Anandamide regulates keratinocyte differentiation by inducing DNA methylation in a CB1 receptor-dependent manner.

Anandamide (arachidonoylethanolamide, AEA) belongs to an important class of endogenous lipids including amides and esters of long chain polyunsaturated fatty acids, collectively termed "endocannabinoids." Recently we have shown that AEA inhibits differentiation of human keratinocytes, by binding to type-1 cannabinoid receptors (CB1R). To further characterize the molecular mechanisms responsible for this effect, we investigated the expression of epidermal differentiation-related genes after AEA treatment. We observed that keratin 1 and 10, transglutaminase 5 and involucrin are transcriptionally down-regulated by AEA. Most importantly, we found that AEA is able to decrease differentiating gene expression by increasing DNA methylation in human keratinocytes, through a p38, and to a lesser extent p42/44, mitogen-activated protein kinase-dependent pathway triggered by CB1R. An effect of AEA on DNA methylation because of CB1R-mediated increase of methyltransferase activity is described here for the first time, and we believe that the importance of this effect clearly extends beyond the regulation of skin differentiation. In fact, the modulation of DNA methylation by endocannabinoids may affect the expression of a number of genes that regulate many cell functions in response to these substances

Endocannabinoids in adipocytes during differentiation and their role in glucose uptake

The molecular basis for the control of energy balance by the endocannabinoidanandamide (AEA) is still unclear. Here, we show that murine 3T3-L1 fibroblastshave the machinery to bind, synthesize and degrade AEA, and that theirdifferentiation into adipocytes increases by approximately twofold the bindingefficiency of cannabinoid receptors (CBR), and by approximately twofold andapproximately threefold, respectively, the catalytic efficiency of the AEAtransporter and AEA hydrolase. In contrast, the activity of the AEA synthetaseand the binding efficiency of vanilloid receptor were not affected by thedifferentiation process. In addition, we demonstrate that AEA increases byapproximately twofold insulin-stimulated glucose uptake in differentiatedadipocytes, according to a CB1R-dependent mechanism that involves nitric oxidesynthase, but not lipoxygenase or cyclooxygenase. We also show that AEA bindingto peroxisome proliferator-activated receptor-gamma, known to inducedifferentiation of 3T3-L1 fibroblasts into adipocytes, is not involved in thestimulation of glucose uptake.[...

Evidence for the intracellular accumulation of anandamide in adiposomes

Anandamide is a lipid messenger that carries out a wide variety of biological functions. It has been suggested that anandamide accumulation involves binding to a saturable cellular component. To identify the structure(s) involved in this process, we analyzed the intracellular distribution of both biotinylated and radiolabeled anandamide, providing direct evidence that lipid droplets, also known as adiposomes, constitute a dynamic reservoir for the sequestration of anandamide. In addition, confocal microscopy and biochemical studies revealed that the anandamide-hydrolase is also spatially associated with lipid droplets, and that cells with a larger adiposome compartment have an enhanced catabolism of anandamide. Overall, these findings suggest that adiposomes may have a critical role in accumulating anandamide, possibly by connecting plasma membrane to internal organelles along the metabolic route of this endocannabinoid.[...

Characterization of the Endocannabinoid System in Human Neuronal Cells and Proteomic Analysis of Anandamide-induced Apoptosis*

Anandamide (AEA) is an endogenous agonist of type 1 cannabinoid receptors (CB1R) that, along with metabolic enzymes of AEA and congeners, compose the “endocannabinoid system.” Here we report the biochemical, morphological, and functional characterization of the endocannabinoid system in human neuroblastoma SH-SY5Y cells that are an experimental model for neuronal cell damage and death, as well as for major human neurodegenerative disorders. We also show that AEA dose-dependently induced apoptosis of SH-SY5Y cells. Through proteomic analysis, we further demonstrate that AEA-induced apoptosis was paralleled by an ∼3 to ∼5-fold up-regulation or down-regulation of five genes; IgG heavy chain-binding protein, stress-induced phosphoprotein-1, and triose-phosphate isomerase-1, which were up-regulated, are known to act as anti-apoptotic agents; actin-related protein 2/3 complex subunit 5 and peptidylprolyl isomerase-like protein 3 isoform PPIL3b were down-regulated, and the first is required for actin network formation whereas the second is still function-orphan. Interestingly, only the effect of AEA on BiP was reversed by the CB1R antagonist SR141716, in SH-SY5Y cells as well as in human neuroblastoma LAN-5 cells (that express a functional CB1R) but not in SK-NBE cells (which do not express CB1R). Silencing or overexpression of BiP increased or reduced, respectively, AEA-induced apoptosis of SH-SY5Y cells. In addition, the expression of BiP and of the BiP-related apoptotic markers p53 and PUMA was increased by AEA through a CB1R-dependent pathway that engages p38 and p42/44 mitogen-activated protein kinases. Consistently, this effect of AEA was minimized by SR141716. In conclusion, we identified BiP as a key protein in neuronal apoptosis induced by AEA

Characterization of the endocannabinoid system in human neuronal cells, and proteomic analysis of anandamide-induced apoptosis

Anandamide (AEA) is a member of an endogenous class of lipid mediators, known as endocannabinoids, which are involved in various biological processes. In particular, AEA regulates cell growth, differentiation, and death. Accumulating evidence demonstrates that AEA controls also epidermal differentiation, one of the best characterized mechanisms of cell specialization. Indeed, the epidermis is a keratinized multistratified epithelium that functions as a barrier to protect the organism from dehydration, mechanical trauma, and microbial insults. Its function is established during embryogenesis and is maintained during the whole life span of the organism, through a complex and tightly controlled program, termed epidermal terminal differentiation (or cornification). Whereas the morphological changes that occur during cornification have been extensively studied, the molecular mechanisms that underlie this process remain poorly understood. In this chapter, we summarize current knowledge about the molecular regulation of proliferation and terminal differentiation in mammalian epidermis. In this context, we show that endocannabinoids are finely regulated by, and can interfere with, the differentiation program. In addition, we review the role of AEA in the control of cornification, and show that it occurs by maintaining a transcriptional repression of gene expression through increased DNA methylation.[...

Characterization of biotin-anandamide, a novel tool for the visualization of anandamide accumulation

Anandamide (N-arachidonoylethanolamide; AEA) acts as an endogenous agonist ofboth cannabinoid and vanilloid receptors. During the last two decades, itsmetabolic pathways and biological activity have been investigated extensively andrelatively well characterized. In contrast, at present, the effective nature and mechanism of AEA transport remain controversial and still unsolved issues. Here, we report the characterization of a biotinylated analog of AEA (b-AEA) that hasthe same lipophilicity of the parent compound. In addition, by means ofbiochemical assays and fluorescence microscopy, we show that b-AEA is accumulatedinside the cells in a way superimposable on that of AEA. Conversely, b-AEA doesnot interact or interfere with the other components of the endocannabinoidsystem, such as type-1 and type-2 cannabinoid receptors, vanilloid receptor, AEA synthetase (N-acylphosphatidylethanolamine-hydrolyzing phospholipase D), or AEAhydrolase (fatty acid amide hydrolase). Together, our data suggest that b-AEAcould be a very useful probe for visualizing the accumulation and intracellulardistribution of this endocannabinoid.[...