Role of cyclooxygenase-2 in cytokine-induced beta-cell dysfunction and damage by isolated rat and human islets

Type I diabetes mellitus is an autoimmune disease characterized by the selective destruction of the insulin-secreting beta-cell found in pancreatic islets of Langerhans. Cytokines such as interleukin-1 (IL-1), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) mediate beta-cell dysfunction and islet degeneration, in part, through the induction of the inducible isoform of nitric-oxide synthase and the production of nitric oxide by beta-cells. Cytokines also stimulate the expression of the inducible isoform of cyclooxygenase, COX-2, and the production of prostaglandin E(2) (PGE(2)) by rat and human islets; however, the role of increased COX-2 expression and PGE(2) production in mediating cytokine-induced inhibition of islet metabolic function and viability has been incompletely characterized. In this study, we have shown that treatment of rat islets with IL-1beta or human islets with a cytokine mixture containing IL-1beta + IFN-gamma +/- TNF-alpha stimulates COX-2 expression and PGE(2) formation in a time-dependent manner. Co-incubation of rat and human islets with selective COX-2 inhibitors SC-58236 and Celecoxib, respectively, attenuated cytokine-induced PGE(2) formation. However, these inhibitors failed to prevent cytokine-mediated inhibition of insulin secretion or islet degeneration. These findings indicate that selective inhibition of COX-2 activity does not protect rat and human islets from cytokine-induced beta-cell dysfunction and islet degeneration and, furthermore, that islet production of PGE(2) does not mediate these inhibitory and destructive effects.     

12/15-Lipoxygenase signaling in the endoplasmic reticulum stress response

Central obesity is associated with chronic inflammation, insulin resistance, β-cell dysfunction, and endoplasmic reticulum (ER) stress. The 12/15-lipoxygenase enzyme (12/15-LO) promotes inflammation and insulin resistance in adipose and peripheral tissues. Given that obesity is associated with ER stress and 12/15-LO is expressed in adipose tissue, we determined whether 12/15-LO could mediate ER stress signals. Addition of 12/15-LO lipid products 12(S)-HETE and 12(S)-HPETE to differentiated 3T3-L1 adipocytes induced expression and activation of ER stress markers, including BiP, XBP-1, p-PERK, and p-IRE1α. The ER stress inducer, tunicamycin, upregulated ER stress markers in adipocytes with concomitant 12/15-LO activation. Addition of a 12/15-LO inhibitor, CDC, to tunicamycin-treated adipocytes attenuated the ER stress response. Furthermore, 12/15-LO-deficient adipocytes exhibited significantly decreased tunicamycin-induced ER stress. 12/15-LO action involves upregulation of interleukin-12 (IL-12) expression. Tunicamycin significantly upregulated IL-12p40 expression in adipocytes, and IL-12 addition increased ER stress gene expression; conversely, LSF, an IL-12 signaling inhibitor, and an IL-12p40-neutralizing antibody attenuated tunicamycin-induced ER stress. Isolated adipocytes and liver from 12/15-LO-deficient mice fed a high-fat diet revealed a decrease in spliced XBP-1 expression compared with wild-type C57BL/6 mice on a high-fat diet. Furthermore, pancreatic islets from 12/15-LO-deficient mice showed reduced high-fat diet-induced ER stress genes compared with wild-type mice. These data suggest that 12/15-LO activity participates in ER stress in adipocytes, pancreatic islets, and liver. Therefore, reduction of 12/15-LO activity or expression could provide a new therapeutic target to reduce ER stress and downstream inflammation linked to obesity.     

Cyclooxygenase-2, Not Microsomal Prostaglandin E Synthase-1, Is the Mechanism for Interleukin-1β-induced Prostaglandin E2 Production and Inhibition of Insulin Secretion in Pancreatic Islets

Arachidonic acid is converted to prostaglandin E(2) (PGE(2)) by a sequential enzymatic reaction performed by two isoenzyme groups, cyclooxygenases (COX-1 and COX-2) and terminal prostaglandin E synthases (cPGES, mPGES-1, and mPGES-2). mPGES-1 is widely considered to be the final enzyme regulating COX-2-dependent PGE(2) synthesis. These generalizations have been based in most part on experiments utilizing gene expression analyses of cell lines and tumor tissue. To assess the relevance of these generalizations to a native mammalian tissue, we used isolated human and rodent pancreatic islets to examine interleukin (IL)-1β-induced PGE(2) production, because PGE(2) has been shown to mediate IL-1β inhibition of islet function. Rat islets constitutively expressed mRNAs of COX-1, COX-2, cPGES, and mPGES-1. As expected, IL-1β increased mRNA levels for COX-2 and mPGES-1, but not for COX-1 or cPGES. Basal protein levels of COX-1, cPGES, and mPGES-2 were readily detected in whole cell extracts but were not regulated by IL-1β. IL-1β increased protein levels of COX-2, but unexpectedly mPGES-1 protein levels were low and unaffected. In microsomal extracts, mPGES-1 protein was barely detectable in rat islets but clearly present in human islets; however, in neither case did IL-1β increase mPGES-1 protein levels. To further assess the importance of mPGES-1 to IL-1β regulation of an islet physiologic response, glucose-stimulated insulin secretion was examined in isolated islets of WT and mPGES-1-deficient mice. IL-1β inhibited glucose-stimulated insulin secretion equally in both WT and mPGES-1(-/-) islets, indicating that COX-2, not mPGES-1, mediates IL-1β-induced PGE(2) production and subsequent inhibition of insulin secretion.     

Stimulation of progesterone and prostaglandin E2 production by lipoxygenase metabolites of arachidonic acid

The role of several lipoxygenase metabolites of arachidonic acid in the action of luteinizing hormone-releasing hormone (LHRH) on ovarian hormone production was investigated. Like LHRH, treatment of rat granulosa cells with 5-HETE, 5-HPETE, 12-HETE, 15-HETE or 15-HPETE stimulated progesterone (P) and prostaglandin E2 (PGE2) production. 12-HEPE was most potent and stimulated P and PGE2 equally well. By contrast, 5-HETE stimulated P better than PGE2, while 15-HETE was a potent stimulator of PGE2 but not of P. Stimulation of P and PGE2 by LHRH or 12-O-tetradecanoylphorbol 13-acetate (TPA) was further augmented by several HETEs and HPETEs. Like protein kinase C, arachidonic acid metabolites appear to mediate the multiple actions of LHRH in the ovary.     

Signal pathways involved in the regulation of prostaglandin E synthase-1 in human gingival fibroblasts

Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme regulating the synthesis of prostaglandin E2 (PGE2) in inflammatory conditions. In this study we investigated the regulation of mPGES-1 in gingival fibroblasts stimulated with the inflammatory mediators interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNFalpha). The results showed that IL-1beta and TNFalpha induce the expression of mPGES-1 without inducing the expression of early growth response factor-1 (Egr-1). Treatment of the cells with the PLA2 inhibitor 4-bromophenacyl bromide (BPB) decreased the cytokine-induced mPGES-1 expression accompanied by decreased PGE2 production whereas the addition of arachidonic acid (AA) upregulated mPGES-1 expression and PGE2 production. The protein kinase C (PKC) activator PMA did not upregulate the expression of mPGES-1 in contrast to COX-2 expression and PGE2 production. In addition, inhibitors of PKC, tyrosine and p38 MAP kinase markedly decreased the cytokine-induced PGE2 production but not mPGES-1 expression. Moreover, the prostaglandin metabolites PGE2 and PGF2alpha induced mPGES-1 expression as well as upregulated the cytokine-induced mPGES-1 expression indicating positive feedback regulation of mPGES-1 by prostaglandin metabolites. The peroxisome proliferator-activated receptor-gamma (PPARgamma) ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), decreased mPGES-1 expression but not COX-2 expression or PGE2 production. The results indicate that the inflammatory-induced mPGES-1 expression is regulated by PLA2 and 15d-PGJ2 but not by PKC, tyrosine kinase or p38 MAP kinase providing new insights into the regulation of mPGES-1.     

