Rat Ovarian Surface Epithelial Research Articles

The Calcium-Sensing Receptor and the Reproductive System.

Active placental transport of maternal serum calcium (Ca2+) to the offspring is pivotal for proper development of the fetal skeleton as well as various organ systems. Moreover, extracellular Ca2+ levels impact on distinct processes in mammalian reproduction. The calcium-sensing receptor (CaSR) translates changes in extracellular Ca2+-concentrations into cellular reactions. This review summarizes current knowledge on the expression of CaSR and its putative functions in reproductive organs. CaSR was detected in placental cells mediating materno-fetal Ca2+-transport such as the murine intraplacental yolk sac (IPYS) and the human syncytiotrophoblast. As shown in casr knock-out mice, ablation of CaSR downregulates transplacental Ca2+-transport. Receptor expression was reported in human and rat ovarian surface epithelial (ROSE) cells, where CaSR activation stimulates cell proliferation. In follicles of various species a role of CaSR activation in oocyte maturation was suggested. Based on studies in avian follicles, the activation of CaSR expressed in granulosa cells may support the survival of follicles after their selection. CaSR in rat and equine sperms was functionally linked to sperm motility and sperm capacitation. Implantation involves complex interactions between the blastocyst and the uterine epithelium. During early pregnancy, CaSR expression at the implantation site as well as in decidual cells indicates that CaSR is important for blastocyst implantation and decidualization in the rat uterus. Localization of CaSR in human extravillous cytotrophoblasts suggests a role of CaSR in placentation. Overall, evidence for functional involvement of CaSR in physiologic mammalian reproductive processes exists. Moreover, several studies reported altered expression of CaSR in cells of reproductive tissues under pathologic conditions. However, in many tissues we still lack knowledge on physiological ligands activating CaSR, CaSR-linked G-proteins, activated intracellular signaling pathway, and functional relevance of CaSR activation. Clearly, more work is required in the future to decode the complex physiologic and pathophysiologic relationship of CaSR and the mammalian reproductive system.

Treatment of Adenosine Activates Mitogen-activated Protein Kinases via Activation of Transcriptional Factor, Elk-1, in Rat Ovarian Surface Epithelial Cells.

Extracellular adenosine has been suggested to play a role in cellular proliferation and intracellular calcium concentrations (Ca2+) in ovarian cells. To investigate the role of adenosine in rat ovarian folliculogenesis or steroidogenesis, we examined the expression of the adenosine receptors and effect of adenosine on growth stimulation in rat ovarian surface epithelial (OSE) cells. Rat OSE cells were isolated and cultured in DMEM media with 10% FBS. Our results indicated that adenosine mRNA was expressed and adenosine exerted a growth-stimulatory effect in rat OSE cells. To investigate the mechanism of the growth-stimulatory effect, the activation of mitogen-activated protein kinases (MAPKs) by adenosine was examined. Treatment with adenosine resulted in MAPK activation in these cells, whereas the stimulatory effect of adenosine in the cellular proliferation and MAPK activation was completely abolished in the presence of PD98059 (a MAPK/ERK Kinase, MEK inhibitor) and staurosporin (a Protein Kinase C, PKC inhibitor), suggesting that the growth stimulatory effect of adenosine may be mediated via PKC-dependent MAPK activation in rat OSE cells. In a time-dependent study, adenosine significantly increased MAPK activity at 5-20 min and the activated MAPK declined to control level after 20 min in these cells. Similarly, treatment with adenosine significantly induced MAPK activation after 5 min and sustained for 60 min in these OSE cells. In addition, treatment with adenosine resulted in substantial phosphorylation of Elk-1, confirming that adenosine action is mediated by activation of MAPK. In conclusion, we demonstrated that adenosine receptor was expressed and adenosine induced a growth-stimulation in rat OSE cells. Furthermore, the treatment with adenosine resulted in the activation of MAPK cascade and phosphorylation of Elk-1 in these cells. Taken together, these results suggest that the MAPK cascade may be involved in the growth stimulation in response to adenosine in rat OSE cells.[This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2010-0003093).] (poster)

