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.     

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is up-regulated by flurbiprofen and other non-steroidal anti-inflammatory drugs in human colon cancer HT29 cells

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to inhibit prostaglandin synthetic enzyme, cyclooxygenases (COXs), as well as to exhibit anti-tumor activity although at much higher concentrations. 15-Hydroxyprostaglandin dehyrogenase (15-PGDH), a key prostaglandin catabolic enzyme, was recently shown to be a tumor suppressor. Effects of NSAIDs on 15-PGDH expression were therefore examined. Flurbiprofen and several other NSAIDs were found to induce 15-PGDH expression in human colon cancer HT29 cells. Flurbiprofen, the most active one, was also shown to induce 15-PGDH expression in other types of cancer cells. Induction of 15-PGDH expression appeared to occur at the stage of mRNA as levels of 15-PGDH mRNA were increased by flurbiprofen in HT29 cells. Levels of 15-PGDH were also found to be regulated at the stage of protein turnover. MEK inhibitors, PD98059 and U-0126, which inhibited ERK phosphorylation were shown to elevate 15-PGDH levels very significantly. These inhibitors did not appear to alter 15-PGDH mRNA levels but down-regulate matrix metalloproteinase-9 (MMP-9). This protease was shown to degrade and inactivate 15-PGDH suggesting that elevation of 15-PGDH levels could be due to inhibition of MMP-9 expression by these inhibitors. Similarly, flurbiprofen was also demonstrated to inhibit ERK activation and to down-regulate MMP-9 expression. Furthermore, flurbiprofen was shown to induce the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), an inhibitor of MMP-9. The turnover of 15-PGDH was found to prolong in the presence of flurbiprofen as compared to that in the absence of this drug. Taken together, these results indicate that flurbiprofen up-regulates 15-PGDH by increasing the expression and decreasing the degradation of 15-PGDH in HT29 cells.     

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.     

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.     

The cyclooxygenase inhibitor indomethacin modulates gene expression and represses the extracellular matrix protein laminin gamma1 in human glioblastoma cells

The induction of cyclooxygenase-2 (COX-2) expression is associated with more aggressive gliomas and poor survival. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX activity and have antitumorigenic properties. In this report, our initial aim was to determine if indomethacin would alter gene expression as measured by suppression subtractive hybridization (SSH). Three up-regulated and four down-regulated genes by indomethacin treatment were identified. Laminin gamma1, an extracellular matrix molecule, was the most significantly repressed gene. The repression of laminin gamma1 by indomethacin was confirmed by Northern and Western blot analyses and occurred in a concentration- and time-dependent manner at the protein level. Among several NSAIDs tested, only sulindac sulfide and indomethacin suppressed laminin gamma1 protein expression, and this repression was observed in both COX-expressing and -deficient cell lines, suggesting that laminin gamma1 repression by COX inhibitors was independent of COX. Indomethacin, at a concentration that represses laminin gamma1, inhibited glioblastoma cell invasion that was partially restored with additional human laminin protein containing gamma1 chain. The repression of laminin gamma1 by NSAIDs may be related to attenuation of invasion of brain tumors. These findings are important in understanding the chemopreventive activity of some NSAIDs and could be relevant for designing therapeutic strategies against glioblastoma.     

Identification of dual cyclooxygenase-eicosanoid oxidoreductase inhibitors: NSAIDs that inhibit PG-LX reductase/LTB(4) dehydrogenase.

Eicosanoids play key roles in many physiologic and disease processes, and their regulation by nonsteroidal anti-inflammatory drugs (NSAIDs) is critical to many therapeutic approaches. These autacoids are rapidly inactivated by specific enzymes such as 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and 15-oxoprostaglandin 13-reductase/leukotriene B(4) 12-hydroxydehydrogenase (PGR/LTB(4)DH) that act on main series of eicosanoids (i.e., leukotrienes, prostaglandins), and recently found to act in lipoxin inactivation. Here, a panel of NSAIDs was assessed to determine each compound's ability to inhibit eicosanoid-directed activities of either the recombinant 15-PGDH or the PG-LXR/LTB(4)DH. The recombinant 15-PGDH that acts on both prostaglandin E(2) (PGE(2)) and lipoxin A(4) (LXA(4)) was not significantly inhibited by the NSAIDs tested. In contrast, several of the widely used NSAIDs were potent inhibitors of the PG-LXR/LTB(4)DH that metabolizes 15-oxo-PGE(2), and LTB(4) as well as 15-oxo-LXA(4). Diclofenac and indomethacin each inhibited PG-LXR/LTB(4)DH-catalyzed conversion of 15-oxo-PGE(2) to 13,14-dihydro-15-oxo-PGE(2) by 70 and 95%, respectively. Also, a COX-2 inhibitor, niflumic acid, inhibited the PG-LXR/LTB(4)DH eicosanoid oxidoreductase (EOR) by 80% while other COX-2 inhibitors such as nimesulide and NS-398 did not inhibit this enzyme. These results indicate that certain clinically useful NSAIDs such as diclofenac and indomethacin, in addition to inhibiting cyclooxygenases (1 and 2), also interfere with eicosanoid degradation by blocking PG-LXR/LTB(4)DH (EOR) and are members of a new class of dual cyclooxygenase (COX)-EOR inhibitors. Moreover, they suggest that the impact of NSAIDs on PG-LXR/LTB(4)DH activities as targets in the local tissue regulation of eicosanoid-mediated processes should be taken into account.     

