Figure 7
SBE445 and CRE461 are functionally important for basal and TGFβ-induced Csrp2 promoter activity. (A) Two putative SBE sites at bp −681 and −445 of the mouse Csrp2 promoter (bold and underlined). The CRE site at −461 is also indicated (italic and underlined). (B) The putative SBE site at −445 and CRE at −461 are important for Csrp2 promoter activity. The −795 Csrp2 wild type and SBE and CRE mutant promoter constructs are schematically depicted in the left panel. VSMCs were transiently transfected with Csrp2 luciferase reporter constructs containing −795, SBE681mut, SBE445mut, CREmut, or CREmut/SBE445mut in triplicate using FuGENE 6 transfection reagent. Two hours after transfection, cells were treated with or without TGFβ (10 ng/ml) for 24 h. Cells were then harvested for luciferase activity and protein assays. Luciferase activity is expressed relative to −795 without TGFβ treatment. Values are mean ± S.E. of at least three experiments. (C) Conservation of the CRE and SBE sites among species. Sequence alignment of the corresponding regions of human and rat promoter sequences to the mouse promoter. The CRE site is in italic and SBE site in bold type. (D) VSMCs were transiently cotransfected with –795Csrp2-luc reporter with empty vector or expression plasmids Smad2, C2/ATF2, or both. Cells were then harvested 24 h later for luciferase activity and protein assays. Luciferase activity is expressed relative to −795 with empty expression vector. Values are mean ± S.E. of at least three experiments. *P < 0.05 vs. empty expression vector. (E) VSMCs were electroporated with control vector or expression plasmids Smad2, C2/ATF2, or both. Total RNA was prepared 12 h later for real-time PCR analysis to assess CRP2 mRNA expression and β-actin was used as an internal control for normalization. Quantification was performed by the comparative CT method. CRP2 mRNA is expressed relative to empty expression vector. Values are mean ± S.E. of at least three experiments. *P < 0.05 vs. empty expression vector.