Portal de Periódicos da CAPES

Keratin 17 Expression in the Hard Epithelial Context of the Hair and Nail, and its Relevance for the Pachyonychia Congenita Phenotype

2000; Elsevier BV; Volume: 114; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.2000.00986.x

1523-1747

Kevin M. McGowan, Pierre A. Coulombe,

Dermatological and Skeletal Disorders

The hard-keratin-containing portion of the murine hair shaft displays a positive immunoreactivity with an antibody against the soft epithelial keratin, K17. The K17-expressing cell population is located in the medulla compartment of the hair. Consistent with this observation, K17-containing cells also occur in the presumptive medulla precursor cells located in the hair follicle matrix. Western blot analysis of hair extracts prepared from a number of mouse strains confirms this observation and suggests that K17 expression in the hair shaft is a general trait in this species. The expression of K17 in human hair extracts is restricted to eyebrow and facial hair samples. These are the major sites for the occurrence of the pili torti (twisted hair) phenotype in the type 2 (Jackson-Lawler) form of pachyonychia congenita, previously shown to arise from inherited K17 mutations. Given that all forms of pachyonychia congenita show an involvement of the nail, we compared the expression of the two other genes mutated in pachyonychia congenita diseases, K6 and K16, with that of K17 in human nail. All three keratins are abundantly expressed within the nail bed epithelium, whereas K17 protein is expressed in the nail matrix, which contains the epithelial cell precursors for the nail plate. Our data suggest a role for K17 in the formation and maintenance of various skin appendages and directly support the concept that pachyonychia congenita is a disease of the nail bed. The hard-keratin-containing portion of the murine hair shaft displays a positive immunoreactivity with an antibody against the soft epithelial keratin, K17. The K17-expressing cell population is located in the medulla compartment of the hair. Consistent with this observation, K17-containing cells also occur in the presumptive medulla precursor cells located in the hair follicle matrix. Western blot analysis of hair extracts prepared from a number of mouse strains confirms this observation and suggests that K17 expression in the hair shaft is a general trait in this species. The expression of K17 in human hair extracts is restricted to eyebrow and facial hair samples. These are the major sites for the occurrence of the pili torti (twisted hair) phenotype in the type 2 (Jackson-Lawler) form of pachyonychia congenita, previously shown to arise from inherited K17 mutations. Given that all forms of pachyonychia congenita show an involvement of the nail, we compared the expression of the two other genes mutated in pachyonychia congenita diseases, K6 and K16, with that of K17 in human nail. All three keratins are abundantly expressed within the nail bed epithelium, whereas K17 protein is expressed in the nail matrix, which contains the epithelial cell precursors for the nail plate. Our data suggest a role for K17 in the formation and maintenance of various skin appendages and directly support the concept that pachyonychia congenita is a disease of the nail bed. green fluorescence protein lymphoid enhancer factor pachyonychia congenita T cell factor Keratins are a group of more than 40 highly insoluble proteins that serve as the subunits for forming intermediate filament polymers in epithelial cells (O'guin et al., 1990O'guin W.M. Schermer A. Lynch M. Sun T-T. Differentiation-specific expression of keratin pairs.in: Goldman R.D. Steinert P.M. Cellular & Molecular Biology of Intermediate Filaments. Plenum, New York1990: 301-334Crossref Google Scholar;Fuchs, 1995Fuchs E. Keratins and the skin.Ann Rev Cell Dev Biol. 1995; 11: 123-153Crossref PubMed Google Scholar). The keratin protein family consists of two groups: the acidic or type I keratins and the basic or type II keratin proteins (Moll et al., 1982Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells.Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4372) Google Scholar). A keratin filament is an obligatory heteropolymer, containing an equimolar amount of type I and type II proteins (Coulombe, 1993Coulombe P.A. The cellular and molecular biology of keratins: beginning a new era.Curr Opin Cell Biol. 1993; 5: 17-29Crossref PubMed Scopus (94) Google Scholar;Steinert, 1993Steinert P.M. Structure, function, and dynamics of keratin intermediate filaments.J Invest Dermatol. 1993; 100: 729-734Abstract Full Text PDF PubMed Google Scholar). To meet this requirement, keratin genes are coordinately expressed as type I-type II pairs (Moll et al., 1982Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells.Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4372) Google Scholar;O'guin et al., 1990O'guin W.M. Schermer A. Lynch M. Sun T-T. Differentiation-specific expression of keratin pairs.in: Goldman R.D. Steinert P.M. Cellular & Molecular Biology of Intermediate Filaments. Plenum, New York1990: 301-334Crossref Google Scholar;Fuchs, 1995Fuchs E. Keratins and the skin.Ann Rev Cell Dev Biol. 1995; 11: 123-153Crossref PubMed Google Scholar). The concept of pairwise regulation is supported by studies that demonstrate tissue- and differentiation-specific expression of keratin genes in the various epithelia of the body. The pairwise regulation of keratin gene transcription (Fuchs, 1995Fuchs E. Keratins and the skin.Ann Rev Cell Dev Biol. 1995; 11: 123-153Crossref PubMed Google Scholar) determines, to a large extent, the resulting filament composition within a given epithelial cell. These filaments are organized into a prominent cytoplasmic network that is anchored at the surface of the nucleus as well as at cell-cell and cell-matrix adhesion complexes, and they typically span the entire cytoplasmic space in between. It is now firmly established, through studies conducted with transgenic mice and with patients suffering from inherited epithelial fragility disorders, that the major function of keratin filaments is to endow epithelial cells with the mechanical resilience they need to withstand the load of mechanical stress to which they are routinely subjected (Coulombe and Fuchs, 1994Coulombe P.A. Fuchs E. Molecular mechanisms of keratin gene disorders and other bullous diseases of the skin.in: Citi S. Molecular Mechanisms in Epithelial Cell Junctions: from Development to Disease. Landes, Austin, TX1994: 259-285Google Scholar;McLean and Lane, 1995McLean W.H.I. Lane E.B. Intermediate filaments in diseases.Curr Opin Cell Biol. 1995; 7: 118-125Crossref PubMed Scopus (209) Google Scholar;Fuchs and Cleveland, 1998Fuchs E. Cleveland D.W. A structural scaffolding of intermediate filaments in health and disease.Science. 1998; 279: 514-519Crossref PubMed Scopus (793) Google Scholar). The notions of tissue- and differentiation-specific regulation of keratin genes suggest that these proteins may impart some degree of specialization to the various epithelia in which they are expressed. Detailed studies of keratin expression patterns support this concept (Moll et al., 1982Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells.Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4372) Google Scholar;Tseng et al., 1982Tseng S.C. Jarvinen M. Nelson W.G. Huang H.W. Woodcock-Mitchell J. Sun T.T. Correlation of specific keratins with different types of epithelial differentiation: monoclonal antibody studies.Cell. 1982; 30: 361-372Abstract Full Text PDF PubMed Scopus (503) Google Scholar;Heid et al., 1986Heid H. Werner E. Franke W.W. The complement of native a-keratin polypetides of hair-forming cells: a subset of eight polypeptides that differ from epithelial cytokeratins.Differentiation. 1986; 32: 101-119Crossref PubMed Scopus (145) Google Scholar). Recent attempts to complement the phenotype of keratin 14 null mice, which die early after birth owing to extensive skin blistering, clearly showed that, even with their high degree of sequence homology, keratin proteins are only partially redundant at a functional level (Hutton et al., 1998Hutton E. Paladini R.D. Yu Q.C. Yen M-Y. Coulombe P.A. Fuchs E. Functional differences between keratins of stratified and simple epithelia.J Cell Biol. 1998; 143: 1-13Crossref PubMed Scopus (88) Google Scholar;Paladini and Coulombe, 1999Paladini R.D. Coulombe P.A. The functional diversity of epidermal keratins revealed by the partial rescue of the keratin 14 null phenotype by keratin 16.J Cell Biol. 1999; 146: 1185-1201Crossref PubMed Scopus (46) Google Scholar). From this it would appear that the multiplicity of keratin genes is related to the functional diversity of epithelial tissues. Thus, the characterization of keratin gene expression in normal and diseased epithelia will continue to play an important role for understanding the biology of complex epithelial systems. The type II keratin K6 isoforms and the type I keratins 16 and/or 17 are coregulated in many complex epithelia, including all major skin appendages (e.g., hair, nail, glands; seeMcGowan et al., 1998bMcGowan K. Coulombe P.A. The wound repair-associated keratins K6, K16 and K17: insights into the role of intermediate filaments in specifying keratinocyte cytoarchitecture.in: Harris J.R. Herrmann H. Subcellular Biochemistry: Intermediate Filament. Plenum, London1998: 173-198Google Scholar, for a review). Unlike most other keratin pairs, however, their distribution cannot be correlated with a well-defined epithelial context, leaving open the issue of their role(s) in vivo. Inherited missense mutations within the coding sequence of K6 isoforms, K16, or K17 cause various forms of ectodermal dysplasias, including pachyonychia congenita (PC). PC refers to a group of genodermatoses that invariably involve dyskeratotic changes in the nail and usually involve related alterations in palmoplantar epidermis and other stratified epithelia depending on the clinical variant (Feinstein et al., 1988Feinstein A. Friedman J. Schewach M. Pachyonychia congenita.J Am Acad Dermatol. 1988; 19: 705-711Abstract Full Text PDF PubMed Scopus (111) Google Scholar). Jadassohn-Lewandowsky or type 1 PC disease (OMIM167200) is clinically distinct by the occurrence of oral leukoplakia (Feinstein et al., 1988Feinstein A. Friedman J. Schewach M. Pachyonychia congenita.J Am Acad Dermatol. 1988; 19: 705-711Abstract Full Text PDF PubMed Scopus (111) Google Scholar;Dahl et al., 1995Dahl P.R. Daoud M.S. Su W.P. Jadassohn–Lewandowski syndrome (pachyonychia congenita).Semin Dermatol. 1995; 14: 129-134Crossref PubMed Google Scholar), and has been associated with mutations in the K6a or K16 sequences (Bowden et al., 1995Bowden P.E. Haley J.L. Kansky A. Rothnagel J.A. Jones D.O. Turner R.J. Mutation of a type II keratin gene (K6a) in pachyonychia congenita.Nat Genet. 1995; 10: 363-365Crossref PubMed Scopus (204) Google Scholar;McLean et al., 1995McLean W.H.I. Rugg E.L. Lunny D.P. et al.Keratin 16 and keratin 17 mutations cause pachyonychia congenita.Nat Genet. 1995; 9: 273-278Crossref PubMed Scopus (280) Google Scholar;Lin et al., 1999Lin M.T. Levy M.L. Bowden P.E. Magro C. Baden L. Baden H.P. Roop D.R. Identification of sporadic mutations in the helix initiation motif of keratin 6 in two pachyonychia congenita patients: further evidence for a mutational hot spot.Exp Dermatol. 1999; 8: 115-119Crossref PubMed Scopus (15) Google Scholar). Jackson-Lawler or type 2 PC disease (MIM 167210) is distinct by the occurrence of neonatal teeth and subcutaneous cysts (Clementi et al., 1986Clementi M. Cardin de Stefani E. Dei Rossi C. Avventi V. Tenconi R. Pachyonychia congenita Jackson Lawler type: a distinct malformation syndrome.Br J Dermatol. 1986; 114: 367-370Crossref PubMed Scopus (29) Google Scholar;Feinstein et al., 1988Feinstein A. Friedman J. Schewach M. Pachyonychia congenita.J Am Acad Dermatol. 1988; 19: 705-711Abstract Full Text PDF PubMed Scopus (111) Google Scholar), and has been associated with mutations in the K6b or K17 sequences (McLean et al., 1995McLean W.H.I. Rugg E.L. Lunny D.P. et al.Keratin 16 and keratin 17 mutations cause pachyonychia congenita.Nat Genet. 1995; 9: 273-278Crossref PubMed Scopus (280) Google Scholar;Smith et al., 1997Smith F.J. Corden L.D. Rugg E.L. et al.Missense mutations in keratin 17 cause either pachyonychia congenita type 2 or a phenotype resembling steatocystoma multiplex.J Invest Dermatol. 1997; 108: 220-223Crossref PubMed Scopus (122) Google Scholar,Smith et al., 1998Smith F.J. Jonkman M.F. van Goor H. Coleman C.M. Covello S.P. Uitto J. McLean W.H. A mutation in human keratin K6b produces a phenocopy of the K17 disorder pachyonychia congenita type 2.Human Mol Genetics. 1998; 7: 1143-1148Crossref PubMed Scopus (144) Google Scholar;Fujimoto et al., 1998Fujimoto W. Nakanishi G. Hirakawa S. Nakanishi T. Shimo T. Takigawa M. Arata J. Pachyonychia congenita type 2: keratin 17 mutation in a Japanese case.J Am Acad Dermatol. 1998; 38: 1007-1009Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Although the tissues affected in type 1 and type 2 PC patients bear an obvious relationship to the expression pattern of the gene affected, a closer look at the issue of genotype-phenotype correlation reveals several idiosyncrasies (McGowan et al., 1998bMcGowan K. Coulombe P.A. The wound repair-associated keratins K6, K16 and K17: insights into the role of intermediate filaments in specifying keratinocyte cytoarchitecture.in: Harris J.R. Herrmann H. Subcellular Biochemistry: Intermediate Filament. Plenum, London1998: 173-198Google Scholar). For instance, mutations in the K16 sequence were also found in the context of a nonepidermolytic form of palmoplantar keratoderma (NEPPK), whereas mutations in K17 were discovered in several instances of steatocystoma multiplex (Shamsher et al., 1995Shamsher M.K. Navsaria H.A. Stevens H.P. et al.Novel mutations in keratin 16 gene underly focal non-epidermolytic palmoplantar keratoderma in two families.Hum Molec Genet. 1995; 4: 1875-1881Crossref PubMed Scopus (107) Google Scholar;Smith et al., 1997Smith F.J. Corden L.D. Rugg E.L. et al.Missense mutations in keratin 17 cause either pachyonychia congenita type 2 or a phenotype resembling steatocystoma multiplex.J Invest Dermatol. 1997; 108: 220-223Crossref PubMed Scopus (122) Google Scholar). These disorders involve minimal alterations to the nail, if any. Thus, significant heterogeneity exists in the clinical picture associated with related mutations in these particular keratin genes, and the underlying basis for this remains unknown. Previous studies have reported that K17 expression in the mature hair follicle is limited to the outer root sheath (Moll et al., 1982Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells.Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4372) Google Scholar;Stark et al., 1987Stark H.J. Breikreutz D. Limat A. Bowden P. Fusenig N.E. Keratins of the human hair follicle: "hyperproliferative" keratins consistently expressed in outer root sheath cells in vivo and in vitro.Differentiation. 