Role for Mucin-5AC in Upper and Lower Airway Pathogenesis in Mice.

Mucin-5AC (MUC5AC) is a major secreted mucin in pathogenic airways. To determine its role in mucus-related airway disorders, Muc5ac-deficient (Muc5ac-/-) and wild-type (Muc5ac+/+) mice were compared in bleomycin-induced pulmonary fibrosis, respiratory syncytial virus (RSV) disease, and ozone toxicity. Significantly greater inflammation and fibrosis by bleomycin were developed in Muc5ac-/- lungs compared to Muc5ac+/+ lungs. More severe mucous cell metaplasia in fibrotic Muc5ac-/- lungs coincided with bronchial Muc2, Muc4, and Muc5b overexpression. Airway RSV replication was higher in Muc5ac-/- than in Muc5ac+/+ during early infection. RSV-caused pulmonary epithelial death, bronchial smooth muscle thickening, and syncytia formation were more severe in Muc5ac-/- compared to Muc5ac+/+. Nasal septal damage and subepithelial mucoserous gland enrichment by RSV were greater in Muc5ac-/- than in Muc5ac+/+. Ozone exposure developed more severe nasal airway injury accompanying submucosal gland hyperplasia and pulmonary proliferation in Muc5ac-/- than in Muc5ac+/+. Ozone caused periodic acid-Schiff-positive secretion only in Muc5ac-/- nasal airways. Lung E-cadherin level was relatively lower in Muc5ac-/- than in Muc5ac+/+ basally and after bleomycin, RSV, and ozone exposure. Results indicate that MUC5AC is an essential mucosal component in acute phase airway injury protection. Subepithelial gland hyperplasia and adaptive increase of other epithelial mucins may compensate airway defense in Muc5ac-/- mice.

Muc5ac null mice are predisposed to spontaneous gastric antro-pyloric hyperplasia and adenomas coupled with attenuated H. pylori-induced corpus mucous metaplasia.

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide and is strongly associated with chronic Helicobacter pylori (Hp) infection. The ability of Hp to closely adhere to the gastric surface protective mucous layer containing mucins (MUC in humans and Muc in animals), primarily Muc5ac, is integral in the stepwise pathogenesis from gastritis to cancer. To probe the role of Muc5ac in Hp-induced gastric pathology, Muc5ac-/- and Muc5ac+/+ (WT) mice were experimentally infected with Hp Sydney strain (SS1). At 16 weeks and 32 weeks post infection (wpi), groups of mice were euthanized and evaluated for the following: gastric histopathological parameters, immunohistochemical expression of mucins (Muc5ac, Muc1, Muc2), Trefoil factor family proteins (Tff1 and Tff2), Griffonia (Bandeiraea) simplicifolia lectin II (GSL II) (mucous metaplasia marker) and Clusterin (Spasmolytic Polypeptide Expressing Metaplasia (SPEM) marker), Hp colonization density by qPCR and gastric cytokine mRNA levels. Our results demonstrate that Muc5ac-/- mice developed spontaneous antro-pyloric proliferation, adenomas and in one case with neuroendocrine differentiation; these findings were independent of Hp infection along with strong expression levels of Tff1, Tff2 and Muc1. Hp-infected Muc5ac-/- mice had significantly lowered gastric corpus mucous metaplasia at 16 wpi and 32 wpi (P = 0.0057 and P = 0.0016, respectively), with a slight reduction in overall gastric corpus pathology. GSII-positive mucous neck cells were decreased in Hp-infected Muc5ac-/- mice compared to WT mice and clusterin positivity was noted within metaplastic glands in both genotypes following Hp infection. Additionally, Hp colonization densities were significantly higher in Muc5ac-/- mice compared to WT at 16 wpi in both sexes (P = 0.05) along with a significant reduction in gastric Tnfα (16 wpi-males and females, P = 0.017 and P = 0.036, respectively and 32 wpi-males only, P = 0.025) and Il-17a (16 wpi-males) (P = 0.025). Taken together, our findings suggest a protective role for MUC5AC/Muc5ac in maintaining gastric antral equilibrium and inhibiting Hp colonization and associated inflammatory pathology.

ß-Arrestin-2 deficiency attenuates abdominal aortic aneurysm formation in mice.

