Abstract for presentation at 6th World Congress on Stress

Objective: To identify mechanisms regulating stress-induced transcriptional control of phenylethanolamine N-methyltransferase (PNMT).
Methods: PC12 cells transfected with a PNMT promoter-luciferase reporter gene construct were exposed to hypoxia (5% oxygen) and luciferase activity determined. Cell signaling pathways were assessed by cell pretreatment with signaling inhibitors. Effects of hypoxia inducible factor (HIF1A, HIF1B and ODD) were determined by expression construct co-transfection or Co+2 or deferoxamine induction. Cytosolic and nuclear extracts prepared from untransfected, hypoxic PC12 cells were respectively assayed for PNMT and signaling kinases and PNMT transcriptional activators and signaling kinases by western analysis. Binding of transcription factors in nuclear extracts to promoter binding elements was assessed by gel mobility shift assays. PNMT and transcription factor mRNA were measured by RT-PCR in total RNA isolated from hypoxic PC12 cells. Changes in hypoxia-induced transcriptional activation of the PNMT promoter following site-directed mutagenesis of transcription factor bindings sites were also examined.
Results: Hypoxia increased PNMT promoter-driven transcription. Associated signaling pathways appeared to include cAMP-protein kinase A, protein kinase C, phospholipase C, PI3/Akt, Erk1/2 MEK and p38 MEK/JNK. Downstream, Egr-1 and Sp1 were activated. HIF also contributed to PNMT transcriptional activation. Changes in transcriptional factor mRNA and protein were consistent with increased transcription. However, the magnitude of change and amount of protein-DNA binding complex were not consistently matched.
Conclusions: Different types of stress likely stimulate common and unique molecular pathways to activate PNMT gene transcription. Post-transcriptional regulatory mechanisms also seem important to stress-induced changes in adrenergic expression.