José Cisneros

Identification of a novel endocannabinoid-hydrolyzing enzyme expressed by microglial cells

The endocannabinoids (eCBs) anandamide and 2-arachidonoyl glycerol (2-AG) are inactivated by a two-step mechanism. First, they are carried into cells, and then anandamide is hydrolyzed by fatty acid amide hydrolase (FAAH) and 2-AG by monoacylglycerol lipase (MGL). Here we provide evidence for a previously undescribed MGL activity expressed by microglial cells. We found that the mouse microglial cell line BV-2 does not express MGL mRNA and yet efficiently hydrolyzes 2-AG. URB597 (3′-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) reduces this hydrolysis by 50%, suggesting the involvement of FAAH. The remaining activity is blocked by classic MGL inhibitors [[1,1-biphenyl]-3-yl-carbamic acid, cyclohexyl ester (URB602) and MAFP (methylarachidonyl fluorophosphate)] and is unaffected by inhibitors of COXs (cyclooxygenases), LOXs (lipooxygenases), and DGLs (diacylglycerol lipases), indicating the involvement of a novel MGL activity. Accordingly, URB602 leads to selective accumulation of 2-AG in intact BV-2 cells. Although MGL expressed in neurons is equally distributed between the cytosolic, mitochondrial, and nuclear fractions, the novel MGL activity expressed by BV-2 cells is enriched in mitochondrial and nuclear fractions. A screen for novel inhibitors of eCB hydrolysis identified several compounds that differentially block MGL, FAAH, and the novel MGL activity. Finally, we provide evidence for expression of the novel MGL by mouse primary microglia in culture. Our results suggest the presence of a novel, pharmacologically distinct, MGL activity that controls 2-AG levels in microglia.

The constitutive production of the endocannabinoid 2-arachidonoylglycerol participates in oligodendrocyte differentiation

Endocannabinoids have recently emerged as instructive cues in the developing central nervous system, and, based on the expression of their receptors, we identified oligodendrocytes as potential targets of these molecules. Here, we show that the enzymes responsible for the synthesis of the endocannabinoid 2‐arachidonoylglycerol (2‐AG), diacylglycerol lipase alpha (DAGLα) and beta (DAGLβ), and degradation, monoacylglycerol lipase (MAGL), can be found in oligodendrocytes at different developmental stages. Moreover, cultured oligodendrocyte progenitor cells (OPCs) express DAGLα and β abundantly, resulting in the stronger production of 2‐AG than in differentiated oligodendrocytes. The opposite is observed with MAGL. CB1 and CB2 receptor antagonists (SR141716 and AM630) impaired OPC differentiation into mature oligodendrocytes and likewise, inhibiting DAGL activity with RHC‐80267 or tetrahydrolipstatin also blocked oligodendrocyte maturation, an effect reversed by the addition of exogenous 2‐AG. Likewise, 2‐AG synthesis disruption using specific siRNAs against DAGLα and DAGLβ significantly reduced myelin protein expression in vitro, whereas a pharmacological gain‐of‐function approach by using cannabinoid agonists or MAGL inhibition had the opposite effects. ERK/MAPK pathway is implicated in oligodendrocyte differentiation because PD98059, an inhibitor of MEK1, abrogated oligodendrocyte maturation. The cannabinoid receptor antagonists and RHC‐80267 all diminished basal ERK1/2 phosphorylation, effects that were partially reversed by the addition of 2‐AG. Overall, our data suggest a novel role of endocannabinoids in oligodendrocyte differentiation such that constitutive release of 2‐AG activates cannabinoid receptors in an autocrine/paracrine way in OPCs, stimulating the ERK/MAPK signaling pathway.

Matrix Metalloproteinase (MMP)-1 Induces Lung Alveolar Epithelial Cell Migration and Proliferation, Protects from Apoptosis, and Represses Mitochondrial Oxygen Consumption

