A Novel Type II Cytokeratin, mK6irs, is Expressed in theHuxley and Henle Layers of the Mouse Inner Root Sheath

Noriaki Aoki, Souhei Sawada, Michael A. Rogers,* JuÈrgen Schweizer,* Yutaka Shimomura,Tomotaka Tsujimoto, Kaoru Ito, and Masaaki ItoDepartment of Dermatology, Niigata University School of Medicine, Asahimachi-dori, Niigata, Japan; *German Cancer Research Center,

Research Programm 2, Heidelberg, Germany

Hair follicle differentiation involves the expression ofboth epithelial-type keratins or cytokeratins and hairkeratins as well as hair keratin-associated proteins. Inthis study, a cDNA clone encoding a cytokeratinfamily member was isolated using RNA differentialdisplay techniques. The predicted amino acidsequence derived from this clone, revealed a homol-ogy with a number of cytokeratins, not only in thecentral a-helical regions but also in the conservedportions of the amino and carboxy terminaldomains, indicating that this protein represents anew member of the mouse type II cytokeratinfamily. Northern blot analysis showed expression inmouse skin, but not in other tissues, including ton-gue, esophagus, and forestomach. One- and two-dimensional western blot analysis showed that thisnew cytokeratin was 57 kDa in size and ran slightlybelow the area of cytokeratin 5, which corresponded

to that of the cytokeratin 6 family members. BothRNA in situ hybridization and immunohistochemicalstudies of mouse anagen hair follicles demonstratedexpression of this cytokeratin in the inner root sheathhair cone during anagen III and in the Henle andHuxley layers of the inner root sheath during anagenVI. The expression of the new cytokeratin began inthe hair bulb and progressed up to the height of thekeratogenous zone. Taken together the sum of thedata analyzed, we have termed this novel cytokeratinmK6irs (mouse gene nomenclature k2-6g) to indicateboth its similar mobility with K6 in two-dimensionalgels and its speci®c expression in the inner rootsheath of the hair follicle. Key words: hair cycle/immu-nohistochemistry/in situ hybridization/RNA differentialdisplay/two-dimensional sodium dodecyl sulfate±polyacryla-mide gel electrophoresis. J Invest Dermatol 116:359±365,2001

Among the skin appendages, the hair follicle has themost complicated structure; composed of several celllayers and displaying dynamic cell kinetics, asindicated by the hair cycle, i.e., the cyclic formationand regression of the hair follicle. During this cycle,

hairs grow in the anagen phase, stop their growth in the catagenphase and rest or fall out during the telogen phase (Ito, 1990).Within the anagen phase, six stages (anagen I±VI) have been furtheridenti®ed histologically.

Morphologically, the anagen hair follicle is divided into threegroups of cell layers, the hair shaft, the inner root sheath (IRS), andthe outer root sheath (ORS). The IRS is further subdivided intothe Huxley and Henle layers as well as the cuticle of the IRS. TheIRS forms in the region of the hair bulb, differentiates, and is shedat the level of the mid-isthmus into the infundibulum of the follicle.The morphologic ®ndings clearly indicate that the IRS plays a partin guiding the developing hair shaft, whereby the IRS forms aninner part of the follicular wall surrounding the lower portion ofthe hair canal (Ito, 1990).

The major structural proteins of the hair follicle have beenidenti®ed as keratins. The keratin multigene family comprises morethan 40 individual but structurally related members that, by virtueof their sites of expression, are traditionally divided into epithelial-type or cytokeratins and hair-type keratins. Two subtypes of keratinhave been further described based on their charge properties. Ingeneral, type I keratins are smaller, with an acidic pkI and includecytokeratins K9 through K20, whereas type II keratins are larger,more basic and include cytokeratins K1±K8 (Moll et al, 1982).Keratin intermediate ®laments are obligatory heterodimers and atleast one member from each type is required to form the two chaincoiled-coil, which is the basic building block of the intermediate®lament (Hatzfeld and Weber, 1990; Steinert, 1990). So far,approximately 40 keratin genes have been isolated from humans,mouse, and sheep. The deduced amino acid sequences show a highsequence identity among the respective keratin subfamilies and thissequence identity is conserved across species boundaries.

