Marc N. Wein

RGS9 modulates dopamine signaling in the basal ganglia.

Regulators of G protein signaling (RGS) modulate heterotrimeric G proteins in part by serving as GTPase-activating proteins for Galpha subunits. We examined a role for RGS9-2, an RGS subtype highly enriched in striatum, in modulating dopamine D2 receptor function. Viral-mediated overexpression of RGS9-2 in rat nucleus accumbens (ventral striatum) reduced locomotor responses to cocaine (an indirect dopamine agonist) and to D2 but not to D1 receptor agonists. Conversely, RGS9 knockout mice showed heightened locomotor and rewarding responses to cocaine and related psychostimulants. In vitro expression of RGS9-2 in Xenopus oocytes accelerated the off-kinetics of D2 receptor-induced GIRK currents, consistent with the in vivo data. Finally, chronic cocaine exposure increased RGS9-2 levels in nucleus accumbens. Together, these data demonstrate a functional interaction between RGS9-2 and D2 receptor signaling and the behavioral actions of psychostimulants and suggest that psychostimulant induction of RGS9-2 represents a compensatory adaptation that diminishes drug responsiveness.

Pharmacologic targeting of a stem/progenitor population in vivo is associated with enhanced bone regeneration in mice.

Drug targeting of adult stem cells has been proposed as a strategy for regenerative medicine, but very few drugs are known to target stem cell populations in vivo. Mesenchymal stem/progenitor cells (MSCs) are a multipotent population of cells that can differentiate into muscle, bone, fat, and other cell types in context-specific manners. Bortezomib (Bzb) is a clinically available proteasome inhibitor used in the treatment of multiple myeloma. Here, we show that Bzb induces MSCs to preferentially undergo osteoblastic differentiation, in part by modulation of the bone-specifying transcription factor runt-related transcription factor 2 (Runx-2) in mice. Mice implanted with MSCs showed increased ectopic ossicle and bone formation when recipients received low doses of Bzb. Furthermore, this treatment increased bone formation and rescued bone loss in a mouse model of osteoporosis. Thus, we show that a tissue-resident adult stem cell population in vivo can be pharmacologically modified to promote a regenerative function in adult animals.

Regulation of Adult Bone Mass by the Zinc Finger Adapter Protein Schnurri-3

Genetic mutations that disrupt osteoblast function can result in skeletal dysmorphogenesis or, more rarely, in increased postnatal bone formation. Here we show that Schnurri-3 (Shn3), a mammalian homolog of the Drosophila zinc finger adapter protein Shn, is an essential regulator of adult bone formation. Mice lacking Shn3 display adult-onset osteosclerosis with increased bone mass due to augmented osteoblast activity. Shn3 was found to control protein levels of Runx2, the principal transcriptional regulator of osteoblast differentiation, by promoting its degradation through recruitment of the E3 ubiquitin ligase WWP1 to Runx2. By this means, Runx2-mediated extracellular matrix mineralization was antagonized, revealing an essential role for Shn3 as a central regulator of postnatal bone mass.

HDAC5 Controls MEF2C‐Driven Sclerostin Expression in Osteocytes

Osteocytes secrete paracrine factors that regulate the balance between bone formation and destruction. Among these molecules, sclerostin (encoded by the gene SOST) inhibits osteoblastic bone formation and is an osteoporosis drug target. The molecular mechanisms underlying SOST expression remain largely unexplored. Here, we report that histone deacetylase 5 (HDAC5) negatively regulates sclerostin levels in osteocytes in vitro and in vivo. HDAC5 shRNA increases, whereas HDAC5 overexpression decreases SOST expression in the novel murine Ocy454 osteocytic cell line. HDAC5 knockout mice show increased levels of SOST mRNA, more sclerostin‐positive osteocytes, decreased Wnt activity, low trabecular bone density, and reduced bone formation by osteoblasts. In osteocytes, HDAC5 binds and inhibits the function of MEF2C, a crucial transcription factor for SOST expression. Using chromatin immunoprecipitation, we have mapped endogenous MEF2C binding in the SOST gene to a distal intergenic enhancer 45 kB downstream from the transcription start site. HDAC5 deficiency increases SOST enhancer MEF2C chromatin association and H3K27 acetylation and decreases recruitment of corepressors NCoR and HDAC3. HDAC5 associates with and regulates the transcriptional activity of this enhancer, suggesting direct regulation of SOST gene expression by HDAC5 in osteocytes. Finally, increased sclerostin production achieved by HDAC5 shRNA is abrogated by simultaneous knockdown of MEF2C, indicating that MEF2C is a major target of HDAC5 in osteocytes.

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.

Regulation of Sclerostin Expression in Multiple Myeloma by Dkk‐1; A Potential Therapeutic Strategy for Myeloma Bone Disease

Sclerostin is a potent inhibitor of osteoblastogenesis. Interestingly, newly diagnosed multiple myeloma (MM) patients have high levels of circulating sclerostin that correlate with disease stage and fractures. However, the source and impact of sclerostin in MM remains to be defined. Our goal was to determine the role of sclerostin in the biology of MM and its bone microenvironment as well as investigate the effect of targeting sclerostin with a neutralizing antibody (scl‐Ab) in MM bone disease. Here we confirm increased sclerostin levels in MM compared with precursor disease states like monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM. Furthermore, we found that a humanized MM xenograft mouse model bearing human MM cells (NOD‐SCID.CB17 male mice injected intravenously with 2.5 million of MM1.S‐Luc‐GFP cells) demonstrated significantly higher concentrations of mouse‐derived sclerostin, suggesting a microenvironmental source of sclerostin. Associated with the increased sclerostin levels, activated β‐catenin expression levels were lower than normal in MM mouse bone marrow. Importantly, a high‐affinity grade scl‐Ab reversed osteolytic bone disease in this animal model. Because scl‐Ab did not demonstrate significant in vitro anti‐MM activity, we combined it with the proteasome inhibitor carfilzomib. Our data demonstrated that this combination therapy significantly inhibited tumor burden and improved bone disease in our in vivo MM mouse model. In agreement with our in vivo data, sclerostin expression was noted in marrow stromal cells and osteoblasts of MM patient bone marrow samples. Moreover, MM cells stimulated sclerostin expression in immature osteoblasts while inhibiting osteoblast differentiation in vitro. This was in part regulated by Dkk‐1 secreted by MM cells and is a potential mechanism contributing to the osteoblast dysfunction noted in MM. Our data confirm the role of sclerostin as a potential therapeutic target in MM bone disease and provides the rationale for studying scl‐Ab combined with proteasome inhibitors in MM.

Schnurri-3 (KRC) interacts with c-Jun to regulate the IL-2 gene in T cells.

The activator protein 1 (AP-1) transcription factor is a key participant in the control of T cell proliferation, cytokine production, and effector function. In the immune system, AP-1 activity is highest in T cells, suggesting that a subset of T cell–specific coactivator proteins exist to selectively potentiate AP-1 function. Here, we describe that the expression of Schnurri-3, also known as κ recognition component (KRC), is induced upon T cell receptor signaling in T cells and functions to regulate the expression of the interleukin 2 (IL-2) gene. Overexpression of KRC in transformed and primary T cells leads to increased IL-2 production, whereas dominant-negative KRC, or loss of KRC protein in KRC-null mice, results in diminished IL-2 production. KRC physically associates with the c-Jun transcription factor and serves as a coactivator to augment AP-1–dependent IL-2 gene transcription.

Sclerostin Antibody Administration Converts Bone Lining Cells into Active Osteoblasts.

