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βig-h3 Induces Keratinocyte Differentiation via Modulation of Involucrin and Transglutaminase Expression through the Integrin α3β1 and the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway*

  • Ju-Eun Oh
    Affiliations
    Department of Oral Biochemistry and Craniomaxillofacial Reconstructive Sciences, Dental Research Institute, and BK21 HLS, Seoul National University College of Dentistry, Seoul 110-749, Korea and the
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  • Joong-Ki Kook
    Affiliations
    Department of Oral Biochemistry, Chosun University College of Dentistry, Gwangju 501-759, Korea
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  • Byung-Moo Min
    Correspondence
    To whom correspondence should be addressed: Dept. of Oral Biochemistry and Craniomaxillofacial Reconstructive Sciences, Seoul National University College of Dentistry, 28 Yeonkun-Dong, Chongno-Ku, Seoul 110-749, Korea. Tel.: 82-2-740-8661; Fax: 82-2-740-8665;
    Affiliations
    Department of Oral Biochemistry and Craniomaxillofacial Reconstructive Sciences, Dental Research Institute, and BK21 HLS, Seoul National University College of Dentistry, Seoul 110-749, Korea and the
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  • Author Footnotes
    * This work was supported by Korea Health 21 R&D Project Grant 02-PJ1-PG3-20507-0038, Ministry of Health & Welfare, Republic of Korea (to B.-M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:March 31, 2005DOI:https://doi.org/10.1074/jbc.M412293200
      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)
      The 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.
      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 (
      • Skonier J.
      • Neubauer M.
      • Madisen L.
      • Bennett K.
      • Plowman G.D.
      • Purchio A.F.
      ,
      • Skonier 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.
      ). 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 (
      • Skonier J.
      • Neubauer M.
      • Madisen L.
      • Bennett K.
      • Plowman G.D.
      • Purchio A.F.
      ,
      • Skonier 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.
      ). 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 (
      • Skonier 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.
      ). 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 (
      • Skonier 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.
      ), little information is available regarding the distribution of the protein in such human tissues as the arteries, eye, kidney, lung, and skin (
      • Gilbert 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.
      ,
      • LeBaron R.G.
      • Bezverkov K.I.
      • Zimber M.P.
      • Pavelec R.
      • Skonier J.
      • Purchio A.F.
      ,
      • O'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.
      ,
      • Rawe I.M.
      • Zhan Q.
      • Burrows R.
      • Bennett K.
      • Cintron C.
      ).
      It is known that βig-h3 acts as a cell adhesion molecule in several cell types (
      • Kim J.-E.
      • Jeong H.-W.
      • Nam J.-O.
      • Lee B.-H.
      • Choi J.-Y.
      • Park R.-W.
      • Park J.Y.
      • Kim I.-S.
      ) and as a bifunctional linker protein to connect various matrix molecules to each other and to cells (
      • Gibson M.A.
      • Kumaratilake J.S.
      • Cleary E.G.
      ,
      • Billings P.C.
      • Herrick D.J.
      • Kucich U.
      • Engelsberg B.N.
      • Abrams W.R.
      • Macarak E.J.
      • Rosenbloom J.
      • Howard P.S.
      ). 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 (
      • Bae 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.
      ,
      • Kim J.-E.
      • Kim S.-J.
      • Lee B.-H.
      • Park R.-W.
      • Kim K.-S.
      • Kim I.-S.
      ), α1β1 (
      • Ohno S.
      • Noshiro M.
      • Makihira S.
      • Kawamoto T.
      • Shen M.
      • Yan W.
      • Kawashima-Ohya Y.
      • Fujimoto K.
      • Tanne K.
      • Kato Y.
      ), and αvβ5 (
      • Kim J.-E.
      • Jeong H.-W.
      • Nam J.-O.
      • Lee B.-H.
      • Choi J.-Y.
      • Park R.-W.
      • Park J.Y.
      • Kim I.-S.
      ). 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 (
      • Bae 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.
      ). It has also been shown to bind in vitro to a number of other matrix components, including fibronectin, laminin, and several collagen types (
      • Billings P.C.
      • Whitbeck J.C.
      • Adams C.S.
      • Abrams W.R.
      • Cohen A.J.
      • Engelsberg B.N.
      • Howard P.S.
      • Rosenbloom J.
      ,
      • Kim J.-E.
      • Park R.-W.
      • Choi J.-Y.
      • Bae Y.-C.
      • Kim K.-S.
      • Joo C.-K.
      • Kim I.-S.
      ). The precise roles of βig-h3 in cell development are currently unknown, but it has been implicated in cell growth (
      • Skonier 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.
      ,
      • Bae 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.
      ), osteoblast differentiation (
      • Dieudonne S.C.
      • Kerr J.M.
      • Xu T.
      • Sommer B.
      • DeRubeis A.R.
      • Kuznetsov S.A.
      • Kim I.-S.
      • Robey P.G.
      • Young M.F.
      ,
      • Kim 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.
      ), and wound healing (
      • Rawe I.M.
      • Zhan Q.
      • Burrows R.
      • Bennett K.
      • Cintron C.
      ). During wound healing, for example, βig-h3 is produced by a range of cell types including activated macrophages, neutrophils, fibroblasts, and keratinocytes (
      • Kane C.J.
      • Hebda P.A.
      • Mansbridge J.N.
      • Hanawalt P.C.
      ).
      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 (
      • LeBaron R.G.
      • Bezverkov K.I.
      • Zimber M.P.
      • Pavelec R.
      • Skonier J.
      • Purchio A.F.
      ). 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 (
      • Min B.-M.
      • Woo K.M.
      • Lee G.
      • Park N.-H.
      ). 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 (
      • Fuchs E.
      ).
      Epidermal and mucosal keratinocytes undergo terminal differentiation when they migrate from the basal layer to the surface (
      • Larjava H.
      ), 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 (
      • Min B.-M.
      • Woo K.M.
      • Lee G.
      • Park N.-H.
      ) and is more similar in some ways with this in vivo process than with calcium-induced differentiation (
      • Lee G.
      • Park B.S.
      • Han S.E.
      • Oh J.-E.
      • You Y.-O.
      • Baek J.-H.
      • Kim G.-S.
      • Min B.-M.
      ). 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 (
      • Min B.-M.
      • Woo K.M.
      • Lee G.
      • Park N.-H.
      ,
      • Oh J.-E.
      • Kook J.-K.
      • Park K.-H.
      • Lee G.
      • Seo B.-M.
      • Min B.-M.
      ). 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.

      EXPERIMENTAL PROCEDURES

      Cell Culture—Normal human oral keratinocytes (NHOKs) were prepared and maintained as previously reported (
      • Min B.-M.
      • Woo K.M.
      • Lee G.
      • Park N.-H.
      ). 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 (
      • Lopez-Bayghen E.
      • Vega A.
      • Cadena A.
      • Granados S.E.
      • Jave L.F.
      • Gariglio P.
      • Alvarez-Salas L.M.
      ) and transglutaminase-1 (
      • Yamanishi K.
      • Inazawa J.
      • Liew F.M.
      • Nonomura K.
      • Ariyama T.
      • Yasuno H.
      • Abe T.
      • Doi H.
      • Hirano J.
      • Fukushima S.
      ). 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 (
      • Jiang C.K.
      • Connolly D.
      • Blumenberg M.
      ) 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 (
      • Jiang C.K.
      • Connolly D.
      • Blumenberg M.
      ). 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 (
      • Kook J.-K.
      • Kim J.H.
      • Min B.-M.
      ). 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 (
      • Kim J.-E.
      • Kim S.-J.
      • Lee B.-H.
      • Park R.-W.
      • Kim K.-S.
      • Kim I.-S.
      ,
      • Kim 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.
      ).
      Flow Cytometric Analysis—Flow cytometric analysis of the cell surface integrin expression level was performed as described previously (
      • Rodeck U.
      • Jost M.
      • Kari C.
      • Shih D.T.
      • Lavker R.M.
      • Ewert D.L.
      • Jensen P.J.
      ). 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 (
      • Okazaki I.
      • Suzuki N.
      • Nishi N.
      • Utani A.
      • Matsuura H.
      • Shinkai H.
      • Yamashita H.
      • Kitagawa Y.
      • Nomizu M.
      ). 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 (
      • Oh J.-E.
      • Kook J.-K.
      • Park K.-H.
      • Lee G.
      • Seo B.-M.
      • Min B.-M.
      ).
      Western Blot Analysis—Western blot analysis was performed as in previous reports (
      • Min B.-M.
      • Woo K.M.
      • Lee G.
      • Park N.-H.
      ) 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 (
      • Fuchs E.
      ,
      • Larjava H.
      ,
      • Lee G.
      • Park B.S.
      • Han S.E.
      • Oh J.-E.
      • You Y.-O.
      • Baek J.-H.
      • Kim G.-S.
      • Min B.-M.
      ,
      • Oh J.-E.
      • Kook J.-K.
      • Park K.-H.
      • Lee G.
      • Seo B.-M.
      • Min B.-M.
      ,
      • Lopez-Bayghen E.
      • Vega A.
      • Cadena A.
      • Granados S.E.
      • Jave L.F.
      • Gariglio P.
      • Alvarez-Salas L.M.
      ,
      • Yamanishi K.
      • Inazawa J.
      • Liew F.M.
      • Nonomura K.
      • Ariyama T.
      • Yasuno H.
      • Abe T.
      • Doi H.
      • Hirano J.
      • Fukushima S.
      ,
      • Jiang C.K.
      • Connolly D.
      • Blumenberg M.
      ,
      • Kook J.-K.
      • Kim J.H.
      • Min B.-M.
      ,
      • Rodeck U.
      • Jost M.
      • Kari C.
      • Shih D.T.
      • Lavker R.M.
      • Ewert D.L.
      • Jensen P.J.
      ,
      • Okazaki I.
      • Suzuki N.
      • Nishi N.
      • Utani A.
      • Matsuura H.
      • Shinkai H.
      • Yamashita H.
      • Kitagawa Y.
      • Nomizu M.
      ,
      • Levine J.H.
      • Moses H.L.
      • Gold L.I.
      • Nanney L.B.
      ,
      • Maruoka Y.
      • Harada H.
      • Mitsuyasu T.
      • Seta Y.
      • Kurokawa H.
      • Kajiyama M.
      • Toyoshima K.
      ,
      • Kang M.K.
      • Guo W.
      • Park N.-H.
      ,
      • Pillai S.
      • Bikle D.D.
      • Elias P.M.
      ) mAb (Sigma). After probing with each antibody, the membrane was stained with 1× Ponceau S stain for 10 min or subjected to immunoblot analysis using a specific antibody to β-actin for determination of the total protein loaded per lane. The relative protein levels were determined as follows. First, the densitometric intensities of the target protein or β-actin from the Western blots and the constitutive protein from the Ponceau S stain were measured with a LAS-1000 Plus (Fuji Photo film, Japan). The ratio of target protein to constitutive protein or β-actin for each sample was then calculated to correct for differences in protein loading. 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).

      RESULTS

      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 cell shape morphology. Cells with PD 17.3 displayed characteristics of terminal differentiation, including highly enlarged and elongated cytoplasm and balling and detaching from the culture dish (Fig. 1A). Expression of the protein involucrin, one of the molecular markers for keratinocyte differentiation (
      • Maruoka Y.
      • Harada H.
      • Mitsuyasu T.
      • Seta Y.
      • Kurokawa H.
      • Kajiyama M.
      • Toyoshima K.
      ,
      • Kang M.K.
      • Guo W.
      • Park N.-H.
      ), increased in NHOKs in conjunction with increasing PD (Fig. 1B). These results are consistent with these authors' previous report (
      • Min B.-M.
      • Woo K.M.
      • Lee G.
      • Park N.-H.
      ) and indicate that serial subculture of primary NHOKs induces keratinocyte differentiation.
      Figure thumbnail gr1
      Fig. 1Expression of βig-h3 and TGF-β is increased during keratinocyte differentiation. Primary NHOKs (PD 13.4) were serially subcultured until they reached the post-mitotic stage of cell proliferation, at which time the culture was maintained for 12 days without further passage. The total cell numbers at the beginning and end of each passage were used to determine the number of PDs. A, microscopic features of NHOKs with increasing PDs. B, levels of involucrin and βig-h3 proteins in NHOKs with different PDs. C and D, levels of βig-h3 and TGF-β1 in the conditioned medium of exponentially proliferating (PD 13.4) and terminally differentiated (PD 17.3) NHOKs, respectively. Serum-free culture medium was collected from the cultures over 48 h and concentrated. A structural protein stained with Ponceau S served as the internal control to account for loading error.
      Because TGF-β is known to induce both βig-h3 expression in keratinocytes (
      • Skonier J.
      • Neubauer M.
      • Madisen L.
      • Bennett K.
      • Plowman G.D.
      • Purchio A.F.
      ,
      • Skonier 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.
      ) and cell differentiation (
      • Min B.-M.
      • Woo K.M.
      • Lee G.
      • Park N.-H.
      ), the expression of TGF-β and βig-h3 was determined in serially subcultured NHOKs. Because TGF-β and βig-h3 are secreted proteins, we determined their levels in cell lysates and the NHOK medium with different PDs. Western blotting of culture media showed that the exponentially proliferating NHOKs with PD 13.4 had very low levels of βig-h3 and TGF-β1 proteins. Expression of both of these proteins was notably increased, however, in the terminally differentiated NHOKs with PD 17.3 (Fig. 1, C and D). Similarly, the level of intracellular 68-kDa βig-h3 was significantly enhanced in terminally differentiated NHOKs (Fig. 1B). These results suggest that variations in βig-h3 expression are closely correlated with keratinocyte differentiation.
      TGF-β Induces βig-h3 Expression and Promotes Keratinocyte Differentiation—βig-h3 is known to be induced by TGF-β1 in many but not all cell types. The present study showed that TGF-β induces βig-h3 expression in NHOKs. When exponentially proliferating NHOKs (PD 13.4) were cultured for 6, 12, 24, 48, 72, or 96 h in the presence of 10 ng/ml TGF-β1, the levels of both βig-h3 protein and βig-h3 transcript were increased in a time-dependent manner compared with untreated cells (Fig. 2A). A second investigation into the effects of TGF-β1 on keratinocyte differentiation and βig-h3 expression had NHOKs with PD 13.4, cells that do not normally show a differentiated phenotype, being treated with 10 or 20 ng/ml TGF-β1 for 96 h. TGF-β1 treatment in keratinocytes resulted in loosening of cell-cell contact as well as cell spreading. Characteristics of differentiation were also exhibited (Fig. 2B). Western blotting showed that TGF-β1 treatment induced βig-h3 expression in cell lysates and culture media and led to significantly increased expression of involucrin and transglutaminase (Fig. 2C); involucrin and transglutaminase are both markers of keratinocyte differentiation (
      • Maruoka Y.
      • Harada H.
      • Mitsuyasu T.
      • Seta Y.
      • Kurokawa H.
      • Kajiyama M.
      • Toyoshima K.
      ,
      • Kang M.K.
      • Guo W.
      • Park N.-H.
      ,
      • Pillai S.
      • Bikle D.D.
      • Elias P.M.
      ). These results indicate that TGF-β1 treatment induces βig-h3 expression in NHOKs and promotes their differentiation. This agrees with a previous study by these authors showing TGF-β expression to be significantly enhanced in and near terminally differentiated NHOKs and to be associated with keratinocyte differentiation (
      • Min B.-M.
      • Woo K.M.
      • Lee G.
      • Park N.-H.
      ).
      Figure thumbnail gr2
      Fig. 2Overexpression of βig-h3 induced by exposure to TGF-β1 leads to keratinocyte differentiation. A, levels of βig-h3 transcript and protein in exponentially proliferating NHOKs treated with 20 ng/ml of TGF-β1. When NHOKs (PD 13.4) reached 20–80% confluence, the cells were cultured for 6, 12, 24, 48, 72, or 96 h in the presence of 20 ng/ml of TGF-β1. The cell density in all groups was ∼80% confluence at harvesting. A structural protein stained with Ponceau S served as the internal control to account for loading error. 28 S RNA was used as control. B, phase-contrast micrographs of NHOKs (PD 13.4) cultured for 96 h in the presence of 10 or 20 ng/ml TGF-β1. C, levels of involucrin, transglutaminase, and βig-h3 proteins in the cell lysates and/or serum-free culture media for NHOKs cultured for 96 h in the presence of 10 or 20 ng/ml TGF-β1.
      Expression of βig-h3 during Epidermal Differentiation, Response of NHEKs to TGF-β, and the Role of TGF-β in Epidermal Differentiation Are Very Similar to That of NHOK Counterparts—There are some biological differences in the oral mucosa versus the skin. Therefore, we determined the expression of βig-h3 during epidermal differentiation and tested the response of NHEKs to TGF-β1 and its role in epidermal differentiation. Primary NHEKs were subcultured at 70% confluence until they reached the post-mitotic stage of proliferation, at which time the culture was maintained for 12 days without further passage. Like primary NHOKs, the serial subculture of primary NHEKs displayed characteristics of terminal differentiation, including cells with highly enlarged and elongated cytoplasm (data not shown). Involucrin expression increased in NHEKs in conjunction with increasing PD (Fig. 3A), indicating that serial subculture of primary NHEKs induces keratinocyte differentiation. Similarly, the level of intracellular βig-h3 was enhanced in terminally differentiated NHEKs (Fig. 3A), suggesting that variations in βig-h3 expression are also correlated with epidermal keratinocyte differentiation. When exponentially proliferating NHEKs (PD 13.5) were cultured for 6, 12, 24, 48, 72, or 96 h in the presence of 10 ng/ml TGF-β1, the levels of both βig-h3 protein and βig-h3 transcript increased in a time-dependent manner compared with untreated cells (Fig. 3B). Western blotting showed that TGF-β1 treatment induced βig-h3 expression in cell lysates and culture media, which led to significantly increased expression of involucrin and transglutaminase (Fig. 3C). Such changes were very similar to that of NHOKs. These results indicate that TGF-β1 treatment induces βig-h3 expression in NHEKs and promotes their differentiation.
      Figure thumbnail gr3
      Fig. 3Expression of βig-h3 during epidermal differentiation, response of NHEKs to TGF-β, and the role of TGF-β in epidermal differentiation are very similar to that of NHOKs. Primary NHEKs (PD 13.5) were serially subcultured until they reached the post-mitotic stage of cell proliferation, at which time the culture was maintained for 12 days without further passage as described in the legend to . A, levels of involucrin and βig-h3 proteins in NHEKs with different PDs. B, levels of βig-h3 transcript and protein in exponentially proliferating NHEKs treated with 10 ng/ml of TGF-β1. Assay conditions were the same as described in the legend to , except that NHEKs (PD 13.5) were used. A structural protein stained with Ponceau S served as the internal control to account for loading error. 28 S RNA was used as a control. C, levels of involucrin, transglutaminase, and βig-h3 proteins in cell lysates and/or serum-free culture media from NHEKs cultured for 96 h in the presence of 10 or 20 ng/ml TGF-β1.
