Journal of Dermatological Science 65 (2012) 141–148

Identification of the C-terminal tail domain of AHF/trichohyalin as the critical sitefor modulation of the keratin filamentous meshwork in the keratinocyte

Takahisa Takase, Yohei Hirai *

Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Sanda, Japan

A R T I C L E I N F O

Article history:

Received 25 August 2011

Received in revised form 21 November 2011

Accepted 20 December 2011

Keywords:

AHF

Trichohyalin

Hair follicle

Keratin

Keratinocyte

A B S T R A C T

Background: AHF/trichohyalin is a large structural protein abundant in the inner root sheath (IRS) of

anagenic hair follicles, which has been thought to mediate the keratin filamentous assembly. However,

its functional mechanism is largely unknown.

Objective: This study aimed at the identification of the key domain in AHF for keratin association and the

establishment of a plausible mechanism for the modulation of the keratin meshwork.

Methods: Several keratinocyte cell lines were introduced with the full length or several mutants of AHF,

together with IRS-specific keratin krt31, and the profile of the AHF granules and the cellular behaviors

were carefully analyzed.

Results: Full length of AHF formed small round granules that clearly bound to and aligned on the

exogenous keratin filaments in the keratinocytes, severely affected cellular growth, mobility and shape.

Intriguingly, the removal of only 6 amino acids around the C-terminal tail of AHF resulted not only in the

complete loss of its keratin adherent ability but also in a dramatic enlargement of the granules.

Conclusion: We propose a model for cytoskeletal modulation in the IRS of anagenic hair follicles: AHF

latches onto the keratin bundles by its C-terminus and rearranges the keratin meshwork by intrinsic

cohesive activity for the granule formation.

� 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights

reserved.

Contents lists available at SciVerse ScienceDirect

Journal of Dermatological Science

jou r nal h o mep ag e: w ww .e lsev ier . co m / jds

1. Introduction

The regulation of cellular mechanical strength directly impactsthe physical characteristics of tissues so as to play vital roles in thestructural changes which occur in the progressively changingepithelia. The hair follicles undergo cyclic tissue morphogenesis,the stages of which roughly consist of growth (anagen), regression(catagen) and resting (telogen) phases. This hair follicle orphogen-esis and cyclic progression were mechanically supported by theIRS, the physical strength of which is directly controlled by thespatio-temporal assembly of the keratin bundles in the IRS [1–4].

AHF, also known as trychohyalin, is a large structural proteinspecifically expressed in the IRS of anagenic hair follicles. It bindsto keratin and is supposed to mediate amorphous–filamentoustransformation of keratin filaments so as to regulate cytoskeletaldynamics [5–7]. We have previously shown that the expression ofAHF/trichohyalin is under the strict control of transcriptionalactivation by bone morphogenic protein (BMP) signaling, and also

* Corresponding author at: Department of Bioscience, School of Science and

Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japan.

Tel.: +81 79 565 7234; fax: +81 79 565 7234.

E-mail address: y-hirai@kwansei.ac.jp (Y. Hirai).

0923-1811/$36.00 � 2012 Japanese Society for Investigative Dermatology. Published b

doi:10.1016/j.jdermsci.2011.12.014

of a rapid protein-elimination mechanism effected by theproteasomes during the progression of the hair cycle [8]. However,the molecular basis underlying its subsequent keratin associationfollowed by cytoskeletal modulation remains to be elucidated.The primary structure of AHF/trichohyalin is composed of twoN-terminal EF hand domains which act as calcium sensors [9],three distinct types of long internal repeats and an unstructuralshort C-terminal tail. Arginine and glutamic acid are unusuallyabundant (25% for both residues), especially in the internal repeatdomains and there are several glutamine residues that arepotential targets of the protein crosslinker transglutaminase(TGase) III [10]. Indeed, a number of amino acid residues indomains 6 and 8 in human trichohyalin have been proven tocovalently bind to keratin molecules [7]. However, the amino acidsequences and the overall domain structures of AHF/trichohyalinare not well conserved among species (�40% of the amino acidresidues are conserved between the human and mouse), and bothhuman and mouse AHF/trichohyalin enable the adherence tokeratin molecules [8]. Thus, we hypothesized that a uniquesequence that is conserved across species is primarily responsiblefor the AHF–keratin association.

In the present study, we identified the small C-terminal tail ofAHF/trichohyalin, which is entirely conserved interspecies, as thecritical site for interaction with keratin filaments. Coincidently, we

y Elsevier Ireland Ltd. All rights reserved.

T. Takase, Y. Hirai / Journal of Dermatological Science 65 (2012) 141–148142

noticed that the rest of the large AHF body possesses an intrinsicpotential for cohesive self-assembly so as to give rise to the largemolecular aggregates. These findings uncover, at least in part, theregulatory mechanism which underlines the physical strength inthe IRS of the anagenic hair follicle, and shed light on the molecularbasis underlying the building up and tearing down of highlyorganized epithelial architecture.

