The adaptor protein Ruk/CIN85 activates plasminogen activator inhibitor-1 (PAI-1) expression via hypoxia-inducible factor-1α

Abstract

SummaryIncreased levels of plasminogen activator inhibitor-1 (PAI-1) indicate an enhanced risk of ischaemic/hypoxic cardiovascular events and a poor prognosis. The expression of PAI-1 can be induced by various stimuli including hypoxia, insulin and insulin-like growth factor 1 (IGF-1). The hypoxia-inducible factor-1 (HIF-1) is critical for hypoxia or insulin/IGF-1 mediated PAI-1 induction, but the components involved in merging the signals are not known so far. The adaptor/scaffold protein Ruk/CIN85 may be a candidate since it plays important roles in the regulation of processes associated with cardiovascular and oncological diseases such as downregulation of receptor tyrosine kinases, apoptosis, adhesion and invasion. Therefore, it was the aim of this study to investigate the involvement of Ruk/CIN85 in the regulation of PAI-1 expression. It was found that Ruk/CIN85 induced PAI-1 mRNA and protein expression both under normoxia and hypoxia. The induction of PAI-1 expression by Ruk/CIN85 occurred at the transcriptional level since the half-life of PAI-1 mRNA was not affected in cells overexpressing Ruk/ CIN85 and reporter gene assays using wild-type and mutant human PAI-1 promoter luciferase constructs showed that the hypoxia responsive element was responsible for Ruk/CIN85 effects. Further, knocking down HIF-1α abolished not only the hypoxia-dependent but also the Ruk/CIN85-dependent PAI-1 induction. In addition, transient or stable overexpression of Ruk/CIN85 also induced HIF-1α protein levels and HIF-1 activity and knocking down Ruk/CIN85 reversed these effects. Thereby, Ruk/CIN85 interfered with the proline hydroxylation-dependent HIF-1α protein destabilisation. Together, these results provide the first evidence that Ruk/CIN85 induces PAI-1 expression via modulation of HIF-1α stability.