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TGFβ family ligands, which include the TGFβs, BMPs, and activins, signal by forming a ternary complex with type I and type II receptors. For TGFβs and BMPs, structures of ternary complexes have revealed differences in receptor assembly. However, structural information for how activins assemble a ternary receptor complex is lacking. We report the structure of an activin class member, GDF11, in complex with the type II receptor ActRIIB and the type I receptor Alk5. The structure reveals that receptor positioning is similar to the BMP class, with no interreceptor contacts; however, the type I receptor interactions are shifted toward the ligand fingertips and away from the dimer interface. Mutational analysis shows that ligand type I specificity is derived from differences in the fingertips of the ligands that interact with an extended loop specific to Alk4 and Alk5. The study also reveals differences for how TGFβ and GDF11 bind to the same type I receptor, Alk5. For GDF11, additional contacts at the fingertip region substitute for the interreceptor interactions that are seen for TGFβ, indicating that Alk5 binding to GDF11 is more dependent on direct contacts. In support, we show that a single residue of Alk5 (Phe84), when mutated, abolishes GDF11 signaling, but has little impact on TGFβ signaling. The structure of GDF11/ActRIIB/Alk5 shows that, across the TGFβ family, different mechanisms regulate type I receptor binding and specificity, providing a molecular explanation for how the activin class accommodates low-affinity type I interactions without the requirement of cooperative receptor interactions. [ABSTRACT FROM AUTHOR]

Wnt3a is a major regulator of bone metabolism however, very few of its target genes are known in bone. Wnt3a preferentially signals through transmembrane receptors Frizzled and co-receptors Lrp5/6 to activate the canonical signaling pathway. Previous studies have shown that the canonical Wnt co-receptors Lrp5 and Lrp6 also play an essential role in normal postnatal bone homeostasis, yet, very little is known about specific contributions by these co-receptors in Wnt3a-dependent signaling. We used high-throughput sequencing technology to identify target genes regulated by Wnt3a in osteoblasts and to elucidate the role of Lrp5 and Lrp6 in mediating Wnt3a signaling. Our study identified 782 genes regulated by Wnt3a in primary calvarial osteoblasts. Wnt3a up-regulated the expression of several key regulators of osteoblast proliferation/ early stages of differentiation while inhibiting genes expressed in later stages of osteoblastogenesis. We also found that Lrp6 is the key mediator of Wnt3a signaling in osteoblasts and Lrp5 played a less significant role in mediating Wnt3a signaling. [ABSTRACT FROM AUTHOR]

Individuals with Hajdu-Cheney syndrome (HCS) present with osteoporosis, and HCS is associated with NOTCH2 mutations causing deletions of the proline-, glutamic acid-, serine-, and threonine-rich (PEST) domain that are predicted to enhance NOTCH2 stability and cause gain-of-function. Previously, we demonstrated that mice harboring Notch2 mutations analogous to those in HCS (Notch2HCS) are severely osteopenic because of enhanced bone resorption. We attributed this phenotype to osteoclastic sensitization to the receptor activator of nuclear factor-κB ligand and increased osteoblastic tumor necrosis factor superfamily member 11 (Tnfsf11) expression. Here, to determine the individual contributions of osteoclasts and osteoblasts to HCS osteopenia, we created a conditional-by-inversion (Notch2COIN) model in which Cre recombination generates a Notch2ΔPEST allele expressing a Notch2 mutant lacking the PESTdomain. Germline Notch2COIN inversion phenocopied the Notch2HCS mutant, validating the model. To activate Notch2 in osteoclasts or osteoblasts, Notch2COIN mice were bred with mice expressing Cre from the Lyz2 or the BGLAP promoter, respectively. These crosses created experimental mice harboring a Notch2ΔPEST allele in Cre-expressing cells and control littermates expressing a wild-type Notch2 transcript. Notch2COIN inversion in Lyz2-expressing cells had no skeletal consequences and did not affect the capacity of bone marrow macrophages to form osteoclasts in vitro. In contrast, Notch2COIN inversion in osteoblasts led to generalized osteopenia associated with enhanced bone resorption in the cancellous bone compartment and with suppressed endocortical mineral apposition rate. Accordingly, Notch2 activation in osteoblast-enriched cultures from Notch2COIN mice induced Tnfsf11 expression. In conclusion, introduction of theHCSmutation in osteoblasts, but not in osteoclasts, causes osteopenia. [ABSTRACT FROM AUTHOR]

