Cornulin, a New Member of the “Fused Gene” Family, Is Expressed During Epidermal Differentiation
2005; Elsevier BV; Volume: 124; Issue: 5 Linguagem: Inglês
10.1111/j.0022-202x.2005.23694.x
ISSN1523-1747
AutoresRomuald Contzler, Bertrand Favre, Marcel Huber, Daniel Hohl,
Tópico(s)Parkinson's Disease Mechanisms and Treatments
ResumoThe protein encoded by the C1orf10 gene was described to be esophageal-specific and a marker for cancer development. This protein, however, has the previously unreported structural features of the "fused gene" family combining sequences and structural similarities of both the S100 proteins and precursor proteins of the cornified cell envelope as in profilaggrin, trichohyalin, and repetin. Since all members of this family are expressed in keratinocytes, we suspected a role in epidermal differentiation and named the protein cornulin. Here, we report that human cornulin mRNA is expressed primarily in the upper layers of differentiated squamous tissues including the epidermis. Using polyclonal peptide antibodies, we show that cornulin is expressed in the granular and lower cornified cell layers of scalp epidermis and foreskin, as well as in calcium-induced differentiated cultured keratinocytes. Ca2+-overlay assay indicated that EF-hand domains of cornulin are functional and bind calcium. In HeLa cells, cornulin, co-transfected with transglutaminase 1, was diffusely distributed throughout the cytoplasm in contrast to small proline-rich 4, which localized to the cell periphery. We conclude that cornulin is a new member of the "fused gene" family, does not appear to be a precursor of the cornified cell envelope by itself, and is a marker of late epidermal differentiation. The protein encoded by the C1orf10 gene was described to be esophageal-specific and a marker for cancer development. This protein, however, has the previously unreported structural features of the "fused gene" family combining sequences and structural similarities of both the S100 proteins and precursor proteins of the cornified cell envelope as in profilaggrin, trichohyalin, and repetin. Since all members of this family are expressed in keratinocytes, we suspected a role in epidermal differentiation and named the protein cornulin. Here, we report that human cornulin mRNA is expressed primarily in the upper layers of differentiated squamous tissues including the epidermis. Using polyclonal peptide antibodies, we show that cornulin is expressed in the granular and lower cornified cell layers of scalp epidermis and foreskin, as well as in calcium-induced differentiated cultured keratinocytes. Ca2+-overlay assay indicated that EF-hand domains of cornulin are functional and bind calcium. In HeLa cells, cornulin, co-transfected with transglutaminase 1, was diffusely distributed throughout the cytoplasm in contrast to small proline-rich 4, which localized to the cell periphery. We conclude that cornulin is a new member of the "fused gene" family, does not appear to be a precursor of the cornified cell envelope by itself, and is a marker of late epidermal differentiation. complementary DNA digoxigenin epidermal differentiation complex phosphate-buffered saline transglutaminase The skin is the first line of defense versus chemical, physical, and microbial insults. Because of these perpetual attacks, the epidermis is continuously regenerating through a process called epidermal differentiation. The terminal differentiation of a keratinocyte in the epidermis involves the cessation of proliferation and subsequent migration from the basal layer to the suprabasal layers with progressive cornification. The cornified outermost cell layer provides a barrier that resists various insults from the external environment and prevents water loss (Kalinin et al., 2002Kalinin A.E. Kajava A.V. Steinert P.M. Epithelial barrier function: Assembly and structural features of the cornified cell envelope.Bioessays. 