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• W2025136908 abstract "In skin, hemidesmosomal protein complexes attach the epidermis to the dermis and are critical for stable connection of the basal epithelial cell cytoskeleton with the basement membrane (BM). In muscle, a similar supramolecular aggregate, the dystrophin glycoprotein complex links the inside of muscle cells with the BM. A component of the muscle complex, dystroglycan (DG), also occurs in epithelia. In this study, we characterized the expression and biochemical properties of authentic and recombinant DG in human skin and cutaneous cells in vitro. We show that DG is present at the epidermal BM zone, and it is produced by both keratinocytes and fibroblasts in vitro. The biosynthetic precursor is efficiently processed to the α- and β-DG subunits; and, in addition, a distinct extracellular segment of the transmembranous β-subunit is shed from the cell surface by metalloproteinases. Shedding of the β-subunit releases the α-subunit from the DG complex on the cell surface into the extracellular space. The shedding is enhanced by IL-1β and phorbol esters, and inhibited by metalloproteinase inhibitors. Deficiency of perlecan, a major ligand of α-DG, enhanced shedding suggesting that lack of a binding partner destabilizes the epithelial DG complex and makes it accessible to proteolytic processing. In skin, hemidesmosomal protein complexes attach the epidermis to the dermis and are critical for stable connection of the basal epithelial cell cytoskeleton with the basement membrane (BM). In muscle, a similar supramolecular aggregate, the dystrophin glycoprotein complex links the inside of muscle cells with the BM. A component of the muscle complex, dystroglycan (DG), also occurs in epithelia. In this study, we characterized the expression and biochemical properties of authentic and recombinant DG in human skin and cutaneous cells in vitro. We show that DG is present at the epidermal BM zone, and it is produced by both keratinocytes and fibroblasts in vitro. The biosynthetic precursor is efficiently processed to the α- and β-DG subunits; and, in addition, a distinct extracellular segment of the transmembranous β-subunit is shed from the cell surface by metalloproteinases. Shedding of the β-subunit releases the α-subunit from the DG complex on the cell surface into the extracellular space. The shedding is enhanced by IL-1β and phorbol esters, and inhibited by metalloproteinase inhibitors. Deficiency of perlecan, a major ligand of α-DG, enhanced shedding suggesting that lack of a binding partner destabilizes the epithelial DG complex and makes it accessible to proteolytic processing. basement membrane dermo-epidermal junction dystroglycan laminin-G like phorbol 12-myristate 13-acetate tissue inhibitor of metalloproteinase Dystroglycan (DG) is a widely expressed cell surface component (Hemler, 1999Hemler M.E. Dystroglycan versatility.Cell. 1999; 97: 543-546Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar; Meier and Ruegg, 2000Meier T. Ruegg M.A. The role of dystroglycan and its ligands in physiology and disease.News Physiol Sci. 2000; 15: 255-259PubMed Google Scholar; Michele and Campbell, 2003Michele D.E. Campbell K.P. Dystrophin–glycoprotein complex: Post-translational processing and dystroglycan function.J Biol Chem. 2003; 278: 15457-15460Crossref PubMed Scopus (372) Google Scholar). In epithelial tissues including skin (Durbeej et al., 1998Durbeej M. Henry M.D. Ferletta M. Campbell K.P. Ekblom P. Distribution of dystroglycan in normal adult mouse tissues.J Histochem Cytochem. 1998; 46: 449-457Crossref PubMed Scopus (155) Google Scholar), it is particularly prominent on the basal side of cells facing basement membranes (BM), together with its ligands laminins 1 and 2, agrin and perlecan, suggesting that DG is a cell surface receptor involved in linking epithelial cells to BM in adult tissues. The ligands contain laminin-G-like (LG) domains, and it is believed that the widely distributed α-DG may be bound to distinct sets of ligands in a tissue-specific manner. The binding of α-DG to perlecan LG-domains is five times stronger than to the most active laminin fragment α-2LG1-3 (Andac et al., 1999Andac Z. Sasaki T. Mann K. Brancaccio A. Deutzmann R. Timpl R. Analysis of heparin, alpha-dystroglycan and sulfatide binding to the G domain of the laminin alpha1 chain by site-directed mutagenesis.J Mol Biol. 1999; 287: 253-264Crossref PubMed Scopus (95) Google Scholar; Talts et al., 1999Talts J.F. Andac Z. Gohring W. Brancaccio A. Timpl R. Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins.EMBO J. 1999; 18: 863-870Crossref PubMed Scopus (398) Google Scholar), raising the possibility that perlecan is a major extracellular partner of α-DG in non-muscle tissues, e.g. at the epidermal BM. DG is initially synthesized as a single polypeptide of 115 kDa, but undergoes post-translational proteolytic conversion to α-DG, a peripheral membrane-associated protein, and β-DG, a transmembrane protein in type I orientation (extracellular N- and intracellular C-terminus) (Holt et al., 2000Holt K.H. Crosbie R.H. Venzke D.P. Campbell K.P. Biosynthesis of dystroglycan: Processing of a precursor propeptide.FEBS Lett. 2000; 468: 79-83Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Whereas β-DG has a molecular weight of 43 kDa in most tissues, the size of α-DG varies (Ibraghimov-Beskrovnaya et al., 1992Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix.Nature. 1992; 355: 696-702Crossref PubMed Scopus (1195) Google Scholar; Holt et al., 2000Holt K.H. Crosbie R.H. Venzke D.P. Campbell K.P. Biosynthesis of dystroglycan: Processing of a precursor propeptide.FEBS Lett. 2000; 468: 79-83Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar; Meier and Ruegg, 2000Meier T. Ruegg M.A. The role of dystroglycan and its ligands in physiology and disease.News Physiol Sci. 2000; 15: 255-259PubMed Google Scholar). The cDNA sequence predicts a molecular weight of 72 kDa for α-DG, but it varies from 120 to 156 kDa in different tissues as a result of post-translational modifications (Durbeej and Campbell, 1999Durbeej M. Campbell K.P. Biochemical characterization of the epithelial dystroglycan complex.J Biol Chem. 1999; 274: 26609-26616Crossref PubMed Scopus (97) Google Scholar). α-DG contains a mucin-like region with extensive O-glycosylation, and several potential N-glycosylation and glycosaminoglycan attachment sites. The proteolytic cleavage between α- and β-DG subunits at the peptide bond Gly 653–Ser 654 occurs intracellularly (Holt et al., 2000Holt K.H. Crosbie R.H. Venzke D.P. Campbell K.P. Biosynthesis of dystroglycan: Processing of a precursor propeptide.FEBS Lett. 2000; 468: 79-83Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), and after the cleavage, α- and β-DG are believed to remain associated with each other through non-covalent interaction of the C-terminal region of the α-subunit with the N-terminal region of β-DG (Sciandra et al., 2001Sciandra F. Schneider M. Giardina B. Baumgartner S. Petrucci T.C. Brancaccio A. Identification of the β-dystroglycan binding epitope within the C-terminal region of alpha-dystroglycan.Eur J Biochem. 2001; 268: 4590-4597Crossref PubMed Scopus (38) Google Scholar). As an exception to this rule, some release of the α-subunit from the cell surface has been observed under pathological conditions (Shimizu et al., 1999Shimizu H. Hosokawa H. Ninomiya H. Miner J.H. Masaki T. Adhesion of cultured bovine aortic endothelial cells to laminin-1 mediated by dystroglycan.J Biol Chem. 1999; 274: 11995-12000Crossref PubMed Scopus (63) Google Scholar; Holt et al., 2000Holt K.H. Crosbie R.H. Venzke D.P. Campbell K.P. Biosynthesis of dystroglycan: Processing of a precursor propeptide.FEBS Lett. 2000; 468: 79-83Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar; Armstrong et al., 2003Armstrong S.C. Latham C.A. Ganote C.E. An ischemic beta-dystroglycan (betaDG) degradation product: Correlation with irreversible injury in adult rabbit cardiomyocytes.Mol Cell Biochem. 2003; 242: 71-79Crossref PubMed Scopus (13) Google Scholar). Intracellularly, β-DG interacts with different utrophin and dystrophin isoforms and other proteins such as caveolin-3 (Henry and Campbell, 1999Henry M.D. Campbell K.P. Dystroglycan inside and out.Curr Opin Cell Biol. 1999; 11: 602-607Crossref PubMed Scopus (253) Google Scholar; Cote et al., 2002Cote P.D. Moukhles H. Carbonetto S. Dystroglycan is not required for localization of dystrophin, syntrophin, and neuronal nitric-oxide synthase at the sarcolemma but regulates integrin alpha 7B expression and caveolin-3 distribution.J Biol Chem. 2002; 277: 4672-4679Crossref PubMed Scopus (49) Google Scholar). DG has gained significance in different areas of human medicine, e.g. in cancer, genetic diseases, and infections (Hemler, 1999Hemler M.E. Dystroglycan versatility.Cell. 1999; 97: 543-546Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar; Meier and Ruegg, 2000Meier T. Ruegg M.A. The role of dystroglycan and its ligands in physiology and disease.News Physiol Sci. 2000; 15: 255-259PubMed Google Scholar; Michele and Campbell, 2003Michele D.E. Campbell K.P. Dystrophin–glycoprotein complex: Post-translational processing and dystroglycan function.J Biol Chem. 2003; 278: 15457-15460Crossref PubMed Scopus (372) Google Scholar). In certain malignant tumors, e.g. prostate and mammary cancer, the expression of DG is reduced (Henry et al., 2001Henry M.D. Cohen M.B. Campbell K.P. Reduced expression of dystroglycan in breast and prostate cancer.Hum Pathol. 2001; 32: 791-795Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar; Muschler et al., 2002Muschler J. Levy D. Boudreau R. Henry M. Campbell K. Bissell M.J. A role for dystroglycan in epithelial polarization: Loss of function in breast tumor cells.Cancer Res. 2002; 6: 7102-7109Google Scholar). The reduction is most pronounced in highly invasive tumors, suggesting that diminished expression of DG in cancers may lead to abnormal epithelial cell polarization (Muschler et al., 2002Muschler J. Levy D. Boudreau R. Henry M. Campbell K. Bissell M.J. A role for dystroglycan in epithelial polarization: Loss of function in breast tumor cells.Cancer Res. 2002; 6: 7102-7109Google Scholar) and cell–matrix interactions and thus contribute to progression of the disease. Similarly, the level of DG is often reduced in hereditary muscular dystrophies. No human neuromuscular disease with a primary DG defect is known; rather, the involvement of DG appears to be secondary to mutations in the dystrophin or sarcoglycan genes, and its induced absence contributes to the aggravation of muscle abnormalities (Yamada et al., 2001Yamada H. Saito F. Fukuta-Ohi H. et al.Processing of beta-dystroglycan by matrix metalloproteinase disrupts the link between the extracellular matrix and cell membrane via the dystroglycan complex.Hum Mol Genet. 2001; 10: 1563-1569Crossref PubMed Google Scholar). In some forms of muscular dystrophies, the secondary abnormalities in α-DG result from hypoglycosylation (Brockington et al., 2001Brockington M. Blake D.J. Prandini P. et al.Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan.Am J Hum Genet. 2001; 69: 1198-1209Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar; Kano et al., 2002Kano H. Kobayashi K. Herrmann R. et al.Deficiency of alpha-dystroglycan in muscle–eye–brain disease.Biochem Biophys Res Commun. 2002; 291: 1283-1286Crossref PubMed Scopus (113) Google Scholar), which leads to functional loss of ligand binding (Michele and Campbell, 2003Michele D.E. Campbell K.P. Dystrophin–glycoprotein complex: Post-translational processing and dystroglycan function.J Biol Chem. 2003; 278: 15457-15460Crossref PubMed Scopus (372) Google Scholar). Intriguingly, α-DG was also identified as a cellular receptor for arenaviruses or Mycobacterium leprae (Hemler, 1999Hemler M.E. Dystroglycan versatility.Cell. 1999; 97: 543-546Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). An interesting point in this context is the mode of transmission and cellular entry point of the viruses and bacteria into the human body. Both arenaviruses and M. leprae are transmitted by contaminated food, body fluids, or direct body contact. Therefore, the microorganisms must penetrate the epithelia of either skin or mucosa, and are likely to use the epithelial α-DG isoform as their primary receptor for entry into the body. Here we investigated the biochemical properties of DG in human skin and epithelial cells in vitro and showed that the relation of α- and β-DG in the epithelial DG complex differs from that in the muscle. Since many transmembrane proteins are proteolytically released from cell surfaces and become extracellular matrix components, we analyzed β-DG in this respect and showed that a distinct portion of the β-DG ectodomain is constitutively shed from the cell surface. For characterization of DG protein, antibodies recognizing specific domains were used Fig 1. As α-DG antibody, an antibody raised against a 20 amino acid peptide spanning the non-glycosylated C-terminus of the α-subunit, was used (Herrmann et al., 2000Herrmann R. Straub V. Blank M. et al.Dissociation of the dystroglycan complex in caveolin-3-deficient limb girdle muscular dystrophy.Hum Mol Genet. 2000; 9: 2335-2340Crossref PubMed Scopus (121) Google Scholar). As β-DG antibody, 43DAG/8D5 which recognizes the intracellular C-terminus of β-DG, and the antibody β-DG-N, raised against a recombinant fragment spanning the extracellular domain of the β-subunit, were employed. The latter antibody reacted with β-DG in immunoblots, but showed no signal in immunofluorescence staining, suggesting that only denatured epitopes were recognized. In contrast, both 43DAG/8D5 and the α-DG antibody functioned also for immunofluorescence staining. Immunofluorescence staining of normal human skin with 43DAG/8D5 exhibited a linear signal along the epidermal BM Fig 2a. The pattern was comparable with that of collagen XVII (Tasanen et al., 2000Tasanen K. Eble J.A. Aumailley M. et al.Collagen XVII is destabilized by a glycine substitution mutation in the cell adhesion domain Col15.J Biol Chem. 2000; 275: 3093-3099Crossref PubMed Scopus (59) Google Scholar), another transmembrane protein in basal keratinocytes Fig 2b. Double staining with 43DAG/8D5 and collagen XVII antibody NC16A revealed a colocalization of both signals (not shown). In situ hybridization produced a positive staining in the epidermis, with an apparently stronger signal in the basal cell layer Fig 2c. In cultured keratinocytes and fibroblasts, both 43DAG/8D5 immunostaining Fig 2e, f and in situ hybridization Fig 2g, h yielded a positive signal, indicating that both cell types can produce DG in vitro. Ultrastructural analysis of human skin, using immunoelectron microscopy with the antibody 43DAG/8D5, showed an immunopositive signal at the dermo-epidermal junction (DEJ), in constant association with hemidesmosomes. The binding of the antibody was low under conditions required for immunoelectron microscopy and, therefore, the staining relatively weak. The gold particles, however, were observed only around the hemidesmosomes and not in association with any other intra- or extracellular structures. Further, the positive reaction was reproducible in a number of sections and over long stretches of the DEJ zone Fig 2i. Overall, several methods produced a distinct, positive signal along the DEJ with the antibody 43DAG/8D5, rendering the probability of a non-specific background reaction unlikely. Rather, the discrete staining indicates that DG is present, but in lower abundance than e.g. collagen XVII. Cell extracts and media of human keratinocytes and fibroblasts, and murine cardiomyocytes were submitted to immunoblotting in order to assess the localization of the DG subunits. As expected, the transmembranous β-DG was found only in the cell extracts. In contrast, α-DG was present in both cell extracts (not shown) and media Fig 3A. The level of α-DG was much lower in keratinocytes than in the two other cell types. Immunoblotting of cell extracts with 43DAG/8D5 revealed two β-DG bands, the 43 kDa full-length β-DG and a smaller fragment of about 30 kDa Fig 3B. Detection of the 30 kDa fragment with 43DAG/8D5 indicated that it contained the intracellular C-terminus. In contrast, antibody β-DG-N, which recognizes the extracellular N-terminus of β-DG, did not react with the fragment, suggesting that at least a major part of the ectodomain of β-DG was missing from the 30 kDa polypeptide Fig 3C. This observation was verified by transfecting HaCaT (not shown) and COS-7 cells with full-length DG cDNA and immunoblotting of the cell extracts Fig 4. The transfected cells contained the αβ-precursor; the intact β-subunit and the 30 kDa fragment, indicating that recombinant DG was processed in a similar