Cyclooxygenase-1 and an alternatively spliced mRNA in the rat stomach: effects of aging and ulcers

Prostaglandins play a critical role in gastric mucosal cytoprotection and decrease progressively with age. Cyclooxygenase (COX), the rate-limiting enzyme for prostaglandin synthesis, exists in two isoforms, COX-1 and COX-2. The rat COX-1 gene expresses an alternatively spliced mRNA COX-1 splice variant (SV) that may, at best, code for a truncated COX-1 protein. With the use of competitive PCR, we determined whether COX gene expression was altered in the stomach with increasing age and after gastric ulcer induction. COX-1 mRNA was significantly reduced in the aged, and COX-1SV mRNA was significantly higher in the adults compared with the young and aged stomach. Levels of COX-1 and COX-2 were similarly expressed in the normal stomach. In acute gastric ulcers, only COX-2 mRNA levels were significantly elevated. When ulcers were undergoing healing and repair, COX-1 and COX-2 mRNA levels were significantly elevated. Age-related changes in COX-1 and COX-1SV but not COX-2 mRNA may alter gastric mucosal cytoprotection. Furthermore, COX-1 and COX-2 may both contribute to the healing of a gastric ulcer.     

Role of the different isoforms of cyclooxygenase and nitric oxide synthase during gastric ulcer healing in cyclooxygenase-1 and -2 knockout mice

Traditional NSAIDs, selective cyclooxygenase (COX)-2 inhibitors, and inhibitors of nitric oxide synthase (NOS) impair the healing of preexisting gastric ulcers. However, the role of COX-1 (with or without impairment of COX-2) and the interaction between COX and NOS isoforms during healing are less clear. Thus we investigated healing and regulation of COX and NOS isoforms during ulcer healing in COX-1 and COX-2 deficiency and inhibition mouse models. In this study, female wild-type COX-1(-/-) and COX-2(-/-) mice with gastric ulcers induced by cryoprobe were treated intragastrically with vehicle, selective COX-1 (SC-560), COX-2 (celecoxib, rofecoxib, and valdedoxib), and unselective COX (piroxicam) inhibitors. Ulcer healing parameters, mRNA expression, and activity of COX and NOS were quantified. Gene disruption or inhibition of COX-1 did not impair ulcer healing. In contrast, COX-2 gene disruption and COX-2 inhibitors moderately impaired wound healing. More severe healing impairment was found in dual (SC-560 + rofecoxib) and unselective (piroxicam) COX inhibition and combined COX impairment (in COX-1(-/-) mice with COX-2 inhibition and COX-2(-/-) mice with COX-1 inhibition). In the ulcerated repair tissue, COX-2 mRNA in COX-1(-/-) mice, COX-1 mRNA in COX-2(-/-) mice, and, remarkably, NOS-2 and NOS-3 mRNA in COX-impaired mice were more upregulated than in wild-type mice. This study demonstrates that COX-2 is a key mediator in gastric wound healing. In contrast, COX-1 has no significant role in healing when COX-2 is unimpaired but becomes important when COX-2 is impaired. As counterregulatory mechanisms, mRNA of COX and NOS isoforms were increased during healing in COX-impaired mice.     

Contrasting effects of cyclooxygenase-1 (COX-1) and COX-2 deficiency on the host response to influenza A viral infection

Influenza is a significant cause of morbidity and mortality worldwide despite extensive research and vaccine availability. The cyclooxygenase (COX) pathway is important in modulating immune responses and is also a major target of nonsteroidal anti-inflammatory drugs (NSAIDs) and the newer COX-2 inhibitors. The purpose of the present study was to examine the effect of deficiency of COX-1 or COX-2 on the host response to influenza. We used an influenza A viral infection model in wild type (WT), COX-1-/-, and COX-2-/- mice. Infection induced less severe illness in COX-2-/- mice in comparison to WT and COX-1-/- mice as evidenced by body weight and body temperature changes. Mortality was significantly reduced in COX-2-/- mice. COX-1-/- mice had enhanced inflammation and earlier appearance of proinflammatory cytokines in the BAL fluid, whereas the inflammatory and cytokine responses were blunted in COX-2-/- mice. However, lung viral titers were markedly elevated in COX-2-/- mice relative to WT and COX-1-/- mice on day 4 of infection. Levels of PGE2 were reduced in COX-1-/- airways whereas cysteinyl leukotrienes were elevated in COX-2-/- airways following infection. Thus, deficiency of COX-1 and COX-2 leads to contrasting effects in the host response to influenza infection, and these differences are associated with altered production of prostaglandins and leukotrienes following infection. COX-1 deficiency is detrimental whereas COX-2 deficiency is beneficial to the host during influenza viral infection.     

