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Updated: Aug 7, 2026
Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis
Published on: April 1, 2022
Functional analysis of CBP/p300 in embryonic orofacial mesenchymal cells
D R Warner1, M M Pisano, R M Greene
1Department of Molecular, Cellular, and Craniofacial Biology, University of Louisville Birth Defects Center, University of Louisville School of Dentistry, Louisville, Kentucky 40292, USA. dennis.warner@louisville.edu
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.
Insights
CREB binding protein (CBP) and p300 are key nuclear coactivators. This study found p300, not CBP, is crucial for TGFbeta signaling in mouse embryonic cells, impacting proliferation and matrix production.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- CREB binding protein (CBP) and p300 are nuclear coactivators vital for embryonic development and cellular homeostasis.
- Transforming growth factor beta (TGFbeta) regulates cell proliferation and inhibits mouse embryonic maxillary mesenchymal (MEMM) cell growth.
Purpose of the Study:
- To investigate the roles of CBP and p300 in TGFbeta-mediated control of MEMM cell proliferation.
- To elucidate the involvement of CBP and p300 in TGFbeta signaling pathways.
Main Methods:
- In vitro gene knockdown using siRNAs targeting CBP and p300.
- TGFbeta reporter assays (p3TP-lux) and MEMM cell proliferation assays.
- Overexpression of a dominant-negative p300 mutant (p300deltaC/H3).
Main Results:
- p300, but not CBP, knockdown reduced TGFbeta response in reporter assays.
- Knockdown of CBP/p300 increased basal MEMM cell proliferation but had limited effect on TGFbeta-mediated growth inhibition.
- p300 inhibition increased basal proliferation and reduced TGFbeta-induced p3TP-lux activation.
- CBP/p300 knockdown partially inhibited TGFbeta-induced matrix metalloproteinase-9 expression.
Conclusions:
- p300 plays a significant role in Smad-mediated transcription of p3TP-lux.
- The roles of CBP and p300 in cell proliferation and extracellular matrix metabolism are complex and may involve mechanisms beyond coactivator recruitment.
