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Activation of Mitogen-Activated Protein Kinase Pathway by Keratinocyte Growth Factor or Fibroblast Growth Factor-10 Promotes Cell Proliferation in Human Endometrial Carcinoma Cells

Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago 683-8504, Japan

Address all correspondence and requests for reprints to: Fuminori Taniguchi, M.D., Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago 683-8504, Japan. E-mail: tani4327{at}grape.med.tottori-u.ac.jp.

	Abstract

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Fibroblast growth factors (FGFs) exert diverse effects resulting from their interaction with cognate receptors on target cells. Our current study was designed to examine the local production and action of two specific stromal-epithelial cell mediatory factors, keratinocyte growth factor (KGF) and FGF-10, in human endometrial carcinoma cells. The RT-PCR method was used to determine gene expression of KGF, FGF-10, and KGF receptor in human endometrial carcinoma cells (HEC-1) and human endometrial stromal cells. KGF mRNAs were expressed in both of these cell types. On the other hand, FGF-10 mRNA was detected only in the endometrial stromal cells, and KGF receptor mRNA was observed in the HEC-1 cells. The novel finding of the present study is that KGF is expressed in carcinoma cells and FGF-10 is expressed in human endometrial stromal cells. The distinct phosphorylation of ERK-1 and -2 (ERK1/2), which are members of the MAPK family, was observed when HEC-1 cells were treated with KGF or FGF-10. KGF and FGF-10 could induce the prompt phosphorylation of ERK1/2 and consequently stimulate DNA synthesis. KGF and FGF-10 did not activate the phosphorylation of Akt, protein kinase C, or signal transducer and activator of transcription-3. Blocking the MAPK pathway with the specific methyl ethyl ketone 1/2 inhibitor (U0126) completely neutralized the enhancement of cell proliferation induced by KGF and FGF-10. In addition, KGF and FGF-10 activated expressions of downstream nuclear transcription factors, such as Elk-1 and c-myc, but not c-fos. These results demonstrate for the first time that KGF and FGF-10 are capable of stimulating the growth of endometrial carcinoma cells via activating MAPK pathway through autocrine/paracrine fashion.

	Introduction

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ENDOMETRIAL CANCER IS a common neoplastic disease among women. Our lack of knowledge about the molecular mechanisms involved in the progression of endometrial cancer has hampered the development of an effective treatment. Growth and differentiation of epithelial cells are known to be directed by adjacent mesenchymal cells during embryonic development and optimally maintained by adjacent stroma in adult tissues. Identifying and characterizing the inducer proteins that mediate stromal-epithelial cell interactions are necessary to understand the underlying mechanisms.

The fibroblast growth factor (FGF) family consists of at least 23 structurally related ligands (1) that can activate the cell surface tyrosine kinase receptors to elicit a wide range of cellular responses (2). FGF receptors (FGFRs) (FGFR1-FGFR4) show overlapping ligand-binding specificities; furthermore, an alternative splicing mechanism generates receptor isoforms with altered ligand-binding properties (3).

Keratinocyte growth factor (KGF) (FGF-7) is primarily produced by stromal- or mesenchymal-derived cells in many tissues and acts as an epithelial cell-specific mitogen (4, 5). In particular, KGF mediates mesenchymal-epithelial interactions in many tissues, including the ovary, placenta, endometrium, and seminal vesicle (6, 7, 8, 9, 10). Among FGFs, KGF is unique because it interacts only through the KGF receptor (KGFR, also known as FGFR2IIIb), which is expressed exclusively by epithelial cells, indicating that it may have target cell specificity. FGF-10 was recently identified in the human lung and prostate (11). FGF-10, like KGF, is considered to be the stromal cell-derived growth factor and exhibits close structural and functional similarities to KGF (12, 13). FGF-10 has the ability to bind only FGFR1IIIb and KGFR.

The mechanism of intracellular signaling of the KGF, FGF-10, and KGFR system has not been investigated. KGFR mediates its signal through a dimerization event and phosphorylation cascade. FGFs activate some downstream signaling pathways, including MAPKs. The key MAPK cascade is ERK, also known as classical Ras/Raf/MAPK kinase/MAPK pathway (14). The activation of ERK-1 and -2 (ERK1/2) occurs via phosphorylation by cytoplasmic dual-specificity MAPK kinases, MEK-1 and -2 (MEK1/2), and is often associated with the stimulation of cell proliferation. In the nucleus, ERK1/2 is supposed to phosphorylate and activate some transcription factors, e.g. Elk-1, c-myc, and c-fos.

Although KGF expression in the uterus of several species has been reported, KGF and FGF-10 expression and their functions have not been determined in human endometrial carcinoma cells. The purpose of the present study was to determine the extent to which KGF or FGF-10 participates in the progression of human endometrial adenocarcinoma cells.

	Materials and Methods

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Cell culture

The human endometrial carcinoma cell (HEC)-1 is a model of a moderately well differentiated human endometrial adenocarcinoma. The BeWo cell is considered a human choriocarcinoma cell line (15). For this study, both HEC-1 and BeWo cells were obtained from Human Science Research Resources Bank (Osaka, Japan). The Ishikawa cell (ISHIKAWA 3-H-12 no. 56), a model of well differentiated endometrial adenocarcinoma, was kindly provided by Dr. M. Nishida (Tsukuba University, Ibaragi, Japan). The HEC-1 cell line was maintained in Eagle minimal essential medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) with 10% fetal bovine serum (Sigma, St. Louis, MO). Human endometrial stromal cells were collected from normal endometrial tissue obtained from cycling women who underwent hysterectomy for uterine myoma (n = 6). We confirmed that pathological examination excluded malignancy and patients did not use hormonal therapy within the 3 months before tissue collection. All patients gave their informed consent before collecting the specimens.

Approval of this project was obtained from the Institutional Review Boards of Tottori University School of Medicine. Stromal cells were collected according to the method of Osteen et al. (16) as described previously (17). We used stromal cells in a monolayer culture after the first passage.

Immunohistochemical analysis of the isolated endometrial stromal cells was performed using cytokeratin, vimentin, and factor VIII (DAKO Corp., Kyoto, Japan) to confirm the purification of the stromal cells. The results showed that the purity of stromal cells was more than 98%.

RT-PCR

We performed the RT-PCR method to determine the gene expression of KGF, FGF-10, and KGFR in the HEC-1, and Ishikawa, and human endometrial stromal cells collected during the proliferative and secretory phases. Total RNA was extracted from the HEC-1, Ishikawa, and the cultured endometrial cells by the guanidinium thiocyanate method described (18). The BeWo cells, which express both KGF and KGFR (15), were used as the positive control. Detailed procedures for RNA extraction followed the manufacturer’s protocol (Isogen, Nippon Gene, Tokyo, Japan). The reverse transcription (RT) of RNA from cultured cells into cDNA and PCR was performed using the Gene Amp RNA PCR core kit (Perkin-Elmer, Branchburg, NJ). RNAs (1 µg) extracted from each of the cells were used. The RT of RNA to cDNA was accomplished with 2.5 U/µl of MuLV RT as follows: 10 min at 30 C, 20 min at 47 C, 5 min at 99 C, and 5 min at 4 C. PCR amplification was carried out in 25 mM MgCl2 solution (2 mM), 10x PCR buffer II (1x), water (65.5 µl), and Ampli Taq DNA polymerase (2.5 U/100 µl). Each sample was amplified for 30 cycles. The cycles consisted of 30 sec at 94 C, 30 sec at 60 C, 90 sec at 72 C.

For the RT-PCR, the specific primers for KGF, FGF-10, KGFR, and the glycerol-3-phosphate dehydrogenase (G3PDH) were used (15, 19, 20). The details of the primers and PCR products are shown in Table 1. The specificity of each PCR product was also confirmed by Southern blot analysis. The PCR products were transferred onto a nylon membrane by means of a vacuum blotter with 0.4 M NaOH and 1 M NaCl. The DNA on the membrane was fixed with a UV cross-linker and hybridized with biotinylated oligonucleotide internal probes (Table 1). We used the RT-PCR probes for each product spanned exon-intron junctions to exclude the possibility of genomic DNA contamination. The membrane was treated with streptavidin-alkaline phosphatase, followed by chemiluminescence detection. The membrane was then exposed to x-ray film for 15 min at room temperature. Densitometric analysis of the PCR products was performed using a public domain NIH image program (written by Wayne Rasband, NIH). The densitometric values of KGF and FGF-10 mRNAs were normalized with the corresponding G3PDH mRNAs. Representative bands from each PCR reaction were isolated from the gels and sequenced to ensure that the correct sequence was being amplified. Sequencing was performed by using a model of genetic analyzer (310, PE Applied Biosystems, Foster City, CA).