Background: In IPF MMP-1 is up-regulated and expressed in alveolar epithelial cells. Result: Transfection of MMP-1 in MLE cells increased proliferation/migration, protected from apoptosis, repressed oxygen consumption ratio and ROS production, and stimulated HIF-1α. Conclusion: MMP-1 inhibits mitochondrial function and contributes to a proliferative/migratory and anti-apoptotic phenotype. Significance: MMP-1 promotes the Warburg effect characterized by increased aerobic glycolysis and HIF-1α during normoxia. Idiopathic pulmonary fibrosis is a devastating lung disorder of unknown etiology. Although its pathogenesis is unclear, considerable evidence supports an important role of aberrantly activated alveolar epithelial cells (AECs), which produce a large variety of mediators, including several matrix metalloproteases (MMPs), which participate in fibroblast activation and lung remodeling. MMP-1 has been shown to be highly expressed in AECs from idiopathic pulmonary fibrosis lungs although its role is unknown. In this study, we explored the role of MMP-1 in several AECs functions. Mouse lung epithelial cells (MLE12) transfected with human Mmp-1 showed significantly increased cell growth and proliferation at 36 and 48 h of culture (p < 0.01). Also, MMP-1 promoted MLE12 cell migration through collagen I, accelerated wound closing, and protected cells from staurosporine- and bleomycin-induced apoptosis compared with mock cells (p < 0.01). MLE12 cells expressing human MMP-1 showed a significant repression of oxygen consumption ratio compared with the cells with the empty vector. As under hypoxic conditions hypoxia-inducible factor-1α (HIF-1α) mediates a transition from oxidative to glycolytic metabolism, we analyzed activation of HIF-1α. Ηigher activation of this factor was detected in MMP-1-transfected cells under normoxia and hypoxia. Likewise, a significant decrease of both total and mitochondrial reactive oxygen species was observed in MMP-1-transfected cells. Paralleling these findings, attenuation of MMP-1 expression by shRNA in A549 (human) AECs markedly reduced proliferation and migration (p < 0.01) and increased the oxygen consumption ratio. These findings indicate that epithelial expression of MMP-1 inhibits mitochondrial function, increases HIF-1α expression, decreases reactive oxygen species production, and contributes to a proliferative, migratory, and anti-apoptotic AEC phenotype.

Dual inhibition of alpha/beta-hydrolase domain 6 and fatty acid amide hydrolase increases endocannabinoid levels in neurons

Agonists at cannabinoid receptors, such as the phytocannabinoid Δ9-tetrahydrocannabinol, exert a remarkable array of therapeutic effects but are also associated with undesirable psychoactive side effects. Conversely, targeting enzymes that hydrolyze endocannabinoids (eCBs) allows for more precise fine-tuning of cannabinoid receptor signaling, thus providing therapeutic relief with reduced side effects. Here, we report the development and characterization of an inhibitor of eCB hydrolysis, UCM710, which augments both N-arachidonoylethanolamine and 2-arachidonoylglycerol levels in neurons. This compound displays a unique pharmacological profile in that it inhibits fatty acid amide hydrolase and α/β-hydrolase domain 6 but not monoacylglycerol lipase. Thus, UCM710 represents a novel tool to delineate the therapeutic potential of compounds that manipulate a subset of enzymes that control eCB signaling.

The Medicinal Chemistry of Agents Targeting Monoacylglycerol Lipase

Monoacylglycerol lipase (MAGL) has been recently proposed as the main enzymatic activity responsible for the in vivo hydrolysis of the most abundant endocannabinoid in the brain, the 2-arachidonoylglycerol (2-AG). The endocannabinoids, mainly anandamide (AEA) and 2-AG, are a class of lipid messengers that modulate a broad number of physiological processes both in the central nervous system and in the periphery. To date, AEA has been by far the most studied endocannabinoid, although increasing evidence is pointing out the prominent, and sometimes underestimated, role of 2-AG in the regulation of different functions. Therefore, it is of outmost importance to dissect the specific cellular pathways in which these two endocannabinoids are involved. Nonetheless, little is known about the structural require-ments of MAGL. Here we review the current knowledge on MAGL, with special focus on its structure and catalytic mechanism as the rational basis for the design of potent and selective compounds able to interact with it; the inhibitors that have been described to date, and the therapeutic applications that make MAGL an attractive therapeutic target.

FEV1 inversely correlates with metalloproteinases 1, 7, 9 and CRP in COPD by biomass smoke exposure

BackgroundMatrix metalloproteinases (MMPs) and C-reactive protein (CRP) are involved in chronic obstructive pulmonary disease (COPD) pathogenesis. The aim of the present work was to determine plasma concentrations of MMPs and CRP in COPD associated to biomass combustion exposure (BE) and tobacco smoking (TS).MethodsPulmonary function tests, plasma levels of MMP-1, MMP-7, MMP-9, MMP-9/TIMP-1 and CRP were measured in COPD associated to BE (n = 40) and TS (n =40) patients, and healthy non-smoking (NS) healthy women (controls, n = 40).ResultsPlasma levels of MMP-1, MMP-7, MMP-9, and MMP-9/TIMP-1 and CRP were higher in BE and TS than in the NS healthy women (p <0.01). An inverse correlation between MMP-1, MMP-7, MMP-9, MMP-9/TIMP-1 and CRP plasma concentrations and FEV1 was observed.ConclusionsIncrease of MMPs and CRP plasma concentrations in BE suggests a systemic inflammatory phenomenon similar to that observed in COPD associated to tobacco smoking, which may also play a role in COPD pathogenesis.

