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We demonstrated that both CRE and SBE sites con tribute to CRP2 upregulation by TGFB. Im portantly, these two sites are completely conserved across species among human, mouse, and rat, suggesting the critical importance of the CRE and SBE in the regulation of buy ABT-888 Csrp2 transcription. The importance of CRE in mediating TGFB target gene induction was shown in a rat intestinal epithelial cell line 4 1 that transcription factors CREB 1, ATF2, c Jun, and Smad3 all bind to the CRE site to activate transcription. Interestingly, ATF2, via its basic leucine zipper region, was shown to bind to the MH1 domain of the Smad proteins. TGFB stimulation further enhances the association of ATF2 and Smad3 4 and increases the CRE containing reporter activity in HepG2 cells.<br><br> Since blocking either the ATF2 CRE or TBRI Smad2 3 axis significantly reduced CRP2 expression and given the proximity of CRE and SBE sites, it is possible that Smad heterotrimer and ATF2 might form a higher order complex Afatinib EGFR 阻害剤 to cooperatively, rather than independently, regulate CRP2 expression. This regulation via two cis elements is similar to that of several SMC marker genes. For example, the TGFB induction of SM actin and SM22 expression is coordinately regulated by a TGFB control element and nearby CArG 6GG elements in the 5 promoter. However, the regulatory mechanism of CRP2 expres sion by TGFB differ from these SMC marker genes in that CRP2 induction is mediated through the CRE and SBE elements whereas TCE and CArG elements medi ate the TGFB induction of SM actin and SM22 expression.<br><br> Conclusion Based on our findings, we propose that two signaling pathways downstream of TGFB converge on Csrp2 pro moter to cooperatively induce Csrp2 gene transcription and protein expression. In the canonical pathway, upon TGFB binding, TBRII trans phosphorylates TBRI to activate its kinase function, buy AG-1478 leading to Smad2 3 phosphorylation. Activated Smad2 3 then cooperate with Smad4 to form a complex, translocates into the nucleus and bind to SBE of the Csrp2 promoter. In the non canonical signaling pathway that does not require TBRI, TGFB binds to TBRII and activates Src family kinase and RhoA signaling. Activated ROCK in turn phosphorylates JNK, resulting in ATF2 activation. Activated ATF2 dimer then binds to the CRE site 8 bp upstream of SBE on the Csrp2 promoter.<br><br> The ATF2 on the CRE site and Smad proteins on the SBE site might associate to form a higher order complex to cooperatively enhance CRP2 expression in VSMCs, which represents a previously unrecognized mechanism of VSMC gene in duction by TGFB. Methods Luciferase reporter and expression constructs The 795Csrp2 luc luciferase reporter plasmid was de scribed previously and used as a template to generate mutant constructs. Site directed mutagenesis was per formed using Pfu polymerase to mutate the putative SBE sites at 681 from GTCTAGAA to CTATC GAA and at 445 from GTCTAGAT to CTATCGAT to generate SBE681mut and SBE445mut constructs, respectively. The CREmut SBE445 mut construct with double mutations at 461CRE and 445SBE was generated using 795CREmutCsrp2 luc as a template to mutate putative 445SBE site as above. All constructs were confirmed by DNA sequen cing.