ABSTRACT
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.
Subject(s)
Calcium Signaling/physiology , Calcium/physiology , Epithelial Cells/cytology , Epithelial Cells/physiology , Receptors, Cell Surface/physiology , Amino Acid Substitution , Animals , Benzoquinones , Calcium/pharmacology , Cell Division , Enzyme Inhibitors/pharmacology , Epithelial Cells/drug effects , Extracellular Space/physiology , Female , Flavonoids/pharmacology , Kidney/metabolism , Lactams, Macrocyclic , Mitogen-Activated Protein Kinases/metabolism , Mutagenesis, Site-Directed , Ovary/cytology , Quinones/pharmacology , Rats , Receptors, Calcium-Sensing , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Rifabutin/analogs & derivatives , Transfection , src-Family Kinases/metabolismABSTRACT
Changes in the concentration of extracellular calcium can affect the balance between proliferation and differentiation in several cell types, including keratinocytes, breast epithelial cells, and fibroblasts. This report demonstrates that elevation of extracellular calcium stimulates proliferation-associated signaling pathways in rat fibroblasts and implicates calcium-sensing receptors (CaR) as mediators of this response. Rat-1 fibroblasts express CaR mRNA and protein and respond to known agonists of the CaR with increased IP3 production and release of intracellular calcium. Agonists of the CaR can stimulate increased c-SRC kinase activity and increased extracellular signal-regulated kinase 1/mitogen-activated protein kinase activity. Both of the increases in SRC activity and mitogen-activated protein kinase activation are blocked in the presence of a nonfunctional mutant of the CaR, R796W. Proliferation of wild-type Rat-1 cells is sensitive to changes in extracellular calcium, but expression of the nonfunctional CaR mutant or inhibition of the calcium-dependent increase in SRC kinase activity block the proliferative response to calcium. These results provide evidence of a novel signal transduction pathway modulating the response of fibroblasts to extracellular calcium and imply that calcium-sensing receptors may play a role in regulating cell growth in response to extracellular calcium, in addition to their well known function in systemic calcium homeostasis.
