[Vitamins & Hormones] Activins and Inhibins Volume 85 || Activin Receptor-Like Kinase and the Insulin Gene

C H A P T E R O N E

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Activin Receptor-Like Kinase and

the Insulin Gene

Rie Watanabe1

Contents

I. In

s and

083-

entnt adRes

troduction

Hormones, Volume 85 # 2011

6729, DOI: 10.1016/B978-0-12-385961-7.00001-9 All rig

of Diabetes and Clinical Nutrition, Kyoto University Graduate School of Medicine, Kdress: Laboratory of Infection and Prevention, Department of Biological Responses,earch, Kyoto University, Kyoto, Japan

Else

hts

yoIns

2

II. T
GF-b Family Receptors: ALK 3
III. A
ctivin Isoforms A, B, and AB 5
A.
A ctivins and ALKs 5
B.
P ancreatic endocrine cells 7
IV. N
odal 9
V. In
sulin Gene Regulation 9
A.
T ranscription regulation: A element 10
B.
T ranscription regulation: GG element 11
C.
T ranscription regulation: cAMP response element (CRE) 12
D.
T ranscription regulation: C element 13
E.
T ranscription regulation: E element 13
F.
T ranscription regulation: Smad-binding element (SBE) 14
VI. C
onclusion 15
Ackn
owledgments 16
Refer
ences 16
Abstract

The biological responses of the transforming growth factor-b (TGF-b) superfamily,

which includes Activins and Nodal, are induced by activation of a receptor complex

andSmads. A type I receptor, which is a component of the complex, is known as an

activin receptor-like kinase (ALK); currently seven ALKs (ALK1–ALK7) have been

identified in humans. Activins signaling, which ismediated byALK4 and 7 together

with ActRIIA and IIB, plays a critical role in glucose-stimulated insulin secretion,

development/neogenesis, and glucose homeostatic control of pancreatic endo-

crine cells; the insulin gene is regulated by these signaling pathways via ALK7,

vier Inc.

reserved.

to, Japantitute for

1

2 Rie Watanabe

which is a receptor for Activins AB and B and Nodal. This review discusses signal

transduction of ALKs in pancreatic endocrine cells and the role of ALKs in insulin

gene regulation. � 2011 Elsevier Inc.

I. Introduction

The transforming growth factor-b (TGF-b) superfamily, whichincludes TGF-bs, Activins, Nodal, Inhibins, the bone morphogenetic pro-teins (BMPs), and growth and differentiation factors (GDFs), regulates awide variety of cellular processes involving proliferation, differentiation,adhesion, apoptosis, and migration. All TGF-b family members are synthe-sized as precursor proteins and form dimeric ligands, some of whichremain inactive as latent forms by binding to their propeptides, for example,TGF-bs and some GDFs, or as trapped forms by extracellular antagonists,for example, follistatin, which inhibits Activins and noggin and chordinwhich inhibit some BMPs (Moustakas and Heldin, 2009). On release fromthese inactive states, the dimeric ligands bind to pairs of membrane receptorserine/threonine kinases, type I (activin receptor-like kinases, ALKs) andtype II receptors, promoting the formation of heterotetrameric receptorcomplexes (Fig. 1.1). Ligand binding induces a link between the constitu-tively active type II receptors and the dormant type I receptors; when thetype II receptor phosphorylates a serine/threonine-rich region, called theGS region, in the cytoplasmic domain of the type I receptor, kinase activityof the type I receptor is stimulated, and ligand-dependent signal transduc-tion then advances. Currently, five type II and seven type I receptors havebeen identified in mammals. In addition, the TGF-b family ligands alsointeract with type III receptors: epidermal growth factor–Cripto–FRL1–Cryptic (EGF–CFC)/Cripto, endoglin, and the proteoglycan betaglycan,which are coreceptors and either facilitate or limit the signaling of thereceptor kinase. In the absence of the ligand, the small proteins FKBP12and FKBP12.6 bind to the GS region and maintain the inactive conforma-tion of TGF-b type I receptor by occluding the site of phosphorylationunder the TGF-b signaling.

The activated type I receptor phosphorylates receptor-regulated Smads(R-Smads) in the cytoplasm; phosphorylated R-Smads associate with com-mon-mediator Smad (Co-Smad), Smad4, and the resulting Smad oligomeris then shuttled into the nucleus. In nucleus, the Smad complexes bind totarget genes and regulate their expression together with other transcriptionfactors (Fig. 1.1; Lonn et al., 2009; Massague and Gomis, 2006, Massagueet al., 2005; Moustakas and Heldin, 2009; Schmierer and Hill, 2007; Zhang,2009).

