Cell culture Human HCC cell lines PLC PRF 5, HepG2, Huh7 and Hep3B were purchas

In addition, Smad2 and C2 ATF2 synergistically induced CRP2 mRNA levels. Discussion CRP2 plays a critical role in attenuating the development of arteriosclerosis. Upregulating ABT-888 912444-00-9 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 CRP2 expression to decrease vascular disease. We show that activation of both Smad2 3 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. Our results demonstrate that two signaling axes downstream of TGFB converge on Csrp2 promoter to co operatively control CRP2 induction in VSMCs.<br><br> Smad proteins function as intracellular TGFB signaling effectors. Following activation and translocation into the nucleus, the heterotrimeric Afatinib BIBW2992 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 Smad2 3 to induce CRP2 expression via a SBE site at bp 445 of the Csrp2 promoter. This Smad2 3 activa tion is dependent on TBRI and its kinase activity, as siTBRI or SB431542 inhibited TGFB induced Smad2 3 phosphorylation. TGFB also activated ATF2 to induce CRP2 expression. The fact that ATF2 phosphorylation was not affected ei ther by inhibiting TBRI kinase activity or knocking down TBRI expression indicated that TBRI was dispensable for ATF2 activation.<br><br> By con trast, TBRII 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 AG-1478 EGFR 阻害剤 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 ERK1 2 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 Smad2 3 activation.<br><br> 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 activated JNK in turn phosphorylates Smad3. In glioblastoma 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 downstream of TGFB that are in contrast to our results that p38 MAPK and ERK1 2 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.