Signal Transduction Mechanisms Linking Increased Extracellular Calcium to Proliferation in Ovarian Surface Epithelial Cells Susan A. Hobson, Scott E. McNeil, 1 Fred Lee, and Karin D. Rodland 2 Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland, Oregon 97201-3098 Although ovarian surface epithelial (OSE) cells are the cell type responsible for malignant ovarian carci- noma, relatively little is known about either the extra- cellular stimuli or the intracellular signaling mecha- nisms 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 re- ceptors (CaR). Here we show that agonists of the CaR increase the kinase activity of Src and ERKs (extracel- lular signal-regulated kinases) in rat OSE cells and promote association between tyrosine-phosphory- lated Shc and p120rasGAP. Expression of an interfer- ing mutant CaR inhibited the proliferative response to elevated extracellular calcium, as well as CaR agonist- induced tyrosine phosphorylation and ERK activa- tion. 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 ki- nases. These results indicate that the ability of OSE cells to proliferate in response to increases in extra- cellular calcium involves cross-talk between the G- protein-coupled CaR and the activation of a tyrosine kinase-dependent Ras–Raf–ERK signaling pathway. © 2000 Academic Press Key Words: ovarian surface epithelial cells; prolifer- ation; calcium; calcium-sensing receptors; MAP ki- nases; tyrosine phosphorylation. INTRODUCTION Ovarian surface epithelial (OSE) cells represent an extension of the peritoneal mesothelium which covers the surface of the ovary as a simple cuboidal epithe- lium. Subjected to repeated wounding at each ovula- tion, these cells retain proliferative potential through- out adult life. This proliferative capacity may play an important role in the progression of these cells to ma- lignant ovarian adenocarcinoma, as the number of ovu- latory cycles experienced by a given woman is one of the major risk factors contributing to ovarian cancer [1, 2]. Although some potential mitogens of OSE cells have been identified, including transforming growth factor , macrophage colony-stimulating factor, and lyso- phosphatidic acid [3– 6], relatively little is known about the downstream signal transduction mechanisms reg- ulating the proliferative response of OSE cells to these mitogens. We have shown that human and rat OSE cells are sensitive to changes in extracellular calcium concentration [7]. In this report we have investigated the signal transduction mechanisms linking increased extracellular calcium to increased proliferation in rat OSE cells (ROSE cells). A novel mechanism mediating the effects of extracel- lular calcium on intracellular events has recently been elucidated through the cloning and characterization of a G-protein-coupled, seven-transmembrane-domain re- ceptor capable of specifically binding calcium, magne- sium, barium, and gadolinum [8 –10]. Activation of the calcium-sensing receptors (CaR) by calcium or other ligands results in the production of inositol trisphos- phates and the release of intracellular calcium [8 –10], presumably as a consequence of G-protein-mediated phospholipase C activation. In parathyroid cells, the release of intracellular calcium is associated with the inhibition of parathormone secretion [11]. Initially characterized as the calcium-sensing recep- tor of parathyroid cells responsible for modulating parathormone release in response to changes in plasma ionized calcium [12], the CaR is also expressed in kidney, brain, bone, gastric epithelial cells, and fi- broblasts [9, 10, 13–16]. We have shown that human OSE cells also express functional CaR, as demon- strated by both protein expression and induction of inositol trisphosphate production and intracellular cal- cium release in response to known agonists of the CaR [7]. Although proliferative signaling from other G-pro- tein-coupled receptors has been shown to involve a 1 Current address: Department of Clinical Investigation, Tripler Army Medical Center, Honolulu, HI 96859. 2 To whom correspondence and reprint requests should be ad- dressed at the Department of Cell and Developmental Biology L215, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland Oregon 97201-3098. Fax: (503) 494-4253. E-mail: rodlandk@ohsu.edu. 0014-4827/00 $35.00 1 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Experimental Cell Research 258, 1–11 (2000) doi:10.1006/excr.2000.4910, available online at http://www.idealibrary.com on