The presence of functional cannabinoid CB 2 receptors in the CNS has provoked considerable controversy over the past few years. Formerly considered as an exclusively peripheral receptor, it is now accepted that it is also present in limited amounts and distinct locations in the brain of several animal species, including humans. Furthermore, the inducible nature of these receptors under neuroinflammatory conditions, in contrast to CB 1 , makes them attractive targets for the development of novel therapeutic approaches. In fact, the undesired psychoactive effects caused by CB 1 activation have largely limited the clinical use of cannabinoid-related compounds that act on these receptors. In this review some recent findings on the antiinflammatory properties of CB 2 receptors are presented, as well as new perspectives that have been obtained based on studies of human postmortem brain samples. In addition, various working hypotheses are also proposed and discussed.

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ABSTRACT:To test the neuroprotective effects of the nonpsychoactive cannabinoid cannabidiol (CBD), piglets received i.v. CBD or vehicle after hypoxia-ischemia (HI: temporary occlusion of both carotid arteries plus hypoxia). Nonhypoxic-ischemic sham-operated piglets remained as controls. Brain damage was studied by near-infrared spectroscopy (NIRS) and amplitudeintegrated electroencephalography (aEEG) and by histologic assessment (Nissl and FluoroJadeB staining). In HIϩvehicle, HI led to severe cerebral hemodynamic and metabolic impairment, as reflected in NIRS by an increase in total Hb index (THI) and a decrease in the fractional tissue oxygenation extraction (FTOE); in HIϩCBD the increase of THI was blunted and FTOE remained similar to SHAM. HI profoundly decreased EEG amplitude, which was not recovered in HIϩvehicle, indicating cerebral hypofunction; seizures were observed in all HIϩvehicle. In HIϩCBD, however, EEG amplitude recovered to 46.4 Ϯ 7.8% baseline and seizures appeared only in 4/8 piglets (both p Ͻ 0.05). The number of viable neurons decreased and that of degenerating neurons increased in HIϩvehicle; CBD reduced both effects by more than 50%. CBD administration was free from side effects; moreover, CBD administration was associated with cardiac, hemodynamic, and ventilatory beneficial effects. In conclusion, administration of CBD after HI reduced short-term brain damage and was associated with extracerebral benefits. (Pediatr Res 64: 653-658, 2008)

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BACKGROUND AND PURPOSEThe endocannabinoid system may regulate glial cell functions and their responses to pathological stimuli, specifically, Alzheimer's disease. One experimental approach is the enhancement of endocannabinoid tone by blocking the activity of degradative enzymes, such as fatty acid amide hydrolase (FAAH). EXPERIMENTAL APPROACHWe examined the role of FAAH in the response of astrocytes to the pathologic form of b-amyloid (Ab). Astrocytes from wild-type mice (WT) and from mice lacking FAAH (FAAH-KO) were incubated with Ab for 8, 24 and 48 h, and their inflammatory responses were quantified by ELISA, western-blotting and real-time quantitative-PCR. KEY RESULTSFAAH-KO astrocytes were significantly more responsive to Ab than WT astrocytes, as shown by the higher production of pro-inflammatory cytokines. Expression of COX-2, inducible NOS and TNF-a was also increased in Ab-exposed KO astrocytes compared with that in WTs. These effects were accompanied by a differential pattern of activation of signalling cascades involved in mediating inflammatory responses, such as ERK1/2, p38MAPK and NFk B. PPAR-a and PPAR-g as well as transient receptor potential vanilloid-1 (TRPV1), but not cannabinoid CB1 or CB2 receptors, mediate some of the differential changes observed in Ab-exposed FAAH-KO astrocytes. The pharmacological blockade of FAAH did not render astrocytes more sensitive to Ab. In contrast, exogenous addition of several acylethanolamides (anandamide, palmitoylethanolamide and oleoylethanolamide) induced an antiinflammatory response. CONCLUSIONSThe genetic deletion of FAAH in astrocytes exacerbated their inflammatory phenotype against Ab in a process involving PPAR-a, PPAR-g and TRPV1 receptors.