Independent of species, the keratin pro®le of the hair follicle, asrevealed by one- and two-dimensional gel electrophoresis, ismainly composed of cytokeratins K5, K6a, K6b, K14, and K16/17(Heid et al, 1986, 1988; Lynch et al, 1986; Stark et al, 1987; Kopanand Fuchs, 1989). In situ hybridization studies and immunohisto-chemical studies have demonstrated that these cytokeratins areexpressed in the ORS of the hair follicle (Coulombe et al, 1989;Kopan and Fuchs, 1989; Wollina, 1992; Asada et al, 1993; Van Baar

Manuscript received June 19, 2000; revised October 19, 2000; acceptedfor publication October 23, 2000.

Reprint requests to: Dr. Noriaki Aoki, Department of Dermatology,Niigata University School of Medicine, Asahimachi-dori, Niigata 951-8510, Japan. Email: rayaoki@med.niigata-u.ac.jp

0022-202X/01/$15.00 ´ Copyright # 2001 by The Society for Investigative Dermatology, Inc.

359

et al, 1994; Panteleyev et al, 1997; McGowan and Coulombe, 1998;Winter et al, 1998).

In this study, we have identi®ed a new mouse type IIcytokeratin, mK6irs, using RNA differential display strategy. Thecharacterization of this cytokeratin and the localization of itstranscript and protein are examined in detail. This is one of the ®rstreports of a cytokeratin whose expression is limited to the IRS ofthe hair follicle.

MATERIALS AND METHODS

Animals and skin samples Syngeneic C57BL/6NCrj female mice(purchased from Charles River, Yokohama, Japan), 21±27 d old, andBALB/CAnNCrj mice, 21±42 d old, were housed under 12 h light/darkcycles and fed ad libitum.

RNA extraction Total RNA were isolated using Isogen (Nippongene,Tokyo, Japan) from dorsal skin sections and various other tissues ofC57BL/6NCrj mice according to the manufacturer's recommendations.All skin samples were examined histologically by light microscopy todetermine their developmental stage of the hair cycle.

RNA differential display A differential display kit (Clontech, PaloAlto, CA) was applied for the reverse transcription of mRNA and forsubsequent polymerase chain reaction (PCR) ampli®cation of thegenerated cDNA. Total RNA samples from dorsal skins of 21 d old(telogen phase), 24 d old (anagen I), and 27 d old (anagen III) C57BL/6NCrj mice were reverse transcribed using an oligo(dT) primer after10 min digestion with RNase-free DNase (Boehringer, Mannheim,Germany). The cDNA were ampli®ed by polymerase chain reaction(PCR) using one set of 10 arbitrary primers (P1±10) and nine speci®coligo(dT) primers (T1±9), four deoxyribonucleoside triphosphate,including [a-33P]deoxyadenosine triphosphate (Amersham PharmaciaBiotech, Bucks, U.K.), and Taq polymerase (Clontech) according to themanufacturer's speci®cations. The PCR products were then separated on a5% denaturing polyacrylamide gel and detected by autoradiography.Differentially expressed PCR products were recovered from the gel andreampli®ed under similar conditions. Reampli®ed PCR products wereligated into pGEM-T Easy vector (Promega, Madison, WI) andtransformed into Escherichia coli JM109 (Promega). DNA was extractedfrom different clones and sequenced with automated sequencer (AmershamPharmacia Biotech). The resulting sequences were used to search forhomology with genes registered in GenBank by BLAST.

5¢-rapid ampli®cation of cDNA ends To identify the full-lengthsequence of the mK6irs clone, 5¢-rapid ampli®cation of cDNA ends wasperformed with a marathon cDNA ampli®cation kit (Clontech). After thesynthesis of the ®rst strand cDNA using an oligo(dT) primer derived fromtotal RNA of a mouse anagen skin (day 27), double strand cDNA wasligated to the cDNA adaptor (5¢-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3¢). 5¢-cDNA end of mK6irs wasampli®ed by PCR using adaptor primer (5¢-CCATCCTAATACGACTCACTATAGGGC-3¢) and internal gene-speci®c primer (5¢-CTGAGGGATCGAGGTCACAAGAAGC-3¢) derived from the 3¢-noncoding regionof the partial mK6irs cDNA, according to the manufacturer's instructions.The ampli®ed cDNA fragment was subcloned into pBluescript, andplasmid DNA from ®ve colonies were prepared and sequenced.

