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Basic Fibroblast Growth Factor Maintains Calcium Homeostasis and Granulosa Cell Viability by Stimulating Calcium Efflux via a PKC-Dependent Pathway

Departments of Physiology (J.J.P., A.P., G.F.) and Obstetrics and Gynecology (J.J.P.), University of Connecticut Health Center, Farmington, Connecticut 06030

Address all correspondence and requests for reprints to: John J. Peluso, Ph.D., Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06030. E-mail: peluso{at}nso2.uchc.edu

Previous studies have demonstrated that basic fibroblast growth factor prevents granulosa cell apoptosis. The following six observations provide insight into the mechanism by which basic fibroblast growth factor mediates its antiapoptotic action. First, loading granulosa cells with 1,2 bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, an intracellular calcium chelator, prevented apoptosis when granulosa cells were deprived of basic fibroblast growth factor. Second, treatment with thapsigargin, an agent known to increase intracellular free calcium, induced granulosa cell apoptosis even in the presence of basic fibroblast growth factor. Third, an activator of PKC mimicked, whereas PKC inhibitors blocked, basic fibroblast growth factor’s antiapoptotic action. Fourth, continuous basic fibroblast growth factor exposure maintained relatively constant levels of intracellular free calcium, and a PKC inhibitor induced a sustained 2- to 3-fold increase in intracellular free calcium. Fifth, granulosa cells, as well as spontaneously immortalized granulosa cells, were shown to express PKC, -, and -. Finally, the PKC-specific inhibitor, rottlerin, blocked basic fibroblast growth factor’s antiapoptotic action in granulosa cells and spontaneously immortalized granulosa cells. These studies suggest that basic fibroblast growth factor regulates intracellular free calcium through a PKC-dependent mechanism and that a sustained increase in intracellular free calcium is sufficient to induce and is required for granulosa cell apoptosis.

Additional studies demonstrated that in spontaneously immortalized granulosa cells, basic fibroblast growth factor increased PKC activity by 60% within 2.5 min compared with serum-free control levels. Rottlerin attenuated basic fibroblast growth factor’s ability to stimulate PKC activity and to maintain intracellular free calcium. Further, intracellular free calcium levels in spontaneously immortalized granulosa cells transfected with a PKC antibody in the presence of basic fibroblast growth factor were 2-fold higher than those spontaneously immortalized granulosa cells transfected with IgG. Similarly, transfecting spontaneously immortalized granulosa cells with a specific PKC-substrate increased intracellular free calcium compared with spontaneously immortalized granulosa cells transfected with a specific substrate for PKC. Moreover, basic fibroblast growth factor increased and rottlerin attenuated ⁴⁵Ca efflux by 50% compared with that in basic fibroblast growth factor-treated cells. Finally, an inhibitor of the plasma membrane calciumadenosine triphosphatase pump suppressed ⁴⁵Ca efflux, elevated intracellular free calcium, and induced apoptosis. Collectively, these studies demonstrate that basic fibroblast growth factor activates PKC, which, in turn, stimulates calcium efflux, accounting in part for basic fibroblast growth factor’s ability to maintain calcium homeostasis and, ultimately, granulosa cell viability.

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