2. Materials and methods

2.1. Cells and antibodies

Human skin squamous carcinoma HSC-1 (JCRB1015, NIHS cellbank), non-transformed mouse keratinocyte C-50 (generous giftfrom Dr. Fisher) [11] and the adenocarcinoma cell line SW-13(JCRB9069) that lacks keratin intermediate filaments [12] weremaintained in a 1:1 mixture of DMEM and F12 (DMEM/F12)(Sigma) supplemented with 20% FCS along with penicillin andstreptomycin. Among these cell lines, HSC-1 displayed a well-developed keratin meshwork composed of exogenously intro-duced IRS-specific type I keratin (krt31) and endogenous Type IIkeratin, and therefore these cells were used for the followingexperiments. Rabbit polyclonal and rat monoclonal antibodiesagainst mouse AHF were generated and purified as describedpreviously [13]. The rat monoclonal antibody (mAb) against krt31was prepared from the supernatant of a hybridoma clone that wasestablished simultaneously with the anti-AHF mAb (usinganagenic mouse hair follicles as the antigen) [13]. The mAbprecipitated with 50% ammonium sulfate was dissolved in PBS,dialyzed against PBS and tested for the antigen specificity byWestern blot analyses of the mouse anagenic hair follicles and arecombinant form of the krt31 protein. Mouse mAbs against the T7peptide-tag and its HRP-labeled form were purchased fromNovagen. The secondary antibodies labeled with Alexa 488, Cy3and HRP were form Invitrogen, GE healthcare and Invitrogen,respectively. The cell images stained for AHF and krt31 werecarefully analyzed under confocal microscopy (Nikon).

2.2. Expression constructs

The expression constructs for full length AHF [8], krt31 and theAHF mutants were used for transfection experiments. For krt31,cDNAs from the anagenic mouse dorsal skin were prepared, andthat of full length krt31 (NM010659) was amplified by PCR as a T7-peptide tagged form and inserted into the EcoRI-Not I site of themammalian expression vector pQCXIN (BD Bioscience Clontech).The expression constructs for the AHF deletion mutants wereprepared using a Kilo-sequence deletion kit (TaKaRa). A pShuttlevector having full length AHF cDNA at the Nhe I/Not I site [8] wasdouble digested with Not I and Kpn I, treated with Exo III nucleasefor various time periods, blunt-ended with Mung Bean nuclease,recircularized with the ligase and then introduced into bacterialcells JM109 (Takara). After roughly checking for the deletion formof the C-terminus in 40 colonies picked at random, 8 representativeclones were selected and sequenced. These clones contained theAHF gene lacking certain sequences for amino acids from the C-terminus to 1507, 1085, 799, 663, 586, 400, 326, and 13 residues(AHFDC1507, AHFDC1085, AHFDC799, AHFDC663, AHFDC586,AHFDC400, AHFDC326, and AHFDC13, respectively). Other AHFmutant constructs, including those lacking 6 C-terminal aminoacid residues (AHFDC6), lacking the 7th–13th C-terminal residues(AHFDC13-7; 6 C-terminal residues remains intact), and one inwhich the glutamine residue at 1596 was changed to glutamic acid(AHFQ1596E), were further generated. For these constructs, smallcDNA fragments form Fsp I restriction site in AHF to the C-terminalsequences of the designed AHF mutants, ending with the Not I

restriction site, were generated by PCR using the followingprimers. The forward primer for all of the mutants was50-CAGTTGCGCAGGGAGAAGCGA-30. The reverse primers forAHFDC6, AHFDC13-7 and AHF Q1596E were 50-TTTTGCGGCCGCTTACTGCTCCTGGATGTACTCAT-30, 50-TTTTGCGGCCGCTTAAGGGCGGTATTGAGACCTGAGAGGGCTGGAACGC-30 and 50-TTTTGCGGCCGCTTAAGGGCGGTATTCAGACCTCTGCTCCTGGATGTA-30, respec-tively. After being double-digested with Fsp I and Not I, the PCRproduct for each AHF mutant was replaced with the Fsp I–Not Ifragment of full length AHF in the pShuttle vector.