Vasoactive prostanoids are generated from arachidonic acid by COX-1 and COX-2 in the mouse

Generation of vasoactive prostanoids from arachidonic acid by cyclooxygenase (COX)-1 and COX-2 was investigated in anesthetized mice. Intravenous injections of the prostanoid precursor arachidonic acid increased pulmonary arterial pressure and decreased systemic arterial pressure. Pulmonary pressor and systemic depressor responses were attenuated by SC-560 and nimesulide, inhibitors of COX-1 and COX-2, in doses that did not alter responses to injected prostanoids. Pulmonary pressor responses to arachidonic acid were blocked and a depressor response was unmasked, whereas systemic depressor responses were not altered, by a thromboxane receptor antagonist. Pulmonary and systemic pressor responses to angiotensin II injections and systemic pressor responses to angiotensin II infusion were not modified by COX-1 or COX-2 inhibitors but were attenuated by losartan. Systemic depressor responses to arachidonic acid were smaller in COX-1 and COX-2 knockout mice, whereas responses to angiotensin II, norepinephrine, U-46619, endothelin-1, and PGE(1) were not different in COX-1 and COX-2 knockout and wild-type control mice. These results suggest that vasoactive prostanoids with pulmonary pressor and systemic vasodepressor activity are formed by COX-1 and COX-2 and are consistent with Western blot analysis and immunostaining showing the presence of COX-1 and COX-2. These data suggest that thromboxane A(2) (TxA(2)) is formed from the precursor by COX-1 and COX-2 in the lung and are in agreement with immunofluorescence studies showing thromboxane synthase. The present data suggest that COX-1- or COX-2-derived prostanoids do not modulate responses to angiotensin II or other vasoactive agents and that prostanoid responses are similar in CD-1 and C57BL/6 and in male and female mice.     

Synthesis and biological evaluation of linear phenylethynylbenzenesulfonamide regioisomers as cyclooxygenase-1/-2 (COX-1/-2) inhibitors

A group of regioisomeric phenylethynylbenzenesulfonamides possessing a COX-2 SO2NH2 pharmacophore at the para-, meta- or ortho-position of the C-1 phenyl ring, in conjunction with a C-2 substituted-phenyl (H, OMe, OH, Me, F) group, were synthesized and evaluated as inhibitors of the cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) isozymes. The target 1,2-diphenylacetylenes were synthesized via a palladium-catalyzed Sonogashira cross-coupling reaction. In vitro COX-1/-2 isozyme inhibition structure-activity data showed that COX-1/-2 inhibition and the COX selectivity index (SI) are sensitive to the regioisomeric placement of the COX-2 SO2NH2 pharmacophore where the COX-2 potency order for the benzenesulfonamide regioisomers was generally meta>para and ortho. Among this group of compounds, the in vitro COX-1/-2 isozyme inhibition studies identified 3-(2-phenylethynyl)benzenesulfonamide (10a) as a COX-2 inhibitor (COX-2 IC50=0.45 microM) with a good COX-2 selectivity (COX-2 SI=70). In contrast, 2-[2-(3-fluorophenyl)ethynyl]benzenesulfonamide (11c) possessing a SO2NH2 COX-2 pharmacophore at the ortho-position of the C-1 phenyl ring exhibited COX-1 inhibition and selectivity (COX-1 IC50=3.6 microM). A molecular modeling study where 10a was docked in the binding site of COX-2 shows that the meta-SO2NH2 COX-2 pharmacophore was inserted inside the COX-2 secondary pocket (Arg513, Phe518, Val523, and His90). Similar docking of 10a within the COX-1 binding site shows that the meta-SO2NH2 pharmacophore is unable to interact with the respective amino acid residues in COX-1 that correspond to those near the secondary pocket in COX-2 due to the presence of the larger Ile523 in COX-1 that replaces Val523 in COX-2.     