      Suppression of βig-h3 Expression Inhibits Keratinocyte Differentiation and Extends the Mitotic Capacity of Cells—To test whether βig-h3 could mediate keratinocyte differentiation, and to study the underlying mechanisms of βig-h3-mediated cell differentiation, expression of βig-h3 was down-regulated by transfecting NHOKs with antisense βig-h3 cDNA constructs. If βig-h3 is required for the induction of keratinocyte differentiation, cells transfected with antisense βig-h3 constructs should fail to induce keratinocyte differentiation. NHOK cultures (PD 11.3) were transfected with antisense βig-h3 constructs, and transfectants were selected with G418 and further cultured for 12 days. The transfected cells showed a significant reduction in expression of βig-h3 versus vector-transfected cells. Furthermore, NHOKs transfected with the antisense βig-h3 retained an undifferentiated phenotype, whereas some cells transfected with the vector alone were enlarged and flattened (Fig. 4, A and B). Similarly, the levels of involucrin and transglutaminase in the antisense-βig-h3-transfected cells were significantly reduced (Fig. 4B), indicating that suppression of βig-h3 inhibited keratinocyte differentiation.
      Figure thumbnail gr4
      Fig. 4Suppression of βig-h3 expression by transfection with the antisense βig-h3 cDNA construct inhibits keratinocyte differentiation by decreasing involucrin and transglutaminase expression. Phase-contrast micrographs (A) and decreased levels of βig-h3, involucrin, and transglutaminase (B) in NHOKs transfected with the antisense βig-h3 construct. NHOK cultures (PD 11.3) were transfected with the antisense βig-h3 construct (A) or pcDNA3.1(+) vector (V) and selected with G418. The selected cells were further cultured for 12 days and then harvested. C, involucrin and transglutaminase promoter activities in antisense βig-h3 construct-transfected keratinocytes. Either the antisense βig-h3 construct or the pcDNA3.1(+) vector was transfected into NHOKs (PD 14.1) with involucrin or transglutaminase promoter-CAT constructs and a β-galactosidase expression vector. Two days after transfection, cell lysates were prepared. Data are expressed as percentages of the value for vector-transfected cells (mean ± S.D., n = 6). *, p < 0.01 versus vector-transfected cells. D, extension of the in vitro lifespan of keratinocytes as a consequence of decreased βig-h3 expression by transfection with the antisense βig-h3 construct. The assay conditions were the same as in A. Selected cells were serially subcultured until they reached the post-mitotic stage of proliferation, and the number of PDs was determined at the end of each passage after selection.
      An explanation of how βig-h3 expression causes inhibition of keratinocyte differentiation was sought by evaluation of involucrin and transglutaminase promoter activity following co-transfection of NHOKs with an antisense βig-h3 construct and involucrin or transglutaminase promoter constructs. As predicted, involucrin and transglutaminase promoter activities were significantly inhibited in the antisense βig-h3 construct-transfected cells compared with vector-transfected cells (Fig. 4C).
      Because decreased βig-h3 expression did inhibit keratinocyte differentiation, it was further investigated whether a decrease of βig-h3 would also affect the mitotic ability of cells, in essence extending the in vitro lifespan of NHOKs. NHOK cultures (PD 12.1) were transfected with an antisense βig-h3 construct, selected with G418, and subcultured until they reached the post-mitotic stage of proliferation. As shown in Fig. 4D, the in vitro lifespan of cells that were transfected with the antisense βig-h3 construct was increased ∼2-fold over vector-transfected cells. This increase in mitotic ability was presumably a consequence of decreased βig-h3 expression. These results indicate that suppression of βig-h3 expression inhibits keratinocyte differentiation by decreasing involucrin and transglutaminase promoter activities and also extends the mitotic capacity of the cells.
      Overexpression of βig-h3 and Recombinant βig-h3 Protein Promote Keratinocyte Differentiation by Increasing Involucrin and Transglutaminase Promoter Activities—The role of βig-h3 in cell differentiation was evaluated by examining the effect of overexpression of βig-h3 in keratinocytes. For this, NHOK cultures (PD 11.8) were transfected with βig-h3 expression plasmids to increase βig-h3 expression and transfectants were selected with G418, further cultured for 12 days, and assayed for their levels of βig-h3 and involucrin. Most cells transfected with vector displayed typical keratinocyte morphology and an undifferentiated phenotype, although some were enlarged and flattened. When NHOK cultures were transfected with the βig-h3 expression plasmids, the majority of cells displayed the characteristics of terminal differentiation, principally a highly enlarged and elongated cytoplasm (Fig. 5A). Furthermore, the levels of involucrin as well as βig-h3 were significantly increased in the cells transfected with βig-h3 expression plasmids compared with vector-transfected cells (Fig. 5B), indicating that overexpression of βig-h3 induces keratinocyte differentiation.
      Figure thumbnail gr5
      Fig. 5Overexpression of βig-h3 by transfection with βig-h3 expression plasmids induces keratinocyte differentiation by increasing involucrin and transglutaminase expression. Phase-contrast micrographs (A) and levels of βig-h3 and involucrin (B) in keratinocytes transfected with βig-h3 expression plasmids. NHOK cultures (PD 11.8) were transfected with βig-h3 expression plasmids (βig-h3) or pcDNA3.1(+) vector (V) and selected with G418. The selected cells were further cultured for 12 days and then harvested. C, involucrin and transglutaminase promoter activities in keratinocytes transfected with βig-h3 expression plasmids. The assay conditions were the same as described in the legend to . Data are expressed as percentages of the value for vector-transfected cells (mean ± S.D., n = 6). *, p < 0.01 versus vector-transfected cells.
      If βig-h3 protein is required for the induction of involucrin and transglutaminase gene expression, their promoter activities should be increased in cells transfected with the βig-h3 expression plasmids. As expected, transfection of cells with the βig-h3 expression plasmids significantly increased both involucrin and transglutaminase promoter activities compared with vector-transfected cells (Fig. 5C).
      A direct causal role for βig-h3 in keratinocyte differentiation was further tested by examining the effect of recombinant βig-h3 protein on NHOKs. When NHOKs (PD 12.4) were cultured for 4 days in the presence of 10 μg/ml recombinant βig-h3, the cells displayed a differentiated phenotype (Fig. 6A) and expressed higher levels of involucrin and transglutaminase than did untreated control cells (Fig. 6B). Generally, cell growth is vital to cell differentiation, so the influence of recombinant βig-h3 on cell growth was also evaluated: treatment with 10 μg/ml recombinant βig-h3 decreased cell growth (Fig. 6C). Taken together, these observations indicate that overexpression of βig-h3 and recombinant βig-h3 promotes keratinocyte differentiation by increasing involucrin and transglutaminase expression and by inhibiting cell growth.
