Reproductive Biology and Endocrinology Open Access Mitogen-activated Protein Kinases in Normal and (pre)neoplastic Ovarian Surface Epithelium

Mitogen-activated protein kinases (MAPKs) are a group of serine/threonine kinases which are activated in response to a diverse array of extracellular stimuli and mediate signal transduction from the cell surface to the nucleus. It has been demonstrated that MAPKs are activated by external stimuli including chemotherapeutic agents, growth factors and reproductive hormones in ovarian surface epithelial cells. Thus, the MAPK signaling pathway may play an important role in the regulation of proliferation, survival and apoptosis in response to these external stimuli in ovarian cancer. In this article, an activation of the MAPK signaling cascade by several key reproductive hormones and growth factors in epithelial ovarian cancer is reviewed.


Introduction
Mitogen-activated protein kinases (MAPKs) are a group of serine/threonine kinases which are activated in response to a diverse array of extracellular stimuli, and mediate signal transduction from the cell surface to the nucleus [1]. As illustrated in Fig. 1, three MAPK family including extracellular signal-regulated kinases (ERK1 and ERK2), c-jun terminal kinase/stress-activated protein kinases (JNK/ SAPK) and p38, have been well characterized [2][3][4]. In addition, other MAPK family members, including ERK3, 4 and 5, four p38-like kinases and p57 MAPK have been cloned, but the biological role of these MAPKs is not well understood [2,4]. The MAPK cascade is activated via two distinct classes of cell surface receptors, receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). The signals transmitted through this cascade can cause an activation of diverse molecules which regulate cell growth, survival and differentiation. ERK1 (p44 MAPK) and ERK2 (p42 MAPK) activated by mitogenic stimuli are a group of the most extensively studied members, whereas JNK/SAPK and p38 are activated in response to stress such as heat shock, osmotic shock, cytokines, protein synthesis inhibitors, antioxidants, ultra-violet, and DNA-damaging agents [5,6]. MAPK family members are directly regulated by the kinases known as MAPK kinases (MAPKKs), which activate the MAPKs by phosphorylation of tyrosine and threonine residues [2,4,6]. At least seven different MAP-KKs have been cloned and characterized [2,4]. The first MAPKKs cloned were MAPK/ERK kinase 1 and 2 (MEK 1/ 2), which specifically activate ERKs. MKK3 and 6 specifically activate p38, whereas MKK5 stimulates the phosphorylation of ERK5. The MKK4 and 7 are known to activate JNK. The MAPKKs are activated by a rapidly expanding group of kinases called MAPKK kinases (MAPKKKs), which activate the MAPKKs by phosphorylation of serine and threonine residues [4,6]. These include Raf-1, A-Raf, The MAPK signaling transduction pathways Figure 1 The MAPK signaling transduction pathways. B-raf, MAPK/ERK kinase 1-4 (MEKK1-4), apoptosis-stimulating kinase-1 (ASK-1), and mixed lineage kinse-3 (MLK-3). The MAPKKKs may be activated by kinases known as MAPKKK kinases (MAPKKKKs), one of which is p21-activated kinase (PAK). In addition to these kinases, low molecular weight GTP-binding (LMWG) proteins regulate the activity of MAPKKKs and MAPKKKKs [2,4]. There are several different families of LMWG proteins, two of which include the Ras (N-Ras, K-Ras, and H-Ras) and Rho (Rac 1, 2 and 3, Cdc42 and Rho A, B and C) families. The activated MAPKs phosphorylate a large number of both cytoplasmic and nuclear proteins, exerting their specific functions. For example, activated ERK1/2 phosphorylate ternary complex factor (TCF) proteins such as Elk-1 and SAP-1, which form transcriptional complexes with serum response factor (SRF) in the promoter region of early response genes (e.g. c-fos, egr-1, junB) and thereby regulate their expression [7]. As shown in Fig. 1, many of these nuclear proteins, as a result of their ability to modulate expression of other proteins, are potential candidates for critical factors involved in the cellular response to stimuli.
It appears that the majority of ovarian tumors arise from the ovarian surface epithelium (OSE), which is a simple squamous-to-cuboidal mesothelium covering the ovary [8]. As mentioned earlier, the MAPK cascade can be activated via both RTKs and GPCRs, which include the receptors of growth factors, gonadotropins and gonadotropinreleasing hormones (GnRH). In ovarian cancer cells, MAPKs are activated and regulated by cisplatin [9], paclitaxel [10], endothelin-1 [11] and GnRH [12] suggesting that the MAPK signaling pathway plays an important role in the regulation of proliferation, survival and apoptosis in response to these external stimuli in ovarian cancer. In this review, we summarize the activation of the MAPK and its signaling cascade induced by hormones, growth factors and chemotherapeutic agents in normal and (pre)neoplastic OSE cells.

