BM-40(Osteonectin, SPARC) Is Expressed Both in the Epidermal and in the Dermal Compartment of Adult Human Skin
1998; Elsevier BV; Volume: 110; Issue: 2 Linguagem: Inglês
10.1046/j.1523-1747.1998.00094.x
ISSN1523-1747
AutoresN. Hunzelmann, Martin Hafner, S Anders, Thomas Krieg, Roswitha Nicht,
Tópico(s)Bone Metabolism and Diseases
ResumoThe glycoprotein BM-40 (also termed SPARC, osteonectin) is a secreted protein widely distributed in human and murine tissues. It has been identified in bone, where it is one of the major noncollagenous glycoproteins, in basement membranes, and in several other extracellular tissues (Termine et al., 1981Termine J.D. Kleinman H.K. Whitson S.W. Conn K.M. McGarvey M.L. Martin G.R. Osteonectin, a bone-specific protein linking mineral to collagen.Cell. 1981; 26: 99105Abstract Full Text PDF Scopus (862) Google Scholar, Dziadek et al., 1986Dziadek M. Paulsson M. Aumailley M. Timpl R. Purification and tissue distribution of a small protein (BM-40) extracted from a basement membrane tumor.Eur J Biochem. 1986; 161: 455-464Crossref PubMed Scopus (111) Google Scholar, Sodek, 1993Sodek J. SPARC/osteonectin.in: Kreis T. Vale R. Guidebook to the Extracellular Matrix and Adhesion Proteins. Oxford Universitiy Press, Oxford1993: 89-91Google Scholar). Several studies describe high affinity binding to calcium (Engel et al., 1987Engel J. Taylor W. Paulsson M. Sage M. Hogan B. Calcium-binding domains and calcium- induced transition in SPARC (osteonectin/BM-40), an extracellular glycoprotein expressed in mineralized bone and nonmineralized tissues.Biochem. 1987; 26: 6958-6965Crossref PubMed Scopus (162) Google Scholar, Maurer et al., 1992Maurer P. Mayer U. Bruch M. Jen’ P, Landwehr R, Engel J, Timpl R: High-affinity and low-affinity calcium binding and stability of the multi-domain extracellular 40-kDa basement membrane glycoprotein (BM-40/SPARC/Osteonectin).Eur J Biochem. 1992; 205: 233-240Crossref PubMed Scopus (66) Google Scholar) and calcium-dependent binding to various collagens (Domenicucci et al., 1988Domenicucci C. Goldberg H.A. Hofmann T. Isenmann D. Wasi S. SodekJ: Characterization of porcine osteonectin extracted from foetal calvariae.Biochem J. 1988; 253: 139-151Crossref PubMed Scopus (69) Google Scholar, Sage et al., 1989bSage H. Vernon R.B. Funk S.E. Everitt E.A. Angello J. SPARC, a secreted protein associated with cellular proliferation, inhibits cell spreading in vitro and exhibits Ca2+- dependent binding to the extracellular matrix.J Cell Biol. 1989 b; 109: 341-356Crossref PubMed Scopus (314) Google Scholar, Mayer et al., 1991Mayer U. Aumailley M. Mann K. Timpl R. Engel J. Calcium-dependent binding of basement membrane protein BM-40 (osteonectin, SPARC) to basemet membrane collagen type IV.Eur J Biochem. 1991; 198: 141-150Crossref PubMed Scopus (94) Google Scholar, Nischt et al., 1991Nischt R. Pottgiesser J. Krieg T. Mayer U. Aumailley M. Timpl R. Recombinant expression and properties of the human calcium-binding extracellular matrix protein BM-40.Eur J Biochem. 1991; 200: 529-536Crossref PubMed Scopus (87) Google Scholar; Kelm and Mann, 1991Kelm R.J. Mann K.G. The collagen binding specifity of bone and platelet osteonectin is related to differences in glycosylation.J Biol Chem. 1991; 266: 9632-9639PubMed Google Scholar), as well as binding to hydroxyapatite (Romberg et al., 1985Romberg R.W. Werness P.G. Lollar P. Riggs B.L. Mann K.G. Isolation and characterization of native adult osteonectin.J Biol Chem. 1985; 260: 14831-14834Google Scholar) and platelet-derived growth factor (Raines et al., 1992Raines E.W. Lane T.F. Iruela-Arispe M.L. Ross R. Sage E.H. The extracellular glycoprotein SPARC interacts with