Molecular mechanisms mediating the anti-proliferative effects of Vitamin D in prostate cancer

Calcitriol (1,25-dihydroxyvitamin D(3)) inhibits the growth and stimulates the differentiation of prostate cancer (PCa) cells. The effects of calcitriol are varied, appear to be cell-specific and result in growth arrest and stimulation of apoptosis. Our goal was to define the genes involved in the multiple pathways mediating the anti-proliferative effects of calcitriol in PCa. We used cDNA microarray analysis to identify calcitriol target genes involved in these pathways in both LNCaP human PCa cells and primary prostatic epithelial cells. Interestingly, two of the target genes that we identified play key roles in the metabolism of prostaglandins (PGs), which are known stimulators of PCa cell growth and progression. The expression of the PG synthesizing cyclooxygenase-2 (COX-2) gene was significantly decreased by calcitriol, while that of PG inactivating 15-prostaglandin dehydrogenase gene (15-PGDH) was increased. We postulate that this dual action of calcitriol would reduce the levels of biologically active PGs in PCa cells decreasing their proliferative stimulus and contribute to the growth inhibitory actions of calcitriol. In addition, we propose that calcitriol can be combined with non-steroidal anti-inflammatory drugs that inhibit COX activity, as a potential therapeutic strategy to improve the potency and efficacy of both drugs in the treatment of PCa.     

15-PGDH/15-KETE plays a role in hypoxia-induced pulmonary vascular remodeling through ERK1/2-dependent PAR-2 pathway

We have established that 15-hydroxyeicosatetraenoic acid is an important factor in regulation of pulmonary vascular remodeling (PVR) associated with hypoxia-induced pulmonary hypertension (PH), which is further metabolized by 15-hydroxyprostaglandin dehydrogenase (15-PGDH) to form 15-ketoeicosatetraenoic acid (15-KETE). However, the role of 15-PGDH and 15-KETE on PH has not been identified. The purpose of this study was to investigate whether 15-PGDH/15-KETE pathway regulates hypoxia-induced PVR in PH and to characterize the underlying mechanisms. To accomplish this, Immunohistochemistry, Ultra Performance Liquid Chromatography, Western blot, bromodeoxyuridine incorporation and cell cycle analysis were preformed. Our results showed that the levels of 15-PGDH expression and endogenous 15-KETE were drastically elevated in the lungs of humans with PH and hypoxic PH rats. Hypoxia stimulated pulmonary arterial smooth muscle cell (PASMC) proliferation, which seemed to be due to the increased 15-PGDH/15-KETE. 15-PGDH/15-KETE pathway was also capable of stimulating the cell cycle progression and promoting the cell cycle-related protein expression. Furthermore, 15-KETE-promoted cell cycle progression and proliferation in PASMCs depended on protease-activated receptor 2 (PAR-2). ERK1/2 signaling was likely required for 15-PGDH/15-KETE-induced PAR-2 expression under hypoxia. Our study indicates that 15-PGDH/15-KETE stimulates the cell cycle progression and proliferation of PASMCs involving ERK1/2-mediated PAR-2 expression, and contributes to hypoxia-induced PVR.     

