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J. Biol. Chem., Vol. 280, Issue 22, 21629-21637, June 3, 2005

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ig-h3 Induces Keratinocyte Differentiation via Modulation of Involucrin and Transglutaminase Expression through the Integrin 31 and the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway^*

Ju-Eun Oh, Joong-Ki Kook, and Byung-Moo Min¶

From the 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 Department of Oral Biochemistry, Chosun University College of Dentistry, Gwangju 501-759, Korea

Received for publication, October 29, 2004 , and in revised form, March 7, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
ig-h3 is an extracellular matrix protein whose expression is highly induced by transforming growth factor (TGF)-1. Whereas ig-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 ig-h3 and TGF-1 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 -h3 expression would affect keratinocyte differentiation. Indeed, down-regulation of ig-h3 by transfection with antisense ig-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 ig-h3, either by transfection with ig-h3 expression plasmids or by exposure to recombinant ig-h3, enhanced keratinocyte differentiation by inhibiting cell proliferation and concomitantly increasing involucrin and transglutaminase expression. Recombinant ig-h3 also promoted keratinocyte adhesion through interaction with integrin 31. Changes in ig-h3 expression did not affect intracellular calcium levels. Subsequent analysis revealed not only induction of Akt phosphorylation by recombinant ig-h3 but also blockage of Akt phosphorylation by LY294002, an inhibitor of phosphatidylinositol 3-kinase. Taken together, these findings indicate that enhanced ig-h3, induced by enhanced TGF- during keratinocyte differentiation, provoked cell differentiation by enhancing involucrin and transglutaminase expression through the integrin 31 and phosphatidylinositol 3-kinase/Akt signaling pathway. Lastly, it was observed that ig-h3-mediated keratinocyte differentiation was caused by promotion of cell adhesion and not by calcium regulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Transforming growth factor- (TGF-)¹-inducible gene-h3 (ig-h3) was first cloned from A549 lung adenocarcinoma cells that had been stimulated with TGF-1 (1, 2). ig-h3 has since been shown to be an extracellular matrix protein that can be highly induced by TGF- in several cell types, including mammary epithelial cells, keratinocytes, and lung fibroblasts (1, 2). With structural homology to the insect protein fasciclin, ig-h3 is a 76- to 78-kDa protein containing four repeat regions and 11 cysteine residues, mostly clustered in a distinct amino terminus. The ig-h3 molecule appears to undergo partial processing at the carboxyl terminus to yield a 68–70-kDa isoform (2). Although the ig-h3 transcript has been detected in a variety of human and mouse tissues, including breast, heart, kidney, liver, stomach, and skeletal muscle (2), little information is available regarding the distribution of the protein in such human tissues as the arteries, eye, kidney, lung, and skin (3–6).

It is known that ig-h3 acts as a cell adhesion molecule in several cell types (7) and as a bifunctional linker protein to connect various matrix molecules to each other and to cells (8, 9). ig-h3 contains multiple cell adhesion motifs within its fasciclin-like domains capable of mediating interactions with a variety of cell types via integrins 31 (10, 11), 11 (12), and v5 (7). It is known to mediate the migration and proliferation of normal human epidermal keratinocytes (NHEKs) through two integrin 31-interacting motifs in the second and fourth fas-1 domains (10). It has also been shown to bind in vitro to a number of other matrix components, including fibronectin, laminin, and several collagen types (13, 14). The precise roles of ig-h3 in cell development are currently unknown, but it has been implicated in cell growth (2, 10), osteoblast differentiation (15, 16), and wound healing (6). During wound healing, for example, ig-h3 is produced by a range of cell types including activated macrophages, neutrophils, fibroblasts, and keratinocytes (18).

ig-h3 has been reported in both the dermis and epidermis, with increased staining intensities in the papillary dermis and granular layer of the epidermis (4). Similarly, TGF- has been localized in the normal dermis and epidermis. The investigators of the present study previously demonstrated that expression of TGF-, a potent inducer of differentiation for normal epithelial cells, is increased in and near terminal differentiation of mucosal keratinocytes (19). 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 (20).