The vitamin D receptor (VDR) normally functions as a ligand-dependent transcriptional activator. Here we show that, in the presence of Ets-1, VDR stimulates the prolactin promoter in a ligand-independent manner, behaving as a constitutive activator. Mutations in the AF2 domain abolish vitamin D-dependent transactivation but do not affect constitutive activation by Ets-1. Therefore, in contrast with the actions of vitamin D, activation by Ets-1 is independent of the AF2 domain. Ets-1 also conferred a ligand-independent activation to the estrogen receptor and to peroxisome proliferator-activated receptor ␣. In addition, Ets-1 cooperated with the unliganded receptors to stimulate the activity of reporter constructs containing consensus response elements fused to the thymidine kinase promoter. There is a direct interaction of the receptors with Ets-1 which requires the DNA binding domains of both proteins. Interaction with Ets-1 induces a conformational change in VDR which can be detected by an increased resistance to proteolytic digestion. Furthermore, a retinoid X receptor-VDR heterodimer in which both receptors lack the core C-terminal AF2 domain can recruit coactivators in the presence, but not in the absence, of Ets-1. This suggests that Ets-1 induces a conformational change in the receptor which creates an active interaction surface with coactivators even in the AF2-defective mutants. These results demonstrate the existence of a novel mechanism, alternative to ligand binding, which can convert an unliganded receptor from an inactive state into a competent transcriptional activator.Nuclear receptors normally act as ligand-inducible transcriptional factors by binding as homodimers or as heterodimers with the retinoid X receptor (RXR) to hormone response elements (HREs) in target genes (15). Transcriptional regulation by nuclear receptors is achieved through autonomous activation functions (AFs): a constitutive N-terminal AF1 and a C-terminal ligand-dependent AF2. Ligand binding causes a conformational change in the receptors that allows recruitment of CREB binding protein (CBP)-and p160-related coactivator proteins with histone acetylase activity (26). The multisubunit coactivator complex TRAC-DRIP also binds the nuclear receptors in a ligand-and AF2-dependent manner and may interact directly with the basic transcriptional machinery (9, 21). However, recent evidence has shown that several receptors may also be activated in a ligand-independent manner. A variety of agents including growth factors and cyclic AMP activate the receptors, presumably by stimulation of cellular protein kinases which cause receptor phosphorylation (3,12,22,24,31). This activation appears to involve a ligand-and AF2-independent recruitment of p160 coactivators (8, 27). Furthermore, the estrogen receptor (ER) can be also stimulated in a ligand-independent manner by association with cyclin D1 (35). Cyclin D1 interacts with p160 coactivators (36) and also with the acetylase p/CAF (CBP-associated factor) (18) and can recruit the coactivators...

We have examined the influence of insulin-like growth factor I (IGF-1) on prolactin gene expression in rat pituitary GH4C1 cells. Incubation with IGF-1 increases prolactin mRNA levels and activates the prolactin promoter in transient transfection assays. A similar degree of activation is observed with constructs extending to -3000 and -176 base pairs of the prolactin 5' flanking region, indicating that the IGF-1 response element is located in the proximal promoter sequences. A plasmid containing 101 base pairs shows a partial stimulation by IGF-1, and the response is lost in a deletion to -76 base pairs. The Ras oncoproteins have been implicated as a critical signaling component in mediating the effect of growth factor receptor tyrosine kinases. Expression of oncogenic RasVal12 mimics the effect of IGF-1 on the prolactin promoter, and a dominant negative Ras, RasAsn17, blocks IGF-1-mediated stimulation. Dominant negative mitogen-activated protein kinase (MAPK) also reduces the activation of the prolactin promoter by IGF-1. Ets transcription factors have been described to lie downstream of Ras and MAPK in the signaling pathway leading to prolactin gene activation. Mutation of two Ets binding sites in the promoter region between -101 and -76 abolishes the response to IGF-1. Furthermore, a dominant negative Ets vector strongly reduces the response of the prolactin promoter to IGF-1 and Ras. The endogenous concentration of Ets-related proteins is not limiting in GH4C1 cells for the IGF-1 effect. However, c-Ets-1 and GHF-1 act synergistically in HeLa cells with the IGF-1 receptor, reconstituting pituitary IGF-1 responsiveness. The response to IGF-1 in GH4C1 cells is still observed after transfection with RasVal12 suggesting that, although Ras is required, IGF-1 could stimulate other pathway/s in addition to Ras. Wortmanin, an inhibitor of phosphatidylinositol-3 kinase (PI-3 kinase), also prevents the response of the prolactin promoter to IGF-1. These results show that both the Ras/MAPK/Ets pathway, as well as the activation of PI-3 kinase are involved in the signaling mechanism leading to prolactin expression by IGF-1 in GH4C1 cells.

Heterodimers of the retinoid X receptor (RXR) with the thyroid hormone receptor (TR) are considered to be nonpermissive. It is believed that within these complexes RXR acts as a "silent partner." We demonstrate here that a permissive heterodimer mediates stimulation of prolactin expression by the thyroid hormone T3 and by 9-cis retinoic acid (9-cis-RA). A response element located in the prolactin distal enhancer mediates transactivation by both ligands in pituitary cells, and RXR recruits coactivators when bound to this element as a heterodimer with TR. Furthermore, transcription by the RXR agonist can be obtained in CV-1 cells only after overexpression of coactivators, and overexpression of corepressors inhibits the response in pituitary cells. Thus, cell type-specific differences in coregulator recruitment can determine the cellular response to both ligands. Coactivator recruitment by 9-cis-RA requires the ligand-dependent transactivation domains (AF-2) of both heterodimeric partners. Interestingly, the presence of the RXR ligand can overcome the deleterious effect of the AF-2 mutation E401Q on association with coactivators and transactivation. These results demonstrate an unexpected role for RXR in TR signaling and show that in particular cellular environments this receptor can act as a "nonsilent" partner of TR, allowing stimulation by RXR agonists.