Glycosylation regulates Notch signalling

Intracellular post-translational modifications such as phosphorylation and ubiquitylation have been well studied for their roles in regulating diverse signalling pathways, but we are only just beginning to understand how differential glycosylation is used to regulate intercellular signalling. Recent studies make clear that extracellular post-translational modifications, in the form of glycosylation, are essential for the Notch signalling pathway, and that differences in the extent of glycosylation are a significant mechanism by which this pathway is regulated.     

Extracellular matrix-induced cyclooxygenase-2 regulates macrophage proteinase expression

Chronic inflammatory diseases are characterized by the persistent presence of macrophages and other mononuclear cells, tissue destruction, cell proliferation, and the deposition of extracellular matrix (ECM). The tissue degradation is mediated, in part, by enhanced proteinase expression by macrophages. It has been demonstrated recently that macrophage proteinase expression can be stimulated or inhibited by purified ECM components. However, in an intact ECM the biologically active domains of matrix components may be masked either by tertiary conformation or by complex association with other matrix molecules. In an effort to determine whether a complex ECM produced by vascular smooth muscle cells (SMC) regulates macrophage degradative phenotype, we prepared insoluble SMC matrices and examined their ability to regulate proteinase expression by RAW264.7 and thioglycollate-elicited peritoneal macrophages. Here we demonstrate that macrophage engagement of SMC-ECM triggers PKC-dependent activation of MAPK(erk1/2) leading to increased expression of cyclooxygenase (COX)-2 and prostaglandin (PG) E(2) synthesis. The addition of PGE(2) to macrophage cultures stimulates their expression of both urokinase-type plasminogen activator and MMP-9, and the selective COX-2 inhibitor NS-398 blocks ECM-induced proteinase expression. Moreover, ECM-induced PGE(2) and MMP-9 expression by elicited COX-2(-/-) macrophages is markedly reduced when compared with the response of either COX-2(+/-) or COX-2(+/+) macrophages. These data clearly demonstrate that SMC-ECM exerts a regulatory role on the degradative phenotype of macrophages via enhanced urokinase-type plasminogen activator and MMP-9 expression, and identify COX-2 as a targetable component of the signaling pathway leading to increased proteinase expression.     

Kupffer cell-derived cyclooxygenase-2 regulates hepatocyte Bcl-2 expression in choledocho-venous fistula rats

We have previously demonstrated that after bile duct ligation hepatocytes express Bcl-2, although the mechanisms regulating Bcl-2 expression were not identified. Our aim was to determine if biliary constituents induce hepatocellular expression of Bcl-2 by a cyclooxygenase-2 (COX-2)-dependent mechanism. We used the choledocho-venous fistula (CVF) rat model for these studies and inhibited COX-2 by feeding the animals nimesulide, a selective inhibitor of COX-2 activity. Serum bile acids were 70-fold greater in CVF animals compared with controls, although liver histology and serum alanine aminotransferase values remained normal for the duration of the study. Neither Bcl-2 nor COX-2 was detected in sham-operated animals. However, Bcl-2 was expressed in hepatocytes but not in other liver cells in the CVF animals. In contrast, COX-2 protein was identified in Kupffer cells but not in hepatocytes of CVF animals. Hepatic Bcl-2 protein expression was fourfold lower in the livers from nimesulide-treated CVF rats. In conclusion, high circulating concentrations of biliary constituents are associated with stimulation of de novo hepatocyte expression of Bcl-2 and Kupffer cell expression of COX-2. These data suggest Kupffer cell-derived prostanoids may regulate Bcl-2 expression in the hepatocyte.     

Angiotensin II bi-directionally regulates cyclooxygenase-2 expression in intestinal epithelial cells

We previously demonstrated that angiotensin II (Ang II) receptor signaling is involved in azoxymethane-induced mouse colon tumorigenesis. In order to clarify the role of Ang II in COX-2 expression in the intestinal epithelium, the receptor subtype-specific effect on COX-2 expression in a rat intestinal epithelial cell line (RIE-1) has been investigated. Ang II dose- and time-dependently increased the expression of COX-2, but not COX-1 mRNA and protein. This stimulation was completely blocked by the AT(1) receptor antagonist but not the AT(2) receptor antagonist. Ang II and lipopolysaccharide (LPS) additively induced COX-2 protein in RIE-1 cells, whereas the LPS-induced COX-2 expression was significantly attenuated by low concentrations of Ang II or the AT(2) agonistic peptide CGP-42112A only in AT(2) over-expressed cells. These data indicate that Ang II bi-directionally regulates COX-2 expression via both AT(1) and AT(2) receptors. Control of COX-2 expression through Ang II signaling may have significance in cytokine-induced COX-2 induction and colon tumorigenesis.     