platelet-derived growth factor (PDGF) -AB and -BB and inhibits the binding of PDGF to its receptors.Proc Natl Acad Sci USA. 1992; 89: 12811285Crossref Scopus (327) Google Scholar) in vitro. In several cell culture studies using fibroblasts and endothelial cells, BM-40 has been shown to influence the control of cell shape, migration, growth control, and gene expression (for review, see Lane and Sage, 1994Lane T.F. Sage H.E. The biology of SPARC, a protein that modulates cell-matrix interactions.FASEB J. 1994; 8: 163-173Crossref PubMed Scopus (477) Google Scholar; Reed and Sage, 1996Reed M.J. Sage E.H. SPARC and the extracellular matrix: implications for cancer and wound repair.Curr Top Microbiol Immunol. 1996; 213: 81-94PubMed Google Scholar). Even though the exact biologic function is still unknown, BM-40 is expressed at high levels during tissue repair, differentiation, mouse development, and growth, where it may act as an anti-adhesive factor for cells (Holland et al., 1987Holland P.W.H. Harper S.J. McVey J.H. Hogan B.L.M. In vivo expression of mRNA for the Ca2 +-binding protein SPARC (osteonectin) revealed by in situ hybridisation.J Cell Biol. 1987; 105: 473-482Crossref PubMed Scopus (219) Google Scholar, Sage et al., 1989aSage H. Vernon R.B. Decker J. Funk S. Iruela-Arispe M.L. Distribution of the calciumbinding protein SPARC in tissues of embryonic and adult mice.J Histochem Cytochem. 1989 a; 37: 819-829Crossref PubMed Scopus (163) Google Scholar, Sage and Bornstein, 1991Sage E.H. Bornstein P. Extracellular proteins that modulate cell-matrix interactions.J Biol Chem. 1991; 266: 14831-14834PubMed Google Scholar, Tremble et al., 1993Tremble P.M. Lane T.F. Sage E.H. Werb Z. SPARC, a secreted protein associated with morphogenesis and tissue remodeling, induces expression of metalloproteinases in fibroblasts through a novel extracellular matrix-dependent pathway.J Cell Biol. 1993; 121: 1433-1444Crossref PubMed Scopus (249) Google Scholar, Reed et al., 1993Reed M.J. Puolakkainen P.A. Lane T.F. Dickerson D. Bornstein P. Sage E.H. Expression of SPARC and thrombospondin 1 in wound repair: immunolocalisation and in situ hybridization.JHistochem Cytochem. 1993; 41: 1467-1477Crossref PubMed Scopus (193) Google Scholar). This suggests a versatile function of BM-40 in the regulation of tissue synthesis and turnover, including a major role as a morpho-regulatory element. Although information regarding the structure and potential biologic functions of BM-40 has increased significantly over the last few years, relatively little is known about the expression and distribution of BM-40 in human tissues in vivo. Therefore the aim of this study was to investigate BM-40 expression and synthesis in adult human skin. We report here that BM-40 is expressed in adult human skin in both the dermal and the epidermal compartment, indicating a new role for BM-40 in epidermal differentiation and growth in vivo. Normal skin was obtained from patients admitted for tattoo excisions following informed consent. Specimens were bisected with one half of the specimen undergoing routine paraffin processing and the other half being snap frozen in liquid nitrogen and stored at —70°C until required. Human skin fibroblasts were obtained from normal human skin using the explant culture technique. Primary keratinocyte cultures and subcultures were established according to the method of Limat et al., 1989Limat A. Hunziker T. Billat C. Bayreuther K. Noser F. Postmitotic human dermal fibroblasts efficiently support the growth of human follicular keratinocytes.J Invest Dermatol. 