Epidermal and mucosal keratinocytes undergo terminal differentiation when they migrate from the basal layer to the surface (21), 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 (19) and is more similar in some ways with this in vivo process than with calcium-induced differentiation (22). 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- plays important roles during wound healing and keratinocyte differentiation. Indeed, previous work by the present authors has shown that TGF- and phospholipase C-1 promote keratinocyte differentiation (19, 23). The importance of TGF- suggests a possible role for ig-h3 as well in the mediation of keratinocyte differentiation and in cell adhesion and spreading. This study sought to examine whether changes in ig-h3 expression would affect keratinocyte differentiation, and to determine the molecular mechanism by which this occurred, in an effort to elucidate the role of ig-h3 in the regulation of keratinocyte differentiation. The findings herein demonstrate that enhanced TGF- during keratinocyte differentiation induced ig-h3 expression and thus provoked cell differentiation by enhancing involucrin and transglutaminase expression through the integrin 31 and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Finally, the present study demonstrates that ig-h3-mediated keratinocyte differentiation was caused by promotion of cell adhesion and not by calcium regulation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture—Normal human oral keratinocytes (NHOKs) were prepared and maintained as previously reported (19). 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 x 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 2^N = (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 ig-h3 and Cloning of the Promoter Regions of Involucrin and Transglutaminase 1—Antisense ig-h3 constructs were made by inserting 375-bp human ig-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. ig-h3 expression plasmids were made by inserting 2204-bp human ig-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 ig-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^TM 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 (24) and transglutaminase-1 (25). 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 ig-h3 or pcDNA3.1(+) vector using a Polybrene/glycerol method (26) 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 ig-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 ig-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 (26). pZeoSVLacZ is a -galactosidase expression vector that contains a -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 -galactosidase activities. Detailed conditions for the enzyme activity assay are described elsewhere (27). 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 ig-h3 Proteins—Monoclonal antibodies (mAbs) against the human integrin 2 (P1E6), 3 (P1B5), 5 (P1D6), 6 (GoH3), 1 (P4C10), and 4 (3E1) subunits were obtained from Chemicon (Temecula, CA). The function-blocking mAbs against the human integrin 2 (P1E6), 3 (P1B5), 5 (P1D6), 6 (GoH3), v (AV1), 1 (6S6), and 4 (3E1) subunits were also purchased from Chemicon. Recombinant ig-h3 protein and polyclonal anti-ig-h3 antiserum against recombinant ig-h3 protein were generously provided by Dr. E. Bae (REGEN Biotech, Korea). The expression plasmid for recombinant ig-h3 protein has been described in detail in previous reports (11, 16).

Flow Cytometric Analysis—Flow cytometric analysis of the cell surface integrin expression level was performed as described previously (28). 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-2, 3, 5, 6, 1, and 4) 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 ig-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 x 10⁵ cells/ml) for 30 min at 37 °C. These preincubated cells were transferred onto plates coated with 10 µg/ml of recombinant ig-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 x100 magnification.

Cell Adhesion Assay—Cell adhesion was assayed as described previously (29). Briefly, 24-well culture plates (Nunc, Roskilde, Denmark) were coated with 10 µg/ml recombinant ig-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 x 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-1 and Recombinant ig-h3 Protein on Keratinocyte Differentiation—To determine the effects of TGF-1 and recombinant ig-h3 protein on keratinocyte differentiation and the PI3K/Akt signaling pathway, the expression of involucrin, transglutaminase, and ig-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-1 or 10 µg/ml recombinant ig-h3 protein.

Measurement of Intracellular Calcium—After cells were transfected with either the antisense ig-h3 cDNA constructs or ig-h3 expression plasmids and exposed to recombinant ig-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 (23).

Western Blot Analysis—Western blot analysis was performed as in previous reports (19) using anti-human involucrin (SY5) mAb (Sigma), anti-human TGF-1 (sc-146) polyclonal antibody (Santa Cruz, Santa Cruz, CA), anti-human transglutaminase (Ab-1) polyclonal antibody (Oncogene, Uniondale, NY), polyclonal anti-ig-h3 antiserum against recombinant ig-h3 protein (REGEN Biotech, Korea), and anti--actin (20-33) mAb (Sigma). After probing with each antibody, the membrane was stained with 1x Ponceau S stain for 10 min or subjected to immunoblot analysis using a specific antibody to -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 -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 -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 -actin ratio of the negative controls to obtain the relative levels of protein.

