Two opposing effects of non-steroidal anti-inflammatory drugs on the expression of the inducible cyclooxygenase. Mediation through different signaling pathways

The efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) is considered to be a result of their inhibitory effect on cyclooxygenase (COX) activity. Here, we report that flufenamic acid shows two opposing effects on COX-2 expression; it induces COX-2 expression in the colon cancer cell line (HT-29) and macrophage cell line (RAW 264.7); conversely, it inhibits tumor necrosis factor alpha (TNFalpha)- or lipopolysaccharide (LPS)-induced COX-2 expression. This inhibition correlates with the suppression of TNFalpha- or LPS-induced NFkappaB activation by flufenamic acid. The inhibitor of extracellular signal-regulated protein kinase, p38, or NFkappaB does not affect the NSAID-induced COX-2 expression. These results suggest that the NSAID-induced COX-2 expression is not mediated through activation of NFkappaB and mitogen-activated protein kinases. An activator of peroxisome proliferator-activated receptor gamma, 15-deoxy-Delta(12,14)-prostaglandin J(2), also induces COX-2 expression and inhibits TNFalpha-induced NFkappaB activation and COX-2 expression. Flufenamic acid and 15-deoxy-Delta(12,14)-prostaglandin J(2) also inhibit LPS-induced expression of inducible form of nitric-oxide synthase and interleukin-1alpha in RAW 264.7 cells. Together, these results indicate that the NSAIDs inhibit mitogen-induced COX-2 expression while they induce COX-2 expression. Furthermore, the results suggest that the anti-inflammatory effects of flufenamic acid and some other NSAIDs are due to their inhibitory action on the mitogen-induced expression of COX-2 and downstream markers of inflammation in addition to their inhibitory effect on COX enzyme activity.     

Anisomycin induces COX-2 mRNA expression through p38(MAPK) and CREB independent of small GTPases in intestinal epithelial cells

Cyclooxygenase (COX)-2 expression in intestinal epithelial cells is associated with colorectal carcinogenesis. COX-2 expression is induced by numerous growth factors and gastrointestinal hormones through multiple protein kinase cascades. Here, the role of mitogen activated protein kinases (MAPKs) and small GTPases in COX-2 expression was investigated. Anisomycin and sorbitol induced COX-2 expression in non-transformed, intestinal epithelial IEC-18 cells. Both anisomycin and sorbitol activated p38(MAPK) followed by phosphorylation of CREB. SB202190 and PD169316 but neither PD98059 nor U0126 blocked COX-2 expression and CREB phosphorylation by anisomycin or sorbitol. Clostridium difficile toxin B inhibition of small GTPases did not affect anisomycin-induced COX-2 mRNA expression or phosphorylation of p38MAPK and CREB but did inhibit sorbitol-dependent COX-2 expression and phosphorylation of p38MAPK and CREB. Angiotensin (Ang) II-dependent induction of COX-2 mRNA and induced phosphorylation of p38MAPK and CREB were inhibited by toxin B. Reduction of CREB protein in cells transfected with CREB siRNAs inhibited anisomycin-induced COX-2 expression. These results indicate that activation of p38MAPK signaling is sufficient for COX-2 expression in IEC-18 cells. Ang II and sorbitol require small GTPase activity for COX-2 expression via p38MAPK while anisomycin-induced COX-2 expression by p38MAPK does not require small GTPases. This places small GTPase activity down-stream of the AT1 receptor and hyperosmotic stress and up-stream of p38MAPK and CREB.     

Protein kinase C alpha and zeta regulate nitric oxide-induced NF-kappa B activation that mediates cyclooxygenase-2 expression and apoptosis but not dedifferentiation in articular chondrocytes

Nitric oxide (NO) regulates differentiation, survival, and cyclooxygenase (COX)-2 expression in articular chondrocytes. NO-induced apoptosis and dedifferentiation are mediated by p38 kinase activity and p38 kinase-independent and -dependent inhibition of protein kinase C (PKC)alpha and zeta. Because p38 kinase also activates NF-kappa B, we investigated the functional relationship between PKC and NF-kappa B signaling and the role of NF-kappa B in apoptosis, dedifferentiation, and COX-2 expression. We found that NO-stimulated NF-kappa B activation was inhibited by ectopic PKC alpha and zeta expression, whereas NO-stimulated inhibition of PKC alpha and zeta activity was not affected by NF-kappa B inhibition. Inhibition of NO-induced NF-kappa B activity did not affect inhibition of type II collagen expression but did abrogate COX-2 expression and apoptosis. Taken together, our results indicate that NO-induced inhibition of PKC alpha and zeta activity is required for the NF-kappa B activity that regulates apoptosis and COX-2 expression but not dedifferentiation in articular chondrocytes.     

