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Cell Growth & Differentiation Vol. 11, 607-614, November 2000
© 2000 American Association for Cancer Research


Articles

Up-Modulation of the Expression of Functional Keratinocyte Growth Factor Receptors Induced by High Cell Density in the Human Keratinocyte HaCaT Cell Line1

Alessandro Capone2, Vincenzo Visco2, Francesca Belleudi, Cinzia Marchese, Giorgia Cardinali, Marinella Bellocci, Mauro Picardo, Luigi Frati and Maria Rosaria Torrisi3

Dipartimento di Medicina Sperimentale e Patologia, Università di Roma "La Sapienza," 00161 Roma [A. C., V. V., F. B., L. F., M. R. T.]; Istituto Nazionale Ricerca sul Cancro di Genova, Sezione di Biotecnologie, 00161 Roma [C. M.]; Istituto Dermatologico San Gallicano, 00153 Roma [G. C., M. B., M. P., M. R. T.]; and Istituto Neurologico Mediterraneo "Neuromed," 86077 Pozzilli [L. F.], Italy

Abstract

Keratinocyte growth factor (KGF) is involved in the control of proliferation and differentiation of human keratinocytes. It binds to, and activates, the tyrosine kinase KGF receptor (KGFR), a splicing transcript variant of the fibroblast growth factor receptor 2. We have previously shown (C. Marchese et al., Cell Growth Differ., 8: 989–997, 1997) that differentiation of primary cultured keratinocytes triggered by high Ca2+ concentrations in the growing medium induced up-regulation of KGFR expression, which suggested that KGFR may play a crucial role in the control of the proliferative/differentiative program during transition from basal to suprabasal cells. Here we analyzed the process of modulation of the expression of KGFRs in the human keratinocyte cell line HaCaT, widely used as a model to study keratinocyte differentiation. Western blot and double immunofluorescence for KGFR and the K1 differentiation marker showed that cell differentiation and stratification induced by confluence and high cell density correlated with an increase in KGFR expression. KGFRs, present on suprabasal differentiated cells, appeared to be efficiently tyrosine-phosphorylated by KGF, which indicated that the receptors up-regulated by differentiation can be functionally activated by ligand binding. Bromodeoxyuridine incorporation assay revealed that a significant portion of suprabasal differentiated cells expressing KGFR seemed to be still able to synthesize DNA and to proliferate in response to KGF, which suggested that increased KGFR expression may be required for retention of the proliferative activity.

Introduction

The KGF4 (KGF/FGF7), a member of the FGF family, acts on epithelial cells (1) and is involved in the control of epithelial proliferation and differentiation. Initially described as a potent mitogen in vitro for murine keratinocytes (2 , 3) and later for human keratinocytes (4) , KGF seems also to promote the early steps of the keratinocyte differentiation program (4) and to inhibit the terminal differentiation and apoptosis of cultured human keratinocyte (5) . In vivo, KGF represents a key mediator in the process of reepithelization as observed in normal human wound healing (6) and in experimental models of wound repair (7, 8, 9) .

KGF elicits its activity through high-affinity binding to the tyrosine kinase KGFR, a splicing transcript variant of the FGFR2 (10 , 11) . KGF binding to the receptor induces receptor activation, phosphorylation of the receptor itself and of substrate molecules (12) , and chlatrin-mediated receptor endocytosis (13) . Histochemical studies on human epithelial tissues and skin, performed using a functional KGF-HFc chimeric protein obtained by fusion of the KGF to the HFc portion of the G immunoglobulin (14) , have revealed that KGFRs are mostly distributed on suprabasal layers and very low expressed on the basal cells (14 , 15) . In addition, we have reported that, in human primary cultured keratinocytes, KGFR expression is up-modulated during differentiation induced by high Ca2+ concentration in the culture medium, which suggests that receptor expression may regulate the proliferative/differentiative balance during differentiation from basal to suprabasal cells.