Akt Phosphorylation Assay—To determine the effects of TGF-1, recombinant ig-h3 protein, and changes in ig-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-1 or 10 µg/ml recombinant ig-h3 proteins. Exponentially proliferating keratinocytes were also transfected with either the antisense ig-h3 cDNA constructs or ig-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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Expression of ig-h3 and TGF-1 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 (31, 32), increased in NHOKs in conjunction with increasing PD (Fig. 1B). These results are consistent with these authors' previous report (19) and indicate that serial subculture of primary NHOKs induces keratinocyte differentiation.

View larger version (84K): [in this window] [in a new window]   FIG. 1. Expression of ig-h3 and TGF- 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 ig-h3 proteins in NHOKs with different PDs. C and D, levels of ig-h3 and TGF-1 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.

TGF- Induces ig-h3 Expression and Promotes Keratinocyte Differentiation—ig-h3 is known to be induced by TGF-1 in many but not all cell types. The present study showed that TGF- induces ig-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-1, the levels of both ig-h3 protein and ig-h3 transcript were increased in a time-dependent manner compared with untreated cells (Fig. 2A). A second investigation into the effects of TGF-1 on keratinocyte differentiation and ig-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-1 for 96 h. TGF-1 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-1 treatment induced ig-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 (31–33). These results indicate that TGF-1 treatment induces ig-h3 expression in NHOKs and promotes their differentiation. This agrees with a previous study by these authors showing TGF- expression to be significantly enhanced in and near terminally differentiated NHOKs and to be associated with keratinocyte differentiation (19).

View larger version (70K): [in this window] [in a new window]   FIG. 2. Overexpression of ig-h3 induced by exposure to TGF-1 leads to keratinocyte differentiation. A, levels of ig-h3 transcript and protein in exponentially proliferating NHOKs treated with 20 ng/ml of TGF-1. 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-1. 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-1. C, levels of involucrin, transglutaminase, and ig-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-1.

Expression of ig-h3 during Epidermal Differentiation, Response of NHEKs to TGF-, and the Role of TGF- in Epidermal Differentiation Are Very Similar to That of NHOK Counterparts—

Suppression of ig-h3 Expression Inhibits Keratinocyte Differentiation and Extends the Mitotic Capacity of Cells—To test whether ig-h3 could mediate keratinocyte differentiation, and to study the underlying mechanisms of ig-h3-mediated cell differentiation, expression of ig-h3 was down-regulated by transfecting NHOKs with antisense ig-h3 cDNA constructs. If ig-h3 is required for the induction of keratinocyte differentiation, cells transfected with antisense ig-h3 constructs should fail to induce keratinocyte differentiation. NHOK cultures (PD 11.3) were transfected with antisense ig-h3 constructs, and transfectants were selected with G418 and further cultured for 12 days. The transfected cells showed a significant reduction in expression of ig-h3 versus vector-transfected cells. Furthermore, NHOKs transfected with the antisense ig-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-ig-h3-transfected cells were significantly reduced (Fig. 4B), indicating that suppression of ig-h3 inhibited keratinocyte differentiation.

An explanation of how ig-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 ig-h3 construct and involucrin or transglutaminase promoter constructs. As predicted, involucrin and transglutaminase promoter activities were significantly inhibited in the antisense ig-h3 construct-transfected cells compared with vector-transfected cells (Fig. 4C).

Because decreased ig-h3 expression did inhibit keratinocyte differentiation, it was further investigated whether a decrease of ig-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 ig-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 ig-h3 construct was increased 2-fold over vector-transfected cells. This increase in mitotic ability was presumably a consequence of decreased ig-h3 expression. These results indicate that suppression of ig-h3 expression inhibits keratinocyte differentiation by decreasing involucrin and transglutaminase promoter activities and also extends the mitotic capacity of the cells.

Overexpression of ig-h3 and Recombinant ig-h3 Protein Promote Keratinocyte Differentiation by Increasing Involucrin and Transglutaminase Promoter Activities—The role of ig-h3 in cell differentiation was evaluated by examining the effect of overexpression of ig-h3 in keratinocytes. For this, NHOK cultures (PD 11.8) were transfected with ig-h3 expression plasmids to increase ig-h3 expression and transfectants were selected with G418, further cultured for 12 days, and assayed for their levels of ig-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 ig-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 ig-h3 were significantly increased in the cells transfected with ig-h3 expression plasmids compared with vector-transfected cells (Fig. 5B), indicating that overexpression of ig-h3 induces keratinocyte differentiation.