2002; 24: 789-800Crossref PubMed Scopus (367) Google Scholar). Epidermal differentiation is a complex process that requires a regulated and sequential expression of a variety of genes. Each epidermal layer is characterized by the expression of specific markers such as keratins K5/K14 in the basal cells (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), K1/K10 in the spinous cells (Fuchs and Green, 1980Fuchs E. Green H. Changes in keratin gene expression during terminal differentiation of the keratinocyte.Cell. 1980; 19: 1033-1042Abstract Full Text PDF PubMed Scopus (780) Google Scholar), and K2e in the granular cells. Several genes involved in epidermal differentiation are found within a 2-Mb region at chromosome band 1q21 called the epidermal differentiation complex (EDC) (Mischke et al., 1996Mischke D. Korge B.P. Marenholz I. Volz A. Ziegler A. Genes encoding structural proteins of epidermal cornification and S100 calcium-binding proteins form a gene complex ("epidermal differentiation complex") on human chromosome 1q21.J Invest Dermatol. 1996; 106: 989-992Crossref PubMed Scopus (397) Google Scholar; Lioumi et al., 1998Lioumi M. Olavesen M.G. Nizetic D. Ragoussis J. High-resolution YAC fragmentation map of 1q21.Genomics. 1998; 49: 200-208Crossref PubMed Scopus (23) Google Scholar; South et al., 1999South A.P. Cabral A. Ives J.H. et al.Human epidermal differentiation complex in a single 2.5 Mbp long continuum of overlapping DNA cloned in bacteria integrating physical and transcript maps.J Invest Dermatol. 1999; 112: 910-918Crossref PubMed Scopus (55) Google Scholar). During the final stages of differentiation, these specific proteins are cross-linked together by the action of transglutaminases (TG) to form the cornified envelope. The EDC contains three clustered families of genes encoding the following: (a) precursor proteins of the cornified cell envelope (involucrin, loricrin, and the small proline-rich (SPRR) proteins), characterized by short tandem peptide repeats in the central region (Mehrel et al., 1990Mehrel T. Hohl D. Rothnagel J.A. et al.Identification of a major keratinocyte cell envelope protein, loricrin.Cell. 1990; 61: 1103-11012Abstract Full Text PDF PubMed Scopus (360) Google Scholar; Haftek et al., 1991Haftek M. Serre G. Mils V. Thivolet J. Immunocytochemical evidence for a possible role of cross-linked keratinocyte envelopes in stratum corneum cohesion.J Histochem Cytochem. 1991; 39: 1531-1538Crossref PubMed Scopus (58) Google Scholar; Marvin et al., 1992Marvin K.W. George M.D. Fujimoto W. Saunders N.A. Bernacki S.H. Jetten A.M. Cornifin, a cross-linked envelope precursor in keratinocytes that is down-regulated by retinoids.Proc Natl Acad Sci USA. 1992; 89: 11026-11030Crossref PubMed Scopus (143) Google Scholar; Steinert and Marekov, 1995Steinert P.M. Marekov L.N. The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope.J Biol Chem. 1995; 270: 17702-17711Crossref PubMed Scopus (464) Google Scholar); (b) calcium-binding proteins (S100 proteins), containing EF-hand domains (Haftek et al., 1991Haftek M. Serre G. Mils V. Thivolet J. Immunocytochemical evidence for a possible role of cross-linked keratinocyte envelopes in stratum corneum cohesion.J Histochem Cytochem. 1991; 39: 1531-1538Crossref PubMed Scopus (58) Google Scholar; Presland et al., 1992Presland R.B. Haydock P.V. Fleckman P. Nirunsuksiri W. Dale B.A. Characterization of the human epidermal profilaggrin gene. Genomic organization and identification of an S-100-like calcium binding domain at the amino terminus.J Biol Chem. 1992; 267: 23772-23781Abstract Full Text PDF PubMed Google Scholar; Lee et al., 1993Lee S.C. Kim I.G. Marekov L.N. O'Keefe E.J. Parry D.A. Steinert P.M. The structure of human trichohyalin. Potential multiple roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and an intermediate filament-associated (cross-linking) protein.J Biol Chem. 1993; 268: 12164-12176Abstract Full Text PDF PubMed Google Scholar; Markova et al., 1993Markova N.G. Marekov L.N. Chipev C.C. Gan S.Q. Idler W.W. Steinert P.M. Profilaggrin is a major epidermal calcium-binding protein.Mol Cell Biol. 1993; 13: 613-625Crossref PubMed Scopus (113) Google Scholar; Steinert and Marekov, 1995Steinert P.M. Marekov L.N. The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope.J Biol Chem. 1995; 270: 17702-17711Crossref PubMed Scopus (464) Google Scholar; Krieg et al., 1997Krieg P. Schuppler M. Koesters R. Mincheva A. Lichter P. Marks F. Repetin (Rptn), a new member of the "fused gene" subgroup within the S100 gene family encoding a murine epidermal differentiation protein.Genomics. 