Focal adhesion kinase mediates TGF-beta1-induced renal tubular epithelial-to-mesenchymal transition in vitro.
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which participates in many important cellular processes such as cell adhesion and migration. However, the role of FAK in renal tubular epithelial-to-mesenchymal transition (EMT) is still unknown. FAK was knocked down by transfection of specific small interfering RNA (siRNA) in cultured HK-2 cells, then the cells were stimulated with transforming growth factor-beta 1 (TGF-beta1). The expression of FAK, alpha-smooth muscle actin (alpha-SMA),E-cadherin, Akt, matrix metallopeptidase-9 (MMP-9),tissue inhibitor of metalloproteinase-1 (TIMP-1), and collagen IV were detected by RT-PCR, Western blot and immunofluorescence methods, respectively. Cell migration was determined by transwell assay. The results suggest that the expression of FAK was up-regulated in HK-2 cells when incubated with TGF-beta1(10 microg/l), which was accompanied by reduced expression of E-cadherin and increased expression of alpha-SMA. All these changes were restored by FAK siRNA. Akt phosphorylation was induced by the treatment with TGF-beta1, which was blocked by FAK siRNA. TGF-beta1-induced down-regulation of E-cadherin was recovered by a PI3K/Akt inhibitor, LY294002, without affecting the expression of FAK. Functionally, TGF-beta1 induced an increase in MMP-9 expression, as well as decreased expression of TIMP-1 and collagen IV, which were all restored by the FAK siRNA transfection. In addition, FAK siRNA significantly reduced TGF-beta1-induced cells migration. In conclusion, FAK may play a crucial role in mediating TGF-beta1-induced EMT through the activation of Akt pathway.
Deng
Bingqing
B
Department of Nephrology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Yang
Xiao
X
Liu
Jianshe
J
He
Fangfang
F
Zhu
Zhonghua
Z
Zhang
Chun
C
D015703:Antigens, CD; D015820:Cadherins / Q000378:metabolism; D002460:Cell Line; D002465:Cell Movement; D054338:Cell Transdifferentiation; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000201:enzymology*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D055785:Gene Knockdown Techniques; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D008648:Mesoderm / Q000201:enzymology*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
D015703:Antigens, CD; C513384:CDH1 protein, human; D015820:Cadherins; D016326:Extracellular Matrix Proteins; D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D051057:Proto-Oncogene Proteins c-akt