View larger version (65K): [in this window] [in a new window]   FIG. 3. Expression of ig-h3 during epidermal differentiation, response of NHEKs to TGF-, and the role of TGF- 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 Fig. 1. A, levels of involucrin and ig-h3 proteins in NHEKs with different PDs. B, levels of ig-h3 transcript and protein in exponentially proliferating NHEKs treated with 10 ng/ml of TGF-1. Assay conditions were the same as described in the legend to Fig. 2A, 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 ig-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-1.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Suppression of ig-h3 expression by transfection with the antisense ig-h3 cDNA construct inhibits keratinocyte differentiation by decreasing involucrin and transglutaminase expression. Phase-contrast micrographs (A) and decreased levels of ig-h3, involucrin, and transglutaminase (B) in NHOKs transfected with the antisense ig-h3 construct. NHOK cultures (PD 11.3) were transfected with the antisense ig-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 ig-h3 construct-transfected keratinocytes. Either the antisense ig-h3 construct or the pcDNA3.1(+) vector was transfected into NHOKs (PD 14.1) with involucrin or transglutaminase promoter-CAT constructs and a -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 ig-h3 expression by transfection with the antisense ig-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.

A direct causal role for ig-h3 in keratinocyte differentiation was further tested by examining the effect of recombinant ig-h3 protein on NHOKs. When NHOKs (PD 12.4) were cultured for 4 days in the presence of 10 µg/ml recombinant ig-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 ig-h3 on cell growth was also evaluated: treatment with 10 µg/ml recombinant ig-h3 decreased cell growth (Fig. 6C). Taken together, these observations indicate that overexpression of ig-h3 and recombinant ig-h3 promotes keratinocyte differentiation by increasing involucrin and transglutaminase expression and by inhibiting cell growth.

Changes in ig-h3 Expression Do Not Affect Intracellular Calcium Levels—Calcium is well known to induce differentiation of human keratinocytes (34). To study the involvement of ig-h3 in calcium regulation, changes in ig-h3 expression were evaluated for their affects on intracellular calcium levels in NHOKs. Neither down-regulation of ig-h3 (transfection with antisense ig-h3) nor overexpression of ig-h3 (transfection with ig-h3 expression plasmids) changed the intracellular calcium level compared with vector-transfected cells (data not shown). Similarly, stimulation with 10 µg/ml recombinant ig-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 ig-h3-mediated keratinocyte differentiation.

ig-h3 Mediates Keratinocyte Adhesion Through Integrin 31—After establishing that ig-h3 mediates keratinocyte differentiation, this study turned to whether ig-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 ig-h3 or BSA. As shown in Fig. 7A, ig-h3 was promotive of cell adhesion of both types of NHOKs. These findings are in accordance with a previous study that showed ig-h3 supported adhesion and spreading of endothelial epithelial cells as well as NHEKs (10, 35). To further investigate whether restraint of ig-h3 expression modifies cell adhesion, expression of ig-h3 was reduced by transfecting NHOKs with antisense ig-h3 cDNA constructs. NHOK cultures (PD 12) were transfected with antisense ig-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 ig-h3, cell adhesion was significantly inhibited in antisense ig-h3 construct-transfected cells compared with vector-transfected cells in both conditions (Fig. 7B). Next, in the absence of ig-h3, the effects of TGF- on keratinocyte adhesion were tested. Exponentially proliferating NHOKs (PD 11) were pretreated with different concentrations of TGF-1 for 24 h and then were used for cell adhesion assay. TGF-1-treated cells adhered to ig-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 ig-h3, in response to TGF-1, mediates cell adhesion to allow expression of keratinocyte differentiation-related genes.

View larger version (65K): [in this window] [in a new window]   FIG. 5. Overexpression of ig-h3 by transfection with ig-h3 expression plasmids induces keratinocyte differentiation by increasing involucrin and transglutaminase expression. Phase-contrast micrographs (A) and levels of ig-h3 and involucrin (B) in keratinocytes transfected with ig-h3 expression plasmids. NHOK cultures (PD 11.8) were transfected with ig-h3 expression plasmids (ig-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 ig-h3 expression plasmids. The assay conditions were the same as described in the legend to Fig. 4C. Data are expressed as percentages of the value for vector-transfected cells (mean ± S.D., n = 6). *, p < 0.01 versus vector-transfected cells.