The mitogenic activity assay with recombinant human KGF or FGF-10 (Pepro Technology, London, UK) was performed on the HEC-1 cells. The HEC-1 cells were diluted with 100 µl Eagle minimal essential medium plus 0.1% BSA (Sigma, Tokyo, Japan) to a seeding density of 2 x 10³/well and cultured overnight in 96-well culture plates. The cells were then treated with various concentrations of KGF or FGF-10 (0–100 ng/ml) for 48 h. In neutralization studies, antibodies for KGF (Austral Biologicals, San Ramon, CA) or FGF-10 (Genzyme-Techne, Cambridge, MA) were added at a concentration of 1 µg/ml. An antibody against human IgG (1 µg/ml monoclonal mouse antihuman IgG1; Cosmo Bio. Co., Ltd., Tokyo, Japan) was used as a control. To examine DNA synthesis in proliferating cells, BrdU incorporation was assessed with a kit, cell proliferation ELISA system (Amersham Pharmacia, Tokyo, Japan). Briefly, BrdU was added to the cells and these were incubated for 4 h. During this labeling period, the pyrimidine analog BrdU was incorporated in place of thymidine into the DNA of proliferating cells. The peroxidase-labeled anti-BrdU bound to the BrdU incorporated in newly synthesized, cellular DNA. The absorbance values correlate directly to the amount of DNA synthesis and thereby to the number of proliferating cells in culture. Absorbance was measured at 450 nm with a microplate reader (Bio-Rad Laboratories, Inc., Richmond, CA). The ratio to the mean value of control was used for comparison. Results are presented as means ± SE of triplicate assays.

Western blot analysis

Subconfluent HEC-1 cells were incubated overnight in the absence of serum and then treated with various materials. The cells were lysed with ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1 mM dithiothreitol, 10 mM NaF, 2 mM Na3VO4, and 1x Complete Protease Inhibitor Cocktail (Roche Molecular Biochemicals, Ingelheim, Germany). The lysate was centrifuged and the supernatant prepared. Protein concentration of the lysate supernatant was determined by Bradford’s assay (21). Fifty micrograms of each soluble protein sample were separated by 12% SDS-PAGE, blocked in 2.5% skim milk/TPBS (1x PBS and 0.1% Tween 20). These samples were probed with each primary antibody overnight at 4 C. After incubation with horseradish peroxidase-conjugated secondary antibody (1:2000), protein signals were detected by using enhanced chemiluminescence (Amersham Pharmacia, Little Chalfont, UK). The immunoblots were quantitated using the NIH image program.

Members of the MAPK family, ERK1/2, are activated via phosphorylation by MEK1/2, which is the upstream activator. This cascade is associated with the stimulatory signals of cell proliferation. The effect of KGF or FGF-10 (100 ng/ml) on phosphorylation was assessed using the following: 1) antiphospho-Raf-1 (detects only when phosphorylated at Ser259); 2) antiphospho-MEK-1/2 (Ser 217/221); 3) antiphospho-ERK1/2 (Thr202/Tyr204); 4) anti-ERK1/2; 5) antiphospho-protein kinase C (PKC) (pan; detects PKC, ß I, ß II, and isoforms only when phosphorylated at a carboxy-terminal residue homologous to Ser660 of PKCß II); 6) PKC-ß; 7) antiphospho-Akt (Ser473); 8) anti-Akt; and 9) antiphospho-STAT3 (signal transducers and activators of transcription) (Tyr705); and 10) anti-STAT3. These reagents were obtained from Cell Signaling Technology (Beverly, MA). Furthermore, we evaluated the effect of an MEK1/2 inhibitor, U0126, (Promega Corp., Madison, WI), on ERK1/2 signaling. The cells were pretreated with 50 µM U0126 for 30 min and then treated with 100 ng/ml KGF or FGF-10. After incubation for 10 min, we harvested the cells for protein extraction and performed Western blot analysis by the above-mentioned method.

Nuclear transcription factors, such as Elk-1, c-myc, and c-fos, are phosphorylated by ERK1/2. These factors have been implicated in cell growth and differentiation. To evaluate the effects on nuclear factor activation of KGF or FGF-10 (100 ng/ml), antiphospho-Elk-1 (Ser383, Santa Cruz Biotechnology, Santa Cruz, CA), anti-c-myc (BD PharMingen, Franklin Lakes, CA), and anti-c-fos (Oncogene, Boston, MA) were used.

Statistical analysis

All experiments were repeated at least three times. Data are presented as means with SE. The ANOVA was used for the statistical analysis of BrdU proliferation assay and densitometric assay. A level of P less than 0.05 was considered statistically significant.

