TGF-beta signaling in murine embryonic palate cells involves phosphorylation of the CREB transcription factor

A number of studies over the last several years have demonstrated a crucial role for TGF-beta in epithelial and mesenchymal differentiation during development of the embryonic palate. Molecular mechanism(s) of signal transduction responsible for eliciting these responses remain unresolved. Since cAMP signaling also modulates the same tissue differentiation in the developing palate and palate-derived cells, we hypothesized that TGF-beta activity may be mediated through cAMP-inducible pathways. We thus examined the effects of TGF-beta on activation of the cAMP regulatory element binding protein CREB, a nuclear transcription factor which mediates transcription of genes containing CRE recognition sequences in their promoters. We examined the ability of TGF-beta-treated murine embryonic palate mesenchymal (MEPM) cells to phosphorylate CREB on the amino acid residue serine 133, phosphorylation of which is indispensable for transcriptional activation. TGF-beta treatment led to increased phosphorylation of CREB ser-133 in a time- and dose-dependent manner. Inhibition of serine-threonine phosphatases by okadaic acid enhanced but did not prolong this response. TGF-beta failed to induce the activity of protein kinase A (PKA), a known CREB kinase. Inhibition of either PKA or calcium/calmodulin kinase II (CaMK II) did not abrogate phosphorylation of CREB by TGF-beta. TGF-beta treatment also did not induce phosphorylation of mitogen-activated protein kinases, erk-1 and erk-2, on tyrosine 185, suggesting that these kinases do not mediate CREB phosphorylation by TGF-beta. Additionally, TGF-beta had no effect on CREB binding to known CREB DNA consensus recognition sequences, CRE and TRE. Together, these data suggest an alternative or novel CREB kinase in MEPM cells through which TGF-beta acts to induce CREB ser-133 phosphorylation and subsequent activation of CRE-containing genes.