      Figure thumbnail gr6
      Fig. 6Recombinant βig-h3 induces keratinocyte differentiation by increasing involucrin and transglutaminase expression, and inhibits cell proliferation. Phase-contrast micrographs (A) and levels of involucrin and transglutaminase (B) in keratinocytes treated with recombinant βig-h3. NHOKs with PD 12.4 were cultured for 4 days in the presence of 10 μg/ml of recombinant βig-h3. C, inhibition of cell proliferation in keratinocytes exposed to recombinant βig-h3. NHOK cultures (PD 13.8; 2 × 104 cells/12-well plate) were cultured for 1, 2, 3, or 4 days in the presence of vehicle or 10 μg/ml recombinant βig-h3. Viable cells were counted in a hemocytometer by trypan blue exclusion. Data are expressed as mean ± S.D. (n = 4). *, p < 0.01 versus vehicle-treated control. D, time-dependent changes of intracellular calcium level (340/380 Fura-2 fluorescence ratio) in response to recombinant βig-h3 (arrow, 10 μg/ml) in keratinocytes.
      Changes in βig-h3 Expression Do Not Affect Intracellular Calcium Levels—Calcium is well known to induce differentiation of human keratinocytes (
      • Hennings H.
      • Kruszewski F.H.
      • Yuspa S.H.
      • Tucker R.W.
      ). To study the involvement of βig-h3 in calcium regulation, changes in βig-h3 expression were evaluated for their affects on intracellular calcium levels in NHOKs. Neither down-regulation of βig-h3 (transfection with antisense βig-h3) nor overexpression of βig-h3 (transfection with βig-h3 expression plasmids) changed the intracellular calcium level compared with vector-transfected cells (data not shown). Similarly, stimulation with 10 μg/ml recombinant βig-h3 for 6 min in serum-free medium did not affect the intracellular calcium level (Fig. 6D). In general, these results suggest that calcium is not involved in βig-h3-mediated keratinocyte differentiation.
      βig-h3 Mediates Keratinocyte Adhesion Through Integrin α3β1—After establishing that βig-h3 mediates keratinocyte differentiation, this study turned to whether βig-h3 induces said differentiation by promoting cell adhesion. Accordingly, a cell adhesion assay was devised. Exponentially proliferating (PD 13) and terminally differentiated (PD 20) NHOKs were seeded onto 24-well plates coated with recombinant βig-h3 or BSA. As shown in Fig. 7A, βig-h3 was promotive of cell adhesion of both types of NHOKs. These findings are in accordance with a previous study that showed βig-h3 supported adhesion and spreading of endothelial epithelial cells as well as NHEKs (
      • Bae 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.
      ,
      • Nam J.-O.
      • Kim J.-E.
      • Jeong H.-W.
      • Lee S.-J.
      • Lee B.-H.
      • Choi J.-Y.
      • Park R.-W.
      • Park J.Y.
      • Kim I.-S.
      ). To further investigate whether restraint of βig-h3 expression modifies cell adhesion, expression of βig-h3 was reduced by transfecting NHOKs with antisense βig-h3 cDNA constructs. NHOK cultures (PD 12) were transfected with antisense βig-h3 constructs, and transfectants were selected with G418, then cultured for 12 days. When the transfected cells were seeded onto 96-well plates coated with or without recombinant βig-h3, cell adhesion was significantly inhibited in antisense βig-h3 construct-transfected cells compared with vector-transfected cells in both conditions (Fig. 7B). Next, in the absence of βig-h3, the effects of TGF-β on keratinocyte adhesion were tested. Exponentially proliferating NHOKs (PD 11) were pretreated with different concentrations of TGF-β1 for 24 h and then were used for cell adhesion assay. TGF-β1-treated cells adhered to βig-h3-uncoated 24-well plates in a dose-dependent manner (Fig. 7C). Taken together, these results suggest that a change in cell adhesion properties is required for keratinocyte differentiation to proceed, and that βig-h3, in response to TGF-β1, mediates cell adhesion to allow expression of keratinocyte differentiation-related genes.
      Figure thumbnail gr7
      Fig. 7Recombinant βig-h3 mediates keratinocyte adhesion through integrin α3β1. A, cell adhesion levels of exponentially proliferating (PD 13) and terminally differentiated (PD 20) NHOKs on surfaces coated with BSA or recombinant βig-h3. One percent of BSA or 10 μg/ml of recombinant βig-h3 were used for coating. The cells were rinsed with PBS, fixed in 10% formalin in PBS, and stained with crystal violet. Data are expressed as percentages of the value for BSA-coated control (mean ± S.D., n = 4). *, p < 0.01 versus BSA-coated control. B, cell adhesion levels of NHOKs transfected with the antisense βig-h3 construct or pcDNA3.1(+) vector on 96-well plates coated with or without recombinant βig-h3 (PS). Assay conditions were the same as described in the legend to , except that NHOKs with PD 12 were used. Ten μg/ml of recombinant βig-h3 was used for coating. Data are expressed as mean ± S.D. (n = 4). *, p < 0.01 versus vector-transfected cells. PS, polystyrene surface only. C, TGF-β1 enhanced the levels of keratinocyte adhesion on βig-h3-uncoated polystylene plates in a dose-dependent manner. Exponentially proliferating NHOKs (PD 11) were pretreated with different concentrations of TGF-β1 for 24 h and then were used for cell adhesion assay. Data are expressed as mean ± S.D. (n = 4). *, p < 0.01 versus vehicle-treated control. D, flow cytometric analyses of the integrin subunits on exponentially replicating and terminally differentiated keratinocytes. Cells were incubated with the mAbs specific to the α2, α3, α5, α6, β1, or β4 integrin subunits and then stained with the fluorescein isothiocyanate-conjugated secondary antibody for flow cytometry. The negative control cells were incubated with the secondary antibody alone (Con). Data are expressed as the cell number (y axis) as a function of fluorescence intensity (x axis). E, expression of integrin subunits in exponentially replicating and terminally differentiated NHOKs. MFI, mean fluorescence intensity. Adhesion of the exponentially proliferating (F) and terminally differentiated (G) NHOKs to βig-h3 is blocked by antibodies to α3β1 integrin. Cells were preincubated with 5 μg/ml of the function-blocking mAbs to integrin α2, α3, α5, α6, αv, β1, and β4 subunits for 30 min at 37 °C and then incubated for 1 h on plates precoated with 10 μg/ml recombinant βig-h3. Unbound cells were washed away and the adherent cells fixed, stained with crystal violet, and counted. Data are expressed as percentages of the value for cells preincubated without integrin antibodies (None; mean ± S.D., n = 3).
