title: Inhibition of KCa3.1 suppresses TGF-β1 induced MCP-1 expression in human proximal tubular cells through Smad3, p38 and ERK1/2 signaling pathways.
abstract: It is well known that TGF-β1 plays a central role in renal fibrosis due in large part to stimulation of inflammatory responses. KCa3.1, a potassium channel protein, has been suggested as a potential therapeutic target for diseases such as sickle cell anemia, autoimmunity, atherosclerosis and more recently, kidney fibrosis. Blockade of KCa3.1 has been shown to ameliorate renal fibrosis in diabetic mice in association with reduced TGF-β1 signaling. However, the centrality of KCa3.1 activation to TGF-β1 induced inflammation remains unknown. In this study, human proximal tubular cells (HK2 cells) were incubated with TGF-β1 (2 ng/ml) for 48 h in the presence or absence of KCa3.1 siRNA or the KCa3.1 inhibitor TRAM34. HK2 cells overexpressing KCa3.1 were studied in parallel. The mRNA and protein expression of monocyte chemoattractant protein-1 (MCP-1) were measured by qRT-PCR and ELISA. Downstream TGF-β1 signaling molecules Smad3, p38 and ERK1/2 were measured by Western blot analysis. Using whole-cell patch clamp techniques we found that TGFβ-1 induced a large KCa3.1 K-current that was inhibited by TRAM34. TGF-β1 also increased MCP-1 mRNA and protein expression in HK2 cells compared to control, an effect that was reversed by in the presence of KCa3.1 siRNA. Similarly, TRAM34 significantly reduced the TGF-β1-mediated increase in MCP-1 at both the mRNA and protein levels. Inhibition of KCa3.1 with KCa3.1 siRNA or TRAM34 also reduced TGF-β1-induced phosphorylation of Smad3, p38 and ERK1/2 MAPK pathways. Conversely overexpression of KCa3.1 induced TGF-β1 signaling cascades and expression of MCP-1. The present study is consistent with a key role for KCa3.1 renal proximal tubular cells in mediating the TGF-β1 induction of MCP-1 expression in HK2 cells via Smad3, p38 and ERK1/2 MAPK signaling pathways.
authors.affiliation: Kolling Institute of Medical Research, Sydney Medical School, University of Sydney, Royal North Shore Hospital, St Leonards, NSW 2065, Australia; Xiamen Center of Clinical Laboratory, Xiamen Zhongshan Hospital, Medical College of Xiamen University, Xiamen 361004, China.
authors.forename: Chunling
authors.initials: C
authors.lastname: Huang
authors.affiliation: School of Medical Sciences, Discipline of Physiology and Bosch Institute, University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Margot L
authors.initials: ML
authors.lastname: Day
authors.affiliation: School of Medical Sciences, Discipline of Physiology and Bosch Institute, University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Philip
authors.initials: P
authors.lastname: Poronnik
authors.affiliation: Kolling Institute of Medical Research, Sydney Medical School, University of Sydney, Royal North Shore Hospital, St Leonards, NSW 2065, Australia. Electronic address: carol.pollock@sydney.edu.au.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Pollock
authors.affiliation: Kolling Institute of Medical Research, Sydney Medical School, University of Sydney, Royal North Shore Hospital, St Leonards, NSW 2065, Australia.
authors.forename: Xin-Ming
authors.initials: XM
authors.lastname: Chen
mesh_terms: D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000201:enzymology / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D010766:Phosphorylation; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics; D015398:Signal Transduction; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
chemical_list: C111835:CCL2 protein, human; D018932:Chemokine CCL2; D005419:Flavonoids; D007093:Imidazoles; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; D011720:Pyrazoles; D011725:Pyridines; D034741:RNA, Small Interfering; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C508960:TGFB1 protein, human; C411671:TRAM 34; D053773:Transforming Growth Factor beta1; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one
keywords: Cell signaling; KCa3.1; MCP-1; TGF-β1