Role of c-fos in hypoxia-induced AP-1 cis-element activity and tyrosine hydroxylase gene expression.

Previous studies have demonstrated that hypoxia stimulates expression of the c-fos gene in intact animals and isolated cells. The purpose of the present study was to assess the functional significance of c-fos activation during hypoxia. Using antisense c-fos strategy, we tested the hypothesis that c-fos is essential for activation of activator protein-1 transcription factor complex (AP-1) and subsequent stimulation of down stream genes such as tyrosine hydroxylase (TH) gene during hypoxia. Experiments were performed on rat pheochromocytoma 12 (PC12) cells. AP-1 activity was determined by a reporter gene assay using a luciferase expression vector driven by two copies of an AP-1 cis-element (AP-1-Luc). Cells transfected with AP-1-Luc construct were exposed to normoxia (21% O2) or to varying intensities and/or durations of hypoxia. AP-1 activity increased in response to hypoxia. The magnitude of the response depended on the intensity and duration of the hypoxic stimulus. Increases in AP-1 activity could not be elicited in neuroblastoma cells, indicating that hypoxia-induced increase in AP-1 activity is a cell selective phenomenon. Antisense c-fos abolished hypoxia-induced AP-1 activation in PC12 cells. Hypoxia increased tyrosine hydroxylase-chloramphenicol acetyl transferase activity (TH-CAT), and antisense c-fos and mutations at AP-1 binding sites in TH promoter abolished this effect. These results provide direct evidence that c-fos is essential for functional activation of AP-1 and subsequent activation of delayed response genes such as TH in PC12 cells.