Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

PMCID: PMC379391

Nicotinic acetylcholine receptors modulate osteoclastogenesis

Background: Our aim was to investigate the role of nicotinic acetylcholine receptors (nAChRs) in in-vitro osteoclastogenesis and in in-vivo bone homeostasis. Methods: The presence of nAChR subunits as well as the in-vitro effects of nAChR agonists were investigated by ex vivo osteoclastogenesis assays, real-time polymerase chain reaction, Western blot and flow cytometry in murine bone marrow-derived macrophages differentiated in the presence of recombinant receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). The bone phenotype of mice lacking various nAChR subunits was investigated by peripheral quantitative computed tomography and histomorphometric analysis. Oscillations in the intracellular calcium concentration were detected by measuring the Fura-2 fluorescence intensity. Results: We could demonstrate the presence of several nAChR subunits in bone marrow-derived macrophages stimulated with RANKL and M-CSF, and showed that they are capable of producing acetylcholine. nAChR ligands reduced the number of osteoclasts as well as the number of tartrate-resistant acidic phosphatase-positive mononuclear cells in a dose-dependent manner. In vitro RANKL-mediated osteoclastogenesis was reduced in mice lacking α7 homomeric nAChR or β2-containing heteromeric nAChRs, while bone histomorphometry revealed increased bone volume as well as impaired osteoclastogenesis in male mice lacking the α7 nAChR. nAChR ligands inhibited RANKL-induced calcium oscillation, a well-established phenomenon of osteoclastogenesis. This inhibitory effect on Ca2+ oscillation subsequently led to the inhibition of RANKL-induced NFATc1 and c-fos expression after long-term treatment with nicotine. Conclusions: We have shown that the activity of nAChRs conveys a marked effect on osteoclastogenesis in mice. Agonists of these receptors inhibited calcium oscillations in osteoclasts and blocked the RANKL-induced activation of c-fos and NFATc1. RANKL-mediated in-vitro osteoclastogenesis was reduced in α7 knockout mice, which was paralleled by increased tibial bone volume in male mice in vivo. © 2016 Mandl et al

Changes in dorsal root ganglion CGRP expression in a chronic inflammatory model of the rat knee joint: differential modulation by rofecoxib and paracetamol.

Neuropeptide-expressing small diameter sensory neurones are thought to be vital in generating inflammatory hyperalgesic responses. Within the dorsal root ganglion (DRG), both the levels of the neuropeptide calcitonin gene-related peptide (CGRP) and the numbers of CGRP-immunoreactive (CGRP-IR) DRG neurones have been shown to increase in a number of acute adjuvant-induced inflammatory pain models. The aim of this study was to look specifically at changes in numbers of CGRP-IR DRG neurones in a chronic model of inflammatory joint pain following complete Freund's adjuvant (CFA) injection into the rat knee. In this model, there were significant increases in the number of ipsilateral CGRP-IR small DRG neurones at days 1, 16 and 35 following intra-articular CFA, compared to saline-injected sham animals. This correlated with the behavioural readouts of hypersensitivity and knee joint inflammation at the same time points. There was also a significant increase in the number of ipsilateral CGRP-IR medium DRG neurones and contralateral CGRP-IR small DRG neurones at day 1. Following dosing of CFA-injected rats with rofecoxib (Vioxx) or paracetamol, there was a significant decrease in the number of ipsilateral CGRP-IR small and medium DRG neurones in rofecoxib- but not paracetamol-treated rats. These data also correlated with behavioural readouts where hypersensitivity and knee joint inflammation were significantly reduced by rofecoxib but not paracetamol treatment. In conclusion, these data show that changes in ipsilateral CGRP expression within small DRG neurones are consistent with behavioural readouts in both time course, rofecoxib and paracetamol studies in this model of chronic inflammatory pain

The putative cannabinoid receptor GPR55 defines a novel autocrine loop in cancer cell proliferation

Recently, the orphan receptor G protein-coupled receptor 55 (GPR55) has been proposed as a potential cannabinoid receptor, although controversy remains on its physiological roles. Current evidence suggests a role for GPR55 as a receptor for the lysophospholipid lysophosphatidylinositol (LPI). In this study, we show that GPR55 is expressed in several prostate and ovarian cancer cell lines, both at the mRNA and at the protein level, and that it has a critical role in regulating proliferation and anchorage-independent growth. We further show that GPR55 mediates the effects of LPI in prostate and ovarian cancer cells. Indeed we demonstrate that LPI is able to induce calcium mobilization and activation of Akt and extracellular signal-regulated kinase (ERK)1/2 in these cells and that both pharmacological blockade of GPR55 and its downregulation using specific small interfering RNA strongly inhibits these processes. We further identify an autocrine loop by which LPI is synthesized by cytosolic phospholipase A2, pumped out of the cell by the ATP-binding cassette transporter ABCC1/MRP1, and is then able to initialize cascades downstream of GPR55. All together, these data demonstrate a role of LPI and its receptor GPR55 in cancer cells in activating an autocrine loop that regulates cell proliferation. These findings may have important implications for LPI as a novel cancer biomarker and for its receptor GPR55 as a potential therapeutic target. © 2011 Macmillan Publishers Limited All rights reserved