1989; 92: 758-762Abstract Full Text PDF PubMed Scopus (105) Google Scholar. The HaCaT cell line had been generously provided by Dr. N. Fusenig (DKFZ, Heidelberg). HeLa cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The cells were grown to confluency in monolayer cultures in Dulbecco’s modified Eagle medium, supplemented with 10% fetal calf serum, 50 mg ascorbate per ml, 300 mg glutamine per ml, 50 mg streptomycin per ml, and 400 U penicillin per ml. Thereafter cells were either subjected to RNA isolation or grown on glass slides for subsequent immunohistochemical analysis. Frozen skin biopsies were cut on dry ice and homogenized in 4 M guanidine-isothiocyanate containing 0.1 M 2-mercapto- ethanol. Alternatively cells were scraped from cell culture dishes using a rubber policeman and dissolved in the same buffer. RNA was isolated by ultracentrifugation over a cesium chloride cushion according to published protocols (Maniatis et al., 1982Maniatis T. Frisch E.F. Sambrook J. Molecular cloning. Cold Spring Harbor Laboratory, New York1982Google Scholar). For northern blot analysis 5 mg total RNA was separated by gel electrophoresis in 1% agarose under denaturing conditions and then blotted onto a nylon membrane (Genescreen, Dupont, Boston, MA). As a control for the amount and integrity of RNA blotted, the membrane was stained with methylene blue, 0.04% in 0.2 M sodium acetate (pH 5.2). Filters were hybridized according to published protocols with 32P-labeled random primed BM-40 cDNA probe. After hybridization filters were washed and exposed at—80°C to a radiosensitive film (Kodak, X-OmatAR, Rochester, NY). Frozen 5 mm sections were investigated using the nonradioactive in situ hybridization technique with digoxigenin labeled oligonucleotides. The following anti-sense and sense 5' digoxigenin labeled oligonucleotides corresponding to nucleotide position 136—169 were used (Lankat-Buttgereit et al., 1988Lankat-Buttgereit B. Mann K. Deutzmann R. Timpl R. Krieg T. Cloning and complete amino acid sequences of human and murine basement membrane protein BM-40 (SPARC, osteonectin).FEBS Lett. 1988; 236: 352-356Crossref PubMed Scopus (63) Google Scholar): anti-sense, 5'-gctcccacagatacct-cagtcacctctgccacag-3'; sense, 5'-Ctgtggcagaggtgac-tgaggtatctgtgggag-3'. The specificity of these oligonucleotides was confirmed by northern blot analysis of fibroblast total RNA (not shown). Briefly, frozen sections were mounted on silane-coated slides and fixed with 4% paraformaldehyde in phosphate-buffered saline for 5 min and treated with 2 mg glycine per ml for 15 min thereafter. Prehybridization and hybridization were performed at 42°C in a mixture containing 6 × sodium chloride/sodium citrate, 1 X Denhardts solution, 100 mg tRNA per ml, 0.25% sodium dodecylsulfate. After hybridization and washing in 6 X sodium chloride/ sodium citrate at 42°C, digoxigenin-labeled probes were visualized as described in the manufacturer’s protocol (Boehringer, Mannheim, Germany). Alternatively, anti-sense and sense digoxigenin labeled riboprobes were prepared using a 490-bp BM-40 cDNA fragment subcloned into the vector pBluescript KS+ (Stratagene, La Jolla, CA) (Lankat-Buttgereit et al., 1988Lankat-Buttgereit B. Mann K. Deutzmann R. Timpl R. Krieg T. Cloning and complete amino acid sequences of human and murine basement membrane protein BM-40 (SPARC, osteonectin).FEBS Lett. 1988; 236: 352-356Crossref PubMed Scopus (63) Google Scholar). The probes were labeled with digoxigenin 11-UTP using a RNA labeling kit (Boehringer) and hybridized in the same mixture with 50% formamide. After stringent washings, sections were counter-stained with eosin or methyl green and mounted. Alkaline phosphatase anti-alkaline phosphatase staining was performed following established procedures (Schaumburg-Lever, 1987Schaumburg-Lever G. The alkaline phosphatase anti-alkaline phosphatase technique in dermatopathology.J Cutan Pathol. 1987; 14: 6-9Crossref PubMed Scopus (38) Google Scholar). Rabbit anti-mouse immunoglobulins and alkaline phosphatase anti-alkaline phosphatase complexes were obtained from Dako (Hamburg, Germany). The alkaline phosphatase reaction was demonstrated by incubation in a solution containing Fast Red TR (1 mg per ml) and naphtol AS-TR phosphate (0.2 mg per ml) (Sigma, St. Louis, MO). Levamisole (0.24 mg per ml) was added to block endogenous alkaline phosphatase activity. Primary keratinocytes, HaCaT, and HeLa cells grown on glass slides were stained using fluoroscein isothiocyanate conjugated goat anti-IgG antibodies following established protocols. In brief, after incubation with 1% normal goat serum/1% bovine serum albumin in phosphate-buffered saline at room temperature, the slides were incubated with the primary polyconal rabbit antiserum (Nischt et al., 1991Nischt R. Pottgiesser J. Krieg T. Mayer U. Aumailley M. Timpl R. Recombinant expression and properties of the human calcium-binding extracellular matrix protein BM-40.Eur J Biochem. 1991; 200: 529-536Crossref PubMed Scopus (87) Google Scholar) (diluted 1:400 in 1% normal goat serum/1% bovine serum albumin in phosphate-buffered saline) for 45 min. Incubation with the secondary antibody (fluoroscein isothiocyanate conjugated goat anti-rabbit 1:400 in the same buffer) was carried out for 20 min. After washing and mounting fluorescence microscopy was performed. Negative controls consisted of the omission of primary antibodies, use of preimmune rabbit serum, and staining with the dye alone. As a positive control a polyclonal antibody directed against keratin was used. The stained sections were reviewed by two independent observers. Trichloracetic acid precipitable proteins from serum free culture medium (primary fibroblasts and keratinocytes) were resolved on a 10% sodium dodecylsulfate/polyacyrylamide gel under reducing conditions. Thereafter proteins were transfered to nitrocellulose (Amersham, Braunschweig, Germany). To block nonspecific antibody binding, the membrane was incubated overnight in tris buffered saline containing 5% skimmed milk powder. Subsequently the membrane was incubated with the polyclonal rabbit-anti-BM-40 anti-serum diluted 1:2000. After washing, the membrane was incubated with an horseradishperoxidase-conjugated swine anti-rabbit antibody (Dako) followed by development using the ECL-system (Amersham). It is known from several in vitro studies that fibroblasts and keratinocytes are expressing BM-40 mRNA (Howe et al., 1990Howe C.C. Kath R. Mancianti M.L. Herlyn M. Mueller S. Chrisofalo V. Expression and structure ofhuman SPARC transcripts: SPARC mRNA is expressed by human cells involved in extracellular matrix production and some ofthese cells show an unusual expression pattern.Exp Cell Res. 1990; 188: 185-191Crossref PubMed Scopus (21) Google Scholar, Lankat-Buttgereit et al., 1991Lankat-Buttgereit B. Kulozik M. Hunzelmann N. Krieg Th. Cytokines alter mRNA steady state levels for basement membrane proteins in human fibroblasts.J Dermat Sci. 1991; 2: 300-307Abstract Full Text PDF PubMed Scopus (9) Google Scholar; Wrana et al., 1991Wrana J.L. Overall C.M. Sodek J. Regulation of the expression of a secreted acidic protein rich in cysteine (SPARC) in human fibroblasts by transforming growth factor beta.Eur J Biochem. 