RATIONALE: Abdominal aortic aneurysms (AAAs) are a chronic inflammatory vascular disease for which pharmacological treatments are not available. A mouse model of AAA formation involves chronic infusion of angiotensin II (AngII), and previous studies indicated a primary role for the AngII type 1a receptor in AAA formation. ß-arrestin (ßarr)-2 is a multifunctional scaffolding protein that binds G-protein-coupled receptors such as AngII type 1a and regulates numerous signaling pathways and pathophysiological processes. However, a role for ßarr2 in AngII-induced AAA formation is currently unknown. OBJECTIVE: To determine whether ßarr2 played a role in AngII-induced AAA formation in mice. METHODS AND RESULTS: Treatment of ßarr2(+/+) and ßarr2(-/-) mice on the hyperlipidemic apolipoprotein E-deficient (apoE(-/-)) background or on normolipidemic C57BL/6 background with AngII for 28 days indicated that ßarr2 deficiency significantly attenuated AAA formation. ßarr2 deficiency attenuated AngII-induced expression of cyclooxygenase-2, monocyte chemoattractant protein-1, macrophage inflammatory protein 1α, and macrophage infiltration. AngII also increased the levels of phosphorylated extracellular signal-regulated kinase 1/2 in apoE(-/-)/ßarr2(+/+) aortas, whereas ßarr2 deficiency diminished this increase. Furthermore, inhibition of extracellular signal-regulated kinase 1/2 activation with CI1040 (100 mg/kg per day) reduced the level of AngII-induced cyclooxygenase-2 expression in apoE(-/-)/ßarr2(+/+) mice to the level observed in apoE(-/-)/ßarr2(-/-) mice. AngII treatment also increased matrix metalloproteinase expression and disruption of the elastic layer in apoE(-/-)/ßarr2(+/+) aortas, and ßarr2 deficiency reduced these effects. CONCLUSIONS: ßarr2 contributes to AngII-induced AAA formation in mice by phosphorylated extracellular signal-regulated kinase 1/2-mediated cyclooxygenase-2 induction and increased inflammation. These studies suggest that for the AngII type 1a receptor, G-protein-independent, ßarr2-dependent signaling plays a major role in AngII-induced AAA formation.

Role of cyclooxygenase-2 in exacerbation of allergen-induced airway remodeling by multiwalled carbon nanotubes.

The emergence of nanotechnology has produced a multitude of engineered nanomaterials such as carbon nanotubes (CNTs), and concerns have been raised about their effects on human health, especially for susceptible populations such as individuals with asthma. Multiwalled CNTs (MWCNTs) have been shown to exacerbate ovalbumin (OVA)-induced airway remodeling in mice. Moreover, cyclooxygenase-2 (COX-2) has been described as a protective factor in asthma. We postulated that COX-2-deficient (COX-2(-/-)) mice would be susceptible to MWCNT-induced exacerbations of allergen-induced airway remodeling, including airway inflammation, fibrosis, and mucus-cell metaplasia (i.e., the formation of goblet cells). Wild-type (WT) or COX-2(-/-) mice were sensitized to OVA to induce allergic airway inflammation before a single dose of MWCNTs (4 mg/kg) delivered to the lungs by oropharyngeal aspiration. MWCNTs significantly increased OVA-induced lung inflammation and mucus-cell metaplasia in COX-2(-/-) mice compared with WT mice. However, airway fibrosis after exposure to allergen and MWCNTs was no different between WT and COX-2(-/-) mice. Concentrations of certain prostanoids (prostaglandin D2 and thromboxane B2) were enhanced by OVA or MWCNTs in COX-2(-/-) mice. No differences in COX-1 mRNA concentrations were evident between WT and COX-2(-/-) mice treated with OVA and MWCNTs. Interestingly, MWCNTs significantly enhanced allergen-induced cytokines involved in Th2 (IL-13 and IL-5), Th1 (CXCL10), and Th17 (IL-17A) inflammatory responses in COX-2(-/-) mice, but not in WT mice. We conclude that exacerbations of allergen-induced airway inflammation and mucus-cell metaplasia by MWCNTs are enhanced by deficiencies in COX-2, and are associated with the activation of a mixed Th1/Th2/Th17 immune response.

Genetic ablation of cyclooxygenase-2 in keratinocytes produces a cell-autonomous defect in tumor formation.

Using a mouse skin tumor model, we reported previously that cyclooxygenase-2 (COX-2) deficiency reduced papilloma formation. However, this model did not differentiate between the effects of systemic COX-2-deficiency and keratinocyte-specific COX-2 deficiency on tumor formation. To determine whether keratinocyte-specific COX-2 deficiency reduced papilloma formation, v-H-ras-transformed COX-2+/+ and COX-2-/- keratinocytes were grafted onto nude mice and tumor development was compared. Transformed COX-2+/+ and COX-2-/- keratinocytes expressed similar levels of H-ras, epidermal growth factor receptor and phospho-extracellular signal-regulated kinase 1/2 in vitro; and COX-2-deficiency did not reduce uninfected or v-H-ras infected keratinocyte replication. In contrast, tumors arising from grafted transformed COX-2+/+ and COX-2-/- keratinocytes expressed similar levels of H-ras, but COX-2 deficiency reduced phospho-extracellular signal-regulated kinase 1/2 and epidermal growth factor receptor levels 50-60% and tumor volume by 80% at 3 weeks. Two factors appeared to account for the reduced papilloma size. First, papillomas derived from COX-2-/- keratinocytes showed about 70% decreased proliferation, as measured by bromodeoxyuridine incorporation, compared with papillomas derived from COX-2+/+ keratinocytes. Second, keratin 1 immunostaining of papillomas indicated that COX-2-/- keratinocytes prematurely initiated terminal differentiation. Differences in the levels of apoptosis and vascularization did not appear to be contributing factors as their levels were similar in tumors derived from COX-2-/- and COX-2+/+ keratinocytes. Overall, the data are in agreement with our previous observations that decreased papilloma number and size on COX-2-/- mice resulted from reduced keratinocyte proliferation and accelerated keratinocyte differentiation. Furthermore, the data indicate that deficiency/inhibition of COX-2 in the initiated keratinocyte is an important determinant of papilloma forming ability.

Multi-walled carbon nanotubes induce COX-2 and iNOS expression via MAP kinase-dependent and -independent mechanisms in mouse RAW264.7 macrophages.

BACKGROUND: Carbon nanotubes (CNTs) are engineered graphene cylinders with numerous applications in engineering, electronics and medicine. However, CNTs cause inflammation and fibrosis in the rodent lung, suggesting a potential human health risk. We hypothesized that multi-walled CNTs (MWCNTs) induce two key inflammatory enzymes in macrophages, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), through activation of extracellular signal-regulated kinases (ERK1,2). METHODS: RAW264.7 macrophages were exposed to MWCNTs or carbon black nanoparticles (CBNPs) over a range of doses and time course. Uptake and subcellular localization of MWCNTs was visualized by transmission electron microscopy (TEM). Protein levels of COX-2, iNOS, and ERK1,2 (total ERK and phosphorylated ERK) were measured by Western blot analysis. Prostaglandin-E(2) (PGE(2)) and nitric oxide (NO) levels in cell supernatants were measured by ELISA and Greiss assay, respectively. RESULTS: MWCNTs, but not CBNPs, induced COX-2 and iNOS in a time- and dose-dependent manner. COX-2 and iNOS induction by MWCNTs correlated with increased PGE(2) and NO production, respectively. MWCNTs caused ERK1,2 activation and inhibition of ERK1,2 (U0126) blocked MWCNT induction of COX-2 and PGE2 production, but did not reduce the induction of iNOS. Inhibition of iNOS (L-NAME) did not affect ERK1,2 activation, nor did L-NAME significantly decrease COX-2 induction by MWCNT. Nickel nanoparticles (NiNPs), which are present in MWCNTs as a residual catalyst, also induced COX-2 via ERK-1,2. However, a comparison of COX-2 induction by MWCNTs containing 4.5 and 1.8% Ni did not show a significant difference in ability to induce COX-2, indicating that characteristics of MWCNTs in addition to Ni content contribute to COX-2 induction. CONCLUSION: This study identifies COX-2 and subsequent PGE(2) production, along with iNOS induction and NO production, as inflammatory mediators involved in the macrophage response to MWCNTs. Furthermore, our work demonstrates that COX-2 induction by MWCNTs in RAW264.7 macrophages is ERK1,2-dependent, while iNOS induction by MWCNTs is ERK1,2-independent. Our data also suggest contributory physicochemical factors other than residual Ni catalyst play a role in COX-2 induction to MWCNT.

The prostaglandin E2 receptor, EP2, stimulates keratinocyte proliferation in mouse skin by G protein-dependent and {beta}-arrestin1-dependent signaling pathways.

The prostaglandin E(2) (PGE(2)) G protein-coupled receptor (GPCR), EP2, plays important roles in mouse skin tumor development (Chun, K. S., Lao, H. C., Trempus, C. S., Okada, M., and Langenbach, R. (2009) Carcinogenesis 30, 1620-1627). Because keratinocyte proliferation is essential for skin tumor development, EP2-mediated signaling pathways that contribute to keratinocyte proliferation were investigated. A single topical application of the EP2 agonist, butaprost, dose-dependently increased keratinocyte replication via activation of epidermal growth factor receptor (EGFR) and PKA signaling. Because GPCR-mediated activation of EGFR can involve the formation of a GPCR-ß-arrestin-Src signaling complex, the possibility of a ß-arrestin1-Src complex contributing to EP2-mediated signaling in keratinocytes was investigated. Butaprost induced ß-arrestin1-Src complex formation and increased both Src and EGFR activation. A role for ß-arrestin1 in EP2-mediated Src and EGFR activation was demonstrated by the observation that ß-arrestin1 deficiency significantly reduced Src and EGFR activation. In agreement with a ß-arrestin1-Src complex contributing to EGFR activation, Src and EGFR inhibition (PP2 and AG1478, respectively) indicated that Src was upstream of EGFR. Butaprost also induced the activation of Akt, ERK1/2, and STAT3, and both ß-arrestin1 deficiency and EGFR inhibition (AG1478 or gefitinib) decreased their activation. In addition to ß-arrestin1-dependent EGFR activation, butaprost increased PKA activation, as measured by phospho-GSK3ß (p-GSK3ß) and p-cAMP-response element-binding protein formation. PKA inhibition (H89 or R(P)-adenosine-3',5'-cyclic monophosphorothioate (R(P)-cAMPS)) decreased butaprost-induced cAMP-response element-binding protein and ERK activation but did not affect EGFR activation, whereas ß-arrestin1 deficiency decreased EGFR activation but did not affect butaprost-induced PKA activation, thus indicating that they were independent EP2-mediated pathways. Therefore, the results indicate that EP2 contributed to mouse keratinocyte proliferation by G protein-independent, ß-arrestin1-dependent activation of EGFR and G protein-dependent activation of PKA.

The prostaglandin E2 receptor, EP2, regulates survivin expression via an EGFR/STAT3 pathway in UVB-exposed mouse skin.

We previously reported that cycloogenase (COX)-2-generated prostaglandin E2 (PGE2) had anti-apoptotic effects in UVB-exposed mouse skin that involved EP2-mediated signaling (Chun et al., Cancer Res. 2007; 67: 2015). Because survivin is a regulator of cell survival, the possible involvement of COX-2 and EP2 in survivin expression following UVB exposure of mouse skin was investigated. In wild type mice, UVB exposure time-dependently increased the levels of survivin and phosphorylated-signal transducer and activator of transcription 3 (p-STAT3), a transcription factor that regulates survivin expression; and COX-2- or EP2-deficiency significantly reduced their induction. Topical application of the COX-2 inhibitor, celecoxib, also reduced UVB-induced survivin levels. To further investigate the roles of PGE2 and EP2 in the regulation of survivin, indomethacin was used to inhibit UVB-induced endogenous PG production. UVB-induced survivin levels were reduced by indomethacin, and PGE2 and the EP2 agonist, butaprost, partially restored survivin levels. The epidermal growth factor receptor (EGFR) is a downstream effector of EP2 and EGFR inhibition (AG1478) significantly reduced UVB activation of STAT3 and survivin levels. UVB-induced epidermal apoptosis in COX-2-/- mice was reduced by butaprost and EGFR inhibition blocked butaprost's protective effects. Furthermore, butaprost in the absence of UVB exposure time-dependently increased p-EGFR, p-STAT3, and survivin levels in naïve mouse skin, whereas the EP4 agonist, PGE1 alcohol, did not significantly increase p-STAT3 or survivin levels. These data suggest that COX-2-generated PGE2 regulates survivin expression in mouse skin, in part, via an EP2-mediated EGFR/STAT3 pathway. Therefore, targeting the EP2/survivin pathway may provide a strategy for the chemoprevention/chemotherapy of skin cancer.

A novel role for the T-box transcription factor Tbx1 as a negative regulator of tumor cell growth in mice.

The T-box transcription factor, Tbx1, an important regulatory gene in development, is highly expressed in hair follicle (HF) stem cells in adult mice. Because mouse models of skin carcinogenesis have demonstrated that HF stem cells are a carcinogen target population and contribute significantly to tumor development, we investigated whether Tbx1 plays a role in skin carcinogenesis. We first assessed Tbx1 expression levels in mouse skin tumors, and found down-regulation in all tumors examined. To study the effect of Tbx1 expression on growth and tumorigenic potential of carcinoma cells, we transfected mouse Tbx1 cDNA into a mouse spindle cell carcinoma cell line that did not express endogenous Tbx1. Following transfection, two cell lines expressing different levels of the Tbx1/V5 fusion protein were selected for further study. Intradermal injection of the cell lines into mice revealed that Tbx1 expression significantly suppressed tumor growth, albeit with no change in tumor morphology. In culture, ectopic Tbx1 expression resulted in decreased cell growth and reduced development into multilayered colonies, compared to control cells. Tbx1-transfectants exhibited a reduced proliferative rate compared to control cells, with fewer cells in S and G2/M phases. The Tbx1 transfectants developed significantly fewer colonies in soft agar, demonstrating loss of anchorage-independent growth. Taken together, our data show that ectopic expression of Tbx1 restored contact inhibition to the skin tumor cells, suggesting that this developmentally important transcription factor may have a novel dual role as a negative regulator of tumor growth. © 2011 Wiley Periodicals, Inc.