Novel pathways that contribute to the anti-proliferative and chemopreventive activities of calcitriol in prostate cancer

Calcitriol, the hormonally active form of Vitamin D, inhibits the growth and development of many cancers through multiple mechanisms. Our recent research supports the contributory role of several new and diverse pathways that add to the mechanisms already established as playing a role in the actions of calcitriol to inhibit the development and progression of prostate cancer (PCa). Calcitriol increases the expression of insulin-like growth factor binding protein-3 (IGFBP-3), which plays a critical role in the inhibition of PCa cell growth by increasing the expression of the cell cycle inhibitor p21. Calcitriol inhibits the prostaglandin (PG) pathway by three actions: (i) the inhibition of the expression of cyclooxygenase-2 (COX-2), the enzyme that synthesizes PGs, (ii) the induction of the expression of 15-prostaglandin dehydrogenase (15-PGDH), the enzyme that inactivates PGs and (iii) decreasing the expression of EP and FP PG receptors that are essential for PG signaling. Since PGs have been shown to promote carcinogenesis and progression of multiple cancers, the inhibition of the PG pathway may add to the ability of calcitriol to prevent and inhibit PCa development and growth. The combination of calcitriol and non-steroidal anti-inflammatory drugs (NSAIDs) result in a synergistic inhibition of PCa cell growth and offers a potential therapeutic strategy. Mitogen activated protein kinase phosphatase 5 (MKP5) is a member of a family of phosphatases that are negative regulators of MAP kinases. Calcitriol induces MKP5 expression in prostate cells leading to the selective dephosphorylation and inactivation of the stress-activated kinase p38. Since p38 activation is pro-carcinogenic and is a mediator of inflammation, this calcitriol action, especially coupled with the inhibition of the PG pathway, contributes to the chemopreventive activity of calcitriol in PCa. Mullerian Inhibiting Substance (MIS) has been evaluated for its inhibitory effects in cancers of the reproductive tissues and is in development as an anti-cancer drug. Calcitriol induces MIS expression in prostate cells revealing yet another mechanism contributing to the anti-cancer activity of calcitriol in PCa. Thus, we conclude that calcitriol regulates myriad pathways that contribute to the potential chemopreventive and therapeutic utility of calcitriol in PCa.     

Indomethacin inhibits cell growth of medullary thyroid carcinoma by reducing cell cycle progression into S phase

Indomethacin, a non-steroidal anti-inflammatory drug (NSAID), has been reported to inhibit the growth of medullary thyroid carcinoma (MTC) cells in vitro. However, the mechanism of inhibition of MTC cell growth by indomethacin and its potency have yet to be revealed. We examined the effect of indomethacin on three different MTC cell lines (TT cells, DRO 81-1 cells and HRO 85-1 cells) and two non-MTC cells. The mechanism of indomethacin action in MTC cells was investigated by analyzing intracellular prostaglandin level, apoptosis, and cell cycle in TT cells. Indomethacin inhibited cell growth of all three MTC cell lines but not normal thyroid cells or anaplastic thyroid carcinoma cells. Indomethacin at 10 microM or greater showed a dose response inhibition of cell growth. Indomethacin at 25 muM, a putative therapeutic serum indomethacin level, showed potency similar to 100 to 200 nM sunitinib, a receptor tyrosine kinase inhibitor. To examine whether prostaglandin depletion might determine the inhibition of MTC cell growth, we created different prostaglandin E2 (PGE2) levels in TT cells using three different NSAIDs. A profound PGE2 depletion by indomethacin-ester, a potent cyclooxygenase (COX) II inhibitor, showed the least inhibition of cell growth. Indomethacin did not increase apoptosis of TT cells. Indomethacin, but not naproxen or indomethacin-ester, reduced cell cycle progression into S phase; this was unrelated to the degree of PGE2 depletion. The expression of phosphorylated retinoblastoma (pRb) protein that shifts cells from G(1) to S phase was reduced after exposure to indomethacin. In conclusion, indomethacin has specific anti-tumor effect on MTC cells, probably by reducing cell cycle progression into S phase rather than by prostaglandin depletion. Since no drug therapy is currently available for MTC, indomethacin may be one of the therapeutic candidates.     

Indomethacin Inhibits Endothelial Cell Proliferation by Suppressing Cell Cycle Proteins and PRB Phosphorylation: A Key to Its Antiangiogenic Action?

