βig-h3 Induces Keratinocyte Differentiation via Modulation of Involucrin and Transglutaminase Expression through the Integrin α3β1 and the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway
2005; Elsevier BV; Volume: 280; Issue: 22 Linguagem: Inglês
10.1074/jbc.m412293200
ISSN1083-351X
AutoresJu-Eun Oh, Joong‐Ki Kook, Byung‐Moo Min,
Tópico(s)Cell Adhesion Molecules Research
Resumoβig-h3 is an extracellular matrix protein whose expression is highly induced by transforming growth factor (TGF)-β1. Whereas βig-h3 is known to mediate keratinocyte adhesion and migration, its effects on keratinocyte differentiation remain unclear. In the present study, it was demonstrated that expression of both βig-h3 and TGF-β1 was enhanced during keratinocyte differentiation and that expression of the former was strongly induced by that of the latter. This study also asked whether changes in β-h3 expression would affect keratinocyte differentiation. Indeed, down-regulation of βig-h3 by transfection with antisense βig-h3 cDNA constructs effectively inhibited keratinocyte differentiation by decreasing the promoter activities and thus expression of involucrin and transglutaminase. The result was a ∼2-fold increase in mitotic capacity of the cells. Conversely, overexpression of βig-h3, either by transfection with βig-h3 expression plasmids or by exposure to recombinant βig-h3, enhanced keratinocyte differentiation by inhibiting cell proliferation and concomitantly increasing involucrin and transglutaminase expression. Recombinant βig-h3 also promoted keratinocyte adhesion through interaction with integrin α3β1. Changes in βig-h3 expression did not affect intracellular calcium levels. Subsequent analysis revealed not only induction of Akt phosphorylation by recombinant βig-h3 but also blockage of Akt phosphorylation by LY294002, an inhibitor of phosphatidylinositol 3-kinase. Taken together, these findings indicate that enhanced βig-h3, induced by enhanced TGF-β during keratinocyte differentiation, provoked cell differentiation by enhancing involucrin and transglutaminase expression through the integrin α3β1 and phosphatidylinositol 3-kinase/Akt signaling pathway. Lastly, it was observed that βig-h3-mediated keratinocyte differentiation was caused by promotion of cell adhesion and not by calcium regulation. βig-h3 is an extracellular matrix protein whose expression is highly induced by transforming growth factor (TGF)-β1. Whereas βig-h3 is known to mediate keratinocyte adhesion and migration, its effects on keratinocyte differentiation remain unclear. In the present study, it was demonstrated that expression of both βig-h3 and TGF-β1 was enhanced during keratinocyte differentiation and that expression of the former was strongly induced by that of the latter. This study also asked whether changes in β-h3 expression would affect keratinocyte differentiation. Indeed, down-regulation of βig-h3 by transfection with antisense βig-h3 cDNA constructs effectively inhibited keratinocyte differentiation by decreasing the promoter activities and thus expression of involucrin and transglutaminase. The result was a ∼2-fold increase in mitotic capacity of the cells. Conversely, overexpression of βig-h3, either by transfection with βig-h3 expression plasmids or by exposure to recombinant βig-h3, enhanced keratinocyte differentiation by inhibiting cell proliferation and concomitantly increasing involucrin and transglutaminase expression. Recombinant βig-h3 also promoted keratinocyte adhesion through interaction with integrin α3β1. Changes in βig-h3 expression did not affect intracellular calcium levels. Subsequent analysis revealed not only induction of Akt phosphorylation by recombinant βig-h3 but also blockage of Akt phosphorylation by LY294002, an inhibitor of phosphatidylinositol 3-kinase. Taken together, these findings indicate that enhanced βig-h3, induced by enhanced TGF-β during keratinocyte differentiation, provoked cell differentiation by enhancing involucrin and transglutaminase