Mutation analysis in the coding sequence of thymidine kinase 1 in breast and colorectal cancer.

We report the first mutational study of thymidine kinase 1 (TK1) performed in human solid tumors. We sequenced cDNAs representing the complete coding region of TK1 in human breast (n=22) and colorectal (n=26) cancer. Codon 106 near the ATP binding site constantly differed (ATG --> GTG; Met --> Val) from the one deposited by Bradshaw and Deininger in the Genbank database (Accession number NM_003258). Silent polymorphisms at codon 11 (CCC --> CCT; Pro --> Pro) and codon 75 (GCG --> GCA; Ala --> Ala) were frequently detected in tumors as well as in normal tissues. In breast cancer the two polymorphisms were observed in 63.6% of the samples analyzed. No significant association could be found between polymorphisms and TK activity. In colorectal cancer the incidence of the two changes was 73.1% and 69.2%, respectively. Interestingly, one colon cancer with high cytosolic TK activity displayed two missense mutations located in and near the putative phosphorylation site by tyrosine kinase (s) (TAT --> CAT; Tyr --> His) and by cAMP-, cGMP-dependent protein kinase (TAC --> TGC; Tyr --> Cys), respectively; adjacent normal mucosa showed no mutation. This may open new avenues that imply TK1 activity in tumor cell proliferation.

SR31747A is a sigma receptor ligand exhibiting antitumoural activity both in vitro and in vivo.

SR31747A is a recently described sigma receptor ligand that binds SR31747A-binding protein 1 (SR-BP) and emopamil-binding protein (EBP) (also called the sigma 1 receptor and the human sterol isomerase (HSI), respectively), and has immunoregulatory and antiproliferative activities. To further investigate its antitumour activity and focusing on cancers, which are sensitive to the molecule, we measured the proliferation of different human epithelial breast or prostate cancer cell lines following in vitro and in vivo SR31747A treatment. Firstly, in vitro, we found that nanomolar concentrations of SR31747A dramatically inhibited cell proliferation in both hormono-responsive and -unresponsive cancer cell lines. Secondly, tumour development was significantly decreased in mice treated with SR31747A. In an attempt to decipher the SR31747A mode of action, we found that the two binding sites may not fully account for this activity. Indeed, while competitive experiments indicated that EBP prevails in mediating SR31747A antiproliferative activity, an analysis of the expression of both receptors indicated that the cellular sensitivity to SR31747A is not correlated with either EBP or SR-BP expression. These data suggest that additional binding sites may exist. Preliminary binding studies demonstrated that SR31747A also binds to sigma 2, a protein that has not yet been cloned, but which is considered as a potential marker of the proliferative status of tumour cells. Altogether, our data demonstrate the antitumoural activity of SR31747A both in vitro and in vivo in two different cancer models, broaden the spectrum of its binding proteins and enhance the potential for further therapeutic development of the molecule.

International Union of Pharmacology. XXVII. Classification of cannabinoid receptors.

Two types of cannabinoid receptor have been discovered so far, CB(1) (2.1: CBD:1:CB1:), cloned in 1990, and CB(2) (2.1:CBD:2:CB2:), cloned in 1993. Distinction between these receptors is based on differences in their predicted amino acid sequence, signaling mechanisms, tissue distribution, and sensitivity to certain potent agonists and antagonists that show marked selectivity for one or the other receptor type. Cannabinoid receptors CB(1) and CB(2) exhibit 48% amino acid sequence identity. Both receptor types are coupled through G proteins to adenylyl cyclase and mitogen-activated protein kinase. CB(1) receptors are also coupled through G proteins to several types of calcium and potassium channels. These receptors exist primarily on central and peripheral neurons, one of their functions being to inhibit neurotransmitter release. Indeed, endogenous CB(1) agonists probably serve as retrograde synaptic messengers. CB(2) receptors are present mainly on immune cells. Such cells also express CB(1) receptors, albeit to a lesser extent, with both receptor types exerting a broad spectrum of immune effects that includes modulation of cytokine release. Of several endogenous agonists for cannabinoid receptors identified thus far, the most notable are arachidonoylethanolamide, 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether. It is unclear whether these eicosanoid molecules are the only, or primary, endogenous agonists. Hence, we consider it premature to rename cannabinoid receptors after an endogenous agonist as is recommended by the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. Although pharmacological evidence for the existence of additional types of cannabinoid receptor is emerging, other kinds of supporting evidence are still lacking.