Activation of MAPK by hormonal factors
There is increasing evidence that gonadotropin-releasing hormone (GnRH) and its agonists (GnRHa) may play a critical role in the inhibition of cell proliferation in gynecological cancers including ovarian and endometrial cancers. However, their biological mechanism remains to be uncovered. The GnRH receptor (GnRH-R) belongs to the family of GPCRs. MAPK has been implicated in the antiproliferative effect of GnRHa in CaOV-3 ovarian cancer cell line [12]. Treatment of CaOV-3 cells with GnRHa resulted in an activation of ERK at 5 min, reached the highest activation at 3 h and sustained until 24 h, whereas GnRHa had no effect on the activation of the JNK. In addition, the ERK kinase was also activated and an increase in phosphorylation of son of sevenless (Sos), and Shc was observed following GnRHa treatment. Treatment with an inhibitor of mitogen-activated protein/ERK kinase, PD98059 reversed the antiproliferative effect of GnRHa and the GnRH-induced dephosphorylation of the retinoblastoma protein.
These results indicate that an activation of ERK may play a role in the antiproliferative effect of GnRHa [12]. In our laboratory, we have shown that an agonist of GnRH (D-Ala 6 )-GnRH, induced a biphasic pattern of ERK-1/-2 activation in OVCAR-3 cells (Fig. 2). A low concentration of GnRHa (10 -10 M) resulted in a significant decrease of MAPK activity, whereas high concentrations (10 -7 and 10 -6 M) induced an activation of MAPK pathway in ovarian and placental cells [13]. It is of interest to note that GnRH signaling appears to involve ERK-1/-2 phosphorylation through the activation of adenylyl cyclase and PKC in rat luteinized ovarian tumors [14].
An involvement of gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), has been proposed in the progression and metastasis of ovarian cancer. Expression of FSH receptor (FSH-R), a member of The effect of GnRH on ERK-1/-2 (p44/p42) activation in ovarian cancer line  (Fig. 3). In IOSE-29 cells, treatment with FSH significantly increased MAPK activity at 5-10 min, and an activated MAPK declined to control level after 20 min in these cells (Fig. 4). In addition, treatment with FSH resulted in substantial phosphorylation of Elk-1, the Ets family transcriptional factor [18]. These results support the hypothesis that the MAPK cascade is involved in cellular function such as growth stimulation in response to FSH in pre-neoplastic OSE cells.
Similar to FSH, adenosine triphosphate (ATP) has been implicated in the regulation of cell proliferation and activation of MAPK pathway in ovarian cancer cells. ATP binds to heterotrimeric G protein-coupled P2 purinocep-tors and extracellular ATP has been suggested to play a role in cellular proliferation and intracellular calcium concentrations (Ca 2+ ) in ovarian cancer cells [19,20]. Our recent results indicated that treatment with ATP resulted in an activation of ERK-1/-2 in IOSE-29 cell line as seen Fig. 5[21]. The stimulatory effect of ATP in the cellular proliferation and MAPK activation was completely abolished in the presence of PD98059 and staurosporin (a PKC inhibitor), suggesting that the growth stimulatory effect of ATP is mediated via PKC-dependent MAPK activation in pre-neoplastic OSE cells (Fig. 5). Treatment with ATP resulted in substantial phosphorylation of Elk-1, further implicating the MAPK cascade in the growth stimulatory effect of ATP in pre-neoplastic OSE cells [21].

Activation of MAPK by growth factors and cytokines
Endothelin-1 (ET-1) is a potential autocrine regulatory factor in ovarian cancer. Treatment of OVCA 433 ovarian cancer with ET-1 resulted in a phosphorylation of ERK-2 and mitogenic responses. The epidermal growth factor receptor (EGF-R)/ras-dependent pathway may contribute to the activation of MAPK/ERK-2 and mitogenic signaling induced by ET-1 in these cells, suggesting that ET-1 induced activation of MAPK is mediated in part by signaling pathways that are initiated by transactivation of the EGF-R [11]. As autocrine regulators, lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have been demonstrated to activate MAPK kinase (MEK) and p38 MAPK via AKT pathway in HEY ovarian cancer cells. The kinase activity and S473 phosphorylation of Akt induced by LPA and S1P required both MEK and p38 MAPK, and MEK is likely to be upstream of p38 in these cells, suggesting that the requirement for both MEK and p38 is cell type-and stimulus-specific [22]. In rhesus ovarian surface epithelial cells in culture, treatment with extracellular calcium induced an activation of MAPK in the response to cell proliferation in these normal cells [23]. Human interleukin-8 (IL-8) rapidly activated ERK-1/-2 pathway via stimulation of the CXCR-1/2 receptors [24]. By using inhibitors such as genestein and herbimycin A, tyrosine kinases have been shown to be involved in the IL-8 activation of ERK-1/-2 in SKOV-3 cells, suggesting an important cross-talk between the chemokine and growth factor pathways in the migration and proliferation in ovarian cancer cells [24].
In addition to ERK1/2, the JNK pathway has been suggested to play a role in the cell proliferation and apoptosis in ovarian cancer. For instance, treatment with tumor necrosis factor (TNF) alpha activated ERK1/2 at 10-20 min, and a maximum threefold induction of ERK1/2 activity was observed after 1 min of treatment [25]. Inhibition of TNF alpha-induced ERK1/2 activity by PD98059 was associated with induction of apoptosis in the TNF Effect of ATP on ERK-1/-2 (p44/p42) and Elk-1 in the absence or presence of PD98059 and staurosporin

Activation of MAPK by chemotherapeutic agents
Cisplatin has been widely used as a chemotherapeutic agent to treat ovarian cancers, although its use is somewhat limited because of cisplatin-resistance. The molecular mechanism of cisplatin-induced biological effect in ovarian cancer is not well understood. Cisplatin caused a late and prolonged induction of both ERK1/2 and JNK1 activity in a dose-dependent manner, whereas no significant difference was observed in p38 activity in SKOV-3 cells [9]. These results suggest that ERK and other signal transduction pathways may play a role in response to cisplatin and be important for the development of new strategies to enhance the therapeutic use of platinum drugs.

Concluding Remarks
There is increasing evidence that the three well-characterized members of the MAPK family, ERK1/2, JNK/SAPK and p38, play an important role in the regulation of proliferation, survival and apoptosis in ovarian cancer in response to the external stimuli including hormones, growth factors, cytokines and chemotherapeutic chemicals. The signaling pathways by which hormones such as FSH, ATP and GnRH exert their effects in the regulation of cell proliferation and apoptosis in ovarian cancer cells are proposed in Fig. 6. As well, MAPK pathways may contribute to the metastasis and chemoresistance of ovarian cancer. A better understanding of the MAPK and other signaling pathways in normal and neoplastic OSE will provide new insights for the development of novel therapeutic approach to ovarian cancer.
Activation of the MAPK signaling pathway by FSH, ATP and GnRH in ovarian cancer Figure 6 Activation of the MAPK signaling pathway by FSH, ATP and GnRH in ovarian cancer.