Warner D R, Pisano M M, Greene R M
Department of Molecular, Cellular, and Craniofacial Biology, University of Louisville Birth Defects Center, University of Louisville School of Dentistry, Louisville, Kentucky 40292, USA.
J Cell Biochem. 2006 Dec 1;99(5):1374-9. doi: 10.1002/jcb.20974.
CREB binding protein (CBP) and the close structural homolog, p300, are nuclear coactivators of multiple signaling pathways that play important roles in embryonic development and cellular homeostasis. TGFbeta regulates the proliferation rate of many cell types and has been demonstrated to inhibit the growth rate of mouse embryonic maxillary mesenchymal (MEMM) cells. The role of CBP and p300 in TGFbeta-mediated control of proliferation of MEMM cells was thus investigated using an in vitro gene knockdown approach. TGFbeta reporter assays demonstrated that p300 mRNA knockdown via targeted siRNAs led to a reduction in the response to TGFbeta, whereas knockdown of CBP by the same approach had an insignificant effect. In MEMM cell proliferation assays, siRNA-mediated knockdown of CBP and/or p300 had little impact upon TGFbeta-mediated growth inhibition; however, the basal rate of proliferation was increased. Inhibition of p300 activity via overexpression of a dominant-negative mutant (p300deltaC/H3) led to significant inhibition of TGFbeta-mediated activation of p3TP-lux. As with the siRNA knockdown approach, p300deltaC/H3 also increased the basal rate of cell proliferation of MEMM cells. CBP/p300 siRNA knockdown had a significant but incomplete inhibition of TGFbeta-induction of matrix metalloproteinase-9 (gelatinase B) expression. These data demonstrate that p300 is involved in Smad-mediated transcription of p3TP-lux, however, its role (and that of CBP) in biological processes such as the control of cell proliferation and extracellular matrix metabolism is more complex and may be mediated via mechanisms beyond coactivator recruitment.
CREB结合蛋白（CBP）和结构紧密的同源物p300是多种信号通路的核共激活因子，在胚胎发育和细胞稳态中发挥重要作用。转化生长因子β（TGFβ）调节多种细胞类型的增殖速率，并已被证明可抑制小鼠胚胎上颌间充质（MEMM）细胞的生长速率。因此，使用体外基因敲低方法研究了CBP和p300在TGFβ介导的MEMM细胞增殖控制中的作用。TGFβ报告基因检测表明，通过靶向小干扰RNA（siRNA）敲低p300 mRNA会导致对TGFβ的反应降低，而通过相同方法敲低CBP则效果不显著。在MEMM细胞增殖检测中，siRNA介导的CBP和/或p300敲低对TGFβ介导的生长抑制影响很小；然而，基础增殖速率增加。通过过表达显性负性突变体（p300deltaC/H3）抑制p300活性会导致对TGFβ介导的p3TP-lux激活的显著抑制。与siRNA敲低方法一样，p300deltaC/H3也增加了MEMM细胞的基础细胞增殖速率。CBP/p300 siRNA敲低对TGFβ诱导的基质金属蛋白酶-9（明胶酶B）表达有显著但不完全的抑制作用。这些数据表明，p300参与了Smad介导的p3TP-lux转录，然而，其在诸如细胞增殖控制和细胞外基质代谢等生物学过程中的作用（以及CBP的作用）更为复杂，可能通过共激活因子募集以外的机制介导。
