Methotrexate remains a cornerstone in the treatment of rheumatoid arthritis and other rheumatic diseases. Folate antagonism is known to contribute to the antiproliferative effects that are important in the action of methotrexate against malignant diseases, but concomitant administration of folic or folinic acid does not diminish the anti-inflammatory potential of this agent, which suggests that other mechanisms of action might be operative. Although no single mechanism is sufficient to account for all the anti-inflammatory activities of methotrexate, the release of adenosine from cells has been demonstrated both in vitro and in vivo. Methotrexate might also confer anti-inflammatory properties through the inhibition of polyamines. The biological effects on inflammation associated with adenosine release have provided insight into how methotrexate exerts its effects against inflammatory diseases and at the same time causes some of its well-known adverse effects. These activities contribute to the complex and multifaceted mechanisms that make methotrexate efficacious in the treatment of inflammatory disorders.

Adenosine is a potent endogenous regulator of inflammation and tissue repair. Adenosine, which is released from injured and hypoxic tissue or in response to toxins and medications, may induce pulmonary fibrosis in mice, presumably via interaction with a specific adenosine receptor. We therefore determined whether adenosine and its receptors contribute to the pathogenesis of hepatic fibrosis. As in other tissues and cell types, adenosine is released in vitro in response to the fibrogenic stimuli ethanol (40 mg dl−1) and methotrexate (100 nM). Adenosine A2A receptors are expressed on rat and human hepatic stellate cell lines and adenosine A2A receptor occupancy promotes collagen production by these cells. Liver sections from mice treated with the hepatotoxins carbon tetrachloride (CCl4) (0.05 ml in oil, 50 : 50 v : v, subcutaneously) and thioacetamide (100 mg kg−1 in PBS, intraperitoneally) released more adenosine than those from untreated mice when cultured ex vivo. Adenosine A2A receptor‐deficient, but not wild‐type or A3 receptor‐deficient, mice are protected from development of hepatic fibrosis following CCl4 or thioacetamide exposure. Similarly, caffeine (50 mg kg−1 day−1, po), a nonselective adenosine receptor antagonist, and ZM241385 (25 mg kg−1 bid), a more selective antagonist of the adenosine A2A receptor, diminished hepatic fibrosis in wild‐type mice exposed to either CCl4 or thioacetamide. These results demonstrate that hepatic adenosine A2A receptors play an active role in the pathogenesis of hepatic fibrosis, and suggest a novel therapeutic target in the treatment and prevention of hepatic cirrhosis. British Journal of Pharmacology (2006) 148, 1144–1155. doi:

.Conclusion. These results demonstrate that adenosine A 2A receptors play an active role in the pathogenesis of dermal fibrosis and suggest a novel therapeutic target in the treatment and prevention of dermal fibrosis in diseases such as scleroderma.Adenosine, a product of ATP catabolism, is released from cells and tissues under conditions of stress or hypoxia and is a potent endogenous physiologic and pharmacologic mediator. Adenosine regulates cellular and organ function via interaction with a family of 4 G protein-coupled receptors, A 1 , A 2A , A 2B , and A 3.

We examined the regulation of matrix metalloproteinase (MMP) production by mitogen-activated protein kinases and cyclooxygenases (COXs) in fibroblast-like synoviocytes (FLSCs). IL-1β and TNF-α stimulated FLSC extracellular signal-regulated kinase (ERK) activation as well as MMP-1 and -13 release. Pharmacologic inhibitors of ERK inhibited MMP-1, but not MMP-13 expression. Whereas millimolar salicylates inhibited both ERK and MMP-1, nonsalicylate COX and selective COX-2 inhibitors enhanced stimulated MMP-1 release. Addition of exogenous PGE1 or PGE2 inhibited MMP-1, reversed the effects of COX inhibitors, and inhibited ERK activation, suggesting that COX-2 activity tonically inhibits MMP-1 production via ERK inhibition by E PGs. Exposure of FLSCs to nonselective COX and selective COX-2 inhibitors in the absence of stimulation resulted in up-regulation of MMP-1 expression in an ERK-dependent manner. Moreover, COX inhibition sufficient to reduce PGE levels increased ERK activity. Our data indicate that: 1) ERK activation mediates MMP-1 but not MMP-13 release from FLSCs, 2) COX-2-derived E PGs inhibit MMP-1 release from FLSCs via inhibition of ERK, and 3) COX inhibitors, by attenuating PGE inhibition of ERK, enhance the release of MMP-1 by FLSC.

Results. MTX increased 27-hydroxylase message and completely blocked NS398-induced down-regulation of 27-hydroxylase (mean ؎ SEM 112.8 ؎ 13.1% for NS398 plus MTX versus 71.1 ؎ 4.3% for NS398 alone; P < 0.01). MTX also negated COX-2 inhibitor-mediated down-regulation of ABCA1. The ability of MTX to reverse inhibitory effects on 27-hydroxylase and ABCA1 was blocked by the adenosine A 2A receptor-specific antagonist ZM241385. MTX also prevented NS398 and IFN␥ from increasing transformation of lipid-laden THP-1 macrophages into foam cells.Conclusion. This study provides evidence supporting the notion of an atheroprotective effect of MTX. Through adenosine A 2A receptor activation, MTX promotes reverse cholesterol transport and limits foam cell formation in THP-1 macrophages. This is the first reported evidence that any commonly used medication can increase expression of antiatherogenic reverse cholesterol transport proteins and can counteract the effects of COX-2 inhibition. Our results suggest that one mechanism by which MTX protects against cardiovascular disease in rheumatoid arthritis patients is through facilitation of cholesterol outflow from cells of the artery wall.Methotrexate (MTX) has a long history of use in the treatment of various immunologic diseases, and has been used to treat rheumatoid arthritis (RA) and psoDr.

