Hallmarks of neuro inflammation including activated microglia, astrogliosis, an

TGFB induced CRP2 protein levels while SP600125 significantly reduced CRP2 levels, suggesting JNK functions upstream of ATF2 to mediate ABT888 CRP2 induction. Since unphosphorylated ATF2 is transcriptionally inactive, we overexpressed a constitutively active C2 ATF2 construct in VSMCs and ex amined CRP2 expression levels. Indeed, overexpression of C2 ATF2 increased CRP2 protein 1. 5 fold. Furthermore, C2 ATF2 rescued the TGFB induction of CRP2 that was inhibited by JNK in hibitor SP600125, demonstrating the import ance of JNK ATF2 pathway in TGFB mediated CRP2 induction. We next wanted to determine whether JNK functioned downstream of ROCK in the signaling pathway leading to ATF2 activation. Indeed, Y 27632 abrogated TGFB induced JNK activation, linking ROCK JNK ATF2 signaling axis.<br><br> Both SBE and CRE sites are functionally important for basal and TGFB induction of the Csrp2 promoter activity It is evident from our studies that Smad2 3 and ATF2 par ticipate in the TGFB induction of CRP2. As ATF2 activates Csrp2 transcription AEB071 価格 via CRE site, we set out to identify elements that are responsible for Smad2 3 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 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.<br><br> We then transiently transfected VSMCs with parental wild type lu ciferase plasmid 795Csrp2 luc and mutant constructs. Mutation of SBE681 did AG-014699 ic50 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. These results indicate that both the SBE at bp 445 and CRE at 461 are functionally important in regulating Csrp2 transcription. Further supporting this notion, these two sites are com pletely conserved across species among human, mouse, and rat. To further demonstrate the critical roles of Smad2 3 and ATF2 in Csrp2 transcriptional regulation, we cotransfected luciferase plasmid 795Csrp2 luc with expression plasmids Smad2, C2 ATF2, or both in VSMCs and examined pro moter activity.<br><br> Smad2 slightly increased Csrp2 promoter ac tivity although it did not reach 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 C2 ATF2 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 C2 ATF2 on CRP2 mRNA levels. Similar to promoter activity, Smad2 slightly in creased while C2 ATF2 increased CRP2 mRNA levels to 2 fold.