1997; 43: 339-348Crossref PubMed Scopus (54) Google Scholar); and (c) a family of proteins (profilaggrin, trichohyalin, hornerin, repetin), which combine EF-hand domains at the N-terminus followed by multiple tandem peptide repeats and thus have been described as "fused genes" (Presland et al., 1992Presland R.B. Haydock P.V. Fleckman P. Nirunsuksiri W. Dale B.A. Characterization of the human epidermal profilaggrin gene. Genomic organization and identification of an S-100-like calcium binding domain at the amino terminus.J Biol Chem. 1992; 267: 23772-23781Abstract Full Text PDF PubMed Google Scholar; Markova et al., 1993Markova N.G. Marekov L.N. Chipev C.C. Gan S.Q. Idler W.W. Steinert P.M. Profilaggrin is a major epidermal calcium-binding protein.Mol Cell Biol. 1993; 13: 613-625Crossref PubMed Scopus (113) Google Scholar; Krieg et al., 1997Krieg P. Schuppler M. Koesters R. Mincheva A. Lichter P. Marks F. Repetin (Rptn), a new member of the "fused gene" subgroup within the S100 gene family encoding a murine epidermal differentiation protein.Genomics. 1997; 43: 339-348Crossref PubMed Scopus (54) Google Scholar). Recently, a novel gene, c1orf10, located on 1q21 was described as an esophageal-specific and cancer-associated gene (Xu et al., 2000Xu Z. Wang M.R. Xu X. et al.Novel human esophagus-specific gene c1orf10: cDNA cloning, gene structure, and frequent loss of expression in esophageal cancer.Genomics. 2000; 69: 322-330Crossref PubMed Scopus (37) Google Scholar; Luo et al, 2003). C1orf10 product is a protein of 495 amino acids with a calcium-binding motif of about 90 residues at its N-terminus and a conserved consecutive repeat sequence of 60 amino acids (Xu et al., 2000Xu Z. Wang M.R. Xu X. et al.Novel human esophagus-specific gene c1orf10: cDNA cloning, gene structure, and frequent loss of expression in esophageal cancer.Genomics. 2000; 69: 322-330Crossref PubMed Scopus (37) Google Scholar). Its structural characteristics are similar to those of "fused gene" family members of the EDC, but were not recognized. Here, we have characterized the expression of c1orf10 gene product, which we named cornulin, in human tissues, particularly the foreskin. We demonstrate that the "fused gene" family member cornulin is a Ca2+-binding protein present in the upper layer of squamous epithelia. Therefore, cornulin might have an important role in epidermal differentiation. It has been shown that c1orf10, encoding cornulin, was located on chromosome 1 in region q21 (Xu et al., 2000Xu Z. Wang M.R. Xu X. et al.Novel human esophagus-specific gene c1orf10: cDNA cloning, gene structure, and frequent loss of expression in esophageal cancer.Genomics. 2000; 69: 322-330Crossref PubMed Scopus (37) Google Scholar). Using the human genome sequence, we have established that it is located within the epidermal differentiation complex, very close to the other members of the "fused gene" family (profilaggrin, trichohyalin, and repetin). This family is organized in a cluster of ∼300 kb in the EDC, between S100A11 and NICE1 (Marenholz et al., 2001Marenholz I. Zirra M. Fischer D.F. Backendorf C. Ziegler A. Mischke D. Identification of human epidermal differentiation complex (EDC)-encoded genes by subtractive hybridization of entire YACs to a gridded keratinocyte cDNA library.Genome Res. 2001; 11: 341-355Crossref PubMed Scopus (84) Google Scholar) genes (Figure 1a). In silico analysis of the region 5′ upstream the transcription start site allowed us to identify a retinoic acid (RA) response element (RAR) at position -23, a TATA-box binding protein (TBP) site at position −41 as well as many SP1 sites, located in intron 1, non-coding exon 1 and all around the TBP site. Moreover, a perfect consensus AP1 regulatory motif, described inJang et al., 1996Jang S.I. Steinert P.M. Markova N.G. Activator protein 1 activity is involved in the regulation of the cell type-specific expression from the proximal promoter of the human profilaggrin gene.J Biol Chem. 1996; 271: 24105-24114Crossref PubMed Scopus (85) Google Scholar, was also detected at position -105 (Figure 1b). C1orf10 gene structure is similar to that of the "fused gene" members, i.e., a short first non-coding exon; a long intron 1; a second exon containing the initiation codon and encoding the first EF-hand motif; intron 2; a third exon containing the second EF-hand motif and the additional coding sequence. The main difference between cornulin and other "fused gene" members reside in the number and length of repetitive units. Cornulin has only two repeats of 60 amino acids while other fused gene members have variable motifs repeated several times. Multiple sequence alignment analysis shows that the N-terminal region (90 amino acid) of cornulin, containing two EF-hand domains, is closely related to the same region of other "fused proteins" such as repetin, profilaggrin, and trichohyalin and, to a lesser extent, to sequences of S100 protein family (Xu et al., 2000Xu Z. Wang M.R. Xu X. et al.Novel human esophagus-specific gene c1orf10: cDNA cloning, gene structure, and frequent loss of expression in esophageal cancer.Genomics. 2000; 69: 322-330Crossref PubMed Scopus (37) Google Scholar) (Figure 2a). All these proteins bind calcium. Sequence alignment with the most similar S100 member S100A8, for which a three-dimensional structure is available (Ishikawa et al., 2000Ishikawa K. Nakagawa A. Tanaka I. Suzuki M. Nishihira J. The structure of human MRP8, a member of the S100 calcium-binding protein family, by MAD phasing at 1.9 A resolution.Acta Crystallogr D. 2000; 56: 559-566Crossref PubMed Scopus (53) Google Scholar), indicates that the residues implicated in the binding of calcium are rather well conserved in cornulin (Figure 2b). Therefore, we tested whether cornulin could bind calcium as well. Thus, cornulin and cornulinΔEF were expressed in bacteria using the pET28a vector. Soluble and particulate fractions from isopropyl β-D-thiogalactoside (IPTG)-induced cultures of p28C and p28C ΔEF were electrophoresed and blotted to a membrane. The presence of calcium-binding proteins was tested by an 45Ca2+ overlay assay, as previously described (Maruyama et al., 1984Maruyama K. Mikawa T. Ebashi S. Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis.J Biochem (Tokyo). 1984; 95: 511-519Crossref PubMed Scopus (624) Google Scholar). Autoradiography of the membrane (Figure 2c) revealed the presence of a radioactive band whose position coincides with the band of cornulin visualized by both Ponceau red staining and western blot analysis (Figure 2c, lane 1). In contrast, no radioactive signal is visible at the position of cornulin lacking the EF-hand domains (Figure 2c, lane 3). Bovine brain calmodulin (Calbiochem Dietikon, Switzerland) was used as positive control (Figure 2c, lane 5). Thus, this result demonstrates that cornulin binds Ca2+. Cornulin Blast searches in the Ensembl databases allowed us to identify a murine homologue on mouse chromosome 3. We cloned this homologue, whose coding sequence was identical to that contained in GenBank (accession number XM_355410). Protein sequence comparisons determined that human cornulin presents 57.8% similarity to mouse cornulin. Furthermore, a putative rat homologue on rat chromosome 2 (accession number XP_227367) and a putative chimp homologue were also found (Figure 3a). Interestingly, multiple sequence alignment analysis showed that the amino-terminal region containing the EF-hand domains is the most conserved among all four species with 82.6% identity (Figure 3b). This result underlines the importance of the calcium-binding domain for the function of cornulin. RT-PCR experiments showed that the cornulin transcript was expressed in fetal bladder, scalp skin, foreskin, and in cultured primary keratinocytes (Figure 4a). Traces of cornulin mRNA were found in fetal brain, adult lung, kidney, uterus, skeletal muscle, and heart. Cornulin mRNA was more abundant in scalp skin and foreskin (Figure 4a) than in other tissues. Semi-quantitative RT-PCR experiments using 25, 30, and 35 PCR cycles showed that cornulin mRNA is more abundant in differentiated cultured keratinocytes, 7 d after shifting from 0.1 to 1.2 mM Ca2+ (Figure 4b). This was confirmed both by northern blot analysis, which showed a strong increase of expression, after the Ca2+ shift, of the 1.9 kb cornulin mRNA (Figure 4c), and by