Smad proteins function as intracellular TGFB directly signaling effectors. Following activation and translocation into the nucleus, the heterotrimeric Smad complex recognizes and binds to SBE site to activate target gene expression. Consistent with this canonical signaling pathway, we found in VSMCs that TGFB activated Smad23 to induce CRP2 expression via a SBE site at bp ?445 of the Csrp2 promoter. This Smad23 activa tion is dependent on TBRI and its kinase activity, as siTBRI or SB431542 inhibited TGFB induced Smad23 phosphorylation. TGFB also activated ATF2 to induce CRP2 expression. The fact that ATF2 phosphorylation was not affected ei ther by inhibiting TBRI kinase activity Inhibitors,Modulators,Libraries or knocking down TBRI expression indicated that TBRI was dispensable for ATF2 activation.

By con trast, TBRII Inhibitors,Modulators,Libraries was required for both Smad2 and ATF2 acti vation. Intriguingly, although it was expected that overexpression of DN TBRII inhibited Smad2 phos phorylation DN TBRII failed to inhibit ATF2 phosphorylation. Taken together, these results suggest that in VSMCs TBRII alone is able to mediate TGFB signaling to ATF2 and the cytoplasmic do main of TBRII is not required. This TBRI independent sig naling to ATF2 is similar to the findings in dermal cells that TBRII directly activates ERK12 without the participation of TBRI and supports the notion that TGFB recep tors can activate non Smad proteins and allow Smad independent TGFB responses. We found JNK functioned upstream of ATF2 in the TGFB induction of CRP2 . however, JNK in hibition did not affect Smad23 activation.

These results are in contrast to the findings in Mv1Lu epithelial cells and T98G glioblastoma cells. In epithelial cells, while TGFB activates both Smad3 and JNK there is an interdependent Smad and JNK sig naling because Inhibitors,Modulators,Libraries activated JNK in turn phosphorylates Smad3. In glioblastoma Inhibitors,Modulators,Libraries cells, TGFB activates p38 MAPK and ATF2 but does not induce JNK phos phorylation. furthermore, inhibition of p38 MAPK de creases TGFB induced phosphorylation of ATF2 and Smad2. Those findings indicate an interaction between Smad and p38 MAPK pathways Inhibitors,Modulators,Libraries downstream of TGFB that are in contrast to our results that p38 MAPK and ERK12 are not involved in ATF2 activation. Our current findings and these previous reports suggest that TGFB signaling pathways are likely to be cell type specific. 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 Csrp2 transcription. The http://www.selleckchem.com/products/PF-2341066.html 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.