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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)
. A parallel increase of K1 expression was also detected
(Fig. 1A)
, 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)
. 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)
. 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.
|
, 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. 2D
2E
2F
, 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.
|
(Fig. 4A)
the signal corresponding to KGFR
(green in Fig. 3B
) appeared very weak. These
cells were mostly negative for K1 (red, Fig. 3C
),
although, occasionally, a few K1-positive cells, basally positioned,
could be detected (Fig. 3C
, arrows), as expected
(19)
. After 7 days of culture, cells were postconfluent,
densely packed and stratified in areas of the culture plate (Fig. 3E)
. Double staining for KGFR and K1 as above revealed that
the suprabasal cells were positive for both KGFR (green in
Fig. 3F
) and K1 (red in Fig. 3G
),
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.
|
|
. At 7 days of culture, cells were stratified in
multilayers (Fig. 4, B and C)
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)
, 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 D
; red) and antiphosphotyrosine
(Fig. 5, B and E
; green) antibodies.
In untreated cells (Fig. 5, A–C)
, the signal for
phosphotyrosine residues was undetectable (Fig. 5B)
, whereas
in KGF-treated cells (Fig. 5
, 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.
|
antibodies or with anti-Bek and
anti-BrdUrd (Fig. 6
, D–F) antibodies. At
3 days after plating (Fig. 6, A and D)
, 
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)
in confluent cells and 32% in
stratified cultures at 11 days (Fig. 6, C and F)
.
Interestingly, a significant portion of the
BrdUrd+ cells at both 6 and 11 days were positive
also for K1 (Fig. 6, B and C)
and KGFR (Fig. 6, E and F)
, 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. 6G
, arrows) compared
with control (untreated) cells (Fig. 6H)
, whereas the K1
expression was virtually unmodified by KGF treatment. Thus, KGF
stimulates BrdUrd incorporation in K1-expressing suprabasal cells.
|
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. 
2 A. C. and V. V. contributed equally to this
work.
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
4 The abbreviations used are: KGF, keratinocyte
growth factor; KGFR, KGF receptor; FGF, fibroblast growth factor; FGFR,
FGF receptor; EGFR, epidermal growth factor receptor.
Received for publication 6/ 9/00. Revision received 9/26/00. Accepted for publication 9/27/00.
References
expression and activate the epidermal growth factor receptor signaling pathway in cultured epidermal keratinocytes. Cell Growth Differ., 5: 1283-1292, 1994.[Abstract]
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