Intracellular degradation of insulin and crinophagy are maintained by nitric oxide and cyclo-oxygenase 2 activity in isolated pancreatic islets

BACKGROUND INFORMATION: Pancreatic beta-cells require an optimal insulin content to allow instantaneous secretion of insulin. This is maintained by insulin biosynthesis and intracellular degradation of insulin. Degradation may be effected by crinophagy, i.e. the fusion of secretory granules with lysosomes. IL-1beta (interleukin 1beta) induces distinct changes of beta-cell lysosomes. To study the mechanisms for intracellular insulin degradation and crinophagy, isolated mouse pancreatic islets were exposed to IL-1beta and known pathways for IL-1beta actions were blocked. Intracellular insulin degradation was determined by following the fate of radioactively labelled insulin. Crinophagy was studied by ultrastructural analysis. The effects of blocking pathways for IL-1beta were monitored by measurements of nitrite and PGE(2) (prostaglandin E(2)). RESULTS: IL-1beta caused an enhancement of islet intracellular insulin degradation and an increase in the lysosomal incorporation of beta-cell secretory granules. The effects of IL-1beta were abolished by aminoguanidine, a selective inhibitor of inducible NOS (nitric oxide synthase), or by rofecoxib, a specific inhibitor of COX-2 (cyclo-oxygenase 2). In the absence of IL-1beta, nitroarginine, which is a selective inhibitor of constitutive NOS, caused a decrease in intracellular degradation of insulin in parallel with a decreased production of NO and PGE(2) by the islets. CONCLUSIONS: The correlation between the enhanced intracellular insulin degradation and lysosomal changes caused by IL-1beta suggests that insulin degradation may be effected by crinophagy. Under physiological conditions, significant beta-cell degradation of insulin may depend on the activity of COX-2, possibly stimulated by endogenous NO.     

Destruction of pancreatic beta-cells by transgenic induction of prostaglandin E2 in the islets

Type 2 diabetes mellitus is characterized by insulin resistance of peripheral tissues and dysfunction of pancreatic beta-cells. Furthermore, the number of pancreatic beta-cells decreases as a secondary effect of advanced type 2 diabetes, although the molecular mechanism has not been elucidated. Recently, it has been shown that hyperglycemic conditions induce the expression of cyclooxygenase-2 in pancreatic islets and increase the downstream product prostaglandin E(2) (PGE(2)). To investigate whether high glucose-induced PGE(2) has an adverse effect on pancreatic beta-cells, we generated transgenic mice (RIP-C2mE) that express cyclooxygenase-2 and microsomal prostaglandin E synthase-1 in their beta-cells using the rat insulin-2 gene promoter (RIP). The homozygous RIP-C2mE (Tg/Tg) mice showed severe hyperglycemia from six weeks of age. Although the heterozygous RIP-C2mE (Tg/-) mice showed normal blood glucose levels throughout their lifetime, this level increased significantly compared with that of wild-type mice when glucose was loaded. The relative number of beta-cells to the total islet cell number was reduced to 54 and 14% in the RIP-C2mE (Tg/-) and (Tg/Tg) mice, respectively, whereas that in the wild-type mice was 84%. Importantly, the proliferation rate in the islets of the RIP-C2mE (Tg/Tg) mice at four weeks of age decreased significantly in comparison to that in the wild-type mice. Because beta-cells replicate not only during the postnatal period but also in the adult pancreas at a basal level, it is possible that increased PGE(2) signaling thus contributes to the reduction of the pancreatic beta-cell mass through inhibition of proliferation, thereby aggravating diabetes further.     

Sensitivity difference to the suppressive effect of prostaglandin E2 among mouse strains: a possible mechanism to polarize Th2 type response in BALB/c mice

PGE2 has been shown to play a prominent role in regulating Th1 and Th2 type responses. We studied the role of PGE2 in IFN-gamma production by Staphylococcus aureus Cowan I-stimulated spleen cells from several mouse strains such as BALB/c, C3H/HeN, and C57BL/6. When spleen cells were pretreated with indomethacin (cyclooxygenase (COX)-1 and COX-2 inhibitor) or NS-398 (COX-2-specific inhibitor), S. aureus Cowan I -induced IFN-gamma production was increased more markedly in spleen cells from BALB/c mice than from C3H/HeN and C57BL/6 mouse. However, PGE2 production was not significantly different among spleen cells from three mouse strains. When various concentrations of PGE2 were exogeneously added to spleen cells, PGE2 showed a stronger suppressive effect on IFN-gamma production in spleen cells from BALB/c mice than from other strains of mice. This suppressive effect of PGE2 in BALB/c mice mainly depended on IL-12p70 production by APCs. More PGE2 binding sites were found in BALB/c spleen cells than in C3H/HeN spleen cells, indicating that the sensitivity difference to the suppressive effect of PGE2 was due to the difference of the number of PGE2 receptors. The administration of NS-398 into BALB/c mice enhanced Ag-specific IFN-gamma production, but not IL-4 production. This effect is the same as IL-12 administration in vivo. From these results, we propose that the modulation of PGE2 is important for Th1 activation via IFN-gamma and IL-12p70 production in vitro and in vivo and that PGE2 is one of the pivotal factors in the Th2-dominant immune response in BALB/c mice.     

12-Lipoxygenase-knockout mice are resistant to inflammatory effects of obesity induced by Western diet

Inflammation is a key pathological process in the progression of atherosclerosis and type 2 diabetes. 12/15-lipoxygenase (12-LO), an enzyme involved in fatty acid metabolism, may contribute to inflammatory damage triggered by stressors such as obesity and insulin resistance. We hypothesized that mice lacking 12-LO are protected against inflammatory-mediated damage associated with a "western" diet. To test this hypothesis, age-matched male 12-LO knockout (12-LOKO) and wild-type C57BL/6 (B6) mice were fed either a standard chow or western diet and assessed for several inflammatory markers. Western-fed B6 mice showed expected reductions in glucose and insulin tolerance compared with chow-fed mice. In contrast, western-fed 12-LOKO mice maintained glucose and insulin tolerance similar to chow-fed mice. Circulating proinflammatory cytokines, tumor necrosis factor-alpha and interleukin-6, were increased in western B6 mice but not 12-LOKO mice, whereas the reported protective adipokine, adiponectin, was decreased only in western B6 mice. 12-LO activity was significantly elevated by western diet in islets from B6 mice. Islets from 12-LOKO mice did not show western-diet-induced islet hyperplasia or increases in caspase-3 apoptotic staining observed in western-fed B6 mice. Islets from 12-LOKO mice were also protected from reduced glucose-stimulated insulin secretion observed in islets from western-fed B6 mice. In visceral fat, macrophage numbers and monocyte chemoattractant protein-1 expression were elevated in western B6 mice but not 12-LOKO mice. These data suggest that 12-LO activation plays a role in western-diet-induced damage in visceral fat and islets. Inhibiting 12-LO may provide a new therapeutic approach to prevent inflammation-mediated metabolic consequences of excess fat intake.     

12-Hydroxyeicosatetraenoic acid participates in angiotensin II afferent arteriolar vasoconstriction by activating L-type calcium channels

The lipoxygenase (LO) metabolite, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], constricts renal vessels, contributes to the vascular response to angiotensin, and has been implicated in cardiovascular and renal diseases. The current studies were performed to determine if renal microvascular 12(S)-HETE production is stimulated by angiotensin and the contribution of L-type calcium channels to the vasoconstriction elicited by 12(S)-HETE. Angiotensin increased renal microvascular 12(S)-HETE production by 64%, whereas cyclooxygenase metabolite production was not altered. Renal microvessels also expressed platelet-type 12-LO and leukocyte-type 12-LO. In the juxtamedullary preparation, afferent arteriolar diameter averaged 21 +/- 1 microm and 12(S)-HETE caused a graded decrease in vessel caliber. The afferent arteriolar response to 12(S)-HETE was abolished during L-type calcium channel inhibition. Renal microvascular smooth muscle cells were studied using fluorescence microscopy. Renal myocyte [Ca2+]i averaged 93 +/- 5 nmol/l. The 12(S)-HETE (5 micromol/l) increased myocyte [Ca2+]i to a peak value of 340 +/- 55 nmol/l. The peak [Ca2+]i response following exposure to 12(S)-HETE was greatly attenuated in the absence of extracellular Ca2+ or calcium channel blockade. These results demonstrate that renal microvascular 12(S)-HETE production is increased in response to angiotensin, and activation of L-type calcium channels is an important mechanism responsible for the afferent arteriolar vasoconstriction elicited by 12(S)-HETE.     

