Membrane-associated prostaglandin E synthase-1 is upregulated by proinflammatory cytokines in chondrocytes from patients with osteoarthritis

Prostaglandin E synthase (PGES) including isoenzymes of membrane-associated PGES (mPGES)-1, mPGES-2, and cytosolic PGES (cPGES) is the recently identified terminal enzyme of the arachidonic acid cascade. PGES converts prostaglandin (PG)H(2 )to PGE(2 )downstream of cyclooxygenase (COX). We investigated the expression of PGES isoenzyme in articular chondrocytes from patients with osteoarthritis (OA). Chondrocytes were treated with various cytokines and the expression of PGES isoenzyme mRNA was analyzed by the reverse transcription–polymerase chain reaction and Northern blotting, whereas Western blotting was performed for protein expression. The subcellular localization of mPGES-1 was determined by immunofluorescent microscopy. Conversion of arachidonic acid or PGH(2 )to PGE(2 )was measured by enzyme-linked immunosorbent assay. Finally, the expression of mPGES-1 protein in OA articular cartilage was assessed by immunohistochemistry. Expression of mPGES-1 mRNA in chondrocytes was significantly induced by interleukin (IL)-1β or tumor necrosis factor (TNF)-α, whereas other cytokines, such as IL-4, IL-6, IL-8, IL-10, and interferon-γ, had no effect. COX-2 was also induced under the same conditions, although its pattern of expression was different. Expression of cPGES, mPGES-2, and COX-1 mRNA was not affected by IL-1β or TNF-α. The subcellular localization of mPGES-1 and COX-2 almost overlapped in the perinuclear region. In comparison with 6-keto-PGF(1α )and thromboxane B(2), the production of PGE(2 )was greater after chondrocytes were stimulated by IL-1β or TNF-α. Conversion of PGH(2 )to PGE(2 )(PGES activity) was significantly increased in the lysate from IL-1β-stimulated chondrocytes and it was inhibited by MK-886, which has an inhibitory effect on mPGES-1 activity. Chondrocytes in articular cartilage from patients with OA showed positive immunostaining for mPGES-1. These results suggest that mPGES-1 might be important in the pathogenesis of OA. It might also be a potential new target for therapeutic strategies that specifically modulate PGE(2 )synthesis in patients with OA

MicroRNA and 3T3-L1 pre-adipocyte differentiation

MicroRNAs (miRNAs) have been suggested to play important roles in cell proliferation, apoptosis, and differentiation. In this study, we examined miRNA expression profiles during 3T3-L1 pre-adipocyte differentiation. We constructed miRNA libraries from pre- and post-differentiated 3T3-L1 cells, and identified the expression of 77 previously reported miRNAs and 3 new miRNAs. Next, we investigated the expression levels of 102 miRNAs, including those identified in the libraries, during adipogenesis by Northern blot analysis. Sixty-five miRNAs were detected and the expression of 21 miRNAs was up- or down-regulated during adipogenesis. Intriguingly, changes in the miRNA expression pattern were observed at day 9, when lipid droplets were visible, but not at days 1, 2, or 5 after the induction of differentiation. Antisense inhibition of the up-regulated miRNAs did not affect 3T3-L1 pre-adipocyte differentiation. Although these miRNAs may be involved in modulating adipocyte function, mild down-modulations of the up-regulated miRNAs do not appear to affect 3T3-L1 pre-adipocyte differentiation

Effect of MK-886 on endogenous prostaglandin E(PGE) production in chondrocytes stimulated with IL-1β (IL-1β)

<p><b>Copyright information:</b></p><p>Taken from "Membrane-associated prostaglandin E synthase-1 is upregulated by proinflammatory cytokines in chondrocytes from patients with osteoarthritis"</p><p>Arthritis Research & Therapy 2004;6(4):R355-R365.</p><p>Published online 8 Jun 2004</p><p>PMCID:PMC464891.</p><p>Copyright © 2004 Kojima et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> Chondrocytes from patients with osteoarthritis were incubated with IL-1β in the presence of MK-886 (1 or 10 μM) for 48 h. The PGEconcentration of the medium was then measured by enzyme-linked immunosorbent assay and calculated as a percentage of the control value (IL-1β alone). Data are means ± SD for triplicate experiments; representative results from three patients are shown. < 0.01, untreated control compared with IL-1β alone; *< 0.01, IL-1β alone compared with IL-1β plus MK-886

Induction of prostaglandin E synthase (PGES) activity in chondrocytes stimulated with IL-1β (IL-1β)

<p><b>Copyright information:</b></p><p>Taken from "Membrane-associated prostaglandin E synthase-1 is upregulated by proinflammatory cytokines in chondrocytes from patients with osteoarthritis"</p><p>Arthritis Research & Therapy 2004;6(4):R355-R365.</p><p>Published online 8 Jun 2004</p><p>PMCID:PMC464891.</p><p>Copyright © 2004 Kojima et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> For estimation of the PGES activity that produces prostaglandin (PG)Efrom PGH, exogenous PGH(2 μg) was added to lysates of chondrocytes that had been incubated with (filled circles) or without (open circles) IL-1β (1 ng/ml) for the indicated durations. The lysates were then incubated at 24°C for 30 s in the presence of reduced glutathione, the reaction was stopped with 100 mM FeCl, and the PGEconcentration was measured by enzyme-linked immunosorbent assay. Spontaneous conversion of PGHto PGEin the absence of cell lysates was subtracted from each result. Data are means ± SD for triplicate cultures; representative results from three patients are shown. *< 0.01 and < 0.05 compared with 0 h. Chondrocytes were incubated with or without IL-1β (1 ng/ml) for 48 h. Then the PGES activity of the cell lysates in the presence or absence of MK-886 (1–100 μM) was measured as described in Materials and methods, and calculated as a percentage of the control value (IL-1β alone). Data are means ± SD for triplicate experiments; representative results from three patients are shown. < 0.01, untreated control compared with IL-1β alone; *< 0.01, IL-1β alone compared with IL-1β plus MK-886

Time course of the expression of prostaglandin E synthase (PGES) and cyclooxygenase (COX) isoenzymes in chondrocytes stimulated with interleukin-1β (IL-1β)

<p><b>Copyright information:</b></p><p>Taken from "Membrane-associated prostaglandin E synthase-1 is upregulated by proinflammatory cytokines in chondrocytes from patients with osteoarthritis"</p><p>Arthritis Research & Therapy 2004;6(4):R355-R365.</p><p>Published online 8 Jun 2004</p><p>PMCID:PMC464891.</p><p>Copyright © 2004 Kojima et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> Chondrocytes from osteoarthritis patients were incubated with IL-1β (1 ng/ml) for the indicated durations. Total RNA from the cells was subjected to Northern blot analysis for membrane-associated PGES-1 (mPGES-1), cytosolic PGES (cPGES), COX-1, COX-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Protein from the cells was subjected to Western blot analysis for mPGES-1, cPGES, COX-1, and COX-2. Representative results from three patients are shown here