The prostaglandin receptor EP2 activates multiple signaling pathways and beta-arrestin1 complex formation during mouse skin papilloma development.

Prostaglandin E(2) (PGE(2)) is elevated in many tumor types, but PGE(2)'s contributions to tumor growth are largely unknown. To investigate PGE(2)'s roles, the contributions of one of its receptors, EP2, were studied using the mouse skin initiation/promotion model. Initial studies indicated that protein kinase A (PKA), epidermal growth factor receptor (EGFR) and several effectors-cyclic adenosine 3',5'-monophosphate response element-binding protein (CREB), H-Ras, Src, protein kinase B (AKT) and extracellular signal-regulated kinase (ERK)1/2-were activated in 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted papillomas and that PKA and EGFR inhibition (H89 and AG1478, respectively) decreased papilloma formation. EP2's contributions to the activation of these pathways and papilloma development were determined by inhibiting endogenous TPA-induced PGE(2) production with indomethacin (Indo) and concomitantly treating with the EP2 agonist, CAY10399 (CAY). CAY treatment restored papilloma formation in TPA/Indo-treated mice and increased cyclic adenosine 3',5'-monophosphate and PKA activation as measured by p-CREB formation. CAY treatment also increased EGFR and Src activation and their inhibition by AG1478 and PP2 indicated that Src was upstream of EGFR. CAY also increased H-Ras, ERK1/2 and AKT activation, and AG1478 decreased their activation indicating EGFR being upstream. Supporting EP2's contribution, EP2-/- mice exhibited 65% fewer papillomas and reduced Src, EGFR, H-Ras, AKT and ERK1/2 activation. G protein-coupled receptor (GPCR) activation of EGFR has been reported to involve Src's activation via a GPCR-beta-arrestin-Src complex. Indeed, immunoprecipitation of beta-arrestin1 or p-Src indicated the presence of an EP2-beta-arrestin1-p-Src complex in papillomas. The data indicated that EP2 contributed to tumor formation via activation of PKA and EGFR and that EP2 formed a complex with beta-arrestin1 and Src that contributed to signaling and/or EP2 desensitization.

Prostaglandin E2 released from activated microglia enhances astrocyte proliferation in vitro.

Microglial activation has been implicated in many astrogliosis-related pathological conditions including astroglioma; however, the detailed mechanism is not clear. In this study, we used primary enriched microglia and astrocyte cultures to determine the role of microglial prostaglandin E(2) (PGE(2)) in the proliferation of astrocytes. The proliferation of astrocytes was measured by BrdU incorporation. The level of PGE(2) was measured by ELISA method. Pharmacological inhibition or genetic ablation of COX-2 in microglia were also applied in this study. We found that proliferation of astrocytes increased following lipopolysaccharide (LPS) treatment in the presence of microglia. Furthermore, increased proliferation of astrocytes was observed in the presence of conditioned media from LPS-treated microglia. The potential involvement of microglial PGE(2) in enhanced astrocyte proliferation was suggested by the findings that PGE(2) production and COX-2 expression in microglia were increased by LPS treatment. In addition, activated microglia-induced increases in astrocyte proliferation were blocked by the PGE(2) antagonist AH6809, COX-2 selective inhibitor DuP-697 or by genetic knockout of microglial COX-2. These findings were further supported by the finding that addition of PGE(2) to the media significantly induced astrocyte proliferation. These results indicate that microglial PGE(2) plays an important role in astrocyte proliferation, identifying PGE(2) as a key neuroinflammatory molecule that triggers the pathological response related to uncontrollable astrocyte proliferation. These findings are important in elucidating the role of activated microglia and PGE(2) in astrocyte proliferation and in suggesting a potential avenue in the use of anti-inflammatory agents for the therapy of astroglioma.

Genetic deletion or pharmacological inhibition of cyclooxygenase-1 attenuate lipopolysaccharide-induced inflammatory response and brain injury.

