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Am J Physiol Cell Physiol 293: C1020-C1031, 2007. First published June 27, 2007; doi:10.1152/ajpcell.00169.2007
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

KGF promotes integrin {alpha}5 expression through CCAAT/enhancer-binding protein-beta

Piyush Koria and Stelios T. Andreadis

Bioengineering Laboratory, Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, Amherst, New York

Submitted 22 April 2007 ; accepted in final form 22 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Keratinocyte growth factor (KGF) and {alpha}5beta1-integrin are not expressed in normal skin but they are both highly upregulated in the migrating epidermis during wound healing. Here we report that KGF increased {alpha}5 mRNA and protein levels in epidermoid carcinoma cells and stratified bioengineered epidermis. Interestingly, KGF increased integrin {alpha}5 in the basal as well as suprabasal cell epidermal layers. Promoter studies indicated that KGF-induced integrin {alpha}5 promoter activation was dependent on the C/EBP transcription factor binding site. Accordingly, KGF induced sustained phosphorylation of C/EBP-beta that was dependent on activation of ERK1/2. In addition, a dominant negative form of C/EBP-beta inhibited {alpha}5 promoter activity and blocking C/EBP-beta with siRNA diminished integrin {alpha}5 expression. Taken together, our data indicate that KGF increased integrin {alpha}5 expression by phosphorylating C/EBP-beta. Interestingly, KGF-induced upregulation of integrin {alpha}5 was more pronounced in three-dimensional tissue analogues than in conventional two-dimensional culture suggesting that stratified epidermis may be useful in understanding the effects of growth factors in the local tissue microenvironment.

wound healing; transcription factors; epidermis; signaling pathways

KERATINOCYTE GROWTH FACTOR (KGF) is a monomeric peptide that belongs to the fibroblast growth factor family (FGF-7) and plays a prominent role in epidermal morphogenesis and wound healing. It is mainly expressed by cells of mesenchymal origin, such as fibroblasts, microvascular endothelial and smooth muscle cells but affects epithelial cells (60, 74). Interestingly, one study reported expression of KGF in activated {gamma}{delta}T cells obtained from the skin and intestine, suggesting a possible role of KGF in immunity (6). KGF promotes proliferation and differentiation of epithelial cells in many organs, including the skin, hair follicles, lung, prostate, breast, uterus, and ovary. This paracrine mode of action of KGF on epithelial cells is mediated through the KGF receptor (KGFR or FGFRIIIb), a splice variant of the FGF-2 receptor encoded by the gene fgfr-2 (44, 45).

KGF was discovered by its mitogenicity for a mouse keratinocyte cell line (54), which indicated its possible role in skin. Indeed, targeting expression of KGF to the basal keratinocytes of the developing mouse epidermis caused hyperthickening and altered the differentiation pattern of epidermal tissue (27). Furthermore, development of engineered skin equivalents with KGF-expressing human keratinocytes showed changes in epidermal structure and morphology, including hyperthickening. Besides increasing proliferation of basal cells, KGF induced proliferation of keratinocytes residing in the normally quiescent suprabasal layers of the epidermis (1).

In addition to its mitogenic effects, KGF was shown to have anti-apoptotic, angiogenic, and antimicrobial effects in many epithelial tissues. Specifically, KGF protected gastrointenstinal tissues from inflammation (10); intestinal, oral, and mucosal epithelia from chemotherapy or radiation (17–19); and the alveolar epithelium from hyperoxic injury (2, 25, 53, 64). These protective effects were mediated through KGF-enhanced active ion transport (8), secretion of surfactants (33, 63), or DNA repair (75). Similarly, KGF promoted survival of prenatal ovarian follicles (43), increased hair follicle survival following cytotoxic insult (7), and even prevented ischemia-induced neuronal cell death (56). KGF also protected the barrier function of microvascular but not aortic endothelial cells from peroxide or VEGF-induced permeability and promoted angiogenesis in the rat cornea (23). Finally, KGF induced high expression of antimicrobial peptides in epidermal keratinocytes, suggesting that it might play a role in the innate immunity of the skin (16).

Several studies have also demonstrated the importance of KGF in wound healing. Although KGF is present at very low levels in homeostatic skin, its expression was highly upregulated within 24 h after injury in fibroblasts close to the wound (72). Expression of KGF was also upregulated in psoriatic skin (20) and upon serum stimulation of fibroblasts in vitro (9). While wound healing of KGF receptor-deficient mice was severely impaired (73), mice lacking KGF healed at normal rates (26), possibly due to the compensatory action by other members of the FGF family, e.g., FGF-10 (4) or FGF-22 (5). Despite such redundancies, exogenous KGF significantly enhanced reepithelialization of full and partial thickness wounds in porcine and rabbit ear wound models (50, 61). In addition to epithelialization, exogenous delivery of KGF enhanced granulation tissue formation in an ischemic rabbit ear wound model (23) and injection of KGF DNA accelerated wound closure and reduced inflammation in a diabetic mouse model (40). Finally, biomimetic, cell-controlled delivery of KGF from fibrin hydrogels doubled the rate of wound healing in a hybrid model of human bioengineered skin implanted onto nude mice (21).

