Jonathan H. Gooi

Osteocyte Wnt/β-Catenin Signaling Is Required for Normal Bone Homeostasis

ABSTRACT β-Catenin-dependent canonical Wnt signaling plays an important role in bone metabolism by controlling differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. To investigate its function in osteocytes, the cell type constituting the majority of bone cells, we generated osteocyte-specific β-catenin-deficient mice (Ctnnb1loxP/loxP; Dmp1-Cre). Homozygous mutants were born at normal Mendelian frequency with no obvious morphological abnormalities or detectable differences in size or body weight, but bone mass accrual was strongly impaired due to early-onset, progressive bone loss in the appendicular and axial skeleton with mild growth retardation and premature lethality. Cancellous bone mass was almost completely absent, and cortical bone thickness was dramatically reduced. The low-bone-mass phenotype was associated with increased osteoclast number and activity, whereas osteoblast function and osteocyte density were normal. Cortical bone Wnt/β-catenin target gene expression was reduced, and of the known regulators of osteoclast differentiation, osteoprotegerin (OPG) expression was significantly downregulated in osteocyte bone fractions of mutant mice. Moreover, the OPG levels expressed by osteocytes were higher than or comparable to the levels expressed by osteoblasts during skeletal growth and at maturity, suggesting that the reduction in osteocytic OPG and the concomitant increase in osteocytic RANKL/OPG ratio contribute to the increased number of osteoclasts and resorption in osteocyte-specific β-catenin mutants. Together, these results reveal a crucial novel function for osteocyte β-catenin signaling in controlling bone homeostasis.

EphrinB2 regulation by PTH and PTHrP revealed by molecular profiling in differentiating osteoblasts.

With the aim of identifying new pathways and genes regulated by PTH(1–34) and PTH‐related protein 1–141 [PTHrP(1–141)] in osteoblasts, this study was carried out using a mouse marrow stromal cell line, Kusa 4b10, that acquires features of the osteoblastic phenotype in long‐term culture conditions. After the appearance of functional PTH receptor 1 (PTHR1) in Kusa 4b10 cells, they were treated with either PTH(1–34) or PTHrP(1–141), and RNA was subjected to Affymetrix whole mouse genome array. The microarray data were validated using quantitative real‐time RT‐PCR on independently prepared RNA samples from differentiated Kusa 4b10, UMR106 osteosarcoma cells, and primary mouse calvarial osteoblasts, as well as in vivo using RNA from metaphyseal bone after a single PTH injection to 3‐wk‐old and 6‐mo‐old ovariectomized rats. Of the 45,101 probes used on the microarray, 4675 were differentially expressed by ≥1.5 fold, with a false discovery rate <0.1. Among the regulated genes, ephrinB2 mRNA was upregulated in response to both PTH and PTHrP. This was confirmed by quantitative real‐time PCR in vitro and in vivo. Increased ephrinB2 protein was also shown in vitro by Western blotting, and immunostaining of femur sections showed ephrinB2 in both osteoclasts and osteoblasts. Production of ephrinB2, as well as other ephrins or Eph family members, did not change during differentiation of Kusa 4b10 cells. Blockade of ephrinB2/EphB4 interaction resulted in inhibition of mineralization of Kusa 4b10 cells. Together with the shown effect of ephrinB2 promoting osteoblast differentiation and bone formation through action on EphB4, the data raise the possibility that PTH or PTHrP might regulate ephrinB2 to act in a paracrine or autocrine manner on EphB4 or EphB2 in the osteoblast, contributing as a local event to the anabolic action of PTH or PTHrP.

Calcitonin impairs the anabolic effect of PTH in young rats and stimulates expression of sclerostin by osteocytes

The therapeutic goal of increasing bone mass by co-treatment of parathyroid hormone (PTH) and an osteoclast inhibitor has been complicated by the undefined contribution of osteoclasts to the anabolic activity of PTH. To determine whether active osteoclasts are required at the time of PTH administration, we administered a low dose of the transient osteoclast inhibitor salmon calcitonin (sCT) to young rats receiving an anabolic PTH regimen. Co-administration of sCT significantly blunted the anabolic effect of PTH as measured by peripheral quantitative computer tomography (pQCT) and histomorphometry in the femur and tibia, respectively. To determine gene targets of sCT, we carried out quantitative real time PCR and microarray analysis of metaphyseal samples 1.5, 4 and 6.5h after administration of a single injection of PTH, sCT or PTH+sCT. Known targets of PTH action, IL-6, ephrinB2 and RANKL, were not modified by co-administration with sCT. Surprisingly, at all time points, we noted a significant upregulation of sclerostin mRNA by sCT treatment, as well as down-regulation of two other osteocyte gene products, MEPE and DMP1. Immunohistochemistry confirmed that sCT administration increased the percentage of osteocytes expressing sclerostin, suggesting a mechanism by which sCT reduced the anabolic effect of PTH. Neither mRNA for CT receptor (Calcr) nor labeled CT binding could be detected in sclerostin-enriched cells differentiated from primary calvarial osteoblasts. In contrast, osteocytes freshly isolated from calvariae expressed a high level of Calcr mRNA. Furthermore immunohistochemistry revealed co-localization of CT receptor (CTR) and sclerostin in some osteocytes in calvarial sections. Taken together these data indicate that co-treatment with sCT can blunt the anabolic effect of PTH and this may involve direct stimulation of sclerostin production by osteocytes. These data directly implicate calcitonin as a negative regulator of bone formation through a previously unsuspected mechanism.