ABSTRACT Joint injury causes post-traumatic osteoarthritis (PTOA). About ∼50% of patients rupturing their anterior cruciate ligament (ACL) will develop PTOA within 1-2 decades of the injury, yet the mechanisms responsible for the development of PTOA after joint injury are not well understood. In this study, we examined whole joint gene expression by RNA sequencing (RNAseq) at 1 day, 1-, 6-, and 12 weeks post injury, in a non-invasive tibial compression (TC) overload mouse model of PTOA that mimics ACL rupture in humans. We identified 1446 genes differentially regulated between injured and contralateral joints. This includes known regulators of osteoarthritis such as MMP3, FN1, and COMP, and several new genes including Suco, Sorcs2, and Medag. We also identified 18 long noncoding RNAs that are differentially expressed in the injured joints. By comparing our data to gene expression data generated using the surgical destabilization of the medial meniscus (DMM) PTOA model, we identified several common genes and shared mechanisms. Our study highlights several differences between these two models and suggests that the TC model may be a more rapidly progressing model of PTOA. This study provides the first account of gene expression changes associated with PTOA development and progression in a TC model. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 35:474-485, 2017. [ABSTRACT FROM AUTHOR]

Vertical sleeve gastrectomy (VSG), the most utilized bariatric procedure in clinical practice, greatly reduces body weight and improves a variety of metabolic disorders. However, one of its long-term complications is bone loss and increased risk of fracture. Elevated circulating sclerostin (SOST) and granulocyte-colony stimulating factor (G-CSF) concentrations have been considered as potential contributors to VSG-associated bone loss. To test these possibilities, we administrated antibodies to SOST or G-CSF receptor and investigated alterations to bone and marrow niche following VSG. Neutralizing either SOST or G-CSF receptor did not alter beneficial effects of VSG on adiposity and hepatic steatosis, and anti-SOST treatment provided a further improvement to glucose tolerance. SOST antibodies partially reduced trabecular and cortical bone loss following VSG by increasing bone formation, whereas G-CSF receptor antibodies had no effects on bone mass. The expansion in myeloid cellularity and reductions in bone marrow adiposity seen with VSG were partially eliminated by treatment with Anti-G-CSF receptor. Taken together, these experiments demonstrate that antibodies to SOST or G-CSF receptor may act through independent mechanisms to partially block effects of VSG on bone loss or marrow niche cells, respectively. [ABSTRACT FROM AUTHOR]

Loss of Sostdc1, a growth factor paralogous to Sost, causes the formation of ectopic incisors, fused molars, abnormal hair follicles, and resistance to kidney disease. Sostdc1 is expressed in the periosteum, a source of osteoblasts, fibroblasts and mesenchymal progenitor cells, which are critically important for fracture repair. Here, we investigated the role of Sostdc1 in bone metabolism and fracture repair. Mice lacking Sostdc1 ( Sostdc1 − / − ) had a low bone mass phenotype associated with loss of trabecular bone in both lumbar vertebrae and in the appendicular skeleton. In contrast, Sostdc1 − / − cortical bone measurements revealed larger bones with higher BMD, suggesting that Sostdc1 exerts differential effects on cortical and trabecular bone. Mid-diaphyseal femoral fractures induced in Sostdc1 − / − mice showed that the periosteal population normally positive for Sostdc1 rapidly expands during periosteal thickening and these cells migrate into the fracture callus at 3 days post fracture. Quantitative analysis of mesenchymal stem cell (MSC) and osteoblast populations determined that MSCs express Sostdc1 , and that Sostdc1 − / − 5 day calluses harbor > 2-fold more MSCs than fractured wildtype controls. Histologically a fraction of Sostdc1 -positive cells also expressed nestin and α-smooth muscle actin, suggesting that Sostdc1 marks a population of osteochondral progenitor cells that actively participate in callus formation and bone repair. Elevated numbers of MSCs in D5 calluses resulted in a larger, more vascularized cartilage callus at day 7, and a more rapid turnover of cartilage with significantly more remodeled bone and a thicker cortical shell at 21 days post fracture. These data support accelerated or enhanced bone formation/remodeling of the callus in Sostdc1 − / − mice, suggesting that Sostdc1 may promote and maintain mesenchymal stem cell quiescence in the periosteum. [ABSTRACT FROM AUTHOR]