Integrin {alpha}5beta1—the classic fibronectin receptor—is also very important in the wound healing process. Similar to KGF, integrin {alpha}5 is not expressed by epidermal keratinocytes under homeostatic conditions but its expression was highly upregulated following injury (22, 31, 35, 38, 48) and under conditions of enhanced inflammation, e.g., psoriasis (3). Consequently, keratinocytes failed to attach to fibronectin immediately after isolation from normal epidermis, but integrin expression and attachment to fibronectin were significantly increased when keratinocytes were isolated from healing wounds (24, 66). Similarly, short-term culture of keratinocytes from normal epidermis in the presence of serum induced integrin-{alpha} expression and promoted binding to fibronectin (51, 66). Conversely, culture of keratinocytes under conditions that promoted differentiation and stratification diminished integrin-{alpha}5 expression at the mRNA and protein levels (36, 47).

In this communication, we report that KGF increased integrin-{alpha}5 expression in the basal as well as suprabasal layers of bioengineered epidermis and blocking this integrin diminished KGF-enhanced wound healing. KGF-induced activation of the integrin-{alpha}5 promoter required activation of the ERK1/2 MAP kinase pathway and phosphorylation of the transcription factor C/EBP-beta at Thr235. Our results suggest that KGF may promote wound healing at least in part by upregulating the integrin-{alpha}5 subunit.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and production of skin equivalents. Isolation and culture of primary human keratinocytes and preparation of skin equivalents were performed as described previously (22, 36). Briefly, primary keratinocytes were isolated from neonatal foreskins and cultured on fibroblast feeder layers. For preparation of skin equivalents, keratinocytes were seeded onto acellular dermis and raised to the air-liquid (A/L) interface. KGF (Amgen, Thousand Oaks, CA) or MAPK inhibitors (PD98059 or U0126; EMD Biosciences, San Diego, CA) were added when the skin equivalents were raised to A/L interface and medium was replenished every 3 days.

Primary keratinocytes were cultured in K-SFM (Invitrogen, Carlsbad, CA); HEK-293T and A431 squamous carcinoma cells (ATCC, Manassas, VA) were cultured in DMEM supplemented with 10% fetal bovine serum. Amphotropic retrovirus producing cells (Phoenix-ampho 293 cells; ATCC) were cultured in DMEM supplemented with 10% heat-inactivated calf serum (Hyclone, Logan, UT).

Wound healing assay. Primary keratinocytes (150,000 cells/well) were seeded in 24-well plates in K-SFM. After they reached confluence, the monolayer was scratched using a 1-ml pipette tip. The cells were washed twice with phosphate-buffered saline to remove cellular debris and K-SFM supplemented with high Ca2+ (2 mM) and the indicated concentrations of KGF was added. Images of the wounds were acquired at x4 magnification on an inverted microscope (Diaphot-TMD, Nikon) using a Retiga 1300 digital camera (QImaging, Burnaby, BC, Canada). The area of the wound was quantified using ImageJ 1.28k software (National Institutes of Health) and percent healing was defined as the area of the wound occupied by cells over the initial wound area.

Plasmids and mutants. The plasmids pcDNA3 and pcDNA3-C/EBP-{alpha} were a kind gift from Dr. Robert Smart (59). The CMV-hLAP and CMV-hLAPT235A plasmids were gifts from Dr. Akira (Osaka University, Japan) courtesy of Dr. Jessica Schwartz (University of Michigan, Ann Arbor, MI) (52). The PCEV-KGFR, plasmid encoding the KGF receptor was a kind gift from Dr. Jeffrey Rubin (National Cancer Institute, Bethesda, MD). The PGL3-based plasmids containing the –23 to +92 promoter fragment of integrin {alpha}5–both wild-type (WT; Pr{alpha}5-Luc) and mutant at the activator protein-1 (AP-1) site—were a generous gift from Dr. Fiona Watt (Cancer Research UK London Research Institute, London, UK) (14). The WT promoter fragment and the luciferase gene were cloned into pQCXIX retroviral vector (self-inactivating SIN vector; Clontech, Mountain View, CA) between the virus packaging sequence, {Psi}, and the CMV promoter using BglII and XbaI sites at the 5'- and 3'-end, respectively. The SV40 polyA tail of the PGL3 plasmid was PCR amplified with primers containing the XbaI site and cloned into the same site of the retroviral plasmid downstream of the luciferase gene. Finally, enhanced green fluorescent protein (EGFP) was excised from pLXCGN (generous gift from Dr. Steven J. Greenberg, Roswell Park Cancer Institute, Buffalo, NY) (57) using AgeI and XhoI and cloned into the same sites downstream of the CMV promoter to create retroviral vector pQ(Pr{alpha}5-Luc)CG. Mutations in the AP1, SP1, or C/EBP transcription factor sites of the {alpha}5 promoter in pQ(Pr{alpha}5-Luc)CG were generated using a site directed mutagenesis kit (Stratagene, La Jolla, CA), as per the manufacturer's recommendations.

siRNA inhibition of C/EBP-beta-siRNA-encoding retrovirus was used to block expression of C/EBP-beta in A431 cells. Oligonucleotide encoding for C/EBP-beta siRNA [target sequence: 5'-GAGCGACGAGTACAAGATG-3' (15)] was cloned between BamH1 and EcoR1 sites of pSIREN-RetroQ vector according to manufacturer's instructions (Clontech, Mountain View, CA).

