The differential regulation of Smad7 in kidney tubule cells by connective tissue growth factor and transforming growth factor-beta1.
OBJECTIVE
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-beta1. 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-beta1 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-beta1 and Smad2 and ELISA was performed to measure active TGF-beta1. CTGF or TGF-beta1 was silenced in HK-2 cells using siRNA methodology.


RESULTS
TGF-beta1 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-beta1 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-beta1 (p<0.005). Both mRNA and protein levels of TGF-beta1 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-beta1 limiting the response to TGF-beta1. CTGF likely plays a key role in promoting TGF-beta1 activity by decreasing the availability of Smad7 and increasing Smad2.
Qi
Weier
W
Kolling Institute, Department of Medicine, Royal North Shore Hospital and University of Sydney, Sydney, New South Wales, Australia.
Chen
Xinming
X
Twigg
Stephen
S
Zhang
Yuan
Y
Gilbert
Richard E
RE
Kelly
Darren J
DJ
Pollock
Carol A
CA
D000704:Analysis of Variance; D000818:Animals; D002460:Cell Line; D055513:Connective Tissue Growth Factor; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D007684:Kidney Tubules / Q000378:metabolism*; D034622:RNA Interference; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D051906:Smad7 Protein / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
C526317:CCN2 protein, human; C526316:CCN2 protein, rat; D017931:DNA Primers; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; C497806:SMAD7 protein, human; D051906:Smad7 Protein; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor