Elucidating the Antagonistic Relationship Between Bone Morphogenetic Protein and Activin Signaling Pathways in Osteoprogenitor Cells

Osteoporosis is a disease characterized by low bone mineral density due to the rate of bone resorption exceeding that of bone formation. Substantial evidence indicates the Bone Morphogenetic Protein (BMP) pathway promotes bone formation through action of the effectors SMAD1/5/8 while the Activin pathway negatively influences bone mass through action of the effectors SMAD2/3. Recent studies from our lab suggest that BMP and Activin ligands regulate bone mass in a see-saw-like mechanism via competition for a shared pool of receptors, i.e. receptor-level competition. In the present study we seek to test this hypothesis in vitro via signaling responsiveness assays using pathway-specific western blot analyses in the osteogenic cell line W-20-17. We first confirmed that W-20-17 cells respond to exogenous stimulation by BMP2 and Activin-A. Then, we administered recombinant versions of naturally-occurring extracellular ligand traps for BMP2 or Activin ligands (Noggin and Follistatin, respectively) to examine basal antagonism between these pathways. This revealed that, under basal conditions, SMAD1/5/8 activation is repressed by Activin signaling; interestingly, the converse relationship was not observed. To determine the molecular mechanism allowing for this relationship, we treated W-20-17 cells with SB431542, which is an intracellular inhibitor of Activin signaling that functions downstream of receptor engagement, and found no effect on SMAD1/5/8 activation. Collectively, our results suggest Activin-mediated repression of BMP signaling is ligand-dependent but occurs upstream of SMAD2/3 activation. Current studies seek to identify the specific Activin ligand(s) responsible for this effect; gene expression analyses indicates that W-20-17 cells express multiple Activin subunits including Inhβa and Inhβb. Additionally, overpression studies are ongoing to determine if receptor-level competition is involved in mediating these effects. Collectively, our study seeks to elucidate the mechanism(s) that regulate antagonism BMP and Activin signaling pathways to identify novel opportunities for safer and more effective therapies for low bone mass in humans

Boolean Networks as Predictive Models of Emergent Biological Behaviors

Interacting biological systems at all organizational levels display emergent behavior. Modeling these systems is made challenging by the number and variety of biological components and interactions (from molecules in gene regulatory networks to species in ecological networks) and the often-incomplete state of system knowledge (e.g., the unknown values of kinetic parameters for biochemical reactions). Boolean networks have emerged as a powerful tool for modeling these systems. We provide a methodological overview of Boolean network models of biological systems. After a brief introduction, we describe the process of building, analyzing, and validating a Boolean model. We then present the use of the model to make predictions about the system's response to perturbations and about how to control (or at least influence) its behavior. We emphasize the interplay between structural and dynamical properties of Boolean networks and illustrate them in three case studies from disparate levels of biological organization.Comment: Review, to appear in the Cambridge Elements serie

Loss of BMPR2 Expression in Skeletal Progenitor Cells Reduces Age-Related Bone Loss

Osteoporosis is a disease of low bone mineral density (BMD) that affects 10 million Americans with an additional 34 million at risk for developing the disease. Current FDA-approved therapies for osteoporosis involve anti-resorptive agents but many patients would benefit from augmenting bone formation as well as inhibiting bone loss. We recently reported that targeted deletion of the type 2 BMP receptor BMPR2 using Prx1-Cre in skeletal progenitor cells in mice leads to dramatically increased bone mass and bone formation rate by ten weeks of age in the absence of changes in osteoclast function (Lowery et al 2015). In the present study, we examined the age-related impact of Bmpr2 deletion and found that, consistent with our previous results, both male and female Bmpr2-cKO mice exhibit high bone mass when compared to control mice at 55 weeks of age. We also found that the age-related decline in bone mass from 15 weeks to 55 weeks of age in Bmpr2-cKO mice is reduced approximately three-fold compared to control mice, with male and female Bmpr2-cKO mice losing on average only 18% and 27%, respectively, while male and female control mice lost 55% and 77%, respectively, over the same time span. High bone mass in aged Bmpr2-cKO mice is associated with elevated serum levels of the bone formation marker Procollagen Type I N-terminal Propeptide (P1NP). In contrast, serum levels of the bone resorption marker Collagen Type I C-telopeptide (CTx) are unchanged in Bmpr2-cKO mice. Collectively, these findings indicate that loss of Bmpr2 in skeletal progenitor cells causes a sustained imbalance in bone formation vs. bone resorption and results in high bone mass in the aging skeleton. Our findings suggest that strategies aimed at controlling signaling through BMPR2 have the potential to impact bone mass in the aging adult skeleton

Identification of Commercially Available Antibodies that Block Ligand Binding by BMPR2