Northern blot analysis Ten micrograms of total RNA waselectrophoresed in a 1.5% formaldehyde gel using RNA molecularweight markers and transferred to a nylon ®lter as previously described(Thomas, 1983). The 3¢ untranslated region of mK6irs cDNA (1715±2193 bp) was labeled with 32P by a hexanucleotide random priming method(Feinberg and Vogelstein, 1983) and used as a probe. Hybridization wasperformed at 68°C using QuikHyb Hybridization Solution (Clontech) andfollowed by washing with 0.1 3 sodium citrate/chloride buffer (150 mMNaCl, 15 mM trisodium citrate), 0.1% sodium dodecyl sulfate (SDS) at68°C. The ®lter was subsequently rehybridized with the glyceraldehyde-3-phosphate dehydrogenase probe after removing the mK6irs signals byboiling in water.

Antibody preparation and immunoblot analysis A monospeci®cantibody to mK6irs was produced in rabbits by immunizing with thesynthetic peptide, KDTLTKGSSLSTPSKKGGR, corresponding to uniqueamino acid sequences of the protein (residues 506±524 of mK6irs) using apreviously described immunization protocol (Roop et al, 1984). The

antibody was puri®ed by af®nity chromatography using the syntheticpeptide coupled to activated Sepharose (Brinkley et al, 1980).

Immunoblots were performed as described previously (Winter et al,1997a; 1998). In brief, keratins were extracted using a SDS±mercaptoetha-nol buffer at pH 8 from total dorsal skin of a 4 d old mouse, and resolved bytwo-dimensional SDS±polyacrylamide gel electrophoresis (nonequilibriumpH gradient gel electrophoresis, NEPHGE). For western blots, gels weretransferred to PVDF membranes (Immobilon-P, Millipore, EschbornGermany) by wet blotting. After staining (0.1% Coomassie blue, 40%methanol, 10% acetic acid), destaining (50% methanol), and blocking with10% nonfat milk powder in Tris-buffered saline, membranes wereincubated with the anti-mK6irs antibody at a dilution of 1:15,000.Peroxidase-coupled secondary goat anti-rabbit IgG antibody (Dianova,Hamburg, Germany) was diluted 1:2000 in 5% nonfat milk powder in Tris-buffered saline. The secondary antibody was detected by enhancedchemiluminescence (Amersham Pharmacia Biotech).

In situ hybridization The BALB/CAnNCrj mouse was chosen becauseC57BL/6NCrj mouse from which cDNA clones were isolated hadabundant hair follicle melanin, which interfered with the detection of thetranscripts (Aoki et al, 2000).

For the production of the anti-sense and sense RNA probes, a mK6irscDNA clone covering the 3¢ noncoding region was linealized with NotIand XhoI, respectively. Digoxigenin-labeled RNA probes were synthesizedfrom the SP6 and T7 promoter using DIG-UTP (Boehringer). In situhybridization analysis was performed according to a previously describedmethod (Powell and Rogers, 1990). Brie¯y, 21±42 d old BALB/CAnNCrjmice skins were ®xed in 4% paraformaldehyde and processed to paraf®nsections. Hybridization was performed at 55°C for 22 h with thedigoxigenin-labeled RNA probes at a concentration of 1 ng per ml. Aftertreatment with RNase A and wash in 0.1 3 sodium citrate/chloride buffer,the tissue sections were treated with an anti-digoxigenin antibody coupledto alkaline phosphatase and were stained for alkaline phosphatase activity.

Immunohistochemical analysis Immunohistochemical study wasperformed according to a previously described method (Aoki et al, 1998).Paraf®n sections of 21±42 d old BALB/C mice were deparaf®nized andincubated sequentially with blocking serum, primary antibody, biotinylatedsecond antibody, and streptavidin±peroxidase following the manufacturer'sprotocol (Nichirei, Tokyo). Primary antibodies, af®nity puri®ed anti-mK6irs antibody and preimmune serum, were diluted 1:2000 inphosphate-buffered saline and incubated overnight at 4°C in ahumidi®ed chamber. Antibody binding was visualized withdiaminobenzidine, and the sections were then counterstained withhematoxylin.

Accession number The mK6irs sequence is available under theaccession number AB033744 of the EMBL/GenBank/DDBJ.