      In conjunction with its role in cell adhesion, βig-h3 has been shown to mediate several cellular functions through integrins (
      • Kim J.-E.
      • Jeong H.-W.
      • Nam J.-O.
      • Lee B.-H.
      • Choi J.-Y.
      • Park R.-W.
      • Park J.Y.
      • Kim I.-S.
      ,
      • Bae 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.
      ,
      • Kim J.-E.
      • Kim S.-J.
      • Lee B.-H.
      • Park R.-W.
      • Kim K.-S.
      • Kim I.-S.
      ,
      • Ohno S.
      • Noshiro M.
      • Makihira S.
      • Kawamoto T.
      • Shen M.
      • Yan W.
      • Kawashima-Ohya Y.
      • Fujimoto K.
      • Tanne K.
      • Kato Y.
      ). A pursuit was therefore set upon to identify the particular integrin or integrins by which adhesion, spreading, and differentiation of NHOKs are mediated by βig-h3. The first step was to determine those integrins that are indeed expressed on the surface of NHOKs. This was accomplished by fluorescence-activated cell sorter using mAbs specific to each integrin type. Fig. 7 (D and E) shows that exponentially proliferating and terminally differentiated NHOKs expressed several integrins, including α3β1 and α6β4. The terminally differentiated NHOKs showed lower surface expressions of α and β integrin subunits than exponentially proliferating NHOKs (Fig. 7E). Next, monoclonal integrin function-blocking antibodies were employed with the result that only antibodies to integrin subunits α3 and β1 significantly inhibited βig-h3-mediated adhesion of both types of NHOKs (Fig. 7, F and G). These findings suggest that βig-h3 mediates adhesion of human oral keratinocytes and might mediate keratinocyte differentiation via integrin α3β1.
      βig-h3 Activates Phosphorylation of Akt—Currently, the identities of integrin α3β1-associated signaling molecules that are responsible for mediating keratinocyte adhesion and/or differentiation in response to βig-h3 are unclear. It has been shown that the activation of integrins upon cell adhesion to extracellular matrix protein leads to an increase in phosphorylation of focal adhesion kinase, which is the major tyrosine-phosphorylated protein (
      • Clark E.A.
      • Brugge J.S.
      ,
      • Giancotti F.G.
      • Ruoslahti E.
      ,
      • Guan J.L.
      ). In fact, the focal adhesion kinase pathway is activated by most integrins. However, the downstream signaling pathways that mediate integrin-focal adhesion kinase survival signaling are diverse, and the factors determining which pathway is utilized remain obscure. To determine the signaling pathways that contribute to NHOK adhesion and differentiation as induced by βig-h3, an examination was conducted into the effects of βig-h3 on the phosphorylation status of Akt and MAPKs (ERK, JNK, and p38). Overexpression of βig-h3 or treatment with 10 μg/ml recombinant βig-h3 was found to induce phosphorylation of Akt (Fig. 8, A and B), although these had no effect on the phosphorylation of ERK, JNK, or p38 (data not shown). To further test whether PI3K/Akt is involved in βig-h3-mediated NHOK adhesion and differentiation, NHOK cultures (PD 12.4) were adapted for 12 h, pretreated with 20 μm LY294002, a synthetic inhibitor of the p110 catalytic subunit of PI3K, for 1 h, and then stimulated with 10 μg/ml recombinant βig-h3 for 5, 10, 20, 30, or 60 min. Whereas expression of total Akt did not appear to change, phospho-Akt was increased in cells treated with βig-h3 without pretreatment by LY294002 compared with untreated cells (Fig. 8C). In contrast, LY294002 dramatically blocked the phosphorylation of Akt when paired with βig-h3 treatment (Fig. 8D). Therefore, this study examined whether βig-h3 affects keratinocyte differentiation when the phospho-Akt level is blocked by LY294002. For this, NHOK cultures (PD 11.6) were adapted for 12 h, pretreated with 20 μm LY294002 for 1 h, stimulated with 10 μg/ml recombinant βig-h3 in the presence of 20 μm LY294002 for 2 days, and then assayed for involucrin and transglutaminase expression and the phosphorylation status of Akt. When LY294002 blocked the phosphorylation of Akt (data not shown), the levels of involucrin and transglutaminase expression were notably decreased in recombinant βig-h3-treated cells pretreated with LY294002 compared with recombinant βig-h3-treated cells without pretreatment (Fig. 8E). Taken together, these data suggest that the PI3K/Akt signaling pathway is associated with βig-h3-mediated keratinocyte differentiation.
      Figure thumbnail gr8
      Fig. 8Overexpression of βig-h3 by transfection with βig-h3 expression plasmids and recombinant βig-h3 activate the PI3K/Akt pathway. A, the effect of overexpression of βig-h3 on the phosphorylation status of Akt in keratinocytes. Assay conditions were the same as those described in the legend to . Cell lysates were immunoblotted with antibodies for phospho-Ser473 Akt or total Akt. B, recombinant βig-h3 activates the phosphorylation of Akt in keratinocytes. NHOKs with PD 12.4 were cultured for 4 days in the presence of 10 μg/ml recombinant βig-h3. C, NHOK cultures (PD 13.8) were adapted for 12 h and then stimulated with 10 μg/ml recombinant βig-h3 for 5, 10, 20, 30, or 60 min in serum-free medium. D, pretreatment with LY294002, a PI3K inhibitor, blocks recombinant βig-h3-induced phosphorylation of Akt in keratinocytes. NHOK cultures (PD 13.8) were adapted for 12 h, pretreated with 20 μm LY294002 for 1 h, and stimulated with 10 μg/ml recombinant βig-h3 for 5, 10, 20, 30, or 60 min in serum-free medium. E, pretreatment with LY294002 blocks recombinant βig-h3-induced expression of involucrin and transglutaminase in keratinocytes. Assay conditions were the same as in D, except that NHOKs with PD 11.6 were used and that LY294002-pretreated cells were stimulated with 10 μg/ml recombinant βig-h3 for 2 days in the presence of LY294002.

      DISCUSSION

      The authors of the present study previously demonstrated that TGF-β, a potent inducer of differentiation for normal epithelial cells known to localize in the normal epidermis (
      • LeBaron R.G.
      • Bezverkov K.I.
      • Zimber M.P.
      • Pavelec R.
      • Skonier J.
      • Purchio A.F.
      ), also induces keratinocyte differentiation (
      • Min B.-M.