Localization of relaxin receptors in arteries and veins, and region-specific increases in compliance and bradykinin-mediated relaxation after in vivo serelaxin treatment

Relaxin is a potent vasodilator of small resistance arteries and modifies arterial compliance in some systemic vascular beds, yet receptors for relaxin, such as RXFP1, have only been localized to vascular smooth muscle. This study first aimed to localize RXFP1 in rat arteries and veins from different organ beds and determine whether receptors are present in endothelial cells. We then tested the hypothesis that region‐specific vascular effects of relaxin may be influenced by the cellular localization of RXFP1 within different blood vessels. The aorta, vena cava, mesenteric artery, and vein had significantly higher (P<0.05) RXFP1 immunostaining in endothelial cells compared with vascular smooth muscle, whereas the femoral artery and vein and small pulmonary arteries had higher (P<0.01) RXFP1 immunostaining in the vascular smooth muscle. Male rats were treated subcutaneously with recombinant human relaxin‐2 (serelaxin; 4 μg/h) for 5 d; vasodilation and compliance in mesenteric and femoral arteries and veins were compared with placebo controls. Serelaxin significantly (P=0.04) reduced wall stiffness and increased volume compliance in mesenteric arteries but not in the other vessels examined. This was associated with changes in geometrical properties, and not compositional changes in the extracellular matrix. Serelaxin treatment had no effect on acetylcholine‐mediated relaxation but significantly (P< 0.001) enhanced bradykinin (BK)‐mediated relaxation in mesenteric arteries, involving enhanced nitric oxide but not endothelium‐derived hyperpolarization or vasodilatory prostanoids. In conclusion, there is differential distribution of RXFP1 on endothelial and smooth muscle across the vasculature. In rats, mesenteric arteries exhibit the greatest functional response to chronic serelaxin treatment.—Jelinic, M., Leo, C‐H., Post Uiterweer, E. P., Sandow, S. L., Gooi, J. H., Wlodek, M. E., Conrad, K. P., Parkington, H., Tare, M., Parry, L. J. Localization of relaxin receptors in arteries and veins, and region‐specific increases in compliance and bradykinin‐mediated relaxation after in vivo serelaxin treatment. FASEB J. 28, 275–287 (2014). www.fasebj.org

The Wnt Inhibitor Sclerostin Is Up-regulated by Mechanical Unloading in Osteocytes in Vitro

Background: Recent studies have suggested osteocytes as key players in mechanosensation and skeletal metabolism. Results: Simulated microgravity induces an autonomous up-regulation of SOST/sclerostin and RANKL/OPG in a novel osteocytic cell line, Ocy454. Conclusion: Mechanical loading regulates intrinsic osteocyte responses in concert with hormonal and cytokine inputs. Significance: Learning how osteocytes sense mechanical loads would enable novel interventions to prevent disuse-induced bone loss. Although bone responds to its mechanical environment, the cellular and molecular mechanisms underlying the response of the skeleton to mechanical unloading are not completely understood. Osteocytes are the most abundant but least understood cells in bones and are thought to be responsible for sensing stresses and strains in bone. Sclerostin, a product of the SOST gene, is produced postnatally primarily by osteocytes and is a negative regulator of bone formation. Recent studies show that SOST is mechanically regulated at both the mRNA and protein levels. During prolonged bed rest and immobilization, circulating sclerostin increases both in humans and in animal models, and its increase is associated with a decrease in parathyroid hormone. To investigate whether SOST/sclerostin up-regulation in mechanical unloading is a cell-autonomous response or a hormonal response to decreased parathyroid hormone levels, we subjected osteocytes to an in vitro unloading environment achieved by the NASA rotating wall vessel system. To perform these studies, we generated a novel osteocytic cell line (Ocy454) that produces high levels of SOST/sclerostin at early time points and in the absence of differentiation factors. Importantly, these osteocytes recapitulated the in vivo response to mechanical unloading with increased expression of SOST (3.4 ± 1.9-fold, p < 0.001), sclerostin (4.7 ± 0.1-fold, p < 0.001), and the receptor activator of nuclear factor κΒ ligand (RANKL)/osteoprotegerin (OPG) (2.5 ± 0.7-fold, p < 0.001) ratio. These data demonstrate for the first time a cell-autonomous increase in SOST/sclerostin and RANKL/OPG ratio in the setting of unloading. Thus, targeted osteocyte therapies could hold promise as novel osteoporosis and disuse-induced bone loss treatments by directly modulating the mechanosensing cells in bone.

Communication Between EphrinB2 and EphB4 Within the Osteoblast Lineage

Members of the ephrin and Eph family are local mediators of cell function through largely contact-dependent processes in development and in maturity. Production of ephrinB2 mRNA and protein are increased by PTH and PTHrP in osteoblasts. Both a synthetic peptide antagonist of ephrinB2/EphB4 receptor interaction and recombinant soluble extracellular domain of EphB4 (sEphB4), which is an antagonist of both forward and reverse EphB4 signaling, were able to inhibit mineralization and the expression of several osteoblast genes involved late in osteoblast differentiation. The findings are consistent with ephrinB2/EphB4 signaling within the osteoblast lineage having a paracrine role in osteoblast differentiation, in addition to the proposed role of osteoclast-derived ephrinB2 in coupling of bone formation to resorption. This local regulation might contribute to control of osteoblast differentiation and bone formation at remodeling sites, and perhaps also in modeling.

Reversing LRP5‐Dependent Osteoporosis and SOST Deficiency–Induced Sclerosing Bone Disorders by Altering WNT Signaling Activity

The bone formation inhibitor sclerostin encoded by SOST binds in vitro to low‐density lipoprotein receptor‐related protein (LRP) 5/6 Wnt co‐receptors, thereby inhibiting Wnt/β‐catenin signaling, a central pathway of skeletal homeostasis. Lrp5/LRP5 deficiency results in osteoporosis‐pseudoglioma (OPPG), whereas Sost/SOST deficiency induces lifelong bone gain in mice and humans. Here, we analyzed the bone phenotype of mice lacking Sost (Sost−/−), Lrp5 (Lrp5−/−), or both (Sost−/−;Lrp5−/−) to elucidate the mechanism of action of Sost in vivo. Sost deficiency–induced bone gain was significantly blunted in Sost−/−;Lrp5−/− mice. Yet the Lrp5 OPPG phenotype was fully rescued in Sost−/−;Lrp5−/− mice and most bone parameters were elevated relative to wild‐type. To test whether the remaining bone increases in Sost−/−;Lrp5−/− animals depend on Lrp6, we treated wild‐type, Sost−/−, and Sost−/−;Lrp5−/− mice with distinct Lrp6 function blocking antibodies. Selective blockage of Wnt1 class–mediated Lrp6 signaling reduced cancellous bone mass and density in wild‐type mice. Surprisingly, it reversed the abnormal bone gain in Sost−/− and Sost−/−;Lrp5−/− mice to wild‐type levels irrespective of enhancement or blockage of Wnt3a class‐mediated Lrp6 activity. Thus, whereas Sost deficiency–induced bone anabolism partially requires Lrp5, it fully depends on Wnt1 class–induced Lrp6 activity. These findings indicate: first, that OPPG syndrome patients suffering from LRP5 loss‐of‐function should benefit from principles antagonizing SOST/sclerostin action; and second, that therapeutic WNT signaling inhibitors may stop the debilitating bone overgrowth in sclerosing disorders related to SOST deficiency, such as sclerosteosis, van Buchem disease, and autosomal dominant craniodiaphyseal dysplasia, which are rare disorders without viable treatment options.

The Chemokine Cxcl1 Is a Novel Target Gene of Parathyroid Hormone (PTH)/PTH-Related Protein in Committed Osteoblasts

The PTH receptor (PTHR1) is expressed on osteoblasts and responds to PTH or PTHrP in an endocrine or autocrine/paracrine manner, respectively. A microarray study carried out on PTHR1-positive osteoblasts (Kusa 4b10 cells) identified the cysteine-X-cysteine (CXC) family chemokine ligand 1 (Cxcl1) as a novel immediate PTH/PTHrP-responsive gene. Cxcl1 is a potent neutrophil chemoattractant with recognized roles in angiogenesis and inflammation, but a role in bone biology has not been described. Cxcl1 mRNA levels were up-regulated 1 h after either PTH or PTHrP treatment of differentiated Kusa 4b10 osteoblasts (15-fold) and mouse calvarial osteoblasts (160-fold) and in rat metaphyseal bone (5-fold) 1 h after a single sc injection of PTH. Furthermore, PTH treatment stimulated a 10-fold increase in secreted Cxcl1 protein by both Kusa 4b10 cells and calvarial osteoblasts. Immunohistochemistry and PCR demonstrated that CXCR2, the receptor for Cxcl1, is highly expressed in osteoclast precursors (hemopoietic cells) but is predominantly undetectable in the osteoblast lineage, suggesting that osteoblast-derived Cxcl1 may act as a chemoattractant for osteoclast precursors. Confirming this hypothesis, recombinant Cxcl1 dose-dependently stimulated migration of osteoclast precursors in cell culture studies, as did conditioned media from Kusa 4b10 cells treated with PTH. These data indicate that local action through the PTHR1 could stimulate cells of the osteoblast lineage to release a chemokine capable of attracting osteoclast precursors to the bone environment.

Aging attenuates the vasodilator response to relaxin

Relaxin, an insulin-like growth factor peptide, increases endothelium-dependent vasodilation and vascular compliance and decreases myogenic reactivity. These vascular effects significantly contribute to the physiological circulatory adaptations in pregnancy, particularly in the mesentery and kidney. Aging predisposes to vascular maladaptation and gestational hypertensive disease. We hypothesized that mild aging reduces the vascular responses to relaxin. In 20 young (10-12 wk) and 20 middle-aged (40-46 wk) female Wistar Hannover rats, vascular responses to chronic exposure of relaxin vs. placebo (5 days) were quantified in isolated mesenteric arteries and kidney. Vascular responses were evaluated using pressure-perfusion myograph, wire myograph, and an isolated perfused rat kidney model. Rxfp1 (relaxin family peptide) gene expression was determined by quantitative polymerase chain reaction. In young rats, relaxin stimulated nitric oxide (NO)-dependent flow-mediated vasodilation (2.67-fold, from 48 ± 9 to 18 ± 4 μl/min), reduced myogenic reactivity (from -1 ± 2 to 7 ± 3 μm/10 mmHg), and decreased mesenteric sensitivity to (28%, from 1.39 ± 0.08 to 1.78 ± 0.10 μM) but did not change compliance and renal perfusion flow (RPFF). In aged rats, relaxin did not affect any of the analyzed mesenteric or renal parameters. In aged compared with young placebo-treated rats, all mesenteric characteristics were comparable, while RPFF was lower (17%, from 6.9 ± 0.2 to 5.7 ± 0.1 ml·min⁻¹·100 g⁻¹) even though NO availability was comparable. Rxfp1 expression was not different among young and aged rats. Our findings suggest that moderate aging involves normal endothelial function but blunts the physiological endothelium-dependent and -independent vasodilator response to relaxin.

A Vasoactive Role for Endogenous Relaxin in Mesenteric Arteries of Male Mice

The peptide hormone relaxin has striking effects on the vascular system. Specifically, endogenous relaxin treatment reduces myogenic reactivity through nitric oxide (NO)-mediated vasorelaxation and increases arterial compliance in small resistance arteries. However, less is known about the vascular roles of endogenous relaxin, particularly in males. Therefore, we used male wild-type (Rln +/+) and relaxin knockout (Rln −/−) mice to test the hypothesis that passive wall properties and vascular reactivity in mesenteric arteries would be compromised in Rln −/− mice. Passive compliance was determined in arteries (n = 8–9) mounted on a pressure myograph and in Ca2+-free Krebs containing 2 mM EGTA. Passive volume compliance was significantly (P = 0.01) decreased in the mesenteric arteries of Rln −/− mice. Vascular reactivity was assessed using wire myography. In mesenteric arteries (n = 5) of Rln −/− mice, there was a significant (P<0.03) increase in sensitivity to the vasoconstrictors phenylephrine and thromboxane-mimetic U41669. This enhanced responsiveness to vasoconstrictors was abolished by endothelial denudation, and attributed to impaired NO and prostanoid pathways in Rln −/− mice. Sensitivity to the endothelial agonist acetylcholine was significantly (n = 7–9, P≤0.03) decreased, and this was abolished in the presence of the cyclooxygenase inhibitor, indomethacin (2 µM). This indicates that prostanoid vasoconstrictor pathways were upregulated in the mesenteric arteries of Rln −/− mice. In summary, we demonstrate endothelial dysfunction and impaired arterial wall remodeling in male mice deficient in relaxin. Thus, our results highlight a role for endogenous relaxin in the maintenance of normal mesenteric artery structure and function in males.