Cytoplasm

Nucleus

Promoter

Element ElementSBE

TFsTFs Smads

Smad4

Smad5

Smad1

TGF-b

TGF-b

Latent TGF-b sFollistatin–Activins Noggin–BMPs

BMP

BMP

CriptoActivin/Nodal

Activin

Smad8

Smad3Smad2

IIII III II

Smad6

: Smad pathwaysNon-Smad pathways

Smad7

P

P PP

P

PPPPPP

P

P Smad

s

Smad4

Gene expression

Figure 1.1 TGF-b, Activin/Nodal, and BMP signaling pathways. Smad pathways areindicated. TGF-b and Activin/Nodal type I receptors phosphorylated Smad2 and 3, andBMP type I receptor phosphorylates Smad1, 5, and 8. The activated R-Smads controlgene expressions with various transcription factors (TFs) and Smad4 (Co-Smad) viaSBE. Smad6 and Smad7, inhibitory Smads (I-Smads), downregulate the Smad path-ways. II, type II receptor; I, type I receptor; - - -, downregulation by I-Smads.

Activin Receptor-Like Kinase and the Insulin Gene 3

II. TGF-b Family Receptors: ALK

In early studies, receptor affinity-labeling analyses using radiolabeledTGF-b revealed TGF-b receptors to comprise three distinct size classes:type I, type II, and type III including proteoglycan betaglycan (Cheifetzet al., 1986; Massague and Like, 1985) and endoglin (Cheifetz et al., 1992);other groups have identified receptors of Activin A (EDF; Hino et al., 1989)and BMP4 (BMP2B; Paralkar et al., 1991) with similar approaches. Expres-sion cloning approaches using degenerate DNA primers (Georgi et al., 1990;Mathews and Vale, 1991) or a probe have identified a number of receptorserine/threonine kinases (Franzen et al., 1993; Ryden et al., 1996; ten Dijkeet al., 1993, 1994; Tsuchida et al., 1996), which are type I receptors knownas ALKs; seven ALKs, ALK1-7, have been identified in mammals to date.

4 Rie Watanabe

Furthermore, many studies identified receptor serine/threonine kinases forTGF-b family members (Attisano et al., 1993; Ebner et al., 1993; He et al.,1993; Kang and Reddi, 1996; Lorentzon et al., 1996; Matsuzaki et al., 1993),type II receptors of TGF-b (Lin et al., 1992), Activins (Attisano et al., 1992;Legerski et al., 1992; Mathews and Vale, 1991; Mathews et al., 1992) andBMPs (Kawabata et al., 1995; Liu et al., 1995; Nohno et al., 1995;Rosenzweig et al., 1995), and type III receptors including endoglin(Gougos and Letarte, 1990; Lopez-Casillas et al., 1991; Wang et al., 1991).These findings showed that ALKs include an extracellular ligand-bindingregion, a single transmembrane domain, intracellular serine/threoninekinase and GS regions, except in type II receptors, in which there is noGS region although they are otherwise structurally similar.

ALK1, 2, 3, and 6 are involved in BMP signaling (Miyazono et al., 2010)in combination with the type II receptors BMPR-II (Kawabata et al., 1995;Liu et al., 1995; Nohno et al., 1995; Rosenzweig et al., 1995), ActRIIA(Mathews and Vale, 1991), and IIB (Attisano et al., 1992). ALK1, 2, 3, and 6activate the R-Smads, Smad1, 5, and 8 (Table 1.1). ALK4 and 7 arestimulated by Activins A, B, and AB, Nodal, and some GDFs togetherwith ActRIIA and IIB (Andersson et al., 2006b, 2008; Reissmann et al.,2001; ten Dijke et al., 1994; Tsuchida et al., 2004); ALK5 is activated byTGF-bs through combination with the type II receptor TbRII (Lin et al.,1992; ten Dijke et al., 1994). GDF8 (Myostatin) and GDF11 bind ActRIIAand IIB together with ALK4 and 5 (Andersson et al., 2006a; Lee et al., 2005;Rebbapragada et al., 2003; Tsuchida et al., 2009). Activated ALK4, 5, and 7phosphorylate Smad2 and 3. However, increasing evidence shows thatTGF-b signaling can also activate Smad1 and 5 in a diversity of cell typesin culture (Daly et al., 2008; Finnson et al., 2008; Goumans et al., 2003; Liuet al., 2009), and it has been suggested that a reevaluation of TGF-b familysignaling is required by elucidating type I receptor and Smad pathways.

In TGF-b signaling through receptor–receptor interactions, TbRIIbinds with high affinity and is responsible for cooperative recruitment andtransphosphorylation of its low-affinity ALK5 pair (Wrana et al., 1992,1994); ALK5 is predicted to be structurally similar to ALK3 (Harrisonet al., 2003) and is anticipated to bind in a mode similar to that of ALK3(Hart et al., 2002; Lin et al., 2006; Shi and Massague, 2003; Zuniga et al.,2005). Although they are structurally similar, recent analysis of TGF-b andBMP ligands bound to their respective type I and type II receptor ectodo-mains shows that TGF-b ligands contact both receptors tightly while theevolutionarily more ancient BMPs associate more loosely with their recep-tors (Groppe et al., 2008). Binding of TGF-b to TbRII creates the interfacerequired for ALK5 recruitment to the complex. Thus, signaling regulationthrough receptor complexes and downstream molecules is still insufficientlyclear.