[Exploitation of expression profiles: examples in oncology]. / Exploitation des profils d'expression: exemples en oncologie.

The analysis of biological processes has been revolutionized by the emergence of the DNA array technology. As cellular biological events are controlled by gene expression, their modulations are markers of the cellular activity. These modulations can be indicative of either a physiological process or a pathological one. Monitoring of the expression levels of thousands of genes simultaneously, the expression profiling method is based upon comparative studies where the identification of the differentially expressed genes in two samples is aimed. The two samples under study may be compared temporally or following drug treatment, they may also originate from different sources, e.g. normal versus pathological samples. In that case, gene expression profiling is conducted for diagnostics purposes or therapy monitoring, and offers an opportunity to identify new drug targets. Using different examples, we describe the potentialities of this approach in oncology.

Expression pattern of sigma receptor 1 mRNA and protein in mammalian retina.

Sigma receptors are nonopiate and nonphencyclidine binding sites that are thought to be neuroprotective due to modulation of N-methyl-D-aspartate (NMDA) receptors. Sigma receptor 1 expression has been demonstrated in numerous tissues including brain. Recently, studies using binding assays have demonstrated sigma receptor 1 in neural retina, however these studies did not demonstrate in which retinal cell type(s) sigma receptor 1 was present nor did they establish unequivocally the molecular identity of the receptor. The present study was designed to address these issues. Reverse transcription-polymerase chain reaction (RT-PCR) analysis amplified sigma receptor 1 in neural retina, RPE-choroid complex, and lens isolated from mice. A similar RT-PCR product was amplified also in three cultured cell lines, rat Müller cells, rat ganglion cells and human ARPE-19 cells. In situ hybridization analysis revealed abundant sigma receptor 1 expression in ganglion cells, cells of the inner nuclear layer, inner segments of photoreceptor cells and retinal pigment epithelial (RPE) cells. Immunohistochemical studies detected the sigma receptor 1 protein in retinal ganglion, photoreceptor, RPE cells and surrounding the soma of cells in the inner nuclear layer. These data provide the first cellular localization of sigma receptor 1 in neural retina and establish the molecular identity of sigma receptor 1 in retinal cells. The demonstration that sigma receptor 1 is present in ganglion cells is particularly noteworthy given the well-documented susceptibility of these cells to glutamate toxicity. Our findings suggest that retinal ganglion cells may be amenable to the neuroprotective effects of sigma ligands under conditions of neurotoxicity such as occurs in diabetes.

Expression pattern of the type 1 sigma receptor in the brain and identity of critical anionic amino acid residues in the ligand-binding domain of the receptor.

The type 1 sigma receptor (sigmaR1) has been shown to participate in a variety of functions in the central nervous system. To identify the specific regions of the brain that are involved in sigmaR1 function, we analyzed the expression pattern of the receptor mRNA in the mouse brain by in situ hybridization. SigmaR1 mRNA was detectable primarily in the cerebral cortex, hippocampus, and Purkinje cells of cerebellum. To identify the critical anionic amino acid residues in the ligand-binding domain of sigmaR1, we employed two different approaches: chemical modification of anionic amino acid residues and site-directed mutagenesis. Chemical modification of anionic amino acids in sigmaR1 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide reduced the ligand-binding activity markedly. Since it is known that a splice variant of this receptor which lacks exon 3 does not have the ability to bind sigma ligands, the ligand-binding domain with its critical anionic amino acid residues is likely to be present in or around the region coded by exon 3. Therefore, each of the anionic amino acids in this region was mutated individually and the influence of each mutation on ligand binding was assessed. These studies have identified two anionic amino acids, D126 and E172, that are obligatory for ligand binding. Even though the ligand-binding function was abolished by these two mutations, the expression of these mutants was normal at the protein level. These results show that sigmaR1 is expressed at high levels in specific areas of the brain that are involved in memory, emotion and motor functions. The results also provide important information on the chemical nature of the ligand-binding site of sigmaR1 that may be of use in the design of sigmaR1-specific ligands with potential for modulation of sigmaR1-related brain functions.

