c-Jun NH2-terminal kinase activation contributes to hypoxia-inducible factor 1alpha-dependent P-glycoprotein...

We previously have shown that hypoxia increases the expression of P-glycoprotein, which in turn increases tumor cell capacity to actively extrude chemotherapeutic agents and may contribute to tumor drug resistance. This event is mediated through the hypoxia-inducible factor (HIF-1). Here, we investigated the role of the stress-activated protein kinase c-Jun NH(2)-terminal kinase (JNK) in the signaling mechanisms underlying these events. Hypoxia activates JNK activity in vitro and in vivo. Overexpression of mitogen-activated protein kinase (MAPK) kinase kinase (MEKK-1), which preferentially activates JNK, mimics, in a nonadditive way, hypoxia-induced activity of the MDR1 promoter and expression of MDR1 mRNA and P-glycoprotein. Furthermore, the JNK inhibitor SP600125 selectively and specifically inhibits hypoxia- and MEKK-1-induced MDR1 promoter activity in a dose-dependent manner. JNK inhibition also reversed hypoxia- and MEKK-1-induced activity of an HIF-1-dependent reporter gene. MEKK-1-induced MDR1 expression depends on a functional HIF-1 binding site (hypoxia-responsive element). Hypoxia- but not cobalt chloride-dependent HIF-1-DNA binding and transcriptional activation was inhibited by SP600125, indicating that hypoxia-induced signaling to HIF-1 depends on JNK activation. Because it has been reported that reactive oxygen species are increased in hypoxia and related to JNK activation, we investigated their role in signaling this response. Whereas exogenous addition of H(2)O(2) was sufficient to activate JNK, reactive oxygen species scavengers were without effect on hypoxia-induced JNK or HIF-1 activation. Thus, hypoxia-elicited MDR1 expression, which depends on HIF-1 activation, depends at least in part on signaling via activation of JNK. Furthermore, these events are independent of the generation of reactive oxygen intermediates. Thus, JNK may represent a therapeutic target in the prevention of tumor resistance to chemotherapeutic treatment.