Cyclooxygenase (COX) -1 and -2 metabolize arachidonic acid to prostanoids and reactive oxygen species, major players in the neuroinflammatory process. While most reports have focused on the inducible isoform, COX-2, the contribution of COX-1 to the inflammatory response is unclear. In the present study, the contribution of COX-1 in the neuroinflammatory response to intracerebroventricular lipopolysaccharide (LPS) was investigated using COX-1 deficient (COX-1(-/-)) mice or wild-type (COX-1(+/+)) mice pretreated with SC-560, a selective COX-1 inhibitor. Twenty-four hours after lipopolysaccharide (LPS) injection, COX-1(-/-) mice showed decreased protein oxidation and LPS-induced neuronal damage in the hippocampus compared with COX-1(+/+) mice. COX-1(-/-) mice showed a significant reduction of microglial activation, proinflammatory mediators, and expression of COX-2, inducible NOS, and NADPH oxidase. The transcriptional down-regulation of cytokines and other inflammatory markers in COX-1(-/-) mice was mediated by a reduced activation of NF-kappaB and signal transducer and activator of transcription 3. Administration of SC-560 prior to LPS injection also attenuated the neuroinflammatory response by decreasing brain levels of prostaglandin (PG)E(2), PGD(2), PGF(2alpha), and thromboxane B(2), as well as the expression of proinflammatory cytokines and chemokine. These findings suggest that COX-1 plays a previously unrecognized role in neuroinflammatory damage.

Cyclooxygenase-2 inhibits UVB-induced apoptosis in mouse skin by activating the prostaglandin E2 receptors, EP2 and EP4.

Cyclooxygenase-2 (COX-2) is induced by UVB light and reduces UVB-induced epidermal apoptosis; however, the mechanism is unclear. Therefore, wild-type (WT) and COX-2-/- mice were acutely treated with UVB (5 kJ/m(2)), and apoptotic signaling pathways were compared. Following exposure, apoptosis was 2.5-fold higher in COX-2-/- compared with WT mice. Because prostaglandin E(2) (PGE(2)) is the major UV-induced prostaglandin and manifests its activity via four receptors, EP1 to EP4, possible differences in EP signaling were investigated in WT and COX-2-/- mice. Following UVB exposure, protein levels of EP1, EP2, and EP4 were elevated in WT mice, but EP2 and EP4 levels were 50% lower in COX-2-/- mice. Activated cyclic AMP-dependent protein kinase (PKA) and Akt are downstream in EP2 and EP4 signaling, and their levels were reduced in UVB-exposed COX-2-/- mice. Furthermore, p-Bad (Ser(136) and Ser(155)), antiapoptotic products of activated Akt and PKA, respectively, were significantly reduced in UVB-exposed COX-2-/- mice. To further study the roles of EP2 and EP4, UVB-exposed CD-1 mice were topically treated with indomethacin to block endogenous PGE(2) production, and PGE(2), the EP2 agonist (butaprost) or EP4 agonist (PGE(1) alcohol), was applied. Indomethacin reduced PKA and Akt activation by approximately 60%, but PGE(2) and the agonists restored their activities. Furthermore, both agonists decreased apoptosis in COX-2-/- mice by 50%. The data suggest that COX-2-generated PGE(2) has antiapoptotic roles in UVB-exposed mouse skin that involves EP2- and EP4-mediated signaling.

Neuroinflammatory response to lipopolysaccharide is exacerbated in mice genetically deficient in cyclooxygenase-2.

BACKGROUND: Cyclooxygenases (COX) -1 and -2 are key mediators of the inflammatory response in the central nervous system. Since COX-2 is inducible by inflammatory stimuli, it has been traditionally considered as the most appropriate target for anti-inflammatory drugs. However, the specific roles of COX-1 and COX-2 in modulating a neuroinflammatory response are unclear. Recently, we demonstrated that COX-1 deficient mice show decreased neuroinflammatory response and neuronal damage in response to lipopolysaccharide (LPS). METHODS: In this study, we investigated the role of COX-2 in the neuroinflammatory response to intracerebroventricular-injected LPS (5 mug), a model of direct activation of innate immunity, using COX-2 deficient (COX-2-/-) and wild type (COX-2+/+) mice, as well as COX-2+/+ mice pretreated for 6 weeks with celecoxib, a COX-2 selective inhibitor. RESULTS: Twenty-four hours after LPS injection, COX-2-/- mice showed increased neuronal damage, glial cell activation, mRNA and protein expression of markers of inflammation and oxidative stress, such as cytokines, chemokines, iNOS and NADPH oxidase. Brain protein levels of IL-1beta, NADPH oxidase subunit p67phox, and phosphorylated-signal transducer and activator of transcription 3 (STAT3) were higher in COX-2-/- and in celecoxib-treated mice, compared to COX-2+/+ mice. The increased neuroinflammatory response in COX-2-/- mice was likely mediated by the upregulation of STAT3 and suppressor of cytokine signaling 3 (SOCS3). CONCLUSION: These results show that inhibiting COX-2 activity can exacerbate the inflammatory response to LPS, possibly by increasing glial cells activation and upregulating the STAT3 and SOCS3 pathways in the brain.

