Regucalcin mediated up‐regulation of Wnt/β‐catenin signaling

Abstract

Senescence marker protein‐30 (SMP30), also known as regucalcin (RGN), functions as a lactonase. The penultimate step in vitamin‐C biosynthesis in nonprimate mammals is catalyzed by this enzyme. RGN also regulates the transcriptional process by binding protein and DNA in the nucleus. There is growing evidence that regucalcin plays a multifunctional role as a regulatory protein in Ca2+dependent and independent signaling processes in many tissues especially liver. Recently, we identified RGN as a novel target of β‐catenin in the liver establishing an important role of β‐catenin in vitamin‐C homeostasis and cell survival. We also identified β‐catenin‐RGN association in liver cells. In the current study we investigated whether regucalcin itself has any impact on the Wnt/β‐catenin signaling. GFP‐coupled full‐length (FL), and amino (N)‐, carboxy (C)‐ or middle domain‐deletion mutants of RGN were generated in the laboratory. These constructs were transiently overexpressed in the HEK293 cells and human hepatoma cells (HepG2) cells. We observed RGN‐GFP to be prominently located in the nucleus of cells after transfection, which was blocked with staurosporine, an inhibitor of protein kinase C. Both human and mouse FL‐RGN showed a 2–3‐fold increase in β‐catenin‐dependent transcriptional activity as measured by TOPflash reporter assay over the empty GFP‐vector‐transfected cells. Moreover, deletion of both the N‐ & C‐ but not the middle domain‐deletion mutants of regucalcin abrogated TOPflash activity after transfection. Thus, we have identified a novel role of regucalcin in stimulating the Wnt pathway through direct interaction with nuclear β‐catenin for which amino‐ and carboxy‐ terminals of RGN are of relevance.