1987; 35: 236-248Crossref PubMed Scopus (112) Google Scholar;Troyanovsky et al., 1989Troyanovsky S.M. Guelstein V.I. Tchipysheva T.A. Krutovskikh V.A. Bannikov G.A. Patterns of expression of keratin 17 in human epithelia: dependency on cell position.J Cell Sci. 1989; 93: 419-426PubMed Google Scholar;Panteleyev et al., 1997Panteleyev A.A. Paus R. Wanner R. et al.Keratin 17 gene expression during the murine hair cycle.J Invest Dermatol. 1997; 108: 324-329Crossref PubMed Scopus (58) Google Scholar). Recently, we described K17 immunoreactivity in both the hard and soft portions of the murine hair follicle (McGowan and Coulombe, 1998aMcGowan K. Coulombe P.A. Onset of keratin 17 expression coincides with the definition of major epithelial lineages during mouse skin development.J Cell Biol. 1998; 143: 469-486Crossref PubMed Scopus (238) Google Scholar). In this report, we expand on this observation and we compare the cellular distribution of K6, K16, and K17 in the human nail. The results that we report have important implications for the function of K17 and the pathogenesis of PC. Materials were obtained from the following sources: Protran nitrocellulose filters were purchased from Schleicher and Schuell (Keane, NH); alkaline phosphatase antibody detection kit was purchased from Bio-Rad (Hercules, CA). All other chemicals were reagent grade and were typically obtained from Sigma (St. Louis, MO). Hair clippings were minced and incubated in a solution consisting of 8 M urea, 200 mM tri(hydroxymethyl)-amino- methane (Tris) HCl (pH 9.5), and 200 mM 2-mercaptoethanol for 2 h at 37°C. The samples were then homogenized with a polytron for 30 s and incubated for an additional 2 h at 37°C. Insoluble proteins were removed by centrifugation at 10,000 ×g for 10 min and the supernatant was recovered and stored at -70°C until use (Lynch et al., 1986Lynch M.H. O'guin W.M. Hardy C. Mak L. Sun T.T. Acidic and basic hair/nail ("hard") keratins: their colocalization in upper cortical and cuticle cells of the human hair follicle and their relationship to "soft" keratins.J Cell Biol. 1986; 103: 2593-2606Crossref PubMed Scopus (289) Google Scholar). All other tissue extractions were performed as described byPaladini and Coulombe, 1999Paladini R.D. Coulombe P.A. The functional diversity of epidermal keratins revealed by the partial rescue of the keratin 14 null phenotype by keratin 16.J Cell Biol. 1999; 146: 1185-1201Crossref PubMed Scopus (46) Google Scholar. The preparation and characterization of antisera to keratins 6, 16, and 17 have been described previously (Takahashi et al., 1994Takahashi K. Folmer J. Coulombe P.A. Increased expression of keratin 16 causes anomalies in cytoarchitecture and keratinization in transgenic mouse skin.J Cell Biol. 1994; 127: 505-520Crossref PubMed Scopus (87) Google Scholar;McGowan and Coulombe, 1998aMcGowan K. Coulombe P.A. Onset of keratin 17 expression coincides with the definition of major epithelial lineages during mouse skin development.J Cell Biol. 1998; 143: 469-486Crossref PubMed Scopus (238) Google Scholar). The mouse monoclonal antibody against type I hair keratins (AE13) was the gift of Dr. Henry Sun (Lynch et al., 1986Lynch M.H. O'guin W.M. Hardy C. Mak L. Sun T.T. Acidic and basic hair/nail ("hard") keratins: their colocalization in upper cortical and cuticle cells of the human hair follicle and their relationship to "soft" keratins.J Cell Biol. 1986; 103: 2593-2606Crossref PubMed Scopus (289) Google Scholar). Mouse monoclonal anti-K14 (LL001) was the gift of Dr. Irene Leigh (Purkis et al., 1990Purkis P.E. Steel J.B. Mackenzie I.C. Nathrath W.B. Leigh I.M. Lane E.B. Antibody markers of basal cells in complex epithelia.J Cell Sci. 1990; 97: 39-50Crossref PubMed Google Scholar). Immunoblot analysis was performed using 10 μg of hair extracts with recombinant keratin proteins as positive controls. The production of recombinant keratins has been described previously (Wawersik et al., 1997Wawersik M. Paladini R.D. Noensie E. Coulombe P.A. A proline residue in the a-helical rod domain of type I keratin 16 destabilizes keratin heterotetramers and influences incorporation into filaments.J Biol Chem. 1997; 272: 32557-32565Crossref PubMed Scopus (40) Google Scholar). Samples were electrophoresed and transferred to nitrocellulose; the blots were incubated with primary antisera diluted in blocking buffer (Tris-buffered saline with 0.5% Tween 20 and 5% powdered milk). Bound primary antibodies were revealed by alkaline-phosphatase-conjugated secondary antibodies as recommended by the manufacturer (Bio-Rad) or through enhanced chemiluminescence detection (Amersham). Immunohistochemical analyses were performed on 5 μm sections prepared from either paraffin-embedded or fresh-frozen tissues. Tissues were fixed in Bouin's fixative overnight at 4°C. The fixed tissues were embedded in paraffin and 5 μm sections were stained with hematoxylin and eosin or immunostained. In addition, freshly isolated skin samples were washed in phosphate-buffered saline and quick frozen in liquid nitrogen using OCT (Sakura Finetek, Torrance, CA). The sections were incubated with the primary antisera and then revealed either with a peroxidase-based reaction (Kirkegaard and Perry Laboratories, Gaithersburg, MD) or by indirect immunofluorescence (Jackson Immunological Reagents, West Grove, PA). All studies involving animals were reviewed by the Johns Hopkins University Animal Use and Care Committee. Hair samples from mouse, cat, monkey, and rat were obtained from animals housed at Johns Hopkins University. Human hair samples were provided by Dr. Stan Miller (Department of Dermatology, Johns Hopkins University). Pig and dog hairs were provided by Dr. Barbara Sollner-Webb (Department of Biological Chemistry, Johns Hopkins University). Paraffin sections taken from mouse skin were immunostained with a rabbit polyclonal antibody to K17. These experiments revealed that, in addition to the previously reported staining in the outer root sheath (Moll et al., 1982Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells.Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4372) Google Scholar;Stark et al., 1987Stark H.J. Breikreutz D. Limat A. Bowden P. Fusenig N.E. Keratins of the human hair follicle: "hyperproliferative" keratins consistently expressed in outer root sheath cells in vivo and in vitro.Differentiation. 1987; 35: 236-248Crossref PubMed Scopus (112) Google Scholar;Troyanovsky et al., 1989Troyanovsky S.M. Guelstein V.I. Tchipysheva T.A. Krutovskikh V.A. Bannikov G.A. Patterns of expression of keratin 17 in human epithelia: dependency on cell position.J Cell Sci. 1989; 93: 419-426PubMed Google Scholar;Panteleyev et al., 1997Panteleyev A.A. Paus R. Wanner R. et al.Keratin 17 gene expression during the murine hair cycle.J Invest Dermatol. 1997; 108: 324-329Crossref PubMed Scopus (58) Google Scholar), the hair shaft also shows a positive immunostaining with the antibody (Figure 1). To localize this signal relative to the hard-keratin-expressing portion of the hair follicle these sections were stained with an antibody that recognizes type I hard keratins, AE13. This dual staining experiment revealed that the K17 and AE13 antigens are both expressed in the hair shaft, but do not colocalize in the subcompartments of this structure (Figure 1a-c). Indeed, double-immunofluorescence staining of transverse sections clearly demonstrates that the K17 antigen is expressed in the medulla whereas the majority of hard keratin proteins are synthesized in the cortex (Figure 1d). We did not detect K5 or K6 staining in this region (data not shown), keeping open the question as to what the type II partner for K17 may be in this compartment. We can readily detect K17 immunoreactivity in the matrix portion of the hair follicle (Figure 1e,f). The distribution of K17 protein in this portion of the hair displays a distinct polarity with the cluster of K17 positive cells located opposite to the direction of hair follicle outgrowth. To further characterize the results obtained in tissue sections, Western blot analysis was performed on protein extracts prepared from mouse hair samples. To avoid contamination by outer root sheath keratins, the extracts were prepared from hair clippings rather than plucked hair. Additionally, to test that this result is not limited to the B6C3F1 mouse strain (Figure 1), hair clippings were obtained from a variety of mouse strains and subjected to Western blot analysis with several antibodies (Figure 2, upper gel). Independent of the mouse strain used, the hair extracts contain an immunoreactive band that comigrates with recombinant mouse K17 protein. The mouse strains examined include three nonpigmented (albino) hair samples and the first hair coat produced by hairless (HRS-/–) mice. Additionally, we do not observe any immunoreactivity with an anti-K14 antibody indicating that any contamination with outer root sheath components was minimal (data not shown). Likewise, antibodies to keratins 4, 5, and 6 fail to detect these proteins in the hair extracts (data not shown). Hair extracts were prepared from rat, dog, cat, monkey, pig, and human hairs using the identical procedure described above for mouse. Western blot analyses (Figure 2, lower gel) show that the hair extracts from all of these species, with the exception of human and pig, contain an antigen related to K17. K17 protein is readily detectable in the outer root sheath of human and pig hair follicles when applied to paraffin-embedded tissue sections (data not shown), confirming that the lack of reactivity in these samples is not due to a failure of the antibody to react with human or pig K17 proteins. Protein extracts prepared from hair clippings obtained from various human body sites including the eyebrow, pubic, underarm, body, and whisker regions were analyzed for K17 protein via Western blot analysis (Figure 3, upper panel). The results indicate that the extracts prepared from eyebrow and whisker hairs contained detectable amounts of K17. The other body sites that were tested were lacking in K17 protein. Samples typical of those used to obtain protein extracts were analyzed by light microscopy (Figure 3, lower panel). Both the eyebrow and whisker specimens contained a visible, continuous medulla compartment (B′, E′). The samples used for Figure 3 were obtained from a single individual; therefore we tested additional eyebrow and facial hair specimens from several individuals (Figure 4). All of the samples tested contained K17 as detected by Western blotting. It should be noted that the level of K17 expression in the eyebrows was not uniform across the population that we tested. This lack of uniformity strongly paralleled the degree to which the medulla compartment was visible in these samples when examined by light microscopy (data not shown). The levels of K17 in the whisker preparations were more uniform among the test population and corresponded to the consistent appearance of a medulla compartment in this hair type (data not shown). The matrix portion of the nail, which contains the precursor cell population for the hard-keratin-containing nail plate, stains positively for K17 (Figure 5d). The expression of K6 and K16 is nearly negligible in this portion of the nail (Figure 5b,c). Contrarily, K6 and K16 immunoreactivity can be readily detected in the ventral portion of the proximal nail fold, whereas K17 synthesis is considerably less in this region. The expression patterns for K6, K16, and K17 display much greater overlap in the nail bed epithelium (Figure 5f-h). Whereas K17 immunoreactivity is detectable throughout the entire nail bed including the basal layer, K6 and K16 are restricted to the suprabasal, postmitotic compartment. A similar phenomenon occurs in the outer root sheath of mouse hair follicles (McGowan and Coulombe, 1998aMcGowan K. Coulombe P.A. Onset of keratin 17 expression coincides with the definition of major epithelial lineages during mouse skin development.J Cell Biol. 1998; 143: 469-486Crossref PubMed Scopus (238) Google Scholar). We have produced a line of transgenic mice harboring a transgene consisting of the mouse K17 promoter driving the expression of the enhanced GFP. A complete characterization of these mice will be reported elsewhere. Hair clippings from these mice were used to produce protein extracts for Western blot analysis with an anti-GFP antibody (Figure 6). The GFP was detectable in these extracts, where it migrated as a doublet when compared with a recombinant control, suggesting that the expression of K17 in the hair shaft is controlled by elements in its promoter. The predominant clinical feature of PC is, as the name suggests, a severe dystrophy of the nail. Although there has been much debate about the origin of this lesion (Fleckman, 1985Fleckman P. Anatomy and physiology of the nail.Dermatol Clin. 1985; 3: 373-381PubMed Google Scholar), it is now widely accepted that the nail phenotype in all forms of PC disease arises from hyperkeratosis of the nail bed (Kelly and Pinkus, 1958Kelly E.W. Pinkus H. Report of a case of pachyonychia congenita.Arch Dermatol. 1958; 77: 724-726Crossref Scopus (13) Google Scholar) and not from alterations in the matrix. In fact,Kelly and Pinkus, 1958Kelly E.W. Pinkus H. Report of a case of pachyonychia congenita.Arch Dermatol. 1958; 77: 724-726Crossref Scopus (13) Google Scholar reported that the nail plate and matrix appear normal in PC. Our results, which demonstrate the coexpression of the K6, K16, and K17 genes in the nail bed epithelium, coupled with the similarity of nail alterations in type 1 and type 2 PC diseases, would support this conclusion. Similar to what we are reporting for the hair follicle, we observed K17 staining in the hard-keratin-synthesizing precursor cells located in the nail matrix. Our data further indicate that K6 and K16 are not synthesized in this portion of the nail but can be detected in the ventral nail fold in close proximity to the dorsal nail plate. It is interesting to note the close association between a hard-keratin-containing structure and the expression of K6, K16, and K17 that occurs in both the hair shaft and nail plate.Zaias, 1965Zaias N. The regeneration of the primate nail. Studies of the squirrel monkey, Saimiri.J Invest Dermatol. 1965; 44: 107-117PubMed Scopus (16) Google Scholar noted that most of the nail bed epithelium remains attached to the nail plate following avulsion. Additionally, labeling studies demonstrated the growth rate and movement of both structures to be identical (Zaias, 1967Zaias N. The movement of the nail bed.J Invest Dermatol. 1967; 48: 402-403Abstract Full Text PDF PubMed Scopus (26) Google Scholar,Zaias, 1980Zaias N. The Nail in Health and Disease. S.P. Medical, New York1980Crossref Google Scholar;Zaias and Alvarez, 1968Zaias N. Alvarez J. The formation of the primate nail plate: an autoradiographic study in the squirrel monkey.J Invest Dermatol. 