Genetic deficiency of cyclooxygenase-2 attenuates abdominal aortic aneurysm formation in mice.

OBJECTIVE: Abdominal aortic aneurysms (AAAs) are characterized by chronic inflammation which contributes to the remodeling and eventual weakening of the vessel wall. Increased cyclooxygenase-2 (COX-2) expression is detected in human aneurysmal tissue and is suggested to contribute to the disease. The aim of the current study was to define the role of COX-2 expression in the development of AAAs, using a model of the disease. METHODS: AAAs were induced in mice by chronic angiotensin II infusion, and were analyzed following 3, 7, 21 or 28 days of the infusion. AAA incidence and severity, together with the expression of inflammatory markers, were compared between abdominal aortas from COX-2-deficient mice and their wild-type littermate controls. RESULTS: The AAA incidence in COX-2 wild-type mice was 54% (13/24), whereas AAAs were not detected in COX-2-deficient mice (0/23) following 28 days of angiotensin II infusion. The genetic deficiency of COX-2 also resulted in a 73% and 90% reduction in AAA incidence following 7 and 21 days of angiotensin II infusion, respectively. In COX-2 wild-type mice, COX-2 mRNA expression in the abdominal aorta was induced by angiotensin II beginning 3 days following initiation of the infusion, which continued throughout progression of the disease. Abundant COX-2 protein expression was detected in medial smooth muscle cells adjacent to the AAAs. The deficiency of COX-2 significantly attenuated mRNA expression in the abdominal aorta of the macrophage marker CD68, and the inflammatory cell recruitment chemokines, monocyte chemotactic protein-1 and macrophage inflammatory protein-1alpha. CONCLUSIONS: Our findings suggest that increased COX-2 expression in smooth muscle cells of the abdominal aorta contributes to AAA formation in mice by enhancing inflammatory cell infiltration.

Cyclooxygenase-1-selective inhibition prolongs gestation in mice without adverse effects on the ductus arteriosus.

Preterm delivery is the leading cause of neonatal mortality and contributes significantly to infant morbidity. Classical cyclooxygenase (COX) inhibitors, such as indomethacin, which inhibit both COX-1 and COX-2, are effective for delaying premature labor, but their use is limited by serious complications to the fetus and neonate, including adverse effects on the ductus arteriosus (DA). Using isoform-selective inhibitors, we characterized the roles of the COX isoforms in the initiation of labor and the regulation of fetal and neonatal DA closure in mice. Chronic inhibition of COX-2 during pregnancy (gestation days 15-18) significantly increased neonatal mortality by preventing closure of the DA after birth, whereas acute COX-2 inhibition near the end of term (gestation day 18) constricted the fetal DA. In contrast, the inhibition of COX-1 during pregnancy lacked these prenatal and postnatal adverse effects on the DA and effectively delayed the initiation of full-term labor and LPS-induced preterm labor. These findings suggest that premature fetal DA closure or neonatal patent DA observed following indomethacin tocolysis in women may result from the inhibition of COX-2. Therefore, COX-1-selective inhibitors may provide effective treatment to delay preterm labor with fewer adverse effects on fetal or neonatal health than nonselective or COX-2-selective inhibitors.

Opposite effects of cyclooxygenase-1 and -2 activity on the pressor response to angiotensin II.

Therapeutic use of cyclooxygenase-inhibiting (COX-inhibiting) nonsteroidal antiinflammatory drugs (NSAIDs) is often complicated by renal side effects including hypertension and edema. The present studies were undertaken to elucidate the roles of COX1 and COX2 in regulating blood pressure and renal function. COX2 inhibitors or gene knockout dramatically augment the pressor effect of angiotensin II (Ang II). Unexpectedly, after a brief increase, the pressor effect of Ang II was abolished by COX1 deficiency (either inhibitor or knockout). Ang II infusion also reduced medullary blood flow in COX2-deficient but not in control or COX1-deficient animals, suggesting synthesis of COX2-dependent vasodilators in the renal medulla. Consistent with this, Ang II failed to stimulate renal medullary prostaglandin E(2) and prostaglandin I(2) production in COX2-deficient animals. Ang II infusion normally promotes natriuresis and diuresis, but COX2 deficiency blocked this effect. Thus, COX1 and COX2 exert opposite effects on systemic blood pressure and renal function. COX2 inhibitors reduce renal medullary blood flow, decrease urine flow, and enhance the pressor effect of Ang II. In contrast, the pressor effect of Ang II is blunted by COX1 inhibition. These results suggest that, rather than having similar cardiovascular effects, the activities of COX1 and COX2 are functionally antagonistic.