Production of retrovirus and transduction of cells. Virus producing phoenix ampho cells were transfected using Fugene-6 reagent (Roche, Indianapolis, IN) as per the manufacturer's recommendations. Briefly, 1 x 106 cells were plated in six-well plates one day before transfection. Plasmid DNA (1 µg) was mixed with Fugene-6 (3 µl) and the volume was brought up to 100 µl with antibiotic-free DMEM. The mixture was incubated for 20 min and then added in the cell culture medium overlaying the cells for 24 h. The next day, the medium was replenished and virus supernatant was harvested 24 h later, aliquoted, and stored at –80°C.

For transduction of A431 cells, 150,000 cells were plated in six-well plates in DMEM containing 10% FBS. The next day, the viral supernatant with 8 µg/ml polybrene was incubated with the cells for 24 h. The virus was removed and fresh DMEM supplemented with 10% FBS was added. Transduced cells encoding for luciferase under the WT or mutant {alpha}5 promoter were expanded and GFP+ cells were sorted using fluorescence-activated cell sorter. Cells transduced with the siRNA-encoding virus were selected with 500 ng/ml puromycin and then routinely passaged in selection media.

Transfection of A431 cells. A431 cells were transfected using Lipofectamine 2000 (Invitrogen) as per manufacturer's recommendations. Briefly 500,000 cells were plated in a six-well plate. The next day, DNA (2 µg) was mixed with Lipofectamine 2000 (8 µl) and the volume was brought to 100 µl with OptiMem (Invitrogen). After incubation for 20 min, the transfection mixture was added to 2 ml of OptiMem and overlaid on top of the target cells. After 6 h of incubation, the medium was replaced with DMEM supplemented with 10% FBS and 2 days later, the medium was supplemented with G418 (0.8 mg/ml) for selection of transfected cells.

Luciferase assay. The sorted A431 cells (encoding for luciferase under the WT or mutant {alpha}5 promoter) or 293T cells cotransfected with four plasmids (0.6 µg Pr{alpha}5-luc; 0.6 µg empty vector pcDNA3 or WT-C/EBP-beta or dn-C/EBP-betaT235A; 0.6 µg pCEV-KGFR; and 0.2 µg pBMN-I-GFP) were plated in 6-well plates (500,000 cells/well). After overnight serum starvation, KGF was added at the indicated concentrations in absence of serum and 1 day (293T cells) or 2 days later (A431 cells) luciferase activity was measured using Luciferase assay kit (Promega, Madison, WI) as per manufacturer's recommendations. Briefly, the cells were washed twice with phosphate-buffered saline (PBS) and treated with lysis buffer (300 µl/well). The monolayer was scraped using a cell scraper and the lysate was transferred into an ice-cold microcentrifuge tube and kept on ice. One-third of the lysate (100 µl) was mixed with 100 µl of LAR (provided in the kit) and luminescence was recorded using a luminometer (BioTek Instruments, Winooski, VT). Another 100 µl of the same lysate was used to measure fluorescence intensity (excitation: 488 nm; emission: 525 nm), as a measure of the number of sorted GFP+ cells. To account for differences in cell number under different concentrations of KGF, the values of luminescence were normalized to the values of fluorescence intensity for each sample.

Growth assay. For cell proliferation experiments, A431 cells were plated (100,000 cells/well) in 24-well plates and serum starved overnight before treatment with KGF at the indicated concentrations. Two days after addition of KGF, the cells were washed twice with phosphate-buffered saline, followed by addition of 300 µl water and three freeze-thaw cycles to induce lysis. Cell lysate (100 µl) was mixed with 100 µl of Hoechst 33258 (1:400 dilution in TNE buffer; Molecular Probes, Eugene, OR) and fluorescence intensity was measured using a fluorescence microplate reader (SpectraMax Gemini, Molecular Devices, Menlo Park, CA).

RNA isolation and real-time PCR. Primary keratinocytes were seeded in T25 culture flasks. When they reached confluence, K-SFM was supplemented with Ca2+ (final concentration: 2 mM) overnight to upregulate the KGF receptor (11). The next day, the cells were treated with KGF (20 ng/ml) and RNA was isolated at 6, 24, and 48 h later using SV total RNA isolation kit (Promega). For quantitation of RNA, real-time RT-PCR was performed as described previously (36, 37).

Immunohistochemistry. For detection of Ki67 and integrin {alpha}5 skin equivalents were fixed with 4% paraformaldehyde in PBS for 4 h at 4°C, followed by treatment with 0.1 M ice-cold glycine for 1 h and overnight incubation in 0.6 M sucrose solution at 4°C. Tissues were embedded in OCT, frozen on dry ice, and kept at –75°C until use. For detection of C/EBP-beta skin equivalents were snap-frozen in OCT. Nonfixed OCT-embedded cryosections were permeabilized by treatment with methanol (2 min at room temperature), followed by treatment with 0.1% Triton X-100 (2 min at room temperature).

Immunohistochemistry was performed as described previously (1, 22) using the following primary antibodies. Rabbit anti-human Ki67 (1:50 dilution, 1 h at room temperature; Zymed, San Francisco, CA); mouse anti-human integrin {alpha}5 (1:100 dilution, 30 min at room temperature; BD Pharmingen, San Jose, CA); mouse anti-human C/EBP-beta (1:50 dilution, 4°C overnight, Santa Cruz Biotechnology). Tissue sections were washed three times and incubated with Alexa Fluor 594 conjugated goat anti-rabbit secondary antibody (1:400 dilution, 30 min at room temperature; Molecular Probes) or AlexaFluor 488 goat anti-mouse secondary antibody (1:200 dilution, 30 min at room temperature; Molecular Probes). Tissue sections were washed three times, counterstained with Hoechst 33258 (1:400 dilution, 5 min at room temperature; Molecular Probes), and mounted with crystal mount (Gel/Mount; Biomeda). Images of tissue sections were acquired using an inverted microscope (Diaphot-TMD; Nikon Instruments) and a Retiga 1300 digital camera (QImaging).

Western blots. Cells were washed once with ice-cold PBS and the monolayer was lysed using lysis buffer (Cell Signaling Technology, Danvers, MA) supplemented with DTT and a cocktail of protease inhibitors (Roche Diagnostics, Mannheim, Germany). The lysate was forced through a needle 5 to 10 times to shear genomic DNA and heated at 95°C for 5 min. After centrifugation at 14,000 rpm for 5 min at 4°C, the lysates were separated by SDS-PAGE (8%) and transferred to nitrocellulose membrane [transfer buffer: 25 mM Tris·HCl, pH 8.3, 192 mM glycine, 20% (vol/vol) methanol] for 1 h at 350 mA using an electrophoretic transfer cell (Mini Trans-Blot; Bio-Rad Laboratories, Hercules, CA).

Membranes were blocked with blocking buffer containing 5% (wt/vol) nonfat dry milk in TBS-Tween (20 mM Tris·HCl, pH 7.2–7.4, 150 mM NaCl, 0.1% vol/vol Tween 20) on a rocker platform for 1 h at room temperature. Membranes were incubated with mouse anti-human phospho ERK1/2 (1:2,000 in 5% milk, Cell Signaling Technology); mouse anti-human C/EBP-beta in 5% milk, clone H-7 (Santa Cruz Biotechnology); rabbit anti-human phosphor C/EBP-beta (1:1,000 in 5% BSA, Cell Signaling Technology); mouse anti-human integrin {alpha}5 (1:5,000 in 5% milk, BD Pharmingen) or mouse anti-human beta-actin (1:5,000 in 5% milk; Sigma, St. Louis, MO) overnight at 4°C. After being washed 3 times for 5 min, the membranes were incubated with HRP-conjugated anti-mouse (1:5,000 dilution in 5% nonfat dry milk, 1 h at room temperature; Cell Signaling Technology) or anti-rabbit (1:2,500 dilution in 5% nonfat dry milk, 1 h at room temperature, Cell Signaling Technology). After being washed three times, the protein bands were detected using chemiluminescence (LumiGLO; KPL, Gaithersburg, MD) as per manufacturer's instructions and exposed to film. For loading control, the membranes were stripped (stripping buffer: 62.5 mM Tris·HCl, 2% SDS, and 0.7% beta-mercaptoethanol) for 30 min at 55°C and reprobed for beta-actin. Protein bands were quantified by densitometry using gel electrophoresis imaging software (Kodak EDAS 290) and were internally normalized by the intensity of beta-actin.

Statistical analysis. Statistical analysis of the data was performed using a two-tailed Student's t-test using Microsoft Excel software. The data were considered statistically different when P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
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KGF-induced keratinocyte migration is dependent on integrins {alpha}5, {alpha}3, and {alpha}v. The similar temporal expression pattern of KGF and integrin {alpha}5 during wound healing prompted us to hypothesize that KGF might promote epidermal wound closure at least in part through integrin {alpha}5. To address this hypothesis, we employed a scratch wound model to examine the effect of KGF on keratinocyte wound healing. Keratinocytes were plated in 24-well plates at high density (150,000 cells/well) and allowed to grow to confluence. Confluent monolayers were then wounded and treated with different concentrations of KGF (0, 10, 20, 50, and 100 ng/ml). At 72 h postwounding, KGF (20 ng/ml) promoted wound closure by ~80%, while untreated controls healed by only ~40% (P < 0.05; Fig. 1, A–C). In agreement with previous studies KGF increased the rate of wound healing in a dose-dependent manner (Fig. 1F).


Figure 1
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  		Fig. 1. Keratinocyte growth factor (KGF)-induced migration was dependent of integrin {alpha}5, {alpha}3, and {alpha}v. Confluent monolayers of primary keratinocytes were wounded with a 1-ml pipette tip (~1.4 mm wide). Representative pictures of in vitro wounds at t = 0 h (A) or at t = 72 h postwounding for cells treated with vehicle DMSO (B); 20 ng/ml KGF (C); 20 ng/ml KGF + 10 µM PD98059 (D); or 20 ng/ml KGF + 250 nM U0126 (E). Wound healing was measured at 72 h postwounding. After wounding was completed, the cells were treated with: the indicated concentrations of KGF (F); KGF (20 ng/ml) in the presence of function blocking antibodies (5 µg/ml of each) for the indicated integrin subunits (G); or KGF (20 ng/ml) in presence of MAPK inhibitors, PD98059 (10 µM) or U0126 (250 nM) (H). The fraction of healing was calculated as the ratio of the area covered by cells to the initial wound area at t = 0. All values are means ± SD of quadruplicate samples in a representative experiment. The results are representative of three independent experiments (n = 3). *P < 0.05 difference between the indicated sample and (F) no KGF control or (G, H) KGF only (no antibodies or inhibitors).

