Abstract
The mitogen-activated protein kinase (MAPK) cascade is believed to function as an important regulator of prostaglandin biosynthesis. Previously we reported that interleukin-1beta induces activation of JNK/SAPK and p38 MAPK with concomitant up-regulation of cyclooxygenase (Cox)-2 expression and prostaglandin E2 (PGE2) synthesis. Our experiments demonstrate that overexpression of DeltaMEKK1 (a constitutively active truncation mutant of MEKK1 containing the C-terminal 324 amino acids) increases Cox-2 expression and PGE2 production which is completely blocked by SC68376, a pharmacologic inhibitor of p38 MAPK. DeltaMEKK1 overexpression results in activation of both c-Jun N-terminal kinases/extracellular signal-regulated kinases (JNK/SAPK) and p38 MAPK. Furthermore, activation of MEKK1 increases SEK1/MKK4 but not MKK3 or MKK6 activity. These findings suggest that MEKK1 --> SEK1/MKK4 may function as an upstream kinase capable of activating both p38 MAPK and JNK/SAPK with subsequent induction of Cox-2 expression and PGE2 production. We also found that overexpression of the constitutively active form of SEK1 (SEK1-ED) increases both p38 MAPK and JNK/SAPK phosphorylation, and increases PGE2 production and Cox-2 expression. By comparison, overexpression of the dominant negative form of SEK1 (SEK1-AL) decreases the phosphorylation of both p38 MAPK and JNK/SAPK and reduces Cox-2 expression. Together, this data suggests a potential role for the MEKK1 --> SEK1/MKK4 --> p38 MAPK -->--> Cox-2 cascade linking members of the MAPK pathway with prostaglandin biosynthesis.
Highlights
Prostaglandins are ubiquitous compounds involved in various homeostatic and inflammatory processes throughout the body
Our experiments demonstrate that overexpression of ⌬MEKK1 increases Cox-2 expression and prostaglandin E2 (PGE2) production which is completely blocked by SC68376, a pharmacologic inhibitor of p38 mitogen-activated protein kinase (MAPK). ⌬MEKK1 overexpression results in activation of both c-Jun N-terminal kinases/ extracellular signal-regulated kinases (JNK/SAPK) and p38 MAPK
Activation of MEKK1 increases SEK1/MKK4 but not MKK3 or MKK6 activity. These findings suggest that MEKK1 3 SEK1/MKK4 may function as an upstream kinase capable of activating both p38 MAPK and JNK/SAPK with subsequent induction of Cox-2 expression and PGE2 production
Summary
Reagents—Myelin basic protein (MBP) was purchased from Sigma. The p38 MAPK inhibitor SC68376 was kindly provided by Dr Joe Portnova PET28-c-Jun, a histidine-tagged fusion protein expression plasmid that encodes c-Jun-(1– 79) which contains the amino-terminal activation domain of c-Jun and a mutant c-Jun-(1–79, S63A/S73A), in which serine 63 and 73 of c-Jun[1–79] were mutated to alanine, were generously provided by Dr Maryann Gruda (Department of Molecular Biology, Bristol Myers Squibb Pharmaceutical Research Institute, Princeton, NJ). Immunecomplex p38 MAPK Activity Assay—The cell extracts were immunoprecipitated by incubation overnight with anti-p38 MAPK antibody and with protein A-Sepharose beads for 3 h at 4 °C. The immunecomplex p38 MAPK activity assay using MBP or GST-ATF-2-(1–96) as the substrate was performed at 30 °C for 20 min in 30 l of kinase reaction buffer (5 g of MBP or GST-ATF-2-(1–96), 20 M ATP, 10 Ci of [␥-32P]ATP, 25 mM HEPES, and 20 mM MgCl2). NIH 3T3 cells were stimulated with 1 mM IPTG for the time periods indicated and ⌬MEKK1 protein expression was detected by Western blot assay using an anti-MEKK1 antibody. A difference with a p value of 0.05 was considered statistically significant
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