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit angiogenesis in vivo and in vitro, but the mechanism of this action is unclear. Angiogenesis—formation of new capillary vessels—requires endothelial proliferation, migration, and tube formation. It is stimulated by basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The cell cycle is regulated positively by cyclins and negatively by cyclin-dependent kinase inhibitors (CKI) and the retinoblastoma protein (pRb). Since the effects of NSAIDs on cell cycle-regulatory proteins in endothelial cells remain unknown, we examined the effect of indomethacin on bFGF-stimulated endothelial cell proliferation and on cell cycle regulatory proteins in rat primary aortic endothelial cells (RAEC). Indomethacin significantly inhibited basal and bFGF-stimulated endothelial cell proliferation. This inhibition correlated significantly with reduced cyclin D1 and increased p21 protein expression. Furthermore, indomethacin reduced pRb phosphorylation. These findings suggest that indomethacin arrests endothelial cell proliferation essential for angiogenesis by modulating cell cycle protein levels.     

Differential effects of COX inhibitors against beta-amyloid-induced neurotoxicity in human neuroblastoma cells

Retrospective epidemiological studies have suggested that chronic treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) provides some degree of protection from Alzheimer's disease (AD). Although most NSAIDs inhibit the activity of cyclooxygenase (COX), the rate-limiting enzyme in the production of prostanoids from arachidonic acid (AA), the precise mechanism through which NSAIDs act upon AD pathology remains to be elucidated. Classical NSAIDs like indomethacin inhibit both the constitutive COX-1 and the inducible COX-2 enzymes. In the present work, we characterize the protective effect of the indomethacin on the neurotoxicity elicited by amyloid-β protein (Aβ, fragments 25–35 and 1–42) alone or in combination with AA added exogenously as well as its effects on COX-2 expression. We also compared the neuroprotective effects of indomethacin with the selective COX-1, COX-2 and 5-LOX inhibitors, SC-560, NS-398 and NDGA, respectively. Our results show that indomethacin protected from Aβ and AA toxicity in naive and differentiated human neuroblastoma cells with more potency than SC-560 while, NS-398 only protected neurons from AA-mediated toxicity. Present results suggest that Aβ toxicity can be reversed more efficiently by the non-selective COX inhibitor indomethacin suggesting its role in modulating the signal transduction pathway involved in the mechanism of Aβ neurotoxicity.     

Inhibition of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) by cyclooxygenase inhibitors and chemopreventive agents

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes NAD(+)-dependent oxidation of 15(S)-hydroxyl group of prostaglandins and has been considered a key enzyme involved in biological inactivation of prostaglandins. This enzyme is markedly induced by androgens in hormone-sensitive human prostate cancer cells (Tong M., Tai H. H. Biochem Biophys Res Commun 2000; 276: 77-81) and may be involved in tumorigenesis. Inhibition of this enzyme may be of value in anticancer therapy. Non-steroidal anti-inflammatory drugs (NSAIDs) which inhibit cyclooxygenases (COXs) have been shown to be chemopreventive in epidemiological and animal-model studies. However, chemoprevention by these drugs may not be directly related to their inhibition of COXs. Other targets may be also involved in their chemopreventive activity. We have examined a variety of NSAIDs including COX-2 selective inhibitors, peroxisome proliferator-activated receptor (PPAR) gamma agonists and phytophenolic compounds which have been shown to be chemopreventive for their effect on 15-PGDH. It was found that most of these compounds were potent inhibitors of 15-PGDH. Among these compounds, ciglitazone appeared to be the most powerful inhibitor (IC(50)=2.7 microM). Inhibition by ciglitazone was non-competitive with respect to NAD(+) and uncompetitive with respect to PGE(2).     

Structure-function relationship and role of tumor necrosis factor-alpha-converting enzyme in the down-regulation of L-selectin by non-steroidal anti-inflammatory drugs

It has been recently described that some non-steroidal anti-inflammatory drugs (NSAIDs) are able to induce the shedding of L-selectin in neutrophils, an adhesion molecule that plays an essential role in the inflammatory response. We have found that, according to this capability, NSAIDs could be grouped into three categories. A high releaser group (flufenamic, meclofenamic, and mefenamic acids, diclofenac and aceclofenac), a group of moderate releasers (aspirin, indomethacin, nimesulide, flurbiprofen, and ketoprofen), and a non-releaser group (phenylbutazone and the oxicams, piroxicam and meloxicam). Only NSAIDs from the high releaser group shared diphenylamine in their chemical structure. The amine group of this chemical agent proved to be essential for the anti-L-selectin activity of diphenylamine-based NSAIDs. The presence of a carboxylic acid group in the diphenylamine (N-phenylanthranilic acid) highly increased its ability to reduce the L-selectin surface expression in neutrophils. Diphenylamine and N-phenylanthranilic acid neither affected COX activity in platelets nor modified the activation state of neutrophils. Diphenylamine-related compounds, which include the diphenylamine-based NSAIDs caused a variable reduction in the neutrophil intracellular ATP concentration, which correlated with the differential ability of such compounds to trigger L-selectin shedding (r = 0.97, p < 0.01). Diphenylamine-related compounds failed to down-regulate L-selectin in a tumor necrosis factor-alpha-converting enzyme (TACE)-deficient murine monocytic cell line. Our data indicate that diphenylamine seems to be the structural core of NSAIDs accounting for their down-regulatory activity of L-selectin leukocyte expression. Diphenylamine and its related compounds exert this action on L-selectin through a prostaglandin-independent, TACE-dependent mechanism that seems to be linked to the capability of these agents to uncouple the mitochondrial oxidative phosphorylation.     

