title: NOX4 mediates BMP4-induced upregulation of TRPC1 and 6 protein expressions in distal pulmonary arterial smooth muscle cells.
abstract: BACKGROUND
  Our previous studies demonstrated that bone morphogenetic protein 4 (BMP4) mediated, elevated expression of canonical transient receptor potential (TRPC) largely accounts for the enhanced proliferation in pulmonary arterial smooth muscle cells (PASMCs). In the present study, we sought to determine the signaling pathway through which BMP4 up-regulates TRPC expression.
  
  
  METHODS
  We employed recombinant human BMP4 (rhBMP4) to determine the effects of BMP4 on NADPH oxidase 4 (NOX4) and reactive oxygen species (ROS) production in rat distal PASMCs. We also designed small interfering RNA targeting NOX4 (siNOX4) and detected whether NOX4 knockdown affects rhBMP4-induced ROS, TRPC1 and 6 expression, cell proliferation and intracellular Ca2+ determination in PASMCs.
  
  
  RESULTS
  In rhBMP4 treated rat distal PASMCs, NOX4 expression was (226.73±11.13) %, and the mean ROS level was (123.65±1.62) % of that in untreated control cell. siNOX4 transfection significantly reduced rhBMP4-induced elevation of the mean ROS level in PASMCs. Moreover, siNOX4 transfection markedly reduced rhBMP4-induced elevation of TRPC1 and 6 proteins, basal [Ca2+]i and SOCE. Furthermore, compared with control group (0.21±0.001), the proliferation of rhBMP4 treated cells was significantly enhanced (0.41±0.001) (P<0.01). However, such increase was attenuated by knockdown of NOX4. Moreover, external ROS (H2O2 100 µM, 24 h) rescued the effects of NOX4 knockdown, which included the declining of TRPC1 and 6 expression, basal intracellular calcium concentration ([Ca2+]i) and store-operated calcium entry (SOCE), suggesting that NOX4 plays as an important mediator in BMP4-induced proliferation and intracellular calcium homeostasis.
  
  
  CONCLUSIONS
  These results suggest that BMP4 may increase ROS level, enhance TRPC1 and 6 expression and proliferation by up-regulating NOX4 expression in PASMCs.
authors.affiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The 1st Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The 1st Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Fu
authors.affiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The 1st Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.
authors.forename: Lichun
authors.initials: L
authors.lastname: Tian
authors.affiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The 1st Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.
authors.forename: Yuqin
authors.initials: Y
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The 1st Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China; Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Kai
authors.initials: K
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The 1st Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.
authors.forename: Xiuqing
authors.initials: X
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The 1st Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The 1st Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.
authors.forename: Wenju
authors.initials: W
authors.lastname: Lu
authors.affiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The 1st Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China; Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, Baltimore, Maryland, United States of America; Department of Pulmonary, Inner Mongolia People's Hospital, Huhhot, Inner Mongolia, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Wang
mesh_terms: D000818:Animals; D055415:Bone Morphogenetic Protein 4 / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D011651:Pulmonary Artery / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D050052:TRPC Cation Channels / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics*
chemical_list: C525809:BMP4 protein, human; D055415:Bone Morphogenetic Protein 4; D017382:Reactive Oxygen Species; D050052:TRPC Cation Channels; C492145:Trpc6 protein, rat; C096351:transient receptor potential cation channel, subfamily C, member 1; D006861:Hydrogen Peroxide; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498911:Nox4 protein, rat; D002118:Calcium