FIGURE 6.

Insulin-mediated VEGF induction is dependent on HMGR activity. Quiescent human HaCaT keratinocytes were stimulated with insulin (a) for 24 h in the absence or presence of simvastatin (10 μm) and subsequently analyzed for VEGF mRNA expression by RNase protection assay. b, VEGF protein production from EGF- and insulin-treated HaCaT cells was analyzed by ELISA. ^**, p < 0.01 (unpaired Student's t test) as compared with control. ##, p < 0.01 (unpaired Student's t test) as indicated by the brackets. Bars indicate the mean ± S.D. obtained from three (n = 3) independent cell culture experiments. c, quiescent HaCaT keratinocytes were treated with insulin (2 μg/ml) in the absence or presence of simvastatin (10 μm), mevalonate (1 mm), or a combination of both as indicated and analyzed for VEGF protein production by ELISA. ^**, p < 0.01; ^*, p < 0.05 (unpaired Student's t test) as compared with control. ##, p < 0.01 (unpaired Student's t test) as compared with insulin stimulation alone. Bars indicate the mean ± S.D. obtained from three (n = 3) independent cell culture experiments. Immunoblots of total cellular protein from insulin-(d) or EGF-(e) treated HaCaT cells were analyzed for phospho-4E-BP1 (Thr-37/46) in the absence or presence of wortmannin (WTM, 200 nm) or simvastatin (sim, 10 μm) at the indicated time points. β-Actin was used to control equal loading of blots.