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Uteroglobin represses allergen-induced inflammatory response by blocking PGD2 receptor-mediated functions.

Mandal AK, Zhang Z, Ray R, Choi MS, Chowdhury B, Pattabiraman N, Mukherjee AB - J. Exp. Med. (2004)

Bottom Line: These effects are abrogated by recombinant UG treatment.Here we report that DP signaling is mediated via p38 mitogen-activated protein kinase, p44/42 mitogen-activated protein kinase, and protein kinase C pathways in a cell type-specific manner leading to nuclear factor-kappaB activation stimulating COX-2 gene expression.Further, we found that recombinant UG blocks DP-mediated nuclear factor-kappaB activation and suppresses COX-2 gene expression.

View Article: PubMed Central - PubMed

Affiliation: Bldg. 10, Rm. 9S241, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA.

ABSTRACT
Uteroglobin (UG) is an antiinflammatory protein secreted by the epithelial lining of all organs communicating with the external environment. We reported previously that UG-knockout mice manifest exaggerated inflammatory response to allergen, characterized by increased eotaxin and Th2 cytokine gene expression, and eosinophil infiltration in the lungs. In this study, we uncovered that the airway epithelia of these mice also express high levels of cyclooxygenase (COX)-2, a key enzyme for the production of proinflammatory lipid mediators, and the bronchoalveolar lavage fluid (BALF) contain elevated levels of prostaglandin D2. These effects are abrogated by recombinant UG treatment. Although it has been reported that prostaglandin D2 mediates allergic inflammation via its receptor, DP, neither the molecular mechanism(s) of DP signaling nor the mechanism by which UG suppresses DP-mediated inflammatory response are clearly understood. Here we report that DP signaling is mediated via p38 mitogen-activated protein kinase, p44/42 mitogen-activated protein kinase, and protein kinase C pathways in a cell type-specific manner leading to nuclear factor-kappaB activation stimulating COX-2 gene expression. Further, we found that recombinant UG blocks DP-mediated nuclear factor-kappaB activation and suppresses COX-2 gene expression. We propose that UG is an essential component of a novel innate homeostatic mechanism in the mammalian airways to repress allergen-induced inflammatory responses.

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PGD2-stimulated COX-2 expression is mediated via DP. DP-mRNA expression determined by RT-PCR analysis followed by hybridization with the respective mouse and human DP-cDNA probes (A, inset) in BSM 2146 (lane 1) and NIH-3T3 cells (lane 2). 3H-PGD2 binding (A) to BSM 2146 (•) and NIH-3T3 (○) cells. Note unlabeled PGD2 displaces bound 3H-PGD2 in a dose-dependent manner. Analysis of COX-2 mRNA expression by real-time quantitative RT-PCR (B). NIH-3T3 cells were treated with DP monoclonal antibody before treatment with PGD2. Results are expressed as the mean of three independent experiments ± SD. Asterisks indicates significance at P < 0.05. Bar 1, untreated control cells; bar 2, PGD2-treated cells. Prior to PGD2 stimulation, cells were treated with DP monoclonal antibody at varying dilutions: bar 3, 1:5,000; bar 4, 1:1,000, and bar 5, 1:100. COX-2 protein expression by Western blot analysis (C). Top row: lane 1, untreated control; lane 2, PGD2-treated cells; lane 3, cells treated with DP monoclonal antibody at 1:5,000 dilution (lane 3); 1:1,000 dilution (lane 4); and 1:100 dilution (lane 5). Inhibition of COX-2 protein expression in cells treated with DP antisense s-oligonucleotide (D). Lane 1, untreated control; lane 2, cells treated with PGD2; lane 3, cells treated with nonspecific s-oligo; and lane 4, cells treated with DP antisense s-oligo before PGD2 stimulation.
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fig3: PGD2-stimulated COX-2 expression is mediated via DP. DP-mRNA expression determined by RT-PCR analysis followed by hybridization with the respective mouse and human DP-cDNA probes (A, inset) in BSM 2146 (lane 1) and NIH-3T3 cells (lane 2). 3H-PGD2 binding (A) to BSM 2146 (•) and NIH-3T3 (○) cells. Note unlabeled PGD2 displaces bound 3H-PGD2 in a dose-dependent manner. Analysis of COX-2 mRNA expression by real-time quantitative RT-PCR (B). NIH-3T3 cells were treated with DP monoclonal antibody before treatment with PGD2. Results are expressed as the mean of three independent experiments ± SD. Asterisks indicates significance at P < 0.05. Bar 1, untreated control cells; bar 2, PGD2-treated cells. Prior to PGD2 stimulation, cells were treated with DP monoclonal antibody at varying dilutions: bar 3, 1:5,000; bar 4, 1:1,000, and bar 5, 1:100. COX-2 protein expression by Western blot analysis (C). Top row: lane 1, untreated control; lane 2, PGD2-treated cells; lane 3, cells treated with DP monoclonal antibody at 1:5,000 dilution (lane 3); 1:1,000 dilution (lane 4); and 1:100 dilution (lane 5). Inhibition of COX-2 protein expression in cells treated with DP antisense s-oligonucleotide (D). Lane 1, untreated control; lane 2, cells treated with PGD2; lane 3, cells treated with nonspecific s-oligo; and lane 4, cells treated with DP antisense s-oligo before PGD2 stimulation.

