As ATF2 activates Csrp2 transcription via CRE site, we set out to identify elements that are responsible for Smad23 mediated induction. Examination of the sequences within the mouse ?795 bp Csrp2 promoter revealed that in addition to the previously identified CRE site at bp ?461 there are two potential Smad binding ele selleck chemical Lenalidomide ments, located at bp ?681 and ?445, each with a base divergent from the consensus SBE. To determine the potential function of these two putative SBE sites, we generated SBE mutant luciferase constructs, ?795SBE681 mut and 795SBE445mut, each with 3 bases mutated within the putative SBE sites. We then transiently transfected VSMCs with parental wild type lu ciferase Inhibitors,Modulators,Libraries plasmid 795Csrp2 luc and mutant constructs. Mutation of SBE681 did not affect either basal or TGFB in duction of Csrp2 promoter activity.
Similar to that of CRE mutation, SBE445 mutation not only decreased basal promoter activity but also reduced TGFB responsive ness. Double mutation of CRE and SBE445 fur ther reduced promoter response. Inhibitors,Modulators,Libraries These results indicate that both the SBE Inhibitors,Modulators,Libraries at bp ?445 and CRE at ?461 are functionally important in regulating Csrp2 transcription. Further supporting this notion, these two sites are Inhibitors,Modulators,Libraries com pletely conserved across species among human, mouse, and rat. To further demonstrate the critical roles of Smad23 and ATF2 in Csrp2 transcriptional regulation, we cotransfected luciferase plasmid 795Csrp2 luc with expression plasmids Smad2, C2ATF2, or both in VSMCs and examined pro moter activity.
Smad2 slightly increased Csrp2 promoter ac tivity although it did not reach Inhibitors,Modulators,Libraries a statistical significance, likely because Smad 2 might be in active without TGFB stimulation. Constitutively active C2 ATF2 significantly increased Csrp2 promoter activity to 3. 4 fold. Interestingly, Smad2 and C2ATF2 together enhanced Csrp2 promoter activity to 19 fold, indicating a synergistic effect of these two factors. Next, we performed real time PCR to examine the effects of Smad2 and C2ATF2 on CRP2 mRNA levels. Similar to promoter activity, Smad2 slightly in creased while C2ATF2 increased CRP2 mRNA levels to 2 fold. In addition, Smad2 and C2ATF2 synergistically induced CRP2 mRNA levels. Discussion CRP2 plays a critical role in attenuating the development of arteriosclerosis. Upregulating CRP2 in the injured ar tery may protect against neointima formation.
The goal of this study was to identify mechanisms and signaling path ways that sustain or upregulate ZD6474 CRP2 expression to decrease vascular disease. We show that activation of both Smad23 and ATF2 are essential for enhancing CRP2 ex pression. Unlike the TBRI dependent Smad pathway, Src family kinase RhoA ROCK JNK signaling axis mediates TBRI independent ATF2 activation. The TGFB induction of CRP2 is mediated through the CRE and SBE promoter ele ments.