 
To determine the role of integrins in KGF-mediated wound healing, we measured wound closure in the presence of a panel of integrin-blocking antibodies and KGF (20 ng/ml). Interestingly, blocking integrin-{alpha}5 inhibited wound healing completely, while blocking integrin subunits {alpha}3 or {alpha}v blocked healing significantly by 86% and 52%, respectively (Fig. 1G). In agreement with previous studies that implicated the ERK1/2 MAPK pathway in KGF-induced migration of corneal cells (39, 58), the effect of KGF on keratinocyte migration was completely reversed by ERK1/2 MAP kinase inhibitors, 10 µM PD98059 and 250 nM U0126 (Fig. 1, D, E, and H). Since blocking integrin {alpha}5 had the most pronounced effect on keratinocyte wound healing, the rest of the experiments focused on the effect of KGF on this integrin subunit.

Keratinocyte growth factor increased integrin {alpha}5 mRNA in primary keratinocytes and squamous carcinoma cells. Next, we measured the level of integrin {alpha}5 mRNA in response to KGF. Primary keratinocytes were treated with KGF (20 ng/ml) for 6, 24, or 48 h and {alpha}5 mRNA was quantified using quantitative real time PCR. We found that KGF treatment induced a transient increase of integrin {alpha}5 mRNA. At 6 h posttreatment, {alpha}5 mRNA increased by 2- or 4-fold (in two independent experiments) but subsided to control levels at later times (Fig. 2A). Also, KGF enhanced {alpha}5 mRNA by 3.3-fold (P < 0.001, n = 3) in epidermoid squamous carcinoma cells (A431) even at 24-h posttreatment.


Figure 2
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  		Fig. 2. KGF upregulated {alpha}5 mRNA in primary keratinocytes and A431 cells. A: primary keratinocytes were treated with KGF and at the indicated times total RNA was isolated and subjected to real-time RT-PCR using {alpha}5-specific primers. The data is plotted as fold upregulation over control cells (not treated with KGF). B: primary keratinocytes were treated with KGF (20 ng/ml) in serum-free medium (SFM). A431 cells were plated in DMEM and serum starved overnight before treatment with KGF (25 ng/ml). Two days after KGF treatment, cells were lysed and the amount of integrin-{alpha}5 was determined using Western blot analysis. Each blot is representative of two independent experiments performed with duplicate samples. Error bars represent SD.

 
We also examined the effect of KGF on the level of {alpha}5 protein using Western blot analysis. Surprisingly, we found that KGF treatment did not increase integrin {alpha}5 in cultures of primary keratinocytes but induced a small (54%) and statistically significant increase of {alpha}5 in A431 cells (Fig. 2B).

KGF increased integrin-{alpha}5 mRNA and protein expression in bioengineered epidermis through the ERK 1/2 MAPK pathway. Although integrin-{alpha}5 is not expressed in normal skin, it was highly upregulated when keratinocytes were placed in cell culture (28, 65, 66), suggesting that the effect of KGF on integrin {alpha}5 protein level may be difficult to quantify in conventional cell culture. On the other hand, epidermal stratification at the A/L interface was accompanied by loss of integrin-{alpha}5 and fibronectin, mimicking native epidermis under homeostatic conditions (36). Since stratification decreased integrin-{alpha}5, we hypothesized that the effect of KGF on integrin {alpha}5 might be more pronounced in stratified bioengineered epidermis.

Indeed, oligonucleotide microarray experiments showed that treatment with KGF for 7 days at the A/L interface increased {alpha}5 mRNA by >3.0-fold (P < 0.01; n = 3) compared with control tissues (n = 3) (P. Koria and S. T. Andreadis, unpublished results). Quantitative real-time RT-PCR verified the microarray experiments and showed that KGF increased {alpha}5 mRNA by 2.75-fold (P < 0.05; n = 3) in skin equivalents grown for 7 days at the air-liquid interface. Most importantly, immunohistochemistry showed {alpha}5 protein expression was very low in control tissues (Fig. 3A) but intense staining was evident in the basal as well as suprabasal layers of KGF-treated tissues (Fig. 3B). Addition of PD98059 (50 µM) or U0126 (50 µM) reversed the effect of KGF and abolished integrin-{alpha}5 (Fig. 3, C and D), suggesting that KGF-induced {alpha}5 upregulation may be mediated through the ERK1/2 MAPK pathway.


Figure 3
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  		Fig. 3. KGF increased integrin {alpha}5 in bioengineered skin through the ERK1/2 MAPK pathway. Skin equivalents were cultured at the air-liquid interface for 7 days. A: control; B: treated throughout the culture period with 100 ng/ml KGF; C: 100 ng/ml KGF + 50 µM PD98059; and D: 100 ng/ml KGF + 50 µM U0126. On day 7, cryosections were stained for integrin {alpha}5. The dotted line denotes basement membrane. The experiment was repeated twice each time with duplicate tissues (x40; bar = 20 µm).

 
In agreement with previous results (1), KGF-treated tissues had more cell layers than controls (Fig. 4, A and B). Interestingly, U0126 reduced KGF-induced growth significantly but PD98059 did not (Fig. 4, C and D). Quantitation of Ki67 positive cells after 7 days at the air-liquid interface showed that in contrast to PD98059, U0126 diminished proliferation and hence decreased the thickness of bioengineered skin (Fig. 4E).