The role of selective cyclooxygenase isoforms in human intestinal smooth muscle cell stimulated prostanoid formation and proliferation.

Intestinal smooth muscle plays a major role in the repair of injured intestine and contributes to the prostanoid pool during intestinal inflammatory states. Cyclooxygenase (COX), which catalyzes the conversion of arachidonic acid to prostanoids exists in two isoforms, COX-1 and COX-2. The purpose of this study was to determine the relative contributions of COX-1 and COX-2 in the production of prostanoids by human intestinal smooth muscle (HISM) cells when stimulated by interleukin-1beta (IL-1beta) and lipopolysaccharide (LPS). Furthermore the effects of specific COX-1 and COX-2 inhibitors on the proliferation of smooth muscle cells was also evaluated. Confluent monolayer cultures of HISM cells were incubated with IL-1beta or LPS for 0-24h while control cells received medium alone. PGE2 and PGI2 as 6-keto-PGF1alpha and LTB4 were measured by a specific radioimmunoassay. COX enzymes were evaluated by Western immunoblotting. Unstimulated and stimulated cells were exposed to the specific COX-1 inhibitor valerylsalicylic acid (VSA) and the COX-2 inhibitors NS-398 and SC-58125. The effects of serum on proliferation were then evaluated in the presence of each of the specific COX inhibitors by incorporation of 3H-thymidine into DNA. IL-1beta and LPS increased both PGE2 and 6-keto-PGF1alpha in a dose dependent fashion with enhanced production detected two hours following exposure. Neither stimulus stimulated LTB4 release. Immunoblot analysis using isoform-specific antibodies showed that both COX-1 and COX-2 were present constitutively. Furthermore, COX-1 was upregulated by each inflammatory stimulus. In a separate set of experiments cells were pretreated with either the selective COX-1 inhibitor VSA or the selective COX-2 inhibitors NS-398 or SC-58125 prior to treatment with IL-1beta or LPS. The COX-1 and COX-2 inhibitors decreased both basal and IL-1beta and LPS stimulated prostanoid release. Spontaneous DNA synthesis was present and serum consistently increased proliferation. 3H-thymidine incorporation, stimulated by serum, was inhibited by both COX-1 and COX-2 inhibitors. This study suggests that the prostanoid response stimulated by proinflammatory agents of gut-derived smooth muscle cells appears to be mediated by both COX-1 and COX-2 enzymes. Proliferation of smooth muscles cells also appears to be influenced by both COX-1 and COX-2.     

Ciglitazone mediates COX-2 dependent suppression of PGE2 in human non-small cell lung cancer cells

BACKGROUND: Cyclooxygenase-2 (COX-2) over-expression and subsequent prostaglandin E2 (PGE2) production are frequently associated with human non-small-cell lung cancer (NSCLC) and are involved in tumor proliferation, invasion, angiogenesis, and resistance to apoptosis. Here, we report that ciglitazone downregulates PGE2 in NSCLC cells. METHODS: PGE2 ELISA assay and COX-2 ELISA assay were performed for measuring PGE2 and COX-2, respectively, in NSCLC. The mRNA level of COX-2 was measured by semi-quantitative RT-PCR. The transient transfection experiments were performed to measure COX-2 and peroxisome proliferator-response element (PPRE) promoter activity in NSCLC. Western blots were unitized to measure PGE synthase (PGES) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) protein expression. RESULTS: COX-2 ELISA assays suggested that ciglitazone-dependent inhibition of PGE2 occurs through the suppression of COX-2. Ciglitazone treatment suppressed COX-2 mRNA expression and COX-2 promoter activity while upregulating PPRE promoter activity. Ciglitazone did not modify the expression of enzymes downstream of COX-2 including PGES and 15-PGDH. Utilization of a dominant-negative PPARgamma showed that the suppression of COX-2 and PGE2 by ciglitazone is mediated via non-PPAR pathways. CONCLUSION: Taken together, our findings suggest that ciglitazone is a negative modulator of COX-2/PGE2 in NSCLC.     

