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The effect of mitogen-activated protein kinase phosphatase-2 (MKP-2) overexpression on lipopolysaccharide and TNF α-mediated induction of inflammatory proteins in endothelial cells Mitogen-activated protein kinase phosphatases (MKPs) are a family of dual specificity phosphatases that regulate the activity of the mitogen-activated protein kinases (MAPKs) (Nishida & Gotoh et al., 1993). MKP-2 was one the earliest MKPs to be identified and is able to regulate phosphorylation of c-Jun N-terminal kinase (JNK) (Cadalbert et al., 2005; Guan et al., 1995) . In this study we examined the effect of MKP-2 overexpression on JNK mediated induction of the cell adhesion molecules, ICAM-1 and VCAM-1, and cyclo-oxygenase–2 (COX-2) in human umbilical vein endothelial cells (HUVECs) stimulated by LPS and TNFα. HUVECs (passage 3-7) were infected with adenoviral MKP-2 (Adv.MKP-2) for 40hrs prior to experiments or as a comparision preincubated with the JNK inhibitor, SP600125 for 30 min. MAP kinase activation and protein expression were determined in whole cell lysates which had been resolved by Western blotting and assessed by specific antibodies. In HUVECs, a transient phosphorylation of JNK MAP kinase was stimulated by both LPS (100 μg/ ml) and TNF-α (20 ng/ml). This phosphorylation was significantly attenuated by overexpression of MKP-2 (% inhibition at 300 p.f.u. = 76.96 ±10.73, p< 0.001,60 min and 98.38 ± 1.47, p< 0.001, 30 min for LPS and TNF-α respectively, n=3). Similarly, pretreatment with SP600125 (20 μM) casused a significant inhibition of JNK phosphorylation (% inhibition = 85.36 ± 12.70, p< 0.001, 60 min and 83.48 ± 6.95 , p< 0.001, 30min for LPS and TNF-α respectively, n=4). LPS and TNF-α induced COX-2 protein expression in a time dependent manner which reached it maximum values at 24 h after stimulation (38.29 ±13.57 and 30 ±16.05 fold of basal values for LPS and TNF-α respectively, n=3). Results showed a substantial attenuation of COX-2 protein expression by infection with Adv.MKP-2 and by pretreatment with SP600125. Both LPS and TNF a also stimulated an increase in ICAM-1 and VCAM-1 protein expression which peaked between 8-24 h after stimulation (fold basal ± s.e.m. : ICAM = 38.15 ± 5.41 and 102.21 ± 29.97, VCAM-1= 43.98 ± 8.29 and 69.60 ±14.24 for LPS and TNF-α respectively, n=3). However in contrast to COX-2, ICAM and VCAM expression was not significantly reduced by either Adv-MKP-2 infection nor SP600125. Taken together, these results suggest that MKP-2 may inhibit COX-2 expression through effects upon JNK signaling and that Adv-MKP-2 may be a useful tool to exploit clinically.
Cadalbert L et al. (2005) Cell signal 17: 1254-64 |
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