expression through the integrin α3β1 and phosphatidylinositol 3-kinase/Akt signaling pathway. Lastly, it was observed that βig-h3-mediated keratinocyte differentiation was caused by promotion of cell adhesion and not by calcium regulation. Transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; βig-h3, TGF-β-inducible gene-h3; PI3K, phosphatidylinositol 3-kinase; NHOKs, normal human oral keratinocytes; NHEKs, normal human epidermal keratinocytes; PDs, population doublings; CAT, chloramphenicol acetyltransferase; BSA, bovine serum albumin; PBS, phosphate-buffered saline; mAb, monoclonal antibody; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase.-inducible gene-h3 (βig-h3) was first cloned from A549 lung adenocarcinoma cells that had been stimulated with TGF-β1 (1Skonier J. Neubauer M. Madisen L. Bennett K. Plowman G.D. Purchio A.F. DNA Cell Biol. 1992; 11: 511-522Crossref PubMed Scopus (505) Google Scholar, 2Skonier J. Bennett K. Rothwell V. Kosowski S. Plowman G.D. Wallace P. Edelhoff S. Disteche C. Neubauer M. Marquardt H. Rodgers J. Puchio A.F. DNA Cell Biol. 1994; 13: 571-584Crossref PubMed Scopus (260) Google Scholar). βig-h3 has since been shown to be an extracellular matrix protein that can be highly induced by TGF-β in several cell types, including mammary epithelial cells, keratinocytes, and lung fibroblasts (1Skonier J. Neubauer M. Madisen L. Bennett K. Plowman G.D. Purchio A.F. DNA Cell Biol. 1992; 11: 511-522Crossref PubMed Scopus (505) Google Scholar, 2Skonier J. Bennett K. Rothwell V. Kosowski S. Plowman G.D. Wallace P. Edelhoff S. Disteche C. Neubauer M. Marquardt H. Rodgers J. Puchio A.F. DNA Cell Biol. 1994; 13: 571-584Crossref PubMed Scopus (260) Google Scholar). With structural homology to the insect protein fasciclin, βig-h3 is a 76- to 78-kDa protein containing four repeat regions and 11 cysteine residues, mostly clustered in a distinct amino terminus. The βig-h3 molecule appears to undergo partial processing at the carboxyl terminus to yield a 68–70-kDa isoform (2Skonier J. Bennett K. Rothwell V. Kosowski S. Plowman G.D. Wallace P. Edelhoff S. Disteche C. Neubauer M. Marquardt H. Rodgers J. Puchio A.F. DNA Cell Biol. 1994; 13: 571-584Crossref PubMed Scopus (260) Google Scholar). Although the βig-h3 transcript has been detected in a variety of human and mouse tissues, including breast, heart, kidney, liver, stomach, and skeletal muscle (2Skonier J. Bennett K. Rothwell V. Kosowski S. Plowman G.D. Wallace P. Edelhoff S. Disteche C. Neubauer M. Marquardt H. Rodgers J. Puchio A.F. DNA Cell Biol. 1994; 13: 571-584Crossref PubMed Scopus (260) Google Scholar), little information is available regarding the distribution of the protein in such human tissues as the arteries, eye, kidney, lung, and skin (3Gilbert R.E. Wilkinson-Berka J.L. Johnson D.W. Cox A. Soulis T. Wu L.L. Kelly D.J. Jerums G. Pollock C.A. Cooper M.E. Kidney Int. 1998; 54: 1052-1062Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 4LeBaron R.G. Bezverkov K.I. Zimber M.P. Pavelec R. Skonier J. Purchio A.F. J. Investig. Dermatol. 1995; 104: 844-849Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 5O'Brien E.R. Bennett K.L. Garvin M.R. Zderic T.W. Hinohara T. Simpson J.B. Kimura T. Nobuyoshi M. Mizgala H. Purchoi A. Schwartz S.M. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 576-584Crossref PubMed Scopus (94) Google Scholar, 6Rawe I.M. Zhan Q. Burrows R. Bennett K. Cintron C. Investig. Ophthalmol. Vis. Sci. 1997; 38: 893-900PubMed Google Scholar). It is known that βig-h3 acts as a cell adhesion molecule in several cell types (7Kim J.-E. Jeong H.-W. Nam J.-O. Lee B.-H. Choi J.-Y. Park R.-W. Park J.Y. Kim I.-S. J. Biol. Chem. 2002; 277: 46159-46165Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) and as a bifunctional linker protein to connect various matrix molecules to each other and to cells (8Gibson M.A. Kumaratilake J.S. Cleary E.G. J. Histochem. Cytochem. 