To investigate the process of modulation of KGFR expression, we used a human keratinocyte cell line HaCaT, spontaneously immortalized from a primary culture of keratinocytes (16) , well characterized (17 , 18) , and widely used as a suitable model to study keratinocyte differentiation (18, 19, 20) . The results here obtained using Western blot analysis and double immunofluorescence techniques demonstrate that the expression of KGFR is up-regulated concomitantly with that of the keratin 1 differentiation marker by cell confluency and stratification and that the KGFRs present on differentiating keratinocytes are efficiently activated by the KGF ligand. In addition, combining BrdUrd incorporation assay to identify DNA synthesizing cells and immunofluorescence for KGFR and K1 expression, we analyzed the possibility that the differentiating keratinocytes are still able to proliferate. Our observations reveal that the KGFR expressing keratinocytes, despite their suprabasal position and K1 expression, may still undergo DNA synthesis and may be further stimulated to proliferate by KGF treatment, which suggests that the increased KGFR expression may be required for retention of such proliferative activity.

Results

We have previously reported that the expression of KGFRs on human primary cultured keratinocytes increased in response to the high Ca2+ signal for differentiation (15) . To analyze the possible modulation of KGFR expression in cultured HaCaT cells, we performed Western blot analysis with anti-Bek polyclonal antibodies, which recognize the intracellular domain shared by the two splicing isoforms FGFR2 and KGFR. Because it has been demonstrated that HaCaT cells cultured at high density produce suprabasal keratins, such as K1, and are able to stratify (19) , we compared KGFR expression in cells cultured for 3, 7, or 11 days after plating. A protein species, reacting with anti-Bek antibodies and corresponding to the molecular weight of the human form of KGFR, and characterized by three immunoglobulin loops in the extracellular region (11) , was detected in all cells; however, a clear increase in the protein level was observed in the cell lysates obtained after 7 days of culture compared with those from 3 days cultures (Fig. 1A)Citation . A parallel increase of K1 expression was also detected (Fig. 1A)Citation , confirming that the high-cell-density conditions induced cell differentiation. Comparison between HaCaT cultures grown for 3 and 11 days provided additional evidence of the parallel increase of KGFR and K1 during density-mediated differentiation of HaCaT keratinocytes (Fig. 1B)Citation . In contrast, the expression of EGFRs seems unmodified or slightly reduced after 11 days of culture as compared with 3 days of culture (Fig. 1B)Citation . Thus, the expression of KGFR, but not that of EGFR, is up-regulated during differentiation induced by cell density and stratification and parallels the up-regulation of the K1 marker of early differentiation.



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Fig. 1. A, expression of KGFRs and K1 on HaCaT cells cultured at low density (preconfluent, 3 days of culture) or high density (postconfluent, 7 days of culture) assessed by Western blot analysis with anti-Bek and anti-K1 polyclonal antibodies. Both KGFR and K1 levels increase with time of culture and growing density. B, expression of KGFR, K1, and EGFR on HaCaT cells cultured at low density (3 days of culture) or at very high density (11 days of culture) assessed by Western blot analysis with anti-Bek and anti-K1 polyclonal antibodies and anti-EGFR monoclonal antibody. KGFR and K1 levels increase with cell density, whereas the expression of EGFR does not appear modified.

 
To confirm that the increase of KGFRs is confined to cells committed to the differentiation program, we performed immunofluorescence of HaCaT cells after 7 days of culture. We double-immunolabeled KGFRs with anti-Bek antibodies, which are directed against the intracellular region of the receptor, and with the KGF-HFc chimeric protein, which is known to bind the KGFR similarly to the native KGF ligand (13 , 14) and has been used as a valid tool in previous immunocytochemical studies on KGFR expression (6 , 14) . Moreover, KGF-HFc permits us to selectively label receptors expressed on the cell plasma membrane, if staining is performed before cell permeabilization, whereas anti-Bek antibodies require permeabilization and, therefore, also label receptors located intracellularly. Double immunofluorescence with anti-Bek (rhodamine staining, red) and KGF-HFc (fluorescein staining, green) revealed that KGFRs were mainly distributed on suprabasal stratified cells (Fig. 2, A–C)Citation , in which the two signals corresponding to the ligand and the receptor colocalize (shown in yellow by overlapping the single images). To demonstrate that the suprabasal cells positively stained for KGFR are expressing the K1 marker of differentiation, we double-labeled with anti-K1 antibodies and with KGF-HFc. As shown in Fig. 2DCitation 2ECitation 2FCitation , the K1 cytosolic expression (red) was mostly confined to the suprabasal cells, in which KGFRs appeared labeled also on the cell plasma membrane (green), confirming the differentiative state of the KGFR-positive cells and indicating that K1 and KGFR are coexpressed in the same cell population.



