Heregulin-induced apoptosis is mediated by down-regulation of Bcl-2 and activation of caspase-7 and is potentiated by impairment of protein kinase C alpha activity

Heregulins are a group of growth factors that play diverse and critical rol es in the signaling network of the human epidermal growth factor receptor ( HER or EGFR) superfamily. Our earlier studies have shown that recombinant h eregulin beta1 (HRG) induces apoptosis in SKBr3 breast cancer cells that ov erexpress HER2. Here we report molecular mechanisms of HRG-induced apoptosi s. HRG treatment of SKBr3 cells for 72 h decreased the level of Bcl-2 prote in. HRG treatment led to degradation of poly (ADP-ribose) polymerase (PARP) and activated both caspase-9 and caspase-7. No significant activation of c aspase-3, -6, or -8 was detected. Expression of exogenous caspase-7 by aden ovirus-caspase-7 (Ad-casp-7) in SKBr3 cells resulted in apoptosis, which mi micked the effect of HRG treatment. Expression of exogenous caspase-7 had n o impact on Bcl-2 expression, but promoted PARP degradation. Two highly sel ective inhibitors of protein kinase C (PKC), GF109203X (GF) and Ro318425 (R o), significantly enhanced HRG-induced apoptosis as determined by flow cyto metric analysis and DNA fragmentation assay. Accordingly, the PKC inhibitor GF further decreased the level of Bcl-2 protein and further degraded PARP in HRG-treated cells. Assay of PKC activity indicated that HRG activated PK C in SKBr3 cells, predominantly affecting the PKC alpha isoform. To confirm which PKC isoform(s) mediated potentiation of HRG-induced apoptosis, the p rofile of PKC isoforms was measured in SKBr3 cells. Five PKC isoforms, PKC alpha, PKCi, PKC zeta, PKC lambda, and PKC delta as well as their receptors (RACK1) were expressed in this cell line. Treatment with PKC inhibitors GF and Ro decreased protein levels of both PKC alpha and PKC delta at 24 h. P KC alpha levels were still depressed at 72 h. GF and Ro had little effect o n the expression of other PKC isoforms. An inhibitor of classical PKC isofo rms (Go6976) enhanced HRG-induced apoptosis, whereas the PKC delta selectiv e inhibitor rottlerin did not. As PKCa was the only classical isoform expre ssed in SKBr3 cells, the effect of Go6976 on HRG-induced apoptosis largely related to inhibition of PKCa. Constitutive expression of wild-type PKC alp ha attenuated the apoptosis produced by HRG and GF. Consequently, HRG-induc ed apoptosis in SKBr3 cells appeared to involve down-regulation of Bcl-2 pr otein, activation of caspase-9 and caspase-7, and degradation of PARP. Inhi bition of PKC function enhanced HRG-induced apoptosis, leading to synergist ic down-regulation of Bcl-2 expression. Impairment of the PKC alpha isoform alone was sufficient to potentiate HRG-induced apoptosis.