RESULTS

Fragment 7l2, a product differentially ampli®ed in anagenIII, is not seen in telogen or anagen I Among the severalthousand DNA fragments ampli®ed by PCR based differentialdisplay using 90 primer pair combinations, we identi®ed severalcDNA fragments whose band intensity increased in 21±27 d oldmice. One of them, clone 7l2, had an insert size of 800 bp, was apartial clone and shared a high homology with human and mousecytokeratin cDNA (results not shown). To con®rm the differentialexpression of clone 7l2 during the hair cycle, northern blot analysiswas performed using total RNA extracted from the dorsal skins of21, 24, and 27 d old mice. Northern blot analysis showed thepresence of two distinct transcripts: one major transcript of 2.2 kband one minor transcript of 1.8 kb (Fig 1). These transcripts weredetected at day 27 (anagen III), but were not seen at day 21 (telogenphase) and 24 (anagen I). We therefore selected clone 7l2 forfurther investigation.

The predicted amino acid sequence of mK6irs shows that itis a novel mouse type II cytokeratin To isolate the 5¢ end ofclone 7l2, we ampli®ed mouse anagen skin cDNA using a 5¢-rapidampli®cation of cDNA ends strategy, and obtained only one cDNAfragment via PCR ampli®cation. DNA sequencing of this fragmentallowed the construction of a full-length cDNA containing a singleopen reading frame, which begins with a methionine initiation

360 AOKI ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

codon at nucleotide 52 and ends with a TGA stop codon atnucleotide 1626. Translation of the open reading frame yielded aprotein of 524 residues with a calculated molecular weight of57.4 kDa and an isoelectric pH value of 6.63 (Fig 2). Databasesearching for homologous protein sequences revealed that thisprotein shared a high identity with members of the mouse andhuman type II cytokeratins, particularly in their central a-helicalrod domain (Table I), including the characteristic helix initiationmotif and helix termination motif (Fig 3), which are crucial for thestructure and function of keratins (Parry and Steinert, 1995). Forreasons explained below, we have termed the novel type IIcytokeratin mK6irs.

mK6irs is expressed speci®cally in skin In order to investigatethe expression of mK6irs in mouse tissues, northern blot analysiswas performed with the total RNA from various mouse tissuesusing the 3¢ untranslated region as a probe. As shown in Fig 4, twotranscripts, 1.8 and 2.2 kb, were detected only in 27 d old anagenmouse skin. Transcripts were not present in the other tissues,including the tongue, esophagus, and forestomach. As the entiremK6irs cDNA encompasses 2193 nucleotides, it most likelycorresponds to the more abundantly expressed 2.2 kb transcript.The 1.8 kb transcript may be the product of a related gene or analternatively spliced form of the mK6irs gene.

mK6irs is detectable in a similar region as cytokeratin K4 orK6 by two-dimensional western blotting of anagen mouseskin An antibody was raised in rabbits against a synthetic peptidecorresponding to residues 506±524 of mK6irs (Fig 2). One-dimensional western blotting using this antibody showed theapparent molecular size of the protein to be approximately 57 kDa(data not shown), which was consistent with the size predictedfrom the amino acid sequence.

To investigate the characteristics further of mK6irs, weperformed two-dimensional western blots. Keratins were extractedfrom the total dorsal skin of a 4 d old mouse, a time in which mosthair follicles are in anagen VI and possess a fully developed ORSand IRS. As shown in Fig 5(A), the keratin pattern in the two-dimensional SDS±polyacrylamide gel electrophoresis consistedpredominantly of epidermal keratins, whereas only traces of typeI hair keratins were visible. Western blotting using the anti-mK6irsantibody revealed a single protein separated clearly into threeisoelectric variants (Fig 5B). mK6irs ran at 57 kDa, i.e., slightlybelow the 58.5±59 kDa basal cell cytokeratin K5 (MK5) (Schwei-zer, 1993), and was located in the more basic part of the gel, whichcorresponded to the area of the cytokeratin K4 or K6 (Wild andMischke, 1986; Schweizer, 1993). This location con®rmed the typeII cytokeratin nature of mK6irs.