Transient protection by peripheral benzodiazepine receptors during the early events of ultraviolet light-induced apoptosis.

The peripheral benzodiazepine receptor (PBR) is a mitochondrial protein involved in the formation of mitochondrial permeability transition (PT) pores which play a critical role during the early events of apoptosis. PBRs are located in many tissues and are strongly expressed in the superficial layers of human epidermis. PBRs play a protective role against free radical damage and PBR ligands modulate apoptosis. To investigate the role of PBR during the early events of ultraviolet (UV)-mediated apoptosis we compared the effects of UVB on PBR-transfected Jurkat cells and their wild type counterparts devoid of any PBR expression. Results indicate that early after UVB exposure (up to 4 h), PBR-transfected cells were more resistant to apoptosis and exhibited a delayed mitochondrial transmembrane potential drop, a diminished superoxide anions production, and a reduced caspase-3 activation. Taken together these findings suggest that PBR may regulate early death signals leading to UV induced apoptosis.

Immunocytochemical assessment of sigma-1 receptor and human sterol isomerase in breast cancer and their relationship with a series of prognostic factors.

The purpose of this study was to immunocytochemically investigate two new markers, the sigma-1 receptor and the human sterol isomerase (hSI), in comparison with a series of clinicopathological and immunocytochemical prognostic factors in a trial including 95 patients with operable primary breast cancers. Our results showed no statistically significant relationship between these two markers and the age of the patients, their menopausal status, the tumour size and its histological grade, the nodal status and the expression of the Ki-67 proliferative marker. However, we evidenced a close correlation between the sigma-1 receptor expression and the hormonal receptor positivity (P = 0.008), essentially due to a link with the progesterone receptor status (P = 0.01). By contrast there was an inverse relationship between hSI expression and the oestrogen receptor and/or progesterone receptor positivity (P = 0.098). A significant relationship was shown between both the sigma-1 receptor, hSI expressions and Bcl2 expression, with P= 0.017 and 0.035 respectively. We also assessed whether the expression of the sigma-1 receptor or hSI might be linked with disease-free survival (DFS) and found that the presence of hSI and the absence of sigma-1 receptor expression were associated with a poorer disease-free survival (P= 0.007). Altogether these results suggest that in primary breast carcinomas in association with the evaluation of the steroid receptor status, the sigma-1 receptor and hSI may be interesting new markers useful to identify those patients who might be able to benefit from an adjuvant therapy.

Characterization of NPY receptors controlling lipolysis and leptin secretion in human adipocytes.

In order to characterize neuropeptide Y (NPY) receptors present in human adipocytes, we used selective ligands together with specific molecular probes able to recognize the different NPY receptor subtypes. RT-PCR experiments revealed the presence of Y(1) receptor transcripts with Y(4) and Y(5) and absence of Y(2) signals. Binding studies, using selective radioiodinated ligands, detected a high number (B(max)=497+/-124 fmol/mg protein) of a high affinity binding site only with [(125)I]peptide YY (PYY) and [(125)I](Leu(31), Pro(34))PYY. These sites exhibited a typical Y(1) profile as indicated by the rank order of affinity of NPY analogs and the high affinity of two selective NPY receptor antagonists, SR120819A and BIBP3226. In [(35)S]GTPgammaS binding experiments, PYY activation was totally inhibited by SR120819A and BIBP3226. Both compounds antagonized, with similar efficiency, the antilipolytic effect exerted by NPY in isolated adipocytes. Finally, PYY and Y(1) ligands enhanced adipocyte leptin secretion, an effect totally prevented by SR120819A. Thus, highly expressed in human adipocytes, the Y(1) receptor sustains the strong antilipolytic effect of NPY and exerts a positive action on leptin secretion.