1968; 51: 120-136Crossref PubMed Scopus (74) Google Scholar). A better understanding of the nature of this relationship, in particular whether it is dependent upon K6, K16, and K17 expression, will contribute significantly to the understanding of the nail phenotype in all forms of PC disease. The distinction in the PC phenotypes produced by inherited mutations in the K17 versus K16 mutations results largely from differences in the expression patterns for these genes. As proof of this principle, we have recently described the expression of K17 in the early stages of epidermal appendage development (McGowan and Coulombe, 1998aMcGowan K. Coulombe P.A. Onset of keratin 17 expression coincides with the definition of major epithelial lineages during mouse skin development.J Cell Biol. 1998; 143: 469-486Crossref PubMed Scopus (238) Google Scholar). This observation substantiates a role for K17 in this context and may help explain some of the typifying features of type 2 or Jackson-Lawler PC disease such as the presence of neonatal teeth (Clementi et al., 1986Clementi M. Cardin de Stefani E. Dei Rossi C. Avventi V. Tenconi R. Pachyonychia congenita Jackson Lawler type: a distinct malformation syndrome.Br J Dermatol. 1986; 114: 367-370Crossref PubMed Scopus (29) Google Scholar;Feinstein et al., 1988Feinstein A. Friedman J. Schewach M. Pachyonychia congenita.J Am Acad Dermatol. 1988; 19: 705-711Abstract Full Text PDF PubMed Scopus (111) Google Scholar). This report extends this principle by describing a unique aspect of the K17 gene, its expression in the medulla portion of the hair shaft. This pattern of expression may help to explain a defining feature of type 2 PC disease, the hair follicle phenotype. The expression of K17 in the medulla may help to resolve two key issues concerning this aspect of the disease: how the twisted hair phenotype arises and how it is restricted to certain hair types. The various cell types that make up the hair follicle are derived from precursor cells located in the hair follicle matrix. Consistent with our observation that K17 is expressed in the medulla, we can detect K17 positive cells among the precursor cell population located in the matrix (Figure 1e-f). Interestingly, the K17 positive cell population displays a polarized pattern of expression in the matrix that is similar to that reported by several groups for the sonic hedgehog molecule (Bitgood and MaMahon, 1995Bitgood M.J. MaMahon A.P. Hedgehog and BMP genes are coexpressed at many diverse sites of cell–cell interaction in the mouse embryo.Dev Biol. 1995; 172: 126-138Crossref PubMed Scopus (1137) Google Scholar;Gat et al., 1998Gat U. Das Gupta R. Degenstein L. Fuchs E. De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin.Cell. 1998; 95: 605-614Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar).Gat et al., 1998Gat U. Das Gupta R. Degenstein L. Fuchs E. De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin.Cell. 1998; 95: 605-614Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar have suggested that disrupting the polarization of the sonic hedgehog molecule in the matrix impacts the orientation of hair follicles relative to the skin surface. Given that we observe that K17 expression is polarized in a similar manner and that mutations in the K17 gene result in a twisted hair phenotype, we would predict that this keratin plays a role in the proper function of this subset of matrix epithelial cells. A second feature of the hair follicle phenotype associated with PC type 2 is that it is more pronounced in the eyebrow and other coarse body hairs (McLean et al., 1995McLean W.H.I. Rugg E.L. Lunny D.P. et al.Keratin 16 and keratin 17 mutations cause pachyonychia congenita.Nat Genet. 1995; 9: 273-278Crossref PubMed Scopus (280) Google Scholar). It is somewhat difficult to reconcile this regional phenotype with the more general expression pattern of K17 in the outer root sheath. Our findings suggest that this aspect of the phenotype may originate from K17 expression in the medulla compartment. Our survey of body hairs suggests that only the coarser body hairs contain an extensive medulla compartment. Interestingly, these samples corresponded to those sites most often implicated in type 2 PC disease. Whether there is a direct correspondence between K17 protein and the presence of a medulla remains an open question. A thorough analysis of hair specimens from type 2 PC patients would provide an interesting test of this hypothesis. The relationship between K17 gene expression and the type 2 PC disease phenotype suggests that the regulation of K17 gene expression is an important component in the pathogenesis of the disease. Panteleyev and colleagues examined the distribution of K17 mRNA during various stages in the murine hair cycle using in situ hybridization (Panteleyev et al., 1997Panteleyev A.A. Paus R. Wanner R. et al.Keratin 17 gene expression during the murine hair cycle.J Invest Dermatol. 1997; 108: 324-329Crossref PubMed Scopus (58) Google Scholar). This group found that K17 message was present in the isthmus, suprainfundibulum, and bulge region, although previous immunostaining experiments did not report the synthesis of K17 protein in these regions. Based upon this lack of correlation between mRNA and protein it was proposed that K17 gene expression may be regulated at a post-transcriptional level via differential mRNA stability (Panteleyev et al., 1997Panteleyev A.A. Paus R. Wanner R. et al.Keratin 17 gene expression during the murine hair cycle.J Invest Dermatol. 