Non-steroidal anti-inflammatory drugs inhibit nitric oxide-induced apoptosis and dedifferentiation of articular chondrocytes independent of cyclooxygenase activity

Nitric oxide (NO) causes apoptosis and dedifferentiation of articular chondrocytes by the modulation of extracellular signal-regulated kinase (ERK), p38 kinase, and protein kinase C (PKC) alpha and -zeta. In this study, we investigated the effects and mechanisms of non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, ketoprofen, ibuprofen, sulindac sulfide, and flurbiprofen, in NO-induced apoptosis and dedifferentiation of articular chondrocytes. We found that all of the examined NSAIDs inhibited apoptosis and dedifferentiation. NO production in chondrocytes caused activation of ERK-1/2 and p38 kinase, which oppositely regulate apoptosis and dedifferentiation. NO production also caused inhibition of PKCalpha and -zeta independent of and dependent on, respectively, p38 kinase, which is required for apoptosis and dedifferentiation. Among the signaling molecules modulated by NO, NSAIDs blocked NO-induced activation of p38 kinase, potentiated ERK activation, and blocked inhibition of PKCalpha and -zeta. NSAIDs also inhibited some of the apoptotic signaling that is downstream of p38 kinase and PKC, such as NFkappaB activation, p53 accumulation, and caspase-3 activation. The inhibitory effects of NSAIDs on apoptosis and dedifferentiation were independent of the inhibition of cyclooxygenase (COX)-2 and prostaglandin E(2) (PGE(2)) production, as evidenced by the observation that specific inhibition of COX-2 activity and PGE(2) production or exogenous PGE(2) did not affect NO-induced apoptosis and dedifferentiation. Taken together, our results indicate that NSAIDs block NO-induced apoptosis and dedifferentiation of articular chondrocytes by the modulation of ERK, p38 kinase, and PKCalpha and -zeta in a manner independent of their ability to inhibit COX-2 and PGE(2) production.     

Hypertonic sodium chloride and mannitol induces COX-2 via different signaling pathways in mouse cortical collecting duct M-1 cells

The kidney cortical collecting duct is an important site for the maintenance of sodium balance. Previous studies have shown that, in renal medullary cells, hypertonic stress induces expression of cyclooxygenase-2 (COX-2) via NF-kappaB activation, but little is known about COX-2 expression in response to hypertonicity in the cortical collecting duct. Therefore, we examined the mechanism of hypertonic induction of COX-2 in M-1 cells derived from mouse cortical collecting duct. Induction of COX-2 protein was detected within 6 h of treatment with hypertonic sodium chloride. The treatment also increased COX-2 mRNA accumulation in a cycloheximide-independent manner, suggesting that ongoing protein synthesis is not required for COX-2 induction. Using reporter plasmids containing 0.2-, 0.3-, and 1.5-kb fragments of the COX-2 promoter, we found that hypertonic induction of COX-2 was due to an increase in promoter activity. The COX-2-inductive effect of hypertonicity was inhibited by SB203580, indicating that the effect is mediated by p38 MAPK. Since p38 MAPK can activate NF-kappaB, we made point mutations in the NF-kappaB binding site within the COX-2 promoter. The mutations did not block the induction of COX-2 promoter activity by hypertonic sodium chloride, and hypertonic sodium chloride failed to activate NF-kappaB binding site-driven reporter gene constructs. In contrast, hypertonic mannitol activated NF-kappaB, indicating that hypertonic mannitol and hypertonic sodium chloride activate COX-2 by different mechanisms. Thus, induction of COX-2 expression in M-1 cells by hypertonic sodium chloride does not involve activation of NF-kappaB. Furthermore, the signal transduction pathways that respond to hypertonic stress vary for different osmolytes in cortical collecting duct cells.     