183 MITOGEN-ACTIVATED PROTEIN KINASES ARE ACTIVATED BY ADENOSINE TRIPHOSPHATE IN RAT OVARIAN SURFACE EPITHELIAL CELLS

Extracellular ATP has been suggested to play a role in cellular proliferation and intracellular calcium concentrations (Ca2+) in ovarian cells. To investigate the role of ATP in rat ovarian folliculogenesis or steroidogenesis, we examined the expression of the P2U purinoceptor (P2U-R) and the effect of ATP on growth stimulation in rat ovarian surface epithelial (OSE) cells. Rat OSE cells were isolated and cultured in DMEM media with 10% fetal bovine serum. Our results indicated that P2U-R mRNA was expressed and that ATP exerted a growth-stimulatory effect in rat OSE cells. To investigate the mechanism of the growth-stimulatory effect, we examined the activation of mitogen-activated protein kinases (MAPK) by ATP. Treatment with ATP resulted in MAPK activation in these cells, whereas the stimulatory effect of ATP in cellular proliferation and MAPK activation was completely abolished in the presence of PD98059 [a MAPK/ERK kinase (MEK) inhibitor] and staurosporin [a protein kinase C (PKC) inhibitor], suggesting that the growth-stimulatory effect of ATP may be mediated via PKC-dependent MAPK activation in rat OSE cells. In a time-dependent study, ATP significantly increased MAPK activity at 5 to 20 min, and the activated MAPK in these cells declined to the control level after 20 min. Similarly, treatment with ATP significantly induced MAPK activation after 5 min and sustained it for 60 min in these OSE cells. In addition, treatment with ATP resulted in substantial phosphorylation of Elk-1, confirming that ATP action is mediated by activation of MAPK. In conclusion, we demonstrated that P2U-R was expressed and that ATP induced growth stimulation in rat OSE cells. Furthermore, treatment with ATP resulted in activation of the MAPK cascade and phosphorylation of Elk-1 in these cells. Taken together, these results suggest that the MAPK cascade may be involved in growth stimulation in response to ATP in rat OSE cells. This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST; No. 2010-0003093).

Ras-mediated Loss of the Pro-apoptotic Response Protein Par-4 Is Mediated by DNA Hypermethylation through Raf-independent and Raf-dependent Signaling Cascades in Epithelial Cells

The apoptosis-promoting protein Par-4 has been shown to be down-regulated in Ras-transformed NIH 3T3 fibroblasts through the Raf/MEK/ERK MAPK pathway. Because mutations of the ras gene are most often found in tumors of epithelial origin, we explored the signaling pathways utilized by oncogenic Ras to down-regulate Par-4 in RIE-1 and rat ovarian surface epithelial (ROSE) cells. We determined that constitutive activation of the Raf, phosphatidylinositol 3-kinase, or Ral guanine nucleotide exchange factor effector pathway alone was not sufficient to down-regulate Par-4 in RIE-1 or ROSE cells. However, treatment of Ras-transformed RIE-1 or ROSE cells with the MEK inhibitors U0126 and PD98059 increased Par-4 protein expression. Thus, although oncogenic Ras utilizes the Raf/MEK/ERK pathway to down-regulate Par-4 in both fibroblasts and epithelial cells, Ras activation of an additional signaling pathway(s) is required to achieve the same outcome in epithelial cells. Methylation-specific PCR showed that the par-4 promoter is methylated in Ras-transformed cells through a MEK-dependent pathway and that treatment with the DNA methyltransferase inhibitor azadeoxycytidine restored Par-4 mRNA transcript and protein levels, suggesting that the mechanism for Ras-mediated down-regulation of Par-4 is by promoter methylation. Support for this possibility is provided by our observation that Ras transformation was associated with up-regulation of Dnmt1 and Dnmt3 DNA methyltransferase expression. Finally, ectopic Par-4 expression significantly reduced Ras-mediated growth in soft agar, but not morphological transformation, highlighting the importance of Par-4 down-regulation in specific aspects of Ras-mediated transformation of epithelial cells.

Enhanced cathepsin L expression is mediated by different Ras effector pathways in fibroblasts and epithelial cells.