2.3. Transfection

To express the full length AHF or one of the AHF mutants and T7tagged-krt31, HSC-1 cells were transfected with these expressionconstructs using lipofectamine plus reagent (Invitrogen). Sinceone or both of these transgene products became detectable after24–72 h, the cells from day 1 after transfection were used for theanalyses. To generate HSC-1 cells that stably express keratinfilaments containing T7 tagged-krt31, retroviral particles, whichhad been prepared from the supernatant of the packaging cellline PT67 transfected with the krt31 construct, were infected intoHSC-1 cells. After long term incubation in the presence of G418(500 mg/ml), the surviving cells were tested for the expression ofT7 tagged-krt31. To express the full length AHF protein in most ofHSC-1 cell populations (>90%), adenoviral particles (108 pfu/ml)carrying the AHF expression cassette (Yamamoto et al. [8]) wereintroduced into HSC-1 cells that were stably expressing krt31.These cells were incubated for 24 or 48 h to investigate the cellularbehavior.

2.4. Immunocytochemistry and Western blot analyses

Cryosections (10 mm thick) of anagenic mouse skin wereprepared from ICR mice as described previously [13]. HSC-1 cellscultured on a chamber glass slide were fixed with �20 8C methanolfor 5 min and permeabilized with 0.1% Triton X in Tris bufferedsaline (TBS). The sections on the glass slides, cells on the chamberslides and the blots onto which protein bands from the cells hadbeen transferred were treated with the blocking solution for 1 h,primary antibody solution for 1 h and the secondary antibodies for1 h, as described previously [14–16]. As the blocking solution, TBScontaining 1% BSA or 5% skim milk was used for immunocyto-chemistry or Western blot analyses, respectively. Novex ECL(Invitrogen) or 3,30-diaminobenzidine (Sigma) was used tovisualize HRP-labeled secondary antibodies. To detect the apopto-tic cells, non-permeabilized cells cultured on the chamber slideswere stained with annexin V-FITC (BioVision) and Hoechst33342(Dojindo).

2.5. Analyses of AHF and krt31 in the detergent-soluble and

insoluble fractions

HSC-1 cells or those stably expressing T7 tagged-krt31 wereinfected with AHF-adenovirus or the control-adenovirus [8]. After24 h, cells were treated with ice-cold 1% Triton X-100 and 1% NP-40in TBS containing a protease inhibitor cocktail (Nacalai Tesque) for10 min, and centrifuged at 13,000 rpm for 30 min. The solubilizedmaterials in the supernatant and the insoluble materials in thepellet were separately collected and dissolved in 1� SDS samplebuffer for the detection of AHF and krt31.

2.6. Quantification of the AHF granules

HSC-1 cells were co-transfected with constructs for krt31 andone of AHF mutants, and stained for both transgene products after

Fig. 1. Expression of AHF and IRS keratin. (A) Immunostaining of AHF in the hair follicle. AHF is especially abundant in the IRS of the anagenic vibrissae hair follicles (arrows)

(bar: 200 mm). (B) Immunostaining of the anagenic hair follicle revealed that the distribution of AHF (green) is roughly overlapped with that of the IRS-specific keratin krt31

(red) (bar: 100 mm). (C) Exogenously introduced AHF forms small granules and clearly aligns on the keratin filaments. Upper left, AHF and T7-tagged krt31 were introduced

into squamous keratinocyte HSC-1 cells and the distribution of the transgenes was analyzed with antibodies against AHF and T7 after 24 h. The introduced AHF formed small

granules and aligned on keratin bundles composed of krt31. Accumulation and the co-localization of AHF and T7-labeled keratin were apparent also in the desmosomal

structures at the cell–cell contact sites (arrow heads). Upper right, Localization of AHF and krt31 were independently visualized in a part of the left image (inset). Co-

localization of AHF and krt31 was apparent. Lower left, exogenously introduced GFP (negative control) was expressed throughout the cytoplasm. Lower center, the introduced

AHF formed small granules and aligned on some of the intermediate filamentous structures in the keratinocyte cell line C-50. Lower right, AHF was expressed as randomly

positioned large granules in SW-13 cells, which are devoid of keratin filaments (bar: 5 mm).

T. Takase, Y. Hirai / Journal of Dermatological Science 65 (2012) 141–148 143

T. Takase, Y. Hirai / Journal of Dermatological Science 65 (2012) 141–148144

24 h. From the three independent transfections, all the cellsexpressing both AHF and krt31 were photographed for eachcategory and used for the quantification. For the keratin association,the ratio of AHF granules aligned on the keratin filaments wascounted. For the granules size, the diameter of AHF granules wasmeasured. Although cells in some categories displayed very largeand low number of the AHF granules, more than fifty granules werequantified for each category. Results were expressed as mean �SDand data were analyzed using the Mann–Whitney U-test. The p-value<0.01 was considered statistically significant.

