Secalonic acid D alters the expression and phosphorylation of the transcription factors and their binding to cAMP...

Secalonic acid D (SAD), a cleft palate-inducing mycotoxin, reduces palatal cyclic AMP (cAMP) levels. cAMP relays its signals via the transcription factors (TF) such as cAMP response element (CRE) binding protein (CREB), CRE modulator (CREM) and activator transcription factor-1 (ATF-1) to CRE-containing genes. These studies tested the hypothesis that these TF are present and functional in the developing palate and that SAD alters their expression and function along with that of the CRE-containing gene, proliferating cell nuclear antigen (PCNA). Electrophoretic mobility shift assays (EMSA), using nuclear extracts of control and SAD-treated developing palate tissues and 32P-labeled CRE revealed the formation of a DNA-protein complex. Supershift/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 throughout palate development. This was in correlation with reductions in pCREB levels on gestational day (GD) 13 and 14 palates. On GD 12, however, SAD dramatically increased CREB phosphorylation. The ontogeny of palatal CREB and CREM (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 pathway is functional in the palate and that SAD alters CREB phosphorylation and inhibits its binding to CRE. leading to altered expression of genes involved in cell proliferation, an event critical for normal palate development.