Mentions: It has been demonstrated that PGD2 mediates allergen-induced airway inflammation via its receptor, DP (18). In this study, we found that PGD2 stimulates COX-2 expression in three different cell types of the respiratory system. To understand the mechanism(s) of PGD2-stimulated COX-2 gene expression and inflammatory lipid mediator production, we first determined DP expression and PGD2 binding using BSM-2146 and NIH-3T3 cells. The results of semiquantitative RT-PCR analyses show that these cells express DP-mRNA (Fig. 3 A, inset). We also performed competition-binding assays using 3H-PGD2 as the ligand. The results show that 3H-PGD2 binding on these cells is saturable, and it is displaced by nonradioactive PGD2 in a dose-dependent manner (Fig. 3 A). Since it has been reported that a chemoattractant receptor, CRTH2, also binds PGD2 in some cell types and may transduce some of the biological effects of this eicosanoid, we tested the expression of CRTH2-mRNA in these cells. The results show that the CRTH2-mRNA level is virtually undetectable (not depicted), indicating that CRTH2 is unlikely to be the predominant pathway for PGD2 signaling in these cell types.


Uteroglobin represses allergen-induced inflammatory response by blocking PGD2 receptor-mediated functions.

Mandal AK, Zhang Z, Ray R, Choi MS, Chowdhury B, Pattabiraman N, Mukherjee AB - J. Exp. Med. (2004)