Cyclooxygenase mRNA expression in human patellar tendon at rest and after exercise

Exercise has been shown to acutely elevate several metabolic processes in tendon tissue, including collagen turnover and blood flow, and chronically induce changes in tendon properties. Many of these acute metabolic responses to exercise are regulated by the cyclooxygenase (COX) enzymes. We measured the expression levels of COX-1 [variants 1 and 2 (COX-1v1 and COX-1v2)], COX-2, and the recently discovered intron 1-retaining COX-1 variants (COX-1b(1), COX-1b(2), and COX-1b(3)) at rest and after resistance exercise (RE). Patellar tendon biopsy samples were taken from six individuals (3 men and 3 women) before and 4 h after a bout of RE (3 sets of 10 repetitions at approximately 70% of 1 repetition maximum) and from a separate group of six individuals (3 men and 3 women) before and 24 h after RE and analyzed by real-time RT-PCR. The COX-1 variants were the most abundant COX mRNAs before exercise and remained unchanged (P > 0.05) after exercise. COX-2 was also expressed in tendon tissue at rest and was unchanged (P > 0.05) after exercise. The intron 1-retaining COX-1 variants were not detectable in tendon tissue before or after exercise. COX-1 and COX-2 were expressed at much higher levels by the patellar tendon than by quadriceps skeletal muscle, although the overall COX mRNA expression patterns were similar in skeletal muscle and tendon (COX-1v2 > COX-1v1, P < 0.05; ratio of COX-1 to COX-2 congruent with 4:1). These results suggest that COX-1 and COX-2 are constitutively expressed at relatively high levels in human patellar tendon and are likely targets of COX-inhibiting drugs at rest and after physical activity.     

Do human platelets express COX-2?

The rate-limiting enzyme in prostaglandin (PG)- and thromboxane (TX)-synthesis is known as cyclooxygenase (COX). The COX-enzyme family consists of the classical COX-1 and the inducible COX-2-enzyme. To investigate whether platelets contain COX-2, we measured thiobarbituric acid reactive substances (TBARS) after either blocking COX-1 or COX-2 or adding compounds known to affect COX-expression. To stimulate platelets' different reagents such as collagen, thrombin and arachidonic acid (AA) were used. The inhibitors used in this study were acetylsalicylic acid (ASA), indomethacin and NS-398. Using the western-blot technique, we failed to detect COX-2 in platelets while COX-1 was detectable. We were not able to discover COX-2 in platelets using the methods we applied. As the amount of COX-2 in platelets might be below the detection limit of the methods used, the biological relevance COX-2 in platelets, if even existing at low amounts, remains to be established.     

COX-2 与血液恶性疾病

环氧化酶(COX-2)是体内前列腺素(PG)合成过程中重要的限速酶,它在正常组织中表达甚少,但在肿瘤和炎性细胞中表达较多,近年来的研究表明COX-2的过表达与肿瘤的发生,发展有关,使COX-2成为肿瘤研究的新热点,但COX-2在实体肿瘤中的研究较多而与血液恶性疾病之间关系国内报道较少,现就COX-2的研究进展以及在血液恶性肿瘤中的作用做一综述。     

QSAR analysis of meclofenamic acid analogues as selective COX-2 inhibitors

The use of quantitative structure-activity relationships, since its advent, has become increasingly helpful in understanding many aspects of biochemical interactions in drug research. This approach was utilized to explain the relationship of structure with biological activity of selective COX-2 inhibitors. The enormity of the COX-2 discovery is reflected in the unprecedented speed at which research laboratories have sought to validate its clinical implications. Presented herein is a series of 21 derivatives of meclofenamic acid with selective COX-2 inhibitory activity. Several statistically significant regression expressions were obtained for both COX-1 and COX-2 inhibition using sequential multiple linear regression analysis method. Two of these models were selected and validated further, which revealed the importance of Kier molecular flexibility index for COX-2 inhibitory activity and the number of hydrogen bond donor atoms for COX-1 inhibitory activity. Additionally, linear correlation of molecular flexibility with COX-1 and COX-2 inhibitory activities revealed that flexibility of molecules at COX-2 active site can improve the selectivity of COX-2 inhibitors.     

Activation of p38 MAPK is involved in endothelin-1-stimulated COX-2 expression in cultured Feline esophageal smooth muscle cells

We investigated the possible role of p38 MAPK and ETB receptors in ET-1 induction of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in cultured feline esophageal smooth muscle cells (ESMC). Confluent layers of ESMC were stimulated with 10 nM ET-1 and expression of COX-1 and COX-2, involvement of receptors, and activation of p38 MAPK, were examined by Western blot analysis. Levels of PGE2 induced by ET-1 were measured by Elisa. Using ETA and ETB antagonists (BQ-123 and BQ-788, respectively), the contribution of the ET receptors to COX-1 and COX-2 expression induced by ET-1 was determined. Western blot analysis revealed that treatment of ESMC with ET-1 resulted in transient expression of COX-2 and activation of p38 MAPK. Activation of p38 MAPK was maximal after 1 h. SB202190, a p38 MAPK inhibitor, reduced expression of COX-2, but not COX-1. ET-1-induced release of PGE2 was also blocked by SB202190. COX-2 expression was upregulated only via the ETB receptor, and COX-1 expression was not affected by either antagonist. Taken together, our data suggest that ET-1 causes p38 MAPK-dependent expression of COX-2 by interacting with ETB receptors on ESMC.     

Resveratrol analogues as selective cyclooxygenase-2 inhibitors: synthesis and structure-activity relationship

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is found in grapes and various medical plants. Among cytotoxic, antifungal, antibacterial cardioprotective activity resveratrol also demonstrates non-selective cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibition. In order to find more selective COX-2 inhibitors a series of methoxylated and hydroxylated resveratrol derivatives were synthesized and evaluated for their ability to inhibit both enzymes using in vitro inhibition assays for COX-1 and COX-2 by measuring PGE(2) production. Hydroxylated but not methoxylated resveratrol derivatives showed a high rate of inhibition. The most potent resveratrol compounds were 3,3',4',5-tetra-trans-hydroxystilbene (COX-1: IC(50)=4.713, COX-2: IC(50)=0.0113 microM, selectivity index=417.08) and 3,3',4,4',5,5'-hexa-hydroxy-trans-stilbene (COX-1: IC(50)=0.748, COX-2: IC(50)=0.00104 microM, selectivity index=719.23). Their selectivity index was in part higher than celecoxib, a selective COX-2 inhibitor already established on the market (COX-1: IC(50)=19.026, COX-2: IC(50)=0.03482 microM, selectivity index=546.41). Effect of structural parameters on COX-2 inhibition was evaluated by quantitative structure-activity relationship (QSAR) analysis and a high correlation was found with the topological surface area TPSA (r=0.93). Docking studies on both COX-1 and COX-2 protein structures also revealed that hydroxylated but not methoxylated resveratrol analogues are able to bind to the previously identified binding sites of the enzymes. Hydroxylated resveratrol analogues therefore represent a novel class of highly selective COX-2 inhibitors and promising candidates for in vivo studies.     

Cyclooxygenase-2 Immunoreactivity and Protein Level in the Gerbil Hippocampus During Normal Aging

Cyclooxygenases-2 (COX-2) is not only related to inflammation but also plays critical roles in brain development and synaptic signaling. In the present study, we investigated age-related changes in COX-2 immunoreactivity and protein levels in the gerbil hippocampus. In the hippocampal CA1 region (CA1) and dentate gyrus (DG), weak COX-2 immunoreactivity was observed at postnatal month 1 (PM 1), and COX-2 immunoreactivity was markedly increased at PM 18 and 24. In the CA2/3, COX-2 immunoreactivity was strong at PM 1. COX-2 immunoreactivities in the PM 3, 6 and 12 groups were decreased compared to that in the PM 1 group, and it was increased at PM 18 and 24. In addition, age-related changes in COX-2 levels were similar with immunohistochemical results in the CA2/3. These results suggest that COX-2 immunoreactivity and levels were high in the hippocampus of aged gerbils.     