Reversible modification of the RNAPII C-terminal domain links transcription with RNA processing and surveillance activities. To better understand this, we mapped the location of RNAPII carrying the five types of CTD phosphorylation on the RNA transcript, providing strand-specific, nucleotide-resolution information, and we used a machine learning-based approach to define RNAPII states. This revealed enrichment of Ser5P, and depletion of Tyr1P, Ser2P, Thr4P, and Ser7P in the transcription start site (TSS) proximal ~150 nt of most genes, with depletion of all modifications close to the poly(A) site. The TSS region also showed elevated RNAPII relative to regions further 3′, with high recruitment of RNA surveillance and termination factors, and correlated with the previously mapped 3′ ends of short, unstable ncRNA transcripts. A hidden Markov model identified distinct modification states associated with initiating, early elongating and later elongating RNAPII. The initiation state was enriched near the TSS of protein-coding genes and persisted throughout exon 1 of intron-containing genes. Notably, unstable ncRNAs apparently failed to transition into the elongation states seen on protein-coding genes. [ABSTRACT FROM AUTHOR]

Type 1 diabetes mellitus (T1DM) patients have osteopenia and impaired fracture healing due to decreased osteoblast activity. Further, no adequate treatments are currently available that can restore impaired healing in T1DM; hence a significant need exists to investigate new therapeutics for treatment of orthopedic complications. Sclerostin (SOST), a WNT antagonist, negatively regulates bone formation, and SostAb is a potent bone anabolic agent. To determine whether SOST antibody (SostAb) treatment improves fracture healing in streptozotocin (STZ) induced T1DM mice, we administered SostAb twice weekly for up to 21 days post-fracture, and examined bone quality and callus outcomes at 21 days and 42 days post-fracture (11 and 14 weeks of age, respectively). Here we show that SostAb treatment improves bone parameters; these improvements persist after cessation of antibody treatment. Markers of osteoblast differentiation such as Runx2, collagen I, osteocalcin, and DMP1 were reduced, while an abundant number of SP7/osterix-positive early osteoblasts were observed on the bone surface of STZ calluses. These results suggest that STZ calluses have poor osteogenesis resulting from failure of osteoblasts to fully differentiate and produce mineralized matrix, which produces a less mineralized callus. SostAb treatment enhanced fracture healing in both normal and STZ groups, and in STZ + SostAb mice, also reversed the lower mineralization seen in S TZ calluses. Micro-CT analysis of calluses revealed improved bone parameters with SostAb treatment, and the mineralized bone was comparable to Controls . Additionally, we found sclerostin levels to be elevated in STZ mice and β-catenin activity to be reduced. Consistent with its function as a WNT antagonist, SostAb treatment enhanced β-catenin activity, but also increased the levels of SOST in the callus and in circulation. Our results indicate that SostAb treatment rescues the impaired osteogenesis seen in the STZ induced T1DM fracture model by facilitating osteoblast differentiation and mineralization of bone. [ABSTRACT FROM AUTHOR]

The bone marrow niche is thought to act as a permissive microenvironment required for emergence or progression of hematologic cancers. We hypothesized that osteoblasts, components of the niche involved in hematopoietic stem cell (HSC) function, influence the fate of leukemic blasts. We show that osteoblast numbers decrease by 55% in myelodysplasia and acute myeloid leukemia patients. Further, genetic depletion of osteoblasts in mouse models of acute leukemia increased circulating blasts and tumor engraftment in the marrow and spleen leading to higher tumor burden and shorter survival. Myelopoiesis increased and was coupled with a reduction in B lymphopoiesis and compromised erythropoiesis, suggesting that hematopoietic lineage/progression was altered. Treatment of mice with acute myeloid or lymphoblastic leukemia with a pharmacologic inhibitor of the synthesis of duodenal serotonin, a hormone suppressing osteoblast numbers, inhibited loss of osteoblasts. Maintenance of the osteoblast pool restored normal marrow function, reduced tumor burden, and prolonged survival. Leukemia prevention was attributable to maintenance of osteoblast numbers because inhibition of serotonin receptors alone in leukemic blasts did not affect leukemia progression. These results suggest that osteoblasts play a fundamental role in propagating leukemia in the marrow and may be a therapeutic target to induce hostility of the niche to leukemia blasts. [ABSTRACT FROM AUTHOR]

Abstract: WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1 −/− mice lack any obvious limb or skeletal defects, Sost −/− mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost −/− ; Sostdc1 −/− mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost −/− and Sost −/− ; Sostdc1 −/− mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling. [Copyright &y& Elsevier]