1991; 197: 519-528Crossref PubMed Scopus (115) Google Scholar; Ford et al., 1993Ford R. Wang G. Jannati P. AdlerD, Racanelli P, Higgins PJ.Staiano-Coico L: Modulation of SPARC expression during butyrate-induced terminal differentiation of cultured human keratinocytes: regulation via a TGF-beta-dependent pathway. Exp Cell Res. 1993; 206: 261-275Google Scholar). As BM-40 expression is usually induced when attachment-dependent cells are grown on plastic, a phenomenon referred to as “culture shock” (Sage et al., 1986Sage H. Tupper J. Bramson R. Endothelial cell injury in vitro is associated with increased secretion of an Mw 43000 glycoprotein ligand.J Cell Physiol. 1986; 127: 373-387Crossref PubMed Scopus (74) Google Scholar; Lane and Sage, 1994Lane T.F. Sage H.E. The biology of SPARC, a protein that modulates cell-matrix interactions.FASEB J. 1994; 8: 163-173Crossref PubMed Scopus (477) Google Scholar), we were interested to compare BM-40 mRNA levels in skin directly with the levels expressed by keratinocytes and fibroblasts grown on plastic. As shown in Fig 1, northern blot analysis revealed the usual hybridization pattern for BM-40 mRNA in both fibroblasts and keratinocytes, with a high level of the 2.2 kb and a lower level of the 3 kb transcript that contains additional 3' untranslated sequences (Swaroop et al., 1988Swaroop A. Hogan B.L.M. Francke U. Molecular analysis of the cDNA for human SPARC/Osteonectin/BM-40: sequence, expression and localization of the gene to chromosome 5q31-q33.Genomics. 1988; 2: 37-47Crossref PubMed Scopus (135) Google Scholar); however, in keratinocytes the level was markedly lower when compared with fibroblasts (Fig 1, lanes 2 and 3). The signal obtained with RNA isolated from skin biopsies is very strong, indicating that the signals obtained with cultured keratinocytes and fibroblasts reflect the BM-40 mRNA level in vivo, although we cannot distinguish the cellular source of BM-40 mRNA in this experiment. Because it is known that BM-40 protein synthesis is increased in human keratinocytes during sodium n-butyrate induced differentiation (Staiano-Coico et al., 1989Staiano-Coico L. Helm R.E. McMehon C.K. Pagan-Charry J. La Bruna A. Piraino V. Higgins P.J. Sodium-N-butyrate induces cytoskeletal rearrangements and formation of cornified envelopes in cultured adult human keratinocytes.Cell Tissue Kinet. 1989; 22: 361-375PubMed Google Scholar, Ford et al., 1993Ford R. Wang G. Jannati P. AdlerD, Racanelli P, Higgins PJ.Staiano-Coico L: Modulation of SPARC expression during butyrate-induced terminal differentiation of cultured human keratinocytes: regulation via a TGF-beta-dependent pathway. Exp Cell Res. 1993; 206: 261-275Google Scholar), we asked the question whether the relatively low levels of BM-40 mRNA in keratinocytes might be due to the nondifferentiating conditions used for RNA isolation. To address this question keratinocytes were cultured in the presence or absence of 3 mM sodium n-butyrate for 24, 48, and 72 h. Northern blot analysis of RNA isolated from these cultures revealed a dramatic increase of BM-40 mRNA in the sodium n-butyrate treated keratinocytes already after 24 h when compared with untreated control cells (Fig 2). This result clearly indicates that the elevated protein level observed by Ford et al., 1993Ford R. Wang G. Jannati P. AdlerD, Racanelli P, Higgins PJ.Staiano-Coico L: Modulation of SPARC expression during butyrate-induced terminal differentiation of cultured human keratinocytes: regulation via a TGF-beta-dependent pathway. Exp Cell Res. 1993; 206: 261-275Google Scholar is due to an increase of the steady state BM-40 mRNA level.Figure 2Increased BM-40 mRNA levels in keratinocytes after induction of differentiation. To induce differentiation in cultured keratinocytes the cells were cultivated in 3 mM sodium n-butyrate (lanes 4—6). Untreated keratinocytes were used as controls (lanes 1—3). For RNA isolation