1997; 45: 1683-1696Crossref PubMed Scopus (70) Google Scholar, 9Billings P.C. Herrick D.J. Kucich U. Engelsberg B.N. Abrams W.R. Macarak E.J. Rosenbloom J. Howard P.S. J. Cell. Biochem. 2000; 79: 261-273Crossref PubMed Scopus (43) Google Scholar). βig-h3 contains multiple cell adhesion motifs within its fasciclin-like domains capable of mediating interactions with a variety of cell types via integrins α3β1 (10Bae J.-S. Lee S.-H. Kim J.-E. Choi J.-Y. Park R.-W. Park J.Y. Park H.-S. Sohn Y.-S. Lee D.-S. Lee E.B. Kim I.-S. Biochem. Biophys. Res. Commun. 2002; 294: 940-948Crossref PubMed Scopus (132) Google Scholar, 11Kim J.-E. Kim S.-J. Lee B.-H. Park R.-W. Kim K.-S. Kim I.-S. J. Biol. Chem. 2000; 275: 30907-30915Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar), α1β1 (12Ohno S. Noshiro M. Makihira S. Kawamoto T. Shen M. Yan W. Kawashima-Ohya Y. Fujimoto K. Tanne K. Kato Y. Biochim. Biophys. Acta. 1999; 1451: 196-205Crossref PubMed Scopus (101) Google Scholar), and αvβ5 (7Kim J.-E. Jeong H.-W. Nam J.-O. Lee B.-H. Choi J.-Y. Park R.-W. Park J.Y. Kim I.-S. J. Biol. Chem. 2002; 277: 46159-46165Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). It is known to mediate the migration and proliferation of normal human epidermal keratinocytes (NHEKs) through two integrin α3β1-interacting motifs in the second and fourth fas-1 domains (10Bae J.-S. Lee S.-H. Kim J.-E. Choi J.-Y. Park R.-W. Park J.Y. Park H.-S. Sohn Y.-S. Lee D.-S. Lee E.B. Kim I.-S. Biochem. Biophys. Res. Commun. 2002; 294: 940-948Crossref PubMed Scopus (132) Google Scholar). It has also been shown to bind in vitro to a number of other matrix components, including fibronectin, laminin, and several collagen types (13Billings P.C. Whitbeck J.C. Adams C.S. Abrams W.R. Cohen A.J. Engelsberg B.N. Howard P.S. Rosenbloom J. J. Biol. Chem. 2002; 277: 28003-28009Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 14Kim J.-E. Park R.-W. Choi J.-Y. Bae Y.-C. Kim K.-S. Joo C.-K. Kim I.-S. Investig. Ophthalmol. Vis. Sci. 2002; 43: 656-661PubMed Google Scholar). The precise roles of βig-h3 in cell development are currently unknown, but it has been implicated in cell growth (2Skonier J. Bennett K. Rothwell V. Kosowski S. Plowman G.D. Wallace P. Edelhoff S. Disteche C. Neubauer M. Marquardt H. Rodgers J. Puchio A.F. DNA Cell Biol. 1994; 13: 571-584Crossref PubMed Scopus (260) Google Scholar, 10Bae J.-S. Lee S.-H. Kim J.-E. Choi J.-Y. Park R.-W. Park J.Y. Park H.-S. Sohn Y.-S. Lee D.-S. Lee E.B. Kim I.-S. Biochem. Biophys. Res. Commun. 2002; 294: 940-948Crossref PubMed Scopus (132) Google Scholar), osteoblast differentiation (15Dieudonne S.C. Kerr J.M. Xu T. Sommer B. DeRubeis A.R. Kuznetsov S.A. Kim I.-S. Robey P.G. Young M.F. J. Cell. Biochem. 1999; 76: 231-243Crossref PubMed Scopus (61) Google Scholar, 16Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (110) Google Scholar), and wound healing (6Rawe I.M. Zhan Q. Burrows R. Bennett K. Cintron C. Investig. Ophthalmol. Vis. Sci. 1997; 38: 893-900PubMed Google Scholar). During wound healing, for example, βig-h3 is produced by a range of cell types including activated macrophages, neutrophils, fibroblasts, and keratinocytes (18Kane C.J. Hebda P.A. Mansbridge J.N. Hanawalt P.C. J. Cell. Physiol. 1991; 148: 157-173Crossref PubMed Scopus (194) Google Scholar). βig-h3 has been reported in both the dermis and epidermis, with increased staining intensities in the papillary dermis and granular layer of the epidermis (4LeBaron R.G. Bezverkov K.I. Zimber M.P. Pavelec R. Skonier J. Purchio A.F. J. Investig. Dermatol. 1995; 104: 844-849Abstract Full Text PDF PubMed Scopus (198) Google Scholar). Similarly, TGF-β has been localized in the normal dermis and epidermis. The investigators of the present study previously demonstrated that expression of TGF-β, a potent inducer of differentiation for normal epithelial cells, is increased in and near terminal differentiation of mucosal keratinocytes (19Min B.-M. Woo K.M. Lee G. Park N.-H. Exp. Cell Res. 1999; 249: 377-385Crossref PubMed Scopus (45) Google Scholar). Moreover, the epidermis was found to display a highly coordinated program of sequential changes in gene expression coincident with the phenotypic evolution from a proliferating basal cell to the mature, nonviable cell (20Fuchs E. Curr. Opin. Cell Biol. 1990; 2: 1028-1035Crossref PubMed Scopus (107) Google Scholar). Epidermal and mucosal keratinocytes undergo terminal differentiation when they migrate from the basal layer to the surface (21Larjava H. Am. J. Med. Sci. 1991; 301: 63-68Crossref PubMed Scopus (20) Google Scholar), and both cell types display a limited number of divisions in vitro. Serial subculture-induced keratinocyte differentiation mimics the physiological maturation process observed in the intact epidermis in vivo (19Min B.-M. Woo K.M. Lee G. Park N.-H. Exp. Cell Res. 1999; 249: 377-385Crossref PubMed Scopus (45) Google Scholar) and is more similar in some ways with this in vivo process than with calcium-induced differentiation (22Lee G. Park B.S. Han S.E. Oh J.-E. You Y.-O. Baek J.-H. Kim G.-S. Min B.-M. Arch. Oral Biol. 2000; 45: 809-818Crossref PubMed Scopus (16) Google Scholar). The in vitro differentiation system is thus useful for investigating the mechanisms of keratinocyte differentiation. Many proteins are known or suspected to be associated with the overall process of keratinocyte differentiation. In particular, the roles of involucrin and transglutaminase have been identified as the substrate and enzyme, respectively, required for cornified envelope formation. Much evidence suggests that TGF-β plays important roles during wound healing and keratinocyte differentiation. Indeed, previous work by the present authors has shown that TGF-β and phospholipase C-γ1 promote keratinocyte differentiation (19Min B.-M. Woo K.M. Lee G. Park N.-H. Exp. Cell Res. 1999; 249: 377-385Crossref PubMed Scopus (45) Google Scholar, 23Oh J.-E. Kook J.-K. Park K.-H. Lee G. Seo B.-M. Min B.-M. Int. J. Mol. Med. 2003; 11: 491-498PubMed Google Scholar). The importance of TGF-β suggests a possible role for βig-h3 as well in the mediation of keratinocyte differentiation and in cell adhesion and spreading. This study sought to examine whether changes in βig-h3 expression would affect keratinocyte differentiation, and to determine the molecular mechanism by which this occurred, in an effort to elucidate the role of βig-h3 in the regulation of keratinocyte differentiation. The findings herein demonstrate that enhanced TGF-β during keratinocyte differentiation induced βig-h3 expression and thus provoked cell differentiation by enhancing involucrin and transglutaminase expression through the integrin α3β1 and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Finally, the present study demonstrates that βig-h3-mediated keratinocyte differentiation was caused by promotion of cell adhesion and not by calcium regulation. Cell Culture—Normal human oral keratinocytes (NHOKs) were prepared and maintained as previously reported (19Min B.-M. Woo K.M. Lee G. Park N.-H. Exp. Cell Res. 1999; 249: 377-385Crossref PubMed Scopus (45) Google Scholar). Briefly, NHOKs were isolated from human gingival tissue specimens obtained from healthy volunteers (age range 20 to 30 years) who were undergoing oral surgery. Oral keratinocytes were isolated from separated epithelial tissue by trypsinization, and primary cultures were established in keratinocyte growth medium containing 0.15 mm calcium and a supplementary growth factor bullet kit (keratinocyte growth medium; Clonetics, San Diego, CA). Primary NHEKs were prepared in a manner similar to the NHOKs from human foreskins obtained from patients (1 to 3 years of age) undergoing surgery. Approximately 70% confluent primary NHOKs and NHEKs were plated at 1 × 105 cells per 60-mm culture dish and cultured until the cells reached 70% confluence. Cells were then subcultured at every 70% confluence until they reached the post-mitotic stage of proliferation, at which time the culture was maintained for 12 days without further passage. Determination of NHOK and NHEK Culture Population