ISH analysis, which indicated that cornulin mRNA is expressed in the upper layers of the epidermis (Figure 4d). To investigate the cellular localization of cornulin, we constructed three expression vectors, giving rise to N-terminus GFP fusion proteins as follows: (a) full-length cornulin (pEGFP-cornulin), (b) deletion of 89 first N-terminus amino acids containing the EF-hands (pEGFP-cornulinΔEF), and finally (c) N-terminus containing EF-hands only (pEGFP-EFh). Furthermore, two other full-length constructs of cornulin were generated with either a HA or a V5 tag at N- or C-terminal position, respectively (Figure 5a). Confocal microscopy of HeLa cells transfected with pEGFP-Cornulin revealed a cytoplasmic distribution of cornulin with a denser staining around the nucleus (Figure 5b). In contrast, pEGFP-CornulinΔEF exhibited mostly a granular pattern in the whole cell. pEGFP-EFh localized both in the cytoplasm and in the nucleus, as observed for GFP control. Concerning the possibility that cornulin was a substrate of TG, no colocalization of TG 1 and cornulin was observed after co-transfection of expression vectors in HeLa cells. This indicates that cornulin is not processed by TG 1 in contrast to SPRR4, shown to be cross-linked by TG 1 and thus localized to the cell periphery (Cabral et al., 2001Cabral A. Sayin A. de Winter S. Fischer D.F. Pavel S. Backendorf C. SPRR4, a novel cornified envelope precursor: UV-dependent epidermal expression and selective incorporation into fragile envelopes.J Cell Sci. 2001; 114: 3837-3843PubMed Google Scholar) (Figure 5b). A polyclonal antibody, SZ1229, was produced against a sequence located in the repeats region (EATNDQNRGTETHGQG) and was affinity purified. The predicted molecular weight of cornulin is 53 kDa. Western blot analysis performed on total extracts from 293T cells, transfected with different tagged-fusion vectors (GFP, HA, and V5), revealed that both anti-tag and anti-cornulin (SZ1229) antibodies recognized the same proteins (Figure 6a). Anti-GFP antibody detected a protein that migrated at about 100 kDa, while anti-HA and anti-V5 antibodies detected a protein at about 70 kDa. As the calculated weight is about 83.5 kDa for the GFP-tagged cornulin, and about 56.5 kDa for HA and V5-cornulin, we can hypothesize that the apparent molecular weight is due either to an aberrant migration or to the fact that our protein is subject to post-translational modifications, the nature of which is not yet known. However, unlike profilaggrin, our experiments did not give any evidence of proteolytic processing in vivo (Figure 6b, left panel). Time-course expression analyses in human keratinocytes revealed a unique 70 kDa immunogenic protein in the d7 cytosolic extract (Figure 6b, right panel). Preincubation of our antibody with a 100 × excess of peptide epitope led to the disappearance of the 70 kDa band, confirming the peptide specificity of SZ1229 (Figure 6c). At d7, keratinocytes were differentiated, as indicated by the expression of K10. Thus, we can say that cornulin, like the other "fused" proteins, is a marker of late differentiation. Expression in different epithelial tissues was investigated by immunofluorescence analysis using the affinity-purified antibody SZ1229 (Figure 7). Experiments performed on foreskin sections (Figure 7a–c) exhibited a peripheral immunolabeling of the granular and lower cornified cells (Figure 7a, b). Cornulin expression was also found in scalp skin (Figure 7d–f) in the granular layer and the inner root sheath of the hair follicle. No staining was visible in control sections (Figure 7c and g). Finally, prominent staining was observed on esophagus sections at the periphery of the cells of the granular and the upper spinous layers (Figure 7h). In this paper, we have analyzed the gene c1orf10 and its protein product, which we called cornulin, previously described as being esophagus-specific (Xu et al., 2000Xu Z. Wang M.R. Xu X. et al.Novel human esophagus-specific gene c1orf10: cDNA cloning, gene structure, and frequent loss of expression in esophageal cancer.Genomics. 