Figure 4
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  		Fig. 4. KGF-induced hyperproliferation is dependent on ERK1/2. Skin equivalents were cultured at the air-liquid interface for 7 days: A: control; B: treated for the culture period with 100 ng/ml KGF; C: 100 ng/ml KGF + 50 µM PD98059; or D: 100 ng/ml KGF + 50 µM U0126. On day 7, paraffin sections were stained (A–D) with hematoxylin and eosin to assess the morphology of the tissues. The dotted line denotes basement membrane (x20; bar = 20 µm). E: immunostaining for the nuclear proliferation antigen, Ki67. Proliferation was measured as the fraction of Ki67+ basal cells per millimeter of basement membrane. Each value represents the average of eight randomly selected fields of view and error bars represent SD. The results are representative of two independent experiments each performed with duplicate tissues (n = 4). *P < 0.05, difference between the indicated and control samples. F: A431 cells were treated with KGF with or without MAPK inhibitors (50 µM) and ERK1/2 phosphorylation was assessed using Western blot analysis. The blot was stripped and reprobed for beta-actin for loading control. This experiment was repeated three times and a representative experiment is shown.

 
To clarify the differential effect of MEK inhibitors on integrin {alpha}5 expression vs. proliferation, we examined the phosphorylation levels of ERK1 and ERK2. We found that PD98059 (50 µM) blocked phosphorylation of ERK1 completely and reduced ERK2 phosphorylation by ~50% (n = 4). In contrast, at the same concentration (50 µM) U0126 completely blocked phosphorylation of both ERK1 and ERK2 (Fig. 4F). Taken together, these data suggest that partial blocking of ERK1/2 may be sufficient to inhibit integrin {alpha}5 without affecting basal cell proliferation.

KGF increased {alpha}5 promoter activity through C/EBP binding site. Next, we attempted to identify the transcription factor binding sites of the integrin {alpha}5 promoter that may be involved in KGF-mediated increase of integrin {alpha}5 expression. The {alpha}5 promoter region contains AP1, SP1, and C/EBP transcription factor binding sites (14). To determine which, if any, of these sites was responsible for the action of KGF, we introduced mutations that were previously shown to prevent binding by the respective transcription factors (Fig. 5A) (14) and the mutated promoters were cloned into a retroviral plasmid upstream of the firefly luciferase reporter gene. This plasmid also contained a CMV promoter driving expression of EGFP that was used to normalize the level of luciferase activity (Fig. 5B).


Figure 5
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  		Fig. 5. KGF increased {alpha}5 promoter activity through the C/EBP binding site. A: partial sequence of the {alpha}5 promoter including the C/EBP, SP1, and AP1 transcription factor binding sites (wild type, WT). Mutations in one or more sites were generated using site-directed mutagenesis and the mutated sequences are shown in bold. B: WT or mutated {alpha}5 promoter sequences were each cloned upstream of the luciferase gene in a self-inactivating retroviral vector, which also encoded for EGFP under a CMV promoter. The presence EGFP allowed for normalization of luciferase luminescence by the green fluorescence intensity of each sample. C: recombinant retrovirus was used to transduce A431 cells and EGFP+ cells were sorted using fluorescence-activated cell sorter. Sorted cells were serum starved overnight and then treated with KGF (25 ng/ml) for 2 days. On day 2, luciferase activity was measured and normalized to EGFP fluorescence intensity to account for differences in the number of cells between control and KGF-treated cultures. All values are the means ± SD of quadruplicate samples in one of three independent experiments (total n = 12 samples for each mutated promoter). *P < 0.05, between the indicated KGF-treated sample and its corresponding control sample (no KGF).

 
Primary keratinocytes can be cultured for limited time (passages 5 and 6) before reaching terminal differentiation and they are not amenable to long-term drug selection required to obtain stable expressers (13). As a result, preparation of skin equivalents from primary cells after drug selection is also difficult. For these reasons, signaling experiments employed A431 cells, which not unlike skin equivalents upregulated {alpha}5 protein in response to KGF (Fig. 2B and Fig. 3).

To this end, A431 cells were transduced with the retroviruses encoding for luciferase under the native or mutated {alpha}5 promoters and EGFP+ cells were sorted using a fluorescence-activated cell sorter. After expansion, the cells were treated with KGF and {alpha}5 promoter activity was measured using the luciferase activity assay. Similar to {alpha}5 protein levels, KGF caused a small (~54%) but statistically significant (P < 0.05; n = 12) increase in {alpha}5 promoter activity (Fig. 5C; WT). Luciferase activity was independent of mutations at the AP1 or SP1 sites but mutating the C/EBP binding site blocked KGF-mediated activation of {alpha}5 promoter (Fig. 5C), suggesting that C/EBP transcription factor(s) may mediate the action of KGF.

KGF induced phosphorylation of C/EBP-beta. Next, we examined whether KGF affected expression of C/EBP-beta. Immunostaining showed that C/EBP-beta was localized in the nucleus of all epidermal cells but the level of expression was higher in suprabasal layers (Fig. 6A). Interestingly, KGF increased staining intensity in basal and also suprabasal cells, which neither proliferated nor expressed integrin {alpha}5. In addition, ERK1/2 inhibitors PD98059 and U0126 that blocked KGF-mediated {alpha}5 expression did not reverse the effect of KGF on C/EBP-beta expression or localization. Note that although tissues treated with U0126 were thinner all cell nuclei stained for C/EBP-beta.