Genomic and functional changes induced by the activation of the peripheral cannabinoid receptor CB2 in the promyelocytic cells HL-60. Possible involvement of the CB2 receptor in cell differentiation.

The function of the peripheral cannabinoid receptor (CB2), which is mainly expressed on hematopoietic cells, remains an enigma. In an attempt to decipher its role, we used Affymetrix DNA chips to investigate the gene expression profile of the promyelocytic cells HL-60 transfected with the CB2 receptor and activated with the cannabinoid agonist CP 55,940. Agonist exposure of these cells led to an activation of a mitogen-activated protein kinase cascade and a receptor desensitization, indicating a functional coupling of the transfected receptors. At the genomic level, activation of the CB2 receptors induced an up-regulation of nine genes involved in cytokine synthesis, regulation of transcription, and cell differentiation. A majority of them are under the control of the transcription factor NF-kappaB, whose nuclear translocation was demonstrated. Many features of the transcriptional events, reported here for the first time, appeared to be related to an activation of a cell differentiation program, suggesting that CB2 receptors could play a role in the initialization of cell maturation. Moreover, we showed that CB2-activated wild-type HL-60 cells developed properties usually found in host defense effector cells such as an enhanced release of chemotactic cytokines and an increased motility, characteristic of more mature cells of the granulocytic-monocytic lineage.

A beneficial aspect of a CB1 cannabinoid receptor antagonist: SR141716A is a potent inhibitor of macrophage infection by the intracellular pathogen Brucella suis.

The psychoactive component of marijuana, delta9-tetrahydrocannabinol (THC) suppresses different functions of immunocytes, including the antimicrobicidal activity of macrophages. The triggering of cannabinoid receptors of CB1 and CB2 subtypes present on leukocytes may account for these effects. We investigated the influence of specific CB1 or CB2 receptor antagonists (SR141716A and SR144528, respectively) and nonselective CB1/CB2 cannabinoid receptor agonists (CP55,940 or WIN 55212-2) on macrophage infection by Brucella suis, an intracellular gram-negative bacteria. None of the compounds tested affected bacterial phagocytosis. By contrast, the intracellular multiplication of Brucella was dose-dependently inhibited in cells treated with 10-500 nM SR141716A and 1 microM SR141716A-induced cells exerted a potent microbicidal effect against the bacteria. SR144528, CP55,940, or WIN 55212-2 did not affect (or slightly potentiated) the growth of phagocytized bacteria. However, CP55,940 or WIN 55212-2 reversed the SR141716A-mediated effect, which strongly suggested an involvement of macrophage CB1 receptors in the phenomenon. SR141716A was able to pre-activate macrophages and to trigger an activation signal that inhibited Brucella development. The participation of endogenous cannabinoid ligand(s) in Brucella infection was discussed. Finally, our data show that SR141716A up-regulates the antimicrobial properties of macrophages in vitro and might be a pharmaceutical compound useful for counteracting the development of intramacrophagic gram-negative bacteria.

Monitoring cellular responses to Listeria monocytogenes with oligonucleotide arrays.

Listeria monocytogenes is a pathogenic intracellular microorganism whose infection induces pleiotropic biological changes associated with host cell gene expression regulation. Here we define the gene expression profiles of the human promyelocytic THP1 cell line before and after L. monocytogenes infection. Gene expression was measured on a large scale via oligonucleotide microarrays with probe sets corresponding to 6,800 human genes. We assessed and discussed the reproducibility of the hybridization signatures. In addition to oligonucleotide arrays, we also performed the large scale gene expression measurement with two high-density membranes, assaying for 588 and 18,376 human genes, respectively. This work allowed the reproducible identification of 74 up-regulated RNAs and 23 down-regulated RNAs as a consequence of L. monocytogenes infection of THP1. The reliability of these data was reinforced by performing independent infections. Some of these detected RNAs were consistent with previous results, while some newly identified RNAs encode gene products that may play key roles in L. monocytogenes infection. These findings will undoubtedly enhance the understanding of L. monocytogenes molecular physiology and may help identify new therapeutic targets.