Prostaglandin E2 EP3 receptor regulates cyclooxygenase-2 expression in the kidney

Cyclooxygenase-2 (COX-2) is constitutively expressed and highly regulated in the thick ascending limb (TAL). As COX-2 inhibitors (Coxibs) increase COX-2 expression, we tested the hypothesis that a negative feedback mechanism involving PGE(2) EP3 receptors regulates COX-2 expression in the TAL. Sprague-Dawley rats were treated with a Coxib [celecoxib (20 mg·kg(-1)·day(-1)) or rofecoxib (10 mg·kg(-1)·day(-1))], with or without sulprostone (20 μg·kg(-1)·day(-1)). Sulprostone was given using two protocols, namely, previous to Coxib treatment (prevention effect; Sulp7-Coxib5 group) and 5 days after initiation of Coxib treatment (regression effect; Coxib10-Sulp5 group). Immunohistochemical and morphometric analysis revealed that the stained area for COX-2-positive TAL cells (μm(2)/field) increased in Coxib-treated rats (Sham: 412 ± 56.3, Coxib: 794 ± 153.3). The Coxib effect was inhibited when sulprostone was used in either the prevention (285 ± 56.9) or regression (345 ± 51.1) protocols. Western blot analysis revealed a 2.1 ± 0.3-fold increase in COX-2 protein expression in the Coxib-treated group, an effect abolished by sulprostone using either the prevention (1.2 ± 0.3-fold) or regression (0.6 ± 0.4-fold vs. control, P < 0.05) protocols. Similarly, the 6.4 ± 0.6-fold increase in COX-2 mRNA abundance induced by Coxibs (P < 0.05) was inhibited by sulprostone; prevention: 0.9 ± 0.3-fold (P < 0.05) and regression: 0.6 ± 0.1 (P < 0.05). Administration of a selective EP3 receptor antagonist, L-798106, also increased the area for COX-2-stained cells, COX-2 mRNA accumulation, and protein expression in the TAL. Collectively, the data suggest that COX-2 levels are regulated by a novel negative feedback loop mediated by PGE(2) acting on its EP3 receptor in the TAL.     

Endothelial cell confluence regulates cyclooxygenase-2 and prostaglandin E2 production that modulate motility

Endothelial cells line the vasculature and, after mechanical denudation during invasive procedures or cellular loss from natural causes, migrate to reestablish a confluent monolayer. We find confluent monolayers of human umbilical vein endothelial cells were quiescent and expressed low levels of cyclooxygenase-2, but expressed cyclooxygenase-2 at levels comparable with cytokine-stimulated cells when present in a subconfluent culture. Mechanically wounding endothelial cell monolayers stimulated rapid cyclooxygenase-2 expression that increased with the level of wounding. Cyclooxygenase-2 re-expression occurred throughout the culture, suggesting signaling from cells proximal to the wound to distal cells. Media from wounded monolayers stimulated cyclooxygenase-2 expression in confluent monolayers, which correlated with the level of wounding of the donor monolayer. Wounded monolayers and cells in subconfluent cultures secreted enhanced levels of prostaglandin (PG) E(2) that depended on cyclooxygenase-2 activity, and PGE(2) stimulated cyclooxygenase-2 expression in confluent endothelial cell monolayers. Cells from subconfluent monolayers migrated through filters more readily than those from confluent monolayers, and the cyclooxygenase-2-selective inhibitor NS-398 suppressed migration. Adding PGE(2) to NS-398-treated cells augmented migration. Endothelial cells also migrated into mechanically denuded areas of confluent monolayers, and this too was suppressed by NS-398. We conclude that endothelial cells not in contact with neighboring cells express cyclooxygenase-2 that results in enhanced release of PGE(2), and that this autocrine and paracrine loop enhances endothelial cell migration to cover denuded areas of the endothelium.     

