Figure 1.

p90RSK activation inhibits ERK5 transcriptional activation and regulates subsequent KLF2-eNOS expression and VCAM-1 expression. (A) HUVECs were treated with 200µM of H₂O₂ for indicated times. Cell lysate were immunoprecipitated with anti-ERK5 or IgG as a negative control. ERK5-bound p90RSK was detected by Western blotting using anti-p90RSK antibody (top). p-p90RSK, p90RSK, p-ERK5, ERK5, p-ERK1/2, and tubulin were detected using each specific antibody. (B, C, D) Densitometric quantification of: ERK5-bound-p90RSK (B), phosphorylation of p90RSK (C) and ERK5 (D). Results were normalized to the lowest association (B) phosphorylation level (C, D) within each set of experiments. Results are expressed relative to untreated cells (0 min), and relative to the corresponding band intensity of p90RSK (3^rd from top) (C) or ERK5 (5^th from top) (D) at each time point. Statistical significance was determined by comparing the average level of the control group with that of each experimental data point. Experiments were carried out in triplicate using 3 different batches of HUVECs (B-D, mean ± SEM, n=3, *P<0.05, **P<0.01 compared to control). (E) HUVECs were co-transfected with an empty vector or plasmids encoding p90RSK-WT, Gal4-ERK5, and the Gal4-responsive luciferase reporter pG5-Luc. Four hours later, Opti-MEM was replaced by supplemented M200 medium. Eight hours post transfection, cells were stimulated with 200µM H₂O₂ for 16hrs, and ERK5 transcriptional activity was assayed by the dual-luciferase reporter assay. p90RSK overexpression inhibited ERK5 transcriptional activity in a dose-dependent manner, and also enhanced H₂O₂-mediated reduction in ERK5 transcriptional activity. Luciferase activity was normalized relative to Renilla luciferase activity. The mean luciferase activity of an empty vector (vehicle treatment) was set as 1 (mean ± SEM, n=3). (F, G) HUVECs were co-transfected with plasmids encoding Gal4-ERK5 and the Gal4-responsive luciferase reporter pG5-Luc (F) or KLF2 promoter with luciferase (KLF2-Luc) and ERK5 wild type (G). Cells were also transfected with an empty vector or CA-MEK5α as indicated. Eight hours post transfection, cells were treated with FMK (10µM) for 3hrs, followed by stimulation with 200µM H₂O₂ for 16hrs. Finally, ERK5 transcriptional activity (F) and KLF2 promoter activity (G) were assayed by a dual-luciferase reporter assay (F-G, mean ± SEM, n=3). (H, I) MECs were pretreated with FMK-MEA (10µM, 3hrs), followed by stimulation with either HG/low H₂O₂ or Mannitol/low H₂O₂ as a control and (H) KLF2 mRNA and (I) VCAM-1 mRNA level were detected by qRT-PCR as described in methods (H-I, mean ± SEM, n=3). (J, K) HUVECs were transduced with either adenovirus vector containing DN-p90RSK or LacZ and 24hrs later, exposed to s-flow for 24 hrs in the presence or absence of a combination of high glucose (25mM) and low dose H₂O₂ (20µM). eNOS, p90RSK, and tubulin expressions were detected by Western blotting with specific antibodies (J). eNOS expression was quantified (mean ± SEM, n=3) (K). (L, M) MECs (passage 3) were transduced with either adenovirus vector containing DN-p90RSK or LacZ and 24hrs later, treated with a combination of high glucose (25mM) and a low dose of H₂O₂ (20µM) for indicated times. VCAM-1, PECAM-1 and p90RSK expressions were detected by Western blotting with specific antibodies (L). VCAM1 expression was quantified (mean ± SEM, n=3) (M).