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.

The Pro-Oncogenic Adaptor CIN85 Acts as an Inhibitory Binding Partner of Hypoxia-Inducible Factor Prolyl Hydroxylase 2

“…Our previous findings indicated that overexpression of CIN85 stabilizes HIF1a levels, and notably, that downregulation of CIN85 reversed this effect (13). The results of this study allow us to conclude that lower HIF1a levels under normoxic conditions in D75-103 PHD2-expressing cells originate from decreased protein stability and cannot be attributed to the differences in HIFa mRNA expression.…”

“…Earlier findings, including our own, showed that CIN85 promotes development of various cancers including breast cancer and displays highest levels in the most hypoxic areas of tumor tissues, which also usually display high HIFa levels (11,12). In addition, we could previously show that CIN85 appears to induce HIF1a stability via a so far unknown mechanism (13).…”

“…Moreover, our previous findings indicated that CIN85 contributes to cancer progression via stabilization of HIF1/2a (as well as the expression of HIF target genes such as PAI-1; ref. 13). This study extended these findings and verified that the direct interaction of CIN85 and PHD2 can inhibit PHD2mediated prolyl-hydroxylation of HIFa subunits.…”

Scientific and practical activity of the Laboratory of Cell Signaling Mechanisms of the Palladin Institute of Biochemistry of NAS of Ukraine

“…Численні дослідження свідчать про те, що високі рівні РАІ-1 є показником несприятливо-го прогнозу при багатьох формах онкологічних захворювань, а також характерні для діабету другого типу і серцево-судинних захворювань. Дослідження механізмів регуляції експресії компонентів системи активації плазміногену може допомогти виявленню причин порушення їх експресії за патології [5][6][7][8][9][10].…”

Transcriptional regulation of NOX genes expression in human breast adenocarcinoma MCF-7 cells is modulated by adaptor protein Ruk/CIN85

“…Using expression vector encoding fluorescent sensor of hydrogen peroxide HyPer fused with adaptor protein Ruk/CIN85 and live cell fluorescence microscopy, the co-localization of Ruk/CIN85 and Н 2 О 2 generation was shown to take place at the edges of representative vesicular structures in transiently transfected MCF-7 cells [18]. In addition, it was demon strated that high levels of Ruk l /CIN85 contribu te to the conversion of breast adenocarcinoma cells into a more malignant phenotype via modulation of the Src/Akt pathway [19], development of chemoresistance [20], constitutive activation of transcription factors HIF-1α [21] and NF-κB (unpublished data).…”