Baldock et al.: Neuropeptides 46(6): 383-394, 2012 The endogenous opioid dynorphin is required for normal bone homeostasis in mice Paul A. Baldock a,c,f , Frank Driessler a , Shu Lin a , Iris P.L. Wong a , Yanchuan Shi a , Ernie Yulyaningsih a , Lesley Castillo a , Sonia Janmaat a , Ronaldo F. Enriquez a , Ayse Zengin a , Brigitte L. Kieffer d , Christoph Schwarzer e , John A. Eisman b,c , Amanda Sainsbury a,g , Herbert Herzog a,c,f a Neuroscience Research Program, Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, Sydney, NSW 2010, Australia b Osteoporosis and Bone Biology Research Program, Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, Sydney, NSW 2010, Australia c Faculty of Medicine, University of NSW, Sydney, NSW, Australia d Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université de Strasbourg, Illkirch, France e Department of Pharmacology, Innsbruck Medical University, Innsbruck, Austria f St. Vincent’s Hospital Clinical School, University of New South Wales, Kensington, NSW, Australia g School of Medical Sciences, University of New South Wales, Kensington, NSW, Australia Abstract Chronic opiate usage, whether prescribed or illicit, has been associated with changes in bone mass and is a recognized risk factor for the development of osteoporosis; however, the mechanism behind this effect is unknown. Here we show that lack of dynorphin, an endogenous opioid, in mice (Dyn−/−), resulted in a significantly elevated cancellous bone volume associated with greater mineral apposition rate and increased resorption indices. A similar anabolic phenotype was evident in bone of mice lacking dynorphin’s cognate receptor, the kappa opioid receptor. Lack of opioid receptor expression in primary osteoblastic cultures and no change in bone cell function after dynorphin agonist treatment in vitro indicates an indirect mode of action. Consistent with a hypothalamic action, central dynorphin signaling induces extracellular signal-regulated kinase (ERK) phosphorylation and c-fos activation of neurons in the arcuate nucleus of the hypothalamus (Arc). Importantly, this signaling also leads to an increase in Arc NPY mRNA expression, a change known to decrease bone formation. Further implicating NPY in the skeletal effects of dynorphin, Dyn−/−/NPY−/− double mutant mice showed comparable increases in bone formation to single mutant mice, suggesting that dynorphin acts upstream of NPY signaling to control bone formation. Thus the dynorphin system, acting via NPY, may represent a pathway by which higher processes including stress, reward/addiction and depression influence skeletal metabolism. Moreover, understanding of these unique interactions may enable modulation of the adverse effects of exogenous opioid treatment without directly affecting analgesic responses. 1. Introduction The opioid family is complex, consisting of more than 20 peptides derived from three independent genes, proopiomelanocortin (POMC), proenkephalin (PENK), and prodynorphin (PDyn) (Boer and Van Minnen, 1985). These genes produce inactive polypeptide precursors that are activated following tissue-specific processing. In the case of the prodynorphin precursor, processing produces six dynorphins: dynorphin A, dynorphin B, alpha- and beta- neoendorphin, leumorphine and big dynorphin (Han and xie, 1982). Dynorphin is produced almost exclusively in the CNS, including the hypothalamus (Lin et al., 2006). Pharmacologically, dynorphins preferentially bind to the kappa-opioid receptor (KOR), but they also bind with reduced affinity to mu-opioid receptors (MOR) and delta-opioid receptors (DOR) (Mansour et al., 1995). While opiate analgesia is the result of activation of MOR signaling (Matthes et al., 1996), both dynorphin and KOR expression are elevated by chronic opiate exposure (Wang et al., 1999) indicating that chronic opiate use may involve responses from multiple opioid receptors, including the dynorphin/KOR system. Opioids are critical agents for pain management but chronic use is associated with adverse side effects including an increased risk for the development of osteoporosis, affecting people of all ages, not just the elderly (Daniell et al., 2006 and Shorr et al., 1992). Several