IL-6, leukemia inhibitory factor (LIF), and oncostatin M (OSM) are IL-6-type cytokines that stimulate osteoclast formation and function. In the present study, the resorptive effects of these agents and their regulation of receptor activator of NF-κB ligand (RANKL), RANK, and osteoprotegerin (OPG) were studied in neonatal mouse calvaria. When tested separately, neither human (h) IL-6 nor the human soluble IL-6R (shIL-6R) stimulated bone resorption, but when hIL-6 and the shIL-6R were combined, significant stimulation of both mineral and matrix release from bone explants was noted. Semiquantitative RT-PCR showed that hIL-6 plus shIL-6R enhanced the expression of RANKL and OPG in calvarial bones, but decreased RANK expression. Human LIF, hOSM, and mouse OSM (mOSM) also stimulated 45Ca release and enhanced the mRNA expression of RANKL and OPG in mouse calvaria, but had no effect on the expression of RANK. In agreement with the RT-PCR analyses, ELISA measurements showed that both hIL-6 plus shIL-6R and mOSM increased RANKL and OPG proteins. 1,25-Dihydroxyvitamin D3 (D3) also increased the RANKL protein level, but decreased the protein level of OPG. OPG inhibited 45Ca release stimulated by RANKL, hIL-6 plus shIL-6R, hLIF, hOSM, mOSM, and D3. An Ab neutralizing mouse gp130 inhibited 45Ca release induced by hIL-6 plus shIL-6R. These experiments demonstrated stimulation of calvarial bone resorption and regulation of mRNA and protein expression of RANKL and OPG by D3 and IL-6 family cytokines as well as regulation of RANK expression in preosteoclasts/osteoclasts of mouse calvaria by D3 and hIL-6 plus shIL-6R.

SUMMARY The natural response to itch sensation is to scratch, which relieves the itch through an unknown mechanism. Interaction between pain and itch has been frequently demonstrated, and the selectivity hypothesis of itch, based on data from electrophysiological and behavioral experiments, postulates the existence of primary pain afferents capable of repressing itch. Here, we demonstrate that deletion of vesicular glutamate transporter (VGLUT) 2 in a subpopulation of neurons partly overlapping with the vanilloid receptor (TRPV1) primary afferents resulted in a dramatic increase in itch behavior accompanied by a reduced responsiveness to thermal pain. The increased itch behavior was reduced by administration of antihistaminergic drugs and by genetic deletion of the gastrin-releasing peptide receptor, demonstrating a dependence on VGLUT2 to maintain normal levels of both histaminergic and nonhistaminergic itch. This study establishes that VGLUT2 is a major player in TRPV1 thermal nociception and also serves to regulate a normal itch response.

Immune cells are thought to play an important role in bone loss caused by inflammation (1, 2). Disabling joint destruction in rheumatoid arthritis and the loss of teeth in periodontal disease are examples of skeletal loss that can occur with inflammation. Resorption of bone by osteoclasts represents the primary mechanism responsible for the loss of bone caused by inflammatory disease.Key factors regulating osteoclastogenesis include macrophage colony-stimulating factor (M-CSF) 2 and receptor activator of nuclear factor-B ligand (RANKL). M-CSF is a secreted product of stromal cells/ osteoblasts that enhances colony expansion of monocyte/osteoclast progenitor cells. RANKL exists both as a transmembrane protein in osteoblasts/stromal cells and as a soluble protein (3-6). RANKL directs the expanded progenitor cell population to the osteoclast lineage by activation of receptor activator of nuclear factor-B (RANK). The interaction between RANKL and RANK can be inhibited by osteoprotegerin (OPG), a decoy receptor released from stromal cells/osteoblasts.RANKL stimulation of RANK causes receptor trimerization and recruitment of tumor necrosis factor receptor-associated factors (TRAFs). TRAF1, 2, 3, and 5 bind to the carboxyl-terminal end of the RANK trimer, whereas TRAF6 binds more closely to the membrane. Downstream intracellular signaling mediated by RANK in osteoclast progenitor cells includes TRAF6-dependent activation of NF-B, mitogen-activated protein kinases (MAP kinases) and AP-1, and activation of c-Src and the phosphatidylinositol 3-kinase/Akt pathway (5-7). In addition, immunoreceptor tyrosine-based activation motif-mediated costimulatory signals have been shown to be required for expression of nuclear factor of activated T-cells 2 (NFAT2), the transcription factor believed to be crucial for osteoclast differentiation (8, 9). Several of these intracellular signaling molecules, including p50/p65, c-Fos, NFAT2, Fc receptor common ␥ subunit (FcR␥)/DNAX-activation protein 12 kD (DAP 12), and TRAF6, as well as RANK, RANKL, OPG, M-CSF, and the M-CSF receptor c-Fms, have been shown by gene deletion studies to be essential for osteoclastogenesis (2, 7, 10 -14).* This work was supported by grants from the Swedish Science Council (project 07525), the Swedish Rheumatism Association, the Royal 80 Year Fund of King Gustav V, the Swedish Dental Society, and the County Council of Västerbotten. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed. Tel.: 46-90-785-6290; Fax: 46-90-139289; E-mail: ulf.lerner@odont.umu.se.2 The abbreviations used are: M-CSF, macrophage colony-stimulating factor; ALP, alkaline phosphatase; ␣-MEM, ␣-modification of minimum essential medium; BMM, bone marrow macrophages; CT, calcitonin; CTR, calcitonin receptor; DAP12, DNAX-activating protein 12; D3, 1,25(OH) 2 -vitamin D 3 ; ERK, e...

Background and purpose: Two compounds, URB602 and URB754, have been reported in the literature to be selective inhibitors of monoacylglycerol lipase, although a recent study has questioned their ability to prevent 2-arachidonoyl hydrolysis by brain homogenates and cerebellar membranes. In the present study, the ability of these compounds to inhibit monoacylglycerol lipase and fatty acid amide hydrolase has been reinvestigated. Experimental approach: Homogenates and cell lines were incubated with test compounds and, thereafter, with either3 H]-anandamide. Labelled reaction products were separated from substrate using chloroform: methanol extraction. Key results: In cytosolic fractions from rat brain, URB602 and URB754 inhibited the hydrolysis of 2-oleoylglycerol with IC 50 values of 25 and 48 mM, respectively. Anandamide hydrolysis by brain membranes was not sensitive to URB754, but was inhibited by URB602 (IC 50 value 17 mM). Hydrolysis of 2-oleoylglycerol by human recombinant monoacylglycerol lipase was sensitive to URB602, but not URB754. The lack of selectivity of URB602 for 2-oleoylglycerol compared to anandamide hydrolysis was also observed for intact RBL2H3 basophilic leukaemia cells. C6 glioma expressed mRNA for monoacylglycerol lipase, and hydrolyzed 2-oleoylglycerol in a manner sensitive to inhibition by methyl arachidonoyl fluorophosphonate but not URB754 or URB597. MC3T3-E1 mouse osteoblastic cells, which did not express mRNA for monoacylglycerol lipase, hydrolyzed 2-oleoylglycerol in the presence of URB597, but the hydrolysis was less sensitive to methyl arachidonoyl fluorophosphonate than for C6 cells. Conclusions and implications:The data demonstrate that the compounds URB602 and URB754 do not behave as selective and/or potent inhibitors of monoacylglycerol lipase.