et al., 2004).

Endothelial cells in lymphatic and blood vessels provide anatomic path-

ways for the trafficking of cancer cells from primary tumor to the sites of

distant metastases. Morphological studies suggest that cancer cells enter

the bloodstream primarily through small veins or sinusoidal neovasculature

of the primary tumor. For this event to occur, it is essential that cancer cells

adhere to the vessel, degrade their matrix, and travel to the site of metastasis

(Wai Wong et al., 2012). Available evidence indicates that Thy-1 is an

inducible CAM on microvascular endothelial cells, which upon interaction

with its ligand aVb3 integrin, favors adhesion of melanoma cells to endothe-

lial cells and promotes their migration (Saalbach et al., 2002, 2005). Thus,

Thy-1–integrin interaction might be relevant to allow transport of cancer

cells via the bloodstream to distant sites.

5.4. As a cell biomarkerThy-1 has been used as a marker for RG cells (Chidlow et al., 2005),

T lymphocytes, fibroblasts, stem cells, and cancer cells (see Section 2.2

for more details). Most experimental procedures aiming to identify bio-

markers for cancer diagnosis are focused on the tumor cells. Now, the

importance of the surrounding stroma in tumor progression has also been

recognized by identifying markers in stromal cells present in the cancerous

microenvironment (Chung et al., 2003; Goetz et al., 2011; Sung and

Chung, 2002). Recent studies show that stromal fibroblasts from prostate

tumors with higher Thy-1 content are stronger tumor promoters than those


with low Thy-1 levels. Also, the factors secreted by the former cells increase

the survival of the epithelial cells of benign prostatic hyperplasia in the pres-

ence of apoptotic agents (True et al., 2010; Zhao and Peehl, 2009).

In addition, when NCCIT cells (pluripotent embryonal carcinoma cell

line) are cocultured with fibroblasts associated with prostate cancer cells,

they alter their gene expression pattern and induce proliferation, malignant

transformation, and tumor progression (Pascal et al., 2011). Thus, it appears

that stromal cells containing high levels of surface Thy-1 have an impaired

ability to direct correct cell-specific differentiation, thereby leading to

abnormal behavior, such as that found on cancer cells. Consequently,

Thy-1 expression levels in cancer stromal fibroblasts may represent a useful

cell biomarker to indicate poor cancer prognosis.

6. CONCLUDING REMARKS

Despite all these years of oblivion, Thy-1 has been able to recapture

the scientific community’s attention. Studies on Thy-1 are slowly revealing

its function in different cell types and its potential importance in health and

disease. Indeed, this molecule, which for a long time was believed to be only

a marker for cell lineages, has exposed many more of its charms in the past

10 years.

Thy-1 is a molecule that is expressed abundantly in many cells and due to

its similarities with the immunoglobulins, was early on described as impor-

tant for cell adhesion. Now, it is known to mediate cell–cell interactions that

could trigger signaling pathways in both cis and trans. Thy-1-dependent cis

interactions occur in lipid rafts due to its GPI-mediated membrane anchor.

Thy-1 may interact with itself, or with integral membrane proteins that

function as transducer molecules, or associate functionally with reggie pro-

teins and kinases of the Src family. These interactions, whether direct or

indirect, allow Thy-1 to signal through the plasma membrane despite lac-

king a transmembrane domain. These views are consistent with the idea

that lipid rafts are platforms for signal transduction that facilitate transmission

of extracellular signals to the cell interior. Thy-1 also elicits responses by trig-

gering signals in trans via integrins that seem to represent its main receptors in

various cell types. For example, the pair Thy-1–integrin mediates neuron–

astrocyte communication, maturation of dendritic cells through interaction

with Thy-1(þ) fibroblast, as well as melanoma- and monocyte/leukocyte-

activated endothelial cell interactions to facilitate invasion and


transmigration of these cells to other target tissues. Despite all this acquired

knowledge, much remains to be elucidated. However, now that the ligand

for Thy-1 has been described, all suspected and nonanticipated roles for

Thy-1 can be critically tested.

ACKNOWLEDGMENTSL. L. is supported by FONDECYT 1110149; Fogarty International Center, National

Institutes of Health, Award Number 5R03TW007810; Iniciativas Cientıficas Milenio:

Biomedical Neuroscience Institute P09-015-F. A. Q. is supported by FONDECYT

1130250; Anillo ACT1111. R. H-M acknowledges fellowships from DAAD, the Journal

of Cell Science, and the CAEN International Society for Neurochemistry; support from the

COST action ECMNet and the State of Saxony-Anhalt, and the “European Regional

Development Fund” (CBBS/ERDF 2007–2013); and excellent advice from Dr. Werner

Zuschratter and Oliver Kofler during STED sessions. The authors like to acknowledge

Walter Waymann for his creative designs (http://www.proyectolumina.cl).

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[International Review of Cell and Molecular Biology] Volume 305 || Thy-1-Interacting Molecules and Cellular Signaling in Cis and Trans