Effects of 2-methoxyestradiol on apoptosis and HIF-1α and HIF-2α expression in lung cancer cells under normoxia and hypoxia

Hypoxic tumor cells are known to be more resistant to conventional chemotherapy and radiation than normoxic cells. However, the effects of 2-methoxyestradiol (2-ME), an anti-angiogenic, antiproliferative and pro-apoptotic drug, on hypoxic lung cancer cells are unknown. The aim of the present study was to compare the effects of 2-ME on cell growth, apoptosis, hypoxia-inducible factor 1α (HIF-1α) and HIF-2α gene and protein expression in A549 cells under normoxic and hypoxic conditions. To establish the optimal 2-ME concentration with which to carry out the apoptosis assay and to examine mRNA and protein expression of HIFs, cell growth analysis was carried out through N-hexa-methylpararosaniline staining assays in A549 cell cultures treated with one of five different 2-ME concentrations at different times under normoxic or hypoxic growth conditions. The 2-ME concentration of 10 mM at 72 h was selected to perform all further experiments. Apoptotic cells were analyzed by flow cytometry. Western blotting was used to determine HIF-1α and HIF-2α protein expression in total cell extracts. Cellular localization of HIF-1α and HIF-2α was assessed by immunocytochemistry. HIF-1α and HIF-2α gene expression was determined by real-time PCR. A significant increase in the percentage of apoptosis was observed when cells were treated with 2-ME under a normoxic but not under hypoxic conditions (p=0.006). HIF-1α and HIF-2α protein expression levels were significantly decreased in cells cultured under hypoxic conditions and treated with 2-ME (p<0.001). Furthermore, 2-ME decreased the HIF-1α and HIF-2α nuclear staining in cells cultured under hypoxia. The HIF-1α and HIF-2α mRNA levels were significantly lower when cells were exposed to 2-ME under normoxia and hypoxia. Our results suggest that 2-ME could have beneficial results when used with conventional chemotherapy in an attempt to lower the invasive and metastatic processes during cancer development due to its effects on the gene expression and protein synthesis of HIFs.

Transmembrane protease, serine 4 (TMPRSS4) is upregulated in IPF lungs and increases the fibrotic response in bleomycin-induced lung injury

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease characterized by epithelial cell activation, expansion of the fibroblast population and excessive extracellular matrix accumulation. The mechanisms are incompletely understood but evidence indicates that the deregulation of several proteases contributes to its pathogenesis. Transmembrane protease serine 4 (TMPRSS4) is a novel type II transmembrane serine protease that may promote migration and facilitate epithelial to mesenchymal transition (EMT), two critical processes in the pathogenesis of IPF. Thus, we hypothesized that over-expression of TMPRSS4 in the lung could promote the initiation and/or progression of IPF. In this study we first evaluated the expression and localization of TMPRSS4 in IPF lungs by real time PCR, western blot and immunohistochemistry. Then we examined the lung fibrotic response in wild-type and TMPRSS4 deficient mice using the bleomycin-induced lung injury model. We found that this protease is upregulated in IPF lungs, where was primarily expressed by epithelial and mast cells. Paralleling the findings in vivo, TMPRSS4 was expressed by alveolar and bronchial epithelial cells in vitro and unexpectedly, provoked an increase of E-cadherin. No expression was observed in normal human or IPF lung fibroblasts. The lung fibrotic response evaluated at 28 days after bleomycin injury was markedly attenuated in the haplodeficient and deficient TMPRSS4 mice. By morphology, a significant reduction of the fibrotic index was observed in KO and heterozygous mice which was confirmed by measurement of collagen content (hydroxyproline: WT: 164±21.1 μg/lung versus TMPRSS4 haploinsufficient: 110.2±14.3 μg/lung and TMPRSS4 deficient mice: 114.1±24.2 μg/lung (p<0.01). As in IPF, TMPRSS4 was also expressed in epithelial and mast cells. These findings indicate that TMPRSS4 is upregulated in IPF lungs and that may have a profibrotic role.