The neuroprotective agent SR 57746A abrogates experimental autoimmune encephalomyelitis and impairs associated blood-brain barrier disruption: implications for multiple sclerosis treatment.

Experimental autoimmune encephalomyelitis (EAE) is a T cell autoimmune disorder that is a widely used animal model for multiple sclerosis (MS) and, as in MS, clinical signs of EAE are associated with blood-brain barrier (BBB) disruption. SR 57746A, a nonpeptide drug without classical immunosuppressive properties, efficiently protected the BBB and impaired intrathecal IgG synthesis (two conventional markers of MS exacerbation) and consequently suppressed EAE clinical signs. This compound inhibited EAE-induced spinal cord mononuclear cell invasion and normalized tumor necrosis factor alpha and IFN-gamma mRNA expression within the spinal cord. These data suggested that pharmacological intervention aimed at inhibiting proinflammatory cytokine expression within the central nervous system provided protection against BBB disruption, the first clinical sign of EAE and probably the key point of acute MS attacks. This finding could lead to the development of a new class of compounds for oral therapy of MS, as a supplement to immunosuppressive agents.

Cannabinoid receptor interactions with the antagonists SR 141716A and SR 144528.

The G protein-coupled cannabinoid receptor subtypes CB1 and CB2 have been cloned from several species. The CB1 receptor is highly conserved across species, whereas the CB2 receptor shows considerable cross-species variations. The two human receptors share only 44% overall identity, ranging from 35% to 82% in the transmembrane regions. Despite this structural disparity, the most potent cannabinoid agonists currently available are largely undiscriminating and are therefore unsatisfactory tools for investigating the architecture of ligand binding sites. However, the availability of two highly specific antagonists, SR 141716A for the CB1 receptor and SR 144528 for the CB2 receptor, has allowed us to adopt a systematic approach to defining their respective binding sites through the use of chimeric CB1 receptor/CB2 receptor constructs, coupled with site-directed mutagenesis. We identified the region encompassed by the fourth and fifth transmembrane helices as being critical for antagonist specificity. Both the wild type human receptors overexpressed in heterologous systems are autoactivated; SR 141716A and SR 144528 exhibit classical inverse agonist properties with their respective target receptors. In addition, through its interaction with the CB1 receptor SR 141716A blocks the Gi protein-mediated activation of mitogen-activated protein kinase stimulated by insulin or insulin-like growth factor I. An in-depth analysis of this discovery has led to a modified three-state model for the CB1 receptor, one of which implicates the SR 141716A-mediated sequestration of Gi proteins, with the result that the growth factor-stimulated intracellular pathways are effectively impeded.

Regulation of peripheral cannabinoid receptor CB2 phosphorylation by the inverse agonist SR 144528. Implications for receptor biological responses.

We recently demonstrated that the selective cannabinoid receptor antagonist SR 144528 acts as an inverse agonist that blocks constitutive mitogen-activated protein kinase activity coupled to the spontaneous autoactivated peripheral cannabinoid receptor (CB2) in the Chinese hamster ovary cell line stably transfected with human CB2. In the present report, we studied the effect of SR 144528 on CB2 phosphorylation. The CB2 phosphorylation status was monitored by immunodetection using an antibody specific to the COOH-terminal CB2 which can discriminate between phosphorylated and non-phosphorylated CB2 isoforms at serine 352. We first showed that CB2 is constitutively active, phosphorylated, and internalized at the basal level. By blocking autoactivated receptors, inverse agonist SR 144528 treatment completely inhibited this phosphorylation state, leading to an up-regulated CB2 receptor level at the cell surface, and enhanced cannabinoid agonist sensitivity for mitogen-activated protein kinase activation of Chinese hamster ovary-CB2 cells. After acute agonist treatment, serine 352 was extensively phosphorylated and maintained in this phosphorylated state for more than 8 h after agonist treatment. The cellular responses to CP-55,940 were concomitantly abolished. Surprisingly, CP-55,940-induced CB2 phosphorylation was reversed by SR 144528, paradoxically leading to a non-phosphorylated CB2 which could then be fully activated by CP-55,940. The process of CP-55,940-induced receptor phosphorylation followed by SR 144528-induced receptor dephosphorylation kept recurring many times on the same cells, indicating that the agonist switches the system off but the inverse agonist switches the system back on. Finally, we showed that autophosphorylation and CP-55, 940-induced serine 352 CB2 phosphorylation involve an acidotropic GRK kinase, which does not use Gibetagamma. In contrast, SR 144528-induced CB2 dephosphorylation was found to involve an okadaic acid and calyculin A-sensitive type 2A phosphatase.

