Inhibition of integrin-linked kinase via a siRNA expression plasmid attenuates connective tissue growth factor-induced human proximal tubular epithelial cells to mesenchymal transition.
BACKGROUND
Increasing evidence suggests that connective tissue growth factor (CTGF) is involved in the epithelial-to-mesenchymal transition (EMT). The exact intracellular events that drive this process, however, are not fully understood. In this study, we investigated the role of integrin-linked kinase (ILK) in mediating CTGF-induced EMT.


METHODS
The expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin upon the stimulation by recombinant human CTGF (rhCTGF) in cultured human tubular epithelial cell line (HK-2) was detected by real-time RT-PCR and Western blot. Subsequently, the role of ILK was determined by using ILK siRNA.


RESULTS
rhCTGF increased the mRNA expression of alpha-SMA significantly in a dose- and time-dependent manner, while E-cadherin mRNA decreased in a dose- and time-dependent manner. alpha-SMA protein was up-regulated after stimulation by 5 ng/ml CTGF for 96 h, and increased further after stimulation by 50 ng/ml. An immunocytochemical study showed that alpha-SMA was initially detectable at 48 h, and increased further at 72 h, while there was almost no alpha-SMA immunostaining observed in the control group at the same time point. E-cadherin protein was also down-regulated in a dose-dependent manner. Transfection of HK-2 cells with ILK-siRNA significantly attenuated rhCTGF-induced alpha-SMA induction and E-cadherin repression.


CONCLUSIONS
Our study suggested that ILK mediated the effect of EMT in proximal tubular epithelial cells stimulated by CTGF.
Liu
Bi-Cheng
BC
Institute of Nephrology, Zhong Da Hospital, Southeast University, Nanjing, PR China. liubc64@yahoo.com.cn
Li
Min-Xia
MX
Zhang
Jian-Dong
JD
Liu
Xiao-Cong
XC
Zhang
Xiao-Liang
XL
Phillips
Aled O
AO
D000199:Actins / Q000235:genetics / Q000378:metabolism; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed; D055513:Connective Tissue Growth Factor; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism* / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism* / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000502:physiology*; D008648:Mesoderm / Q000166:cytology; D009395:Nephritis, Interstitial / Q000473:pathology* / Q000503:physiopathology; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection; D000099200:Scaffold Protein ILK
D000199:Actins; D015820:Cadherins; D055513:Connective Tissue Growth Factor; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D000099200:Scaffold Protein ILK; C526317:CCN2 protein, human