TGF-β Signaling in Murine Embryonic Palate Cells Involves Phosphorylation of the CREB Transcription Factor

Abstract

A number of studies over the last several years have demonstrated a crucial role for TGF-β 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-β activity may be mediated through cAMP-inducible pathways. We thus examined the effects of TGF-β 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-β-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-β treatment led to increasedphosphorylation 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-β 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-β. TGF-β 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-β. Additionally, TGF-β 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-β acts to induce CREB ser-133 phosphorylation and subsequent activation of CRE-containing genes.