3. Results

3.1. Expression of AHF and IRS keratin

In the hair follicles, AHF was abundantly detectable in the IRSduring the anagenic phase (Fig. 1A) and exhibited an apparentpattern of a co-distribution with IRS-specific keratin krt31(Fig. 1B). To clarify the localization and the possible intermolecu-

Fig. 2. Effect of AHF on the keratinocyte behaviors. (A) HSC-1 cells were infected with con

and the expression of AHF transgene product was analyzed after 48 h. Left upper, Wester

in the SDS-PAGE gel after blotting were stained with Coomassie Brilliant Blue. Infection o

Immunostaining of the cells confirmed the effective infection (cells of more than 90% expr

the AHF transgene. Left, the cells stably expressing krt31 were infected with control aden

48 h. Forced expression of AHF resulted in an abnormally flattened morphology and the d

cells appeared to be severely suppressed by AHF expression as judge by WST-1 assay [16]

and annexin V without cell permeabilization. AHF led to the irregular shape of nuclei but n

expressing krt31 (krt31+) or the parental HSC-1 cells (krt31�) were infected with AHF-AV

1% Triton X-100 and the soluble (sup) and insoluble (ppt) fractions were analyzed for AHF

and krt31 in each category. The exogenously introduced AHF appeared to be insoluble

lar interaction in detail, we first introduced the expressionconstruct for T7-labeled keratin31 into several different kerati-nocyte cell lines to choose the appropriate cell model displaying aclear filamentous keratin meshwork labeled with the T7-tag. Wefound that human squamous keratinocyte HSC-1 cells stably andsuccessfully incorporate exogenous krt31 in keratin filaments andassemble clear T7-labeled keratin bundles. When HSC-1 cellswere infected with AHF, the cells formed small rounded AHFgranules which clearly bound to and aligned on the keratinmeshwork, and formed an undercoat on desmosomal structures(Fig. 1C, upper). While negative control GFP was uniformlydistributed in the cytoplasmic space, the keratinocyte cell lineC-50, which possesses other types of epidermal keratin, alsoproduced small AHF granules, and these were distributed alongcytoplasmic filamentous structures (Fig. 1C, lower). In SW-13cells, which are devoid of keratin filaments, AHF formedsignificantly larger granules that were distributed randomly ina disordered pattern (Fig. 1C, lower right). These observationssuggest that the IRS-specific AHF molecule interacts with

trol adenoviral particles (Control AV) or those carrying the AHF transgene (AHF AV),

n blot analysis of the transgene product. Left lower, total cellular proteins remaining

f the AHF-adenovirus led to an abundant expression of AHF in the HSC-1 cells. Right,

essed AHF) (bar: 120 mm). (B) Phenotypic appearance of HSC-1 cells with expressing

oviral particles or those carrying the AHF transgene, followed by the cultivation for

isorganized cell–cell junctional structures (insets) (bar: 30 mm). Right, the growth of

. (C) The cells infected and incubated for 48 h as (B) were stained with Hoechst 33342

ot the apoptotic cell death. (D) AHF altered the solubility of krt31. HSC-1 cells stably

(AHF+) or Control-AV (AHF�). After 24 h, the cells were treated with 1% NP-40 and

and krt31. Total cell lysate (total) was also analyzed to know the total amount of AHF

and clearly pulled down detergent soluble-krt31.

Fig. 3. The AHF mutants prepared in this study. AHF consists of 1599 amino acid

residues and is composed of N-terminal EF hand domains, three types of repeat

domains and finally the C-terminal tail (A). (B) The constructs for the several AHF

deletion mutants containing AHF that lacks C-terminal 13 a.a. (AHFDC13), 326 a.a.

(AHFDC326), 400 a.a. (AHFDC400), 586 a.a. (AHFDC586), 663 a.a. (AHFDC663), 799

a.a. (AHFDC799),1085 a.a. (AHFDC1085) and 1507 a.a. (AHFDC1507) were

generated by the partial deletions from full length AHF gene at the C-terminus.

(C) AHF mutants lacking 6 C-terminal a.a. (AHFDC6), the 7th–13th a.a (AHFDC13-7)

and one in which glutamine at position 1596 was replaced with glutamic acid (AHF

Q1596E) were also prepared by PCR-based techniques.

T. Takase, Y. Hirai / Journal of Dermatological Science 65 (2012) 141–148 145

intermediate filaments of several different keratin subsets andimply that AHF is endowed with the capacity to generatemolecular aggregations.