Expression of the cDNA for NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase as a catalytically active enzyme in Escherichia coli.

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme involved in the catabolism of the prostaglandins. The cDNA for human placental 15-PGDH has been expressed in Escherichia coli as a catalytically active protein. The polymerase chain reaction was used to introduce restriction endonuclease sites at each end of the 15-PGDH coding sequence. The 15-PGDH DNA was then inserted into the bacterial expression plasmids pUC-18 and pUC-19 which contain the isopropyl-l-thio-beta-D-galactopyranoside (IPTG) inducible lacZ promoter. Extracts from E. coli containing these expression plasmids exhibited 15-PGDH activity which was inducible with (IPTG). Crude extracts from E. coli expressing 15-PGDH activity were found to contain proteins of the predicted sizes in stained SDS-polyacrylamide gels and in Western blots using human placental 15-PGDH antiserum. The specific activity in E. coli extracts was several hundred-fold higher than that seen in extracts from human placenta.     

In vitro antiproliferative activity against human colon cancer cell lines of representative 4-thiazolidinones. Part I

The characterization of two cyclooxygenase isoforms (COX), the rate-limiting enzyme for the synthesis of prostaglandins (PGs) from arachidonic acid, has allowed the development of COX-2 selective inhibitors as non-steroidal anti-inflammatory drugs (NSAIDs) with significant gastric tolerability. However, PGs are also important in cancer pathogenesis. Thus, there is an increasing interest in studying COX-2 inhibitors as potential drugs aimed at the prevention and treatment of cancer, especially colorectal cancer. The purpose of this study was to determine the inhibitory effects of some representative 4-thiazolidinones, already widely investigated as potential NSAIDs, on the growth of five human colon carcinoma cell lines with a different COX-2 expression, and to correlate them with COX-2 inhibitory properties. Our results preliminarily revealed that 2-phenylimino derivative 3 and 2,4-thiazolidindione 4 were the most active compounds. In particular, 3 mainly inhibited the HT29 cell line characterized by a high COX-2 expression, whereas 4 showed antiproliferative properties on all tested cell lines, suggesting molecular targets other than COX-2 inhibition.     

Prostaglandin E2 synthesis in cartilage explants under compression: mPGES-1 is a mechanosensitive gene

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. 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. For both COX and PGES, three isoforms have been described: in cartilage, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. COX-3 (a variant of COX-1) and mPGES-2 have been recently cloned but little is known about their expression and regulation in cartilage, as is also the case for 15-PGDH. We investigated the regulation of the genes encoding COX and PGES isoforms during mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) for 2 to 24 hours. After determination of the amount of PGE₂ released in the media (enzyme immunoassay), mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blotting 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 interleukin 1 receptor antagonist (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, whereas COX-3 and mPGES-2 mRNA expression was not modified. 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. We conclude that, along with COX-2, dynamic compression induces mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.     

Regulation of synthesis and activity of NAD(+)-dependent 15-hydroxy-prostaglandin dehydrogenase (15-PGDH) by dexamethasone and phorbol ester in human erythroleukemia (HEL) cells

Dexamethasone stimulated 15-PGDH activity in HEL cells in a time and concentration dependent manner. Increase in 15-PGDH activity by dexamethasone was found to be accompanied by an increase in enzyme synthesis as revealed by Western blot and [35S]methionine labeling studies. In addition to dexamethasone, other anti-inflammatory steroids also increased 15-PGDH activity in the order of their glucocorticoid activity. Among sex steroids only progesterone increased significantly 15-PGDH activity. 12-0-Tetradecanoylphorbol-13-acetate (TPA) also induced the synthesis of 15-PGDH but inhibited the enzyme activity. It appears that TPA caused a time dependent inactivation of 15-PGDH by a protein kinase C mediated mechanism.