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Basic Fibroblast Growth Factor Inhibits Apoptosis of Spontaneously Immortalized Granulosa Cells by Regulating Intracellular Free Calcium Levels through a Protein Kinase C-Dependent Pathway¹

Departments of Physiology (G.F., A.P., J.J.P.) and Obstetrics and Gynecology (K.L., 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 shown that basic fibroblast growth factor (bFGF) inhibits primary granulosa cells from undergoing apoptosis. The present studies were designed to determine whether spontaneously immortalized granulosa cells (SIGCs) undergo apoptosis when deprived of growth factors and whether bFGF prevents apoptosis. In the absence of serum, the SIGCs lost cell contact and underwent apoptosis as indicated by the presence of annexin V binding, DNA ladders, and nuclear fragmentation. Basic FGF maintained cell contact and reduced the percentage of apoptotic cells. This antiapoptotic action was not observed if bFGF was added 30 min after serum withdrawal. Further, intracellular free calcium ([Ca²⁺]_i) levels gradually increased 3- to 4-fold within 10 min of serum withdrawal. This increase was inhibited by bFGF. The intracellular calcium chelator, BAPTA, completely prevented the SIGCs from undergoing apoptosis in the absence of serum. These observations suggest that bFGF’s ability to regulate [Ca²⁺]_i is an essential component of its antiapoptotic action. The phorbol ester TPA, an activator of protein kinase C (PKC), blocked apoptosis due to serum deprivation. Conversely, bisindolylmaleimide II, an inhibitor of PKC, completely attenuated, whereas bisindolylmaleimide V, an inactive bisindolylmaleimide analog, did not influence bFGF’s antiapoptotic action. Also, treatment with the PKC inhibitor, chelerythrine chloride, interfered with bFGF’s ability to maintain calcium homeostasis. Western blot analysis revealed that SIGCs expressed PKC, , , and . Of these PKC isoforms, only PKC has been shown to be activated by TPA. In apoptotic SIGCs, PKC levels were depleted. When PKC levels were reduced by pretreatment with 500 nM TPA, neither bFGF nor 10 nM TPA suppressed apoptosis. Collectively, these observations suggest that bFGF maintains [Ca²⁺]_i and thereby SIGC viability through a PKC-dependent pathway.

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