| Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for mesenchymal cells. The PDGF B-chain (c-sis proto-oncogene) homodimer (PDGF BB) and v-sis, its viral counterpart, activate both alpha- and beta-receptor subunits (alpha-PDGFR and beta-PDGFR) and mediate phenotypic transformation, which is characterized by anchorage-independent growth. In contrast, the PDGF A chain homodimer (PDGF AA) activates alpha-PDGFR only and fails to induce phenotypic transformation. In the present study, we investigated alpha- and beta-PDGFR specific signaling pathways that are responsible for the differences between the transforming ability of PDGF AA and BB. To study PDGF BB activation of beta-PDGFR, we established NIH3T3 clones in which alpha-PDGFR signaling is inhibited by a dominant-negative alpha-PDGFR, or an anti-sense construct of alpha-PDGFR. Here, we demonstrated that alpha-PDGFR activation alone is sufficient for PDGF BB-mediated anchorage-independent cell growth. More importantly, inhibition of alpha-PDGFR signaling enhanced PDGF BB-mediated phenotypic transformation, suggesting that alpha-PDGFR antagonizes beta-PDGFR-induced transformation. While both alpha- and beta-receptors effectively activate ERKs, alpha-PDGFR, but not beta-PDGFR, activates stress-activated protein kinase-1/c-Jun NH2-terminal kinase-1 (SAPK1/JNK-1). Inhibition of JNK-1 activity using a dominant-negative JNK-1 mutant markedly enhanced PDGF BB-mediated anchorage-independent cell growth, demonstrating an antagonistic role for JNK-1 in PDGF-induced transformation. Consistently, overexpression of wild-type JNK-1 reduced PDGF BB-mediated transformation. We also provided evidence that alpha-PDGFR activates stress-activated protein kinase-1/c-Jun NH2-terminal kinase (SAPK1/JNK) leading to PDGF-induced p21 WAF1/CIP1 expression, an inhibitor of cyclin-dependent kinases and a known downstream mediator of the tumor suppressor gene p53. Over-expression of dominant negative JNK-1 downregulated PDGF induced p21WAF1/CIP1 expression, whereas overexpression of wild type JNK-1 upregulated or restored p21WAF1/CIP1 expression in control cells or antisense and dominant negative alpha-PDGFR stably-transfected clones. Analysis of deletion mutant of p21WAF1/CIP1 promoter suggested a region, rich in AP-1 and c-jun sites, are JNK-1 responsive. These findings are in consistent with the known functional roles of JNK-1 in phosphorylation of c-Jun, a component of AP-1 complex. Take together, the present study proves that alpha-PDGFR negatively regulates PDGF BB induced transformation through activation of JNK-1, which, in turn, up-regulates p21WAF1/CIP1 expression. alpha-PDGFR activates both survival promoting and suppressing activities, whereas beta-PDGFR has transformation and survival prone activities. The overall effects explain why PDGF BB is more transforming and survival-prone than PDGF AA is in NIH3T3 cells. |
