Effect of MKP2 overexpression on 7KCh-mediated inflammation.

<p>Overexpression was achieved by transducing the ARPE19 cells with a commercially available adenovirus expressing MPK2. ARPE19 cells were treated with 8 µM 7KCh for 24 hr and the mRNA inductions of the inflammatory markers were measured by qRT-PCR. <b>(a)</b> Immunoblots demonstrating the overexpression of MKP2 and the effects on the phosphorylation of JNK and ERK1/2 and P38 MAPK<b>.</b> JNK and p38 were induced by 7KCh treatment but there was no significant increase of phosphorylated ERK1/2 at the 24 hr time point. MKP2 significantly reduced the formation p-JNK and p-ERK1/2 but had no effect on p38. <b>(b)</b> QRT-PCR of the inflammatory markers (mean ± s.d., <i>n</i>≥3) with and without the overexpression of MKP2. The overexpression of MKP2 suppressed the induction of IL-1β (8.8 to 0.4 fold), IL-6 (8.1 to 2.0 fold), IL-8 (22.5 to 4.3 fold), VEGF (4.1 to 1.7 fold), CHOP (40.9 to 20.4 fold), and GRP78 (5.4 to 2.2 fold). <b>(c)</b> The secreted cytokine levels were measured using the Luminex XMAP technology in the conditioned medium after treatment with 6 µM 7KCh for 48 hr (VEGF, <i>n</i> = 3) or 8 µM 7KCh for 24 hr (IL-6 and IL-8, <i>n</i> = 4) with and without MKP2 overexpression (mean ± s.d.). MKP2 overexpression reduced the secreted cytokine level for VEGF (2145 pg/ml to 1442 pg/ml), IL-6 (337 pg/ml to 188 pg/ml) and IL-8 (1523 pg/ml to 662 pg/ml). <b>(d)</b> MKP2 overexpression reduced the induction of CHOP and GRP78. GFP overexpression was used as control. *<i>p</i><0.05, two-tailed Student's t-test.