mK6irs transcripts are expressed in the Huxley and Henlelayers of the hair follicle inner root sheath To determine thelocalization of mK6irs transcripts in skin, we analyzed mouse skinsamples from day 21 to day 42, which corresponded to their secondhair cycle. mK6irs transcripts were not detected in mouse skin fromtelogen to anagen II (days 21 and 24) (data not shown), appeared inanagen III±IV (days 27 and 30) (Fig 6a), and was abundantlyexpressed in anagen V and VI (days 32, 36, and 39) (Fig 6b±f).Expression ceased after re-entry into catagen phase (day 42) (datanot shown). An examination of sections in anagen III, the time atwhich the proliferating epithelial cells complete formation of afollicle bulb surrounding the dermal papilla and hair follicleelongation has commenced, showed mK6irs transcript expression inthe IRS, which appears cone-shaped lying medial to the ORS(Fig 6a). In anagen VI when the follicle has fully elongated, thetranscripts were predominantly restricted to the Huxley and Henlelayers of the IRS. The expression of mK6irs transcripts in the Henlelayer commenced at the lower hair bulb (Fig 6b), and was detectedup to the lower portion of the hair follicle keratogenous zone, a

Figure 2. Amino acid sequence of mK6irs. The central a-helical roddomain is indicated by hatched boxes. The sequence is available fromEMBL/GenBank/DDBJ (accession number AB033744).

Figure 1. The gene fragment expressed differentially by RNAdifferential display, clone 7l2, can be detected in mouse skins atday 27 but not at days 21 and 24 by northern blot. Total RNA (10 mg)prepared from the dorsal skins of C57BL/6 mouse at days 21, 24, and 27was electrophoresed and transferred to nylon ®lter. The ®lter washybridized with 32P-labeled mK6irs cDNA probe and followed by washingwith 0.1 3 sodium citrate/chloride buffer, 0.1% SDS at 68°C. The blot wassubsequently rehybridized with the glyceraldehyde-3-phosphate dehydro-genase (GAPDH) probe. The RNA markers are indicated at left side.

Table I. Similarity of mK6irs to other cytokeratins

Cytokeratins Identity (%)

Full length Rod domain

Mouse type II K1 49 64K4 56 67K6 55 63K8 48 60

Human type II K1 49 67K2e 51 67K2p 52 69K3 53 71K4 59 71K5 55 70K6a 57 70K6b 57 70K6c 56 70K6e 56 70K6f 57 70K6hf 57 67K7 51 65K8 49 62

Mouse type I K10, K12, K13,K15, K18, K19

23±28 27±30

Human type I K9, K10,K12±K20

22±27 23±29

VOL. 116, NO. 3 MARCH 2001 NOVEL CYTOKERATIN IN MOUSE HAIR FOLLICLE 361

region where the Henle layer has completely keratinized (Fig 6c).Subsequently, its expression was detectable continuously in theHuxley layer at the height of the keratogenous zone (Fig 6b±d).Transverse sections showed signals in the Huxley and Henle layersbut not in the hair shaft, including the medulla, and ORS(Fig 6e,f). During the hair growth cycle, staining was not detectedin epidermis, sebaceous gland, and sweat gland (data not shown),whereas weak staining appeared in the upper cortical layer anddermis (Fig 6b,d). In situ hybridization using a sense probe alsoshowed the weak upper cortical layer and dermal staining (Fig 6h),thus indicating an unspeci®c reaction.

mK6irs protein is expressed in the Huxley and Henle layersof the IRS We performed immunohistochemical experimentsusing the dorsal skins of 21±42 d old BALB/C mice to investigatethe localization of mK6irs protein in the mouse skin throughoutthe different hair growth phases. No staining was detected intelogen and anagen I±IV animals (data not shown). In anagen V andVI, anti-mK6irs antibody reacted consistently with the IRSinitiating in the upper hair bulb to a region just below thesebaceous gland of the hair follicle (Fig 7a). In the upper portion ofthe hair bulb, the staining commenced in the Henle layer andcontinued until the complete keratinization of the Henle layeroccurred (Fig 7b). Similar staining was detected in the Huxley layerfrom the suprabulbar portion up to the height of the keratogenouszone, where the Huxley layer completely keratinizes (Fig 7b±d).mK6irs staining in the IRS was detectable from anagen V to themid-catagen phase, the time when the IRS moves up the follicleahead of the cortical layer, the cortex forming the brush end of theclub hair by interdigitating with the transformed ORS (Fig 7e).Immunostaining was not observed in the other cells of the hairfollicle and the epidermis (data not shown). Preimmune serum alsoshowed no positive staining in mouse skin (Fig 7f).

