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The differential regulation of Smad7 in kidney tubule cells by connective tissue growth factor and transforming growth factor‐beta1
Author(s) -
QI WEIER,
CHEN XINMING,
TWIGG STEPHEN,
ZHANG YUAN,
GILBERT RICHARD E,
KELLY DARREN J,
POLLOCK CAROL A
Publication year - 2007
Publication title -
nephrology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.752
H-Index - 61
eISSN - 1440-1797
pISSN - 1320-5358
DOI - 10.1111/j.1440-1797.2007.00788.x
Subject(s) - ctgf , transforming growth factor , growth factor , smad , connective tissue , endocrinology , transforming growth factor beta , medicine , basal (medicine) , transfection , biology , cell culture , receptor , pathology , insulin , genetics
Summary: Aims: Smad7 is an inhibitory Smad that regulates transforming growth factor‐β (TGF‐β) signaling. Connective tissue growth factor (CTGF) is recognized as a potent downstream mediator of the fibrogenic effects of TGF‐β1. SMAD binding sites have been identified in both TGF‐β and CTGF promoters. The effect of CTGF on Smad7 expression and its role in the regulation of Smad7 induced by TGF‐β1 in renal tubular cells is unknown. Methods: Human model of proximal tubular cells (HK‐2 cells) was used and confirmed using a diabetic rat model. RT‐PCR was performed to measure Smad7, TGF‐β1 and Smad2 and ELISA was performed to measure active TGF‐β1. CTGF or TGF‐β1 was silenced in HK‐2 cells using siRNA methodology. Results: TGF‐β1 induced Smad7 in a time‐dependent manner, peaking at 30 min (P < 0.0005) but sustained up to 24 hrs (p < 0.005). Conversely, CTGF reduced Smad7, which was maximal at 24 hrs (p < 0.05). This was supported by our in vivo data demonstrating that CTGF protein significantly increased while Smad7 mRNA level was reduced in a diabetic rat model. The basal expression level of Smad7 decreased in TGF‐β1 silenced cells compared to cells transfected with non‐specific siRNA (p < 0.0005). The basal expression level of Smad7 increased in CTGF silenced cells (p < 0.05), which was increased by TGF‐β1 (p < 0.005). Both mRNA and protein levels of TGF‐β1 decreased in CTGF silenced cells (p < 0.05 and p < 0.005 respectively) accompanied by reduction in Smad2 mRNA level in CTGF silenced cells. Conclusions: Smad7 is induced rapidly by TGF‐β1 limiting the response to TGF‐β1. CTGF likely plays a key role in promoting TGF‐β1 activity by decreasing the availability of Smad7 and increasing Smad2.