cells were harvested after 24 h (lanes 1, 4), 48 h (lanes 2, 5), and 72 h (lanes 3, 6). The RNA samples were separated in a 1% agarose gel, blotted onto Genescreen, and hybridized to 32P-labeled BM-40 cDNA as described in Materials and Methods. In the lower part methylen blue staining of rRNA is shown to confirm even loading of the RNA samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In order to analyze BM-40 protein expression in keratinocytes and fibroblasts, cells were grown to confluency in normal growth medium and were subsequently cultivated for 48 h in serum- free medium. Western blot analysis of these conditioned media show that both cell types secrete BM-40 into the extracellular space (Fig 3). In contrast to melanoma cells that show specific cleavage of BM-40 (Ledda et al., 1997Ledda M.F. Bravo A.I. Adris S. Bover L. Mordoh J. Podhajcer O.L. The expression of the secreted protein acidic and rich in cysteine (SPARC) is associated with the neoplastic progression of human Melanoma.J Invest Dermatol. 1997; 108: 210-214Abstract Full Text PDF PubMed Scopus (149) Google Scholar), only one band of about 42 kDa was detected in the supernatants of keratinocytes and fibroblasts. Deposition of BM-40 was studied by indirect immunofluorescence with the BM-40 polyclonal anti-serum on unfixed keratinocytes and the keratinocyte-like cell line HaCaT (not shown) grown for 72 h on coverslides. Under these culture conditions keratinocytes only show signs of early differentiation. As seen in Fig 4(a), the most prominent staining ofBM-40 protein is detectable in the periphery ofkeratinocytes with suprabasal morphology, suggesting that BM-40 is either associated with the plasma membrane of these cells or secreted in the intercellular space. This is in contrast to the observation of Ford et al., 1993Ford R. Wang G. Jannati P. AdlerD, Racanelli P, Higgins PJ.Staiano-Coico L: Modulation of SPARC expression during butyrate-induced terminal differentiation of cultured human keratinocytes: regulation via a TGF-beta-dependent pathway. Exp Cell Res. 1993; 206: 261-275Google Scholar who found BM-40 deposition restricted to cornified envelopes. HeLa cells, as a negative control cell line lacking BM-40 expression (Hafner et al., 1994Hafner M. Zimmermann K. Pottgiesser J. Krieg T. Nischt R. A purine-rich sequence in the human BM-40 gene promoter region is a prerequisite for maximum transcription.Matrix Biol. 1994; 14: 733-741Crossref Scopus (25) Google Scholar), were negative (Fig 4b). To identify the cellular sources in the skin that are responsible for the high BM-40 mRNA level obtained by northern blot analysis, nonradioactive in situ hybridization using both digoxigenin labeled oligonucleotides (Fig 5) or anti-sense and sense strand riboprobes (results not shown) was performed. In the dermis expression was detected in fibroblasts throughout the whole compartment as well as in endothelial cells, smooth muscle cells, and glandular epithelial cells (Fig 5a,c). In the epidermal compartment BM-40 mRNA expression is seen throughout the epidermis, being partly more pronounced in the basal cell layer (Fig 5a,b). Immunohistochemistry with the BM-40 anti-serum revealed staining in the whole dermal compartment, showing a clearly more pronounced signal directly below the basement membrane and in the papillary dermis (Fig 6a). This finding coincides with the increased density of endothelial and fibroblast-like cells in the papillary dermis showing BM-40 mRNA expression. An increase in staining intensity could also be noted around vascular structures. In the epidermis, staining for BM-40 was detected intercellularly most prominently in the suprabasal spinous and to