MiRNA-26b regulates the expression of cyclooxygenase-2 in desferrioxamine-treated CNE cells

Here we report that miR-26b is involved in COX-2 overexpression in desferrioxamine (DFOM)-treated carcinoma of nasopharyngeal epithelial (CNE) cells. The level of miR-26b in DFOM-treated CNE cells is inversely proportional to the expression level of the COX-2 protein. Overexpression of miR-26b in DFOM-treated CNE cells inhibits cell proliferation. A luciferase reporter gene experiment suggests that the 3′ untranslated region of COX-2 carries a binding site for miR-26b. Overexpression of miR-26b marginally reduces the levels of COX-2 protein in DFOM-treated CNE cells. Moreover, knockdown of COX-2 expression had a similar effect to overexpression of miR-26b. Taken together, these results suggest that miR-26b regulates COX-2 expression in DFOM-treated cells.     

Cytosolic phospholipase A2alpha regulates induction of brain cyclooxygenase-2 in a mouse model of inflammation

The products of arachidonic acid metabolism are key mediators of inflammatory responses in the central nervous system, and yet we do not know the mechanisms of their regulation. The phospholipase A(2) enzymes are sources of cellular arachidonic acid, and the enzymes cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) are essential for the synthesis of inflammatory PGE(2) in the brain. These studies seek to determine the function of cytosolic phospholipase A(2)alpha (cPLA(2)alpha) in inflammatory PGE(2) production in the brain. We wondered whether cPLA(2)alpha functions in inflammation to produce arachidonic acid or to modulate levels of COX-2 or mPGES-1. We investigated these questions in the brains of wild-type mice and mice deficient in cPLA(2)alpha (cPLA(2)alpha(-/-)) after systemic administration of LPS. cPLA(2)alpha(-/-) mice had significantly less brain COX-2 mRNA and protein expression in response to LPS than wild-type mice. The reduction in COX-2 was most apparent in the cells of the cerebral blood vessels and the leptomeninges. The brain PGE(2) concentration of untreated cPLA(2)alpha(-/-) mice was equal to their wild-type littermates. After LPS treatment, however, the brain concentration of PGE(2) was significantly less in cPLA(2)alpha(-/-) than in cPLA(2)alpha(+/+) mice (24.4 +/- 3.8 vs. 49.3 +/- 11.6 ng/g). In contrast to COX-2, mPGES-1 RNA levels increased equally in both mouse genotypes, and mPGES-1 protein was unaltered 6 h after LPS. We conclude that cPLA(2)alpha regulates COX-2 levels and modulates inflammatory PGE(2) levels. These results indicate that cPLA(2)alpha inhibition is a novel anti-inflammatory strategy that modulates, but does not completely prevent, eicosanoid responses.     

Cytoskeletal architecture regulates cyclooxygenase-2 in human endothelial cells: Autocrine modulation by prostacyclin

Endothelium is a highly dynamic tissue that controls vascular homeostasis. This requires constant rearrangements of the shape or function of endothelial cells that cannot set aside the role of the cytoskeleton. The aim of this study was to determine the mechanisms by means of which cytoskeletal alterations induce cyclooxygenase‐2 (Cox‐2) expression in human endothelial cells using compounds that interfere with microtubule or actin architecture. Microtubule disruption by nocodazole markedly increased Cox‐2 expression and activity, and provoked paracellular gap formation, a cardinal feature of endothelial barrier dysfunction. The Cox‐2 metabolite prostacyclin down‐regulated Cox‐2 through an autocrine receptor‐mediated mechanism, and partially prevented the disassembly of endothelial monolayers. There was also an interaction between microtubules and actin filaments in nocodazole‐induced Cox‐2 expression. Nocodazole provoked the dissolution of the F‐actin cortical ring and stress fiber formation, increased actin glutathionylation, and concomitantly lowered intracellular levels of reduced glutathione. The restoration of glutathione levels by N‐acetylcysteine opposed Cox‐2 expression and preserved the integrity of endothelial monolayers. Among the signaling pathways connecting microtubule disruption with Cox‐2 up‐regulation, crucial roles are played by Src family kinase activation, serine/threonine phosphatase 2A inhibition, and the phosphorylation of mitogen activated protein kinase p38. Our findings provide a mechanistic insight into the observation that Cox‐2 is induced in endothelial cells under cytoskeleton‐perturbing conditions such as those occurring in the presence of atherogenic/inflammatory stimuli and oxidative stress. In this scenario, Cox‐2 up‐regulation by endothelia exposed to noxious conditions can be considered protective of the vasodilatory and anti‐thrombotic properties of the vessel wall. J. Cell. Physiol. 227: 3847–3856, 2012. © 2012 Wiley Periodicals, Inc.     

Methenyltetrahydrofolate synthetase regulates folate turnover and accumulation

Cellular folate deficiency impairs one-carbon metabolism, resulting in decreased fidelity of DNA synthesis and inhibition of numerous S-adenosylmethionine-dependent methylation reactions including protein and DNA methylation. Cellular folate concentrations are influenced by folate availability, cellular folate transport efficiency, folate polyglutamylation, and folate turnover specifically through degradation. Folate cofactors are highly susceptible to oxidative degradation in vitro with the exception of 5-formyltetrahydrofolate, which may be a storage form of folate. In this study, we determined the effects of depleting cytoplasmic 5-formyltetrahydrofolate on cellular folate concentrations and folate turnover rates in cell cultures by expressing the human methenyltetrahydrofolate synthetase cDNA in human MCF-7 cells and SH-SY5Y neuroblastoma. Cells with increased methenyltetrahydrofolate synthetase activity exhibited: 1) increased rates of folate turnover, 2) elevated generation of p-aminobenzoylglutamate in culture medium, 3) depressed cellular folate concentrations independent of medium folic acid concentrations, and 4) increased average polyglutamate chain lengths of folate cofactors. These data indicate that folate catabolism and folate polyglutamylation are competitive reactions that influence cellular folate concentrations, and that increased methenyltetrahydrofolate synthetase activity accelerates folate turnover rates, depletes cellular folate concentrations, and may account in part for tissue-specific differences in folate accumulation.     

Identification of a serum component that regulates cyclooxygenase-2 gene expression in cooperation with 4-hydroxy-2-nonenal

Atherosclerosis is a disorder of lipid metabolism as well as a chronic inflammatory disease. Cyclooxygenase-2 (COX-2), an inducible isoform responsible for high levels of prostaglandin production during inflammation and immune responses, mediates a variety of biological actions involved in vascular pathophysiology. We have previously shown that COX-2 gene expression is dramatically induced by a lipid-derived endogenous electrophile, 4-hydroxy-2-nonenal (HNE) (Kumagai, T., Matsukawa, N., Kaneko, Y., Kusumi, Y., Mitsumata, M., and Uchida, K. (2004) J. Biol. Chem. 279, 48389-48396). In the present study, based on the finding that HNE induced COX-2 expression only in the serum-containing media, we characterized a serum component essential for the HNE-induced COX-2 induction and found that low density lipoprotein (LDL) that had been denatured by freeze-thawing or oxidized LDL might be involved in the COX-2 induction. Moreover, we characterized the cellular events triggered by the combined stimulus of HNE and oxidized LDL and established that COX-2 induction is regulated by two sets of signaling mechanisms, one for the up-regulation of the scavenger receptor CD36 by HNE and one for the CD36-mediated COX induction by oxidized LDL. These findings represent a demonstration of a link between lipoprotein modification and activation of the inflammatory potential of macrophages.     

Glycosylation regulates specific induction of rice immune responses by Acidovorax avenae flagellin

Plants have a sensitive system that detects various pathogen-derived molecules to protect against infection. Flagellin, a main component of the bacterial flagellum, from the rice avirulent N1141 strain of the Gram-negative phytopathogenic bacterium Acidovorax avenae induces plant immune responses including H₂O₂ generation, whereas flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. To clarify the molecular mechanism that leads to these differing responses between the K1 and N1141 flagellins, recombinant K1 and N1141 flagellins were generated using an Escherichia coli expression system. When cultured rice cells were treated with recombinant K1 or N1141 flagellin, both flagellins equally induced H₂O₂ generation, suggesting that post-translational modifications of the flagellins are involved in the specific induction of immune responses. Mass spectrometry analyses using glycosyltransferase-deficient mutants showed that 1,600- and 2,150-Da glycans were present on the flagellins from N1141 and K1, respectively. A deglycosylated K1 flagellin induced immune responses in the same manner as N1141 flagellin. Site-directed mutagenesis revealed that glycans were attached to four amino acid residues (Ser¹⁷⁸, Ser¹⁸³, Ser²¹², and Thr³⁵¹) in K1 flagellin. Among mutant K1 flagellins in which each glycan-attached amino acid residue was changed to alanine, S178A and S183A, K1 flagellin induced a strong immune response in cultured rice cells, indicating that the glycans at Ser¹⁷⁸ and Ser¹⁸³ in K1 flagellin prevent epitope recognition in rice.