title: Divergent signaling pathways cooperatively regulate TGFβ induction of cysteine-rich protein 2 in vascular smooth muscle cells.
abstract: BACKGROUND
  Vascular smooth muscle cells (VSMCs) of the arterial wall play a critical role in the development of occlusive vascular diseases. Cysteine-rich protein 2 (CRP2) is a VSMC-expressed LIM-only protein, which functionally limits VSMC migration and protects against pathological vascular remodeling. The multifunctional cytokine TGFβ has been implicated to play a role in the pathogenesis of atherosclerosis through numerous downstream signaling pathways. We showed previously that TGFβ upregulates CRP2 expression; however, the detailed signaling mechanisms remain unclear.
  
  
  RESULTS
  TGFβ treatment of VSMCs activated both Smad2/3 and ATF2 phosphorylation. Individually knocking down Smad2/3 or ATF2 pathways with siRNA impaired the TGFβ induction of CRP2, indicating that both contribute to CRP2 expression. Inhibiting TβRI kinase activity by SB431542 or TβRI knockdown abolished Smad2/3 phosphorylation but did not alter ATF2 phosphorylation, indicating while Smad2/3 phosphorylation was TβRI-dependent ATF2 phosphorylation was independent of TβRI. Inhibiting Src kinase activity by SU6656 suppressed TGFβ-induced RhoA and ATF2 activation but not Smad2 phosphorylation. Blocking ROCK activity, the major downstream target of RhoA, abolished ATF2 phosphorylation and CRP2 induction but not Smad2 phosphorylation. Furthermore, JNK inhibition with SP600125 reduced TGFβ-induced ATF2 (but not Smad2) phosphorylation and CRP2 protein expression while ROCK inhibition blocked JNK activation. These results indicate that downstream of TβRII, Src family kinase-RhoA-ROCK-JNK signaling pathway mediates TβRI-independent ATF2 activation. Promoter analysis revealed that the TGFβ induction of CRP2 was mediated through the CRE and SBE promoter elements that were located in close proximity.
  
  
  CONCLUSIONS
  Our results demonstrate that two signaling pathways downstream of TGFβ converge on the CRE and SBE sites of the Csrp2 promoter to cooperatively control CRP2 induction in VSMCs, which represents a previously unrecognized mechanism of VSMC gene induction by TGFβ.
authors.forename: Meng-Ling
authors.initials: ML
authors.lastname: Wu
authors.forename: Chung-Huang
authors.initials: CH
authors.lastname: Chen
authors.forename: Yung-Tsang
authors.initials: YT
authors.lastname: Lin
authors.forename: Yuan-Jyun
authors.initials: YJ
authors.lastname: Jheng
authors.forename: Yen-Chun
authors.initials: YC
authors.lastname: Ho
authors.forename: Liang-Tung
authors.initials: LT
authors.lastname: Yang
authors.forename: Linyi
authors.initials: L
authors.lastname: Chen
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Layne
authors.affiliation: Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan. syet@nhri.org.tw.
authors.forename: Shaw-Fang
authors.initials: SF
authors.lastname: Yet
mesh_terms: D051698:Activating Transcription Factor 2 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D060588:LIM Domain Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D054460:rho-Associated Kinases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D051698:Activating Transcription Factor 2; C497106:Atf2 protein, mouse; D002352:Carrier Proteins; C507808:Crip2 protein, mouse; D060588:LIM Domain Proteins; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D016212:Transforming Growth Factor beta; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein