A Novel Type II , mK6irs, is Expressed in the Huxley 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 Ito Department 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 of to that of the cytokeratin 6 family members. Both both epithelial-type or and hair RNA in situ hybridization and immunohistochemical keratins as well as hair -associated proteins. In studies of mouse anagen hair follicles demonstrated this study, a cDNA clone encoding a cytokeratin expression of this cytokeratin in the inner root sheath family member was isolated using RNA differential hair cone during anagen III and in the Henle and display techniques. The predicted amino acid Huxley layers of the inner root sheath during anagen sequence derived from this clone, revealed a homol- VI. The expression of the new cytokeratin began in ogy with a number of cytokeratins, not only in the the hair bulb and progressed up to the height of the central a-helical regions but also in the conserved keratogenous zone. Taken together the sum of the portions of the amino and carboxy terminal data analyzed, we have termed this novel cytokeratin domains, indicating that this protein represents a mK6irs (mouse gene nomenclature k2-6g) to indicate new member of the mouse type II cytokeratin both its similar mobility with K6 in two-dimensional family. Northern blot analysis showed expression in gels and its speci®c expression in the inner root mouse skin, but not in other tissues, including ton- sheath of the hair follicle. Key words: hair cycle/immu- gue, esophagus, and forestomach. One- and two- nohistochemistry/in situ hybridization/RNA differential dimensional western blot analysis showed that this display/two-dimensional sodium dodecyl sulfate±polyacryla- new cytokeratin was 57 kDa in size and ran slightly mide gel electrophoresis. J Invest Dermatol 116:359±365, below the area of cytokeratin 5, which corresponded 2001

mong the skin appendages, the hair follicle has the The major structural proteins of the hair follicle have been most complicated structure; composed of several cell identi®ed as keratins. The keratin multigene family comprises more layers and displaying dynamic cell kinetics, as than 40 individual but structurally related members that, by virtue A indicated by the hair cycle, i.e., the cyclic formation of their sites of expression, are traditionally divided into epithelial- and regression of the hair follicle. During this cycle, type or cytokeratins and hair-type keratins. Two subtypes of keratin hairs grow in the anagen phase, stop their growth in the catagen have been further described based on their charge properties. In phase and rest or fall out during the telogen phase (Ito, 1990). general, type I keratins are smaller, with an acidic pkI and include Within the anagen phase, six stages (anagen I±VI) have been further cytokeratins K9 through K20, whereas type II keratins are larger, identi®ed histologically. more basic and include cytokeratins K1±K8 (Moll et al, 1982). Morphologically, the anagen hair follicle is divided into three Keratin intermediate ®laments are obligatory heterodimers and at groups of cell layers, the hair shaft, the inner root sheath (IRS), and least one member from each type is required to form the two chain the outer root sheath (ORS). The IRS is further subdivided into coiled-coil, which is the basic building block of the intermediate the Huxley and Henle layers as well as the cuticle of the IRS. The ®lament (Hatzfeld and Weber, 1990; Steinert, 1990). So far, IRS forms in the region of the hair bulb, differentiates, and is shed approximately 40 keratin genes have been isolated from humans, at the level of the mid-isthmus into the infundibulum of the follicle. mouse, and sheep. The deduced amino acid sequences show a high The morphologic ®ndings clearly indicate that the IRS plays a part sequence identity among the respective keratin subfamilies and this in guiding the developing hair shaft, whereby the IRS forms an sequence identity is conserved across species boundaries. inner part of the follicular wall surrounding the lower portion of Independent of species, the keratin pro®le of the hair follicle, as the hair canal (Ito, 1990). revealed