Table 1.1 Component regulators in TGF-b family member pathways

Type I Type II Ligand R-Smads I-Smads Co-Smad

ALK1 BMPR-II/

ActRIIA/

ActRIIB

BMPs Smad1 Smad6 Smad4

ALK2 Smad5 Smad7

ALK3 Smad8

ALK6

ALK4 ActRIIA/

ActRIIB

Activin A,

B, AB/

Nodal

Smad2 Smad7 Smad4

ALK7 Smad3

ALK1 TbR-II TGF-bs Smad1 Smad7 Smad4

ALK2 Smad5

ALK3 Smad8

ALK5 TbR-II TGF-bs Smad2 Smad7 Smad4

Smad3

ALK2 BMPR-II/

ActRIIA/

ActRIIB

GDFs Smad1 Smad6 Smad4

ALK3 Smad5 Smad7

ALK6 Smad8

ALK4 ActRIIA/

ActRIIB

Smad2 Smad7

ALK5 Smad3

ALK7

ActRIIA also is known as ActRII. ALK3 andALK6 denote BMPR-IA andBMPR-IB, respectively. ALK4 isActR-IB,andALK5isTbR-I.TypeI, type I receptor;TypeII, type II receptor; I-Smads, the inhibitorySmads.


III. Activin Isoforms A, B, and AB

A. Activins and ALKs

Activins are disulfide-linked homo- or heterodimers of the b subunits ofInhibin/Activin A and B; Activin A (bAbA), Activin B (bBbB), andActivin AB (bAbB) and multifunctional proteins were originally identifiedas factors in ovarian fluid that stimulated the secretion of follicle stimulatinghormone from pituitary cells (Ling et al., 1986; Vale et al., 1986). Activinshave potent mesoderm-inducing activity in Xenopus laevis (McDowell andGurdon, 1999); Nodal is also an authentic mesoderm inducer in manyspecies, including mammals (Shen, 2007). Activins are expressed in a widevariety of tissues, and three isoforms A, B, and AB have been isolated from

6 Rie Watanabe

natural sources (Ling et al., 1986; Nakamura et al., 1992; Vale et al., 1986).Activin A and AB have equivalent biological activity levels in various assaysystems, whereas the biological activity of native Activin B is significantlylower than those of Activin A and AB (Nakamura et al., 1992). AdditionalInhibin/Activin b subunit genes (bC and bE) have been identified inmammals (Fang et al., 1996, 1997; Hashimoto et al., 2002; Hotten et al.,1995; Lau et al., 1996; O’Bryan et al., 2000; Schmitt et al., 1996; Vejda et al.,2002).

Gene disruption studies have shown that phenotypes of the Inhibin bA-and the Inhibin bB-deficient mouse clearly differ, indicating a lack offunctional redundancy between Activins A and B during embryogenesis(Matzuk et al., 1995; Vassalli et al., 1994). Furthermore, replacement of themature region in the gene of Inhibin bA with the corresponding matureregion of Inhibin bB compensates for the Inhibin bA phenotype but evokesadditional phenotypes (Brown et al., 2000). These findings indicate that thesignalings via Activin A and B have disparate behavior, suggesting a lack ofeffective compensatory mechanisms.

ALK4 and 7 utilize Activins: ALK4 is activated throughActivin A, B, andAB, and ALK7 is stimulated by Activin B and AB (Tsuchida et al., 2004).ALK4 is ubiquitously expressed while ALK7 is expressed in embryonic brain(Lorentzon et al., 1996; Tsuchida et al., 1996), adult central nervous system(Kang and Reddi, 1996; Lorentzon et al., 1996; Ryden et al., 1996; Tsuchidaet al., 1996), prostate (Kang and Reddi, 1996), adipose tissue (Kang andReddi, 1996; Lorentzon et al., 1996), kidney (Ryden et al., 1996; Tsuchidaet al., 1996), testis (Tsuchida et al., 1996), gastrointestinal tract (Bondestamet al., 2001; Lorentzon et al., 1996), liver (Lorentzon et al., 1996; Tsuchidaet al., 1996), heart (Bondestam et al., 2001), thymus (Lorentzon et al., 1996),coagulating gland (Kang and Reddi, 1996), nasal cavity epithelium(Lorentzon et al., 1996), fetal and adult pancreatic islets (Watanabe et al.,1999), MIN6 (Watanabe et al., 1999), and INS-1 (Zhang et al., 2006) cells.

Pancreatic b-cell line MIN6 cells, in which ALK4 and ActRIIB expres-sion is barely detectable and weak, respectively, while ALK7 and ActRIIAare abundantly expressed (Tsuchida et al., 2004; Watanabe et al., 1999), arehighly sensitive to Activin AB and modestly to Activin B (Tsuchida et al.,2004). Furthermore, Activin AB and B augment DNA-binding transcrip-tional activities of Smads in a dose-dependent manner, whereas dominantnegative ALK7 (ALK7D/N) expression strongly reduces the activities;glucose-stimulated insulin secretion (GSIS) also is enhanced by ActivinAB and B (Tsuchida et al., 2004) but not by Activin A (Shibata et al.,1996; Tsuchida et al., 2004), and it has been shown that Activin AB bindsto a combination of ALK7 and ActRIIA in MIN6 cells (Tsuchida et al.,2004).