	Results

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Gene expressions of KGF, FGF-10, and KGFR in HEC-1 and human endometrial stromal cells

RT-PCR was performed to analyze the mRNA expression of KGF, FGF-10, and KGFR in the HEC-1 and human endometrial stromal cells. Southern hybridization of RT-PCR products showed that KGF transcripts were detected in all cell types. Sequencing revealed that the amplified bands were targeting products. On the other hand, FGF-10 mRNA was detected only in the endometrial stromal cells. KGFR mRNAs were observed in the endometrial carcinoma cells but not in the endometrial stromal cells (Fig. 1). Expressions of KGF and FGF-10 mRNAs were higher in the endometrial stromal cells collected during the secretory phase than in the proliferative phase by densitometric analysis (Fig. 2).

View larger version (35K): [in this window] [in a new window]   Figure 1. The expressions of KGF, FGF-10, and KGFR mRNAs in HEC-1 and human endometrial stromal cells. The PCR products for KGF (196 bp), FGF-10 (304 bp), KGFR (505 bp), and internal control G3PDH (450 bp) are indicated. Specificity of each PCR product was confirmed by Southern blot analysis. P-ESC, Proliferative endometrial stromal cells; S-ESC, secretory endometrial stromal cells; NC, reverse transcriptase (-). BeWo cells were used as the positive control of KGF and KGFR. These results represented pooled data.

View larger version (25K): [in this window] [in a new window]   Figure 2. Comparison of the expressions of KGF (A) and FGF-10 (B) mRNAs in human endometrial stromal cells (n = 6). Relative densitometric units of the bands are shown in the bottom panel. The levels of intensities of KGF and FGF-10 mRNAs were normalized with the corresponding ethidium bromide-stained G3PDH signals. Each bar represents the means with SE. Significant differences are shown by asterisks. *, P < 0.05.

Adding KGF or FGF-10 to the medium significantly increased cell proliferation in a dose-dependent manner. KGF or FGF-10 at a concentration of 100 ng/ml enhanced the level of BrdU incorporation up to 143% or 135% of the control, respectively (Fig. 3). Antibodies for KGF or FGF-10 specifically abrogated these stimulatory effects. Adding anti-KGF antibody alone decreased the proliferation of HEC-1 cells to 84% of the control (Fig. 3), implying that KGF works as an autocrine growth factor. An antihuman IgG antibody did not influence cell proliferation.

View larger version (18K): [in this window] [in a new window]   Figure 3. Comparison of mitogenic activity assay of recombinant human KGF and FGF-10 on HEC-1 cells. BrdU incorporation assay was performed. HEC-1 cells were cultured overnight and then treated with KGF or FGF-10 (0–100 ng/ml) for 48 h. Effects of KGF and FGF-10 were neutralized by 5 µg/ml of respective antibodies (KGF Ab and FGF-10 Ab). Results represent the mean ± SE of three different experiments. Significant differences are shown by asterisks. *, P < 0.05; **, P < 0.01 vs. control.

Time-course analyses of both 44 kDa kinase (ERK 1) and 42 kDa kinase (ERK 2) in HEC-1 cells during treatment with KGF or FGF-10 were examined using antiphospho-ERK1/2 antibody. KGF or FGF-10 rapidly activated ERK1/2. The activation of ERK1/2 was transient, decreasing gradually within 1 h. After the cells were exposed to KGF or FGF-10 (100 ng/ml), the early phosphorylation of ERK1/2 reached a maximal level after 15 min and 5 min, respectively (Fig. 4). The levels of 42 kDa MAPK were higher than those of the 44 kDa MAPK. The consecutive phosphorylations of Raf-1, MEK1/2, and ERK1/2 were observed by KGF or FGF-10 (Fig. 5A). In contrast to the activation of MAPK pathway, KGF and FGF-10 had no effect on the activation of phospho-PKC (pan; 78, 80, 82, and 85 kDa) and phospho-Akt (62 kDa). Phospho-STAT3 (92 kDa) was not detected (Fig. 5B).

View larger version (26K): [in this window] [in a new window]   Figure 4. The time course of ERK1/2 phosphorylation induced by KGF or FGF-10 in HEC-1 cells. KGF or FGF-10 (100 ng/ml) was added to the medium for 0–120 min as indicated. Phosphorylated and total ERK1/2 (44 and 42 kDa) were detected by Western blot analysis.

View larger version (31K): [in this window] [in a new window]   Figure 5. The effects of KGF or FGF-10 on phosphorylation of signal transduction pathways in HEC-1 cells by Western blot analysis. Cells were treated with KGF or FGF-10 (100 ng/ml) for 10 min. A, Phosphorylated Raf-1 (74 kDa), phospho-MEK1/2 (45 kDa), phospho- and total-ERK1/2 (44 and 42 kDa). B, Phospho-PKC (pan; 78, 80, 82, and 85 kDa), PKC-ß (80 kDa), phospho- and total-Akt (62 kDa), phospho- and total-STAT3 (92 kDa) were examined by each specific antibody. In STAT3, total cell extracts from Hela cells prepared with interferon- treatment serve as positive control.

To confirm that the activation of the MAPK pathway is required for KGF- or FGF-10-induced cell proliferation, HEC-1 cells were incubated with KGF or FGF-10 in the presence of U0126, a potent MEK1/2 inhibitor. Pretreating with U0126 completely blocked phosphorylation of the ERK1/2 activated by KGF or FGF-10 (Fig. 6A). In the BrdU proliferation assay, U0126 abolished the mitogenic activity of HEC-1 cells exerted by KGF or FGF-10 (Fig. 6B), suggesting that KGF and FGF-10 induced cell proliferation through the MAPK pathway.

View larger version (22K): [in this window] [in a new window]   Figure 6. The effects of kinase inhibitors on induced ERK1/2 activation and cell proliferation by KGF or FGF-10 in HEC-1 cells. Cells were preincubated with U0126 (50 µM) for 30 min before 10-min stimulation by growth factors. A, Phosphorylated and total ERK1/2 (44 and 42 kDa) were assessed by Western blot analysis. B, DNA synthesis was evaluated by BrdU incorporation assay. •, with U0126; , without U0126. Significant differences are shown by asterisks. *, P < 0.05; **, P < 0.01.

The phosphorylation of Elk-1 by KGF or FGF-10 in HEC-1 cells was examined. KGF or FGF-10 increased the expression of phospho-Elk-1 (68 kDa). FGF-10 dramatically stimulated the expression of c-myc protein (62 kDa), although the activation by KGF was weak. On the other hand, the expression of c-fos protein (62 kDa) was not changed (Fig. 7).

View larger version (29K): [in this window] [in a new window]   Figure 7. The effects of KGF or FGF-10 on the activation of nuclear transcription factors in HEC-1 cells by Western blot analysis. To analyze the phosphorylation of Elk-1 (68 kDa), cells were treated with KGF or FGF-10 (100 ng/ml) for 10 min. To analyze the expressions of c-myc (62 kDa) and c-fos protein (62 kDa), cells were treated for 24 h. Comparison of expression of phospho-Elk-1, c-myc, and c-fos was presented. Relative densitometric units of the bands are shown in the bottom panel, with the density of the control bands set arbitrarily at 1.0. Each bar represents the means with SE. Significant differences are shown by asterisks. *, P < 0.05; **, P < 0.01.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The expressions of KGF and FGF-10 have been investigated in the endometrium of experimental animals as well as in humans. For example, in the rhesus monkey, KGF mRNA expression was progesterone dependent in the uterine endometrium (8). As shown in Fig. 2, KGF and FGF-10 mRNAs were pronounced in the human endometrial stromal cells collected during the secretory phase, compared with the proliferative phase. It has been reported that gene expression of leukemia inhibitory factor, macrophage colony-stimulating factor, IL-1ß, and IL-15 increase during the secretory phase of human endometrium (22, 23, 24, 25). These findings, together with ours, suggested a possible role of KGF or FGF-10 as a factor that mediated the action of progesterone. KGF or FGF-10, which is derived from the uterine stromal cells, may be novel paracrine mediators of endometrial epithelial function.

Only two reports have investigated the role of KGF on endometrial carcinoma. Although Pekonen et al. (26) failed to show KGF expression in human endometrial adenocarcinoma cells, in our study we detected the presence of KGF transcripts in endometrial cancer cell lines, such as HEC-1 and Ishikawa cells. Siegfried et al. (27) suggested that KGF mRNA expression was significantly lower in endometrial adenocarcinoma tissue, compared with cycling endometrial tissue. However, as shown in Fig. 1, we could not observe a difference between the levels of KGF mRNA expression in HEC-1 and Ishikawa cells and that of cultured endometrial stromal cells during the secretory phase. This discrepancy may be due to the inevitable contamination of stromal or epithelial cells in uterine specimens collected during gynecologic surgery.