Sclerostin antibody (Scl‐Ab) increases osteoblast activity, in part through increasing modeling‐based bone formation on previously quiescent surfaces. Histomorphometric studies have suggested that this might occur through conversion of bone lining cells into active osteoblasts. However, direct data demonstrating Scl‐Ab‐induced conversion of lining cells into active osteoblasts are lacking. Here, we used in vivo lineage tracing to determine if Scl‐Ab promotes the conversion of lining cells into osteoblasts on periosteal and endocortical bone surfaces in mice. Two independent, tamoxifen‐inducible lineage‐tracing strategies were used to label mature osteoblasts and their progeny using the DMP1 and osteocalcin promoters. After a prolonged “chase” period, the majority of labeled cells on bone surfaces assumed a thin, quiescent morphology. Then, mice were treated with either vehicle or Scl‐Ab (25 mg/kg) twice over the course of the subsequent week. After euthanization, marked cells were enumerated, their thickness quantified, and proliferation and apoptosis examined. Scl‐Ab led to a significant increase in the average thickness of labeled cells on periosteal and endocortical bone surfaces, consistent with osteoblast activation. Scl‐Ab did not induce proliferation of labeled cells, and Scl‐Ab did not regulate apoptosis of labeled cells. Therefore, direct reactivation of quiescent bone lining cells contributes to the acute increase in osteoblast numbers after Scl‐Ab treatment in mice.

Osteocyte‐Secreted Wnt Signaling Inhibitor Sclerostin Contributes to Beige Adipogenesis in Peripheral Fat Depots

Cells of the osteoblast lineage are increasingly identified as participants in whole‐body metabolism by primarily targeting pancreatic insulin secretion or consuming energy. Osteocytes, the most abundant bone cells, secrete a Wnt‐signaling inhibitor called sclerostin. Here we examined three mouse models expressing high sclerostin levels, achieved through constitutive or inducible loss of the stimulatory subunit of G‐proteins (Gsα in mature osteoblasts and/or osteocytes). These mice showed progressive loss of white adipose tissue (WAT) with tendency toward increased energy expenditure but no changes in glucose or insulin metabolism. Interestingly beige adipocytes were increased extensively in both gonadal and inguinal WAT and had reduced canonical β‐catenin signaling. To determine if sclerostin directly contributes to the increased beige adipogenesis, we engineered an osteocytic cell line lacking Gsα which has high sclerostin secretion. Conditioned media from these cells significantly increased expression of UCP1 in primary adipocytes, and this effect was partially reduced after depletion of sclerostin from the conditioned media. Similarly, treatment of Gsα‐deficient animals with sclerostin‐neutralizing antibody partially reduced the increased UCP1 expression in WAT. Moreover, direct treatment of sclerostin to wild‐type mice significantly increased UCP1 expression in WAT. These results show that osteocytes and/or osteoblasts secrete factors regulating beige adipogenesis, at least in part, through the Wnt‐signaling inhibitor sclerostin. Further studies are needed to assess metabolic effects of sclerostin on adipocytes and other metabolic tissues.

SIKs control osteocyte responses to parathyroid hormone

Parathyroid hormone (PTH) activates receptors on osteocytes to orchestrate bone formation and resorption. Here we show that PTH inhibition of SOST (sclerostin), a WNT antagonist, requires HDAC4 and HDAC5, whereas PTH stimulation of RANKL, a stimulator of bone resorption, requires CRTC2. Salt inducible kinases (SIKs) control subcellular localization of HDAC4/5 and CRTC2. PTH regulates both HDAC4/5 and CRTC2 localization via phosphorylation and inhibition of SIK2. Like PTH, new small molecule SIK inhibitors cause decreased phosphorylation and increased nuclear translocation of HDAC4/5 and CRTC2. SIK inhibition mimics many of the effects of PTH in osteocytes as assessed by RNA-seq in cultured osteocytes and following in vivo administration. Once daily treatment with the small molecule SIK inhibitor YKL-05-099 increases bone formation and bone mass. Therefore, a major arm of PTH signalling in osteocytes involves SIK inhibition, and small molecule SIK inhibitors may be applied therapeutically to mimic skeletal effects of PTH.