We identified a glycoprotein hormone β-subunit (OGH, also called GPB5) that as a heterodimer with the α-subunit GPA2, serves as a second ligand for the thyroid-stimulating hormone receptor. Mice in which the OGH gene is deleted (OGH-/- are indistinguishable from WT littermates in body weight, response to high-fat diet, metabolic parameters, body composition, and insulin tolerance. Mice engineered to transgenically globally overexpress OGH (OGH- TG) develop ≈2-fold elevations in their basal thyroid levels and weigh slightly less than WI littermates despite increased food intake because of an increase in their metabolic rates. Moreover, when OGH-TE mice are challenged with a high-fat diet, they gain significantly less weight and body fat than their WT littermates. The OGH-TG mice also have reduced blood glucose, insulin, cholesterol, and triglycerides. In contrast to other approaches in which the thyroid axis is activated, OGH-IG mice exhibit only minor changes in heart rate and blood pressure. Our findings suggest that constitutive low-level activation of the thyroid axis (via OGH or other means) may provide a beneficial therapeutic approach for combating diet-induced obesity. [ABSTRACT FROM AUTHOR]

Investigates the effect of one multiple synostosis syndrome and two milder disorder proximal symphalangism disease-causing missense mutations on the structure and function of noggin. Effect of noggin missense mutations on the protein's ability to form secreted disulfide-linked dimers in COS-7 cells; Interaction of noggin with intracellular bone morphogenetic protein-14.

Heterotopic ossification (HO), the formation of ectopic bone in soft tissues, has been extensively studied in its two primary forms: post-traumatic HO (tHO) typically found in patients who have experienced musculoskeletal or neurogenic injury and in fibrodysplasia ossificans progressiva (FOP), where it is genetically driven. Given that in both diseases HO arises via endochondral ossification, the molecular mechanisms behind both diseases have been postulated to be manifestations of similar pathways including those activated by BMP/TGFβ superfamily ligands. A significant step towards understanding the molecular mechanism by which HO arises in FOP was the discovery that FOP causing ACVR1 variants trigger HO in response to activin A, a ligand that does not activate signaling from wild type ACVR1, and that is not inherently osteogenic in wild type settings. The physiological significance of this finding was demonstrated by showing that activin A neutralizing antibodies stop HO in two different genetically accurate mouse models of FOP. In order to explore the role of activin A in tHO, we performed single cell RNA sequencing and compared the expression of activin A as well as other BMP pathway genes in tHO and FOP HO. We show that activin A is expressed in response to injury in both settings, but by different types of cells. Given that wild type ACVR1 does not transduce signal when engaged by activin A, we hypothesized that inhibition of activin A will not block tHO. Nonetheless, as activin A was expressed in tHO lesions, we tested its inhibition and compared it with inhibition of BMPs. We show here that anti-activin A does not block tHO, whereas agents such as antibodies that neutralize ACVR1 or ALK3-Fc (which blocks osteogenic BMPs) are beneficial, though not completely curative. These results demonstrate that inhibition of activin A should not be considered as a therapeutic strategy for ameliorating tHO. [ABSTRACT FROM AUTHOR]

Pw1/Peg3 is an imprinted gene expressed from the paternally inherited allele. Several imprinted genes, including Pw1/Peg3, have been shown to regulate overall body size and play a role in adult stem cells. Pw1/Peg3 is expressed in muscle stem cells (satellite cells) as well as a progenitor subset of muscle interstitial cells (PICs) in adult skeletal muscle. We therefore examined the impact of loss-of-function of Pw1/Peg3 during skeletal muscle growth and in muscle stem cell behavior. We found that constitutive loss of Pw1/Peg3 function leads to a reduced muscle mass and myofiber number. In newborn mice, the reduction in fiber number is increased in homozygous mutants as compared to the deletion of only the paternal Pw1/Peg3 allele, indicating that the maternal allele is developmentally functional. Constitutive and a satellite cell-specific deletion of Pw1/Peg3, revealed impaired muscle regeneration and a reduced capacity of satellite cells for self-renewal. RNA sequencing analyses revealed a deregulation of genes that control mitochondrial function. Consistent with these observations, Pw1/Peg3 mutant satellite cells displayed increased mitochondrial activity coupled with accelerated proliferation and differentiation. Our data show that Pw1/Peg3 regulates muscle fiber number determination during fetal development in a gene-dosage manner and regulates satellite cell metabolism in the adult. [ABSTRACT FROM AUTHOR]