Mechanism of secalonic acid D-induced inhibition of transcription factor binding to cyclic AMP response element in the developing murine palate.
Regulation of gene expression via the protein kinase A (PKA) pathway is mediated through Ser133 phosphorylation of the transcription factor (TF), cAMP response element (CRE) binding protein (CREB). Secalonic acid D (SAD), a mycotoxin causing cleft palate (CP), induces phosphorylation of palatal CREB in vivo. SAD-induced increase in phosphoCREB (pCREB), however, is associated with decreased binding of TF to CRE in vivo. Mechanism(s) involved in these two effects of SAD were studied using palatal nuclear extracts (PNE). Stimulation of CREB phosphorylation by SAD was confirmed in vitro in both cell culture and cell-free systems, and this phosphorylation was not altered by currently known CREB kinase (PKA, CaMK, MEK, p38MAPK, PKC) or phosphatase inhibitors. SAD-induced increase in pCREB, however, was associated with decreased TF binding to CRE in vitro. Two-dimensional gel analysis ruled out additional inhibitory phosphorylations. Addition of SAD to PNE following an increase in PKA-phosphorylated CREB resulted in reduced TF binding to CRE. Further, SAD was shown to bind directly to phosphorylated nuclear proteins (pCREB) with greater affinity. In addition, the inhibitory effect of SAD occurred with CRE of proliferating cell nuclear antigen (PCNA) gene. These studies confirm that stimulation of CREB phosphorylation by SAD does not involve sites other than Ser133 and is mediated by a novel kinase. They also indicate that SAD directly binds to CREB to inhibit its binding to CRE of genes such as PCNA. This effect could lead to reduced palatal mesenchymal cell number, smaller palatal shelf, and thus CP.
Hanumegowda
Umesh M
UM
Department of Veterinary Biomedical Sciences, University of Missouri, W148 Vet Med Bldg, 1600 E Rollins, Columbia, Missouri 65211, USA.
Dhulipala
Vamsidhara C
VC
Reddy
Chada S
CS
D000818:Animals; D050882:CREB-Binding Protein; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010159:Palate / Q000002:abnormalities / Q000196:embryology / Q000378:metabolism; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D015398:Signal Transduction / Q000235:genetics; D013723:Teratogens / Q000494:pharmacology* / Q000633:toxicity; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014966:Xanthenes / Q000494:pharmacology* / Q000633:toxicity; D044004:Xanthones
D017362:Cyclic AMP Response Element-Binding Protein; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D013723:Teratogens; D015534:Trans-Activators; D014157:Transcription Factors; D014966:Xanthenes; D044004:Xanthones; C009218:secalonic acid; D050882:CREB-Binding Protein; C494364:Crebbp protein, mouse; D017868:Cyclic AMP-Dependent Protein Kinases