Ras expression induces increased expression and altered targeting of lysosomal proteases in multiple cell types, but the specific downstream cytoplasmic signaling pathways mediating these changes have not been identified. In this study, we compared the involvement of 3 major Ras effectors, Raf, phosphatidylinositol 3-kinase (PI3K) and Ral guanine nucleotide exchange factor (RalGEF) in the Ras-mediated alteration of lysosomal protease protein expression and targeting in rat 208F fibroblasts and rat ovarian surface epithelial (ROSE) cells. Effector domain mutants of Ras, constitutively activated variants of Raf, PI3K and RalGEF and pharmacologic inhibitors of MEK and PI3K were utilized to determine the role of these downstream pathways in mediating fibroblast transformation and lysosomal protease regulation in the fibroblasts and epithelial cells. We found that Raf activation of the ERK mitogen-activated protein kinase pathway alone was sufficient to cause morphologic and growth transformation of the fibroblasts and was necessary and sufficient to alter cathepsin L expression and targeting. In contrast, transformation and upregulation of cathepsin L expression in the epithelial cells required the activity of all 3 Ras effectors. Increased protease secretion from the epithelial cells was not observed on ectopic expression of Ras, as it was from the fibroblasts, consistent with the utilization of different signaling pathways in the 2 cell types. In neither cell type did Ras expression increase the expression, processing or secretion of 2 other major lysosomal proteases, cathepsin B and cathepsin D. Thus, Ras utilizes different effectors to mediate transformation and to deregulate cathepsin L expression and secretion in fibroblast and epithelial cells.

Identification of epidermal growth factor-responsive genes in normal rat ovarian surface epithelial cells

Alteration in epidermal growth factor receptor (EGFR) family signaling is among the most frequently implicated effectors of human oncogenesis. Overexpression of members of this family of receptors has often been detected in many epithelial tumors and is believed to be associated with an overall poor prognosis in patients with cancer. Therefore, we hypothesized that identification of potential EGF target genes in normal cells will provide a basis for unbiased genetic analysis of this signaling pathway in cancer. We utilized Atlas Rat 1.2 nylon cDNA arrays (Clontech) to determine gene expression changes in normal rat ovarian surface epithelial (ROSE) cells following EGF treatment. The results indicate activation of genes involved in a wide variety of cellular mechanisms, including regulation of cell cycle and proliferation, apoptosis, and protein turnover. In addition, using an in vitro model of ovarian cancer, we demonstrated that malignant transformation of ROSE cells resulted in alteration of downstream effectors of the EGFR pathway, as exemplified by aberrant expression of p66Shc, c-Jun, c-Myc, c- Fos, Lot1, p21Cip/Waf, and cdc25A. These data suggest that knowledge of the downstream genetic lesions, which may result in loss of growth factor requirement of the affected cells, will be crucial for the selection of the EGFR pathway as an effective target for cancer therapy.

Signal Transduction Mechanisms Linking Increased Extracellular Calcium to Proliferation in Ovarian Surface Epithelial Cells

Although ovarian surface epithelial (OSE) cells are the cell type responsible for malignant ovarian carcinoma, relatively little is known about either the extracellular stimuli or the intracellular signaling mechanisms responsible for regulating proliferation in these cells. We have demonstrated that OSE cells proliferate in response to elevation of extracellular calcium and that OSE cells express functional calcium-sensing receptors (CaR). Here we show that agonists of the CaR increase the kinase activity of Src and ERKs (extracellular signal-regulated kinases) in rat OSE cells and promote association between tyrosine-phosphorylated Shc and p120rasGAP. Expression of an interfering mutant CaR inhibited the proliferative response to elevated extracellular calcium, as well as CaR agonist-induced tyrosine phosphorylation and ERK activation. Transfection with dominant negative mutants of Ras, Raf, and MKK1 also inhibited the increase in ERK activity in response to calcium, as did treatment with herbimycin, a selective inhibitor for Src family kinases. These results indicate that the ability of OSE cells to proliferate in response to increases in extracellular calcium involves cross-talk between the G-protein-coupled CaR and the activation of a tyrosine kinase-dependent Ras–Raf–ERK signaling pathway.

Opposing actions of hepatocyte growth factor and basic fibroblast growth factor on cell contact, intracellular free calcium levels, and rat ovarian surface epithelial cell viability.