Apobec-1 Increases Cyclooxygenase-2 and Aggravates Injury in Oxygen-Deprived Neurogenic Cells and Middle Cerebral Artery Occlusion Rats

Given that cyclooxygenase-2 (COX-2) plays a crucial role during cerebral ischemia and Apobec-1 is a critical regulator of COX-2 mRNA stabilization in gastrointestinal settings, the correlation of COX-2 and Apobec-1 was investigated in neurogenic cells and rat model of cerebral ischemia. After neurogenic SH-SY5Y, NG108-15 and PC12 cells were exposed to oxygen-glucose deprivation, cell viability, LDH leakage and Apobec-1 expression were determined. The effect of Apobec-1 overexpression on injury severity of oxygen-glucose deprivation, COX-2 expression, C-to-U editing of COX-2 mRNA were measured in vitro. Then the correlation of Apobec-1 level and injury severity was analyzed in cells with oxygen-glucose deprivation and in rats with middle cerebral artery occlusion. Apobec-1 expression was elevated along with upregulation of COX-2 and injury severity of oxygen-glucose deprivation in the three cell lines. Apobec-1 overexpression aggravated injury of oxygen-glucose deprivation in vitro and could be correlated to injury severity in vivo. Meanwhile, Apobec-1 increased COX-2 expression and COX-2 mRNA stabilization in neurogenic cells, and failed to catalyze C-to-U editing of COX-2 mRNA. Apobec-1 could upregulate COX-2 expression in neurogenic cells by stabilizing COX-2 mRNA, and might aggravate injury of oxygen-glucose deprivation in neurogenic cells as well as in rats with cerebral ischemia.     

COX-2 disruption leads to increased central vasopressin stores and impaired urine concentrating ability in mice

It was hypothesized that cyclooxygenase-2 (COX-2) activity promotes urine concentrating ability through stimulation of vasopressin (AVP) release after water deprivation (WD). COX-2-deficient (COX-2(-/-), C57BL/6) and wild-type (WT) mice were water deprived for 24 h, and water balance, central AVP mRNA and peptide level, AVP plasma concentration, and AVP-regulated renal transport protein abundances were measured. In male COX-2(-/-), basal urine output and water intake were elevated while urine osmolality was decreased compared with WT. Water deprivation resulted in lower urine osmolality, higher plasma osmolality in COX-2(-/-) mice irrespective of gender. Hypothalamic AVP mRNA level increased and was unchanged between COX-2(-/-) and WT after WD. AVP peptide content was higher in COX-2(-/-) compared with WT. At baseline, plasma AVP concentration was elevated in conscious chronically catheterized COX-2(-/-) mice, but after WD plasma AVP was unchanged between COX-2(-/-) and WT mice (43 ± 11 vs. 70 ± 16 pg/ml). Renal V2 receptor abundance was downregulated in COX-2(-/-) mice. Medullary interstitial osmolality increased and did not differ between COX-2(-/-) and WT after WD. Aquaporin-2 (AQP2; cortex-outer medulla), AQP3 (all regions), and UT-A1 (inner medulla) protein abundances were elevated in COX-2(-/-) at baseline and further increased after WD. COX-2(-/-) mice had elevated plasma urea and creatinine and accumulation of small subcapsular glomeruli. In conclusion, hypothalamic COX-2 activity is not necessary for enhanced AVP expression and secretion in response to water deprivation. Renal medullary COX-2 activity negatively regulates AQP2 and -3. The urine concentrating defect in COX-2(-/-) is likely caused by developmental glomerular injury and not dysregulation of AVP or collecting duct aquaporins.