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Fig. 2. Immunofluorescence analysis of the distribution of KGFR on HaCaT cells cultured at high density (7 days of culture). In A–C, double immunolabeling of KGFRs with anti-Bek antibodies (rhodamine staining, red) and with KGF-HFc chimeric protein (fluorescein staining, green) shows that KGFRs are mainly present on suprabasal stratified cells. In D–F, a significant portion of suprabasal cells positively labeled with KGF-HFc (green) appears also positive for the K1 differentiation marker (red). Arrows, cells positive for both KGFR and K1. Bar, 40 µm.

 
To determine whether KGFR and K1 up-regulation that is induced by cell density occurs with similar kinetics and in the same single cells, we double-stained HaCaT cells, cultured for 3 and 7 days after plating, with KGF-HFc and K1 as above. At 3 days after plating, when cells were preconfluent Citation (Fig. 4A)Citation the signal corresponding to KGFR (green in Fig. 3BCitation ) appeared very weak. These cells were mostly negative for K1 (red, Fig. 3CCitation ), although, occasionally, a few K1-positive cells, basally positioned, could be detected (Fig. 3CCitation , arrows), as expected (19) . After 7 days of culture, cells were postconfluent, densely packed and stratified in areas of the culture plate (Fig. 3E)Citation . Double staining for KGFR and K1 as above revealed that the suprabasal cells were positive for both KGFR (green in Fig. 3FCitation ) and K1 (red in Fig. 3GCitation ), which provided further evidence of such coexpression in differentiating cells and which demonstrated parallel up-modulation of KGFR and K1 in HaCaT cells induced to stratify.



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Fig. 3. Phase-contrast microscopy (A, E) and double immunofluorescence with KGF-HFc chimeric protein (B, F; green) and anti-K1 antibodies (C, G; red) of HaCaT cells cultured at low density (3 days of culture) or at high density (7 days of culture). At low density (A–D), cells appear preconfluent and all basally located (A); the signal corresponding to KGFR (green) is very weak and only a few cells (C, arrowheads) appear positive for K1. At high density (E–H), cells are confluent, densely packed and partially stratified (E, left side). Double immunofluorescence for KGFR (F, green) and K1 (G, red) shows that suprabasal stratified cells (F–H, left side) are positive for both KGFR and K1 as assembled by overlapping the single images (H, yellow), whereas in basal cells (E–H, right side) both signals are virtually undetectable (F–H). Bar, 40 µm.

 


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Fig. 4. Ultrastructural analysis of HaCaT cells grown for 3 days (A) shows a basal layer of undifferentiated cells. When cells are cultured for 7 days at confluency (B and C), they appear to stratify in multilayers, and the suprabasal cells show typical features of keratinocyte differentiation, such as bundles of cytoplasmic keratin filaments and desmosomes at the intercellular contacts. At 12 days of culture, the increase in stratification and differentiation is shown by an evident increase in the number of desmosomes interconnecting the cells (D). Bars: A, 2 µm; B and C, 1 µm; D, 0.5 µm. Nu, nucleus; M, mitochondria; k, keratin filaments; d, desmosomes.

 
Because in HaCaT cells, K1 synthesis is dependent on cell packaging and superimposition (19) and may not be strictly related to stratification, ultrastructural examination of HaCaT cells grown at different densities was performed by conventional thin-section electron microscopy. Our observations confirm that after 3 days of culture, HaCaT cells all appeared basally located and undifferentiated (Fig. 4A)Citation . At 7 days of culture, cells were stratified in multilayers (Fig. 4, B and C)Citation and the cells on suprabasal position showed ultrastructural features of keratinocyte differentiation, i.e., bundles of tonofilaments and intercellular junctions. Eleven days after plating, the number of desmosomes and the amount of cytosolic keratin filaments were further increased (Fig. 4D)Citation , which indicated that HaCaT cells in our growing conditions display a normal differentiation potential and may undergo efficient stratification.