2000; 69: 322-330Crossref PubMed Scopus (37) Google Scholar). We present several lines of evidence that cornulin belongs to the "fused gene" family of proteins and plays a role in squamous differentiation. C1orf10 is located within the EDC and possesses the genomic and structural characteristics of a "fused gene" family member: (1) it is located on chromosome 1q21, (2) it contains three exons (exon 1 is non-translated), and (3) it encodes a protein with the fused protein structure, i.e., an amino-terminal Ca2+-binding domain and repeats in the carboxy-terminal region. Structurally, cornulin contains two EF-hand Ca2+ binding domains in its amino-terminal region highly comparable with those of profilaggrin (Presland et al., 1992Presland R.B. Haydock P.V. Fleckman P. Nirunsuksiri W. Dale B.A. Characterization of the human epidermal profilaggrin gene. Genomic organization and identification of an S-100-like calcium binding domain at the amino terminus.J Biol Chem. 1992; 267: 23772-23781Abstract Full Text PDF PubMed Google Scholar), trichohyalin (Lee et al., 1993Lee S.C. Kim I.G. Marekov L.N. O'Keefe E.J. Parry D.A. Steinert P.M. The structure of human trichohyalin. Potential multiple roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and an intermediate filament-associated (cross-linking) protein.J Biol Chem. 1993; 268: 12164-12176Abstract Full Text PDF PubMed Google Scholar), repetin 1Huber et al, manuscript submitted., and mouse hornerin (Makino et al., 2001Makino T. Takaishi M. Morohashi M. Huh N.H. Hornerin, a novel profilaggrin-like protein and differentiation-specific marker isolated from mouse skin.J Biol Chem. 2001; 276: 47445-47452Crossref PubMed Scopus (46) Google Scholar). Cornulin also bears two repeats of 60 amino acids in the C-terminal part (Xu et al., 2000Xu Z. Wang M.R. Xu X. et al.Novel human esophagus-specific gene c1orf10: cDNA cloning, gene structure, and frequent loss of expression in esophageal cancer.Genomics. 2000; 69: 322-330Crossref PubMed Scopus (37) Google Scholar), which are rich in glutamine (∼24%) and threonine (∼21%). They are mainly made of polar (∼57%) and charged (∼25%) residues. The function of these two repeats remains unknown. Concerning its amino-acid composition, cornulin is more similar to repetin, another member of the "fused gene" family, known to be implicated in epidermal differentiation (Krieg et al., 1997Krieg P. Schuppler M. Koesters R. Mincheva A. Lichter P. Marks F. Repetin (Rptn), a new member of the "fused gene" subgroup within the S100 gene family encoding a murine epidermal differentiation protein.Genomics. 1997; 43: 339-348Crossref PubMed Scopus (54) Google Scholar). Despite strong expression in the esophagus, cornulin is not specific for this tissue and both transcript and protein are found in scalp skin and foreskin, as well as in cultured keratinocytes. Furthermore, as for profilaggrin and other "fused gene" proteins, Ca2+ could play a role in the regulation of its expression because cornulin is expressed at higher levels in Ca2+-induced differentiated keratinocytes. Sequence alignment of N-terminus of cornulin and S100A8 protein showed that residues involved in the Ca2+ binding (Ishikawa et al., 2000Ishikawa K. Nakagawa A. Tanaka I. Suzuki M. Nishihira J. The structure of human MRP8, a member of the S100 calcium-binding protein family, by MAD phasing at 1.9 A resolution.Acta Crystallogr D. 2000; 56: 559-566Crossref PubMed Scopus (53) Google Scholar) are well conserved (Figure 2). Effectively, Ca2+-binding assay proved that EF-hand domains of cornulin are functional as observed for the other "fused" proteins, trichohyalin, repetin, and profilaggrin, which also bind calcium (Lee et al., 1993Lee S.C. Kim I.G. Marekov L.N. O'Keefe E.J. Parry D.A. Steinert P.M. The structure of human trichohyalin. Potential multiple roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and an intermediate filament-associated (cross-linking) protein.J Biol Chem. 1993; 268: 12164-12176Abstract Full Text PDF PubMed Google Scholar; Markova et al., 1993Markova N.G. Marekov L.N. Chipev C.C. Gan S.Q. Idler W.W. Steinert P.M. Profilaggrin is a major epidermal calcium-binding protein.Mol Cell Biol. 1993; 13: 613-625Crossref PubMed Scopus (113) Google Scholar; Presland et al., 1995Presland R.B. Bassuk J.A. Kimball J.R. Dale B.A. Characterization of two distinct calcium-binding sites in the amino-terminus of human profilaggrin.J Invest Dermatol. 