In contrast, in HEK293 and HT22 cells that express ActRIIs and ALK4but not ALK7 and are highly sensitive to Activin A and AB and


intermediately to Activin B, it has been shown that ALK7 overexpressioninduces a dramatic augmentation of Activin B sensitivity for DNA-bindingtranscriptional activity and that Activin AB modestly enhances the activity,although Activin A sensitivity appears to remain unchanged, indicating thatALK7 is an Activin B-preferring receptor in those cell lines (Tsuchida et al.,2004). In addition, coexpression of ALK7 and ALK7D/N reduces ALK7-enhanced activity via Activin AB in HEK293 cells.

Taken together, these findings suggest that ALK4 is favored by ActivinA; although ALK4 is able to respond to Activin isoforms A as well as B,ALK7 prefers Activin B to Activin A, and Activin AB is more effective thanthe other two isoforms in ALK4- and ALK7-sensitive signal transduction.However, it remains unknown whether ALK4/Activin B- and ALK4/Activin AB-mediated signaling mechanisms are similar to that throughALK7/Activins B and AB.

B. Pancreatic endocrine cells

Activins A and B are expressed in pancreatic islets (Furukawa et al., 1995;La Rosa et al., 2004; Ogawa et al., 1993, 1995; Tsuchida et al., 2004; Wadaet al., 1996; Yasuda et al., 1993) including a-, b-, and d-cells, suggesting anautocrine and/or paracrine system of Activin signals within islets, althoughtheir actual secretion levels have not been evaluated. Many studies haveshown the importance of Activin signals to physiological functions anddevelopment/neogenesis of pancreatic endocrine cells.

With regard to glucose homeostasis, it has been found that Activin Astimulates GSIS (Florio et al., 2000; Totsuka et al., 1988; Tsuchida et al.,2004; Verspohl et al., 1993) in a concentration-dependent manner (Florioet al., 2000; Verspohl et al., 1993; Yasuda et al., 1993)mediated byCa2þ entry(Mogami et al., 1995; Shibata et al., 1993) and counteracted by reduction ofextracellular Ca2þ (Shibata et al., 1993). Consistently, MIN6 cells, in whichexpression of ALK4, the Activin A-preferring receptor, is barely detectable,lack the Activin A effect on GSIS (Shibata et al., 1996; Tsuchida et al., 2004)although Activin AB and B augment GSIS. However, HIT-T15 insulinomacells, in which ALK7 expression is not detectable (Watanabe et al., 1999),exhibit such Activin A effects (Shibata et al., 1996). These findings indicatethat ALK4 plays an essential role in GSIS and Ca2þ-mediated mechanismsvia Activin A in pancreatic b-cells, which also suggests a role of a combina-tion of Activin B and ALK7 in the control of GSIS. More recently, it hasbeen shown that Activins have opposite responses to Ca2þ influx in pancre-atic islets (Bertolino et al., 2008): Activin A increases glucose-stimulatedCa2þ influx whereas Activin B reduces it. In addition, pancreatic islets showdifferent gene-expression profiles of ALK7, Inhibin bA and Inhibin bB atvarious glucose concentrations (Bertolino et al., 2008; Zhang et al., 2006),indicating that the extracellular glucose condition regulates the expression of

8 Rie Watanabe

the genes of Activins and ALKs related to the glucose homeostasis inpancreatic islets. These findings indicate that in pancreatic islets, Activin Aand B exhibit contrary behavior in GSIS control, which may be essential tocontrol glucose homeostasis precisely. Because Activin B in pancreaticb-cells can also stimulate GSIS (Tsuchida et al., 2004), other endocrinecells (e.g., a-cells and d-cells) and/or a novel mechanism may be related toGSIS control of islets, and the Activin AB signal might augment the action ofActivins A and B in certain glucose conditions in islets.

In addition, recent mutant mice studies have shown that TGF-b familymembers also play an important role in pancreatic islet functions and glucosehomeostasis. It has been found that mice lacking follistatin-like 3 (FSTL3),which is an Activins and GDF8 (Myostatin) antagonist, exhibit an alteredmetabolic phenotype that includes increased pancreatic islet number andsize, improved glucose tolerance and enhanced insulin sensitivity(Mukherjee et al., 2007), while conditional adult overexpression ofSmad7, a potent cytoplasmic inhibitor of TGF-bs and Activins signaling,reduces pancreatic insulin content and produces severe hypoinsulinemia(Smart et al., 2006). Mice with attenuated ALK3 (BMPR-IA) signaling inb-cells show decreased expression of key genes involved in insulin geneexpression and glucose sensing and develop diabetes due to impaired insulinsecretion, and further transgenic expression of BMP4 in b-cells enhancesGSIS and glucose clearance (Goulley et al., 2007).

