Mitogen-activated protein kinases facilitate many cellular processes and are essential for immune cell function. Their activity is controlled by kinases and dual-specificity phosphatases. A comprehensive microarray analysis of human leukocytes identified DUSP2 (encoding the phosphatase PAC-1) as one of the most highly induced transcripts in activated immune cells. We generated Dusp2(-/-) mice and found considerably reduced inflammatory responses in the 'K/BxN' model of rheumatoid arthritis. PAC-1 deficiency led to increased activity of Jun kinase (Jnk) but unexpected impairment of the activity of extracellular signal-regulated kinase (Erk) and the kinase p38, reduced activity of the transcription factor Elk1 and a complex of mobilized transcription factor NFAT and the AP-1 transcription factor and decreased effector immune cell function. Thus, PAC-1 is a key positive regulator of inflammatory cell signaling and effector functions, mediated through Jnk and Erk mitogen-activated protein kinase crosstalk.

Partial hepatectomy (PH) triggers a rapid regenerative response in the remaining tissue to reinstate the organ function and the cell numbers. Among the molecules that change in the course of regeneration is an accumulation of prostaglandin E2 in the sera of rats with PH. Analysis of the cyclooxygenase (COX) isoenzymes in the remnant liver showed the preferential expression of COX-2 in hepatocytes. Cultured regenerating hepatocytes expressed significant levels of COX-2, a process that was not observed in the sham counterparts. Maximal expression of COX-2 was detected 16 h after PH with increased levels present even at 96 h. Pharmacological inhibition of COX-2 activity with NS398 shunted the up-regulation of cell proliferation after PH, which suggests a positive interaction of prostaglandins with the progression of the cell cycle. Similar results were obtained after PH of mice lacking the COX-2 gene. The expression of COX-2 in regenerating liver was concomitant with a decrease in CCAAT-enhancer binding protein (C/EBP-a) level and an increase in the expression of C/EBP-b and C/EBP-d. These results suggest a contribution of the enhanced synthesis of prostaglandins to liver regeneration observed after PH.

Treatment of primary cultures of fetal hepatocytes with proinflammatory cytokines, lipopolysaccharide (LPS), and hepatocyte growth factor promoted the expression of cyclooxygenase-2 (COX-2) and the synthesis of high amounts of prostaglandins (PGs). Under these conditions, the active forms of the matrix metalloproteinases-2 and -9 (MMPs) were released to the extracellular medium. This process was inhibited when the synthesis of PGs was suppressed pharmacologically with COX-2 inhibitors. Addition to the cell cultures of PGE 2 promoted the release of MMPs through a mechanism that involved the expression of COX-2 and the synthesis of additional PGs. Kinetic analysis of the secretion of MMPs in response to LPS and PGE 2 showed a similar time course, with a lag period of 6 hours, which suggests that PGE 2 does not act directly on the mechanism of MMP processing and release. Inhibitors of protein kinase A, p38 MAP kinase, phosphatidylinositol-3-kinase, and nuclear factor B Prostaglandin H synthase, also known as cyclooxygenase (COX), catalyzes the synthesis of prostaglandin H 2 from arachidonate and constitutes a key regulatory step in the biosynthesis of prostanoids. 1-3 Two distinct isoforms of COX are known: COX-1 and COX-2. The two isoenzymes are encoded by different genes, and the control of their expression, regulation of enzyme activity, and physiologic functions are very different. 1,4 COX-1 seems to be involved in the house keeping function of prostaglandins (PGs) 4 and is expressed constitutively. In contrast, COX-2 is inducible, is expressed as an immediate early gene, and is involved in the onset of inflammation and mitogenic responses. 1,3,5,6 COX-2 expression is positively regulated by lipopolysaccharide (LPS), interleukin 1␤, tumor necrosis factor ␣, and reactive oxygen intermediates in different cells. 7,8 In addition to these stimuli, it has been described that hepatocyte growth factor (HGF) and epidermal growth factor (EGF) triggered the expression of COX-2 in rat gastric epithelial cells, in human amnion-derived WISH cells, and in human gingival fibroblasts, and this effect was mediated through the activation of the ERK2 signaling pathway. [9][10][11] Our previous results showed that adult hepatocytes failed to express COX-2 regardless of the proinflammatory factors used. However, fetal hepatocytes, which exhibit a liver phenotype distinct from the adult counterpart, were able to express COX-2 upon challenge with LPS and proinflammatory cytokines. 8 Regarding the mechanism responsible for the suppression of COX-2 inducibility in adult hepatocytes, our data suggest the existence of a close relationship between COX-2 induction and the decrease of C/EBP␣ levels that bind to the nuclear factor-interleukin 6 site of the COX-2 promoter. 12 In addition to inflammation and cell growth regulation, COX-2 expression has been associated with carcinogenesis and tumor development. 3,6 Several population-based studies have detected a 40% to 50% decrease in relative risk for colorectal cancer in persons who regularl...