DISCUSSION

The hair follicle contains three major classes of proteins:cytokeratins and hair keratins as well as the hair keratin-associatedhigh-glycine/tyrosine proteins and cysteine-rich proteins. In the

anagen phase hair follicles, both types of keratin genes are initiallyactivated, followed by the subsequent expression of high-glycine/tyrosine proteins and thereafter, by cysteine-rich proteins (Aokiet al, 1997; Powell and Rogers, 1997). In the telogen phase, theexpression of all of these genes decreases signi®cantly and ®nallyceases. This scenario implies that cDNA encoding hair folliclekeratins can be isolated by differentially screening mouse skinsbetween the anagen and telogen phase.

Using this differential display strategy in this study, we were ableto isolate one cDNA clone, which encoded a novel cytokeratin.Multiple sequence comparisons showed that the new keratinbelonged to the type II cytokeratin subfamily. Regarding itsdesignation, we followed the reasoning that has previously beenapplied for the numerical integration into the cytokeratin catalog(Moll et al, 1982) of another, recently discovered type IIcytokeratins, K6hf (Winter et al, 1998). The respective criteriawere: (i) the isoelectric pH values of the keratin that determines itsclassi®cation as acidic, type I and basic to neutral, type IIcytokeratin; (ii) its relative molecular mass on SDS±polyacrylamidegels, which de®nes the type-speci®c numerical designationaccording to decreasing molecular weights; and (iii) its tissueexpression. Indeed, our two-dimensional western blots con®rmed

Figure 3. Amino acid alignment of helix initiation and helixtermination motifs of mK6irs and human and mouse type I andtype I cytokeratins reported to date. Black boxes represent identicalresidues.

Figure 4. mK6irs transcript can be detected abundantly in theanagen skin. Total RNA was extracted from various tissues of C57BL/6NCrj, separated by electrophoresis in 1.5% agarose, transferred to a nylon®lter, and hybridized with 32P-labeled mK6irs cDNA probe. mK6irstranscript is detected only in the skin from 27 d old mice.

Figure 5. Two-dimensional SDS±polyacrylamide gel electrophor-esis and western blot reveal that mK6irs runs at the region ofcytokeratin K4 or K6. (A) Coomassie-stained two-dimensional keratinpattern of 4 d old mouse skin. The keratin proteins are designated accordingto the catalog of human keratins (Moll et al, 1982). The arrowhead indicatesthe major 62 kDa stratum corneum equivalents of the weak MK1 band(Schweizer, 1993). (B) Western blot of (A) with anti-mK6irs antibody,which resolves the protein into three isoelectric variants. The arrowsdenotes the position of MK5. Proteins were separated by nonequilibriumpH gradient gel electrophoresis in the ®rst dimension (N), and bySDS±polyacrylamide gel electrophoresis (8% gel) in the second dimension(S).

362 AOKI ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

that the new keratin is a member of the type II cytokeratin family.Its position on these blots is very close to the theoretical position ofK4 or K6 (Wild and Mischke, 1986; Schweizer et al, 1988;Schweizer, 1993). Sequence comparisons of the new keratin withall known mouse type II keratins revealed the highest homologywith mK4 as well as a slightly lower homology with mK6. Previousstudies indicated that cytokeratin 4 occurs in the tongue,esophagus, and forestomach, but not fundamentally in the skin(Moll et al, 1982; Wild and Mischke, 1986; Schweizer et al, 1988).In contrast, K6 has been shown to be expressed in the hair follicle(Stark et al, 1987; Winter et al, 1998). Finally, the new keratin wasspeci®cally expressed in the IRS of the hair follicle. These data ledus to designate the new mouse keratin mK6irs. In this context it isworth mentioning that mK6irs also exhibits a high homology withthe human companion layer speci®c keratin K6hf. The K6hf genehas recently been shown to be located adjacent to the K6b gene(Rogers et al, 2000), thus indicating that it may be part of theputative K6 gene cluster. It remains to be seen whether the mK6irsgene or its still unknown human counterpart are also located in therespective K6 gene cluster.

mK6irs adds to cytokeratins K5, K6, K14, and K16/17 known tobe expressed in the ORS of the hair follicle (Coulombe et al, 1989;Kopan and Fuchs, 1989; Panteleyev et al, 1997; McGowan andCoulombe, 1998; Winter et al, 1998). Whereas the latter also showsexpression in epidermis, mK6irs expression is restricted to the IRSof the hair follicle. Previous reports claimed the expression of othertype I and type II cytokeratins in the IRS, such as K1, K10, K13,and K16 (Stark et al, 1990). Again these cytokeratins are not present

exclusively in the IRS and their IRS expression needs furthercon®rmation. From this it follows that, up to now, mK6irs is theonly IRS-speci®c cytokeratin. Similar to the human type Icompanion layer speci®c keratin K6hf (Winter et al, 1998), thetype II partner for mK6irs, necessary for the biologic functionalityof the type II keratin, is not yet known.