      • Woo K.M.
      • Lee G.
      • Park N.-H.
      ). Here, it was observed that βig-h3 and TGF-β1 expression were significantly enhanced during keratinocyte differentiation and that TGF-β1 exposure induced keratinocyte differentiation as well as βig-h3 mRNA and protein expression. This study thus examined whether changes in βig-h3 expression would affect keratinocyte differentiation.
      The role of βig-h3 in mediating the differentiation of keratinocytes was demonstrated as follows: (i) a decrease in βig-h3 expression led to inhibition of keratinocyte differentiation and a ∼2-fold increase in mitotic capacity; (ii) down-regulation of βig-h3 expression resulted in decreased promoter activities and thus expression of the involucrin and transglutaminase genes; (iii) overexpression of βig-h3 significantly promoted keratinocyte differentiation and increased promoter activities and thus expression of the involucrin and transglutaminase genes. Together, these results demonstrate that βig-h3 induces keratinocyte differentiation by increasing promoter activities and expression of involucrin and transglutaminase, markers of keratinocyte differentiation (
      • Maruoka Y.
      • Harada H.
      • Mitsuyasu T.
      • Seta Y.
      • Kurokawa H.
      • Kajiyama M.
      • Toyoshima K.
      ,
      • Kang M.K.
      • Guo W.
      • Park N.-H.
      ,
      • Pillai S.
      • Bikle D.D.
      • Elias P.M.
      ) that serve as substrate and enzyme for cornified envelope formation.
      A growing body of reports identifies βig-h3 as a cell adhesion substrate, regulator of cell growth, linker of matrix components, and transducer of TGF-β-mediated signaling (
      • Skonier J.
      • Neubauer M.
      • Madisen L.
      • Bennett K.
      • Plowman G.D.
      • Purchio A.F.
      ,
      • LeBaron R.G.
      • Bezverkov K.I.
      • Zimber M.P.
      • Pavelec R.
      • Skonier J.
      • Purchio A.F.
      ,
      • Kim J.-E.
      • Jeong H.-W.
      • Nam J.-O.
      • Lee B.-H.
      • Choi J.-Y.
      • Park R.-W.
      • Park J.Y.
      • Kim I.-S.
      ,
      • Gibson M.A.
      • Kumaratilake J.S.
      • Cleary E.G.
      ). Although the precise functions of βig-h3 in cell development are currently unknown, it has been associated with inhibition of osteoblast differentiation (
      • Dieudonne S.C.
      • Kerr J.M.
      • Xu T.
      • Sommer B.
      • DeRubeis A.R.
      • Kuznetsov S.A.
      • Kim I.-S.
      • Robey P.G.
      • Young M.F.
      ,
      • Kim 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.
      ,
      • Ohno S.
      • Doi T.
      • Fujimoto K.
      • Ijuin C.
      • Tanaka N.
      • Tanimoto K.
      • Honda K.
      • Nakahara M.
      • Kato Y.
      • Tanne K.
      ). Reports show the level of βig-h3 mRNA to be decreased in human bone marrow stromal cells treated with dexamethasone, a promoter of osteogenic cell differentiation (
      • Dieudonne S.C.
      • Kerr J.M.
      • Xu T.
      • Sommer B.
      • DeRubeis A.R.
      • Kuznetsov S.A.
      • Kim I.-S.
      • Robey P.G.
      • Young M.F.
      ). βig-h3 is also reported to inhibit bone nodule formation of mouse osteoblasts (
      • Kim 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.
      ) and human periodontal ligament cells in vitro (
      • Ohno S.
      • Doi T.
      • Fujimoto K.
      • Ijuin C.
      • Tanaka N.
      • Tanimoto K.
      • Honda K.
      • Nakahara M.
      • Kato Y.
      • Tanne K.
      ). Nevertheless, the role of βig-h3 on keratinocyte differentiation remains unknown.
      Whereas one published study reports in vivo expression of βig-h3 in the granular layer of the epidermis (
      • LeBaron R.G.
      • Bezverkov K.I.
      • Zimber M.P.
      • Pavelec R.
      • Skonier J.
      • Purchio A.F.
      ), another reports that in situ hybridization could not detect βig-h3 expression in that tissue but rather detected βig-h3 transcripts in basal keratinocytes (
      • Bae 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.
      ). Numerous studies in various cell types, including keratinocytes, involve TGF-β1 in the induction of βig-h3 (
      • Skonier J.
      • Neubauer M.
      • Madisen L.
      • Bennett K.
      • Plowman G.D.
      • Purchio A.F.
      ,
      • Skonier 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.
      ). TGF-β has been localized in the normal dermis and epidermis and TGF-β1 is constitutively expressed in suprabasal keratinocytes (
      • Levine J.H.
      • Moses H.L.
      • Gold L.I.
      • Nanney L.B.
      ). Although there is no direct evidence showing that TGF-β1 produced by suprabasal keratinocytes stimulates basal keratinocytes to synthesize βig-h3 in normal skin and mucosa, expression of βig-h3 might be regulated by TGF-β in the epidermis. Participation of βig-h3 in keratinocyte differentiation is supported by our results, namely that overexpression of βig-h3 by TGF-β1 treatment promoted keratinocyte differentiation.
      The present findings agree with these authors previous report that expression of TGF-β is significantly enhanced in and near terminally differentiated NHOKs and is associated with keratinocyte differentiation (
      • Min B.-M.
      • Woo K.M.
      • Lee G.
      • Park N.-H.
      ). These results were confirmed using recombinant βig-h3 protein: βig-h3 treatment resulted in both an increase of involucrin and transglutaminase expression and a differentiated phenotype in normal human keratinocytes. Here, it was also shown that βig-h3 was capable of decreasing cell growth. As has been reported by others, overexpression of βig-h3 was found to decrease cellular growth rate (
      • Skonier 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.
      ). This further suggests that βig-h3 might be involved in differentiation.
      One mechanism of action for βig-h3 in keratinocyte differentiation would be to promote cell adhesion. In fact, βig-h3 has been reported to mediate cell adhesion in several cell types such as skin fibroblasts (
      • LeBaron R.G.
      • Bezverkov K.I.
      • Zimber M.P.
      • Pavelec R.
      • Skonier J.
      • Purchio A.F.
      ), epidermal keratinocytes (
      • Bae 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.
      ), corneal epithelial cells (
      • Rawe I.M.
      • Zhan Q.
      • Burrows R.
      • Bennett K.
      • Cintron C.
      ,
      • Kim J.-E.
      • Jeong H.-W.