To demonstrate that the KGFRs that are up-regulated by differentiation might be functionally tyrosine-phosphorylated and activated in response to KGF binding, we treated HaCaT cells, cultured for 6 days after plating, with KGF for 10 min at 37°C in the presence of 0.3 M NaCl to decrease KGF interaction with low-affinity binding sites and to increase ligand binding to the high-affinity KGFRs (19 , 21) . Cells were then double-labeled with anti-Bek (Fig. 5, A and DCitation ; red) and antiphosphotyrosine (Fig. 5, B and ECitation ; green) antibodies. In untreated cells (Fig. 5, A–C)Citation , the signal for phosphotyrosine residues was undetectable (Fig. 5B)Citation , whereas in KGF-treated cells (Fig. 5Citation , D-F) colocalization of the two signals on suprabasal cells revealed receptor phosphorylation and activation. Thus, the increase in KGFR expression that occurs during cell differentiation corresponds to a parallel increase in the ability of these receptors to be triggered by their ligand KGF.



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Fig. 5. Tyrosine phosphorylation of up-regulated KGFRs induced by KGF. Double staining of HaCaT cells, cultured at high density (6 days after plating) with anti-Bek (A, D) and antiphosphotyrosine (Ptyr; B and E) antibodies. In untreated cells (A–C), the phosphotyrosine signal on both KGFR-positive and KGFR-negative cells (A) appears undetectable; in cells treated with KGF for 10 min at 37°C, the KGFR-positive suprabasal cells (D) are positively stained also for phosphotyrosine (E). The extent of colocalization of the two signals is assessed by overlapping the single images (F, yellow). Bar, 40 µm.

 
To analyze whether the cells that expressed increased levels of KGFR were still capable of DNA synthesis and proliferative activity, we performed a BrdUrd incorporation assay and observed the labeled nuclei at immunofluorescence. Cells, grown at different densities, were incubated with BrdUrd for 24 h at 37°C and fixed in formaldehyde; incorporated BrdUrd was then visualized with anti-BrdUrd antibody followed by fluorescein-conjugated secondary antibodies. To correlate the proliferating activity with the KGFR and K1 expression, we performed double immunofluorescence staining with anti-K1 and anti-BrdUrd (Fig. 6, A–C)Citation antibodies or with anti-Bek and anti-BrdUrd (Fig. 6Citation , D–F) antibodies. At 3 days after plating (Fig. 6, A and D)Citation , ~90% of the preconfluent cells showed intense BrdUrd staining in the nuclei. With increased time of culture, the percentage of cells showing nuclei that were positively stained for BrdUrd decreased as did cell density and stratification, becoming 81% after 6 days (Fig. 6, B and E)Citation in confluent cells and 32% in stratified cultures at 11 days (Fig. 6, C and F)Citation . Interestingly, a significant portion of the BrdUrd+ cells at both 6 and 11 days were positive also for K1 (Fig. 6, B and C)Citation and KGFR (Fig. 6, E and F)Citation , as shown by the presence of the two signals (nuclear for BrdUrd and cytosolic for K1 or KGFR) in the same individual cells suprabasally located. In control experiments, performed by omission of BrdUrd from the culture medium during the 24-h incubation, the cell nuclei were all negative. In some experiments, a shorter incubation with BrdUrd, given as a pulse of 1 h, was also performed, and the results obtained (not shown) were overlapping with those observed after 24-h BrdUrd exposure. Thus, a significant portion of differentiating cells, expressing KGFR and the K1 marker, appeared to be still able to incorporate BrdUrd and, therefore, synthesize DNA. To investigate whether KGF treatment of stratified cultures could induce DNA synthesis in suprabasal K1-expressing cells, we performed double immunofluorescence with anti-K1 and anti-BrdUrd, as above, of cells cultured for 9 days after plating, treated for an additional 48 h with KGF, and incubated with BrdUrd for 1 h or for 12 h. In KGF-stimulated cultures, the number of BrdUrd+ cells that also expressed K1 appeared drastically increased (Fig. 6GCitation , arrows) compared with control (untreated) cells (Fig. 6H)Citation , whereas the K1 expression was virtually unmodified by KGF treatment. Thus, KGF stimulates BrdUrd incorporation in K1-expressing suprabasal cells.



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Fig. 6. A–F, BrdUrd incorporation (24 h) in HaCaT cells grown at different densities. Double immunofluorescence with antiBrdUrd (green) antibodies and anti-K1 (A–C, red) or anti-Bek (D–F, red) antibodies of cells cultured for 3 days (A, D), 6 days (B, E) and 11 days (C, F). Codistribution of the nuclear BrdUrd and the cytosolic K1 signals, as well as of BrdUrd and KGFR signals, visible by overlapping the single images, is evident in a significant portion of suprabasal cells expressing K1 and KGFR after either 6 days or 11 days of culture (B, C, E, F). Arrows (A), basal cells expressing K1. Arrowheads (D and E), cells in mitosis. G and H, BrdUrd incorporation (12 h) and K1 expression in HaCaT cells cultured for 9 days and treated with KGF (G) for an additional 2 days or kept untreated (H). The number of cells double-positive for the BrdUrd nuclear staining (green) and for the cytosolic K1 signal (red) is increased in KGF-treated cultures (G, arrows) compared with the untreated cultures (H). The number of K1-expressing cells appears unmodified by the KGF treatment. Bar, 40 µm.

 
Discussion

Modulation in the number of functional growth factor receptors exposed on the cell surface to the action of the cognate ligands represents a key strategy used by the cellular machinery to regulate the proliferation rate and the differentiation process. The mechanisms responsible for such modulation may operate at different transcriptional, translational, and posttranslational levels, including ligand-induced down-regulation by endocytosis and subsequent intracellular receptor degradation. The receptor for KGF, among the growth factor receptors expressed on keratinocytes, is thought to play a unique crucial role in controlling epithelial proliferation and differentiation, and modulation of KGFR has been observed both in vivo and in vitro (6 , 15 , 22) . However, the functional roles and the mechanisms involved in such KGFR modulation still remain to be elucidated. To this purpose, the establishment and evaluation of useful cellular model systems naturally expressing the receptors and able to grow and differentiate in controlled culture conditions is of fundamental need. We analyzed here the nontransformed human keratinocyte cell line HaCaT, a widely used model to study keratinocyte differentiation both in vitro (18, 19, 20 , 23) and in surface transplants (17) , capable of producing a pseudo-stratified epithelium in culture. Our results demonstrate that, similarly to human primary cultured keratinocytes, HaCaT cells express KGFR and that the receptor expression is up-modulated during differentiation from basal to suprabasal cells. Both the coexpression of the K1 differentiation marker with the up-regulated KGFRs and the parallel morphological and ultrastructural observations of the cells grown at different densities clearly indicate that the increase in KGFR expression occurs during HaCaT differentiation and stratification. These findings parallel our previous observations reporting that KGFR expression increased during Ca2+induced differentiation of keratinocytes (15) ; however, here we demonstrated that KGFR up-modulation can be induced by a density-dependent process of differentiation and stratification, which is considered a more physiological stimulus for keratinocyte differentiation compared with that triggered by high Ca2+ concentrations in the growing medium (24) . In fact, in the normal epidermis also, the confluence of the keratinocytes in the basal tissue layer seems to represent the most relevant factor in determining growth arrest and commitment to differentiation; moreover, wounds of the epidermal tissues and subsequent loss of integrity and cell confluence in the basal layer are known to trigger hyperproliferation and reepithelialization.

Comparing KGFR expression at different HaCaT cell densities (corresponding to distinct stratification steps) with the expression of EGFR, we found that up-regulation occurs only for KGFR, whereas the amount of EGFR protein appears unmodified or slightly reduced. These results are consistent with: (a) the different distribution of KGFR and EGFR in normal human skin, because KGFRs are mainly present in the suprabasal spinous layers (6 , 14) , whereas EGFRs are predominantly distributed on the basal layer (25 , 26) ; (b) the different effects of KGF and EGF in response to the Ca2+ differentiative signal, because KGF promotes the expression in vitro of differentiation markers, whereas EGF blocks such expression (4 , 15) ; and (c) the different mitogenic action of KGF and EGF on confluent cells, because KGF exerts its mitogenic effect mostly after keratinocytes reach confluence, whereas the EGF effect decreases with confluence (5) . Taken together, all of these findings demonstrate that KGF and EGF play different roles in the control of keratinocyte proliferation and differentiation and that the expression of the receptors for these growth factors may be crucial for such control.

Furthermore, we showed here that KGFRs up-regulated during differentiation are functionally tyrosine-phosphorylated by the usual brief treatment with KGF used to activate the receptor (10 , 13) . However, it is noteworthy that this receptor activation seems to occur in suprabasal cells already committed to the differentiation program, as revealed by the presence of the K1 marker. Therefore, either the up-regulated KGFRs may control the residual proliferative activity in suprabasal cells or may play a role in some keratinocyte differentiative steps. The BrdUrd incorporation, which indicates active DNA synthesis in the suprabasal cells expressing KGFRs, seems to suggest that up-regulation of KGFRs in cells committed to differentiate may sustain proliferation. Consistent with this hypothesis, our findings that KGF treatment induces BrdUrd incorporation in K1-expressing cells strongly support a role of the KGF and its receptor in controlling proliferation in differentiating keratinocytes. Our results are also consistent with the observation that KGFRs, unlike other growth factor receptors, act as a potent mitogen on keratinocytes only after they attain confluence (5) , implying that cell-density plays a fundamental role in controlling KGFR function. Work is in progress to further elucidate the possible distinct roles of KGFRs during keratinocyte differentiation, which may depend not only on the number and expression of the receptors but probably on the differential activation of substrates and signal transduction pathways.

Materials and Methods

Cells.
HaCaT cells were cultured in DMEM supplemented with 10% FCS and antibiotics. To reach different levels of confluence and stratification, HaCaT cells were plated on coverslips previously coated with 2% gelatin (Sigma Chemicals Co., St. Louis, MO; 5000 cells/coverslip) onto 24-well plates. Cells were allowed to grow for 3 (preconfluent), 7 (confluent), and 11 (postconfluent) days. For KGF treatment in KGFR phosphorylation experiments, cells were serum-starved for 12 h and were incubated with KGF (Upstate Biotechnology, Lake Placid, NY; 100 ng/ml in medium containing 0.3 M NaCl) for 10 min. at 37°C or were kept untreated as control after starvation.

Immunofluorescence.
Cells were fixed in 4% paraformaldehyde in PBS for 30 min at 25°C and permeabilized with 0.1% Triton X-100 in PBS for 10 min. For double-staining experiments, cells were incubated with KGF-HFc 0.3 M NaCl and anti-Bek polyclonal antibodies (1:10 in PBS; C-17; Santa Cruz Biotechnology Inc, Santa Cruz, CA) or anti-HK1 polyclonal antibodies (1:50 in PBS; Berkeley Antibody Company, Richmond, CA).

To analyze KGFR phosphorylation, cells untreated or treated with KGF as above, were fixed in methanol at -20°C for 4 min and then incubated with anti-Bek polyclonal antibodies and antiphosphotyrosine monoclonal antibody (1:100 in PBS; UBI) for 1 h at 25°C.

The primary antibodies were visualized with goat antimouse IgG-FITC (1:10 in PBS; Cappel Research Products, Durham, NC) and goat antirabbit IgG-Texas Red (1:100 in PBS; Jackson Immuno Research Laboratories Inc., West Grove, PA), after appropriate washing with PBS.