by one- and two-dimensional gel electrophoresis, is mainly composed of cytokeratins K5, K6a, K6b, K14, and K16/17 Manuscript received June 19, 2000; revised October 19, 2000; accepted (Heid et al, 1986, 1988; Lynch et al, 1986; Stark et al, 1987; Kopan for publication October 23, 2000. and Fuchs, 1989). In situ hybridization studies and immunohisto- Reprint requests to: Dr. Noriaki Aoki, Department of Dermatology, chemical studies have demonstrated that these cytokeratins are Niigata University School of Medicine, Asahimachi-dori, Niigata 951- expressed in the ORS of the hair follicle (Coulombe et al, 1989; 8510, Japan. Email: [email protected] Kopan and Fuchs, 1989; Wollina, 1992; Asada et al, 1993; Van Baar

0022-202X/01/$15.00 ´ Copyright # 2001 by The Society for Investigative Dermatology, Inc. 359 360 AOKI ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY et al, 1994; Panteleyev et al, 1997; McGowan and Coulombe, 1998; antibody was puri®ed by af®nity chromatography using the synthetic Winter et al, 1998). peptide coupled to activated Sepharose (Brinkley et al, 1980). In this study, we have identi®ed a new mouse type II Immunoblots were performed as described previously (Winter et al, cytokeratin, mK6irs, using RNA differential display strategy. The 1997a; 1998). In brief, keratins were extracted using a SDS±mercaptoetha- characterization of this cytokeratin and the localization of its nol buffer at pH 8 from total dorsal skin of a 4 d old mouse, and resolved by two-dimensional SDS±polyacrylamide gel electrophoresis (nonequilibrium transcript and protein are examined in detail. This is one of the ®rst pH gradient gel electrophoresis, NEPHGE). For western blots, gels were reports of a cytokeratin whose expression is limited to the IRS of transferred to PVDF membranes (Immobilon-P, Millipore, Eschborn the hair follicle. Germany) by wet blotting. After staining (0.1% Coomassie blue, 40% methanol, 10% acetic acid), destaining (50% methanol), and blocking with MATERIALS AND METHODS 10% nonfat milk powder in Tris-buffered saline, membranes were Animals and skin samples Syngeneic C57BL/6NCrj female mice incubated with the anti-mK6irs antibody at a dilution of 1:15,000. (purchased from Charles River, Yokohama, Japan), 21±27 d old, and Peroxidase-coupled secondary goat anti-rabbit IgG antibody (Dianova, BALB/CAnNCrj mice, 21±42 d old, were housed under 12 h light/dark Hamburg, Germany) was diluted 1:2000 in 5% nonfat milk powder in Tris- cycles and fed ad libitum. buffered saline. The secondary antibody was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech). RNA extraction Total RNA were isolated using Isogen (Nippongene, Tokyo, Japan) from dorsal skin sections and various other tissues of In situ hybridization The BALB/CAnNCrj mouse was chosen because C57BL/6NCrj mice according to the manufacturer's recommendations. C57BL/6NCrj mouse from which cDNA clones were isolated had All skin samples were examined histologically by light microscopy to abundant hair follicle melanin, which interfered with the detection of the determine their developmental stage of the hair cycle. transcripts (Aoki et al, 2000). For the production of the anti-sense and sense RNA probes, a mK6irs RNA differential display A differential display kit (Clontech, Palo cDNA clone covering the 3¢ noncoding region was linealized with NotI Alto, CA) was applied for the reverse transcription of mRNA and for and XhoI, respectively. Digoxigenin-labeled RNA probes were synthesized subsequent polymerase chain reaction (PCR) ampli®cation of the from the SP6 and T7 promoter using DIG-UTP (Boehringer). In situ generated cDNA. Total RNA samples from dorsal skins of 21 d old hybridization analysis was performed according to a previously described (telogen phase), 24 d old (anagen I), and 27 d old (anagen III) C57BL/ method (Powell and Rogers, 1990). Brie¯y, 21±42 d old BALB/CAnNCrj 6NCrj mice were reverse transcribed using an oligo(dT) primer after mice skins were ®xed in 4% paraformaldehyde and processed to paraf®n 10 min digestion with RNase-free DNase (Boehringer, Mannheim, sections. Hybridization was performed at 55°C for 22 h with the Germany). The cDNA were ampli®ed by