Figure 6
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  		Fig. 6. KGF induced phosphorylation of C/EBP-beta. Skin equivalents were cultured at the air-liquid interface for 7 days. A: control; B: treated for the culture period with 100 ng/ml KGF; C: 100 ng/ml KGF + 50 µM PD98059; D: 100 ng/ml KGF + U0126. On day 7, cryosections were stained for C/EBP-beta (A–D). The dotted line denotes basement membrane. The experiment was repeated twice each time with duplicate tissues (x40; bar = 20 µm). E: skin equivalents were treated as described above, and on day 7, the tissues were lysed and analyzed for phosphor C/EBP-beta by Western blot analysis. The membranes were stripped and reprobed for beta-actin that served as loading control. This experiment was repeated twice each time with duplicate tissues and a representative blot is shown. Error bars represent SD.

 
Previous studies showed that phosphorylation of C/EBP-beta at threonine 235 by ERK1/2 was critical for its activation in response to growth hormone (46, 52). These results prompted us to investigate whether C/EBP-beta was phosphorylated in response to KGF. Indeed, KGF induced phosphorylation of C/EBP-beta in bioengineered epidermis. Phosphor C/EBP-beta decreased by PD98059 (~50%; P < 0.05) and was completely blocked by U0126 (Fig. 6E), suggesting that phosphorylation of C/EBP-beta may be dependent on ERK1/2. This data raised the possibility that KGF might increase {alpha}5 promoter activity through phosphorylation of C/EBP-beta.

C/EBP-beta phosphorylation was necessary for {alpha}5 promoter activation by KGF. To address this hypothesis, we examined the kinetics of KGF-induced phosphorylation of C/EBP-beta in A431 cells using Western blots. Interestingly, KGF induced sustained phosphorylation of C/EBP-beta, which followed phosphorylation of ERK1/2 (Fig. 7A). The level of phosphorylated ERK1/2 peaked as early as 5 min after treatment and subsided after 24 h, whereas phosphor-C/EBP-beta increased by 30-min posttreatment and remained at a similar level even after 2 days.


Figure 7
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  		Fig. 7. Phosphorylation of C/EBP-beta at MAPK site (Thr235) is necessary for KGF stimulated {alpha}5 promoter activation. A: A431 cells were serum starved overnight and the next day treated with KGF (25 ng/ml) for the indicated times. Phosphorylated C/EBP-beta, total C/EBP-beta, and Erk1/2 were measured using Western blot analysis and beta-actin served as loading control. B: A431 cells were transduced with retrovirus encoding for C/EBP-beta siRNA (si-C/EBP-beta) or a scrambled siRNA (control) and selected in puromycin. After selection, control or si-C/EBP-beta cells were plated in six-well plates (5 x 105 cells/well) and 2 days later, lysates were prepared and C/EBP-beta was measured using Western blot analysis. C: si-C/EBP-beta or control cells were serum starved overnight and the next day treated with 25 ng/ml KGF. On day 2 after KGF treatment, the amount of integrin-{alpha}5 was measured using Western blot analysis. Each blot is representative of two independent experiments with similar results. D: 293T cells were cotransfected with four plasmids: P{alpha}5-luc; pCEV-KGFR; empty vector (pcDNA), wild-type C/EBP-beta (WT), or dominant negative C/EBP-beta (dnT235A); and EGFP-encoding plasmid. Serum-starved cells were treated with KGF and luciferase activity was measured 24 h later and luminescence values were normalized to GFP fluorescence intensity of each sample. The plot shows one of three independent experiments each carried out with duplicate samples (n = 6) and error bars represent SD. *P < 0.05 between the indicated and corresponding control (no KGF) sample. #P < 0.05 between the indicated WT and pcDNA samples.

 
Next, we employed siRNA to block expression of the C/EBP protein. Using siRNA-encoding retrovirus and puromycin selection, we established stable A431 cells (si-C/EBP-beta cells) that exhibited >75% reduction of C/EBP-beta (Fig. 7B). Notably, si-C/EBP-beta cells diminished expression of {alpha}5 integrin in control and KGF-treated samples (Fig. 7C).

Finally, we examined whether phosphorylation of C/EBP-beta at the MAPK consensus site (Thr235) was necessary for KGF mediated {alpha}5 promoter activation. To this end, we employed 293T cells that were co-transfected with four plasmids encoding for: 1) empty vector (pcDNA) or WT C/EBP-beta or the dominant negative (dnT235A); 2) a luciferase reporter gene driven by the integrin {alpha}5 promoter; 3) KGF receptor; and 4) EGFP. Transfected cells were treated with KGF and {alpha}5 promoter activity was measured by luciferase assay and normalized to transfection efficiency by EGFP fluorescence intensity (Fig. 7D). As expected, KGF increased promoter activity by ~2.0-fold in control cells (not transfected with C/EBP-beta). Overexpression of C/EBP-beta increased promoter activity, which was further enhanced by ~2-fold with KGF. In contrast, {alpha}5 promoter activity decreased significantly by overexpression of the dominant negative C/EBP-beta (dnT235A) and was not restored by KGF. These results suggest that phosphorylation of C/EBP-beta at Thr235 was necessary for basal as well as KGF-stimulated {alpha}5 promoter activation.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The crosstalk of growth factors with integrins has been demonstrated in several cell types and is thought to be critical for basic cell functions, such as proliferation, migration, and survival. EGF was shown to promote keratinocyte migration on collagen by increasing expression of the integrin {alpha}2 subunit (12). HGF/SF promoted adhesion of malignant B cells to fibronectin and collagen and this effect was mediated by integrins {alpha}4beta1 and {alpha}5beta1 (71). Other studies showed that HGF/SF activated integrin {alpha}vbeta3 in epithelial cells and triggered clustering of integrins beta1, beta3, beta4, and beta5 in actin-rich adhesive sites and lamellipodia (68, 69). HGF/SF was also shown to modulate integrin function by inducing direct association of its receptor (met) with integrin {alpha}6beta4 (67). Similar to HGF/SF, KGF also increased keratinocyte migration on fibronectin and vitronectin and this effect depended on protein kinase C (PKC) (32). Recent evidence suggested that a growth factor can also decrease integrin expression. Specifically, KGF induced differentiation of prostate epithelial cells by decreasing the level of integrin {alpha}2beta1 (30). In contrast to integrin {alpha}2, we discovered that KGF increased expression of integrin {alpha}5 at the mRNA and protein level and that this increase was predominantly observed in stratified epidermis. Moreover, we showed that the effect of KGF was mediated through phosphorylation of C/EBP-beta.