a lesser degree in the basal and prickle cell layers (Fig 6b). No staining was detected in the stratum corneum. This staining pattern clearly demonstrates that BM-40 expression in vivo is not restricted to the cornified cell layers of the epidermis. Due to the origin of its purification, it had previously been assumed that the expression of BM-40 is mainly restricted to connective tissue and bone (Termine et al., 1981Termine J.D. Kleinman H.K. Whitson S.W. Conn K.M. McGarvey M.L. Martin G.R. Osteonectin, a bone-specific protein linking mineral to collagen.Cell. 1981; 26: 99105Abstract Full Text PDF Scopus (862) Google Scholar, Lankat-Buttgereit et al., 1988Lankat-Buttgereit B. Mann K. Deutzmann R. Timpl R. Krieg T. Cloning and complete amino acid sequences of human and murine basement membrane protein BM-40 (SPARC, osteonectin).FEBS Lett. 1988; 236: 352-356Crossref PubMed Scopus (63) Google Scholar). As has been implicated from several in vitro studies (Howe et al., 1990Howe C.C. Kath R. Mancianti M.L. Herlyn M. Mueller S. Chrisofalo V. Expression and structure ofhuman SPARC transcripts: SPARC mRNA is expressed by human cells involved in extracellular matrix production and some ofthese cells show an unusual expression pattern.Exp Cell Res. 1990; 188: 185-191Crossref PubMed Scopus (21) Google Scholar, Wrana et al., 1991Wrana J.L. Overall C.M. Sodek J. Regulation of the expression of a secreted acidic protein rich in cysteine (SPARC) in human fibroblasts by transforming growth factor beta.Eur J Biochem. 1991; 197: 519-528Crossref PubMed Scopus (115) Google Scholar, Reed et al., 1993Reed M.J. Puolakkainen P.A. Lane T.F. Dickerson D. Bornstein P. Sage E.H. Expression of SPARC and thrombospondin 1 in wound repair: immunolocalisation and in situ hybridization.JHistochem Cytochem. 1993; 41: 1467-1477Crossref PubMed Scopus (193) Google Scholar), BM-40 mRNA expression could be detected in vivo in fibroblasts throughout the dermis, in endothelial, smooth muscle and glandular epithelial cells. Increased deposition, as shown by immunohistochemistry, seems thereby to take place in areas supposedly characterized by an increased turnover of the surrounding connective tissue, i.e., papillary dermis, perivascular areas, and adnexal structures. In the epidermis, as shown by in situ hybridization, the highest BM- 40 mRNA expression was detected in cells of the basal layer. This indicates that the increase ofBM-40 mRNA levels observed in sodium n-butyrate differentiated keratinocytes might be due either to a higher expression rate or to stabilization of BM-40 mRNA mainly taking place in basal keratinocytes after induction of the differentiation process. In contrast, as shown by immunohistochemistry, there was no or only little protein detectable in this layer, indicating either that BM-40 mRNA is mainly translated in suprabasal cells or that the protein is readily deposited in a polarized fashion beneath the basal cell layer in the basement membrane zone or above in the extracellular space of the suprabasal cell layers. From our studies we cannot exclude that BM-40 is also localized intracellularly as has been recently shown by Porter et al., 1995Porter P.L. Sage E.H. Lane T.F. Fuchs S.E. Gown A.M. Distribution of SPARC in normal and neoplastic human tissue.J Histochem Cytochem. 