polymerase chain reaction digoxigenin-labeled RNA probes at a concentration of 1 ng per ml. After (PCR) using one set of 10 arbitrary primers (P1±10) and nine speci®c treatment with RNase A and wash in 0.1 3 sodium citrate/chloride buffer, oligo(dT) primers (T1±9), four deoxyribonucleoside triphosphate, the tissue sections were treated with an anti-digoxigenin antibody coupled including [a-33P]deoxyadenosine triphosphate (Amersham Pharmacia to alkaline phosphatase and were stained for alkaline phosphatase activity. Biotech, Bucks, U.K.), and Taq polymerase (Clontech) according to the Immunohistochemical analysis Immunohistochemical study was manufacturer's speci®cations. The PCR products were then separated on a performed according to a previously described method (Aoki et al, 1998). 5% denaturing polyacrylamide gel and detected by autoradiography. Paraf®n sections of 21±42 d old BALB/C mice were deparaf®nized and Differentially expressed PCR products were recovered from the gel and incubated sequentially with blocking serum, primary antibody, biotinylated reampli®ed under similar conditions. Reampli®ed PCR products were second antibody, and streptavidin±peroxidase following the manufacturer's ligated into pGEM-T Easy vector (Promega, Madison, WI) and protocol (Nichirei, Tokyo). Primary antibodies, af®nity puri®ed anti- transformed into Escherichia coli JM109 (Promega). DNA was extracted mK6irs antibody and preimmune serum, were diluted 1:2000 in from different clones and sequenced with automated sequencer (Amersham phosphate-buffered saline and incubated overnight at 4°C in a Pharmacia Biotech). The resulting sequences were used to search for humidi®ed chamber. Antibody binding was visualized with homology with genes registered in GenBank by BLAST. diaminobenzidine, and the sections were then counterstained with hematoxylin. 5¢-rapid ampli®cation of cDNA ends To identify the full-length sequence of the mK6irs clone, 5¢-rapid ampli®cation of cDNA ends was Accession number The mK6irs sequence is available under the performed with a marathon cDNA ampli®cation kit (Clontech). After the accession number AB033744 of the EMBL/GenBank/DDBJ. synthesis of the ®rst strand cDNA using an oligo(dT) primer derived from total RNA of a mouse anagen skin (day 27), double strand cDNA was RESULTS ligated to the cDNA adaptor (5¢-CTAATACGACTCACTATAGGGCT CGAGCGGCCGCCCGGGCAGGT-3¢). 5¢-cDNA end of mK6irs was Fragment 7l2, a product differentially ampli®ed in anagen ampli®ed by PCR using adaptor primer (5¢-CCATCCTAATACGACTC III, is not seen in telogen or anagen I Among the several ACTATAGGGC-3¢) and internal gene-speci®c primer (5¢-CTGAGGGA thousand DNA fragments ampli®ed by PCR based differential TCGAGGTCACAAGAAGC-3¢) derived from the 3¢-noncoding region display using 90 primer pair combinations, we identi®ed several of the partial mK6irs cDNA, according to the manufacturer's instructions. cDNA fragments whose band intensity increased in 21±27 d old The ampli®ed cDNA fragment was subcloned into pBluescript, and plasmid DNA from ®ve colonies were prepared and sequenced. mice. One of them, clone 7l2, had an insert size of 800 bp, was a partial clone and shared a high homology with human and mouse Northern blot analysis Ten micrograms of total RNA was cytokeratin cDNA (results not shown). To con®rm the differential electrophoresed in a 1.5% formaldehyde gel using RNA molecular expression of clone 7l2 during the hair cycle, northern blot analysis weight markers and transferred to a nylon ®lter as previously described was performed using total RNA extracted from the dorsal skins of (Thomas, 1983). The 3¢ untranslated region of mK6irs cDNA (1715± 21, 24, and 27 d old mice. Northern blot analysis showed the 2193 bp) was labeled with 32P by a hexanucleotide random priming method presence of two distinct transcripts: one major transcript of 2.2 kb (Feinberg and Vogelstein, 1983) and used as a probe. Hybridization was and one minor transcript of 1.8 kb (Fig 1). These transcripts were performed at 68°C using QuikHyb Hybridization Solution (Clontech) and detected at day 27 (anagen III), but were not seen at day 21 (telogen followed by washing with 0.1 3 sodium citrate/chloride buffer (150 mM phase) and 24 (anagen I). We therefore selected clone 7l2 for NaCl, 15 mM trisodium citrate), 0.1% sodium dodecyl sulfate (SDS) at 68°C. The ®lter was subsequently rehybridized with the glyceraldehyde-3- further investigation. phosphate dehydrogenase probe after removing the mK6irs signals by The predicted amino acid sequence of mK6irs shows that it boiling in water. is a novel mouse type II cytokeratin To isolate the 5¢ end of Antibody preparation and immunoblot analysis A monospeci®c clone 7l2, we ampli®ed mouse anagen skin cDNA using a 5¢-rapid antibody to mK6irs was produced in rabbits by immunizing with the ampli®cation of cDNA ends strategy, and obtained only one cDNA synthetic peptide, KDTLTKGSSLSTPSKKGGR, corresponding to unique fragment via PCR ampli®cation. DNA sequencing of this fragment amino acid sequences of the protein (residues 506±524 of mK6irs) using a allowed the construction of a full-length cDNA containing a single previously described immunization protocol (Roop et al, 1984). The open reading frame, which begins with a methionine initiation VOL. 116, NO. 3 MARCH 2001 NOVEL CYTOKERATIN IN MOUSE HAIR FOLLICLE 361

Table I. Similarity of mK6irs to other cytokeratins

Cytokeratins Identity (%)

Full length Rod domain

Mouse type II K1 49 64 K4 56 67 K6 55 63 K8 48 60 Human type II K1 49 67 K2e 51 67 K2p 52 69 K3 53 71 K4 59 71 Figure 1. The gene fragment expressed differentially by RNA K5 55 70 differential display, clone 7l2, can be detected in mouse skins at K6a 57 70 day 27 but not at days 21 and 24 by northern blot. Total RNA (10 mg) K6b 57 70 prepared from the dorsal skins of C57BL/6 mouse at days 21, 24, and 27 K6c 56 70 was electrophoresed and transferred to nylon ®lter. The ®lter was K6e 56 70 32 hybridized with P-labeled mK6irs cDNA probe and followed by washing K6f 57 70 with 0.1 3 sodium citrate/chloride buffer, 0.1% SDS at 68°C. The blot was K6hf 57 67 subsequently rehybridized with the glyceraldehyde-3-phosphate dehydro- K7 51 65 genase (GAPDH) probe. The RNA markers are indicated at left side. K8 49 62 Mouse type I K10, K12, K13, 23±28 27±30 K15, K18, K19 Human type I K9, K10, 22±27 23±29 K12±K20

mK6irs is detectable in a similar region as cytokeratin K4 or K6 by two-dimensional western blotting of anagen mouse skin An antibody was raised in rabbits against a synthetic peptide corresponding to residues 506±524 of mK6irs (Fig 2). One- dimensional western blotting using this antibody showed the apparent molecular size of the protein to be approximately 57 kDa (data not shown), which was consistent with the size predicted from the amino acid sequence. To investigate the characteristics further of mK6irs, we performed two-dimensional western blots. Keratins were extracted from the total dorsal skin of a 4 d old mouse, a time in which most hair follicles are in anagen VI and possess a fully developed ORS and IRS. As shown in Fig 5(A), the keratin pattern in the two- dimensional SDS±polyacrylamide gel electrophoresis consisted Figure 2. Amino acid sequence of mK6irs. The central a-helical rod domain is indicated by hatched boxes. The sequence is available from predominantly of epidermal keratins, whereas only traces of type EMBL/GenBank/DDBJ (accession number AB033744). I hair keratins were visible. Western blotting using the anti-mK6irs antibody revealed a single protein separated clearly into three isoelectric variants (Fig 5B). mK6irs ran at 57 kDa, i.e., slightly codon at nucleotide 52 and ends with a TGA stop codon at below the 58.5±59 kDa basal cell cytokeratin K5 (MK5) (Schwei- nucleotide 1626. Translation of the open reading frame yielded a zer, 1993), and was located in the more basic part of the gel, which protein of 524 residues with a calculated molecular weight of corresponded to the area of the cytokeratin K4 or K6 (Wild and 57.4 kDa and an isoelectric pH value of 6.63 (Fig 2). Database Mischke, 1986; Schweizer, 1993). This location con®rmed the type searching for homologous protein sequences revealed that this II cytokeratin nature of mK6irs. protein shared a high identity with members of the mouse and mK6irs transcripts are expressed in the Huxley and Henle human type II cytokeratins, particularly