Induction of Renal Fibrotic Genes by TGF-β1 Requires EGFR Activation, p53 and Reactive Oxygen Species

Cell Signal. 2013 Nov;25(11):2198-209. doi: 10.1016/j.cellsig.2013.07.007. Epub 2013 Jul 18.

Abstract

While transforming growth factor-β (TGF-β1)-induced SMAD2/3 signaling is a critical event in the progression of chronic kidney disease, the role of non-SMAD mechanisms in the orchestration of fibrotic gene changes remains largely unexplored. TGF-β1/SMAD3 pathway activation in renal fibrosis (induced by ureteral ligation) correlated with epidermal growth factor receptor(Y845) (EGFR(Y845)) and p53(Ser15) phosphorylation and induction of disease causative target genes plasminogen activator inhibitor-1 (PAI-1) and connective tissue growth factor (CTGF) prompting an investigation of the mechanistic involvement of EGFR and tumor suppressor p53 in profibrotic signaling. TGF-β1, PAI-1, CTGF, p53 and EGFR were co-expressed in the obstructed kidney localizing predominantly to the tubular and interstitial compartments. Indeed, TGF-β1 activated EGFR and p53 as well as SMAD2/3. Genetic deficiency of either EGFR or p53 or functional blockade with AG1478 or Pifithrin-α, respectively, effectively inhibited PAI-1and CTGF induction and morphological transformation of renal fibroblasts as did SMAD3 knockdown or pretreatment with the SMAD3 inhibitor SIS3. Reactive oxygen species (ROS)-dependent mechanisms initiated by TGF-β1 were critical for EGFR(Y845) and p53(Ser15) phosphorylation and target gene expression. The p22(Phox) subunit of NADPH oxidase was also elevated in the fibrotic kidney with an expression pattern similar to p53 and EGFR. EGF stimulation alone initiated, albeit delayed, c-terminal SMAD3 phosphorylation (that required the TGF-β1 receptor) and rapid ERK2 activation both of which are necessary for PAI-1 and CTGF induction in renal fibroblasts. These data highlight the extensive cross-talk among SMAD2/3, EGFR and p53 pathways essential for expression of TGF-β1-induced fibrotic target genes.

Keywords: (mouse embryo fibroblasts); 3,3′-diaminobenzidine; ALK; BMP; CDK; CTGF; DAB; DPI; ECM; EGF; EGFR; ERK; HB-EGF; IHC; MEFs; MEK; N-acetyl cysteine; NAC; NADPH oxidase; NOX; Obstructive nephropathy; PAI-1; ROS; Renal fibrosis; SMAD signaling; TGF-β1; UUO; activin-like kinase; bone morphogenic protein; chronic kidney disease; connective tissue growth factor; diphenyleneiodonium chloride; epidermal growth factor; epidermal growth factor receptor; extracellular matrix; extracellular signal-regulated kinases; heparin-binding EGF; immunohistochemistry; mitogen-activated protein kinase kinase; p53; plasminogen activator inhibitor-1; reactive oxygen species; transforming growth factor-β1; unilateral ureteral obstruction; α-SMA; α-smooth muscle actin.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Benzothiazoles / pharmacology
  • Connective Tissue Growth Factor / genetics
  • Connective Tissue Growth Factor / metabolism
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / drug effects
  • Endothelium, Vascular / metabolism
  • ErbB Receptors / agonists
  • ErbB Receptors / genetics*
  • ErbB Receptors / metabolism
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism*
  • Fibrosis
  • Gene Expression Regulation
  • Isoquinolines / pharmacology
  • Mice
  • Mink
  • Myocytes, Smooth Muscle / cytology
  • Myocytes, Smooth Muscle / drug effects
  • Myocytes, Smooth Muscle / metabolism
  • Pyridines / pharmacology
  • Pyrroles / pharmacology
  • Quinazolines / pharmacology
  • Rats
  • Reactive Oxygen Species / metabolism*
  • Renal Insufficiency, Chronic / genetics*
  • Renal Insufficiency, Chronic / metabolism
  • Renal Insufficiency, Chronic / pathology
  • Serpin E2 / genetics
  • Serpin E2 / metabolism
  • Signal Transduction
  • Smad2 Protein / genetics
  • Smad2 Protein / metabolism
  • Smad3 Protein / genetics
  • Smad3 Protein / metabolism
  • Toluene / analogs & derivatives
  • Toluene / pharmacology
  • Transforming Growth Factor beta1 / pharmacology*
  • Tumor Suppressor Protein p53 / genetics*
  • Tumor Suppressor Protein p53 / metabolism
  • Tyrphostins / pharmacology

Substances

  • 6,7-dimethyl-2-(2E)-3-(1-methyl-2-phenyl-1H-pyrrolo(2,3-b)pyridin-3-yl-prop-2-enoyl)-1,2,3,4-tetrahydroisoquinoline hydrochloride
  • Benzothiazoles
  • CCN2 protein, mouse
  • Isoquinolines
  • Pyridines
  • Pyrroles
  • Quinazolines
  • Reactive Oxygen Species
  • Serpin E2
  • Serpine2 protein, mouse
  • Smad2 Protein
  • Smad2 protein, mouse
  • Smad3 Protein
  • Smad3 protein, mouse
  • Transforming Growth Factor beta1
  • Tumor Suppressor Protein p53
  • Tyrphostins
  • Connective Tissue Growth Factor
  • RTKI cpd
  • Toluene
  • pifithrin
  • EGFR protein, mouse
  • ErbB Receptors