manner as its normal counterpart. The above observation suggested that the ectodomain of β-DG is constitutively shed from the cell surface. A mainly cytoplasmic fragment of β-DG has been described in the literature (Losasso et al., 2000Losasso C. Di Tommaso F. Sgambato A. et al.Anomalous dystroglycan in carcinoma cell lines.FEBS Lett. 2000; 484: 194-198Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar; Yamada et al., 2001Yamada H. Saito F. Fukuta-Ohi H. et al.Processing of beta-dystroglycan by matrix metalloproteinase disrupts the link between the extracellular matrix and cell membrane via the dystroglycan complex.Hum Mol Genet. 2001; 10: 1563-1569Crossref PubMed Google Scholar; Armstrong et al., 2003Armstrong S.C. Latham C.A. Ganote C.E. An ischemic beta-dystroglycan (betaDG) degradation product: Correlation with irreversible injury in adult rabbit cardiomyocytes.Mol Cell Biochem. 2003; 242: 71-79Crossref PubMed Scopus (13) Google Scholar), but its composition and biosynthesis remained unknown. To facilitate the analysis of β-DG and its fragments, an expression vector was constructed in which a His-tag and an Xpress-epitope were fused to the N-terminus of β-DG Fig 5. Forty-eight hours after transfection of COS-7 cells with this construct, medium and cells were harvested and recombinantly expressed proteins and released fragments purified via the His-tag. Immunoblotting with different domain-specific antibodies identified the soluble ectodomain of β-DG in the medium, with an apparent molecular weight of 25 kDa Fig 5. Control blots of the fragment with an antibody to the intracellular domain of β-DG remained negative. Full-length β-DG was present in cell extracts. Since partially purified His-tagged β-DG was analyzed in this experiment, β-DG30 was not detected in the experiment. For characterization of the enzymes involved in shedding of β-DG, keratinocytes and transfected HaCaT cells were cultured in the presence of protease inhibitors Table I. Cleavage of β-DG was completely inhibited by the MMP- and sheddase-targeting hydroxamate derivates BB3103, BB3241, and IC-3 (Franzke et al., 2002Franzke C.W. Tasanen K. Schacke H. et al.Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs.EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (188) Google Scholar) and to about 70%–80% with the metal chelator ortho-phenantroline. Partial inhibition was detected with 500 nM TIMP-3 (∼30%), but none with 500 nM TIMP-1 and TIMP-2. In contrast, no effect was observed with the aspartate protease inhibitor pepstatin A, the cystein peptidase inhibitor E-64, the serine protease inhibitors AEBSF or aprotinin or a chloromethylketone furin-inhibitor. This inhibitor profile strongly indicated that metalloproteinases, especially members of the a disintegrin and metallo-proteinase (ADAM) family, were involved.Table IEffect of proteinase inhibitors on shedding of β-DGInhibitor(μM)Keratinocytes Inhibition (%) Transfected HaCaT cellsNone001,10 ortho-phenantroline1007283IC-3 (TAPI)259591BB3241109099BB3103109899TIMP-10.5–1TIMP-20.5–2TIMP-30.53031Pepstatin A100611E-6410041Aprotinin5072AEBSF100021Furin-inhibitoraDecanoyl-RVKR-chloromethyl ketone. Shedding in the prescence or absence of inhibitors was analyzed in normal human keratinocytes and HaCaT cells transfected with cDNA of DG by immunoblotting followed by semiquantitative densitometry of the signals. The inhibiton is expressed in % of controls. DG, dystroglycan.10032a Decanoyl-RVKR-chloromethyl ketone.Shedding in the prescence or absence of inhibitors was analyzed in normal human keratinocytes and HaCaT cells transfected with cDNA of DG by immunoblotting followed by semiquantitative densitometry of the signals. The inhibiton is expressed in % of controls. DG, dystroglycan. Open table in a new tab For further identification of these sheddases, the effect of two well-known ADAM stimulators, IL-1β and phorbol 12-myristate 13-acetate (PMA), on β-DG release was analyzed. In untreated cells, constitutive low level shedding was observed. IL-1β increased the shedding up to 10-fold within 2 h Fig 6A. But the effect was transient since the amount of β-DG30 declined with longer incubation time. The induction by PMA was seen already