TGFβ-induced deptor suppression recruits mTORC1 and not mTORC2 to enhance collagen I (α2) gene expression.

Falguni Das,Nandini Ghosh-Choudhury,Hanna E Abboud,Balakuntalam S Kasinath,Amit Bera,Goutam Ghosh Choudhury,Soumitro Pal

doi:10.1371/journal.pone.0109608

Abstract

Enhanced TGFβ activity contributes to the accumulation of matrix proteins including collagen I (α2) by proximal tubular epithelial cells in progressive kidney disease. Although TGFβ rapidly activates its canonical Smad signaling pathway, it also recruits noncanonical pathway involving mTOR kinase to regulate renal matrix expansion. The mechanism by which chronic TGFβ treatment maintains increased mTOR activity to induce the matrix protein collagen I (α2) expression is not known. Deptor is an mTOR interacting protein that suppresses mTOR activity in both mTORC1 and mTORC2. In proximal tubular epithelial cells, TGFβ reduced deptor levels in a time-dependent manner with concomitant increase in both mTORC1 and mTORC2 activities. Expression of deptor abrogated activity of mTORC1 and mTORC2, resulting in inhibition of collagen I (α2) mRNA and protein expression via transcriptional mechanism. In contrast, neutralization of endogenous deptor by shRNAs increased activity of both mTOR complexes and expression of collagen I (α2) similar to TGFβ treatment. Importantly, downregulation of deptor by TGFβ increased the expression of Hif1α by increasing translation of its mRNA. TGFβ-induced deptor downregulation promotes Hif1α binding to its cognate hypoxia responsive element in the collagen I (α2) gene to control its protein expression via direct transcriptional mechanism. Interestingly, knockdown of raptor to specifically block mTORC1 activity significantly inhibited expression of collagen I (α2) and Hif1α while inhibition of rictor to prevent selectively mTORC2 activation did not have any effect. Critically, our data provide evidence for the requirement of TGFβ-activated mTORC1 only by deptor downregulation, which dominates upon the bystander mTORC2 activity for enhanced expression of collagen I (α2). Our results also suggest the presence of a safeguard mechanism involving deptor-mediated suppression of mTORC1 activity against developing TGFβ-induced renal fibrosis.

Highlights

Renal tubulointerstitial fibrosis represents the best predictor of clinical outcome of end-stage renal disease [1]
As deptor is an inhibitor of mTOR, we examined activation of mTORC1 employing phosphorylation of S6 kinase (Thr-389) and 4EBP-1 (Thr-37/46) as indicators
Note that phosphorylation of Akt at Thr-308 was increased by TGFb (Fig. 1D). These results suggest that TGFbinduced deptor downregulation activates both mTORC1 and mTORC2 in a prolonged manner

Summary

Introduction

Renal tubulointerstitial fibrosis represents the best predictor of clinical outcome of end-stage renal disease [1]. The initiation of phase of fibrosis involves infiltration of inflammatory cells that secrete profibrogenic growth factors and cytokines. One such factor, TGFb, acts on various renal cells including the proximal tubular epithelial cells to increase expression of matrix proteins, which significantly contribute to the fibrotic process. Activated type I receptor phosphorylates the receptor-specific Smads (Smad 3 and 2) at the C-terminus, which is released from the type I receptor and SARA, a Smad-recruiting protein to the plasma membrane [4]. Smad 2 and 3 act downstream of TGFb receptor function, a recent study indicated a protective function of Smad 2 in renal fibrosis and matrix protein expression in proximal tubular epithelial cells [8]

Methods

Results

Conclusion