On the other hand, it has been demonstrated that TGF-b signaling, whichincludes Activin A, induces definitive endoderm in mouse and humanembryonic stem cells (D’Amour et al., 2005; Kubo et al., 2004; Yasunagaet al., 2005). Activin A and B are able to induce the transformation ofembryonic stem cells into insulin-producing cells together with other variousstimuli (D’Amour et al., 2006; Jiang et al., 2007; Ku et al., 2004; Ricordi andEdlund, 2008; Shi et al., 2005), and a recent report shows that Activin B ismore potent than Activin A in inducing expression of PDX-1, which plays anessential role in the development of pancreas during differentiation of humanembryonic stem cells (Frandsen et al., 2007). In addition, in X. laevis, Activinor mature Vg1, a TGF-b-related factor, also induces the expression ofXlHbox8, a PDX-1 homolog (Gamer and Wright, 1995; Henry et al.,1996). Early pancreatic-bud explants treated with TGF-b1 in vitro enhancethe formation of endocrine cells and inhibit the development of acinar tissue(Sanvito et al., 1994), and further treatment of early buds with follistatin, anActivin antagonist, enhances acinar development while inhibiting that ofendocrine cells (Miralles et al., 1998). In dorsal development of the chickpancreas, a notochord signal (comprising Activin B and FGF2) represses sonichedgehog expression and generates larger insulin-secreting islets (Hebroket al., 1998), and experiments in Xenopus embryos have shown that transientexposure to Activin and RA can induce pancreas development from isolatedanimal cap ectoderm (Moriya et al., 2000). Activin A associated with HGF or


betacellulin induces the conversion of pancreatic AR42J cells derived from arat pancreatic acinar carcinoma into insulin-secreting cells (Mashima et al.,1996a,b). Furthermore, developing pancreata of mice lacking the Activintype IIB receptor have severely reduced islet mass but apparently normalacinar tissue (Kim et al., 2000), while transgenic mice with mutated Activintype II receptors have smaller islet area (Shiozaki et al., 1999; Yamaoka et al.,1998), lower survival rate, and lower insulin content in the whole pancreaswith impaired glucose tolerance (Yamaoka et al., 1998).

Thus, evidence strongly suggests that stimulation byActivins plays a criticalrole in pancreatic b-cell development and production of insulin-positive cellsand b-cell functions. However, the molecular mechanisms by which Activinsinduce development/neogenesis and regulate b-cell functions remain unclear.

IV. Nodal

Nodal signaling also involves ALK4 and 7 together with ActRIIA andIIB. Unlike Activins, however, Nodal signaling requires additional core-ceptors from the EGF–CFC protein family such as Cripto to assembleits receptor complexes (Schier and Shen, 2000). Cripto has importantroles during development and oncogenesis, and independently bindsNodal and ALK4/7 to promote signaling (Reissmann et al., 2001; Yeoand Whitman, 2001). Recently, it has been shown that Activins signalingis inhibited by Cripto overexpression (Adkins et al., 2003; Gray et al., 2003),and two binding mechanisms have been demonstrated: one involves directinteraction between soluble Cripto and Activin B but not Activin A(Adkins et al., 2003); the other involves type II receptor (IIA and IIB)associated binding between Cripto and Activins A and B (Gray et al.,2003) in blocking Activin signaling. In addition, it has been indicated thatCripto functions as a noncompetitive Activin A antagonist (Kelber et al.,2008).

Further experiments are required to elucidate the molecular mechanismsof Cripto function, Nodal signaling and other inhibitory reactions toActivins signaling, and dynamic relations within TGF-b family membersand/or cell-to-cell signals could well play an important role in regulation ofpancreatic b-cell function and action.

V. Insulin Gene Regulation

Insulin is a polypeptide hormone critically involved in the control ofglucose homeostasis and is synthesized exclusively in pancreatic islet b-cellsby various stimuli. The cloning and sequencing of the human insulin gene

10 Rie Watanabe

was reported in 1980 (Bell et al., 1980), and gene mapping studies assignedthe human insulin gene to chromosome 11 (p15.5; Harper et al., 1981;Owerbach et al., 1980). Currently, insulin genes have been identifiedamong a number of mammalian species (Steiner et al., 1985; Watanabeet al., 2008). Most animals have a single copy of the insulin gene, whereas inmouse and rat, two nonallelic insulin genes are present (Soares et al., 1985;Steiner et al., 1985). In postnatal life, the insulin gene is expressed almostexclusively in pancreatic b-cells, although low levels of insulin are detectedin a number of extrapancreatic tissues (Kojima et al., 2004; Rosenzweig et al.,1980) including brain (Devaskar et al., 1994), thymus ( Jolicoeur et al., 1994;Pugliese et al., 1997; Smith et al., 1997; Vafiadis et al., 1997), lachrymal glands(Cunha et al., 2005), and salivary glands (Vallejo et al., 1984). In thymus thatectopically expresses a broad range of tissue-specific genes for negativeselection of autoreactive T cells, variations of insulin expression may beespecially relevant to diabetes (Pugliese, 1998). However, there is littleunderstanding of the regulatory sequences and their signaling pathwaysthat control insulin gene expression in non-b-cells, and the role of insulinexpression in those cells remains largely unclear.