in their central a-helical layers of the hair follicle inner root sheath To determine the rod domain (Table I), including the characteristic helix initiation localization of mK6irs transcripts in skin, we analyzed mouse skin motif and helix termination motif (Fig 3), which are crucial for the samples from day 21 to day 42, which corresponded to their second structure and function of keratins (Parry and Steinert, 1995). For hair cycle. mK6irs transcripts were not detected in mouse skin from reasons explained below, we have termed the novel type II telogen to anagen II (days 21 and 24) (data not shown), appeared in cytokeratin mK6irs. anagen III±IV (days 27 and 30) (Fig 6a), and was abundantly expressed in anagen V and VI (days 32, 36, and 39) (Fig 6b±f). mK6irs is expressed speci®cally in skin In order to investigate Expression ceased after re-entry into catagen phase (day 42) (data the expression of mK6irs in mouse tissues, northern blot analysis not shown). An examination of sections in anagen III, the time at was performed with the total RNA from various mouse tissues which the proliferating epithelial cells complete formation of a using the 3¢ untranslated region as a probe. As shown in Fig 4, two follicle bulb surrounding the dermal papilla and hair follicle transcripts, 1.8 and 2.2 kb, were detected only in 27 d old anagen elongation has commenced, showed mK6irs transcript expression in mouse skin. Transcripts were not present in the other tissues, the IRS, which appears cone-shaped lying medial to the ORS including the tongue, esophagus, and forestomach. As the entire (Fig 6a). In anagen VI when the follicle has fully elongated, the mK6irs cDNA encompasses 2193 nucleotides, it most likely transcripts were predominantly restricted to the Huxley and Henle corresponds to the more abundantly expressed 2.2 kb transcript. layers of the IRS. The expression of mK6irs transcripts in the Henle The 1.8 kb transcript may be the product of a related gene or an layer commenced at the lower hair bulb (Fig 6b), and was detected alternatively spliced form of the mK6irs gene. up to the lower portion of the hair follicle keratogenous zone, a 362 AOKI ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 3. Amino acid alignment of helix initiation and helix termination motifs of mK6irs and human and mouse type I and type I cytokeratins reported to date. Black boxes represent identical residues.

Figure 4. mK6irs transcript can be detected abundantly in the anagen 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. mK6irs transcript is detected only in the skin from 27 d old mice. region where the Henle layer has completely keratinized (Fig 6c). Subsequently, its expression was detectable continuously in the Huxley layer at the height of the keratogenous zone (Fig 6b±d). Transverse sections showed signals in the Huxley and Henle layers but not in the hair shaft, including the medulla, and ORS Figure 5. Two-dimensional SDS±polyacrylamide gel electrophor- (Fig 6e,f). During the hair growth cycle, staining was not detected esis and western blot reveal that mK6irs runs at the region of cytokeratin K4 or K6. (A) Coomassie-stained two-dimensional keratin in epidermis, sebaceous gland, and sweat gland (data not shown), pattern of 4 d old mouse skin. The keratin proteins are designated according whereas weak staining appeared in the upper cortical layer and to the catalog of human keratins (Moll et al, 1982). The arrowhead indicates dermis (Fig 6b,d). In situ hybridization using a sense probe also the major 62 kDa stratum corneum equivalents of the weak MK1 band showed the weak upper cortical layer and dermal staining (Fig 6h), (Schweizer, 1993). (B) Western blot of (A) with anti-mK6irs antibody, thus indicating an unspeci®c reaction. which resolves the protein into three isoelectric variants. The arrows denotes the position of MK5. Proteins were separated by nonequilibrium mK6irs protein is expressed in the Huxley and Henle layers pH gradient gel electrophoresis in the ®rst dimension (N), and by of the IRS We performed immunohistochemical experiments SDS±polyacrylamide gel electrophoresis (8% gel) in the second dimension using the dorsal skins of 