Colocalization of sterol isomerase and sigma(1) receptor at endoplasmic reticulum and nuclear envelope level.

SR31747A is a sigma ligand previously described as having original immunosuppressive properties. Two SR31747A targets were recently identified and termed sigma(1) or SR-BP-1 (SR31747A-binding protein-1) and hSI (human sterol isomerase). In order to characterize these proteins further, we examined their expression and localization at the subcellular level. Based on the amino acid sequence deduced from the cloned hSI, anti-hSI polyclonal antibody was raised against the N-terminal fragment of the protein. Using this antibody, we performed Western-blot experiments to demonstrate the presence of hSI in various B and T cell lines, and hSI expression was quantified in these cell lines by flow cytometry and estimated at 15 000-30 000 sites per cell. Subcellular localization studies by both confocal and electron microscopy, performed on THP1 cells with anti-hSI antibody and with the previously described anti-(SR-BP-1) monoclonal antibody, demonstrated that: (a) hSI was colocalized with SR-BP-1; (b) hSI and SR-BP-1 were associated with the endoplasmic reticulum and with the outer and inner membranes of the nuclear envelope; (c) both proteins were delocalized during the cell cycle at the mitosis step when the nuclear membranes disappeared. Taken together our results suggest that both SR31747A-binding proteins not only play a role in sterol metabolism but indirectly affect lipoprotein functions.

The expression of peripheral benzodiazepine receptors in human skin: the relationship with epidermal cell differentiation.

The peripheral benzodiazepine receptor (PBR) is a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands. PBR is part of a heteromeric receptor complex involved in the formation of mitochondrial permeability transition pores and in the early events of apoptosis. PBR may function as an oxygen-dependent signal generator; recent data indicate that these receptors may preserve the mitochondria of haematopoietic cell lines from damage caused by oxygen radicals. To identify PBRs in human skin, we used a specific monoclonal antibody directed against the C-terminus fragment of the human receptor. PBR immunoreactivity was found in keratinocytes, Langerhans cells, hair follicles and dermal vascular endothelial cells. Interestingly, confocal microscopic examination of skin sections revealed that PBR expression was strongly upregulated in the superficial differentiated layers of the epidermis. Ultrastructurally, PBRs were distributed throughout the cytoplasm but were selectively expressed on the mitochondrial membranes of epidermal cells. The elevated level of PBRs in the spinous layer was not associated with an increased number of mitochondria nor with an increased amount of mRNA as assessed by in situ hybridization on microautoradiographed skin sections. The present work provides, for the first time, evidence of PBR immunoreactivity in human skin. This mitochondrial receptor may modulate apoptosis in the epidermis; its increased expression in differentiated epidermal layers may represent a novel mechanism of natural skin protection against free radical damage generated by ultraviolet exposure.

Cannabinoid receptor CB1 activates the Na+/H+ exchanger NHE-1 isoform via Gi-mediated mitogen activated protein kinase signaling transduction pathways.