Atherogenic diet causes lethal ileo-ceco-colitis in cyclooxygenase-2 deficient mice.

Cyclooxygenases (COX) regulate a variety of inflammatory diseases, including inflammatory bowel disease (IBD). While the pathological effects of COX-1 inhibition by NSAIDs on intestinal ulceration are well established, the role of COX-2 on intestinal inflammation remains under investigation. In this paper, we report a protective role for COX-2 against diet-mediated intestinal inflammation in mice. COX-2(-/-) mice fed an atherogenic diet or diet containing cholate, but not chow or fat alone, had a high mortality whereas COX-1(-/-) mice and wild-type mice were unaffected by the dietary changes. Histological analysis identified the cause of death in COX-2(-/-) mice due to severe intestinal inflammation that was surprisingly limited to the ileo-ceco-colic junction. COX-2 expression is induced in the cecum of wild-type mice fed an atherogenic diet. Our findings show that COX-2 plays an anti-inflammatory role at the ileo-ceco-colic junction in mice, and the pathology of diet-mediated intestinal inflammation in COX-2(-/-) mice offers an excellent model system to elucidate the molecular mechanisms of intestinal inflammation.

A novel anti-atherogenic role for COX-2--potential mechanism for the cardiovascular side effects of COX-2 inhibitors.

Atherosclerosis, the underlying cause of cardiovascular disease, is characterized by lipid accumulation, lipoprotein oxidation, and inflammation. Products of the cyclooxygenase (COX) pathway participate in acute and chronic inflammation. The inducible form of COX, COX-2, generates lipid mediators of inflammation that are pro-inflammatory and COX-2-selective inhibitors are potent anti-inflammatory agents. However, clinical data suggest an increased risk of cardiovascular side effects in patients using COX-2-selective inhibitors. In this paper, we sought to determine the effect of COX-2 deficiency on atherosclerosis-related lipoprotein metabolism in mice. We demonstrate that COX-2 deficiency resulted in (i) accumulation of lipids in circulation and liver, (ii) pro-inflammatory properties of HDL as measured by HDL's increased reactive oxygen species (ROS) content, decreased paraoxonase 1 (PON1) activity, decreased serum apoA-1, reduced ability to efflux cholesterol and to prevent LDL oxidizability, and (iii) increased TXB(2) in circulation. Moreover, when placed on an atherogenic diet, COX-2 deficiency resulted in (i) increased lipid deposition in the aorta, (ii) a further dramatic imbalance in circulating eicosanoids, i.e. decreased serum PGI(2) coupled with increased PGE(2) and TXB(2), and (iii) a marked elevation of pro-inflammatory cytokines, TNF and IL-6. Our results suggest, for the first time, that COX-2 deficiency contributes to the pro-atherogenic properties of HDL in mice.

MPP+-induced COX-2 activation and subsequent dopaminergic neurodegeneration.

The importance of cyclooxygenase-2 (COX-2) in mediating Parkinson's disease (PD) was suggested in reports, indicating that COX-2 selective inhibitors or genetic knockout reduce 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic (DA) neurotoxicity in a mouse model of PD. However, cell types and mechanisms underlying the activation of COX-2 have not been clearly elucidated in these animal studies. Using primary neuron-glia cultures, we aimed to determine 1) whether microglia participate in 1-methyl-4-phenylpryridinium (MPP)-induced COX-2 activation and 2) whether the activation of COX-2 contributes to subsequent neurotoxicity. MPP, in a concentration-dependent manner, increased prostaglandin E2 (PGE2) production in mixed neuron-microglia cultures but not in enriched neuron, microglia, or astroglia cultures nor in mixed neuron-astroglia cultures. MPP-induced PGE2 increase was completely abolished by treatment with DuP697, a COX-2 selective inhibitor. DuP697 also significantly reduced MPP-induced DA neurotoxicity as determined by DA uptake assay. Immunocytochemistry and confocal microscopy studies showed enhanced COX-2 expression in both microglia and neurons after MPP treatment. However, neuronal increase in COX-2 expression was not totally dependent on the production of PGE2 from microglia, since microglia deficient in COX-2 only attenuated, but did not completely block, MPP-increased PGE2 production in mixed neuron-microglia cultures, suggesting that part of PGE2 production was originated from neurons. Together, these results indicate that MPP-induced COX-2 expression and subsequent PGE2 production depend on interactions between neurons and microglia. Microgliosis may also be responsible for the COX-2 activation in neurons, leading to the enhanced DA neurotoxicity, which, in turn, reinforces microgliosis. Thus inhibition of microgliosis and COX-2 activity may stop this vicious circle and be valuable strategies in PD therapy.