Mechanical stress and prostaglandin E2 synthesis in cartilage

Knee osteoarthritis (OA) results, at least in part, from overloading and inflammation leading to cartilage degradation. Prostaglandin E2 (PGE2) is one of the main catabolic factors involved in OA in which metalloproteinase (MMP) is crucial for cartilage degradation. Its synthesis is the result of cyclooxygenase (COX) and prostaglandin E synthase (PGES) activities whereas NAD+-dependent 15 hydroxy-prostaglandin dehydrogenase (15-PGDH) is the key enzyme implicated in the catabolism of PGE2. Among the isoforms described, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. We investigated the regulation of the COX, PGES and 15-PGDH and MMP-2, MMP-9 and MMP-13 genes by mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) from 2 to 24 h. After determination of the PGE2 release in the media, mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blot respectively. Mechanical compression of cartilage explants significantly increased PGE2 production in a time dependent manner. This was not due to the synthesis of IL-1, since pretreatment with IL1-Ra did not alter the PGE2 synthesis. Interestingly, COX-2 and mPGES-1 mRNA expression significantly increased after 2 hours, in parallel with protein expression. Moreover, we observed a delayed overexpression of 15-PGDH just before the decline of PGE2 synthesis after 18 hours suggesting that PGE2 synthesis could be altered by the induction of 15-PGDH expression. MAPK are involved in signaling, since specific inhibitors partially inhibited COX-2 and mPGES-1 expressions. Lastly, compression induced MMP-2, -9, -13 mRNA expressions in cartilage. We conclude that dynamic compression induces pro-inflammatroy mediators release and matrix degradating enzymes synthesis. Notably, compression increases mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.     

15-Hydroxyprostaglandin-dehydrogenase is involved in anti-proliferative effect of non-steroidal anti-inflammatory drugs COX-1 inhibitors on a human medullary thyroid carcinoma cell line

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit prostaglandin (PG) synthesis enzymes, the cyclooxygenases (COX-1 and 2). It is suggested that these enzymes are not their only targets. We reported that in tumoral TT cell, indomethacin, in vivo and in vitro, decreases proliferation and increases activity of 15-hydroxyprostaglandin-dehydrogenase (15-PGDH), the PG catabolism key enzyme. Here, we show that the COX-1 inhibitors, selective or not, and sulindac sulfone, a non-COX inhibitor, increased 15-PGDH activity and reduced PGE2 levels. This increase was negatively correlated to the decrease in cell proliferation and suggested that 15-PGDH could be implicated in NSAIDs anti-proliferative effect. Indeed, the silencing of 15-PGDH expression by RNA interference using 15-PGDH specific siRNA enhanced TT cell proliferation and abolished the anti-proliferative effect of a representative non-selective inhibitor, ibuprofen. Moreover, a specific inhibitor of 15-PGDH activity, CAY 10397, completely reversed the effect of ibuprofen on proliferation. Consequently our results demonstrate that, at least in TT cells, 15-PGDH is implicated in proliferation and could be a target for COX-1 inhibitors specific or not. NSAIDs defined by their COX inhibition should also be defined by their effect on 15-PGDH.     

Indomethacin increases 15-PGDH mRNA expression in HL60 cells differentiated by PMA

We previously reported an induction of 15-hydroxyprostaglandin dehydrogenase type I mRNA (15-PGDH) expression accompanied by a decrease in prostaglandin E2(PGE2) levels during cord blood monocytes differentiation into preosteoclastic cells by 1,25 dihydroxyvitamin D3 (1,25 (OH)2D3). These results suggested a role of prostaglandin (PG) enzymes in adhesion and/or differentiation of monocytes.In the present work, we studied modulation of gene expression of PG metabolism enzymes mRNAs in HL60 cells differentiated by phorbol myristate acetate (PMA) into the monocyte/macrophage lineage. We showed that adhesion of HL60 induced by PMA causes an increase of cyclooxygenase 2 (COX 2) and 15-PGDH mRNAs. When adding indomethacin, a non steroidal antiinflammatory drug known to inhibit COX activity, the cells remained attached and expressed large amounts of 15-PGDH mRNA while COX 2 mRNA expression remained unchanged. Indomethacin, in association with PMA can consequently exert a dual control on key enzymes of PGE2 metabolism without modifying adhesion of the cells.     

Regulation of epithelial syndecan-1 expression by inflammatory cytokines

Syndecan-1 is expressed on the basolateral surface of columnar epithelium and contributes to wound repair by facilitating increased growth factor binding. Inflammatory bowel disease (IBD) is associated with reduced syndecan-1 expression in areas of inflamed mucosa that is likely to impair mucosal healing. Reduced syndecan-1 expression in IBD may be related to the presence of increased inflammatory cytokines. To test this hypothesis, monolayers of HT29 and T84 colonic epithelial cells were stimulated with tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta or IL-6. Stimulation of HT29 cells with TNF-alpha and IL-1beta resulted in reversible down-regulation of syndecan-1 at both protein and mRNA levels but little effect was observed with IL-6. Loss of syndecan-1 expression was caused by shedding of the ectodomain as revealed by increased levels of soluble syndecan-1 measured in the conditioned medium of stimulated cells. No increase in cytoplasmic staining accompanied the loss of cell surface syndecan-1 expression. TNF-alpha and IL-1beta are capable of down-regulating syndecan-1 expression and may account in part for the reduced expression of syndecan-1 seen in IBD.     

Prostaglandin catabolism in fetal and maternal tissues--a study of 15-hydroxyprostaglandin dehydrogenase and delta 13 reductase with specific assay methods

Recent studies have demonstrated that extraductal tissues such as lung are important sources of prostaglandin E2 which maintains the patency of ductus arteriosus in fetuses and prematurely-born infants. Also, organs such as lung are known to be active in the catabolism of PGE2. Earlier studies of enzymes involved in the catabolism of PGE2 such as 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and delta 13 reductase all used non-specific methods. In the present report, we studied 15-PGDH in fetal and maternal rat lung, kidney, and fetal lamb lung, kidney and ductus arteriosus with the use of a specific substrate (15-S)-[15(3)H-PGE2]. In addition, we measured the activity of delta 13 reductase in these tissues by measuring the conversion of [1-14C]-15-keto PGE2 to [1-14C]-15-keto-13,14-dihydro PGE2. The results from these studies demonstrated that in fetal rat lung and kidney, 15-PGDH activities increased rapidly while delta 13 reductase remained unchanged during late gestation. Ductus arteriosus possessed little 15-PGDH activities. These results strongly suggest that extraductal regulation of PGE2 metabolism is important in determining ductal caliber in fetuses and prematurely delivered neonates.     

白细胞介素23受体、白细胞介素17A在炎症性肠病患者中的表达及临床意义

目的：通过检测白细胞介素23受体（1L-23R）及白细胞介素17A（IL-17A）在炎症性肠病（IBD）患者肠黏膜及血清中的表达水平,探讨其在IBD发病过程中的作用及意义。方法：收集32例克罗恩病（CD）患者、29例溃疡性结肠炎（UC）患者及27例对照者的内镜肠黏膜活检标本,采用荧光定量PCR技术检测肠黏膜内IL-23R、IL-17AmRNA的表达情况,免疫组化技术分析IL-23R、IL-17A在肠黏膜中的原位表达。结果：与健康对照组相比,CD及UC患者肠黏膜组织内IL-23RmRNA表达显著增高（P〈0．05）,CD及UC组间的表达量差异无统计学意义（P〉0．05）。CD及UC患者肠黏膜组织内IL-17AmRNA表达显著增高（P〈0．05）,CD组肠黏膜组织内IL．17AmRNA表达显著高于uc组（P〈0．05）。免疫组化分析显示IL-23R阳性细胞在CD与uc肠黏膜固有层内有较多表达,较正常黏膜内的肠上皮细胞相比,CD及UC患者肠黏膜IL-23R蛋白表达量最著增高（P〈0．05）,UC及CD组间的表达量差异无统计学意义（P〉0．05）。IL-17A阳性细胞在CD与UC肠黏膜固有层内有较多表达,较正常黏膜内的肠上皮细胞相比,CD及UC患者肠黏膜IL-17A蛋白表达量最著增高（P〈0．05）。结论：IL．23R及IL-17A在IBD患者肠黏膜中表达显著增高,提示IL-23R及IL-17A表达异常与IBD的发生发展密切相关,有可能成为IBD治疗的新靶点。     

Spatial heterogeneity of TNF-alpha-induced T cell migration to colonic mucosa is mediated by MAdCAM-1 and VCAM-1

Relatively little is known about how recirculation of lymphocytes through the inflamed intestinal mucosa is regulated. The aim of this study was to investigate the dynamic process of T lymphocyte-endothelial cell adhesion in TNF-alpha-challenged murine colonic mucosa by intravital microscopy. T lymphocytes from spleen (SPL) and intestinal lamina propria (LPL) were fluorescence labeled, and their adhesion to microvessels in the colonic mucosa was observed. In TNF-alpha (25 microg/kg)-stimulated colonic venules, an enhanced adhesion of SPL and LPL was demonstrated, with dominant recruitment of LPLs. The magnitude of the increased LPL adhesion was more significant in the colon than in the small intestine. These T lymphocyte interactions in the colonic mucosa were significantly reduced by blocking MAbs against either mucosal addressin cell adhesion molecule-1 (MAdCAM-1), VCAM-1, alpha(4)-integrin, or beta(7)-integrin but not by anti-ICAM-1. Immunohistochemistry revealed significant MAdCAM-1 expression in the lamina propria and VCAM-1 expression in the submucosa of TNF-alpha-treated colon. Spatial heterogeneity of MAdCAM-1 and VCAM-1 activation following TNF-alpha challenge may promote specific T lymphocyte recruitment in the inflamed colonic mucosa.     

Involvement of Endoplasmic Reticulum Stress in Inflammatory Bowel Disease: A Different Implication for Colonic and Ileal Disease?

Background

Endoplasmic reticulum (ER) stress has been suggested to play a role in inflammatory bowel disease (IBD). The three branches (ATF6, IRE1 and PERK) of the unfolded protein response (UPR) have different roles and are not necessarily activated simultaneously.

Methodology/Principal Findings

Expression of UPR-related genes was investigated in colonic and ileal biopsies of 23 controls, 15 ulcerative colitis (UC) and 54 Crohn''s disease (CD) patients. This expression was confirmed at protein level in colonic and ileal samples of five controls, UC and CD patients. HSPA5, PDIA4 and XBP1s were significantly increased in colonic IBD at mRNA and/or protein levels, indicating activation of the ATF6 and IRE1 branch. Colonic IBD was associated with increased phosphorylation of EIF2A suggesting the activation of the PERK branch, but subsequent induction of GADD34 was not observed. In ileal CD, no differential expression of the UPR-related genes was observed, but our data suggested a higher basal activation of the UPR in the ileal mucosa of controls. This was confirmed by the increased expression of 16 UPR-related genes as 12 of them were significantly more expressed in ileal controls compared to colonic controls. Tunicamycin stimulation of colonic and ileal samples of healthy individuals revealed that although the ileal mucosa is exhibiting this higher basal UPR activation, it is still responsive to ER stress, even more than colonic mucosa.

Conclusions/Significance

Activation of the three UPR-related arms is seen in colonic IBD-associated inflammation. However, despite EIF2A activation, inflamed colonic tissue did not increase GADD34 expression, which is usually involved in re-establishment of ER homeostasis. This study also implies the presence of a constitutive UPR activation in healthy ileal mucosa, with no further activation during inflammation. Therefore, engagement of the UPR differs between colon and ileum and this could be a factor in the development of ileal or colonic disease.     

Involvement of the constitutive prostaglandin E synthase cPGES/p23 in expression of an initial prostaglandin E2 inactivating enzyme, 15-PGDH

We previously showed that cytosolic prostaglandin (PG) E synthase (cPGES/p23) which isomerizes PGH(2) to PGE(2), is essential for fetal mouse development. Embryonic fibroblasts derived from cPGES/p23 knockout mice generated higher amounts of PGE(2) in culture supernatants than wild-type-derived cells. In order to elucidate this apparent conflict that absence of PGE(2) synthetic enzyme caused facilitation of PGE(2) biosynthesis, we examined expression of the PGE(2) degrading enzyme in embryonic fibroblasts. We report here that embryonic fibroblasts deficient in cPGES/p23 decreased the expression of the PGE(2) degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which catalyzes the inactivating conversion of the PGE(2) 15-OH to a 15-keto group, compared with that of wild-type. In addition, rat fibroblastic 3Y1 cells harboring cPGES/p23 siRNA exhibited lower 15-PGDH expression than mock-transfected cells. Furthermore, forcible expression of cPGES/p23 in 3Y1 cells resulted in facilitation of 15-PGDH promoter activity. These results suggest that the PGE(2)-inactivating pathway is controlled by the PGE(2) biosynthetic enzyme, cPGES/p23.     

Increased Expression of 15-Hydroxyprostaglandin Dehydrogenase in Spinal Astrocytes During Disease Progression in a Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive, and fatal neurodegenerative disease caused by selective loss of motor neurons. Both ALS model mice and patients with sporadic ALS have increased levels of prostaglandin E2 (PGE2). Furthermore, the protein levels of microsomal PGE synthase-1 and cyclooxygenase-2, which catalyze PGE2 biosynthesis, are significantly increased in the spinal cord of ALS model mice. However, it is unclear whether PGE2 metabolism in the spinal cord is altered. In the present study, we investigated the protein level of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme in prostaglandin metabolism, in ALS model mice at three different disease stages. Western blotting revealed that the 15-PGDH level was significantly increased in the lumbar spinal cord at the symptomatic stage and end stage. Immunohistochemical staining demonstrated that 15-PGDH immunoreactivity was localized in glial fibrillary acidic protein (GFAP)-positive astrocytes at the end stage. In contrast, 15-PGDH immunoreactivity was not identified in NeuN-positive large cells showing the typical morphology of motor neurons in the anterior horn. Unlike 15-PGDH, the level of PGE2 in the spinal cord was increased only at the end stage. These results suggest that the significant increase of PGE2 at the end stage of ALS in this mouse model is attributable to an imbalance of the synthetic pathway and 15-PGDH-dependent scavenging system for PGE2, and that this drives the pathogenetic mechanism responsible for transition from the symptomatic stage.     

Prostanoid receptors in intestinal epithelium: selective expression, function, and change with inflammation

The tissue concentration of PGE(2)is heightened during mucosal inflammation. Nevertheless, the cellular targets of this prostanoid and its effects on epithelial cell physiology are incompletely understood. We used a panel of specific immunoglobulin and mRNA probes in order to localize and quantitate the four member EP family of prostanoid receptors for binding PGE(2)on cells of histologically normal and inflamed human colonic mucosa, and then examined the physiological consequences for the epithelial component of intestine, with special attention to its barrier function. Prostanoid receptors were selectively expressed on a limited number of human colonic mucosal cells, and differed markedly between normal and inflamed tissue. In non-inflamed mucosa, EP(2)and EP(3)were expressed on epithelia at the apex of crypts; while EP(4)was expressed on surface and lateral crypt epithelia. Dual immunostaining and in situ hybridization with digoxygenin-labelled RNA probes largely confirmed the epithelial localization of EP(4). On the other hand, during inflammation, lateral crypt (non-surface) epithelial cells newly and significantly expressed prostanoid receptors EP(2)and EP(3)(p<0.05, by computer-assisted densitometry). Functionally, exogenous E series prostanoids applied to epithelial monolayers in nM concentrations brought about a 24% increase in the level of barrier function; an associated rise in intracellular cAMP (EC(50)of 281); and protection of epithelium from the effects of T cell cytokines. A major perturbation in the number and distribution of functional eicosonoid receptors on epithelia occurs in chronic inflammation of human colonic mucosa.     

p38 mitogen-activated protein kinase is activated and linked to TNF-alpha signaling in inflammatory bowel disease

Inflammatory bowel diseases (IBD)--Crohn's disease and ulcerative colitis--are relapsing chronic inflammatory disorders which involve genetic, immunological, and environmental factors. The regulation of TNF-alpha, a key mediator in the inflammatory process in IBD, is interconnected with mitogen-activated protein kinase pathways. The aim of this study was to characterize the activity and expression of the four p38 subtypes (p38alpha-delta), c-Jun N-terminal kinases (JNKs), and the extracellular signal-regulated kinases (ERK)1/2 in the inflamed intestinal mucosa. Western blot analysis revealed that p38alpha, JNKs, and ERK1/2 were significantly activated in IBD, with p38alpha showing the most pronounced increase in kinase activity. Protein expression of p38 and JNK was only moderately altered in IBD patients compared with normal controls, whereas ERK1/2 protein was significantly down-regulated. Immunohistochemical analysis of inflamed mucosal biopsies localized the main expression of p38alpha to lamina propria macrophages and neutrophils. ELISA screening of the supernatants of Crohn's disease mucosal biopsy cultures showed that incubation with the p38 inhibitor SB 203580 significantly reduced secretion of TNF-alpha. In vivo inhibition of TNF-alpha by a single infusion of anti-TNF-alpha Ab (infliximab) resulted in a highly significant transient increase of p38alpha activity during the first 48 h after infusion. A significant infliximab-dependent p38alpha activation was also observed in THP-1 myelomonocytic cells. In human monocytes, infliximab enhanced TNF-alpha gene expression, which could be inhibited by SB 203580. In conclusion, p38alpha signaling is involved in the pathophysiology of IBD.     