We previously showed that the cannabinoid receptor CB1 stably transfected in Chinese hamster ovary cells was constitutively active and could be inhibited by the inverse agonist SR 141716A. In the present study, we demonstrate that the cannabinoid agonist CP-55940 induced cytosol alkalinization of CHO-CB1 cells in a dose- and time-dependent manner via activation of the Na+/H+ exchanger NHE-1 isoform. By contrast, the inverse agonist SR 141716A induced acidification of the cell cytosol, suggesting that the Na+/H+ exchanger NHE-1 was constitutively activated by the CB1 receptor. CB1-mediated NHE1 activation was prevented by both pertussis toxin treatment and the specific MAP kinase inhibitor PD98059. NHE-1 and p42/p44 MAPK had a similar time course of activation in response to the addition of CP-55940 to CHO-CB1 cells. These results suggest that CB1 stimulates NHE-1 by G(i/o)-mediated activation of p42/p44 MAP kinase and highlight a cellular physiological process targeted by CB1.

Stimulation of peripheral cannabinoid receptor CB2 induces MCP-1 and IL-8 gene expression in human promyelocytic cell line HL60.

Using the recently developed methodology of nucleic acid microarrays spotted with specific cDNAs probes belonging to different gene families, we showed for the first time that nanomolar concentrations of the cannabinoid ligand CP-55940 upregulated the expression of two different members of the chemokine gene family: the alpha-chemokine interleukin-8 (IL-8) and the beta-chemokine monocyte chemotactic protein-1 (MCP-1), in the promyelocytic cell line HL60 transfected with peripheral cannabinoid receptors (CB2). These genomic modulations observed on large-scale cDNA arrays were first confirmed by Northern blot studies. Furthermore, ELISA evaluations in culture supernatants indicated that the cannabinoid-induced activation of these two chemokine genes was followed by enhanced expression and secretion of the corresponding proteins. These upregulations initially observed in transfected HL60 cells overexpressing CB2 receptors, also occurred in normal non-transfected HL60 cells. The enhancement of IL-8 and MCP-1 gene transcription and protein production was shown to be pertussis toxin sensitive attesting that this phenomenon was a Gi protein-coupled receptor-mediated process as expected for cannabinoid receptors. More specifically, the abolition of the cannabinoid-induced effect by the specific CB2 antagonist SR 144528 indicated a strict peripheral cannabinoid-mediated process. Altogether, our data highlight a possible new function of peripheral cannabinoid receptors in the modulation of immune and inflammatory responses.

Gi protein modulation induced by a selective inverse agonist for the peripheral cannabinoid receptor CB2: implication for intracellular signalization cross-regulation.

The peripheral cannabinoid receptor (CB2) is a G protein-coupled receptor that is both positively and negatively coupled to the mitogen-activated protein kinase (MAPK) and cAMP pathways, respectively, through a Bordetella pertussis toxin-sensitive G protein. CB2 receptor-transfected Chinese hamster ovary cells exhibit high constitutive activity blocked by the CB2-selective ligand, SR 144528, working as an inverse agonist. We showed here that in addition to the inhibition of autoactivated CB2 in this model, we found that SR 144528 inhibited the MAPK activation induced by Gi-dependent receptors such as receptor-tyrosine kinase (insulin, insulin-like growth factor 1) or G protein-coupled receptors (lysophosphatidic acid), but not by Gi-independent receptors such as the fibroblast growth factor receptor. We showed that this SR 144528 inhibitory effect on Gi-dependent receptors was mediated by a direct Gi protein inhibition through CB2 receptors. Indeed, we found that through binding to the CB2 receptors, SR 144528 blocked the direct activation of the Gi protein by mastoparan analog in Chinese hamster ovary CB2 cell membranes. Furthermore, we described that sustained treatment with SR 144528 induced an up-regulation of the cellular Gi protein level as shown in Western blotting as well as in confocal microscopic experiments. This up-regulation occurred with a concomitant loss of SR 144528 ability to inhibit the insulin or lysophosphatidic acid-induced MAPK activation. This inverse agonist-induced modulation of the Gi strongly suggests that the modulated protein is functionally associated with the complex SR 144528/CB2 receptors, and that the Gi level may account for the heterologous desensitization phenomena.