title: Basic fibroblast growth factor inhibits apoptosis of spontaneously immortalized granulosa cells by regulating intracellular free calcium levels through a protein kinase Cdelta-dependent pathway.
abstract: 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 ifbFGF 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 PKCdelta, tau, lambda, and zeta. Of these PKC isoforms, only PKCdelta has been shown to be activated by TPA. In apoptotic SIGCs, PKCdelta levels were depleted. When PKCdelta 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 PKCdelta-dependent pathway.
authors.affiliation: Department of Obstetrics and Gynecology, University of Connecticut Health Center, Farmington 06030, USA.
authors.forename: K
authors.initials: K
authors.lastname: Lynch
authors.forename: G
authors.initials: G
authors.lastname: Fernandez
authors.forename: A
authors.initials: A
authors.lastname: Pappalardo
authors.forename: J J
authors.initials: JJ
authors.lastname: Peluso
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D002461:Cell Line, Transformed; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D006107:Granulosa Cells / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D016222:Fibroblast Growth Factor 2; D004533:Egtazic Acid; D011493:Protein Kinase C; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium