PGE2 induces IL-6 in orbital fibroblasts through EP2 receptors and increased gene promoter activity: implications to thyroid-associated ophthalmopathy

Background

IL-6 plays an important role in the pathogenesis of Graves'' disease and its orbital component, thyroid-associated ophthalmopathy (TAO). Orbital tissues become inflamed in TAO, a process in which prostanoids have been implicated. Orbital fibroblasts both generate and respond to PGE₂, underlying the inflammatory phenotype of these cells.

Methodology/Principal Findings

Using cultured orbital and dermal fibroblasts, we characterized the effects of PGE₂ on IL-6 expression. We found that the prostanoid provokes substantially greater cytokine synthesis in orbital fibroblasts, effects that are mediated through cell-surface EP₂ receptors and increased steady-state IL-6 mRNA levels. The pre-translational up-regulation of IL-6 results from increased gene promoter activity and can be reproduced with the PKA agonist, Sp-cAMP and blocked by interrupting the PKA pathway. PGE₂-induced production of cAMP in orbital fibroblasts was far greater than that in dermal fibroblasts, resulting from higher levels of adenylate cyclase. PGE₂ provokes CREB phosphorylation, increases the pCREB/CREB ratio, and initiates nuclear localization of the pCREB/CREB binding protein/p300 complex (CBP) preferentially in orbital fibroblasts. Transfection with siRNAs targeting either CREB or CBP blunts the induction of IL-6 gene expression. PGE₂ promotes the binding of pCREB to its target DNA sequence which is substantially greater in orbital fibroblasts.

Conclusion/Significance

These results identify the mechanism underlying the exaggerated induction of IL-6 in orbital fibroblasts and tie together two proinflammatory pathways involved in the pathogenesis of TAO. Moreover, they might therefore define an attractive therapeutic target for the treatment of TAO.     

The role of cyclic-3′,5′-adenosine monophosphate in prostaglandin-mediated inhibition of basic calcium phosphate crystal-induced mitogenesis and collagenase induction in cultured human fibroblasts

Synovial fluid basic calcium phosphate (BCP) crystals are associated with severe destructive arthropathies characterised by synovial proliferation and non-inflammatory degradation of intra-articular collagenous structures. BCP crystals stimulate fibroblast and chondrocyte mitogenesis, metalloprotease secretion and prostaglandin production. As a tissue protective effect of prostaglandins has been suggested, we recently studied the effect of PGE₁ on BCP crystal-induced mitogenesis and collagenase mRNA accumulation in human fibroblasts (HF). We demonstrated a dose-dependent inhibition of BCP crystal-induced mitogenesis and collagenase mRNA accumulation. The mechanism of PGE₁ inhibition of BCP crystal-induced mitogenesis and collagenase mRNA accumulation was therefore explored. PGE₁ (100 ng/ml) increased HF intracellular cAMP 40-fold over control. BCP alone caused no such change but inhibited the PGE₁-induced increase in intracellular cAMP by at least 60%. The PGE₁-induced increase in intracellular cAMP was also blocked by the adenyl cyclase inhibitor, 2′,5′-dideoxyadenosine (ddA) (10 μM) and ddA reversed the PGE₁-mediated inhibition of BCP crystal-induced mitogenesis. Dibutyrul cAMP also inhibited BCP crystal-induced mitogenesis in a concentration-dependent manner. Agents which increase intracellular cAMP levels such as the adenyl cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) mimicked the effect of PGE₁ on HF collagenase mRNA levels. PGE₁ inhibits the biologic effects of BCP crystals through the cAMP signal transduction pathway and such inhibition may have significant therapeutic implications.     

Prostaglandin E2 inhibits the proliferation of human gingival fibroblasts via the EP2 receptor and Epac

Elevated levels of prostaglandins such as PGE₂ in inflamed gingiva play a significant role in the tissue destruction caused by periodontitis, partly by targeting local fibroblasts. Only very few studies have shown that PGE₂ inhibits the proliferation of a gingival fibroblast (GF) cell line, and we expanded this research by using primary human GFs (hGFs) and looking into the mechanisms of the PGE₂ effect. GFs derived from healthy human gingiva were treated with PGE₂ and proliferation was assessed by measuring cell number and DNA synthesis and potential signaling pathways were investigated using selective activators or inhibitors. PGE₂ inhibited the proliferation of hGFs dose‐dependently. The effect was mimicked by forskolin (adenylate cyclase stimulator) and augmented by IBMX (a cAMP‐breakdown inhibitor), pointing to involvement of cAMP. Indeed, PGE₂ and forskolin induced cAMP generation in these cells. Using selective EP receptor agonists we found that the anti‐proliferative effect of PGE₂ is mediated via the EP₂ receptor (which is coupled to adenylate cyclase activation). We also found that the effect of PGE₂ involved activation of Epac (exchange protein directly activated by cAMP), an intracellular cAMP sensor, and not PKA. While serum increased the amount of phospho‐ERK in hGFs by ～300%, PGE₂ decreased it by ～50%. Finally, the PGE₂ effect does not require endogenous production of prostaglandins since it was not abrogated by two COX‐inhibitors. In conclusion, in human gingival fibroblasts PGE₂ activates the EP₂—cAMP—Epac pathway, reducing ERK phosphorylation and inhibiting proliferation. This effect could hamper periodontal healing and provide further insights into the pathogenesis of inflammatory periodontal disease. J. Cell. Biochem. 108: 207–215, 2009. © 2009 Wiley‐Liss, Inc.     

Suppression of prostaglandin synthesis by analogues of cyclic AMP and forskolin in chondrocyte monolayer cultures

Recent experimental studies indicated that prostaglandin E₂ (PGE₂) is the most abundant prostanoid synthesized by rabbit articular chondrocytes. Exogenous PGE₂ stimulates cyclic AMP (cAMP) synthesis in these cells. Analogues of cAMP and forskolin have now been shown to suppress the biosynthesis of PGE₂ in the presence of serum in a time-dependent manner. The most abundant prostanoid, PGE₂ was most markedly affected. PGF_2α was unaffected. These results indicated that intracellular accumulation of cAMP in chondrocytes and relative resistance of cAMP to phosphodiesterases control prostanoid synthesis in a negative feedback loop.     

Role of prostaglandin H synthase-2 in prostaglandin E2 formation in rat carrageenin-induced pleurisy

Rat carrageenin-induced pleurisy was used to clarify the role of prostaglandin H synthase (PGHS)-2 in acute inflammation. Intrapleural injection of 0.2 ml of 2% λ-carrageenin induced accumulation of exudate and infiltration of leukocytes into the pleural cavity. When PGHS-1 and -2 proteins in the pleural exudate cells were analyzed by Western blot analysis, PGHS-2 was detectable from 1 hr after carrageenin injection. Its level rose sharply, remained high from 3 to 7 hr after injection, and then fell to near the detection limit. PGHS-1 was also detected, but kept almost the same level throughout the course of the pleurisy. Levels of prostaglandin (PG) E₂ and thromboxane (TX) B₂ in the exudate increased from hour 3 to hour 7, and then declined. Thus, the changes of the level of PGE₂ were closely paralleled those of PGHS-2.The selective PGHS-2 inhibitors NS-398, nimesulide and SC-58125 suppressed the inflammatory reaction and caused a marked decrease in the level of PGE₂ but not in those of TXB₂ and 6-keto-PGF_1α. These results suggest that the PGHS-2 expressed in the pleural exudate cells may be involved in PGE₂ formation at the site of inflammation.     

THE NEUROPEPTIDE CALCITONIN GENE-RELATED PEPTIDE INHIBITS TNF-α BUT POORLY INDUCES IL-6 PRODUCTION BY FETAL RAT OSTEOBLASTS

Tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6) are potent inflammatory cytokines produced by osteoblasts and whose contribution to bone loss occurring in oestrogen deficiency is well documented. Calcitonin gene-related peptide (CGRP) is a neuropeptide abundantly concentrated in sensory nerve endings innervating bone metaphyses and periosteum suggesting that it controls bone homeostasis locally. Since CGRP was shown to inhibit TNF-α production by T cells and stimulate IL-6 expression by fibroblasts, this study was designed to investigate whether CGRP regulated TNF-α and IL-6 production by osteoblasts. We show that CGRP inhibits the production of TNF-α by both lipopolysaccharide (LPS)- and IL-1-stimulated fetal rat osteoblasts. Like CGRP, the cAMP agonists prostaglandin E₂(PGE₂), dibutyryl cAMP (Bt₂cAMP) and forskolin inhibit TNF-α production by osteoblasts. Exposure of osteoblasts to a high dose of phorbol myristoyl acetate (PMA) to deplete PKC activity abolished CGRP-mediated TNF-α suppression. In contrast with its potent inhibition of TNF-α production, we show that CGRP is a weak inducer of IL-6 when compared to PGE₂, Bt₂cAMP and forskolin. However, in presence of isobutylmethylxanthine (IBMX) CGRP stimulates the production of IL-6. Collectively, these data suggest that the inhibition of TNF-α CGRP is cAMP dependent and PMA sensitive and that the concentration of intracellular cAMP may be a regulatory mechanism for IL-6 expression in osteoblasts.     

Cytokine-mediated PGE2 expression in human colonic fibroblasts

We investigatedprostanoid biogenesis in human colonic fibroblasts (CCD-18Co and 5 primary fibroblast cultures) and epithelial cell lines (NCM460, T84,HT-29, and LS 174T) and the effect of PGE₂ on fibroblast morphology.Cytokine-stimulated PGE₂production was measured. PGH synthase-1 and -2 (PGHS-1 and -2) proteinand mRNA expression were evaluated. BasalPGE₂ levels were low in all celltypes (0.15-6.47 ng/mg protein). Treatment for 24 h with interleukin-1 (IL-1; 10 ng/ml) or tumor necrosis factor- (50 ng/ml), respectively, elicited maximal 25- and 6-fold inductions ofPGE₂ synthesis in CCD-18Cocultures and similar results in primary fibroblast cultures; maximalinductions with IL-1 in colonic epithelial cell lines were from zeroto fivefold. Treatment of CCD-18Co fibroblasts with IL-1 causedmaximal 21- and 53-fold increases, respectively, in PGHS-2 protein andmRNA levels without altering PGHS-1 expression.PGE₂ (0.1 µmol/l) elicited adramatic shape change in selected fibroblasts. Colonic fibroblasts are potentially important as cytokine targets and a source of and targetfor colonic prostanoids in vivo.

     

Regulation of human monocyte matrix metalloproteinases by SPARC

SPARC (secreted protein, acidic and rich in cysteine), also called osteonectin or BM-40, is a collagen-binding glycoprotein secreted by a variety of cells and is associated with functional responses involving tissue remodeling, cell movement and proliferation. Because SPARC and monocytes/macrophages are prevalent at sites of inflammation and remodeling in which there is connective tissue turnover, we examined the effect of SPARC on monocyte matrix metalloproteinase (MMP) production. Treatment of human peripheral blood monocytes with SPARC stimulated the production of gelatinase B (MMP-9) and interstitial collagenase (MMP-1). Experiments with synthetic peptides indicated that peptide 3.2, belonging to the alpha helical domain III of SPARC, is the major peptide mediating the MMP production by monocytes. SPARC and peptide 3.2 were also shown to induce prostaglandin synthase (PGHS)-2 as determined by Western and Northern blot analyses. The increase in PGHS-2 stimulated by SPARC or peptide 3.2 correlated with substantially elevated levels of prostaglandin E₂ (PGE₂) and other arachidonic acid metabolites as measured by radioimmunoassay and high performance liquid chromatography (HPLC), respectively. Moreover, the synthesis of MMP was dependent on the generation of PGE₂ by PGHS-2, since indomethacin inhibited the production of these enzymes and their synthesis was restored by addition of exogenous PGE₂ or dibutyryl cAMP (Bt₂cAMP). These results demonstrate that SPARC might play a significant role in the modulation of connective tissue turnover due to its stimulation of PGHS-2 and the subsequent release of PGE₂, a pathway that leads to the production of MMP by monocytes. J. Cell. Physiol. 173:327–334, 1997. Published 1997 Wiley-Liss, Inc.

1 This article was prepared by a group of United States government employees and non-United States government employees, and as such is subject to 17 U.S.C. Sec. 105.

     

Prostaglandin E2 production and induction of prostaglandin endoperoxide synthase-2 is inhibited in a murine macrophage-like cell line,RAW 264.7, by Mallotus japonicus phloroglucinol derivatives

An aqueous acetone extract obtained from the pericarps of Mallotus japonicus (MJE) was observed to inhibit prostaglandin (PG) E₂ production in a lipopolysaccharide (LPS)-activated murine macrophage-like cell line, RAW 264.7. Six phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against PGE₂ production. Among these phloroglucinol derivatives, isomallotochromanol showed the strongest inhibitory activity, with an IC₅₀ of 1.0 μM. MJE and its phloroglucinol derivatives did not effect the enzyme activity of either prostaglandin endoperoxide synthase (PGHS)-1 or PGHS-2. However, induction of PGHS-2 in LPS-activated macrophages was inhibited by MJE and its phloroglucinol derivatives, whereas the level of PGHS-1 protein was not affected. Moreover, RT-PCR analysis showed that MJE and its phloroglucinol derivatives significantly suppressed PGHS-2 mRNA expression. Therefore, the observed inhibition of PGHS-2 induction by MJE and its phloroglucinol derivatives was likely due to a suppression of PGHS-2 mRNA expression. These results suggest that MJE and its phloroglucinol derivatives have the pharmacological ability to suppress PGE₂ production by activated macrophages.     

Role of prostaglandin H synthase-2-mediated conversion of arachidonic acid in controlling 3T6 fibroblast growth

The specific role(s) of arachidonic acid (AA) and itsmetabolites in the signaling pathways that regulated fibroblast growth was studied. A Western blot analysis demonstrated that prostaglandin Hsynthase-2 (PGHS-2) was expressed by 3T6 fibroblast cultures in RPMI1640 supplemented with fetal calf serum (10%). Dexamethasone, whichinhibits AA release and PGHS-2 expression, significantly reduced cellproliferation. Ketoprofen, a dual cyclooxygenase inhibitor, andCGP-28238, a specific PGHS-2 inhibitor, reduced fibroblastproliferation in a dose-dependent manner. These drugs also reduced[³H]thymidineincorporation into the DNA of fibroblasts. These effects werecorrelated with a decrease in prostaglandin (PG)E₂ levels in the cell medium.However, piroxicam at doses that selectively inhibit PGHS-1 did nothave a significant effect on fibroblast proliferation. Finally, weshowed that the antiproliferative effect of dexamethasone and PGHS-2inhibitors was significantly antagonized whenPGE₂ was added to the culturemedium. Our results suggest that PGHS-2 and prostaglandins such asPGE₂ might play an important rolein the regulation of 3T6 fibroblast growth stimulated by growth factorsof serum.

     

Interleukin-4 inhibits lipopolysaccharide-induced expression of prostaglandin H synthase-2 in human alveolar macrophages

Several studies have shown that interleukin-4 (IL-4) down-regulates synthesis of prostaglandin E₂ (PGE₂). We evaluated the mechanisms for this suppression in human alveolar macrophages (HAMs). Normal HAMs were obtained from healthy nonsmoking volunteers. The cells either remained unstimulated, or were exposed to 10 μg/ml of lipopolysaccharide (LPS) and/or various amounts of IL-4. LPS alone induced the synthesis of large amounts of PGE₂ and prostaglandin H synthase-2 (PGHS-2) protein. This effect of LPS was suppressed by increasing amounts of IL-4. Expression of LPS-induced PGHS-2 mRNA was also inhibited by IL-4. In addition, IL-4 inhibited expression of CD14, which is a receptor for LPS bound to the LPS-binding protein (LBP). We conclude that IL-4 down-regulates LPS-induced release of PGE₂, by reducing expression of the enzyme, PGHS-2. One potential mechanism for this effect of IL-4 is a reduced expression of CD14, which is the LPS-LBP receptor. © 1995 Wiley-Liss Inc.     

Effect of Prostaglandins on Hepatic Cyclic Nucleotide Concentration,Carbohydrate and Lipid Metabolism

The effects of exogenous prostaglandin E₁ (PGE₁) or prostaglandin E₂ (PGE₂) were studied in the isolated perfused rat liver and in the intact canine liver in order to determine the possible physiological role of prostaglandins on hepatic carbohydrate and lipid metabolism. The data indicate that PGE₁ and PGE₂ did not stimulate cyclic AMP (cAMP) and cyclic GMP (cGMP) concentrations in intact dog liver and PGE₁ failed to stimulate cAMP or cGMP in fed or fasted perfused rat liver. PGE₁ did not promote hyperglycemia, glycogenolysis, lipolysis, or prevent epinephrine-induced hyperglycemia in the isolated perfused rat liver. Other known glycogenolytic agents including glucagon and epinephrine increased cAMP and glycogenolysis in the same perfusion system. This study does not support a physiologic role for PGE₁ on hepatic glycogenolysis or lipolysis. If PGE₁ subsequently is found to influence other metabolic parameters such as lipogenesis, gluconeogenesis, ureogenesis or amino acid transport in isolated perfused liver, such alterations would probably occur independent of changes in cyclic nucleotide activity.     

Direct Melanoma Cell Contact Induces Stromal Cell Autocrine Prostaglandin E2-EP4 Receptor Signaling That Drives Tumor Growth,Angiogenesis, and Metastasis

The stromal cells associated with tumors such as melanoma are significant determinants of tumor growth and metastasis. Using membrane-bound prostaglandin E synthase 1 (mPges1^−/−) mice, we show that prostaglandin E₂ (PGE₂) production by host tissues is critical for B16 melanoma growth, angiogenesis, and metastasis to both bone and soft tissues. Concomitant studies in vitro showed that PGE₂ production by fibroblasts is regulated by direct interaction with B16 cells. Autocrine activity of PGE₂ further regulates the production of angiogenic factors by fibroblasts, which are key to the vascularization of both primary and metastatic tumor growth. Similarly, cell-cell interactions between B16 cells and host osteoblasts modulate mPGES-1 activity and PGE₂ production by the osteoblasts. PGE₂, in turn, acts to stimulate receptor activator of NF-κB ligand expression, leading to osteoclast differentiation and bone erosion. Using eicosanoid receptor antagonists, we show that PGE₂ acts on osteoblasts and fibroblasts in the tumor microenvironment through the EP4 receptor. Metastatic tumor growth and vascularization in soft tissues was abrogated by an EP4 receptor antagonist. EP4-null Ptger4^−/− mice do not support B16 melanoma growth. In vitro, an EP4 receptor antagonist modulated PGE₂ effects on fibroblast production of angiogenic factors. Our data show that B16 melanoma cells directly influence host stromal cells to generate PGE₂ signals governing neoangiogenesis and metastatic growth in bone via osteoclast erosive activity as well as angiogenesis in soft tissue tumors.     

Uncoordinate regulation of collagenase,stromelysin, and tissue inhibitor of metalloproteinases genes by prostaglandin E2: Selective enhancement of collagenase gene expression in human dermal fibroblasts in culture

The degradative effects of interleukin-1 (IL-1) on the extracellular matrix of connective tissue are mediated primarily by metalloproteinases and prostaglandins. Clinical observations suggest that these effects can be prevented, to some extent, by the use of non-steroidal anti-inflammatory drugs. We have examined the role of prostaglandin E₂ (PGE₂) in IL-1-induced gene expression by human skin fibroblasts in culture. Incubation of confluent fibroblast cultures with varying concentrations (0.01–1.0 μg/ml) of PGE₂ led to a dose-dependent elevation of collagenase mRNA steady-state levels, the promoter activity, and the secretion of the protein, whereas relatively little effect was observed on stromelysin and TIMP gene expression. Exogenous PGE₂ had no additive or synergistic effect with IL-1 on collagenase gene expression. Furthermore, commonly used non-steroidal anti-inflammatory drugs (indomethacin, acetyl salicylic acid and ibuprofen), at doses which block prostaglandin synthesis in cultured fibroblasts, failed to counteract IL-1-induced collagenase and stromelysin gene expression, nor did they affect TIMP expression. Although the effects of PGE₂ did not potentiate those of IL-1 on collagenase gene expression in vitro, one could speculate that massive production of PGE₂ by connective tissue cells in vivo in response to inflammatory mediators such as IL-1 or tumor necrosis factor-α, could lead to sustained expression of collagenase in connective tissue cells after clearance of the growth factors.     

The effect of PGE2 on the activation of quiescent lung fibroblasts

The effect of prostaglandin E₂ (PGE₂) on fibroblast proliferation was examined. The presence of PGE₂ for 24 h inhibited the growth of quiescent cells stimulated with serum, platelet-derived growth factor and macrophage-derived factors. Maximal inhibition of nuclear labeling with [³H]thymidine occurred at concentrations greater than 10⁻⁷ M. The inhibitory effect of PGE₂ was less potent in exponentially growing cells and was not the result of conversion of PGE₂ to PGA₂ during incubation in growth medium. The G₁ phase was determined to be 12–14 h in untreated cultures. The extent of growth inhibition by PGE₂ was similar with addition of PGE₂ at 0, 3, 6, or 9 h following restimulation of quiescent cell cultures. Approximately 25% of the cells that enter S phase are refractory to PGE₂-induced growth inhibition. Short-term exposure to PGE₂ (5 min and 30 min) caused substantial growth inhibition. The serum-induced proliferation was also inhibited by the cAMP analogue, dibutyrl cAMP. Our results suggest that PGE₂ affects a distinct subpopulation of cells. Restimulation of quiescent cells treated with PGE₂ for 24 h, indicated that release from PGE₂ exposure is associated with prolongation of the G₁ phase of the cell cycle.     

β‐Adrenergic‐induced CD40 overexpression on gingival fibroblasts: Role of PGE2

CD40, a member of the tumour necrosis factor‐α receptor family, is constitutively expressed by cells of haematopoietic and non‐haematopoietic origin, including fibroblasts. Signalling through this receptor molecule regulates inflammatory mediator secretion by many cell types. The work has been performed in healthy subjects and the authors studied, by cellular culture, flow cytometric analysis and ELISA assay, the expression of CD40 and PGE₂ (prostaglandin E₂) generation on gingival fibroblasts stimulated by β‐AR (β‐adrenoceptor) agonists. Herein, the authors demonstrate that β‐AR subtype activation via their own specific agonists markedly increased CD40 expression on human gingival fibroblasts. This effect was prevented by β‐AR subtype‐specific antagonists. In addition, gingival fibroblast β‐AR stimulation resulted in an increase in PGE₂ generation. The inhibition of PLA₂ (phospholipase A₂) and COX‐1 (cyclo‐oxygenase‐1) but not COX‐2 impaired β‐AR increase of PGE₂, an effect that was restored by the addition of low concentrations of PGE₂, suggesting that PGE₂ generation is implicated in the mechanism underlying β‐AR‐agonist‐mediated CD40 overexpression. Our work has revealed an endogenous β‐AR mediator network involving gingival fibroblasts.     

Role of prostaglandins in fibroblast activation and fibrosis

Fibroblasts release prostaglandins and express a range of prostanoid receptors. However the importance of prostaglandins in fibroblast biology have not been fully explored. Our studies showed that the prostaglandin metabolite PGI₂ blocks the activation of fibroblasts, antagonising the induction of Ras/MEK/ERK signalling by TGFβ. Endogenous PGI₂ acts so as to limit the activation of fibroblasts following tissue injury. By contrast PGE₂ induced in injured tissues or disease states may promote recruitment of inflammatory cells and lead to secondary activation of fibroblasts. The effects of PGI₂ on cell signaling could be manipulated to inhibit fibrosis in patients.