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1 Cyclooxygenase-2 (COX-2) is involved in the biosynthesis of prostanoids in the course of in¯ammatory reactions. This isoenzyme is regulated at the transcription level and many cells express COX-2 upon challenge with lipopolysaccharide (LPS) or pro-in¯ammatory cytokines. 2 Since hepatocytes respond to LPS and pro-in¯ammatory stimuli, we investigated the expression of COX-2 in foetal and adult hepatocytes upon challenge with these substances. 3 COX-2 was expressed in foetal hepatocytes incubated with LPS, tumour necrosis factor-a and interleukin-1b. This response rapidly decreased after birth and was absent in hepatocytes from animals aged 2 days or more and treated under identical conditions. The expression of COX-2 was determined at the mRNA, protein and enzyme activity levels using Northern and Western blot, and following the synthesis of prostaglandin E 2 , respectively. The use of NS 398, a speci®c pharmacological inhibitor of COX-2, con®rmed the expression of this isoenzyme in activated foetal hepatocytes. 4 Synergism in COX-2 expression was observed between LPS, tumour necrosis factor-a and interleukin-1b. Interleukin-6 and permeant analogues of cyclic AMP failed to induce COX-2 or to synergize with LPS. Also, transforming growth factor-b inhibited the LPS-and pro-in¯ammatory cytokines-dependent expression of COX-2. 5 These results indicate that foetal hepatocytes are competent to express COX-2 upon challenge with pro-in¯ammatory stimuli, a process lost completely in hepatocytes isolated from animals aged 2 days.

Stimulation of fetal hepatocytes with proinflammatory cytokines and lipopolysaccharide promotes the expression of cyclooxygenase-2 (COX-2) and nitric oxide synthase-2 (NOS-2), whereas the hepatoma cell line HepG2 exhibits a behavior similar to that described for adult hepatocytes and only expresses NOS-2. The effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on the inflammatory onset was analyzed in these cells since in addition to the inhibition of cyclooxygenase activity, these drugs interfere with other signaling pathways related with the inflammatory response. Inhibition of nuclear factor B (NF-B) activation by aspirin and salicylate has been described in many cells. C yclooxygenase (COX) catalyzes the synthesis of prostaglandin H 2 from arachidonate and is a key regulatory step in the biosynthesis of prostanoids. [1][2][3] There are two isoenzymes: COX-1 and COX-2 encoded by different genes and with distinct physiological functions. 1,4 COX-1 seems to be involved in the homeostatic function of prostaglandins (PGs) 4 and is expressed constitutively. COX-2 is inducible, is expressed as an immediate early gene, and is involved in the onset of inflammation and mitogenic responses. 5,6 In addition to inflammation and cell growth regulation, COX-2 expression has been associated with carcinogenesis and tumor development. 3,6 Studies in a variety of animal models of colon cancer (both genetic and carcinogen induced) and in human colon cancer cells have also indicated a significant reduction in tumor multiplicity and metastatic potential by nonsteroidal anti-inflammatory drug (NSAID) treatment. 3,7 However, there is cumulative evidence suggesting that most of these effects are independent of COX activity and PG synthesis inhibition. This is based on the fact that high concentrations of NSAIDs, 2 or 3 orders of magnitude higher than those required to inhibit PG production, are necessary for the inhibition of cell growth and induction of apoptosis. 8 Alternatively, exogenous addition of PGs contribute to tumor growth by inhibiting apoptosis and favoring angiogenesis. [8][9][10] Indeed, recent work indicates that COX-2 may play a significant role in the regulation of angiogenesis associated with neoplastic tumor cells, and COX-2 selective inhibitors may block the growth of blood vessels into developing tumors. 9 Therefore, inhibition of COX activity may account for part of the antitumor activity of NSAIDs and, at the same time, it appears that these drugs modulate the signal transduction pathways involved in the regulation of inflammation and tumor growth. These COXindependent anti-inflammatory effects of NSAIDs include the inhibition of the MAP kinases, IB kinase (IKK), and cyclin-dependent kinase, which results in the inhibition of transcription dependent on nuclear factor B (NF-B) and activating protein 1 (AP-1). 8 For example, the anti-inflammatory and antiproliferative properties of aspirin and sulindac are mediated in part by their specific inhibition of IKK-␤, the kinase that phosphorylates IB leading to i...

The effect of rofecoxib, a selective cyclooxygenase-2 inhibitor, on inflammatory signaling has been investigated in elicited murine peritoneal macrophages. Macrophages treated with 10 M rofecoxib exhibited an important inhibition in the early activation of nuclear factor B (NF-B) and the mitogen-activated protein kinase p38, the extracellular-regulated kinase p44, and the c-Jun N-terminal kinase. Moreover, this drug decreased the protein levels of nitric-oxide synthase-2 and cyclooxygenase-2 in lipopolysaccharide (LPS)-treated macrophages. Rofecoxib delayed and attenuated NF-B activation, which impaired significantly the expression of B-dependent genes. This drug and related coxibs did not affect cell viability and protected against LPS-induced apoptosis through the impairment of the inflammatory response. These data show an additional anti-inflammatory mechanism of selective cyclooxygenase-2 inhibitors through the attenuation of macrophage activation.

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