The differential regulation of Smad7 in kidney tubule cells by connective tissue growth factor and transforming growth factor-beta1

Aims: Smad7 is an inhibitory Smad that regulates transforming growth factor-beta (TGF-beta) signaling. Connective tissue growth factor (CTGF) is recognized as a potent downstream mediator of the fibrogenic effects of TGF-beta 1. SMAD binding sites have been identified in both TGF-beta and CTGF promoters. The effect of CTGF on Smad7 expression and its role in the regulation of Smad7 induced by TGF-beta 1 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-beta 1 and Smad2 and ELISA was performed to measure active TGF-beta 1, CTGF or TGF-beta 1 was silenced in HK-2 cells using siRNA methodology. Results: TGF-beta 1 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-beta 1 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-beta 1 (p < 0.005). Both mRNA and protein levels of TGF-beta 1 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-beta 1 limiting the response to TGF-beta 1. CTGF likely plays a key role in promoting TGF-beta 1 activity by decreasing the availability of Smad7 and increasing Smad2.