Phospholipase D isozymes mediate epigallocatechin gallate-induced cyclooxygenase-2 expression in astrocyte cells

Little is known about the effect of epigallocatechin-3 gallate (EGCG), a major constituent of green tea, on the expression of cyclooxygenase (COX)-2. Here, we studied the role of phospholipase D (PLD) isozymes in EGCG-induced COX-2 expression. Stimulation of human astrocytoma cells (U87) with EGCG induced formation of phosphatidylbutanol, a specific product of PLD activity, and synthesis of COX-2 protein and its product, prostaglandin E(2) (PGE(2)). Pretreatment of cells with 1-butanol, but not 3-butanol, suppressed EGCG-induced COX-2 expression and PGE synthesis. Furthermore, evidence that PLD was involved in EGCG-induced COX-2 expression was provided by the observations that COX-2 expression was stimulated by overexpression of PLD1 or PLD2 isozymes and treatment with phosphatidic acid (PA), and that prevention of PA dephosphorylation by 1-propranolol significantly potentiated COX-2 expression induced by EGCG. EGCG induced activation of p38 mitogen-activated protein kinase (p38 MAPK), and specific inhibition of p38 MAPK dramatically abolished EGCG-induced PLD activation, COX-2 expression, and PGE(2) formation. Moreover, protein kinase C (PKC) inhibition suppressed EGCG-induced p38 MAPK activation, COX-2 expression, and PGE(2) accumulation. The same pathways as those obtained (2)in the astrocytoma cells were active in primary rat astrocytes, suggesting the relevance of the findings. Collectively, our results demonstrate for the first time that PLD isozymes mediate EGCG-induced COX-2 expression through PKC and p38 in immortalized astroglial line and normal astrocyte cells.     

Vitamin C suppresses proliferation of the human melanoma cell SK-MEL-2 through the inhibition of cyclooxygenase-2 (COX-2) expression and the modulation of insulin-like growth factor II (IGF-II) production

Vitamin C plays a crucial role in the suppression of proliferation of several types of cancer. Over-expression of cyclooxygenase (COX)-2 and type I insulin-like growth factor (IGF) receptor are important for proliferation and protection from apoptosis in malignancies. However, its specific mechanisms, especially the interaction between COX-2 expression and IGF-I axis mediated by vitamin C, remain yet to be clarified. Therefore, we investigated the effects of vitamin C on the proliferation of melanoma cells via the modulation of COX-2 expression and IGF-I axis. As a result, we found that 1.0 mM vitamin C inhibits the proliferation of SK-MEL-2 without induction of apoptosis. At that moment, IGF-II production was decreased, followed by the inhibition of COX-2 activity. IGF-IR expression was also down-regulated by vitamin C treatment. It coincided with the result from the inhibition of COX-2 by NS-398 and COX-2 siRNA. In addition, the decreased IGF-IR expression by vitamin C was restored by the treatment of recombinant prostaglandin E2. Finally, we determined whether the signal pathway would be involved in vitamin C-induced IGF-II and IGF-IR down-regulation. When the cells were exposed to SB203580, a specific inhibitor of p38 MAPK, COX-2 expression was dramatically recovered. In addition, phosphorylated p38 MAPK was increased after vitamin C treatment. Taken together, vitamin C suppresses proliferation of the human melanoma cell line SK-MEL2 via the down-regulation of IGF-II production and IGF-IR expression, which is followed by the activation of p38 MAPK and the inhibition of COX-2 expression.     

Role of MAPK in the regulation of double-stranded RNA- and encephalomyocarditis virus-induced cyclooxygenase-2 expression by macrophages

In response to virus infection or treatment with dsRNA, macrophages express the inducible form of cyclooxygenase-2 (COX-2) and produce proinflammatory prostaglandins. Recently, we have shown that NF-kappaB is required for encephalomyocarditis virus (EMCV)- and dsRNA-stimulated COX-2 expression in mouse macrophages. The dsRNA-dependent protein kinase R is not required for EMCV-stimulated COX-2 expression, suggesting the presence of protein kinase R-independent pathways in the regulation of this antiviral gene. In this study, the role of MAPK in the regulation of macrophage expression of cyclooxygenase-2 (COX)-2 in response to EMCV infection was examined. Treatment of mouse macrophages or RAW-264.7 cells with dsRNA or infection with EMCV stimulates the rapid activation of the MAPKs p38, JNK, and ERK. Inhibition of p38 and JNK activity results in attenuation while ERK inhibition does not modulate dsRNA- and EMCV-induced COX-2 expression and PGE2 production by macrophages. JNK and p38 appear to selectively regulate COX-2 expression, as inhibition of either kinase fails to prevent dsRNA- or EMCV-stimulated inducible NO synthase expression by macrophages. Using macrophages isolated from TLR3-deficient mice, we show that p38 and JNK activation and COX-2 expression in response to EMCV or poly(IC) does not require the presence the dsRNA receptor TLR3. These findings support a role for p38 and JNK in the selective regulation of COX-2 expression by macrophages in response to virus infection.     