3.2. Effect of AHF on keratinocyte cellular behaviors

To investigate the effect of the expression and keratinassociation of AHF on cellular behaviors, an attempt was madeto find an appropriate model cells that express AHF protein inculture. We did not find any cell lines expressing detectableamount of endogenous AHF, and the number of AHF-positive cellpopulation in primary keratinocyte appeared to be dramaticallydecreased upon passage (data not shown). We were unable toisolate cells stably expressing the AHF transgene product evenafter several trials, implying an inhibitory effect of AHF protein onthe growth of cells. Thus, we introduced a high-titer adenoviruscarrying the AHF expression cassette into HSC-1 cells havingkeratin filaments containing the T7 tagged-krt31. By thistreatment we obtained cells with more than 90% transientlyproduced AHF protein, an amount that reached �10% of the totalcellular proteins, and this was visualized even with CoomassieBrilliant Blue staining of the cell samples (Fig. 2A). The expressionof the infected AHF molecule led to a more flattened cellmorphology, abnormal cell–cell junctional apparatus, irregularshaped-nuclei and severe growth arrest (Fig. 2B and C). Time-lapseanalyses made the cell behavior and cell fate evident; the growth ofthe cells was suppressed in tandem with cell shape change (thesame areas at t = 0 and 48 h were shown in the left panel of Fig. 2B).The AHF triggered-growth arrest may not be due to the apoptoticcell death, since no nuclear fragmentation was evident andannexin V did not detect the membrane translocation ofphosphatidylserine (Fig. 2C). As the immunolocalization experi-ments clearly demonstrated the interaction between AHF andkeratin bundles, we next try to confirm this molecular associationby another method. We noticed that most of the exogenous AHFprotein in HSC-1 cells could not be solubilized in buffer containing2% NP-40, 2% Triton X-100, 2% empigen BB, 1% Chaps, 2%b-octylglucoside or 1% Tween 20, so that an investigation of theAHF–keratin interaction in this cell model in solutions, such as co-immunoprecipitation, was not feasible. Furthermore, the primaryhuman keratinocytes that produce an immunoprecipitation-soluble subpopulation of AHF [8] did not express the antigenrecognized by the mAb against mouse krt31. However, we foundthat major population of krt31 that was solubilized in 1% NP-40and 1% Triton X-100 became abundant in the detergent-insolublefraction when insoluble AHF was present, indicating that AHF hadpulled down the keratin filaments or conferred the characteristicof insolubility upon them (Fig. 2D). These observations underlinethe key role of AHF in the regulation of the cytoskeletal dynamicsand led us to pursue the molecular elements required for theintermolecular interaction between AHF and keratin.

3.3. Identification of keratin binding site in AHF

The AHF primary structure is composed of N-terminal EF handdomains, three types of internally repeating domains and a short C-terminal tail (Fig. 3A). To identify the indispensable domain forkeratin binding, HSC-1 cells were introduced with one of theconstructs for the AHF deletion mutants and krt31 to investigatetheir cytoplasmic distribution pattern (Fig. 3B). Among AHFmutant constructs, AHFDC1507 failed to express detectabletransgene products. Surprisingly, however, we found all the restof the mutants generated, including that lacking only 13 aminoacid residues from the C-terminus (i.e., 99% intact) exhibitedprofoundly different behavior from that of full length AHF: theyfailed to co-localize with and align on the keratin filaments (Fig. 4A

and B). Concomitantly with the keratin dissociation, these AHFmutants gave rise to much larger granules, suggesting that the restof the large AHF N-terminal body possesses the potential forcohesive assembly and self-aggregation (Fig. 4A and B). The largestsize of the granules was observed in the shortest AHF mutants thatstill possess the EF hand domains, AHFDC799 and AHFDC1085,along with all of the first and part of the second repeat domains(Fig. 3B). Despite the fact that the primary structure of AHF/trichohyalin is not well conserved in the human, mouse, sheep andbovine forms [17], this C-terminal 13 amino acid sequence(YEYIQEQRSQYRP) is nevertheless shared by all of these species.To narrow down the key sequence in this C-terminal tail domain,we generated additional AHF mutants in which a 6 amino acid C-terminal sequence and the 7th–13th amino acids of the C-terminalwere removed (Fig. 3C). Both of the AHF mutants again failed toadhere to the keratin bundles while also exhibiting large granules,proving that the AHF C-terminal tail is indispensable for bindingkeratin molecules (Fig. 4A and B). Since AHF/trichohyalin has beenreported to be a substrate of the protein crosslinker TGase III [10],and the glutamine residue in this C-terminal sequence (QYRP)matches the consensus motif of the TGase target (QXR/KC), it isimplied that the covalent crossbridge between AHF and keratin isinvolved in this intermolecular association. To further investigatethis possibility, we prepared a distinct AHF mutant in which thisglutamine residue has been changed to glutamic acid

Fig. 4. Cytoplasmic distribution patterns of the AHF mutants and krt31 in HSC-1 cells. (A) One of the expression constructs for AHF mutants and that for krt31 were co-

transfected into HSC-1 cells, and the transgene products were analyzed after 24 h. Green, AHF mutant; red, krt31 (bar: 10 mm). (B) Quantitative analyses of the keratin

association (left lower) and the size of the granules (left upper) formed in the AHF mutants. For the keratin association, the percent of the granules that bound to and aligned

on the keratin bundles, which was visualized with an anti-T7 tag mAb, were measured. #, The granules size was too large to evaluate the keratin association. For the granular

size, the diameter of the granules on the photo images was measured for each category. All the C-terminally deleted mutants appeared to lose the ability to associate with the

keratin bundles, whereas AHF Q1596E retains this ability. Data are the mean � SD; **p < 0.01. Concomitantly with keratin dissociation, the granules of the AHF mutants appeared

to be significantly larger (summarized in the right panel).

