Functional analysis of CBP/p300 in embryonic orofacial mesenchymal cells

Abstract

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.