Previous studies demonstrated that cell-to-cell contact stimulates a tyrosine phosphorylation signal transduction pathway that prevents rat ovarian surface epithelial (ROSE) cells from undergoing apoptosis. Hepatocyte growth factor (HGF), also know as scatter factor (SF), is expressed by ovarian stromal and thecal cells and has been shown to reduce cell contact in nonovarian tissues. The present studies were designed to determine whether HGF/SF promotes ROSE cells to dissociate and subsequently become apoptotic. Because an increase in intracellular free calcium ([Ca2+]i) is often an early event in the apoptotic cascade, the effects of HGF/SF on [Ca2+]i levels were also assessed. ROSE cells were cultured in serum-free medium with HGF/SF, basic fibroblast growth factor (bFGF), thapsigargin, Bay K, actinomycin D, cycloheximide, and/or BAPTA depending on the experimental design. Cell contact was assayed by time-lapse photography; [Ca2+]i levels were measured with Fluo-3, and apoptosis was assessed by in situ DNA staining. HGF/SF decreased cell contact within 1 h, increased [Ca2+]i levels by 3 h, and induced apoptosis by 6 h of culture. bFGF inhibited these HGF/SF-induced responses. The increase in [Ca2+]i appears to represent a point in the apoptotic cascade that commits ROSE cells to die. This concept is based on the observations that: 1) in the presence of the calcium chelator BAPTA, HGF/SF decreased cell contact but did not increase [Ca2+]i or apoptosis; 2) bFGF blocked HGF/SF-induced increase in [Ca2+]i; 3) bFGF did not attenuate HGF/SF's apoptotic action if exposed to cells after the increase in [Ca2+]i; and 4) RNA and protein synthesis were required for HGF/SF to increase [Ca2+]i, whereas the thapsigargin- and Bay K-induced increase in [Ca2+]i and apoptosis were independent of RNA/protein synthesis. These observations indicate that the components of the apoptotic cascade distal to the increase in [Ca2+]i are present within ROSE cells and are activated by a sustained elevation of [Ca2+]i. The present studies also show that when ROSE cells establish contact with 3T3 cells that express N-cadherin, [Ca2+]i levels are maintained at low basal levels. In contrast, cell contact with 3T3 cells that do not express N-cadherin results in elevated [Ca2+]i levels. Similarly, a synthetic N-cadherin peptide, which inhibits homophilic N-cadherin binding, increases [Ca2+]i levels. Taken together, these data indicate that homophilic N-cadherin binding between adhering cells plays an important role in maintaining calcium homeostasis. Further, these data support the concept that HGF/SF's ability to promote the dissociation of ROSE cells accounts in part for its ability to increase [Ca2+]i levels.

Basic Fibroblast Growth Factor and N-Cadherin Maintain Rat Granulosa Cell and Ovarian Surface Epithelial Cell Viability by Stimulating the Tyrosine Phosphorylation of the Fibroblast Growth Factor Receptors

Both granulosa cells (GCs) and ovarian surface epithelial cells undergo apoptosis in vivo. Although basic fibroblast growth factor (bFGF) and N-cadherin-mediated cell contact inhibit GC apoptosis, little is known about the factors that influence rat ovarian surface epithelial (ROSE) cell apoptosis. The present studies were designed to determine whether bFGF and N-cadherin maintain the viability of both GC and ROSE cells by stimulating separate signaling pathways. For the GC studies, large GCs were collected from immature rat ovaries after Percoll gradient centrifugation and placed in serum-free culture for 24 h. These studies confirmed that about 10% of the aggregated GCs and more than 50% of single GCs were apoptotic after culture. bFGF reduced the percentage of apoptotic single GCs, but did not influence aggregated GCs. A neutralizing antibody to bFGF blocked bFGF’s antiapoptotic action, but did not alter the percentage of apoptotic aggregated GCs. The antibody to N-cadherin not only increased the percentage of aggregated apoptotic GCs, but also blocked bFGF’s ability to maintain the viability of single GCs. The effect of the FGF receptor antibody was similar to that of the N-cadherin antibody. Like GCs, ROSE cells also undergo apoptosis in serum-free medium. Exposure to either the N-cadherin or FGF receptor antibody, even in the presence of serum, increased the percentage of apoptotic aggregated ROSE cells. As tyrosine kinase activity is involved in maintaining cell viability, the pattern of tyrosine-phosphorylated proteins was examined after culture in control (ascites) or N-cadherin antibody-supplemented medium. Exposure to the N-cadherin antibody altered the pattern of tyrosine-phosphorylated proteins, decreasing the tyrosine phosphorylation of proteins in the 130- to 180-kDa range and increasing the tyrosine phosphorylation of one or more proteins of about 50 kDa. The identity of the 50-kDa protein is unknown. However, immunoprecipitation studies demonstrated that the N-cadherin antibody reduced the amount of tyrosine-phosphorylated FGF receptor in both GCs and ROSE cells by 50%. This decrease corresponds to an increase in apoptosis among aggregated cells. Taken together, these data suggest that homophilic N-cadherin binding and bFGF-FGF receptor binding activate signal transduction pathways that converge at the level of the FGF receptor and subsequently promote the viability of both GC and ROSE cells.