Previous studies of FGF-10 suggested that its expression is restricted to the mesenchymal cells, compared with other FGF family members. FGF-10 is expressed by a limited number of embryonic tissues and ductal organs, such as fetal lung, limb bud, and developing prostate, and by even fewer adult tissues (28, 29). The present study is the first report showing the involvement of FGF-10 in the growth of endometrial carcinoma cells. The distinct nonoverlapping patterns of expression of KGF and FGF-10 in HEC and endometrial carcinoma cells suggest independent roles in the progress of endometrial carcinoma.

We also showed a cell-specific system of regulation that restricted KGF expression to the stromal cells and KGFR expression to the epithelial cells in the endometrium. Derangement of this regulatory system may bring disastrous consequences. Miki et al. (19) reported that the forced expression of the KGFR in the fibroblast cells producing KGF led to malignant transformation of the cells. The finding of the present study is that the expression of KGF together with KGFR mRNAs in the HEC-1 cells may be one such example in human cancer cells.

KGF or FGF-10 also works as a paracrine growth-promoting factor in androgen-dependent human prostate (11), supporting our findings that KGF or FGF-10 significantly induced the mitogenic activity in HEC-1 cells. Based on knowledge of the involvement of a number of growth factors and their receptors in the progression of malignancy, it will be important to further investigate the role of the KGF or FGF-10/KGFR in human epithelial tumors as well as mechanisms that may lead to its oncogenic activation.

The precise mechanisms of intracellular KGF/FGF-10 signaling remain to be elucidated. Epidermal growth factor has been reported to activate the MAPK, the phosphatidyl inositol 3-kinase, and the JAK-STAT pathways for exerting its effects on target cells (30, 31, 32). We investigated possible pathways for KGF or FGF-10 stimulation. Interestingly, a transient dual phosphorylation of ERK1/2 induced by KGF or FGF-10 was specifically observed in HEC-1 cells. Like most receptor tyrosine kinase-activating growth factors, the present results demonstrated that KGFR signal passed through the MAPK pathway. Recently it was reported that KGF activated ERK1/2 in a porcine trophectoderm cell line (33). KGF and FGF-10 recognizes only KGFR with high affinity, precluding the possibility that ERK 1/2 is activated by other members of the FGFR family.

MEKs are the only known upstream activators of ERKs. As seen from a representative Western blot analysis (Fig. 5A), U0126, the specific MEK1/2 inhibitor, completely blocked the phosphorylation of ERK1/2. Strikingly, U0126 also inhibited completely the cell proliferation induced by KGF or FGF-10. Therefore, our results clearly demonstrated that the phosphorylation of ERK1/2 is essential for the proliferation of HEC-1 cells. Elk-1, c-myc, and c-fos as nuclear transcription factors are probably major downstream targets in MAPK pathway. We indicated that KGF or FGF-10 stimulated expression of phospho-Elk-1 and c-myc protein, although the activation of the factors by KGF was weak. The activator protein-1 complex of transcription factors, which is composed of the Jun and Fos proteins, is related to many oncogenic signal transduction pathways. We confirmed by luciferase reporter assay that KGF or FGF-10 did not activate the activator protein-1 site (data not shown).

Like most epithelial malignancies, endometrial carcinoma results from the accumulation of several genetic and epigenetic alterations in oncogenes (K-ras), tumor suppressor genes (PTEN), or genes involved in DNA repair. The accumulative effect of these abnormalities is responsible for the transition from normal endometrial cells to hyperplasia and carcinoma. Once the tumor has developed, several additional molecular abnormalities occur in different neoplastic subclones; these new alterations are responsible for tumor heterogeneity, tumor invasion, and metastasis. Characterization of the distinct signaling pathway and the mechanism of gene activation of KGF or FGF-10 may lead to identifying specific targets for novel cancer therapy.

In conclusion, our findings demonstrated that KGF and FGF-10 may play significant roles in the growth of endometrial carcinoma cells via the activating MAPK pathway.

	Acknowledgments

 

	Footnotes

 
Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; FGF, fibroblast growth factor; FGFR, FGF receptor; G3PDH, glycerol-3-phosphate dehydrogenase; HEC, human endometrial carcinoma; KGF, keratinocyte growth factor; KGFR, KGF receptor; PKC, protein kinase C; RT, reverse transcription; STAT, signal transducers and activators of transcription.

Received July 8, 2002.

Accepted November 3, 2002.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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