Mechanisms of strain-dependent development of mast cells from mouse splenocytes

Mast cell development from spleen cells was not triggered by prostaglandin E1 (PGE1) or dibutyryl cAMP (db-cAMP) during a 12 day culture when the spleen cells were obtained from C57BL/6N and DBA/1 mice, but mast cells did develop when the spleen cells were obtained from C3H/HeN, BALB/c and ICR mice. A lack of endogenous IFN-gamma in the initial 2 days of the culture period was responsible for the failure. This was confirmed by adding neutralizing anti-IFN-gamma antibody and rIFN-gamma to the cultures and by determining IFN-gamma levels in the spleen cell cultures. Th1 cells in the spleens of C57Bl and DBA/1 mice were much more sensitive to PGE1 and db-cAMP than Th1 cells from other inbred mice strains, and consequently, IFN-gamma production was inhibited in spleen cell cultures of C57BL and DBA/1 mice on addition of PGE1 or db-cAMP. Furthermore, the different sensitivities of Th1 cells to PGE and db-cAMP were dependent on the different levels of IL-12 p40 monomers or homodimers in the spleen cell cultures. As the endogenous specific inhibitors of IL-12 p70 (heterodimers of p40 and p35), large amounts of IL-12 p40 monomers or homodimers in the spleen cell cultures of C57BL and DBA/1 mice enhanced the ability of PGE1 and db-cAMP to inhibit IFN-gamma production by antagonizing the activity of IL-12 heterodimers. These results indicate that the strain-dependent development of mast cells from mouse splenocytes is related to endogenous IFN-gamma levels, which are regulated by PGE, db-cAMP, IL-12 p70 and IL-12 p40.     

TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) regulate CD4+ T cell interferon-gamma (IFN-gamma) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4-/- granuloma cells were stimulated to produce IFN-gamma when cultured with IL-10 or TGF-beta neutralizing monoclonal antibody. In comparison, C57BL/6 wild-type (WT) control granuloma cells produced less IFN-gamma. IL-12, IL-18, and soluble egg antigen stimulated IFN-gamma release from C57BL/6 IL-4-/- and WT mice. IFN-gamma production in C57 IL-4-/- and WT granulomas was IL-12 dependent, because IL-12 blockade partly abrogated IFN-gamma secretion after stimulation. All granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-beta, anti-IL-10, recombinant IL-18, or antigen stimulation. C57 WT and IL-4-/- mouse granuloma cells expressed IL-12 receptor (IL-12R) beta1-subunit mRNA but little beta2 mRNA. TGF-beta or IL-10 blockade did not influence beta1 or beta2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-gamma, produced IL-12 p70 and little p40, and expressed IL-12R beta2 and little beta1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and IL-4-/- mice, cells produce IL-12 (for IFN-gamma production) and IL-10 and TGF-beta modulate IFN-gamma secretion via mechanisms independent of IL-12 and IL-12R mRNA regulation. We found substantial differences in control of granuloma IFN-gamma production and IL-12 circuitry in C57BL/6 and CBA mice.     

Prostaglandin E(2) mediates inhibition of insulin secretion by interleukin-1beta.

Interleukin-1beta (IL-1beta) and prostaglandin E(2) (PGE(2)), frequently co-participants in inflammatory states, are two well recognized inhibitors of glucose-induced insulin secretion. Previous reports have concluded that the inhibitory effects of these two autacoids on pancreatic beta cell function are not related because indomethacin, a potent prostaglandin synthesis inhibitor, does not prevent IL-1beta effects. However, indomethacin is not a specific cyclooxygenase inhibitor, and its other pharmacologic effects are likely to inhibit insulin secretion independently. Since we recently observed that IL-1beta induces cyclooxygenase-2 (COX-2) gene expression and PGE(2) synthesis in islet beta cells, we have reassessed the possibility that PGE(2) mediates IL-1beta effects on beta function. By using two cell lines (HIT-T15 and betaHC13) as well as Wistar rat isolated pancreatic islets, we examined the ability of two COX-2-specific antagonists, NS-398 and SC-236, to prevent IL-1beta inhibition of insulin secretion. Both drugs prevented IL-1beta from inducing PGE(2) synthesis and inhibiting insulin secretion; adding back exogenous PGE(2) re-established inhibition of insulin secretion in the presence of IL-1beta. We also found that EP3, the PGE(2) receptor subtype whose post-receptor effect is to decrease adenylyl cyclase activity and, thereby, insulin secretion, is the dominant mRNA subtype expressed. We conclude that endogenous PGE(2) mediates the inhibitory effects of exogenous IL-1beta on beta cell function.     