21±42 d old BALB/C mice to investigate (S). the localization of mK6irs protein in the mouse skin throughout the different hair growth phases. No staining was detected in telogen and anagen I±IV animals (data not shown). In anagen V and anagen phase hair follicles, both types of keratin genes are initially VI, anti-mK6irs antibody reacted consistently with the IRS activated, followed by the subsequent expression of high-glycine/ initiating in the upper hair bulb to a region just below the tyrosine proteins and thereafter, by cysteine-rich proteins (Aoki sebaceous gland of the hair follicle (Fig 7a). In the upper portion of et al, 1997; Powell and Rogers, 1997). In the telogen phase, the the hair bulb, the staining commenced in the Henle layer and expression of all of these genes decreases signi®cantly and ®nally continued until the complete keratinization of the Henle layer ceases. This scenario implies that cDNA encoding hair follicle occurred (Fig 7b). Similar staining was detected in the Huxley layer keratins can be isolated by differentially screening mouse skins from the suprabulbar portion up to the height of the keratogenous between the anagen and telogen phase. Using this differential display strategy in this study, we were able zone, where the Huxley layer completely keratinizes (Fig 7b±d). to isolate one cDNA clone, which encoded a novel cytokeratin. mK6irs staining in the IRS was detectable from anagen V to the Multiple sequence comparisons showed that the new keratin mid-catagen phase, the time when the IRS moves up the follicle belonged to the type II cytokeratin subfamily. Regarding its ahead of the cortical layer, the cortex forming the brush end of the designation, we followed the reasoning that has previously been club hair by interdigitating with the transformed ORS (Fig 7e). applied for the numerical integration into the cytokeratin catalog Immunostaining was not observed in the other cells of the hair (Moll et al, 1982) of another, recently discovered type II follicle and the epidermis (data not shown). Preimmune serum also cytokeratins, K6hf (Winter et al, 1998). The respective criteria showed no positive staining in mouse skin (Fig 7f). were: (i) the isoelectric pH values of the keratin that determines its classi®cation as acidic, type I and basic to neutral, type II DISCUSSION cytokeratin; (ii) its relative molecular mass on SDS±polyacrylamide The hair follicle contains three major classes of proteins: gels, which de®nes the type-speci®c numerical designation cytokeratins and hair keratins as well as the -associated according to decreasing molecular weights; and (iii) its tissue high-glycine/tyrosine proteins and cysteine-rich proteins. In the expression. Indeed, our two-dimensional western blots con®rmed VOL. 116, NO. 3 MARCH 2001 NOVEL CYTOKERATIN IN MOUSE HAIR FOLLICLE 363

Figure 6. mK6irs transcripts are expressed in the Huxley and Henle layers of the IRS. Digoxigenin-labeled sense and anti-sense RNA probes prepared from mK6irs cDNA were hybridized to paraf®n sections of BALB/CAnNCrj mouse skin at days 27 (a) and 36 (b± h), and counterstained with methyl 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 demonstrate that 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 occasionally weak 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. that the new keratin is a member of the type II cytokeratin family. exclusively in the IRS and their IRS expression needs further Its position on these blots is very close to the theoretical position of con®rmation. From this it follows that, up to now, mK6irs is the K4 or K6 (Wild and Mischke, 1986; Schweizer et al, 1988; only IRS-speci®c cytokeratin. Similar to the human type I Schweizer, 1993). Sequence comparisons of the new keratin with companion layer speci®c keratin K6hf (Winter et al, 1998), the all known mouse type II keratins revealed the highest homology type II partner for mK6irs, necessary for the biologic functionality with mK4 as well as a slightly lower homology with mK6. Previous of the type II keratin, is not yet known. studies indicated that cytokeratin 4 occurs in the tongue, This study has shown, that mK6irs is expressed predominantly in esophagus, and