1995; 104: 218-223Crossref PubMed Scopus (51) Google Scholar). Previous studies have shown that expression of late markers of differentiation could be mediated/regulated by RA (Asselineau et al., 1989Asselineau D. Bernard B.A. Bailly C. Darmon M. Retinoic acid improves epidermal morphogenesis.Dev Biol. 1989; 133: 322-335Crossref PubMed Scopus (185) Google Scholar; Hohl et al., 1991Hohl D. Lichti U. Breitkreutz D. Steinert P.M. Roop D.R. Transcription of the human loricrin gene in vitro is induced by calcium and cell density and suppressed by retinoic acid.J Invest Dermatol. 1991; 96: 414-418Abstract Full Text PDF PubMed Google Scholar, Hohl et al., 1995Hohl D. de Viragh P.A. Amiguet-Barras F. Gibbs S. Backendorf C. Huber M. The small proline-rich proteins constitute a multigene family of differentially regulated cornified cell envelope precursor proteins.J Invest Dermatol. 1995; 104: 902-909Crossref PubMed Scopus (148) Google Scholar; Krieg et al., 1997Krieg P. Schuppler M. Koesters R. Mincheva A. Lichter P. Marks F. Repetin (Rptn), a new member of the "fused gene" subgroup within the S100 gene family encoding a murine epidermal differentiation protein.Genomics. 1997; 43: 339-348Crossref PubMed Scopus (54) Google Scholar). The position of a RAR site in the promoter region of c1orf10 suggests that cornulin expression is likely mediated by RA. Moreover, the presence of an AP1 site indicates that cornulin expression could be regulated through a PKC-mediated pathway. In fact, PKC has been shown to be a fundamental regulator of the coordinated changes in keratinocyte gene expression that occur during the spinous to granular cell transition in the epidermis (Dlugosz and Yuspa, 1993Dlugosz A.A. Yuspa S.H. Coordinate changes in gene expression which mark the spinous to granular cell transition in epidermis are regulated by protein kinase C.J Cell Biol. 1993; 120: 217-225Crossref PubMed Scopus (214) Google Scholar). Interestingly, several AP1 sites were described to be functional and essential for high-level transcriptional activity of many late markers (DiSepio et al., 1995DiSepio D. Jones A. Longley M.A. Bundman D. Rothnagel J.A. Roop D.R. The proximal promoter of the mouse loricrin gene contains a functional AP-1 element and directs keratinocyte-specific but not differentiation-specific expression.J Biol Chem. 1995; 270: 10792-10799Crossref PubMed Scopus (94) Google Scholar; Welter et al., 1995Welter J.F. Crish J.F. Agarwal C. Eckert R.L. Fos-related antigen (Fra-1), junB, and junD activate human involucrin promoter transcription by binding to proximal and distal AP1 sites to mediate phorbol ester effects on promoter activity.J Biol Chem. 1995; 270: 12614-12622Crossref PubMed Scopus (172) Google Scholar). The identical AP1 site in both cornulin and profilaggrin promoters (Jang et al., 1996Jang S.I. Steinert P.M. Markova N.G. Activator protein 1 activity is involved in the regulation of the cell type-specific expression from the proximal promoter of the human profilaggrin gene.J Biol Chem. 1996; 271: 24105-24114Crossref PubMed Scopus (85) Google Scholar) suggests a similar mechanism for their transcriptional regulation. Results obtained by western blotting with affinity-purified polyclonal anti-cornulin antibody indicate that cornulin might undergo post-translational modifications because the apparent molecular weight is about 70 kDa, whereas the calculated weight is about 53 kDa. This is consistent with in silico prediction results (not shown), indicating that five Thr and 14 Ser residues could be phophorylated and two Asn residues could be N-glycosylated. The final conclusion of whether cornulin is a substrate of TG needs further investigations. Co-transfection assays in HeLa cells, using both cornulin and TG 1, showed no colocalization at the cell periphery suggesting that cornulin is not a substrate of this enzyme. However, cornulin is mainly localized in the periphery of cells in vivo. To explain these results, we hypothesize that HeLa cells do not contain "cross-linking" partners to whom cornulin could bind. Residues important in the cr
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