Doublings— Individual keratinocytes isolated from gingival epithelial specimens and foreskins were plated in 60-mm culture dishes. Three days after seeding, the number of cells that had been originally plated was determined by colony counting. Cells were cultured until they reached 70% confluence and then the cell numbers in the various dishes were counted in a hemocytometer. This count allowed determination of the number of population doublings (PDs) of the primary cultures. Harvested primary cultures were subcultured until they reached the post-mitotic stage of proliferation. The PD was calculated at the end of each passage according to the formula 2N = (Cf/Ci), where N denotes the number of PDs; Cf, the total number of cells harvested at the end of a passage; and Ci, the total number of attached cells at seeding. Construction of Vectors Expressing Antisense RNA and Wild-type βig-h3 and Cloning of the Promoter Regions of Involucrin and Transglutaminase 1—Antisense βig-h3 constructs were made by inserting 375-bp human βig-h3 cDNA fragments (nucleotides 879 to 1253) containing the ATG start codon, in an antisense orientation, into the EcoRI sites of a pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA), which expresses a neomycin (G418) resistance gene. βig-h3 expression plasmids were made by inserting 2204-bp human βig-h3 cDNA fragments (nucleotides -5 to 2199) containing the ATG start codon, in a sense orientation, into the NotI sites of the same pcDNA3.1(+) vector. The human βig-h3 cDNA encompassing nucleotides -5 to 2199 was amplified by reverse transcriptase-PCR using sense (5′-GCACCATGGCGCTCTTCGTGC-3′) and antisense (5′-TGCACAAGGCTCACATCTCATTA-3′) primers. Similarly, the human involucrin promoter region (nucleotides -2063 to 1325) was amplified with sense (5′-GAAGCTTAGACCGGTGTGCTTCCTTGACTTGA-3′) and antisense primers (5′-GGTCGACCTGATGGGTATTGACTGGAGGAGGAAC-3′), and the human transglutaminase-1 promoter region (nucleotides -2269 to 70) with sense (5′-TGCATGCTCCTTCTTTCTGCGCCATCCTATTGTTT-3′) and antisense primers (5′-TGTCGACGCAGTACTGAGTCCTGGGGCTGAGATGG-3′). Amplification was performed with an ABI Prism™ 310 Genetic Analyzer (PerkinElmer Life Sciences) and the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit (PerkinElmer Life Sciences). The resulting sequences were compared with published sequences of the promoter regions of involucrin (24Lopez-Bayghen E. Vega A. Cadena A. Granados S.E. Jave L.F. Gariglio P. Alvarez-Salas L.M. J. Biol. Chem. 1996; 271: 512-520Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and transglutaminase-1 (25Yamanishi K. Inazawa J. Liew F.M. Nonomura K. Ariyama T. Yasuno H. Abe T. Doi H. Hirano J. Fukushima S. J. Biol. Chem. 1992; 267: 17858-17863Abstract Full Text PDF PubMed Google Scholar). The amplified 3.4-kb fragment of human involucrin promoter and the 2.3-kb fragment of human transglutaminase-1 promoter were subcloned into a pCAT-basic vector (Promega, Madison, WI), linking them to the chloramphenicol acetyltransferase (CAT) gene. The correct orientation of the inserts with respect to the CAT sequence was verified by restriction enzyme analysis. Transfection, Selection, and CAT Assay—NHOKs were transfected in suspension with antisense βig-h3 or pcDNA3.1(+) vector using a Polybrene/glycerol method (26Jiang C.K. Connolly D. Blumenberg M. J. Investig. Dermatol. 1991; 97: 969-973Abstract Full Text PDF PubMed Scopus (90) Google Scholar) and then incubated in keratinocyte growth medium. Beginning 2 days after transfection, cells were incubated for 4 days in 70 μm G418 (Invitrogen). Selected cells were serially subcultured until they reached the post-mitotic stage of proliferation and then harvested. The intracellular levels of βig-h3, involucrin, transglutaminase, Akt, phospho-Akt, and calcium were quantitated by Western blotting and dual-wavelength fluorescence imaging, as described elsewhere. In other experiments of the present study, exponentially proliferating keratinocytes, plated in 35-mm culture dishes, were co-transfected with antisense βig-h3 or pcDNA3.1(+) vector, involucrin- or transglutaminase-1-CAT chimeric plasmid promoter constructs, and 1.0 μg of pZeoSVLacZ using the Polybrene/glycerol method (26Jiang C.K. Connolly D. Blumenberg M. J. Investig. Dermatol. 