View larger version (56K): [in this window] [in a new window]   FIG. 6. Recombinant ig-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 ig-h3. NHOKs with PD 12.4 were cultured for 4 days in the presence of 10 µg/ml of recombinant ig-h3. C, inhibition of cell proliferation in keratinocytes exposed to recombinant ig-h3. NHOK cultures (PD 13.8; 2 x 104 cells/12-well plate) were cultured for 1, 2, 3, or 4 days in the presence of vehicle or 10 µg/ml recombinant ig-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 ig-h3 (arrow, 10 µg/ml) in keratinocytes.

View larger version (39K): [in this window] [in a new window]   FIG. 7. Recombinant ig-h3 mediates keratinocyte adhesion through integrin 31. A, cell adhesion levels of exponentially proliferating (PD 13) and terminally differentiated (PD 20) NHOKs on surfaces coated with BSA or recombinant ig-h3. One percent of BSA or 10 µg/ml of recombinant ig-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 ig-h3 construct or pcDNA3.1(+) vector on 96-well plates coated with or without recombinant ig-h3 (PS). Assay conditions were the same as described in the legend to Fig. 4A, except that NHOKs with PD 12 were used. Ten µg/ml of recombinant ig-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-1 enhanced the levels of keratinocyte adhesion on ig-h3-uncoated polystylene plates in a dose-dependent manner. Exponentially proliferating NHOKs (PD 11) were pretreated with different concentrations of TGF-1 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 2, 3, 5, 6, 1, or 4 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 ig-h3 is blocked by antibodies to 31 integrin. Cells were preincubated with 5 µg/ml of the function-blocking mAbs to integrin 2, 3, 5, 6, v, 1, and 4 subunits for 30 min at 37 °C and then incubated for 1 h on plates precoated with 10 µg/ml recombinant ig-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).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The role of ig-h3 in mediating the differentiation of keratinocytes was demonstrated as follows: (i) a decrease in ig-h3 expression led to inhibition of keratinocyte differentiation and a 2-fold increase in mitotic capacity; (ii) down-regulation of ig-h3 expression resulted in decreased promoter activities and thus expression of the involucrin and transglutaminase genes; (iii) overexpression of ig-h3 significantly promoted keratinocyte differentiation and increased promoter activities and thus expression of the involucrin and transglutaminase genes. Together, these results demonstrate that ig-h3 induces keratinocyte differentiation by increasing promoter activities and expression of involucrin and transglutaminase, markers of keratinocyte differentiation (31–33) that serve as substrate and enzyme for cornified envelope formation.

View larger version (43K): [in this window] [in a new window]   FIG. 8. Overexpression of ig-h3 by transfection with ig-h3 expression plasmids and recombinant ig-h3 activate the PI3K/Akt pathway. A, the effect of overexpression of ig-h3 on the phosphorylation status of Akt in keratinocytes. Assay conditions were the same as those described in the legend to Fig. 5A. Cell lysates were immunoblotted with antibodies for phospho-Ser473 Akt or total Akt. B, recombinant ig-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 ig-h3. C, NHOK cultures (PD 13.8) were adapted for 12 h and then stimulated with 10 µg/ml recombinant ig-h3 for 5, 10, 20, 30, or 60 min in serum-free medium. D, pretreatment with LY294002, a PI3K inhibitor, blocks recombinant ig-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 ig-h3 for 5, 10, 20, 30, or 60 min in serum-free medium. E, pretreatment with LY294002 blocks recombinant ig-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 ig-h3 for 2 days in the presence of LY294002.

Whereas one published study reports in vivo expression of ig-h3 in the granular layer of the epidermis (4), another reports that in situ hybridization could not detect ig-h3 expression in that tissue but rather detected ig-h3 transcripts in basal keratinocytes (10). Numerous studies in various cell types, including keratinocytes, involve TGF-1 in the induction of ig-h3 (1, 2). TGF- has been localized in the normal dermis and epidermis and TGF-1 is constitutively expressed in suprabasal keratinocytes (30). Although there is no direct evidence showing that TGF-1 produced by suprabasal keratinocytes stimulates basal keratinocytes to synthesize ig-h3 in normal skin and mucosa, expression of ig-h3 might be regulated by TGF- in the epidermis. Participation of ig-h3 in keratinocyte differentiation is supported by our results, namely that overexpression of ig-h3 by TGF-1 treatment promoted keratinocyte differentiation.