      • Nam J.-O.
      • Lee B.-H.
      • Choi J.-Y.
      • Park R.-W.
      • Park J.Y.
      • Kim I.-S.
      ), and chondrocytes (
      • Ohno S.
      • Noshiro M.
      • Makihira S.
      • Kawamoto T.
      • Shen M.
      • Yan W.
      • Kawashima-Ohya Y.
      • Fujimoto K.
      • Tanne K.
      • Kato Y.
      ). In studies with NHOKs, βig-h3 was also found to promote keratinocyte adhesion. To further investigate whether reduction of βig-h3 expression and TGF-β1 modify cell adhesion, NHOKs transfected with antisense βig-h3 cDNA constructs and NHOKs pretreated with TGF-β1 were examined. Cell adhesion was significantly inhibited in cells with reduced βig-h3 compared with vector-transfected cells, but cell adhesion was significantly enhanced in TGF-β1-pretreated cells. This suggests that βig-h3-mediated keratinocyte differentiation is caused by the promotion of cell adhesion. The results presented here further indicate that βig-h3 might mediate NHOK adhesion and differentiation by interacting with integrin α3β1. Integrin α3β1 is a known receptor for βig-h3 (
      • Kim J.-E.
      • Jeong H.-W.
      • Nam J.-O.
      • Lee B.-H.
      • Choi J.-Y.
      • Park R.-W.
      • Park J.Y.
      • Kim I.-S.
      ,
      • Bae 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.
      ). Recently, TGF-β1 has been shown to increase affinity of the integrin α3β1 for βig-h3, resulting in enhanced adhesion and migration of epidermal keratinocytes toward βig-h3 (
      • Jeong H.-W.
      • Kim I.-S.
      ).
      Calcium action is another possible mechanism for keratinocyte differentiation, as it is well known to induce differentiation of human keratinocytes. Specifically, calcium concentrations higher than 0.1 mm in epidermal keratinocytes or 0.15 mm in oral keratinocytes induce terminal differentiation of human basal keratinocytes in vitro. Furthermore, a gradient of extracellular and intracellular calcium across the epidermis, with a lower concentration of calcium in the basal cell compartment than in the granular cell layer, is recognized as a principle regulator of keratinocyte maturation in vivo (
      • Pillai S.
      • Bikle D.D.
      • Elias P.M.
      ,
      • Hennings H.
      • Kruszewski F.H.
      • Yuspa S.H.
      • Tucker R.W.
      ). These investigators have previously shown that intracellular calcium concentration gradually increases during NHOK differentiation (
      • Oh J.-E.
      • Kook J.-K.
      • Park K.-H.
      • Lee G.
      • Seo B.-M.
      • Min B.-M.
      ) and that treatment with calcium induces said differentiation (
      • Lee G.
      • Park B.S.
      • Han S.E.
      • Oh J.-E.
      • You Y.-O.
      • Baek J.-H.
      • Kim G.-S.
      • Min B.-M.
      ). In terms of intracellular calcium level, changes in βig-h3 expression or exposure to recombinant βig-h3 were found to have no effect, suggesting that βig-h3-mediated keratinocyte differentiation is not caused by calcium regulation.
      The phosphorylation status of MAPKs (ERK, JNK, p38) and Akt, a downstream target of PI3K at Ser473, was examined in cells transfected with the wild-type βig-h3 gene and in cells treated with recombinant βig-h3 protein so as to elucidate something of the signaling pathway of βig-h3-mediated keratinocyte differentiation. Both types of cells up-regulated for βig-h3 showed a higher level of phosphorylated Akt than did vector-transfected and βig-h3-untreated cells, respectively. The phosphorylation status of MAPKs in the βig-h3 up-regulated cells was similar to that of the control (data not shown). Subsequent analysis revealed blocking of Akt phosphorylation by LY294002, an inhibitor of PI3K. The notion that exogenous calcium administration might induce keratinocyte differentiation by activating PI3K/Akt was tested and found not to be the case. Calcium did not affect Akt phosphorylation (data not shown).
      It has been reported that, because the βig-h3 gene is induced in several cell lines whose proliferation was affected by TGF-β, βig-h3 might be involved in mediating some of the signals of TGF-β (
      • Skonier J.
      • Neubauer M.
      • Madisen L.
      • Bennett K.
      • Plowman G.D.
      • Purchio A.F.
      ). One recent study reports that TGF-β1 activates focal adhesion kinase and Akt in epidermal keratinocytes and that PI3K could be a common downstream factor that transduces signals from TGF-β1 and leads to activation of the integrin α3β1 by βig-h3 (
      • Jeong H.-W.
      • Kim I.-S.
      ). Because overexpression of βig-h3 or treatment with recombinant βig-h3 was found to activate the PI3K/Akt pathway, decrease cell proliferation, and promote keratinocyte differentiation, we suggest that βig-h3 serves as a downstream factor of TGF-β in keratinocyte differentiation. Whether TGF-β1-induced βig-h3 expression completely accounts for the increase in involucrin and transglutaminase levels observed with TGF-β1 remains unknown. Nevertheless, exogenous recombinant βig-h3 administration in exponentially proliferating keratinocytes, which do not normally show the differentiation phenotype, was sufficient to induce differentiation. We propose that βig-h3 affects keratinocyte differentiation by promoting involucrin and transglutaminase expression through a molecular pathway that involves both integrin α3β1 and PI3K/Akt (Fig. 9).
      Figure thumbnail gr9
      Fig. 9Proposed pathway for induction of keratinocyte differentiation by βig-h3, illustrating the central role of the cell surface receptor integrin α3β1 and PI3K/Akt and its upstream regulator βig-h3.
      Considering that βig-h3 and TGF-β1 expression are significantly enhanced during keratinocyte differentiation, and that TGF-β1 exposure induces said differentiation in addition to βig-h3 mRNA and protein expression, βig-h3 must be granted an important role in keratinocyte differentiation. This role is at least partially explained by the findings of the present study: (i) enhanced TGF-β during keratinocyte differentiation induced βig-h3 expression and led to keratinocyte differentiation by enhancing involucrin and transglutaminase expression, (ii) involucrin and transglutaminase expression were mediated through the integrin α3β1 and PI3K/Akt signaling pathway, and (iii) βig-h3-mediated keratinocyte differentiation was caused by enhanced cell adhesion, not by calcium regulation.

      Acknowledgments

      We thank Dr. Eunhee Bae for providing recombinant βig-h3 protein and polyclonal anti-βig-h3 antiserum against recombinant βig-h3 protein.

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