1997; 108: 324-329Crossref PubMed Scopus (58) Google Scholar). We have observed K17 protein in the regions in question, however: isthmus, suprainfundibulum, and bulge (McGowan and Coulombe, 1998aMcGowan K. Coulombe P.A. Onset of keratin 17 expression coincides with the definition of major epithelial lineages during mouse skin development.J Cell Biol. 1998; 143: 469-486Crossref PubMed Scopus (238) Google Scholar). Thus, we believe that the regulation of K17 gene expression is largely transcriptional. Previous studies have implicated members of the lymphoid enhancer factor/T cell factor (LEF/TCF) family of transcriptional complexes as key regulators of epidermal appendage development (Powell et al., 1991Powell B.C. Nesci A. Rogers G.E. Regulation of keratin gene expression in hair follicle differentiation.Ann NY Acad Sci. 1991; 642: 1-20Crossref PubMed Scopus (73) Google Scholar;van Genderen et al., 1994van Genderen C. Okamura R.M. Farinas I. Quo R-G. Parslow T.G. Bruhn L. Grosscheld R. Development of several organs that require inductive epithelial–mesenchymal interactions is impaired in Lef-1-deficient mice.Genes Dev. 1994; 8: 2691-2703Crossref PubMed Scopus (788) Google Scholar;Zhou et al., 1994Zhou P. Byrne C. Jacobs J. Fuchs E. Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate.Genes Dev. 1994; 9: 700-713Crossref Scopus (276) Google Scholar). A recent study byDas Gupta and Fuchs, 1999Das Gupta R. Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation.Development. 1999; 126: 4557-4568PubMed Google Scholar clearly defines the temporal and spatial activity of these transcription factors in the epidermis. In their study, mice harboring a transgene containing a β-galactosidase gene under the control of an LEF/TCF inducible promoter demonstrated the activation of this promoter in the hard keratin precursor cells located in the hair follicle matrix. The K17 promoter contains binding sites for this family of transcription factors. We have previously shown that the ectopic expression of LEF-1 in the basal layer of the epidermis leads to K17 synthesis in this compartment (McGowan and Coulombe, 1998aMcGowan K. Coulombe P.A. Onset of keratin 17 expression coincides with the definition of major epithelial lineages during mouse skin development.J Cell Biol. 1998; 143: 469-486Crossref PubMed Scopus (238) Google Scholar), suggesting that K17 expression may be an LEF/TCF responsive gene. Similar to the experiments ofDas Gupta and Fuchs, 1999Das Gupta R. Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation.Development. 1999; 126: 4557-4568PubMed Google Scholar, we have constructed a transgene containing 2.0 kb of the mouse K17 promoter regulating the expression of the GFP cDNA. Hair extracts prepared from these mice contained detectable amounts of the reporter transgene product, indicating that the 5′ upstream region of the mouse K17 gene is transcriptionally active within the hair shaft and provides a molecular mechanism for K17 expression in this context. We have provided experimental evidence that the soft keratin, K17, can also be expressed in the predominantly hard keratin portion of the hair shaft. The expression of K17 in this context may reflect a requirement for this protein in determining hair shape and orientation. Additionally, K17-containing filaments may provide unique mechanical properties required of coarser body hairs. Whether K17 plays an essential role in the formation and maintenance of these structures awaits the results of gene inactivation experiments and the function of K17 protein in the medulla remains an open question. Given its diverse pattern of expression, compared with other keratin genes expressed in the hair follicle, K17 may be uniquely suited for addressing the important questions of how keratin genes are regulated and how they impart functional diversity to epithelial tissues. Note added in proof After the submission of our manuscript we became aware of a paper entitled "Keratin expression in the normal nail unit: markers of regional differentiation", by De Berker, Wojnarowska, Sviland, Westgate, Dawber and Leigh, which appeared in the British Journal of Dermatology 142:89–96, 2000. We refer readers to this paper for additional information regarding keratin gene expression in the nail. We are very grateful to Dr. Henry Sun (New York University, New York) and Dr. Irene Leigh (Royal School of Medicine and Dentistry, London) for their generous gift of antibodies, and to Dr. George Rogers (University of Adelaide, Australia) for his expert advice on hair protein extraction. We thank Pig Newton for the donation of hair samples. K.M. was supported by an NRSA fellowship from the National Cancer Institute. This work was supported by NIH Grant AR44232 to P.A.C.