A TGFβ1‐BIGH3‐Apoptosis Axis Comprising Peptidase and Integrin α3β1 Promotes Renal Cell Death

Abstract

Transforming growth factor‐β1 (TGFβ1) helps contribute to diabetes complications in various organs including the renal system. TGFβ1 signaling activates genes encoding various extracellular matrix (ECM) molecules that accumulate and promote renal cell injury, increasing the probability of end stage renal disease (ESRD). The mechanisms underlying development of ESRD are complex and not known completely. We have shown previously that TGFβ1 strongly stimulates renal proximal tubule epithelial (PTE) cells to express the gene that encodes an ECM protein called BIGH3. This newly‐synthesized endogenous BIGH3 protein increased PTE cell apoptosis. Treating PTE cells with exogenous recombinant BIGH3 protein increased apoptosis. Anti‐BIGH3 antibody blocked endogenous and exogenous BIGH3‐mediated apoptosis. These findings signify a TGFβ1‐BIGH3‐Apoptosis axis that augments kidney disease progression. BIGH3’s unstructured C‐terminus encodes integrin‐binding sequences EPDIM and RGD. In the kidney, BIGH3 C‐terminal proteolysis (CTP) provides bioactive C‐terminal fragments (BCTF) that comprise EPDIM and RGD and account for PTE cell apoptosis. The EPDIM motif in BIGH3 induced two‐fold greater apoptosis of PTE cells when compared to apoptosis induced by the RGD motif. In order to provide evidence that BIGH3 CTP and integrin interaction with EPDIM is required for PTE cell apoptosis, we used recombinant BIGH3, PTE cells, BIGH3 mutagenesis, protease inhibitors, TUNEL, immunoblots, integrin α3 antibody and siRNA. Homology modeling and molecular docking software Raptor X, ZDOCK and AutoDock Vina were used to generate monomeric and heterodimer models of α3β1 integrin; followed by in silico analysis of EPDIM interaction with α‐integrin subunit. Results show that a serine peptidase accomplishes BIGH3 CTP, which we regard as mechanistic in the proposed axis. A 21% chemical inhibition of BIGH3 CTP resulted in a 43% decrease in PTE cell apoptosis. In silico analysis indicates that the BCTF encoding EPDIM, bound to α3 subunit, prevents full activation and disrupts integrin‐mediated pro‐survival signals. We conclude that BIGH3 mediates at least some of the destructive arm of TGFβ1 signaling in diabetic nephropathy. BIGH3 CTP provides local mediator BCTFs that act through paracrine or autocrine pathways to block α3β1 pro‐survival signaling leading to PTE cell apoptosis. The data indicate that molecules of the axis pathway, including BIGH3, BIGH3 peptidase, and BIGH3 BCTFs are potential therapeutic targets to treat diabetic nephropathy. In a broader perspective, we have evidence that a similar mechanism is active in retinal endothelial and retinal pericyte apoptosis in diabetic complications of the ocular system.