Limited effect of anti-rheumatic treatment on 15-prostaglandin dehydrogenase in rheumatoid arthritis synovial tissue

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which prostaglandin E₂ (PGE₂) displays an important pathogenic role. The enzymes involved in its synthesis are highly expressed in the inflamed synovium, while little is known about 15- prostaglandin dehydrogenase (15-PGDH) that metabolizes PGE₂. Here we aimed to evaluate the localization of 15-PGDH in the synovial tissue of healthy individuals or patients with inflammatory arthritis and determine the influence of common RA therapy on its expression.

Methods

Synovial tissue specimens from healthy individuals, psoriatic arthritis, ostheoarthritis and RA patients were immunohistochemically stained to describe the expression pattern of 15-PGDH. In addition, the degree of enzyme staining was evaluated by computer analysis on stained synovial biopsies from two groups of RA patients, before and after RA specific treatment with either intra-articular glucocorticoids or oral methotrexate therapy. Prostaglandins derived from the cyclooxygenase (COX) pathway were determined by liquid-chromatography mass spectrometry in supernatants from interleukin (IL) 1β-activated fibroblast-like synoviocytes (FLS) treated with methotrexate.

Results

15-PGDH was present in healthy and inflamed synovial tissue, mainly in lining macrophages, fibroblasts and vessels. Intra-articular glucocorticoids showed a trend towards reduced 15-PGDH expression in RA synovium (p = 0.08) while methotrexate treatment left the PGE₂ pathway unaltered both in biopsies ex vivo and in cultured FLS.

Conclusions

Early methotrexate therapy has little influence on the expression of 15-PGDH and on any of the PGE₂ synthesizing enzymes or COX-derived metabolites. Thus therapeutic strategies involving blocking induced PGE₂ synthesis may find a rationale in additionally reducing local inflammatory mediators.     

Enhanced transferrin receptor expression by proinflammatory cytokines in enterocytes as a means for local delivery of drugs to inflamed gut mucosa

Therapeutic intervention in inflammatory bowel diseases (IBDs) is often associated with adverse effects related to drug distribution into non-diseased tissues, a situation which attracts a rational design of a targeted treatment confined to the inflamed mucosa. Upon activation of immune cells, transferrin receptor (TfR) expression increases at their surface. Because TfR is expressed in all cell types we hypothesized that its cell surface levels are regulated also in enterocytes. We, therefore, compared TfR expression in healthy and inflamed human colonic mucosa, as well as healthy and inflamed colonic mucosa of the DNBS-induced rat model. TfR expression was elevated in the colonic mucosa of IBD patients in both the basolateral and apical membranes of the enterocytes. Increased TfR expression was also observed in colonocytes of the induced colitis rats. To explore the underlying mechanism CaCo-2 cells were treated with various proinflammatory cytokines, which increased both TfR expression and transferrin cellular uptake in a mechanism that did not involve hyper proliferation. These findings were then exploited for the design of targetable carrier towards inflamed regions of the colon. Anti-TfR antibodies were conjugated to nano-liposomes. As expected, iron-starved Caco-2 cells internalized anti-TfR immunoliposomes better than controls. Ex vivo binding studies to inflamed mucosa showed that the anti-TfR immunoliposomes accumulated significantly better in the mucosa of DNBS-induced rats than the accumulation of non-specific immunoliposomes. It is concluded that targeting mucosal inflammation can be accomplished by nano-liposomes decorated with anti-TfR due to inflammation-dependent, apical, elevated expression of the receptor.     

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.     

15-Hydroxyprostaglandin dehydrogenase is down-regulated in colorectal cancer

Prostaglandin E2 (PGE2) can stimulate tumor progression by modulating several proneoplastic pathways, including proliferation, angiogenesis, cell migration, invasion, and apoptosis. Although steady-state tissue levels of PGE2 stem from relative rates of biosynthesis and breakdown, most reports examining PGE2 have focused solely on the cyclooxygenase-dependent formation of this bioactive lipid. Enzymatic degradation of PGE2 involves the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The present study examined a range of normal tissues in the human and mouse and found high levels of 15-PGDH in the large intestine. By contrast, the expression of 15-PGDH is decreased in several colorectal carcinoma cell lines and in other human malignancies such as breast and lung carcinomas. Consistent with these findings, we observe diminished 15-Pgdh expression in ApcMin+/- mouse adenomas. Enzymatic activity of 15-PGDH correlates with expression levels and the genetic disruption of 15-Pgdh completely blocks production of the urinary PGE2 metabolite. Finally, 15-PGDH expression and activity are significantly down-regulated in human colorectal carcinomas relative to matched normal tissue. In summary, these results suggest a novel tumor suppressive role for 15-PGDH due to loss of expression during colorectal tumor progression.