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 α activity

Heregulins are a group of growth factors that play diverse and critical roles in the signaling network of the human epidermal growth factor receptor (HER or EGFR) superfamily. Our earlier studies have shown that recombinant heregulin beta1 (HRG) induces apoptosis in SKBr3 breast cancer cells that overexpress HER2. Here we report molecular mechanisms of HRG-induced apoptosis. HRG treatment of SKBr3 cells for 72 h decreased the level of Bcl-2 protein. HRG treatment led to degradation of poly (ADP-ribose) polymerase (PARP) and activated both caspase-9 and caspase-7. No significant activation of caspase-3, -6, or -8 was detected. Expression of exogenous caspase-7 by adenovirus-caspase-7 (Ad-casp-7) in SKBr3 cells resulted in apoptosis, which mimicked the effect of HRG treatment. Expression of exogenous caspase-7 had no impact on Bcl-2 expression, but promoted PARP degradation. Two highly selective inhibitors of protein kinase C (PKC), GF109203X (GF) and Ro318425 (Ro), significantly enhanced HRG-induced apoptosis as determined by flow cytometric 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 PKC in SKBr3 cells, predominantly affecting the PKC alpha isoform. To confirm which PKC isoform(s) mediated potentiation of HRG-induced apoptosis, the profile 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. PKC alpha levels were still depressed at 72 h. GF and Ro had little effect on the expression of other PKC isoforms. An inhibitor of classical PKC isoforms (Go6976) enhanced HRG-induced apoptosis, whereas the PKC delta selective inhibitor rottlerin did not. As PKCa was the only classical isoform expressed in SKBr3 cells, the effect of Go6976 on HRG-induced apoptosis largely related to inhibition of PKCa. Constitutive expression of wild-type PKC alpha attenuated the apoptosis produced by HRG and GF. Consequently, HRG-induced apoptosis in SKBr3 cells appeared to involve down-regulation of Bcl-2 protein, activation of caspase-9 and caspase-7, and degradation of PARP. Inhibition of PKC function enhanced HRG-induced apoptosis, leading to synergistic down-regulation of Bcl-2 expression. Impairment of the PKC alpha isoform alone was sufficient to potentiate HRG-induced apoptosis.