Phytanic acid activates NADPH oxidase through transactivation of epidermal growth factor receptor in vascular smooth muscle cells.

BACKGROUND

Phytanic acid (PA) has been implicated in development of cancer and its defective metabolism is known to cause life-threatening conditions, such as Refsum disease, in children. To explore molecular mechanisms of phytanic acid-induced cellular pathology, we investigated its effect on NADPH oxidase (NOX) and epidermal growth factor receptor (EGFR) in rat aortic smooth muscle cells (RASMC).

METHODS

Smooth muscle cells were isolated from rat aortae using enzymic digestion with collagenase and elastase. Cultured RASMC were treated with varying concentrations (0.5-10 μg/ml) of phytanic acid in the presence/absence of fetal bovine serum (FBS) and/or EGFR inhibitor, AG1478. Following treatment with experimental agents, NOX activity was assayed in RASMC cultures by luminescence method. Gene expression of NOX-1 and p47phox was assessed using RT-PCR. NOX-1, p47phox and, total EGFR protein and its phosphorylated form were measured by Western blotting.

RESULTS

Treatment of RASMC with supraphysiological concentrations (>2.5 μg/ml) of PA significantly (p < 0.01) increased the NOX activity. PA also significantly increased gene/protein expression of NOX-1 and p47phox whereas p22phox and p67phox remained unaffected. Interestingly, PA (2.5-10 μg/ml) markedly (2-3 folds) increased the total and phosphorylated EGFR. Treatment of cells with EGFR inhibitor, AG1478, significantly blocked the PA-induced enhancement of NOX activity.

CONCLUSIONS

Our findings that PA transactivates EGFR and induces NOX activity in vascular smooth muscle cells provide new insights into molecular mechanisms of PA's role in cancer and Refsum disease.