CREB mediated events in normal and secalonic acid D altered palate development in mice

Secalonic acid D (SAD), a teratogenic mycotoxin, produced by Penicillium oxalicum in contaminated corn, induces cleft palate (CP) as the only defect in the offspring of exposed mothers, when administered as a single intra-peritoneal injection of 30 mg/kg body weight on gestation day (GD) 11. Secalonic acid D is shown to reduce 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. Electrophoretic mobility shift assays (EMSA) and supershift assays with palatal nuclear extracts (PNE), showed the presence of CREB, CREM and another unidentified TF but not ATF-1 in the complex with CRE. Subsequent 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, while, exposure to SAD significantly reduced CREB binding throughout palate development. However, SAD dramatically increased CREB phosphorylation which was correlated with decreased binding to CRE. Phosphorylation induced by SAD was not altered by currently known CREB kinase (PKA, CaMK, MEK, p38MAPK, PKC) or phosphatase inhibitors. Two-dimensional gel analysis suggested that SAD induced phosphorylation was similar to that induced by PKA, ruling out inhibitory phosphorylations. Addition of SAD to PNE following PKA-stimulation resulted in reduced TF binding to CRE. Further, SAD was not only shown to bind to nuclear proteins but to bind to phosphorylated proteins with greater affinity, indicating that SAD-induced decrease in TF binding to CRE is due to direct inhibition by SAD and not due to inhibitory phosphorylation.; Exposure to SAD resulted in a significant and dose-dependent decrease in proliferation of mesenchymal cells. Cell death seen following exposure to SAD was unrelated to apoptosis. Synthesis of either, hyaluronic acid (HA), sulfated glycosaminoglycans (sGAG) or fibronectin were not reduced by SAD. Expression of proliferating cell nuclear antigen, a CRE-containing gene was inhibited by SAD in vivo and in vitro in a dose dependent manner. These results show that 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.