Notch4-dependent antagonism of canonical TGF-β1 signaling defines unique temporal fluctuations of SMAD3 activity in sheared proximal tubular epithelial cells

Abstract

Transforming growth factor-β1 (TGF-β1) is thought to drive fibrogenesis in numerous organ systems. However, we recently established that ectopic expression of TGF-β1 abrogates collagen accumulation via canonical SMAD signaling mechanisms in a shear-induced model of kidney fibrosis. We herein delineate the temporal control of endogenous TGF-β1 signaling that generates sustained synchronous fluctuations in TGF-β1 cascade activation in shear-stimulated proximal tubule epithelial cells (PTECs). During 8-h exposure to physiological shear stress (0.3 dyn/cm²), PTECs experience in situ oscillatory concentrations of active endogenous TGF-β1 that are ~10-fold greater than those detected under higher stress regimes (2-4 dyn/cm²). The elevated levels of intrinsic TGF-β1 maturation observed under physiological conditions are accompanied by persistent downstream SMAD3 activation. Pathological shear stresses (2 dyn/cm²) first elicit temporal variations in phosphorylated SMAD3 with an apparent period of ~6 h, whereas even higher stresses (4 dyn/cm²) abolish SMAD3 activation. These divergent patterns of SMAD3 activation are attributed to varying levels of Notch4-dependent phospho-SMAD3 degradation. Depletion of Notch4 in shear-stimulated PTECs eventually increases the levels of active TGF-β1 protein by approximately fivefold, recovers stable SMAD phosphorylation and ubiquitinated SMAD species, and attenuates collagen accumulation. Collectively, these data establish Notch4 as a critical mediator of shear-induced fibrosis and further reinforce the renoprotective effects of canonical TGF-β1 signaling.

Comprehensive transcriptome analysis of fluid shear stress altered gene expression in renal epithelial cells

“…A heat map of all 8 PTEC samples shows a clear distinction between fluid shear stress treated samples and static controls (Figure b). Furthermore, our genome wide RNA sequencing analysis confirmed genes known to be altered by fluid shear stress in renal epithelial cells, including Ptgs2 ( Cox2 ), Ccl2 ( Mcp1 ), Edn1 , Egr1, Snai1 , Cdh1 , and Tgfb1 (Flores, Battini, Gusella, & Rohatgi, ; Flores, Liu, Liu, Satlin, & Rohatgi, ; Grabias & Konstantopoulos, , ; Maggiorani et al, ; Pandit et al, ; Schwachtgen, Houston, Campbell, Sukhatme, & Braddock, ).…”

“…Thus far we applied fluid shear stress of 2.0 dyn/cm 2 , which is known to be an increased physio‐pathological shear stress (Essig, Terzi, Burtin, & Friedlander, ; Grabias & Konstantopoulos, , ; Weinbaum et al, ). To compare the gene expression to physiological levels of shear, we exposed the cells to a shear stress range of 0.25–2.0 dyn/cm 2 .…”

“…In this study we used RNA sequencing to get a comprehensive overview of the transcriptome alterations upon fluid shear stress in proximal tubular epithelial cells. Physiological shear stress in renal epithelial cells is ranging from 0.05–1.0 dyn/cm 2 , where proximal tubular cells experience the highest range of shear stress (Essig et al, ; Grabias & Konstantopoulos, , ; Weinbaum et al, ). We applied a fluid shear stress of 2.0 dyn/cm 2 , which is known to be an increased physio‐pathological shear stress, mimicking hyperfiltration after renal mass reduction or during progression of renal disease.…”

Notch in fibrosis and as a target of anti-fibrotic therapy

“…In addition, TGFβ induces renal epithelial Jagged-1 expression in fibrosis [46], while Notch mediated EMT is associated with increased expression of the Snail transcription factor [17]. Depletion of Notch4 in shear-stimulated proximal tubule epithelial cells attenuates collagen accumulation via effects on TGFβ signaling [97]. The vitamin D analogue, paricalcitol attenuates TGF-β1 induced Smad2 phosphorylation and upregulation of the Notch ligand Jagged-1, ACTA2 and thrombospondin-1 and prevented the TGF-β1-mediated loss of E-Cadherin [98].…”

The physical basis of renal fibrosis: effects of altered hydrodynamic forces on kidney homeostasis

“…We also identified dynamic Notch4-dependent regulation of the TGF-␤1 signaling axis in part via targeted degradation of downstream SMAD3 protein (47) (Fig. 3C).…”