The insulin promoter is located within a region spanning about 400 bpthat flanks the transcriptional start site (Edlund et al., 1985; German et al.,1995; Hay and Docherty, 2006; Melloul et al., 2002; Walker et al., 1983).This region contains many ciselements that bind transcription factors, someof which are expressed mainly in pancreatic b-cells and a few other endo-crine or neural cell types, while others have widespread tissue distribution(German et al., 1992; Glick et al., 2000; Hay and Docherty, 2006; Qiu et al.,2002; Watanabe et al., 2008). This chapter focuses on representative regu-latory elements and a Smad-related element within the human insulinpromoter (Fig. 1.2).

A. Transcription regulation: A element

A elements containing the core sequence, 50-TAAT-30 (A1, A3, and A5),bind homeodomain proteins (Rudnick et al., 1994). Among these proteins,the pancreatic and duodenal homeobox factor-1 (PDX-1; Offield et al.,1996), also called IPF1 (Ohlsson et al., 1993), STF-1 (Leonard et al., 1993),IDX-1 (Miller et al., 1994), IUF-1 (Boam and Docherty, 1989), and GSF(Marshak et al., 1996; Melloul et al., 1993) is a well-characterized home-odomain protein expressed in pancreatic islets that plays an essential role indevelopment of pancreas and regulates insulin and somatostatin gene pro-moters (Liberzon et al., 2004; Ohneda et al., 2000). Recent studies show theglucose-responsive region includes the A3 element (da Silva Xavier et al.,2004; MacFarlane et al., 1994; Marshak et al., 1996; Petersen et al., 1994).

A5 G1

-336 -170 -87

TATA

PDX1

SP1 A1E1C1GG2SBECRE2CRE1A3E2G2C2NRECore GG1/A2

-319 -216 -82

ATF2 ATF2PDX1 PDX1 PDX1 PDX1MafA BETA2

c-Jun c-JunPax4

CREMCREM

Smads

(+)

(-)

-58

Figure 1.2 The major cisacting elements in the human insulin promoter. Transcriptionfactors binding to representative elements are shown. The upper transcription factorsupregulate the gene expression (þ), the lower factors downregulate it (�).


Itwas reported thatHNF-1a and -1b also bind to theA3element and stimulatethe transactivation in the human insulin promoter (Okita et al., 1999).

B. Transcription regulation: GG element

In the human insulin promoter, PDX-1 also responds to the core sequences,50-GGAAAT-30 (called the GG elements, GG1 and GG2; Boam et al.,1990; Hay and Docherty, 2006; Le Lay et al., 2004; Tomonari et al.,1999) and regulates expression of the gene. GG1 also has been designatedthe A2 element (German et al., 1995).

GG2 is by far the more conserved, being present in the insulinpromoter of all mammals except rodent. The human GG2 element isunder positive control of PDX-1 (Le Lay et al., 2004), whereas thecorresponding region in the rodent gene is negatively regulated byNkx2.2, a homeodomain transcription factor of the NK2 class (Cissellet al., 2003), which demonstrates a fundamental difference in the regula-tion of the human and rodent insulin genes. In the human insulinpromoter, although the GG2 element displays a lower PDX-1 bindingaffinity than A3 and A1 elements in gel mobility shift assays, it is morecritical to transcriptional activation in b-cell transfection assays (Le Layand Stein, 2006). Comparison analyses between the GG elementsshow that a mutation of the GG1 element drastically decreases thetranscriptional activity of the human insulin promoter in MIN6 cells,suggesting that the GG1 element may play the more critical role inb-cell-specific transcriptional activity than the GG2 element (Tomonariet al., 1999). In addition, PDX-1-dependent (Watanabe et al., 2008) andglucose-induced (da Silva Xavier et al., 2004) transactivation of the humaninsulin promoter is also strongly decreased by a mutation of GG1 element.

The early study on transacting factors for GG elements of the humaninsulin gene by DNase footprint analysis shows transaction of a ubiquitousfactor with the GG1 element and of a b-cell-specific factor with the GG2

12 Rie Watanabe

element (Boam et al., 1990). It has been shown that the transcriptionfactor binding to the GG1 element interacts with a transcription factorthat binds to the adjacent C1 element (Tomonari et al., 1999), whichbinds the basic leucine zipper (bZIP) factor MafA (Matsuoka et al., 2003),while the GG2 element also contributes to synergistic activation byPDX-1 and MafA (Le Lay and Stein, 2006).

Taken together, these findings suggest that both of the GG regulatoryelements have a function in insulin expression, and that PDX-1 plays amajor role in GG regulation together with proximate transcription factors.However, the signaling mechanisms remain unclear.

C. Transcription regulation: cAMP response element (CRE)

In the pancreatic b-cell, glucose (Grill and Cerasi, 1974) and hormonesincluding incretins increase intracellular cAMP (Drucker et al., 1987) andCa2þ. The human insulin promoter has four CREs, which bind to theCREB/ATF family (Inagaki et al., 1992), and all of these sites are transcrip-tionally active (Hay et al., 2005; Inagaki et al., 1992). CRE1 and CRE2 arefound in the promoter region, CRE3 is in the first exon and CRE4 is in thefirst intron. Recombinant CREB and ATF2 bind to CRE sites in rat andhuman insulin promoters (Inagaki et al., 1992; Oetjen et al., 1994), and ChIPanalysis demonstrates that CREB binds to mouse insulin 2 promoter (Kurodaet al., 2009), whereas only ATF2 markedly enhances glucose-induced trans-activation of the human insulin promoter (Ban et al., 2000). In addition, it alsohas been shown that siRNA-mediated knockdown of ATF-2 diminishes thestimulatory effects of cAMP-related signaling on insulin promoter activity,suggesting that ATF-2 may be a key regulator of the human insulin promoter(Hay et al., 2007). Furthermore, the c-jun protooncogene product (c-Jun),which was able to form a heterodimer with ATF2 and bind to the CRE sitewith high affinity (Macgregor et al., 1990), represses cAMP-induced activity ofthe human insulin promoter (Inagaki et al., 1992).

The human insulin promoter also has nine CpG sequences located atpositions �357, �345, �234, �206, �180, �135, �102, �69, and �19bp relative to the transcription start site (Kuroda et al., 2009), and the CpGsites at �206 bp and �180 bp are parts of CRE1 and CRE2, respectively.Methylation of the human insulin promoter also suppresses reporter geneexpression, suggesting that DNA methylation/demethylation may play acrucial role in insulin gene regulation by ATF2 and CREB. Indeed, in themouse Insulin 2 gene, specific methylation of the CpG site in CRE alonesuppresses promoter activity, and ChIP analysis shows that methylationincreases the binding of methyl-CpG-binding protein 2 (MeCP2) andconversely inhibits the binding of ATF2 and CREB (Kuroda et al., 2009).


D. Transcription regulation: C element

The bZIP protein MafA has been identified as the rat insulin promoterelement 3b1 (RIPE3b1) factor (Shieh and Tsai, 1991), which is a transcrip-tion factor that binds to the C1/RIPE3b1 element and positively regulatesthe transcriptional activity of the insulin promoters in mouse (Matsuokaet al., 2003), rat (Kajihara et al., 2003; Matsuoka et al., 2003; Olbrot et al.,2002), and human (Kataoka et al., 2002). The C1/RIPE3b1 element alsohas been shown to play a critical role in b-cell-specific insulin gene tran-scription as well as in its glucose-regulated expression (Kataoka et al., 2002;Sharma and Stein, 1994), and the expression and binding of MafA to theC1/RIPE3b1 element is upregulated in a glucose-sensitive manner(Kataoka et al., 2002; Sharma and Stein, 1994; Sharma et al., 1995). It hasbeen found that the transcription factors PDX-1, MafA, and BETA2, whichbind to the A3, C1, and E1 elements, respectively, synergistically controlglucose-regulated transcription of the insulin gene in rat (Zhao et al., 2005),whereas there is no indication of any synergistic effect between PDX-1,MafA, or BETA2 on the human insulin promoter (Docherty et al., 2005).In addition, the additive effect of PDX-1 and MafA is known (Dochertyet al., 2005). In the human insulin gene, ATF2 also enhances glucose-induced transactivation, and c-Jun, which is able to form a heterodimerwith ATF2, represses it (Ban et al., 2000; Inagaki et al., 1992). These findingsuggest that cooperative regulation among these transcription factors mayplay a major role in glucose-dependent transcription of the human insulingene. Indeed, MafA but not MafB also can heterodimerize with c-Jun(Benkhelifa et al., 1998; Kerppola and Curran, 1994).

The human insulin promoter also has the C2 element (Read et al., 1997),the DNA-binding activity of which is regulated in a redox-dependentmanner (Cakir and Ballinger, 2005; Sen and Packer, 1996). The C2 elementis able to bind PAX4, which negatively regulates transcriptional activity(Campbell et al., 1999). Another member of the Pax gene family, Pax6, theone most closely related to Pax4, has no significant effect on the transcrip-tional activity of the human insulin gene (Campbell et al., 1999), althoughPax6 binds to the C2 element and acts as a transactivator of the rat insulin Ipromoter (Fujitani et al., 1999; Sander et al., 1997).