1991; 97: 969-973Abstract Full Text PDF PubMed Scopus (90) Google Scholar). pZeoSVLacZ is a β-galactosidase expression vector that contains a β-galactosidase gene driven by a SV40 promoter and enhancer; this was used as an internal control for normalization of transfection efficiency. The cells were lysed 48 h after co-transfection and the cell extracts assayed for CAT and β-galactosidase activities. Detailed conditions for the enzyme activity assay are described elsewhere (27Kook J.-K. Kim J.H. Min B.-M. Int. J. Oncol. 1998; 13: 765-771PubMed Google Scholar). A pCAT-control vector (Promega) containing a SV40 promoter and enhancer, shown to be unresponsive, was included as a control in each transfection experiment. Antibodies and Recombinant βig-h3 Proteins—Monoclonal antibodies (mAbs) against the human integrin α2 (P1E6), α3 (P1B5), α5 (P1D6), α6 (GoH3), β1 (P4C10), and β4 (3E1) subunits were obtained from Chemicon (Temecula, CA). The function-blocking mAbs against the human integrin α2 (P1E6), α3 (P1B5), α5 (P1D6), α6 (GoH3), αv (AV1), β1 (6S6), and β4 (3E1) subunits were also purchased from Chemicon. Recombinant βig-h3 protein and polyclonal anti-βig-h3 antiserum against recombinant βig-h3 protein were generously provided by Dr. E. Bae (REGEN Biotech, Korea). The expression plasmid for recombinant βig-h3 protein has been described in detail in previous reports (11Kim J.-E. Kim S.-J. Lee B.-H. Park R.-W. Kim K.-S. Kim I.-S. J. Biol. Chem. 2000; 275: 30907-30915Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 16Kim J.-E. Kim E.-H. Han E.-H. Park R.-W. Park I.-H. Jun S.-H. Kim J.-C. Young M.F. Kim I.-S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (110) Google Scholar). Flow Cytometric Analysis—Flow cytometric analysis of the cell surface integrin expression level was performed as described previously (28Rodeck U. Jost M. Kari C. Shih D.T. Lavker R.M. Ewert D.L. Jensen P.J. J. Cell Sci. 1997; 110: 113-121Crossref PubMed Google Scholar). Briefly, NHOKs were detached by gentle treatment with 0.05% trypsin and 0.53 mm EDTA in phosphate-buffered saline (PBS), washed, and incubated with anti-integrin mAbs (anti-α2, α3, α5, α6, β1, and β4) for 45 min at 4 °C. After washing, cells were incubated with fluorescein isothiocyanate-labeled secondary antibodies for 45 min at 4 °C. Finally, cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences). Adhesion Inhibition Assay—To identify the βig-h3 receptor on NHOKs, 5 μg/ml of mAbs to different types of integrins were incubated with exponentially proliferating (PD 13) or terminally differentiated (PD 20) NHOKs in 0.5 ml of incubation solution (2 × 105 cells/ml) for 30 min at 37 °C. These preincubated cells were transferred onto plates coated with 10 μg/ml of recombinant βig-h3 protein (plates had been protein-coated and stored overnight at 4 °C) and then incubated for an additional 1 h at 37 °C. After incubation, unattached cells were removed by two rinses with PBS. Attached cells were fixed with 10% formalin in PBS for 15 min, rinsed twice with PBS, and stained with 0.005% crystal violet for 1 h. The plates were gently rinsed with double distilled water three times. To ensure a representative count, each plate was divided into quarters and two fields per quarter were photographed using an Olympus BX51 microscope at ×100 magnification. Cell Adhesion Assay—Cell adhesion was assayed as described previously (29Okazaki I. Suzuki N. Nishi N. Utani A. Matsuura H. Shinkai H. Yamashita H. Kitagawa Y. Nomizu M. J. Biol. Chem. 2002; 277: 37070-37078Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Briefly, 24-well culture plates (Nunc, Roskilde, Denmark) were coated with 10 μg/ml recombinant βig-h3 protein and held overnight at 4 °C before being blocked with