BMP6 reverses TGF-beta1-induced changes in HK-2 cells: implications for the treatment of renal fibrosis.
OBJECTIVE
The aim of the study was to investigate the potential role of BMP6 in TGF-beta1-mediated changes in HK-2 cells.


METHODS
BMP6 was purified via heparin affinity and reverse phase liquid chromatography. The purity, specificity, and bioactivity of BMP6 were determined by SDS-PAGE, Western blot assays, and the induction of alkaline phosphatase (ALP) activity, respectively. Cell proliferation, morphology, and expression levels of alpha-SMA and E-cadherin were assessed by cell viability, microscopy, and Western blot assays, respectively. In addition, cell adhesion abilities were determined by counting the number of attached cells. The expression of fibronectin, collagen IV, matrix metalloproteinases 2 (MMP-2), and tissue inhibitors of matrix metalloproteinases 2 (TIMP-2) were analyzed using RT-PCR. MMP-2 activity was analyzed by zymography, whereas the activation of the MAPKs and Smad signaling were analyzed using Western blot assays and a reporter gene assay, respectively.


RESULTS
Our results indicated that recombinant BMP6 induced ALP activity in a dose-dependent and time-course-dependent manner. Treatment with TGF-beta1 reduced both the cell proliferation and the expression of E-cadherin, induced a morphological transformation, decreased the expression and activity of MMP-2, and increased the expression levels of alpha-SMA, fibronectin, and TIMP-2 in HK-2 cells. All of these effects were inhibited when cells were treated with TGF-beta1 in combination with rhBMP6, whereas rhBMP6 alone demonstrated no such effect. Treatment with TGF-beta1, rhBMP6, or a combination of both had no effect on the expression of collagen IV. In addition, the administration of rhBMP6 prevented the enhanced adhesion behavior triggered by TGF-beta1. Furthermore, the addition of rhBMP6 abrogated the JNK and Smad2/3 signaling that was activated by TGF-beta1.


CONCLUSIONS
BMP6 ameliorated the TGF-beta1-induced changes in HK-2 cells. The suppression of TGF-beta1-mediated JNK and Smad2/3 signaling activation were implicated in these effects.Acta Pharmacologica Sinica (2009) 30: 994-1000; doi: 10.1038/aps.2009.56; published online 22 June 2009.
Yan
Ji-Dong
JD
Medical College of Nankai University, Tianjin 300071, China.
Yang
Shuang
S
Zhang
Jie
J
Zhu
Tian-Hui
TH
D000818:Animals; D055418:Bone Morphogenetic Protein 6 / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002460:Cell Line; D049109:Cell Proliferation; D006224:Cricetinae; D003412:Cricetulus; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005355:Fibrosis / Q000188:drug therapy / Q000473:pathology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D007674:Kidney Diseases / Q000188:drug therapy / Q000473:pathology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D051785:Smad Proteins / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism*
D055418:Bone Morphogenetic Protein 6; D016326:Extracellular Matrix Proteins; D011994:Recombinant Proteins; D051785:Smad Proteins; D053773:Transforming Growth Factor beta1; D048031:JNK Mitogen-Activated Protein Kinases