E. Transcription regulation: E element

In the insulin gene, E elements, sharing the consensus sequence 50-CANNTG-30 (Boam et al., 1990; Crowe and Tsai, 1989; Karlsson et al.,1987, 1989; Ohlsson and Edlund, 1986; Whelan et al., 1989), bind tran-scription factors (Boam et al., 1990; Moss et al., 1988; Nelson et al., 1990;Ohlsson et al., 1988; Peyton et al., 1994; Walker et al., 1990) of the basichelix-loop-helix (bHLH) class that function as potent transcriptional

14 Rie Watanabe

activators of tissue-specific genes by forming heterodimers between bothubiquitous and cell-restricted family members (Dumonteil et al., 1998; Nayaet al., 1995). Mutagenesis of the E1 element in the human insulin promoterreduces basal (Docherty et al., 2005) and glucose-induced (da Silva Xavieret al., 2004; Odagiri et al., 1996) transcriptional activity. The heterodimerbetween ubiquitous E47 and neuroendocrine cell specific BETA2/NeuroDbinds to the E1 element and induces transactivation in rat (Dumonteil et al.,1998; Naya et al., 1995).

Although the E1 element is highly conserved, in the human insulinpromoter the E2 element is the homologous sequence not the consensussequence (Boam et al., 1990); the human E2 sequence is able to bind theubiquitous transcription factor USF (Read et al., 1993).

F. Transcription regulation: Smad-binding element (SBE)

The structures of Smad2, Smad3, and Smad4 include two conserveddomains in the amino (MH1) and carboxyl (MH2) termini, connected bya proline-rich nonconserved linker region (Massague et al., 2005). Smad2and Smad3 are phosphorylated by ALK4, 5, and 7; the phosphorylatedSmad complexes translocate into the nucleus and interact with DNA-binding proteins and coactivators (Fig. 1.1). The MH1 domains of Smad3and 4 but not that of Smad2 can act directly on the DNA sequence50-GTCT-30 or its complement 50-AGAC-30, called the SBE. ManySmad-responsive promoter regions contain one or more SBEs, which inmany instances contain an extra base as 50-CAGAC-30.

The human insulin promoter has a highly conserved SBE, 50-CAGAC-30, and Activin AB/B and Nodal signaling pathways, which activate ALK7,induce DNA binding of Smad3 and stimulate the transcriptional activity ofthe human insulin gene (Watanabe et al., 2008). Mutagenesis of the SBEdramatically reduces ALK7/Smad3-induced transcription (Watanabe et al.,2008), suggesting that the SBE plays a crucial role in human insulin geneexpression induced by these signals. In addition, PDX-1 is able to predomi-nantly interact with phosphorylated Smad3, and then bind to the promoterin an Activin AB/Nodal-sensitive manner and synergistically upregulatetransactivation; this synergy is completely abolished by mutations of theelements A2/GG1, A3, or SBE (Watanabe et al., 2008; Fig. 1.3). Thus,these findings suggest that association between the cell/tissue-specific tran-scription factor PDX-1 and ubiquitous factors, at least Smad3, on the insulinpromoter specifically controls insulin gene expression via Activin AB/B andNodal signals.

Pancreatic b -cell

Follistatin–Activins

Cytoplasm

Nucleus

Promoter

SBE

PDX-1

PDX-1

PDX-1

PDX-1 Smad3

Smad3

Smad3

Cripto

PP

P

P

P

PP

P

P P

P

Activin/Nodal

NodalActivin

Smad3

Smad3

Smad2

Smad7

Smad2Smad2

Smad2

Smad2Smad2

II I

Smad4

Smad3Smad2

A2/GG1A3 Insulin

Figure 1.3 Activin AB and Nodal signaling in the pancreatic b-cell. Activated ALK7phosphorylates Smad2 and 3, and the activated Smads bind to the SBE in the humaninsulin promoter, resulting in stimulation of transcription of the gene together withPDX-1.


VI. Conclusion

Growing evidence demonstrates the importance of TGF-b familymember signaling as well as that of Activins in physiological functions anddevelopment/neogenesis of pancreatic endocrine cells. The insulin pro-moter is precisely regulated by various stimuli and complex signalings thatcontrol b-cell functions and action. Insulin gene transcription is directlystimulated by combination of Smad2/3 and PDX-1 via Activin AB/Nodal-associated ALK7 signalings in pancreatic b-cells, and the highly conservedSBE within the insulin promoter is related to this process. In human,Activins, Nodal, TGF-bs, and some GDFs utilize Smad2 and 3 in controlof many cellular processes, suggesting that this SBE of the insulin gene alsomay be involved in the various signaling pathways through these familymembers in pancreatic b-cells.

16 Rie Watanabe

ACKNOWLEDGMENTS

The authors’ work was supported in part by a Grant-in-Aid for JSPS Fellows and Establish-ment of International COE for Integration of Transplantation Therapy and RegenerativeMedicine from the Ministry of Education, Culture, Sports, Science, and Technology(MEXT), Japan.

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[Vitamins & Hormones] Activins and Inhibins Volume 85 || Activin Receptor-Like Kinase and the Insulin Gene