after 15 min and increased to 2.5-fold within 1 h Fig 6B, suggesting involvement of a protein kinase C (PKC)-mediated process. In agreement with this hypothesis, bisindolylmaleimide I (BIM), a potent and selective inhibitor of PKC (Toullec et al., 1991Toullec D. Pianetti P. Coste H. et al.The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C.J Biol Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar) completely reversed the stimulatory effect of PMA. No modulation of β-DG shedding was observed with TNF-α or TGF-β2 (not shown). As a control, the effect of the stimulators on the expression of β-DG at mRNA and protein level was analyzed, but no difference was observed (not shown). Taken together, the inhibitor and stimulator profiles implicate metalloproteinases as candidate proteases. Since only TIMP-3, but not the typical MMP-inhibitors TIMP-1 and TIMP-2, influenced β-DG shedding, members of the ADAM family are very likely candidate sheddases. The fact that perlecan is a strong binding partner for α-DG (Talts et al., 1999Talts J.F. Andac Z. Gohring W. Brancaccio A. Timpl R. Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins.EMBO J. 1999; 18: 863-870Crossref PubMed Scopus (398) Google Scholar) prompted us to analyze the effects of ligand interactions on shedding. Skin fibroblasts isolated from perlecan-deficient E 13.5 mouse embryos were used to determine the impact of a missing α-DG binding partner on the stability of the protein complex. These cells contained increased amounts of the 30 kDa β-DG, accompanied by a higher level of α-DG in the cell culture media Fig 7A. In parallel, skin fibroblasts from laminin α2-deficient mice (kindly provided by E. Engvall and M. Ruegg) were prepared and analyzed in the same way. In these cells, however, no difference in β-DG shedding was observed compared with normal cells (not shown). This suggested that lack of perlecan has a specific, destabilizing effect on the DG complex that results in increased shedding of β-DG and subsequent release of α-DG into the media. Further, the deposition of α-DG into the extracellular matrix of the different cells was analyzed. In normal cells, pericellular fibers contained both perlecan and α-DG (Fig 7B, panels a and b). In contrast, in perlecan-deficient fibroblasts α-DG was nearly absent from the pericellular fibers (Fig 7B, panel d), suggesting that the lack of a binding partner leads to a greatly reduced deposition of α-DG into the extracellular matrix. DG is best known for its function in muscle, but it also occurs in non-muscle tissues within diverse supramolecular aggregates (Hemler, 1999Hemler M.E. Dystroglycan versatility.Cell. 1999; 97: 543-546Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar; Meier and Ruegg, 2000Meier T. Ruegg M.A. The role of dystroglycan and its ligands in physiology and disease.News Physiol Sci. 2000; 15: 255-259PubMed Google Scholar; Michele and Campbell, 2003Michele D.E. Campbell K.P. Dystrophin–glycoprotein complex: Post-translational processing and dystroglycan function.J Biol Chem. 2003; 278: 15457-15460Crossref PubMed Scopus (372) Google Scholar). For example, DG is enriched towards the basal side of epithelial cells that are in close contact with BM in several developing and adult tissues, such as kidney, liver, small intestine, brain, retina, trachea, salivary gland, and skin (Ibraghimov-Beskrovnaya et al., 1993Ibraghimov-Beskrovnaya O. Milatovich A. Ozcelik T. Yang B. Koepnick K. Francke U. Campbell K.P. Human dystroglycan: Skeletal muscle cDNA, genomic structure, origin of tissue specific isoforms and chromosomal localization.Hum Mol Genet. 1993; 2: 1651-1657Crossref PubMed Scopus (208) Google Scholar; Gorecki et al., 1994Gorecki D.C. Derry J.M. Barnard E.A. Dystroglycan: Brain localisation and chromosome mapping in the mouse.Hum Mol Genet. 1994; 3: 1589-1597Crossref PubMed Scopus (66) Google Scholar; Durbeej et al., 1998Durbeej M. Henry M.D. Ferletta M. Campbell K.P. Ekblom P. Distribution of dystroglycan in normal adult mouse tissues.J Histochem Cytochem. 1998; 46: 449-457Crossref PubMed Scopus (155) Google Scholar). Despite expanding knowledge about DG in non-muscle tissues, a number of questions remain to be answered about the composition, components, and functions of the DG complex in different epithelia. Here we show, using antibodies to the cytoplasmic domain of β-DG that in human skin DG is localized at the dermal–epidermal BM zone. Double-immunofluorescence stainings with antibodies specific to β-DG and collagen XVII showed a nearly complete colocalization of both proteins. Immunoelectron microscopy suggested that DG is associated with the hemidesmosomes, similarly to collagen XVII. In situ hybridization demonstrated DG mRNA expression in the epidermis, with the strongest signal in the basal cell layer. Interestingly, in vitro DG was synthesized in both keratinocytes and fibroblasts indicating that its expression may be differentially regulated in situ and in vitro. A similar observation has been made with endothelial cells. Cultured endothelial cells expressed DG in vitro, whereas minimal or no expression was found in normal blood vessels in situ (Hosokawa et al., 2002Hosokawa H. Ninomiya H. Kitamura Y. Fujiwara K. Masaki T. Vascular endothelial cells that express dystroglycan are involved in angiogenesis.J Cell Sci. 2002; 115: 1487-1496PubMed Google Scholar), rendering the authors to postulate that DG expression depended on the proliferation state of the cells. Proteolytic processing and maturation of structural proteins are emerging as an important aspect of BM biology. A wide spectrum of extracellular matrix molecules is proteolytically cleaved to yield mature biosynthetic products, and to release functionally important domains or biologically active fragments. For example, processing of procollagen VII to mature collagen (Rattenholl et al., 2002Rattenholl A. Pappano W.N. Koch M. et al.Proteinases of the bone morphogenetic protein-1 family convert procollagen VII to mature anchoring fibril collagen.J Biol Chem. 2002; 277: 26372-26378Crossref PubMed Scopus (107) Google Scholar), shedding of transmembrane collagen XVII from keratinocyte surfaces (Franzke et al., 2002Franzke C.W. Tasanen K. Schacke H. et al.Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs.EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (188) Google Scholar) or cleavage of laminin 5 (Veitch et al., 2003Veitch D.P. Nokelainen P. McGowan K.A. et al.Mammalian Tolloid metalloproteinase, and not matrix metalloprotease 2 nor membrane type 1 metalloprotease, processes laminin-5 in keratinocytes and skin.J Biol Chem. 2003; 278: 15661-15668Crossref PubMed Scopus (123) Google Scholar) influence cell adhesion, migration, and assembly of the DEJ (Gagnoux-Palacios et al., 2001Gagnoux-Palacios L. Allegra M. Spirito F. Pommeret O. Romero C. Ortonne J.P. Meneguzzi G. The short arm of the laminin gamma2 chain plays a pivotal role in the incorporation of laminin 5 into the extracellular matrix and in cell adhesion.J Cell Biol. 2001; 153: 835-850Crossref PubMed Scopus (100) Google Scholar; Tunggal et al., 2002Tunggal L. Ravaux J. Pesch M. et al.Defective laminin 5 processing in cylindroma cells.Am J Pathol. 2002; 160: 459-468Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In this study, we provide evidence that also β-DG is proteolytically released from the surface of normal cutaneous cells. Previously, a β-DG fragment of about 30 kDa instead of the normal 43 kDa molecule, was observed under pathological conditions, e.g. in muscular dystrophies, in certain cancer cells or in ischemic cardiomyocytes (Losasso et al., 2000Losasso C. Di Tommaso F. Sgambato A. et al.Anomalous dystroglycan in carcinoma cell lines.FEBS Lett. 2000; 484: 194-198Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar; Yamada et al., 2001Yamada H. Saito F. Fukuta-Ohi H. et al.Processing of beta-dystroglycan by matrix metalloproteinase disrupts the link between the extracellular matrix and cell membrane via the dystroglycan complex.Hum Mol Genet. 2001; 10: 1563-1" @default.
• W2025136908 created "2016-06-24" @default.
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• W2025136908 date "2004-06-01" @default.
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• W2025136908 title "Dystroglycan in Skin and Cutaneous Cells: β-Subunit Is Shed from the Cell Surface" @default.
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