forestomach, but not fundamentally in the skin the Henle and Huxley layers of the IRS during the hair follicle (Moll et al, 1982; Wild and Mischke, 1986; Schweizer et al, 1988). growth phase. Interestingly, in anagen III, the protein could not be In contrast, K6 has been shown to be expressed in the hair follicle detected despite the apparent expression of the transcript in the (Stark et al, 1987; Winter et al, 1998). Finally, the new keratin was IRS. The inability to demonstrate mK6irs during the early anagen speci®cally expressed in the IRS of the hair follicle. These data led phase might be due to determinant masking of the protein. In us to designate the new mouse keratin mK6irs. In this context it is addition, the presence of the mK6irs protein in the IRS continues worth mentioning that mK6irs also exhibits a high homology with until mid-catagen, a time in which mK6irs transcripts are not the human companion layer speci®c keratin K6hf. The K6hf gene detected, indicating a temporal discrepancy of expression. has recently been shown to be located adjacent to the K6b gene A preliminary BLAST database search failed to show the (Rogers et al, 2000), thus indicating that it may be part of the presence of a human ortholog of mK6irs. The identi®cation of putative K6 gene cluster. It remains to be seen whether the mK6irs the human ortholog of mK6irs is, however, important for the gene or its still unknown human counterpart are also located in the following reason. It is now ®rmly established that mutations in respective K6 gene cluster. keratins can cause a large number of hereditary dermatoses, which mK6irs adds to cytokeratins K5, K6, K14, and K16/17 known to are characterized by keratin intermediate ®lament disruption (for a be expressed in the ORS of the hair follicle (Coulombe et al, 1989; review, Irvine and McLean, 1999). Interestingly, transgenic mice Kopan and Fuchs, 1989; Panteleyev et al, 1997; McGowan and expressing a mutant cytokeratin K6 have been shown to develop Coulombe, 1998; Winter et al, 1998). Whereas the latter also shows progressive cicatricial alopecia due to the gradual destruction of the expression in epidermis, mK6irs expression is restricted to the IRS ORS (Rothnagel et al, 1994). More recently, mutations in hair of the hair follicle. Previous reports claimed the expression of other keratin genes hHb1 and hHb6 have been shown to be present in type I and type II cytokeratins in the IRS, such as K1, K10, K13, the congenital hair disease monilethrix, which is characterized by and K16 (Stark et al, 1990). Again these cytokeratins are not present beaded and fragile hairs (Winter et al, 1997b, 2000; Zlotogorski et al, 364 AOKI ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 7. mK6irs is expressed in the Huxley and Henle layers. Paraf®n sections from BALB/ C mouse skin at days 36 (anagen VI) (a±d, f) and 42 (catagen phase) (e), were incubated with af®- nity puri®ed anti-mK6irs antibody (a±e) or preim- mune serum (f), and counterstained with hematoxylin. (b, c) Higher magni®cations of the lower and upper parts of a. Arrowheads indicate onset and cessation of mK6irs synthesis in the Huxley layer, and arrows indicate those in the Henle layer. Note that mK6irs is expressed in the IRS in the catagen phase (open arrows)(e). The preimmune 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.

1998; Korge et al, 1999; Oetting et al, 1999; Pearce et al, 1999). The Kopan R, Fuchs E: A new look into an old problem: keratins as tools to investigate production of transgenic mice expressing a mutated form of the determination, morphogenesis, and differentiation in skin. Genes Deve 3:1±15, 1989 IRS-speci®c K6irs cytokeratin is likely to provide insight into its Korge BP, Hamm H, Jury CS, et al: Identi®cation of novel mutations in basic hair function in the hair follicles, and may represent a putative model for keratins hHb1 and hHb6 in monilethrix: implications for protein structure and a human hair disorder. Therefore, the discovery of the human K6irs clinical phenotype. J Invest Dermatol 113:607±612, 1999 gene will probably allow further analysis of hereditary hair diseases. 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