Colocalization of the two fluorescence signals was analyzed by recording and merging single-stained images using a cooled CCD color digital camera SPOT-2 (Diagnostic Instruments Incorporated, Sterling Heights, MI) and FISH 2000/H1 software (Delta Sistemi, Roma, Italy).

Electron Microscopy.
HaCaT cells, grown on coverslips as above, were washed three times in PBS and fixed with 2% glutaraldehyde in the same buffer for 60 min at 4°C. Samples were postfixed in 1% osmium tetroxide in veronal acetate buffer (pH 7.4) for 2 h at 4°C and were stained with uranyl acetate (5 mg/ml), dehydrated in acetone, and embedded in Epon 812. Thin sections were examined unstained or poststained with uranyl acetate and lead hydroxide.

BrdUrd Incorporation.
For BrdUrd incorporation assay, cells grown on tissue culture chamber slides (Nunc Inc., Naperville, IL), were incubated with 100 µM BrdUrd (Sigma) for 24 h at 37°C to allow BrdUrd incorporation. BrdUrd incorporation was also performed in some experiments by incubation with 100 µM BrdUrd as above for 1 h at 37°C. Cells were then fixed in 4% formaldehyde in PBS for 30 min at 25°C, followed by treatment with 0.1 M glycine for 20 min. at 25°C and with 0.5% HCl/0.1% Triton X-100 for an additional 45 min at 25°C to allow permeabilization. After extensive washing in PBS, cells were buffered with 0,1 M Na2B4O7 and incubated with anti-BrdUrd monoclonal antibody [1:50 in blocking buffer (PBS 0.5% BSA and 0.5% Tween 20; Sigma] for 30 min at 25°C in humidity chamber, followed by goat antimouse IgG-Texas Red (1:50 in blocking buffer; Jackson Immuno Research Laboratories Inc., PA). For double-staining experiments, cells were then incubated with anti-Bek or anti-HK1 polyclonal antibodies and by the secondary antibodies as above. For KGF treatment, cells cultured for 9 days after plating were incubated with KGF (20 ng/ml) for an additional 48 h before BrdUrd incubation (1 h or 12 h at 37°C).

Western Blot Analysis.
Cells, cultured for 3, 6, and 11 days after plating, were lysed in radioimmunoprecipitation assay buffer [10 mM Tris (pH 7.4), 50 mM NaCl, 1 mM EDTA, 10 mM KCl, 1% NP40, 0.1% SDS, and 0.05% Tween 20) supplemented with protease inhibitors (10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin). Total proteins (25 µg) were resolved under reducing conditions by 10% SDS-PAGE and transferred to reinforced nitrocellulose (PROTRAN; Schleider & Schuell, Keene, NH). The membrane was blocked with 3% nonfat dry milk in PBS with 0.05% Tween 20 for 4 h at room temperature. The membrane was incubated with anti-K1 polyclonal antibodies (1:500 in PBS, 0.05% Tween 20, and 3% nonfat dried milk; Berkeley Antibody Company), with anti-Bek monoclonal antibody, (1:200 as above; C8; Santa Cruz) or with anti-EGFR 1005 monoclonal antibody (1:500 as above; 1005; Santa Cruz) for 1 h at room temperature, followed by enhanced chemiluminescence detection (Amersham Corp., Arlington Heights, IL).

Footnotes

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.

1 This work was partially supported by grants from Ministero dell’Università e Ricerca Scientifica e Tecnologica, from Associazione Italiana per la Ricerca sul Cancro, and from Consiglio Nazionale delle Ricerche (Target Project on "Biotechnology"), Italy. Back

2 A. C. and V. V. contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Università di Roma "La Sapienza," Dipartimento Medicina Sperimentale e Patologia, Viale Regina Elena 324, 00161 Roma, Italy. Phone: 3906-4468450; Fax: 3906-4468450; E-mail: torrisi{at}uniroma1.it Back

4 The abbreviations used are: KGF, keratinocyte growth factor; KGFR, KGF receptor; FGF, fibroblast growth factor; FGFR, FGF receptor; EGFR, epidermal growth factor receptor. Back

Received for publication 6/ 9/00. Revision received 9/26/00. Accepted for publication 9/27/00.

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