This study has shown, that mK6irs is expressed predominantly inthe Henle and Huxley layers of the IRS during the hair folliclegrowth phase. Interestingly, in anagen III, the protein could not bedetected despite the apparent expression of the transcript in theIRS. The inability to demonstrate mK6irs during the early anagenphase might be due to determinant masking of the protein. Inaddition, the presence of the mK6irs protein in the IRS continuesuntil mid-catagen, a time in which mK6irs transcripts are notdetected, indicating a temporal discrepancy of expression.

A preliminary BLAST database search failed to show thepresence of a human ortholog of mK6irs. The identi®cation ofthe human ortholog of mK6irs is, however, important for thefollowing reason. It is now ®rmly established that mutations inkeratins can cause a large number of hereditary dermatoses, whichare characterized by keratin intermediate ®lament disruption (for areview, Irvine and McLean, 1999). Interestingly, transgenic miceexpressing a mutant cytokeratin K6 have been shown to developprogressive cicatricial alopecia due to the gradual destruction of theORS (Rothnagel et al, 1994). More recently, mutations in hairkeratin genes hHb1 and hHb6 have been shown to be present inthe congenital hair disease monilethrix, which is characterized bybeaded and fragile hairs (Winter et al, 1997b, 2000; Zlotogorski et al,

Figure 6. mK6irs transcripts are expressed in the Huxley and Henle layers of the IRS. Digoxigenin-labeled sense and anti-sense RNA probesprepared from mK6irs cDNA were hybridized to paraf®n sections of BALB/CAnNCrj mouse skin at days 27 (a) and 36 (b± h), and counterstained withmethyl green. (c, d) Higher magni®cations of the middle and upper parts of the follicle in b. The longitudinal (a±d) and transverse (e, f) sections demonstratethat mK6irs transcripts are located in the Huxley and Henle layers of the IRS. Note that mK6irs transcripts appear in the hair cone (open arrows) in anagen III(a). Arrowheads indicate onset and cessation of mK6irs mRNA expression in the Huxley layer, and arrows indicate those in the Henle layer. The occasionallyweak staining seen in the upper cortical layer and dermis is an artifact, as it is also found in sections hybridized with the sense probe (g, h). Hu, Huxley layer;He, Henle layer; Co, cortex. Scale bars: (a, e±g) 50 mm; (b, h) 100 mm; (c, d) 25 mm.

VOL. 116, NO. 3 MARCH 2001 NOVEL CYTOKERATIN IN MOUSE HAIR FOLLICLE 363

1998; Korge et al, 1999; Oetting et al, 1999; Pearce et al, 1999). Theproduction of transgenic mice expressing a mutated form of theIRS-speci®c K6irs cytokeratin is likely to provide insight into itsfunction in the hair follicles, and may represent a putative model fora human hair disorder. Therefore, the discovery of the human K6irsgene will probably allow further analysis of hereditary hair diseases.

We thank Ulrike Beckhaus for excellent technical assistance. We are grateful to Dr

Takako Abe and Dr Ryozo Kuwano (Research Laboratory for Molecular Genetics,

Niigata University) for their helpful suggestions.

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Figure 7. mK6irs is expressed in the Huxleyand Henle layers. Paraf®n sections from BALB/C mouse skin at days 36 (anagen VI) (a±d, f) and42 (catagen phase) (e), were incubated with af®-nity puri®ed anti-mK6irs antibody (a±e) or preim-mune serum (f), and counterstained withhematoxylin. (b, c) Higher magni®cations of thelower and upper parts of a. Arrowheads indicateonset and cessation of mK6irs synthesis in theHuxley layer, and arrows indicate those in theHenle layer. Note that mK6irs is expressed in theIRS in the catagen phase (open arrows) (e). Thepreimmune serum control section (f) shows no la-beling. Hu, Huxley layer; He, Henle layer; Co,cortex. Scale bars: (a, e, f) 100 mm; (b±d) 25 mm.

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