The present findings agree with these authors previous report that expression of TGF- is significantly enhanced in and near terminally differentiated NHOKs and is associated with keratinocyte differentiation (19). These results were confirmed using recombinant ig-h3 protein: ig-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 ig-h3 was capable of decreasing cell growth. As has been reported by others, overexpression of ig-h3 was found to decrease cellular growth rate (2). This further suggests that ig-h3 might be involved in differentiation.

One mechanism of action for ig-h3 in keratinocyte differentiation would be to promote cell adhesion. In fact, ig-h3 has been reported to mediate cell adhesion in several cell types such as skin fibroblasts (4), epidermal keratinocytes (10), corneal epithelial cells (6, 7), and chondrocytes (12). In studies with NHOKs, ig-h3 was also found to promote keratinocyte adhesion. To further investigate whether reduction of ig-h3 expression and TGF-1 modify cell adhesion, NHOKs transfected with antisense ig-h3 cDNA constructs and NHOKs pretreated with TGF-1 were examined. Cell adhesion was significantly inhibited in cells with reduced ig-h3 compared with vector-transfected cells, but cell adhesion was significantly enhanced in TGF-1-pretreated cells. This suggests that ig-h3-mediated keratinocyte differentiation is caused by the promotion of cell adhesion. The results presented here further indicate that ig-h3 might mediate NHOK adhesion and differentiation by interacting with integrin 31. Integrin 31 is a known receptor for ig-h3 (7, 10). Recently, TGF-1 has been shown to increase affinity of the integrin 31 for ig-h3, resulting in enhanced adhesion and migration of epidermal keratinocytes toward ig-h3 (17).

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 (33, 34). These investigators have previously shown that intracellular calcium concentration gradually increases during NHOK differentiation (23) and that treatment with calcium induces said differentiation (22). In terms of intracellular calcium level, changes in ig-h3 expression or exposure to recombinant ig-h3 were found to have no effect, suggesting that ig-h3-mediated keratinocyte differentiation is not caused by calcium regulation.

View larger version (13K): [in this window] [in a new window]   FIG. 9. Proposed pathway for induction of keratinocyte differentiation by ig-h3, illustrating the central role of the cell surface receptor integrin 31 and PI3K/Akt and its upstream regulator ig-h3.

It has been reported that, because the ig-h3 gene is induced in several cell lines whose proliferation was affected by TGF-, ig-h3 might be involved in mediating some of the signals of TGF- (1). One recent study reports that TGF-1 activates focal adhesion kinase and Akt in epidermal keratinocytes and that PI3K could be a common downstream factor that transduces signals from TGF-1 and leads to activation of the integrin 31 by ig-h3 (17). Because overexpression of ig-h3 or treatment with recombinant ig-h3 was found to activate the PI3K/Akt pathway, decrease cell proliferation, and promote keratinocyte differentiation, we suggest that ig-h3 serves as a downstream factor of TGF- in keratinocyte differentiation. Whether TGF-1-induced ig-h3 expression completely accounts for the increase in involucrin and transglutaminase levels observed with TGF-1 remains unknown. Nevertheless, exogenous recombinant ig-h3 administration in exponentially proliferating keratinocytes, which do not normally show the differentiation phenotype, was sufficient to induce differentiation. We propose that ig-h3 affects keratinocyte differentiation by promoting involucrin and transglutaminase expression through a molecular pathway that involves both integrin 31 and PI3K/Akt (Fig. 9).

Considering that ig-h3 and TGF-1 expression are significantly enhanced during keratinocyte differentiation, and that TGF-1 exposure induces said differentiation in addition to ig-h3 mRNA and protein expression, ig-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- during keratinocyte differentiation induced ig-h3 expression and led to keratinocyte differentiation by enhancing involucrin and transglutaminase expression, (ii) involucrin and transglutaminase expression were mediated through the integrin 31 and PI3K/Akt signaling pathway, and (iii) ig-h3-mediated keratinocyte differentiation was caused by enhanced cell adhesion, not by calcium regulation.

	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.

^¶ 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; E-mail: bmmin{at}snu.ac.kr.

¹ The abbreviations used are: TGF-, transforming growth factor-; ig-h3, TGF--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.

	ACKNOWLEDGMENTS

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

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