PBS containing 1% heat-inactivated bovine serum albumin (BSA; Sigma) for 1 h at 37 °C. Cells were detached by treatment with 0.05% trypsin and 0.53 mm EDTA in PBS, resuspended in the culture media (1 × 105 cells/500 μl), added to each plate, and incubated for 1 h at 37 °C. Removal of unattached cells and fixing, staining, and imaging of attached cells was identical as in the adhesion inhibition assay. Effects of Exogenous TGF-β1 and Recombinant βig-h3 Protein on Keratinocyte Differentiation—To determine the effects of TGF-β1 and recombinant βig-h3 protein on keratinocyte differentiation and the PI3K/Akt signaling pathway, the expression of involucrin, transglutaminase, and βig-h3; the promoter activities of involucrin and transglutaminase; the intracellular calcium level; and the phosphorylation status of Akt were determined in exponentially proliferating NHOKs, which were cultured for 4 days in the presence of 10 or 20 ng/ml TGF-β1 or 10 μg/ml recombinant βig-h3 protein. Measurement of Intracellular Calcium—After cells were transfected with either the antisense βig-h3 cDNA constructs or βig-h3 expression plasmids and exposed to recombinant βig-h3 protein, the intracellular calcium level was measured by digital video microfluorimetry using an intensified CCD camera coupled to a microscope (Olympus 1X71S8F-2, Japan) supported by Metafluor software on a Pentium computer (Shutter Instrument, Novato, CA). This procedure has been described previously (23Oh J.-E. Kook J.-K. Park K.-H. Lee G. Seo B.-M. Min B.-M. Int. J. Mol. Med. 2003; 11: 491-498PubMed Google Scholar). Western Blot Analysis—Western blot analysis was performed as in previous reports (19Min B.-M. Woo K.M. Lee G. Park N.-H. Exp. Cell Res. 1999; 249: 377-385Crossref PubMed Scopus (45) Google Scholar) using anti-human involucrin (SY5) mAb (Sigma), anti-human TGF-β1 (sc-146) polyclonal antibody (Santa Cruz, Santa Cruz, CA), anti-human transglutaminase (Ab-1) polyclonal antibody (Oncogene, Uniondale, NY), polyclonal anti-βig-h3 antiserum against recombinant βig-h3 protein (REGEN Biotech, Korea), and anti-β-actin (20Fuchs E. Curr. Opin. Cell Biol. 1990; 2: 1028-1035Crossref PubMed Scopus (107) Google Scholar, 21Larjava H. Am. J. Med. Sci. 1991; 301: 63-68Crossref PubMed Scopus (20) Google Scholar, 22Lee G. Park B.S. Han S.E. Oh J.-E. You Y.-O. Baek J.-H. Kim G.-S. Min B.-M. Arch. Oral Biol. 2000; 45: 809-818Crossref PubMed Scopus (16) Google Scholar, 23Oh J.-E. Kook J.-K. Park K.-H. Lee G. Seo B.-M. Min B.-M. Int. J. Mol. Med. 2003; 11: 491-498PubMed Google Scholar, 24Lopez-Bayghen E. Vega A. Cadena A. Granados S.E. Jave L.F. Gariglio P. Alvarez-Salas L.M. J. Biol. 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This ratio was normalized to the target protein/constitutive protein or β-actin ratio of the negative controls to obtain the relative levels of protein. Akt Phosphorylation Assay—To determine the effects of TGF-β1, recombinant βig-h3 protein, and changes in βig-h3 expression on the phosphorylation status of Akt, exponentially proliferating NHOKs were cultured for 4 days in the presence of 10 or 20 ng/ml TGF-β1 or 10 μg/ml recombinant βig-h3 proteins. Exponentially proliferating keratinocytes were also transfected with either the antisense βig-h3 cDNA constructs or βig-h3 expression plasmids. The phosphorylation status of Akt was determined by Western blot analysis using anti-phospho-Akt (Ser473, 9271) and anti-Akt (9272) (Cell signaling Technology, Beverly, MA). Expression of βig-h3 and TGF-β1 Is Enhanced during Keratinocyte Differentiation—Primary NHOKs were subcultured at every 70% confluence until they reached the post-mitotic stage of proliferation, at which time the culture was maintained for 12 days without further passage. Primary NHOKs (PD 13.4) and cells with lower PDs (PD 15.8) displayed the typical keratinocyte morphology and retained an undifferentiated phenotype. Some cells with PD 16.9, meanwhile, maintained their squamous
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