PGD2-stimulated COX-2 expression is mediated via DP. DP-mRNA expression determined by RT-PCR analysis followed by hybridization with the respective mouse and human DP-cDNA probes (A, inset) in BSM 2146 (lane 1) and NIH-3T3 cells (lane 2). 3H-PGD2 binding (A) to BSM 2146 (•) and NIH-3T3 (○) cells. Note unlabeled PGD2 displaces bound 3H-PGD2 in a dose-dependent manner. Analysis of COX-2 mRNA expression by real-time quantitative RT-PCR (B). NIH-3T3 cells were treated with DP monoclonal antibody before treatment with PGD2. Results are expressed as the mean of three independent experiments ± SD. Asterisks indicates significance at P < 0.05. Bar 1, untreated control cells; bar 2, PGD2-treated cells. Prior to PGD2 stimulation, cells were treated with DP monoclonal antibody at varying dilutions: bar 3, 1:5,000; bar 4, 1:1,000, and bar 5, 1:100. COX-2 protein expression by Western blot analysis (C). Top row: lane 1, untreated control; lane 2, PGD2-treated cells; lane 3, cells treated with DP monoclonal antibody at 1:5,000 dilution (lane 3); 1:1,000 dilution (lane 4); and 1:100 dilution (lane 5). Inhibition of COX-2 protein expression in cells treated with DP antisense s-oligonucleotide (D). Lane 1, untreated control; lane 2, cells treated with PGD2; lane 3, cells treated with nonspecific s-oligo; and lane 4, cells treated with DP antisense s-oligo before PGD2 stimulation.
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fig3: PGD2-stimulated COX-2 expression is mediated via DP. DP-mRNA expression determined by RT-PCR analysis followed by hybridization with the respective mouse and human DP-cDNA probes (A, inset) in BSM 2146 (lane 1) and NIH-3T3 cells (lane 2). 3H-PGD2 binding (A) to BSM 2146 (•) and NIH-3T3 (○) cells. Note unlabeled PGD2 displaces bound 3H-PGD2 in a dose-dependent manner. Analysis of COX-2 mRNA expression by real-time quantitative RT-PCR (B). NIH-3T3 cells were treated with DP monoclonal antibody before treatment with PGD2. Results are expressed as the mean of three independent experiments ± SD. Asterisks indicates significance at P < 0.05. Bar 1, untreated control cells; bar 2, PGD2-treated cells. Prior to PGD2 stimulation, cells were treated with DP monoclonal antibody at varying dilutions: bar 3, 1:5,000; bar 4, 1:1,000, and bar 5, 1:100. COX-2 protein expression by Western blot analysis (C). Top row: lane 1, untreated control; lane 2, PGD2-treated cells; lane 3, cells treated with DP monoclonal antibody at 1:5,000 dilution (lane 3); 1:1,000 dilution (lane 4); and 1:100 dilution (lane 5). Inhibition of COX-2 protein expression in cells treated with DP antisense s-oligonucleotide (D). Lane 1, untreated control; lane 2, cells treated with PGD2; lane 3, cells treated with nonspecific s-oligo; and lane 4, cells treated with DP antisense s-oligo before PGD2 stimulation.
Mentions: It has been demonstrated that PGD2 mediates allergen-induced airway inflammation via its receptor, DP (18). In this study, we found that PGD2 stimulates COX-2 expression in three different cell types of the respiratory system. To understand the mechanism(s) of PGD2-stimulated COX-2 gene expression and inflammatory lipid mediator production, we first determined DP expression and PGD2 binding using BSM-2146 and NIH-3T3 cells. The results of semiquantitative RT-PCR analyses show that these cells express DP-mRNA (Fig. 3 A, inset). We also performed competition-binding assays using 3H-PGD2 as the ligand. The results show that 3H-PGD2 binding on these cells is saturable, and it is displaced by nonradioactive PGD2 in a dose-dependent manner (Fig. 3 A). Since it has been reported that a chemoattractant receptor, CRTH2, also binds PGD2 in some cell types and may transduce some of the biological effects of this eicosanoid, we tested the expression of CRTH2-mRNA in these cells. The results show that the CRTH2-mRNA level is virtually undetectable (not depicted), indicating that CRTH2 is unlikely to be the predominant pathway for PGD2 signaling in these cell types.

Bottom Line: These effects are abrogated by recombinant UG treatment.Here we report that DP signaling is mediated via p38 mitogen-activated protein kinase, p44/42 mitogen-activated protein kinase, and protein kinase C pathways in a cell type-specific manner leading to nuclear factor-kappaB activation stimulating COX-2 gene expression.Further, we found that recombinant UG blocks DP-mediated nuclear factor-kappaB activation and suppresses COX-2 gene expression.

View Article: PubMed Central - PubMed

Affiliation: Bldg. 10, Rm. 9S241, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA.

ABSTRACT
Uteroglobin (UG) is an antiinflammatory protein secreted by the epithelial lining of all organs communicating with the external environment. We reported previously that UG-knockout mice manifest exaggerated inflammatory response to allergen, characterized by increased eotaxin and Th2 cytokine gene expression, and eosinophil infiltration in the lungs. In this study, we uncovered that the airway epithelia of these mice also express high levels of cyclooxygenase (COX)-2, a key enzyme for the production of proinflammatory lipid mediators, and the bronchoalveolar lavage fluid (BALF) contain elevated levels of prostaglandin D2. These effects are abrogated by recombinant UG treatment. Although it has been reported that prostaglandin D2 mediates allergic inflammation via its receptor, DP, neither the molecular mechanism(s) of DP signaling nor the mechanism by which UG suppresses DP-mediated inflammatory response are clearly understood. Here we report that DP signaling is mediated via p38 mitogen-activated protein kinase, p44/42 mitogen-activated protein kinase, and protein kinase C pathways in a cell type-specific manner leading to nuclear factor-kappaB activation stimulating COX-2 gene expression. Further, we found that recombinant UG blocks DP-mediated nuclear factor-kappaB activation and suppresses COX-2 gene expression. We propose that UG is an essential component of a novel innate homeostatic mechanism in the mammalian airways to repress allergen-induced inflammatory responses.

Show MeSH
Related in: MedlinePlus