Negative regulation of interleukin-2 and p38 mitogen-activated protein kinase during T-cell activation by the adaptor ALX

Activation of na?ve T cells requires synergistic signals produced by the T-cell receptor (TCR) and by CD28. We previously identified the novel adaptor ALX, which, upon overexpression in Jurkat T cells, inhibited activation of the interleukin-2 (IL-2) promoter by TCR/CD28, suggesting that it is a negative regulator of T-cell activation. To further understand the physiological role of ALX, ALX-deficient mice were generated. Purified T cells from ALX-deficient mice demonstrated increased IL-2 production, CD25 expression, and proliferation in response to TCR/CD28 stimulation. Enhanced IL-2 production and proliferation were also observed when ALX-deficient mice were primed in vivo with ovalbumin-complete Freund's adjuvant and then restimulated ex vivo. Consistent with our initial overexpression studies, these data demonstrate that ALX is a negative regulator of T-cell activation. While TCR/CD28-mediated activations of phosphotyrosine induction, extracellular signal-regulated kinase 1/2, Jun N-terminal protein kinase, IkappaB kinase alpha/beta, and Akt were unaltered, constitutive activation of p38 mitogen-activated protein kinase and its upstream regulators MKK3/6 were observed for ALX-deficient splenocytes. The phenotype of ALX-deficient mice resembled the phenotype of those deficient in the transmembrane adaptor LAX, and an association between ALX and LAX proteins was demonstrated. These results suggest that ALX, in association with LAX, negatively regulates T-cell activation through inhibition of p38.     

Regulation of the maintenance of peripheral T-cell anergy by TAB1-mediated p38 alpha activation

In anergic T cells, T-cell receptor (TCR)-mediated responses are functionally inactivated by negative regulatory signals whose mechanisms are poorly understood. Here, we show that CD4(+) T cells anergized in vivo by superantigen Mls-1(a) express a scaffolding protein, transforming growth factor beta-activated protein kinase 1-binding protein 1 (TAB1), that negatively regulates TCR signaling through the activation of mitogen-activated protein kinase p38 alpha. TAB1 was not expressed in naive and activated CD4(+) T cells. Inhibition of p38 activity in anergic T cells by a chemical inhibitor resulted in the recovery of interleukin 2 (IL-2) and the inhibition of IL-10 secretion. T-cell hybridoma 2B4 cells transduced with TAB1-containing retrovirus (TAB1-2B4 cells) showed activated p38 alpha, inhibited extracellular signal-regulated kinase (ERK) activity, culminating in reduced IL-2 levels and increased IL-10 production. The use of a p38 inhibitor or cotransfection of a dominant-negative form of p38 in TAB1-2B4 cells resulted in the recovery of ERK activity and IL-2 production. These results imply that TAB1-mediated activation of p38 alpha in anergic T cells regulates the maintenance of T-cell unresponsiveness both by inhibiting IL-2 production and by promoting IL-10 production.     

Inhibition of the expression and activity of cyclooxygenase-2 by chicory extract

Chicory is a major source of fructans with reported prebiotic-bifidogenic properties. In the present study, the potential anti-inflammatory activities of chicory were investigated. Ethyl acetate chicory root extract produced a marked inhibition of prostaglandin E(2) (PGE(2)) production in human colon carcinoma HT29 cells treated with the pro-inflammatory agent TNF-alpha. Two independent mechanisms of action were identified: (1) a drastic inhibition of the induction by TNF-alpha of cyclooxygenase 2 (COX-2) protein expression and (2) a direct inhibition of COX enzyme activities with a significantly higher selectivity for COX-2 activity. The inhibition of TNF-alpha-dependent induction of COX-2 expression was mediated by an inhibition of NF-kappaB activation. A major sesquiterpene lactone of chicory root, the guaianolide 8-deoxylactucin, was identified as the key inhibitor of COX-2 protein expression present in chicory extract. Altogether, the data presented strongly support chicory root as a promising source of functional food ingredient, combining prebiotic and anti-inflammatory properties.