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.     

Diallyl disulfide inhibits proliferation and transdifferentiation of lung fibroblasts through induction of cyclooxygenase and synthesis of prostaglandin E2

Platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) are critically involved in idiopathic pulmonary fibrosis by inducing the proliferation and transdifferentiation of lung fibroblasts. In the present study, we examined the impact of diallyl disulfide (DADS), a garlic-derived compound, on such pathological conditions. DADS showed profound inhibitory effects on the PDGF-BB-induced proliferation of human and mouse lung fibroblasts. DADS also abrogated the TGF-β1-induced expression of α-smooth muscle actin, type I collagen and fibronectin. Following treatment with DADS, the expression of cyclooxygenase-2 (COX-2) and the synthesis of prostaglandin E₂ (PGE₂) were found to be markedly enhanced, which in turn led to elevated cAMP levels in lung fibroblasts. Notably, the effect of DADS was largely abolished in the presence of either COX inhibitor indomethacin or siRNA-targeting COX-2, or in the absence of the PGE₂ receptor EP2, supporting an essential role for the COX-2–PGE₂–cAMP autocrine loop. Furthermore, we demonstrated that the upregulated expression of COX-2 was a result of increased level of histone 3 acetylation at COX-2 locus in DADS-treated cells. Together, these results suggest that DADS, by inducing COX-2 expression, may have therapeutic potential in treating lung fibrosis.     

Aggravation of Alzheimer's disease due to the COX‐2‐mediated reciprocal regulation of IL‐1β and Aβ between glial and neuron cells

Alzheimer's disease (AD) is the most common form of dementia and displays the characteristics of chronic neurodegenerative disorders; amyloid plaques (AP) that contain amyloid β‐protein (Aβ) accumulate in AD, which is also characterized by tau phosphorylation. Epidemiological evidence has demonstrated that long‐term treatment with nonsteroidal anti‐inflammatory drugs (NSAIDs) markedly reduces the risk of AD by inhibiting the expression of cyclooxygenase 2 (COX‐2). Although the levels of COX‐2 and its metabolic product prostaglandin (PG)E₂ are elevated in the brain of AD patients, the mechanisms for the development of AD remain unknown. Using human‐ or mouse‐derived glioblastoma and neuroblastoma cell lines as model systems, we delineated the signaling pathways by which COX‐2 mediates the reciprocal regulation of interleukin‐1β (IL‐1β) and Aβ between glial and neuron cells. In glioblastoma cells, COX‐2 regulates the synthesis of IL‐1β in a PGE₂‐dependent manner. Moreover, COX‐2‐derived PGE₂ signals the activation of the PI3‐K/AKT and PKA/CREB pathways via cyclic AMP; these pathways transactivate the NF‐κB p65 subunit via phosphorylation at Ser 536 and Ser 276, leading to IL‐1β synthesis. The secretion of IL‐1β from glioblastoma cells in turn stimulates the expression of COX‐2 in human or mouse neuroblastoma cells. Similar regulatory mechanisms were found for the COX‐2 regulation of BACE‐1 expression in neuroblastoma cells. More importantly, Aβ deposition mediated the inflammatory response of glial cells via inducing the expression of COX‐2 in glioblastoma cells. These findings not only provide new insights into the mechanisms of COX‐2‐induced AD but also initially define the therapeutic targets of AD.     

Cooperation of Epac1/Rap1/Akt and PKA in prostaglandin E(2) -induced proliferation of human umbilical cord blood derived mesenchymal stem cells: Involvement of c-Myc and VEGF expression

Prostaglandin E₂ (PGE₂) is well known to regulate cell functions through cAMP; however, the role of exchange protein directly activated by cAMP (Epac1) and protein kinase A (PKA) in modulating such functions is unknown in human umbilical cord blood‐derived mesenchymal stem cells (hUCB‐MSCs). Therefore, we investigated the relationship between Epac1 and PKA during PGE₂‐induced hUCB‐MSC proliferation and its related signaling pathways. PGE₂ increased cell proliferation, and E‐type prostaglandin (EP) 2 receptor mRNA expression level and activated cAMP generation, which were blocked by EP2 receptor selective antagonist AH 6809. PGE₂ increased Epac1 expression, Ras‐related protein 1 (Rap1) activation level, and Akt phosphorylation, which were inhibited by AH 6809, adenylyl cyclase inhibitor SQ 22536, and Epac1/Rap1‐specific siRNA. Also, PGE₂ increased PKA activity, which was inhibited by AH 6809, SQ 22536, and PKA inhibitor PKI. HUCB‐MSCs were incubated with the Epac agonist 8‐pCPT‐cAMP or the PKA agonist 6‐phe‐cAMP to examine whether Epac1/Rap1/Akt activation was independent of PKA activation. 8‐pCPT‐cAMP increased Akt phosphorylation but not PKA activity. 6‐Phe‐cAMP increased PKA activity, but not Akt phosphorylation. Additionally, an Akt inhibitor or PKA inhibitor (PKI) did not block the PGE₂‐induced increase in PKA activity or Akt phosphorylation, respectively. Moreover, PGE₂ increased glycogen synthase kinase (GSK)‐3β phosphorylation and nuclear translocation of active‐β‐catenin, which were inhibited by Akt inhibitor or/and PKI. PGE₂ increased c‐Myc and vascular endothelial growth factor (VEGF) expression levels, which were blocked by β‐catenin siRNA. In conclusion, PGE₂ stimulated hUCB‐MSC proliferation through β‐catenin‐mediated c‐Myc and VEGF expression via Epac/Rap1/Akt and PKA cooperation. J. Cell. Physiol. 227: 3756–3767, 2012. © 2012 Wiley Periodicals, Inc.     

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.     

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.     

Epigallocatechin‐3‐O‐gallate up‐regulates microRNA‐199a‐3p expression by down‐regulating the expression of cyclooxygenase‐2 in stimulated human osteoarthritis chondrocytes

Osteoarthritis (OA) is a most common form of arthritis worldwide leading to significant disability. MicroRNAs (miRNAs) are non‐coding RNAs involved in various aspects of cartilage development, homoeostasis and pathology. Several miRNAs have been identified which have shown to regulate expression of target genes relevant to OA pathogenesis such as matrix metalloproteinase (MMP)‐13, cyclooxygenase (COX)‐2, etc. Epigallocatechin‐3‐O‐gallate (EGCG), the most abundant and active polyphenol in green tea, has been reported to have anti‐arthritic effects, however, the role of EGCG in the regulation of miRNAs has not been investigated in OA. Here, we showed that EGCG inhibits COX‐2 mRNA/protein expression or prostaglandin E₂ (PGE₂) production via up‐regulating microRNA hsa‐miR‐199a‐3p expression in interleukin (IL)‐1β‐stimulated human OA chondrocytes. This negative co‐regulation of hsa‐miR‐199a‐3p and COX‐2 by EGCG was confirmed by transfection of OA chondrocytes with anti‐miR‐199a‐3p. Transfection of OA chondrocytes with anti‐miR‐199a‐3p significantly enhanced COX‐2 expression and PGE₂ production (P < 0.001), while EGCG treatment significantly inhibited anti‐miR‐199a‐3p transfection‐induced COX‐2 expression or PGE₂ production in a dose‐dependent manner. These results were further re‐validated by co‐treatment of these transfection OA chondrocytes with IL‐1β and EGCG. EGCG treatment consistently up‐regulated the IL‐1β‐decreased hsa‐miR‐199a‐3p expression (P < 0.05) and significantly inhibited the IL‐1β‐induced COX‐2 expression/PGE₂ production (P < 0.05) in OA chondrocytes transfected with anti‐hsa‐miR‐199a‐3p. Taken together, these results clearly indicate that EGCG inhibits COX‐2 expression/PGE₂ production via up‐regulation of hsa‐miR‐199a‐3p expression. These novel pharmacological actions of EGCG on IL‐1β‐stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG‐derived compounds inhibit cartilage breakdown or pain by up‐regulating the expression of microRNAs in human chondrocytes.     

The differential impact of PDE4 subtypes in human lung fibroblasts on cytokine-induced proliferation and myofibroblast conversion

Lung fibroblast proliferation and differentiation into myofibroblasts are pathological key events during development of lung fibrosis. Cyclic nucleotide signaling is described as a negative modulator of these cellular processes, and cyclic nucleotide degrading type 4 phosphodiesterases (PDE4) are important regulators of these pathways. In this study, we elucidated expression and the role of individual subtypes of PDE4 in primary normal human lung fibroblast (NHLF) in controlling cytokines-induced proliferation and conversion to myofibroblasts by short-interfering RNAs (siRNAs) induced knockdown. We verified the expression of PDE4A, B, and D, while PDE4C was only minor or even not expressed in NHLF. An efficient liposome-mediated transfection method for mRNA silencing and a knockdown of the expressed PDE4 subtypes was achieved in these cells. This knockdown was further validated by PDE4 protein expression analysis and PDE4 activity measurements. Functionally, the knockdown of PDE4A and PDE4B inhibited proliferation induced by the cytokine combination of bFGF and IL-1β, whereas knockdown of PDE4D was ineffective. In contrast, TGF-β induced differentiation into myofibroblasts was affected by knockdown of PDE4B and PDE4D, but not by PDE4A knockdown. In summary, our data allow to assign different PDE4 subtypes to distinct functions of human lung fibroblasts and highlight the predominant role of PDE4B in controlling pathophysiological processes of human lung fibroblasts. This provides a scientific rationale for focused therapeutic targeting of PDE4B to treat respiratory diseases with fibrotic lesions in the lung.     

Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression

Tissue stiffening is a hallmark of fibrotic disorders but has traditionally been regarded as an outcome of fibrosis, not a contributing factor to pathogenesis. In this study, we show that fibrosis induced by bleomycin injury in the murine lung locally increases median tissue stiffness sixfold relative to normal lung parenchyma. Across this pathophysiological stiffness range, cultured lung fibroblasts transition from a surprisingly quiescent state to progressive increases in proliferation and matrix synthesis, accompanied by coordinated decreases in matrix proteolytic gene expression. Increasing matrix stiffness strongly suppresses fibroblast expression of COX-2 (cyclooxygenase-2) and synthesis of prostaglandin E₂ (PGE₂), an autocrine inhibitor of fibrogenesis. Exogenous PGE₂ or an agonist of the prostanoid EP2 receptor completely counteracts the proliferative and matrix synthetic effects caused by increased stiffness. Together, these results demonstrate a dominant role for normal tissue compliance, acting in part through autocrine PGE₂, in maintaining fibroblast quiescence and reveal a feedback relationship between matrix stiffening, COX-2 suppression, and fibroblast activation that promotes and amplifies progressive fibrosis.     

Modulation of mitogenic activity and cellular binding of basic fibroblast growth factor by basic proteins

Polycationic molecules were studied either for their ability to displace the binding of basic fibroblast growth factor (bFGF) to high- and low-affinity membrane interaction sites and/or to modulate bFGF-induced proliferation of fibroblasts. Heparin-binding polypeptides, such as polylysine, protamine, histones, and thrombin-displaced [125I]bFGF bound to bovine brain membrane receptors. The most displacing polypeptides were those with the strongest affinity to heparin. Two of these polypeptides, protamine and polylysine, inhibited (at 5 microM) by more than 90% the mitogenic effect induced by bFGF on Chinese hamster lung fibroblast cells (CCL39). At the same dose, no effect was observed with basic proteins that do not bind to heparin, such as cytochrome C and lysozyme. An interesting observation was that protamine at 1 microM potentiated by 1.5-fold the mitogenic activity of bFGF, while it acted as an inhibitor at higher concentration.     

Interleukin-1β induction of tissue inhibitor of metalloproteinase (TIMP-1) is functionally antagonized by prostaglandin E2 in human synovial fibroblasts

Elevated levels of tissue inhibitor of metalloproteases-1 (TIMP-1) have been demonstrated in inflamed synovial membranes, and it is believed that the inhibitor may play a critical role in the regulation of connective tissue degradation. The present study was undertaken to define the cellular mechanism of action of the inflammatory mediators, interleukin-1β (IL-1β) and prostaglandin E₂ (PGE₂), in the control of TIMP-1 synthesis and expression in human synovial fibroblasts. Recombinant human IL-1β induced a time- and dose-dependent saturable response in terms of TIMP-1 mRNA expression (effective concentration for 50% maximal response, EC₅₀ = 31.5 ± 3.3 pg/ml) and protein synthesis (EC₅₀ = 30 ± 3.3 pg/ml). The protein kinase C (PKC) inhibitors, H-7, staurosporine, and calphostin C, reversed the rhIL-1β induction of TIMP-1 mRNA. PGE₂ also inhibited rhIL-1β-stimulated TIMP-1 mRNA expression and protein secretion in a dose-dependent fashion. The concentration of PGE₂ necessary to block 50% of rhIL-1β-stimulated TIMP-1 secretion, IC₅₀, was 1.93 ng/ml (4.89 nM). Forskolin, and other stable derivatives of cAMP, mimicked, to a large extent, the effects of PGE₂. The phorbol ester, PMA, up-regulated considerably the mRNA expression of TIMP-1 but had no effect on protein production. Calphostin C substantially reduced PMA-activated TIMP-1 expression. Staurosporine, calphostin C, H-7, and substances that elevate cellular levels of cAMP, like PGE₂, also reduced basal expression and synthesis of TIMP-1. Taken together, the data suggest that PKA and C may mediate opposing effects in terms of TIMP-1 expression and secretion in human synovial fibroblasts.     

The action of the mast cell product tryptase on cyclooxygenase-2 (COX2) and subsequent fibroblast proliferation involves activation of the extracellular signal-regulated kinase isoforms 1 and 2 (erk1/2)

The mast cell product tryptase, via protease-activated receptor 2 (PAR2), induces cyclooxygenase-2 (COX2) and 15-deoxy-prostaglandin J2 (15d-PGJ2) synthesis. 15d-PGJ2, through the nuclear peroxisome proliferator activated receptor gamma (PPARgamma), subsequently causes fibroblast proliferation. In this study we attempted to determine initial events of the tryptase/PAR2 signaling pathway leading to COX2 induction and fibroblast proliferation. In human fibroblasts (HFFF2), cDNA array, RT-PCR and Western blotting studies demonstrated that tryptase, but not 15d-PGJ2, up-regulates c-jun, c-fos and COX2 expression, and phosphorylates the extracellular signal-regulated kinase isoforms 1 and 2 (erk1/2). Furthermore, tryptase effects on erk1/2, c-jun, c-fos, COX2 and cell proliferation were prevented by PD98059, an inhibitor of the mitogen-activated protein kinase kinase (MEK). Other kinases [P38, stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JUNK), erk5], intracellular Ca(2+) or cAMP were not affected by tryptase/PAR2. Our study identifies crucial intracellular events leading to induction of COX2 and fibroblast proliferation, i.e. a cornerstone of fibrosis.     

Promotion of the prehierarchical follicle growth by postovulatory follicles involving PGE2–EP2 signaling in chickens

The postovulatory follicle (POF) in birds is an enigmatic structure, the function of which remains largely unknown. Previous studies on chickens have shown that removal of POFs leads to the postponement of oviposition and the disturbance of broody behavior. One suggestion is that POFs may secrete some crucial hormones or cytokines to act on reproductive organs. However, such secretions and their specific target organs remain to be identified. Here, we investigate the putative functions of POFs in promoting the development of prehierarchical follicles in chickens and explore the possible signaling mechanisms controlling these processes. Results show that POFs express steroidogenic acute regulatory protein (STAR), cholesterol side‐chain cleavage enzyme (CYP11A1), cyclooxygenase 1 (COX1), and COX2 in granulosa cells (GCs), and, most notably, that POF1 produces more prostaglandin E2 (PGE₂) or prostaglandin F2α than do the F1 follicle or the other POFs. Using coculture systems, we also found that POF1 or GCs from POF1 (POF1‐GCs) significantly promote the proliferation of theca externa cells of small white follicles (SWFs, one phase of the prehierarchical follicle). Treatment with PGE₂ significantly facilitates theca externa cell proliferation in SWFs. This POF‐stimulating effect on SWF growth was prevented by treatment with indomethacin (COX inhibitor) or TG6‐10‐1 (PGE₂ type 2 receptor [EP2] antagonist). Therefore, POF1 may secrete PGE₂ to stimulate the progression of SWF by PGE₂–EP2 signaling. These results indicate that POF1 may serve as a transient supplementary endocrine gland in the chicken ovary that stimulates the development of the prehierarchical follicles through PGE₂–EP2 signaling.     

Arachidonic acid metabolism in growth control of A549 human lung adenocarcinoma cells

The role of individual eicosanoids of the arachidonic acid (AA) cascade in the growth control of A549 human lung adenocarcinoma cells has been studied. Cyclooxygenase and lipoxygenase metabolites of [¹⁴C]AA incorporated were actively synthesized in the cultures of tumor cells with full confluence unaccomplished. In such cultures inhibitors of AA metabolism (indomethacin and esculetin) and also a lipoxygenase metabolite of AA, 15-hydroxyeicosatetraenoic acid (15-HETE), significantly suppressed the incorporation of [³H]thymidine and biosynthesis of prostaglandin E₂(PGE₂). Other lipoxygenase metabolites of AA (5-HETE and 12-HETE) had no effect on these parameters. The basic fibroblast growth factor (bFGF) had practically no affect on the growth of A549 cells and the PGE₂ production in cultures with 5% fetal calf serum (FCS); however, in the presence of 0.5% FCS this factor significantly increased the number of tumor cells. The growth-stimulating effect of bFGF was completely abolished by a cyclooxygenase inhibitor indomethacin. The data suggest a key role of PGE₂ in the growth control of A549 cells with an active synthesis of cyclooxygenase and lipoxygenase metabolites of AA, its importance in realization of the mitogenic effect of bFGF, and specific features of 15-HETE as a down-regulator of the PGE₂-dependent proliferation.     

Assessment of Rapid Morphological Changes Associated with Elevated cAMP Levels in Human Orbital Fibroblasts

Orbital fibroblasts exhibit a phenotype distinct from that of other types of fibroblasts. Addition of prostaglandin E₂(PGE₂) to culture medium elicits a dramatic change in orbital fibroblast morphology. That response is mediated through the generation of cAMP. Orbital fibroblasts can generate high levels of PGE₂through induction by proinflammatory cytokines of prostaglandin endoperoxide H synthase-2 (PGHS-2). Here we compare the influence on fibroblast morphology of exogenous PGE₂, forskolin, and 8-br-cAMP to that mediated through PGHS-2 induction by a lymphocyte-derived cytokine. Within a few hours, orbital fibroblasts treated with any of these test compounds appear under phase-contrast microscopy to exhibit a stellate morphology. When these changes were assessed quantitatively by electric cell–substrate impedance sensing (ECIS), it became evident that 8-br-cAMP, forskolin, and PGE₂initiated shape changes within 30 min of addition to the culture medium, while effects of the cytokine were first evident after approximately 3.5 h. Dermal fibroblasts failed to respond to any of these compounds with regard to changes in cellular morphology. Analysis of micromotion, manifested as small impedance fluctuations, revealed that orbital fibroblasts treated with 8-br-cAMP exhibit less motion than did untreated cells. These results suggest that orbital fibroblast shape can be altered by several compounds known to alter intracellular cAMP levels. They demonstrate the utility of ECIS in the assessment of very rapid and dynamic cellular events associated with changes in cell morphology.     

TLR4‐dependent induction of vascular adhesion molecule‐1 in rheumatoid arthritis synovial fibroblasts: Roles of cytosolic phospholipase A2α/cyclooxygenase‐2

Lipopolysaccharide (LPS)/Toll‐like receptor 4 (TLR4)‐mediated signaling pathways have caught the attention of strategies designed for rheumatoid arthritis (RA). In this study, we identified that cPLA₂α acted as a modulator of LPS‐induced VCAM‐1 expression and THP‐1 (human acute monocytic leukemia cell line) adherence. Treatment of RA synovial fibroblasts (RASFs) with LPS, a TLR4 agonist, promoted the VCAM‐1 expression and THP‐1 adherence which were decreased by pretreatment with a selective cytosolic phospholipase A₂ (cPLA₂) inhibitor (AACOCF₃), implying the involvement of cPLA₂α in these responses. This notion was further confirmed by knockdown of cPLA₂α expression by transfection with cPLA₂α small interfering RNA (siRNA) leading to a decrease in VCAM‐1 expression and THP‐1 adherence induced by LPS. Subsequently, the LPS‐stimulated cPLA₂α phosphorylation was attenuated by pretreatment with a MEK1/2 inhibitor (U0126), suggesting that LPS‐stimulated cPLA₂α phosphorylation and activity are mediated through an ERK‐dependent mechanism. Moreover, COX‐2‐derived PGE₂ production appeared to involve in LPS‐induced VCAM‐1 expression which was attenuated by pretreatment with selective COX‐2 inhibitors (NS‐398 and celecoxib), transfection with COX‐2 siRNA, or PGE₂ receptor antagonists. In addition, pretreatment with ecosapentaenoic acid (EPA), a substrate competitor of arachidonic acid (AA), also blocked LPS‐induced VCAM‐1 mRNA and protein expression, and THP‐1 adherence. Collectively, these results suggest that LPS‐induced VCAM‐1 expression and adhesion of THP‐1 cells are mediated through the TLR4/ERK/cPLA₂α phosphorylation and COX‐2 expression/PGE₂ synthesis in RASFs. J. Cell. Physiol. 223: 480–491, 2010. © 2010 Wiley‐Liss, Inc.     

Prostaglandin E Receptor Subtypes in Cultured Rat Microglia and Their Role in Reducing Lipopolysaccharide-Induced Interleukin-1 β Production

Abstract : Prostaglandins (PGs) are potent modulators of brain function under normal and pathological conditions. The diverse effects of PGs are due to the various actions of specific receptor subtypes for these prostanoids. Recent work has shown that PGE₂, while generally considered a proinflammatory molecule, reduces microglial activation and thus has an antiinflammatory effect on these cells. To gain further insight to the mechanisms by which PGE₂ influences the activation of microglia, we investigated PGE receptor subtype, i.e., EP1, EP2, EP3, and EP4, expression and function in cultured rat microglia. RT-PCR showed the presence of the EP1 and EP2 but not EP3 and EP4 receptor subtypes. Sequencing confirmed their identity with previously published receptor subtypes. PGE₂ and the EP1 agonist 17-phenyl trinor PGE₂ but not the EP3 agonist sulprostone elicited reversible intracellular [Ca²⁺] increases in microglia as measured by fura-2. PGE₂ and the EP2/EP4-specific agonists 11-deoxy-PGE₁ and 19-hydroxy-PGE₂ but not the EP4-selective agonist 1-hydroxy-PGE₁ induced dose-dependent production of cyclic AMP (cAMP). Interleukin (IL)-1β production, a marker of activated microglia, was also measured following lipopolysaccharide exposure in the presence or absence of the receptor subtype agonists. PGE₂ and the EP2 agonists reduced IL-1β production. IL-1β production was unchanged by EP1, EP3, and EP4 agonists. The adenylyl cyclase activator forskolin and the cAMP analogue dibutyryl cAMP also reduced IL-1β production. Thus, the inhibitory effects of PGE₂ on microglia are mediated by the EP2 receptor subtype, and the signaling mechanism of this effect is likely via cAMP. These results show that the effects of PGE₂ on microglia are receptor subtype-specific. Furthermore, they suggest that specific and selective manipulation of the effects of PGs on microglia and, as a result, brain function may be possible.     

Prostaglandin E(2) inhibits collagen expression and proliferation in patient-derived normal lung fibroblasts via E prostanoid 2 receptor and cAMP signaling

Uncontrolled fibroblast activation is one of the hallmarks of fibrotic lung disease. Prostaglandin E(2) (PGE(2)) has been shown to inhibit fibroblast migration, proliferation, collagen deposition, and myofibroblast differentiation in the lung. Understanding the mechanisms for these effects may provide insight into the pathogenesis of fibrotic lung disease. Previous work has focused on commercially available fibroblast cell lines derived from tissue whose precise origin and histopathology are often unknown. Here, we sought to define the mechanism of PGE(2) inhibition in patient-derived fibroblasts from peripheral lung verified to be histologically normal. Fibroblasts were grown from explants of resected lung, and proliferation and collagen I expression was determined following treatment with PGE(2) or modulators of its receptors and downstream signaling components. PGE(2) inhibited fibroblast proliferation by 33% and collagen I expression by 62%. PGE(2) resulted in a 15-fold increase in intracellular cAMP; other cAMP-elevating agents inhibited collagen I in a manner similar to PGE(2). These effects were reproduced by butaprost, a PGE(2) analog selective for the cAMP-coupled E prostanoid (EP) 2 receptor, but not by selective EP3 or EP4 agonists. Fibroblasts expressed both major cAMP effectors, protein kinase A (PKA) and exchange protein activated by cAMP-1 (Epac-1), but only a selective PKA agonist was able to appreciably inhibit collagen I expression. Treatment with okadaic acid, a phosphatase inhibitor, potentiated the effects of PGE(2). Our data indicate that PGE(2) inhibits fibroblast activation in primary lung fibroblasts via binding of EP2 receptor and production of cAMP; inhibition of collagen I proceeds via activation of PKA.