Secalonic acid D alters the expression and phosphorylation of the transcription factors and their binding to cAMP response element in developing murine secondary palate

Secalonic acid D (SAD), a cleft palate-inducing mycotoxin, reduces palatal cyclic AMP (cAMP) levels, cAMP relays its signals via the transcription fac tors (TF) such as cAMP response element (CRE) binding protein (CRES), CRE m odulator (CREM) and activator transcription factor-1 (ATF-1) to CRE-contain ing genes. These studies tested the hypothesis that these TF are present an d functional in the developing palate and that SAD altars their expression and function along with that of the CRE-containing gene, proliferating cell nuclear antigen (PCNA). Electrophoretic mobility shift assays (EMSA), usin g nuclear extracts of control and SAD-treated developing palate tissues and P-32-labeled CRE revealed the formation of a DNA-protein complex, Supershi ft/ablation assays with TF antibodies showed the presence of CREB, CREM and another unidentified TF but not ATF-1 in the complex, Western analyses of the DNA-protein complex from preparative EMSA revealed increased binding of CREB to CRE in direct correlation with increase in phospho-CREB (pCREB) in the controls. Exposure to SAD significantly reduced CREB binding throughou t palate development. This was in correlation with reductions in pCREB leve ls on gestational day (GD) 13 and 14 palates. On GD 12, however, SAD dramat ically increased CREB phosphorylation. The ontogeny of palatal CREB and CRE M (several isoforms) expression remained unchanged in controls whereas SAD increased that of the repressor isoform of CREM. The expression of PCNA was inhibited by SAD on GD 12. These results show that the cAMP signaling path way is functional in the palate and that SAD alters CREB phosphorylation an d inhibits its binding to CRE, leading to altered expression of gents invol ved in cell proliferation, an event critical for normal palate development.