T. Takase, Y. Hirai / Journal of Dermatological Science 65 (2012) 141–148146

(AHFQ1596E) in full length AHF (Fig. 3C). This point mutation didnot affect the keratin-adherent distribution of the transgeneproduct, suggesting that the covalent bridge between AHF/trichohyalin and keratin via the AHF C-terminus is not primaryresponsible for this intermolecular interaction (Fig. 4A and B).

Taking these results together, we propose a model for themodulation of the keratin meshwork and the subsequent cellularphysical strength induced by AHF. A strictly controlled pattern ofexpression of AHF in linked to the keratin bundles by the AHF C-

terminal tail and this results in a rearrangement of the keratinmeshwork by the cohesive activity of its N-terminal EF hand andrepeating domains (Fig. 5).

4. Discussion

In the present study, we started with the distribution of AHF/trichohyalin, a potent modulator of keratin filamentous assembly.This protein has been shown to form round granules of several

Fig. 5. A model of the function of AHF in the keratin filamentous meshwork. AHF

possesses functionally distinct domains, that is, a C-terminal tail domain for keratin

binding and N-terminal EF hands and repeat domains for molecular aggregation.

AHF may both bind and draw in the keratin filaments, leading to a modulation of the

cytoskeletal meshwork and consequently mechanical support in epidermal tissues.

T. Takase, Y. Hirai / Journal of Dermatological Science 65 (2012) 141–148 147

different sizes and to be abundantly expressed in the IRS ofanagenic hair follicles along with co-distribution to the keratinfilaments [7,18–20]. These molecular profiles were clearly visual-ized in a squamous keratinocyte HSC-1 cell line introduced withexpression constructs for AHF and the IRS-specific keratin krt31tagged with the T7 peptide, where the small round AHF granulesbound to and aligned on the keratin meshwork labeled with theT7-tag. However, we revealed that AHF also recognizes non-IRSepidermal keratin filaments and that the lack of AHF–keratininteraction results in the formation of much larger AHF granules.While such larger AHF granules may physically punish the cells,the keratin-associated form of the small granules led to anunusually flattened cellular appearance and the deformed nucleiwithout inducing the apoptotic cell death. Concomitant with thechange in cellular behavior, the physical features of the keratinfilaments were influenced by the AHF granules, which wasreflected by the detergent insolubility. Considering the specificexpression in the anagenic IRS that directly contact with the rapid-growing hair shaft, AHF may contribute to stabilize the keratinmeshworks to maintain or reinforce the IRS structures in vivo.Indeed, we noticed that the cultured epidermal sheets of mouseembryos (13 g.d.) often became stiff in response to the forcedexpression of AHF (data not shown). It is noteworthy, however, wecould not detect the activity for the compaction or intramolecularcrosslink of the keratin filament in AHF, which have been shownfor another keratin binding protein filaggrin [21,22].

By analyzing the AHF mutants, we identified the AHFC-terminus as the critical keratin association site. The removalof only six amino acids from the C-terminal at the 1st–6th or 7th–13th amino acid residues resulted in the complete loss of keratin-adherent activity. There are reports that TGase III in the hairfollicles induces covalent crosslinks between the AHF/trichohyalinand keratin molecules [7], and the AHF’s C-terminal tail contains aglutamine residue (at 1596) that meets the consensus of this TGaseIII target (QXR/KC) [10]. However, we were able to exclude the

possible involvement of crosslinks between AHF and keratin, sincethe point mutation at 1596 of glutamine to glutamic acid did notimpact this intermolecular association. Given the strict controlexercised over AHF [8] and its unstable nature (estimated as135.89 on an instability index) [23], the intermolecular crosslinksidentified by the previous works may function to stabilize thekeratin–AHF complex on the keratin meshwork. However, we didnot detect any covalent cross-bridges between transientlyexpressed AHF and keratin, e.g., Western blot analyses revealedthat the size of the transiently expressed AHF in keratin-producingor non-producing cells was the same, and neither an antibodyagainst the T7-tag nor pan-keratin reacted to the protein band ofintroduced AHF (data not shown). On the other hand, while theAHF C-terminal tail is clearly indispensable for keratin association,this 13 amino acid sequence may not be sufficient by itself, sinceGFP fused with this sequence never co-precipitated with thekeratin molecules in detergent-containing buffer, even though thisprotein often co-localized with the keratin filaments (data notshown).

The AHF molecule is expressed in the form of rounded granules,the size of which became dramatically larger when set free fromthe keratin association, and these granules were retrieved in theinsoluble fraction of the cells treated with detergent-containingbuffer, despite the low level of hydrophobic amino acid content inthe AHF sequence (hydropathicity was calculated as �2.39). Theseobservations indicate that AHF possesses intrinsically criticalinformation for the inter- and intra-molecular assembly in its largeN-terminal body, which is composed of EF hand domains forcalcium binding and three distinct types of long internal repeats.All of the AHF deletion-mutants containing the EF hand domainsand the first two repetitive domains produced granules ofsignificantly larger sizes, indicating the importance of thesedomains for molecular aggregation. Of note, the EF hand domainis known to functions as a calcium sensor [9], and the positive(arginine) and negative (glutamic acid) residues abundant in theinternal repetitive domains are evenly distributed. This molecularprofile is reminiscent of the recently engineered amphiphilicpolypeptides that give rise to the stable molecular aggregates inthe presence of cations [24–26]. To clarify the role of the regularlyarranged charged residues in this domain, testing the molecularprofiles of synthetic peptides corresponding to the unit of eachrepetitive sequence in AHF, with or without the EF hand domain ormultiple amino acid mutations will be necessary.

The lack of AHF-expressing model cells and the insoluble natureof AHF have been practical obstacles to the elucidation of the role ofAHF, but the finding that AHF possesses molecular domains forkeratin association and molecular aggregation may provide a novelmodel of the control of cytoskeletal dynamics and the IRS physicalstrength in anagenic hair follicles. Several reports have pointed outthat the expression of AHF/trichohyalin is not restricted to theprogressive keratinocytes in the IRS but also weakly occurs in otherepidermal tissues such as the nail, tongue and certain epidermallayers so as to participate in the strength of these tissues [27,28].Indeed, we observed that AHF displays a dot-like expression patternalong with the cytoplasmic filamentous structures in epidermal celllines that are devoid of IRS keratin. If the other keratin-bundlemodulators in epidermal tissues, such as filaggrin and keratohyalin[18,29], lack any sequences which resemble the AHF’s C-terminus,this observation indicates the distinct and synergetic roles for thesekeratin-associated molecules [18,29]. Attempts to clarify whetherthis is indeed the case are now underway.

Acknowledgments

We thank to Drs. Susan M Fisher for C-50 keratinocytes,Motomu Manabe and Kiyotaka Hitomi for critical comments and

T. Takase, Y. Hirai / Journal of Dermatological Science 65 (2012) 141–148148

Sachiya Yamamoto for technical supports. We are grateful toKyoko Hirai for the preliminary indications and all members ofHirai laboratory for helpful discussions. Part of this work wassupported by Hyogo COE Program Promotion Project.

References

[1] Kikkawa Y, Oyama A, Ishii R, Miura I, Amano T, Ishii Y, et al. A small deletionhotspot in the type II keratin gene mK6irs1/Krt2-6g on mouse chromosome 15,a candidate for causing the wavy hair of the caracul (Ca) mutation. Genetics2003;165:721–33.

[2] Peters T, Sedlmeier R, Bussow H, Runkel F, Luers GH, Korthaus D, et al. Alopeciain a novel mouse model RCO3 is caused by mK6irs1 deficiency. J InvestDermatol 2003;121:674–80.

[3] Powell BC, Rogers GE. Cyclic hair-loss and regrowth in transgenic mice over-expressing an intermediate filament gene. EMBO J 1990;9:1485–93.

[4] Schweizer J, Langbein L, Rogers MA, Winter H. Hair follicle-specific keratinsand their diseases. Exp Cell Res 2007;313:2010–20.

[5] Alibardi L. Fine structure and immunocytochemistry of monotreme hairs, withemphasis on the inner root sheath and trichohyalin-based cornification duringhair evolution. J Morphol 2004;261:345–63.

[6] Rothnagel JA, Rogers GE. Trichohyalin, an intermediate filament-associatedprotein of the hair follicle. J Cell Biol 1986;102:1419–29.

[7] Steinert PM, Parry DA, Marekov LN. Trichohyalin mechanically strengthens thehair follicle: multiple cross-bridging roles in the inner root shealth. J Biol Chem2003;278:41409–1.

[8] Yamamoto S, Hirai K, Hasegawa-Oka Y, Hirai Y. Molecular elements of theregulatory control of keratin filament modulator AHF/trichohyalin in the hairfollicle. Exp Dermatol 2009;18:152–9.

[9] Grabarek Z. Insights into modulation of calcium signaling by magnesium incalmodulin, troponin C and related EF-hand proteins. Biochim Biophys Acta2011;1813:913–21.

[10] Yamane A, Fukui M, Sugimura Y, Itoh M, Alea MP, Thomas V, et al. Identifica-tion of a preferred substrate peptide for transglutaminase 3 and detection of insitu activity in skin and hair follicles. FEBS J 2010;277:3564–74.

[11] Ruggeri B, Caamano J, Goodrow T, DiRado M, Bianchi A, Trono D, et al.Alterations of the p53 tumor suppressor gene during mouse skin tumorprogression. Cancer Res 1991;51:6615–21.

[12] Schweitzer SC, Klymkowsky MW, Bellin RM, Robson RM, Capetanaki Y, EvansRM. Paranemin and the organization of desmin filament networks. J Cell Sci2001;114:1079–89.

[13] Takebe K, Oka Y, Radisky D, Tsuda H, Tochigui K, Koshida S, et al. Epimorphinacts to induce hair follicle anagen in C57BL/6 mice. FASEB J 2003;17:2037–47.

[14] Aono Y, Hirai Y. A culture system for the live analysis of successive develop-mental processes and the morphological control of mammalian vertebralcartilage. Cytotechnology 2011;63:269–77.

[15] Hirai Y, Nose A, Kobayashi S, Takeichi M. Expression and role of E- andP-cadherin adhesion molecules in embryonic histogenesis. I. Lung epithelialmorphogenesis. Development (Cambridge England) 1989;105:263–70.

[16] Okugawa Y, Hirai Y. Overexpression of extracellular epimorphin leads toimpaired epidermal differentiation in HaCaT keratinocytes. J Invest Dermatol2008;128:1884–93.

[17] Alibardi L. Comparative aspects of the inner root sheath in adult and develop-ing hairs of mammals in relation to the evolution of hairs. J Anat 2004;205:179–200.

[18] Manabe M, O’Guin WM. Keratohyalin, trichohyalin and keratohyalin–tricho-hyalin hybrid granules: an overview. J Dermatol 1992;19:749–55.

[19] O’Guin WM, Sun TT, Manabe M. Interaction of trichohyalin with intermediatefilaments: three immunologically defined stages of trichohyalin maturation.J Invest Dermatol 1992;98:24–32.

[20] Steinert PM, Marekov LN. Multiple roles for trichohyalin in the inner rootsheath. Exp Dermatol 1999;8:331–2.

[21] Kuechle MK, Presland RB, Lewis SP, Fleckman P, Dale BA. Inducible expressionof filaggrin increases keratinocyte susceptibility to apoptotic cell death. CellDeath Differ 2000;7:566–73.

[22] Manabe M, Sanchez M, Sun TT, Dale BA. Interaction of filaggrin with keratinfilaments during advanced stages of normal human epidermal differentiationand in ichthyosis vulgaris. Differentiation 1991;48:43–50.

[23] Guruprasad K, Reddy BV, Pandit MW. Correlation between stability of a proteinand its dipeptide composition: a novel approach for predicting in vivo stability ofa protein from its primary sequence. Protein Eng 1990;4:155–61.

[24] Caplan MR, Schwartzfarb EM, Zhang S, Kamm RD, Lauffenburger DA. Effects ofsystematic variation of amino acid sequence on the mechanical properties of aself-assembling, oligopeptide biomaterial. J Biomater Sci Polym Ed 2002;13:225–36.

[25] Zhang S, Lockshin C, Herbert A, Winter E, Rich A. Zuotin, a putative Z-DNAbinding protein in Saccharomyces cerevisiae. EMBO J 1992;11:3787–96.

[26] Zhang S, Marini DM, Hwang W, Santoso S. Design of nanostructured biologicalmaterials through self-assembly of peptides and proteins. Curr Opin Chem Biol2002;6:865–71.

[27] O’Keefe EJ, Hamilton EH, Lee SC, Steinert P. Trichohyalin: a structural protein ofhair, tongue, nail, and epidermis. J Invest Dermatol 1993;101:65S–71S.

[28] Manabe M, O’Guin WM. Existence of trichohyalin-keratohyalin hybrid gran-ules: co-localization of two major intermediate filament-associated proteinsin non-follicular epithelia. Differentiation 1994;58:65–75.

[29] O’Regan GM, Irvine AD. The role of filaggrin loss-of-function mutations inatopic dermatitis. Curr Opin Allergy Clin Immunol 2008;8:406–10.

Takahisa Takase Graduate student of Bioscience department, Kwansei GakuinUniversity, Japan.

Yohei Hirai Professor of Bioscience department, Kwansei Gakuin University, Japan. Heearned BS/MS/Ph.D degrees from Kyoto University, Japan.