Involvement of tyrosine kinases on cyclooxygenase expression and prostaglandin E2 production in human gingival fibroblasts stimulated with interleukin-1beta and epidermal growth factor

The purpose of the present study was to investigate the involvement of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and tyrosine kinase on prostaglandin E2 (PGE2) production in human gingival fibroblasts stimulated by interleukin-1beta (IL-1beta) and/or epidermal growth factor (EGF). The cytokine IL-1beta and to a lesser extent EGF, enhanced COX-2 mRNA levels in gingival fibroblasts. Simultaneous treatment with EGF and IL-1beta resulted in enhanced COX-2 mRNA levels accompanied by a synergistic stimulation of PGE2 biosynthesis compared to the cells treated with IL-1beta or EGF alone. Neither IL-1beta EGF nor the combination of IL-1beta and EGF enhanced COX-1 mRNA levels in gingival fibroblasts. The tyrosine kinase inhibitors, Herbimycin A and PD 153035 hydrochloride, reduced COX-2 mRNA levels as well as PGE2 production induced by IL-1beta or the combination of IL-1beta and EGF whereas COX-1 mRNA levels were not affected. Furthermore, the COX-2 specific inhibitor, NS-398, abolished the PGE2 production induced by IL-1beta, EGF, or the combination. These results indicate that the synergy between IL-1beta and EGF on PGE2 production is due to an enhanced gene expression of COX-2 and that tyrosine kinase(s) are involved in the signal transduction of COX-2 in gingival fibroblasts.     

The shunt from the cyclooxygenase to lipoxygenase pathway in human osteoarthritic subchondral osteoblasts is linked with a variable expression of the 5-lipoxygenase-activating protein

Osteoarthritis (OA) is characterized by articular cartilage degradation and hypertrophic bone changes with osteophyte formation and abnormal bone remodeling. Two groups of OA patients were identified via the production of variable and opposite levels of prostaglandin E2 (PGE2) or leukotriene B4 (LTB4) by subchondral osteoblasts, PGE2 levels discriminating between low and high subgroups. We studied whether the expression of 5-lipoxygenase (5-LO) or 5-LO-activating protein (FLAP) is responsible for the shunt from prostaglandins to leukotrienes. FLAP mRNA levels varied in low and high OA groups compared with normal, whereas mRNA levels of 5-LO were similar in all osteoblasts. Selective inhibition of cyclooxygenase-2 (COX-2) with NS-398-stimulated FLAP expression in the high OA osteoblasts subgroup, whereas it was without effect in the low OA osteoblasts subgroup. The addition of PGE2 to the low OA osteoblasts subgroup decreased FLAP expression but failed to affect it in the high OA osteoblasts subgroup. LTB4 levels in OA osteoblasts were stimulated about twofold by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plus transforming growth factor-beta (TGF-beta), a situation corresponding to their effect on FLAP mRNA levels. Treatments with 1,25(OH)2D3 and TGF-beta also modulated PGE2 production. TGF-beta stimulated PGE2 production in both OA osteoblast groups, whereas 1,25(OH)2D3 alone had a limited effect but decreased the effect of TGF-beta in the low OA osteoblasts subgroup. This modulation of PGE2 production was mirrored by the synthesis of COX-2. IL-18 levels were only slightly increased in a subgroup of OA osteoblasts compared with normal; however, no relationship was observed overall between IL-18 and PGE2 levels in normal and OA osteoblasts. These results suggest that the shunt from the production of PGE2 to LTB4 is through regulation of the expression of FLAP, not 5-LO, in OA osteoblasts. The expression of FLAP in OA osteoblasts is also modulated differently by 1,25(OH)2D3 and TGF-beta depending on their endogenous low and high PGE2 levels.