1995; 43: 791-800Crossref PubMed Scopus (194) Google Scholar. The role of BM-40 as an integral part of the epidermal intercellular space is unclear. But it is intriguing to speculate that BM-40, which has been classified as an “anti-adhesin” (Sage and Bornstein, 1991Sage E.H. Bornstein P. Extracellular proteins that modulate cell-matrix interactions.J Biol Chem. 1991; 266: 14831-14834PubMed Google Scholar; Lane and Sage, 1994Lane T.F. Sage H.E. The biology of SPARC, a protein that modulates cell-matrix interactions.FASEB J. 1994; 8: 163-173Crossref PubMed Scopus (477) Google Scholar), might be involved in the migration process during epidermal differentiation by diminishing the contact ofthe basal keratinocytes to the underlying basement membrane and cell-cell contacts in the suprabasal layers. Whether BM-40, as a Ca2+ binding protein, is contributing to the Ca2+ gradient in the epidermis (Forslind, 1987Forslind B. Quantitative X-ray microanalysis of skin.Acta Derm Venereol (Stockh) (Suppl). 1987; 134: 1-8PubMed Google Scholar) is unknown, and it remains to be seen whether the EF-hands in BM-40 fulfil a strictly structural role or whether they are used to transmit a Ca2+-coupled signal as seen in the cytosolic homologs of the EF-hand family (for a review see Maurer et al., 1996Maurer P. Hohenester E. Engel J. Extracellular calcium binding proteins.Curr Op Cell Biol. 1996; 8: 609-617Crossref PubMed Scopus (78) Google Scholar). Because BM-40 has been recently shown to be a substrate for transglutaminase catalyzed cross-linking in differentiating cartilage (Aeschlimann et al., 1995Aeschlimann D. Kaupp O. Paulsson M. Transglutaminase-catalyzed matrix cross-linking in differentiating cartilage: identification of osteonectin as a major glutaminyl substrate.J Cell Biol. 1995; 129: 881-892Crossref PubMed Scopus (182) Google Scholar, Hohenadl et al., 1995Hohenadl C. Mann K. Mayer U. Timpl R. Paulsson M. Aeschlimann D. Two adjacent N-terminal glutamines of BM-40 (Osteonectin, SPARC) act as amine acceptor sites in transglutaminase catalyzed modification.J Biol Chem. 1995; 270: 23415-23420Crossref PubMed Scopus (54) Google Scholar), it is of interest to point out that the expression pattern in skin of an antibody generated to transglutaminase cross-links (Aeschlimann et al., 1995Aeschlimann D. Kaupp O. Paulsson M. Transglutaminase-catalyzed matrix cross-linking in differentiating cartilage: identification of osteonectin as a major glutaminyl substrate.J Cell Biol. 1995; 129: 881-892Crossref PubMed Scopus (182) Google Scholar) parallels the expression pattern in the skin described herein for BM-40. This supports the possibility that BM-40 in the epidermis might be a target for transglutaminase catalyzed cross-linking, thereby altering biologic functions of this protein. In summary, this study demonstrates that BM-40 is not only a component of the connective tissue of different organs but also part of epithelial structures such as the epidermis of adult human skin. Because BM-40 is detected in different epithelial tumors (Porter et al., 1995Porter P.L. Sage E.H. Lane T.F. Fuchs S.E. Gown A.M. Distribution of SPARC in normal and neoplastic human tissue.J Histochem Cytochem. 1995; 43: 791-800Crossref PubMed Scopus (194) Google Scholar, Bellahcene and Castronovo, 1995Bellahcene A. Castronovo V. Increased expression of osteonectin and osteopontin, two bone matrix proteins, in human breast cancer.Am J Pathol. 1995; 146: 95-100PubMed Google Scholar), an understanding of the precise function of this protein in epithelial differentiation would also provide insight into the role of BM-40 in processes underlying tumor growth and invasion. The expert technical assistance of Caroline Hartmann and Marion Schmoll is gratefully acknowledged. Keratinocyte RNA was obtained in cooperation with A. Limat. Parts of this study were presented at the XXVth Annual Meeting of ESDR 1996. This work was supported by Koln Fortune Program and a grant (90.010.3) from the Wilhelm Sander Stiftung, Germany.
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