Basic fibroblast growth factor and N-cadherin maintain rat granulosa cell and ovarian surface epithelial cell viability by stimulating the tyrosine phosphorylation of the fibroblast growth factor receptors.

Both granulosa cells (GCs) and ovarian surface epithelial cells undergo apoptosis in vivo. Although basic fibroblast growth factor (bFGF) and N-cadherin-mediated cell contact inhibit GC apoptosis, little is known about the factors that influence rat ovarian surface epithelial (ROSE) cell apoptosis. The present studies were designed to determine whether bFGF and N-cadherin maintain the viability of both GC and ROSE cells by stimulating separate signaling pathways. For the GC studies, large GCs were collected from immature rat ovaries after Percoll gradient centrifugation and placed in serum-free culture for 24 h. These studies confirmed that about 10% of the aggregated GCs and more than 50% of single GCs were apoptotic after culture. bFGF reduced the percentage of apoptotic single GCs, but did not influence aggregated GCs. A neutralizing antibody to bFGF blocked bFGF's antiapoptotic action, but did not alter the percentage of apoptotic aggregated GCs. The antibody to N-cadherin not only increased the percentage of aggregated apoptotic GCs, but also blocked bFGF's ability to maintain the viability of single GCs. The effect of the FGF receptor antibody was similar to that of the N-cadherin antibody. Like GCs, ROSE cells also undergo apoptosis in serum-free medium. Exposure to either the N-cadherin or FGF receptor antibody, even in the presence of serum, increased the percentage of apoptotic aggregated ROSE cells. As tyrosine kinase activity is involved in maintaining cell viability, the pattern of tyrosine-phosphorylated proteins was examined after culture in control (ascites) or N-cadherin antibody-supplemented medium. Exposure to the N-cadherin antibody altered the pattern of tyrosine-phosphorylated proteins, decreasing the tyrosine phosphorylation of proteins in the 130- to 180-kDa range and increasing the tyrosine phosphorylation of one or more proteins of about 50 kDa. The identity of the 50-kDa protein is unknown. However, immunoprecipitation studies demonstrated that the N-cadherin antibody reduced the amount of tyrosine-phosphorylated FGF receptor in both GCs and ROSE cells by 50%. This decrease corresponds to an increase in apoptosis among aggregated cells. Taken together, these data suggest that homophilic N-cadherin binding and bFGF-FGF receptor binding activate signal transduction pathways that converge at the level of the FGF receptor and subsequently promote the viability of both GC and ROSE cells.

Determinants of Cisplatin Sensitivity in Normal versus Spontaneously Transformed Rat Ovarian Surface Epithelial Cells

