Functions of vasopressin and oxytocin in bone mass regulation

Prior studies show that oxytocin (Oxt) and vasopressin (Avp) have opposing actions on the skeleton exerted through high-affinity G protein-coupled receptors. We explored whether Avp and Oxtr can share their receptors in the regulation of bone formation by osteoblasts. We show that the Avp receptor 1α (Avpr1α) and the Oxt receptor (Oxtr) have opposing effects on bone mass: Oxtr^−/− mice have osteopenia, and Avpr1α^−/− mice display a high bone mass phenotype. More notably, this high bone mass phenotype is reversed by the deletion of Oxtr in Oxtr^−/−:Avpr1α^−/− double-mutant mice. However, although Oxtr is not indispensable for Avp action in inhibiting osteoblastogenesis and gene expression, Avp-stimulated gene expression is inhibited when the Oxtr is deleted in Avpr1α^−/− cells. In contrast, Oxt does not interact with Avprs in vivo in a model of lactation-induced bone loss in which Oxt levels are high. Immunofluorescence microscopy of isolated nucleoplasts and Western blotting and MALDI-TOF of nuclear extracts show that Avp triggers Avpr1α localization to the nucleus. Finally, a specific Avpr2 inhibitor, tolvaptan, does not affect bone formation or bone mass, suggesting that Avpr2, which primarily functions in the kidney, does not have a significant role in bone remodeling.Over the past decade, we have described direct actions of anterior and posterior pituitary hormones on the skeleton (1–8). We have shown that these actions are exerted via G protein-coupled receptors resident on both osteoblasts and osteoclasts. We also find that the skeleton is highly sensitive to the action of posterior pituitary hormones; for example, mice haploinsufficient in oxytocin (Oxt) have osteopenic bones, but lactation is normal; lactation is impaired only in Oxt^−/− mice (2). Likewise, Tshr haploinsufficient mice are completely euthyroid with normal thyroid follicles but display significant osteopenia (4). The exquisite sensitivity of the skeleton to pituitary hormones comes as no surprise, considering that the pituitary gland and the skeleton are both evolutionarily more primitive than target endocrine organs (7).Apart from the known actions of growth hormone on the skeleton, Tsh, Fsh, Acth, Oxt, and vasopressin (Avp) have all been shown to regulate the formation and/or function of both osteoblasts and osteoclasts and thus to control bone remodeling in vivo (2–4, 6–8). The two neurohypophyseal hormones Oxt and Avp have opposing functions (2, 3). Oxt stimulates and Avp inhibits osteoblast formation. Consequently, the genetic deletion of the Oxt receptor (Oxtr) and Avp receptor 1α (Avpr1α) yields opposing phenotypes, notably osteopenia in Oxtr^−/− mice and high bone mass in Avpr1α^−/− mice (2, 3). These findings may explain the rapid recovery of bone loss at weaning when plasma Oxt levels are high (9) and also the profound loss of bone noted in chronic hyponatremic states, such as the syndrome of inappropriate antidiuretic hormone secretion (SIADH), in which serum Avp levels are elevated (3).We find high levels of Oxtr expression on both osteoclasts and osteoblasts (2, 10), in addition to their abundant expression in breast and uterine tissue, where they regulate lactation and parturition, respectively (11). Avpr1αs, in contrast, are distributed more ubiquitously, whereas Avpr2s are localized mainly in the kidney, where they regulate free water excretion (12). Osteoblasts express both Avpr1α and Avrpr2 (3). The only other known isoform, Avpr1β, is expressed predominantly in the pancreas and pituitary; it regulates ACTH secretion from pituitary corticotrophs (13). Sequence alignment shows that the binding sites of the Oxtr and Avprs are highly conserved, with specific amino acids within the predicted binding pocket providing ligand selectivity (14–16). The respective ligands Oxt and Avp also are homologous nonapeptides, differing in only two amino acids, and are known to interact with the other’s receptor with different affinities (17).To our knowledge, osteoblasts and osteoclasts are the only cells in which Oxtr, Avpr1α, and Avpr2 are coexpressed. We also have shown that osteoblastic Oxtrs undergo internalization and nuclear translocation upon binding to Oxt and that this action is independent of cytosolic Erk phosphorylation (18). Avpr1α activation by Avp also activates Erk phosphorylation within minutes (3). The homology between the ligands and their respective receptors and converging downstream signals suggest that Avp and Oxtr may share receptors with opposing or convergent signals. Here, we have explored these interactions in the regulation of osteoblastic bone formation by using mice lacking one or both receptors, chemical inhibitors, and physiological models of high bone turnover.