KGF induced sustained phosphorylation of C/EBP-beta, which was inhibited by both PD98059 and U0126. Suppression of C/EBP-beta by siRNA resulted in significant reduction of integrin {alpha}5. In addition, overexpression of a dominant negative C/EBP-beta prevented activation of integrin {alpha}5 promoter by KGF. Altogether, these data suggest that KGF may promote {alpha}5 integrin expression by phosphorylation of C/EBP-beta, implicating this transcription factor in keratinocyte migration and wound healing. C/EBP-beta has been implicated in differentiation and tumor development of several epithelial cells, including mammary epithelial cells and keratinocytes (34). In the epidermis, C/EBP-beta induced expression of early keratinocyte differentiation markers keratin-1 and -10 through the transcription factor, AP-2 (42). C/EBP-beta was also implicated in epidermal tumorigenesis and knockout mice lacking C/EBP-beta were completely resistant to tumor development in response to chemical treatment (62, 77). Nonetheless, this is the first report implicating C/EBP-beta as a key mediator of KGF signaling, possibly promoting epidermal cell migration during the early phases of wound healing.

The two inhibitors of the ERK1/2 pathway showed differential effects on proliferation vs. integrin {alpha}5 expression. Specifically, both U0126 and PD98059 blocked KGF-induced integrin {alpha}5 upregulation but only U0126 inhibited proliferation. Western blots of phosphor ERK1/2 showed that U0126 (50 µM) blocked both ERK1 and ERK2, whereas PD98059 (50 µM) blocked ERK1 completely but blocked ERK2 only partially (Fig. 4F). Previous studies demonstrated that ERK1 knockout mice were viable but showed a deficit in thymocyte maturation (49). On the other hand, ERK2 knockouts died at early stages of embryonic development, suggesting that ERK2 may be essential for tissue development (29, 55, 76). A recent study (70) provided further evidence that ERK2 was necessary for cell proliferation, but ERK1 decreased ERK2-mediated cell growth by competing with ERK2 for binding to the upstream kinase MEK. Taken together, this data may suggest that PD98059 did not inhibit proliferation possibly because it did not inhibit ERK2 phosphorylation. On the other hand, PD98059 blocked ERK1 phosphorylation and integrin {alpha}5 expression, suggesting that KGF-induced {alpha}5 upregulation might be mediated through ERK1. This is an interesting hypothesis that awaits further experimental evidence.

Previous work showed that upregulation of C/EBPs during keratinocyte differentiation inhibited integrin transcription, while AP-1 transcription factors induced integrin-{alpha}5 expression (14). In another study, KGF enhanced lipogenesis in type II alveolar epithelial cells by increasing C/EBP-{alpha} and -{delta} but had no effect on C/EBP-beta (41). Interestingly, our work showed that KGF, which was previously shown to delay keratinocyte differentiation (1), increased integrin {alpha}5 expression by phosphorylating C/EBP-beta. Collectively, these studies suggest that different C/EBP isoforms may have diverse roles in various cellular processes. They also suggest that KGF may mediate its effects through different C/EBP isoforms depending on cell type and tissue context.

Interestingly, KGF had modest effects on the protein level of integrin-{alpha}5 in primary keratinocytes in conventional cell culture, but its effects on three-dimensional bioengineered epidermis were dramatic. Keratinocytes expressed high levels of integrin-{alpha}5 in conventional cell culture, and therefore, the effects of KGF were difficult to detect in that system. By contrast, culture at the A/L interface promoted stratification and diminished expression of integrin {alpha}5 (36). These results suggest that bioengineered epidermis may be approaching homeostasis and that treatment with a single growth factor, i.e., KGF promotes integrin {alpha}5 expression and proliferation mimicking the state of injured epidermis (22, 24). Consequently, tissue-engineered skin may be a better system to study the effects of KGF and possibly other wound healing factors by providing a more physiological tissue context and a three-dimensional architecture that affords resolution of spatial differences.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grant R01-EB000876-01 and National Science Foundation Grant BES-0354626 (to S. T. Andreadis).


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. T. Andreadis, Bioengineering Laboratory, Dept. of Chemical and Biological Engineering, 908 Furnas Hall, Univ. at Buffalo, State Univ. of New York, Amherst, NY 14260 (e-mail: sandread{at}eng.buffalo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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