title: 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Bryan M
authors.initials: BM
authors.lastname: Grabias
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Konstantopoulos
mesh_terms: D002460:Cell Line; D003094:Collagen / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D005355:Fibrosis; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000378:metabolism* / Q000473:pathology; D040542:Mechanotransduction, Cellular; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D000074562:Receptor, Notch4; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D013314:Stress, Mechanical; D013997:Time Factors; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: C106075:NOTCH4 protein, human; D011518:Proto-Oncogene Proteins; D000074562:Receptor, Notch4; D051880:Receptors, Notch; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C508960:TGFB1 protein, human; D053773:Transforming Growth Factor beta1; D003094:Collagen
keywords: chronic kidney disease; fluid shear; frequency-dependent signaling; tubulointerstitial fibrosis