The interaction of HIF‐1a and MUC1 is GSK3b‐dependent during Ischemia‐Reperfusion Injury

Abstract

BackgroundHypoxia inducible factor‐1 (HIF‐1) is a transcription factor regarded as the most significant mediator of cellular adaptive responses to hypoxic insults including acute kidney injury (AKI). The inducible subunit HIF‐1a is targeted to proteasomal degradation by binding to the von Hippel‐Lindau (VHL) ubiquitin ligase (oxygen‐dependent), or by GSK3b phosphorylation (VHL‐independent). Now there is emerging evidence that the transmembrane glycoprotein mucin 1 (human MUC1 or rodent Muc1) expressed on the apical surface of polarized kidney epithelia plays a novel and important role under pathologic conditions. We previously reported that Muc1 plays a protective role in a mouse model of ischemia‐reperfusion injury (IRI). We found that Muc1 induction in kidneys after ischemia is associated with increased HIF‐1a and b‐catenin levels and downstream signaling. The small cytoplasmic tail of Muc1/MUC1 exhibits canonical binding sites (i) for b‐catenin (SxxxxxSSL59 numbering from the transmembrane domain) and (ii) for phosphorylation by GSK3b (SxxxS44). While GSK3b phosphorylation of b‐catenin enhances its degradation, MUC1 binding stabilizes b‐catenin and enhances its nuclear trafficking. GSK3b phosphorylation of MUC1 blocks MUC1 co‐IP with b‐catenin. As GSK3b phosphorylation of HIF‐1a also enhances HIF‐1a degradation, and HIF‐1a is stabilized by binding to MUC1, we tested the hypothesis that inhibition of GSK3b activity enhances HIF‐1a binding to MUC1.MethodsMuc1 KO mice and littermates were subjected to IRI using hanging‐weight protocol of 20 min ischemia and 48 h recovery. In addition, in vitro pull‐down assays and co‐IP studies in cultured human kidney cell lines with wild type and mutant constructs were conducted to fully characterize the MUC1‐HIF‐1a complex.ResultsWe observed reduced kidney function after IRI, and a significant increase in the levels of activated energy sensor AMP‐activated protein kinase (pAMPK) in Muc1 KO mice when compared to controls. This was associated with a significant damage in the proximal tubules as indicated by a significant two‐fold higher level of urinary KIM‐1 (PT injury marker). Although Muc1 is normally expressed in the thick ascending limb and distal nephron, we discovered that Muc1 is induced in the PT after ischemic injury. Furthermore, using human kidney cell lines, we observed co‐IP (i) between endogenous HIF‐1a and MUC1 in HK‐2 cells, and (ii) between endogenous HIF‐1a and Tac‐MUC1 chimeras in HEK293 cells. Moreover, we found that the co‐IP of Tac‐MUC1 with HIF‐1a in HEK293 cells was enhanced by (i) mutation of the GSK3b phosphorylation site on MUC1 (S44A), (ii) chemical inhibition of GSK3b activity with TDZD8 (10 mM, overnight), or (iii) both, suggesting that the interaction of HIF‐1a and MUC1 is GSK3b‐dependent.ConclusionsThese results suggest that Muc1 protects the kidney during IRI by transactivation of the HIF‐1 adaptive response via binding to HIF‐1a and inhibits its GSK3b‐mediated degradation.Support or Funding InformationThis work was supported by the National Institutes of Health (K01 DK109038, and P30‐DK‐079307), Dialysis Clinic, Inc. and Pittsburgh Center for Kidney Research.