Basic fibroblast growth factor inhibits apoptosis of spontaneously immortalized granulosa cells by regulating intracellular free calcium levels through a protein kinase C delta-dependent pathway

Previous studies have shown that basic fibroblast growth factor (bFGF) inhi bits primary granulosa cells from undergoing apoptosis. The present studies were designed to determine whether spontaneously immortalized granulosa ce lls (SIGCs) undergo apoptosis when deprived of growth factors and whether b FGF prevents apoptosis. In the absence of serum, the SIGCs lost cell contac t 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 w as not observed if bFGF was added 30 min after serum withdrawal. Further, i ntracellular free calcium ([Ca2+](i)) levels gradually increased 3- to 4-fo ld within 10 min of serum withdrawal. This increase was inhibited by bFGF. The intracellular calcium chelator, BAPTA, completely prevented the SIGCs f rom undergoing apoptosis in the absence of serum. These observations sugges t that bFGF's ability to regulate [Ca2+](i) is an essential component of it s antiapoptotic action. The phorbol ester TPA, an activator of protein kina se C (PKC), blocked apoptosis due to serum deprivation. Conversely, bisindo lylmaleimide II, an inhibitor of PKC, completely attenuated, whereas bisind olylmaleimide V, an inactive bisindolylmaleimide analog, did not influence bFGF's antiapoptotic action. Also, treatment with the PKC inhibitor, cheler ythrine chloride, interfered with bFGF's ability to maintain Calcium homeos tasis. Western blot analysis revealed that SIGCs expressed PKC delta, tau, lambda, and zeta. Of these PKC isoforms, only PKC delta has been shown to b e activated by TPA. In apoptotic SIGCs, PKC delta levels were depleted. Whe n PKC delta levels were reduced by pretreatment with 500 nM TPA, neither bF GF nor 10 nM TPA suppressed apoptosis. Collectively, these observations sug gest that bFGF maintains [Ca2+]i and thereby SIGC viability through a PKC d elta -dependent pathway.