Prior studies indicate that adenosine and the adenosine A 2A receptor play a role in hepatic fibrosis by a mechanism that has been proposed to involve direct stimulation of hepatic stellate cells (HSCs). The objective of this study was to determine whether primary hepatic stellate cells produce collagen in response to adenosine (via activation of adenosine A 2A receptors) and to further determine the signaling mechanisms involved in adenosine A 2A receptor-mediated promotion of collagen production. Cultured primary HSCs increase their collagen production after stimulation of the adenosine A 2A receptor in a dose-dependent fashion. Likewise, LX-2 cells, a human HSC line, increases expression of procollagen ␣I and procollagen ␣III mRNA and their translational proteins, collagen type I and type III, in response to pharmacological stimulation of adenosine A 2A receptors. Based on the use of pharmacological inhibitors of signal transduction, adenosine A 2A receptor-mediated stimulation of procollagen ␣I mRNA and collagen type I collagen expression were regulated by signal transduction involving protein kinase A, src, and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (erk), but surprisingly, adenosine A 2A receptor-mediated stimulation of procollagen ␣III mRNA and collagen type III protein expression depend on the activation of p38 mitogen-activated protein kinase (MAPK), findings confirmed by small interfering RNA-mediated knockdown of src, erk1, erk2, and p38 MAPK. These results indicate that adenosine A 2A receptors signal for increased collagen production by multiple signaling pathways. These results provide strong evidence in support of the hypothesis that adenosine receptors promote hepatic fibrosis, at least in part, via direct stimulation of collagen expression and that signaling for collagen production proceeds via multiple pathways.

266 AICAR = aminoimidazolecarboxamidoribonucleotide; Fc = crystallizable fragment (of antibody); IFN = interferon; IL = interleukin; RA = rheumatoid arthritis; Th = T helper (cells); TNF = tumor necrosis factor. Arthritis Research Vol 4 No 4 Chan and Cronstein IntroductionThe demonstration in 1985 that low-dose, intermittent methotrexate is a potent and effective therapy for rheumatoid arthritis (RA) [1] led to a dramatic change in the way that patients with RA are treated. Indeed, methotrexate is no less efficacious than specific anti-tumor-necrosis-factor (anti-TNF) therapy for the relief of symptomatic joint inflammation in early RA, and the difference between methotrexate and etanercept with respect to protection from structural injury in RA is probably not biologically significant [2]. Thus, methotrexate remains the cornerstone of therapy for RA, and understanding the mechanism(s) responsible for the therapeutic efficacy of this agent may lead to the development of new therapies. History and clinical pharmacologyMethotrexate was first developed in the 1940s as a specific antagonist of folic acid. This drug inhibits the proliferation of malignant cells, primarily by inhibiting the de novo synthesis of purines and pyrimidines. Because administration of high doses of reduced folic acid (folinic acid) or even folic acid itself can reverse the antiproliferative effects of methotrexate, it is clear that methotrexate does act as an antifolate agent. Interestingly, although not originally designed as such, methotrexate appears to be a 'pro-drug', i.e. a compound that is converted to the active agent after uptake. Methotrexate is taken up by cells via the reduced folate carrier and then is converted within the cells to polyglutamates [3]. Methotrexate polyglutamates are longlived metabolites that retain some of the antifolate activities of the parent compound, although the potency for inhibition of various folate-dependent enzymes is shifted [3][4][5][6]. Proposed mechanisms of action of methotrexateLow-dose methotrexate was introduced for the treatment of RA because of its presumed antiproliferative properties, although it was unclear how inhibiting proliferation of the lymphocytes thought to be responsible for synovial inflammation in RA for one day a week might lead to effective suppression of disease activity. However, it soon became clear that inhibition of folic acid metabolism could not be completely responsible for the anti-inflammatory effect of methotrexate. During the past 15 years, it has become clear that administration of folic acid in doses of 1-5 mg per day helps to prevent much of the toxicity of methotrexate without interfering with the anti-inflammatory efficacy of the drug, whereas very high doses of folinic acid also prevent methotrexate toxicity but may interfere with its efficacy [7][8][9][10][11][12][13][14][15][16][17][18][19][20]. There are two potential explanations for the Review Molecular action of methotrexate in inflammatory diseases AbstractDespite the recent introduction of biologic...

Activation of adenosine A2A receptor (A2AR) promotes fibrosis and collagen synthesis. However, the underlying mechanism is still unclear, not least because cAMP, its principal effector, has been found to inhibit TGFβ1-induced collagen synthesis. Here, we show that in primary normal human dermal fibroblasts, A2AR stimulation with CGS21680 elicits a modest cAMP increase (150 ± 12% of control; EC50 54.8 nM), which stimulates collagen1 (Col1) and collagen3 (Col3), but maximal cAMP resulting from direct activation of adenylyl cyclase by forskolin (15,689 ± 7038% of control; EC50 360.7 nM) inhibits Col1 and increases Col3. Similar to Col1 expression, fibroblast proliferation increased following physiological cAMP increases by CGS21680 but was inhibited by cAMP increases beyond the physiological range by forskolin. The A2AR-mediated increase of Col1 and Col3 was mediated by AKT, while Col3, but not Col1, expression was dependent on p38 and repressed by ERK. TGFβ1 induced phosphorylation of Smad2/3 and increased Col3 expression, which was prevented by Smad3 depletion. In contrast, CGS21680 did not activate Smad2/3, and Smad2/3 knockdown did not prevent CGS21680-induced Col1 or Col3 increases. Our results indicate that cAMP is a concentration-dependent switch for collagen production via noncanonical, AKT-dependent, Smad2/3-independent signaling. These observations explain the paradoxical effects of cAMP on collagen expression.