Urocortin Is a Novel Regulator of Osteoclast Differentiation and Function Through Inhibition of a Canonical Transient Receptor Potential 1-Like Cation Channel
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187 Urocortin is a novel regulator of osteoclast differentiation and function through inhibition of a canonical transient receptor potential 1-like cation channel Charlotte E Combs, Karen Fuller, Hashethra Kumar, Anthony P Albert, Grisha Pirianov, James McCormick1, Ian C Locke2, Timothy J Chambers and Kevin M Lawrence Department of Cellular Pathology, St George’s, University of London, Cranmer Terrace, London SW17 ORE, UK 1Medical Molecular Biology Unit, Institute of Child Health, University College London, London WC1N 1EH, UK 2Department of Biomedical Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK (Correspondence should be addressed to K M Lawrence; Email: [email protected]) Abstract This study investigated the role of urocortin (UCN), a expressed CRF receptor 2b alone. Unstimulated osteoclasts member of the corticotrophin-releasing factor (CRF) family contained constitutively active cation channel currents with of peptides, in osteoclast maturation and function. We found a unitary conductance of 3–4 pS, which were inhibited by K K that 10 7 M UCN significantly (P!0.05) suppressed over 70% with UCN (10 7 M). Compounds that regulate osteoclast differentiation from bone marrow precursor cells calcium signalling and energy status of the cell, both crucial in culture and reduced the expression of several osteoclastic for osteoclast activity were investigated. The non-selective C markers. Furthermore, UCN potently suppressed osteoclast cation channel blockers, lanthanum (La3 ) and gadolinium C bone resorption, by significantly inhibiting both the plan (Gd3 ), inhibited actin ring formation in osteoclasts, C area of bone resorbed by osteoclasts and actin ring formation whereas modulators of voltage-dependent Ca2 channels K9 within osteoclasts at 10 M(P!0.05), with complete and KATP channels had no effect. These findings show for K inhibition at 10 7 M(P!0.001). UCN also inhibited the first time that UCN is a novel anti-resorptive molecule K osteoclast motility (10 7 M) but had no effect on osteoclast that acts through a direct effect on osteoclasts and their survival. Osteoclasts expressed mRNA encoding both UCN precursor cells. and the CRF receptor 2b subtype. Pre-osteoclasts however, Journal of Endocrinology (2012) 212, 187–197 Introduction receptor diversity produced by alternative RNA splicing (Chen et al. 1993, Lovenberg et al. 1995, Perrin & Vale 1999, Urocortin (UCN) is a member of the corticotrophin- Hauger et al. 2003, Fekete & Zorrilla 2007). Radioligand releasing factor (CRF) family of peptides. It was originally binding studies have demonstrated that CRF and UCN cloned from a rat brain cDNA library, using a probe can bind to both CRFR1 and CRFR2, whereas UCN 2 and 3 homologous to CRF (Vaughan et al. 1995). Since then, two bind exclusively to CRFR2. UCN has been shown to act new members have been isolated, UCN 2 and 3, which are predominantly through both Gas and Gaq G-protein subunits equivalent to human stresscopin-related peptide and stressco- to stimulate diverse signalling cascades, through respectively, pin respectively (Hsu & Hsueh 2001, Lewis et al. 2001, Reyes adenylate cyclase (AC) and phospholipase C (PLC) pathways, et al. 2001, Fekete & Zorrilla 2007). All three UCNs are depending on the tissue type (Grammatopoulos et al. synthesised as precursors of w200 amino acids, which are 2000, Cantarella et al. 2001, Graziani et al. 2002, McEvoy then subsequently cleaved to yield the active peptides, which et al.2002, Bayatti et al.2003, Karteris et al.2005). in the case of UCN is 40 amino acids (Fekete & Zorrilla UCN-mediated activation of these signal transduction 2007). The level of functional availability of UCN is closely pathways modulates a variety of end effectors including ion regulated by a high affinity CRF-binding protein (CRF-BP), channels involved in calcium signalling and energy expressed at both release and activation sites of UCN, which metabolism, such as L-type voltage-gated calcium channels, acts as a decoy receptor to terminate the ligand signal (Behan store-operated channels and KATP channels (Lawrence et al. et al. 1995, Seasholtz et al. 2002). 