Osteoporosis, a disease of low bone mineral density, affects 10 million Americans and triggers significant health problems and considerable socioeconomic burdens. Current treatments for osteoporosis have significant limitations, necessitating identifying new treatment strategies via building a better understanding of the endogenous mechanisms regulating bone mass. A recent study demonstrated that removal of the BMP type 2 receptor (BMPR2) in skeletal progenitor cells of Bmpr2-cKO mice during embryonic development leads to reduced age-related bone loss by sustained elevation in bone formation rate. This present study sought to advance the translational potential of the genetic model by identifying antibodies that neutralize the ligand-binding function of the BMPR2 extracellular domain (BMPR2-ECD). This study first established a modified, cell-free immunoprecipitation assay wherein the ligand BMP2 was pulled-down by BMPR2-ECD conjugated to Protein G beads; the unbound BMP2 (found in the supernatant) was subsequently quantified by ELISA. This yielded a standard assay wherein approximately 2 ug BMPR2-ECD leads to a 70% reduction in BMP2 signal. Next, the neutralizing ability of 3F6, a mouse monoclonal antibody raised against the ligand-binding region of BMPR2, was examined and was found to cause a dose-dependent inhibition of BMPR2-ECD ligand-binding. Given the ascites preparation of 3F6, specificity of this assay was confirmed by demonstrating that ligand-binding activity of BMPR2-ECD is unchanged in the presence of non-specific, negative-control ascites. Using these results as a guide, 1F12, another mouse monoclonal antibody raised against the ligand-binding region of BMPR2, was evaluated and was also found to neutralize the ligand-binding function of BMPR2-ECD. In contrast, no effect on ligand-binding function of BMPR2-ECD was observed with 9A10 even though this mouse monoclonal antibody is also raised against the ligand-binding region of BMPR2. These results provide proof-of-concept data for future studies evaluating inhibition of BMPR2 function in vivo as a means to reduce age-related bone loss

Antagonism Between Bone Morphogenetic Protein and Activin Signaling Pathways in Osteoprogenitor Cells

Osteoporosis is a disease characterized by low bone mineral density due to the rate of bone resorption exceeding that of bone formation. Evidence indicates the Bone Morphogenetic Protein (BMP) pathway promotes bone formation through action of effectors SMAD1/5/8 while the Activin pathway negatively influences bone mass through effectors SMAD2/3. Studies suggest that BMPs and Activins regulate bone mass in a see-saw-like mechanism. We seek to test this hypothesis in vitro via signaling responsiveness assays using pathway-specific western blot analyses in the osteogenic murine bone marrow stromal cell line W-20-17. We first confirmed that W-20-17 cells exhibit basal activation of SMAD1/5/8 and SMAD2/3 under serum-restricted conditions. Treatment with Follistatin, which sequesters Activin ligands in the extracellular environment, leads to an increase in BMP pathway activation. To determine the mechanism allowing for this, we treated W-20-17 cells with SB431542, an intracellular inhibitor of Activin signaling that functions downstream of receptor engagement, and found no effect on BMP pathway activation. In contrast, treatment with BMP pathway inhibitor Noggin had no effect on Activin pathway activation despite robust inhibition of BMP signaling. Our results suggest Activin-mediated repression of BMP signaling in these cells is ligand-dependent but occurs upstream of SMAD2/3 activation. Gene expression analyses indicate that W-20-17 cells express Activin A and its receptors ALK4, ACVR2A, and ACVR2B. Given that ACVR2A and ACVR2B also have high affinity for BMP ligands, this raises the possibility that Activin-mediated repression of BMP signaling may occur via competition for a shared pool of receptors. Over-expression studies and osteoblast activity assays are underway to examine this hypothesis. Our work seeks to elucidate the mechanism(s) that regulate antagonism of BMP and Activin signaling pathways to identify novel opportunities for treating low bone mass in humans

Identification of a bone morphogenetic protein type 2 receptor neutralizing antibody

The bone morphogenetic protein (BMP) signaling pathway comprises the largest subdivision of the transforming growth factor (TGFβ) superfamily. BMP signaling plays essential roles in both embryonic development and postnatal tissue homeostasis. Dysregulated BMP signaling underlies human pathologies ranging from pulmonary arterial hypertension to heterotopic ossification. Thus, understanding the basic mechanisms and regulation of BMP signaling may yield translational opportunities. Unfortunately, limited tools are available to evaluate this pathway, and genetic approaches are frequently confounded by developmental requirements or ability of pathway components to compensate for one another. Specific inhibitors for type 2 receptors are poorly represented. Thus, we sought to identify and validate an antibody that neutralizes the ligand-binding function of BMP receptor type 2 (BMPR2) extracellular domain (ECD)

Loss of the Nutrient Sensor Tas1R3 Leads to Reduced Bone Resorption

Background: The Taste receptor, type 1 (TAS1R) family of heterotrimeric G protein-coupled receptors participates in monitoring energy and nutrient needs. TAS1R member 3 (TAS1R3) either recognizes amino acids such as glycine and L-glutamate or sweet molecules such as sucrose and fructose when dimerized with TAS1R member 1 (TAS1R1) or TAS1R member 2 (TAS1R2), respectively. Loss of TAS1R3 expression can cause impaired mTORC1 signaling and increased autophagy, indicating that signaling through this receptor is critical for assessing nutrient needs. Recently, it was reported that global deletion of TAS1R3 expression in Tas1R3 mutant mice leads to increased cortical bone mass and trabecular remodeling but the underlying cellular mechanism leading to this phenotype remains unclear. Results: To address this open question, we quantified bone turnover markers in serum from 20-week-old wild type and Tas1R3 mutant mice and found that levels of the resorption marker Collagen Type I C-telopeptide (CTx) were reduced on average by \u3e60% in the absence of TAS1R3 expression. Levels of the bone formation marker Procollagen Type I N-terminal Propeptide (P1NP) tend to be higher in Tas1R3 mutant mice but this finding did not reach statistical significance (

Are Thiel-embalmed Cadavers Effective Tools in Educating Medical Students to Perform Knee Arthrocentesis?

INTRODUCTION: The purposes of this study are to determine whether Thiel-embalmed cadavers are an effective educational tool in teaching medical students to perform knee arthrocentesis, to compare the use of Thiel-embalmed cadavers to formalin-embalmed cadavers in arthrocentesis education, and to determine whether the use of Thiel-embalmed cadavers is potentially generalizable to the instruction of other orthopedic procedures. METHODS: Sixty-eight third-year medical students participated in the study. The participants first completed a pre-survey to assess their prior experience with arthrocentesis procedures and Thiel-embalmed cadavers. Participants then attended an instructional session where the knee arthrocentesis procedure was demonstrated on a Thiel-embalmed cadaver. Participants then individually performed the simulated knee arthrocentesis procedure twice: once on a Thiel-embalmed cadaver and once on a formalin-embalmed cadaver. Success of each attempt was determined through the visualization of aspirated joint fluid. Following the laboratory session, each participant completed a post-survey to determine whether the session improved their perceived confidence in performing knee arthrocentesis, if they preferred the use of Thiel-embalmed cadavers or formalin-embalmed cadavers as a teaching tool, and if they believed simulated practice using Thiel-embalmed cadavers would be effective for learning other orthopedic procedural skills. RESULTS: Sixty-eight students participated in the laboratory session and successfully completed both pre- and post-course surveys. 96% of participants reported that they felt confident performing knee arthrocentesis under physician supervision following their participation in the laboratory session (versus 15% of participants in the pre-survey). 96% of participants reported that the Thiel-embalmed cadavers provided a more realistic teaching model than formalin-embalmed cadavers for learning knee arthrocentesis. 100% of participants believed the incorporation of simulated practice using Thiel-embalmed cadavers is an effective method in teaching students to perform knee arthrocentesis. 100% of participants reported that they would participate in future sessions using Thiel-embalmed cadavers to learn and practice other orthopedic procedural techniques. DISCUSSION: This study used a moderate sample size of third-year medical students to provide data regarding the suitability of using Thiel cadavers in arthrocentesis education. Results indicate that Thiel cadavers are effective tools in teaching medical students to perform knee arthrocentesis, that students preferred the Thiel cadavers to the formalin cadavers, and that the use of Thiel cadavers is a safe, engaging, and high-quality teaching modality for demonstrating proper arthrocentesis procedural technique to medical students. Since this study looked specifically at teaching knee arthrocentesis to medical students, it is uncertain whether the benefits of Thiel cadavers are generalizable to the education of other orthopedic procedures and subject groups such as residents, fellows, and practicing physicians. Further studies should be performed to assess whether Thiel cadavers are beneficial in teaching other orthopaedic procedures and if these benefits extend to other subject groups

Loss of the nutrient sensor TAS1R3 leads to reduced bone resorption

The taste receptor type 1 (TAS1R) family of heterotrimeric G protein-coupled receptors participates in monitoring energy and nutrient status. TAS1R member 3 (TAS1R3) is a bi-functional protein that recognizes amino acids such as L-glycine and L-glutamate or sweet molecules such as sucrose and fructose when dimerized with TAS1R member 1 (TAS1R1) or TAS1R member 2 (TAS1R2), respectively. It was recently reported that deletion of TAS1R3 expression in Tas1R3 mutant mice leads to increased cortical bone mass but the underlying cellular mechanism leading to this phenotype remains unclear. Here, we independently corroborate the increased thickness of cortical bone in femurs of 20-week-old male Tas1R3 mutant mice and confirm that Tas1R3 is expressed in the bone environment. Tas1R3 is expressed in undifferentiated bone marrow stromal cells (BMSCs) in vitro and its expression is maintained during BMP2-induced osteogenic differentiation. However, levels of the bone formation marker procollagen type I N-terminal propeptide (PINP) are unchanged in the serum of 20-week-old Tas1R3 mutant mice as compared to controls. In contrast, levels of the bone resorption marker collagen type I C-telopeptide are reduced greater than 60% in Tas1R3 mutant mice. Consistent with this, Tas1R3 and its putative signaling partner Tas1R2 are expressed in primary osteoclasts and their expression levels positively correlate with differentiation status. Collectively, these findings suggest that high bone mass in Tas1R3 mutant mice is due to uncoupled bone remodeling with reduced osteoclast function and provide rationale for future experiments examining the cell-type-dependent role for TAS1R family members in nutrient sensing in postnatal bone remodeling