The chemosensitivity of spontaneously transformed rat ovarian surface epithelial (ROSE) cell lines was compared to that of the parental cells from which they were derived. Cisplatin cytotoxicity was determined in three nontransformed (early passage) and three transformed (late passage) ROSE cell lines. Transformed cells were uniformly more sensitive to cisplatin than parental cells (1.5- to 2.6-fold) and grew more rapidly (doubling time range 15-22 hr) than parental cells (28-37 hr). Increased doubling time correlated with decreased cisplatin sensitivity (ρ = 0.771). Cisplatin accumulation did not correlate with cisplatin sensitivity. Glutathione (GSH) levels were higher in two of three early passage cell lines, but the correlation between GSH and decreased cisplatin sensitivity in the overall panel of cell lines was modest (ρ = 0.549). No statistically significant differences in DNA-platinum binding were observed between early and late passage cell lines. However, initial levels of DNA-bound platinum correlated with cisplatin sensitivity (ρ = 0.812) in the six-cell-line panel. GSH levels were inversely correlated with cisplatin accumulation (ρ = -0.829) and DNA platination (ρ = -0.786). Increased cisplatin sensitivity in spontaneously transformed ROSE cell lines showed a weak, inverse relationship with GSH levels, but more strongly correlated with their increased growth kinetics and to DNA platination.

An in vitro model of ovarian epithelial carcinogenesis: changes in cell-cell communication and adhesion occurring during neoplastic progression.

To investigate the cellular mechanisms of ovarian epithelial carcinogenesis, a series of progressively transformed rat ovarian surface epithelial (ROSE) cell lines were developed and studied. Transfection of primary ROSE cells and an immortalized ROSE line (ROSE 199) with the pSV3neo plasmid (SV40 T-antigen) yielded transformed lines which retained epithelial morphology. In vivo selection of these pSV3neo cell populations resulted in further phenotypic transformation. Transfection of ROSE 199 with pSV2neo/c-H-rasEJ (rasEJp21) resulted in a malignant line which appeared fibroblast-like and formed invasive sarcomas both in athymic mice and in immunocompetent rats. Gap junctional intercellular communication (GJIC) and cell-cell adhesion were studied in this series of ROSE lines. Both c-H-rasEJ-transformation and in vivo selection resulted in a significant reduction of GJIC between adjoining cells and a transition of in vitro migration as continuous epithelial sheets to the dissociation of individual cells. This apparent shift in cell adhesiveness was associated with reduced expression of the E-cadherin adhesion molecule. Our data suggest that neoplastic progression of the ovarian surface epithelium may be associated with concomitant reductions in GJIC, E-cadherin expression and functional adhesiveness.

Intermediate filaments in rat ovarian surface epithelial cells: changes with neoplastic progression in culture.

Interrelationships between neoplastic progression and the expression of intermediate filaments were examined in primary cultures, immortal lines, and Kirsten murine sarcoma virus (KiMSV) transformed lines of rat ovarian surface epithelial (ROSE) cells. Immunofluorescence microscopy revealed abundant keratin filaments in all cells of primary cultures. In immortal, nontumorigenic lines, keratin filaments were detected in fewer cells, in smaller numbers, and in microscopically altered forms. The percentage of keratin-positive cells ranged from 4 to 54%. Its expression was inversely proportional to cell density. Keratin expression was similar in the two immortal lines, although one had retained a monolayered epithelial growth pattern resembling primary cultures, while in the other the growth pattern of the cells was more atypical. The two KiMSV-transformed lines were previously shown to produce tumors in vivo that resemble human ovarian endometrioid stromal sarcomas. In spite of this histologic appearance, the proportion of keratin-positive cells in these cells was increased over the immortal lines. Keratin expression was unrelated to cell density, and keratin in most virally transformed cells was limited to few, fine filaments. In thymidine-labelled immortal and virus-transformed cultures stained for keratin, no correlation was found between keratin expression and proliferative activity. The keratin profiles of primary and immortal cultures were identical on Western blots, with subtypes ranging from 52 to 66 kDa. The two virally transformed lines lacked some of the subtypes. Vimentin networks were faint or absent in primary cultures. In the immortal and the virus-transformed lines, neoplastic progression was associated with increasing vimentin expression but with no changes in filament morphology and distribution. The results show that the abnormalities in intermediate filament expression that accompany immortalization do not preclude the retention of a normal epithelial morphology and growth pattern in this cell type. Furthermore, the number of intermediate filaments and their intracellular distribution appear to be altered at an earlier stage in neoplastic progression than those mechanisms that select for specific keratin subtypes, or those that respond to regulation by cell density. Finally, the presence of keratin in the KiMSV-transformed lines examined in this study supports the hypothesis that human ovarian stromal sarcomas can arise in the OSE.