2002, Tao et al. 2004, 2006, Smani et al. 2007). UCN binds to two G-protein-coupled receptor sub- The CRF family members have been linked to a number of types, CRF receptor 1 (CRFR1) and CRFR2, with further physiological and pathological conditions. CRF is the classic Journal of Endocrinology (2012) 212, 187–197 DOI: 10.1530/JOE-11-0254 0022–0795/12/0212–187 q 2012 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/01/2021 09:58:39PM via free access 188 C E COMBS and others . Effect of urocortin on osteoclasts mediator of the hypothalamic stress response, initiating an performed at 37 8Cin5%CO2 in humidified air, unless stated anxiogenic, neuroendocrine and behavioural stress pheno- otherwise. Slices of bovine cortical bone were prepared as type. In contrast, UCN plays a more anxiolytic role in this previously described (Fuller et al. 2006). response and is also involved in appetite suppression and energy metabolism (Spina et al. 1996, Smagin et al. 1998, Tanaka et al. 2009). The peptides are also expressed in Generation of osteoclasts peripheral tissue, where they appear to be cytoprotective Osteoclast suspensions were prepared from murine bone through CRFR2, in models of cellular stress (Brar et al. 2002, marrow cells as previously described (Fuller et al. 2006). Lawrence & Latchman 2006). Recently, UCN has been Briefly, MF1 mice (4–8 weeks old) were killed by cervical found to be elevated in the synovial fluid of patients with dislocation in accord with Home Office Ethical Guidelines. rheumatoid arthritis (Kohno et al. 2001, Uzuki et al. 2001) Femora and tibiae were aseptically removed and dissected free and has also been linked to a reduction in inflammation and of adherent soft tissue. The bone ends were removed and the bone erosion in a mouse model of this disease (Gonzalez-Rey marrow cavity was flushed out into a Petri dish by slowly et al. 2007). However, there is little information on the role of injecting PBS at one end of the bone using a sterile 25-gauge UCN in the physiology or pathology of bone. Because UCN needle. The bone marrow suspension was passed repeatedly is the only member of this family capable of binding to both through a 21-gauge needle to obtain a single cell suspension. CRFR1 and 2, its use would give the best chance of detecting Bone marrow cells were then washed, re-suspended in an effect on osteoclasts. MEM/FCS, and incubated at a density of 3!105 cells/ml Osteoclasts are derived from the monocyte macrophage for 24 h in a 75 cm2 flask (Greiner Bio-One, Stonehouse, lineage; they are large multinucleated cells and are the only Gloucestershire, UK) containing M-CSF (50 ng/ml), to cells of the body capable of resorbing bone. They mature to deplete the cell preparations of stromal cells. Non-adherent form large tartrate-resistant acid phosphatase (TRAP) cells were collected by centrifugation and used immediately positive, multinucleated cells in the presence of receptor (pre-osteoclasts) or added to 90 mm diameter cell culture k activator of NF B ligand (RANKL) and macrophage colony dishes (Greiner) in MEM/FCS, containing M-CSF (50 ng/ml), stimulating factor (M-CSF). Upon activation, osteoclasts 6 RANKL (30 ng/ml) and TGF-b1(0.1 ng/ml) (7.2!10 locate to the bone surface and initiate resorption by creating cells in 25 ml for each dish). Cultures were incubated for a sealing zone, which is accompanied by a ruffled border 5 days, when osteoclast numbers are maximal. Cells were fed and actin reorganisation (Va¨a¨na¨nen et al. 2000). Excessive every 2–3 days by replacing 15 ml of culture medium with resorption by these cells is the cause of several common an equal volume of fresh medium and cytokines. When diseases of bone, including osteoporosis. multinuclear cells had formed, the medium was removed and In this study we investigated the effects of UCN on the the cell layer washed three times with PBS without calcium formation and function of osteoclasts. Our results clearly . demonstrate that UCN is a regulator of osteoclast resorption, and magnesium. Six millilitres of 0 02% EDTA were added to by suppressing osteoclast maturation and potently inhibiting the dish and cells incubated for 20 min at room temperature. osteoclastic function. Furthermore, we provide evidence that The EDTA was then removed from the dish and replaced the inhibitory action of UCN on osteoclasts is mediated by with 4 ml calcium/magnesium-free PBS. A cell scraper suppression of constitutively active cation channels that have (Greiner) was used to scrape the cells into the PBS, and the properties characteristic of a canonical transient receptor resulting cell suspension was agitated using a pipette to ensure potential 1 (TRPC1) channel. uniform cell dispersal for further use as described below. Assessment of bone resorption and actin ring formation Materials and Methods Seventy-five microlitres of osteoclast-containing cell suspension were added to wells of a 96-well plate (Greiner), Culture media and reagents each of which contained a bone slice in 75 ml MEM/FCS. Cells were incubated in minimum essential medium (MEM) Cells were allowed to sediment for 20 min at 37 8C with Earle’s salts, supplemented with 10% FCS, 2 mM before the bone slices were washed in PBS and transferred glutamine, 100 IU/ml benzylpenicillin and 100 mg/ml to fresh wells. Cells for assessment of resorption were streptomycin, UCN (all from Sigma), unless stated otherwise. incubated for 4 h in 200 ml MEM/FCS in the presence of Recombinant human M-CSF and soluble recombinant M-CSF (50 ng/ml), RANKL (30 ng/ml), IL1a (10 ng/ml) K K murine RANKL were purchased from PeproTech EC with UCN (10 7–10 10 M) or vehicle.