Activin and BMP Signaling Mechanics in Myogenic Cells

The Activin and BMP signaling pathways exert reciprocal effects on myogenesis and skeletal muscle regeneration after injury. Signal transduction in both pathways is mediated by ligand- induced activation of transcriptional regulators called SMAD proteins, with Activins leading to phosphorylation of SMAD2/3 and BMPs leading to phosphorylation of SMAD1/5/8. These ligands bind to their own type I receptors but can also compete for shared type II receptors (ACVR2A and/or ACVR2B). However, it is unclear how these pathways interact in skeletal muscle progenitor cells. To address this deficiency, we investigated the effects of the ligands BMP2 and Activin-A on C2C12 cells, which are an immortalized mouse myoblast cell line with myogenic potential, using a sequence of pre-treatment and co-treatment assays followed by western blot analyses to examine the activation level of their respective SMAD proteins. As expected, treatment with exogenous BMP2 or Activin A led to phosphorylation of SMAD1/5/8 and SMAD2/3, respectively. Moreover, pre-treatment of C2C12 cells with Activin-A before BMP2 delivery resulted in an attenuated phosphorylation of SMAD1/5/8; this effect seems to be specific to Activin ligands, though, since the converse relationship was not observed with respect to phosphorylation of SMAD2/3. Interestingly, seemingly contradictory results were observed when Activin-A and BMP2 were delivered to C2C12 cells simultaneously; this co-treatment scenario results in a higher level of SMAD1/5/8 phosphorylation than delivery of either ligand alone. Ongoing experiments seek to elucidate the mechanism(s) impacting the cellular responses under sequential versus simultaneous activation by Activin and BMP ligands. A better understanding of these mechanisms could lead to novel therapeutic strategies for regeneration of skeletal muscle tissue after injury

Clinical Relevance and Mechanisms of Antagonism Between the BMP and Activin/TGF-β Signaling Pathways.

The transforming growth factor β (TGF-β) superfamily is a large group of signaling molecules that participate in embryogenesis, organogenesis, and tissue homeostasis. These molecules are present in all animal genomes. Dysfunction in the regulation or activity of this superfamily\u27s components underlies numerous human diseases and developmental defects. There are 2 distinct arms downstream of the TGF-β superfamily ligands-the bone morphogenetic protein (BMP) and activin/TGF-β signaling pathways-and these 2 responses can oppose one another\u27s effects, most notably in disease states. However, studies have commonly focused on a single arm of the TGF-β superfamily, and the antagonism between these pathways is unknown in most physiologic and pathologic contexts. In this review, the authors summarize the clinically relevant scenarios in which the BMP and activin/TGF-β pathways reportedly oppose one another and identify several molecular mechanisms proposed to mediate this interaction. Particular attention is paid to experimental findings that may be informative to human pathology to highlight potential therapeutic approaches for future investigation

Identification of Gene Signature Associated with Elevated Bone Formation Rate in Aging Mice

Osteoporosis is a disease of low bone mass resulting from bone resorption exceeding bone formation that places individuals at enhanced risk for fracture, disability, and death. There is an urgent and unmet need for novel targets in treating osteoporosis, requiring a better understanding of the endogenous mechanisms regulating bone formation. Recent work indicates that deletion of the Bmpr2 gene in skeletal progenitor cells of mice using Prx1-Cre leads to substantially elevated bone mass in young adulthood due to increased bone formation rate. Additionally, unpublished work suggests that the age-related decline in bone mass of female Bmpr2 mutant mice is reduced approximately two-fold compared to control mice and quantification of serum bone turnover markers reveals this is associated with a sustained increase in bone formation to at least 35 weeks of age (but not 55 weeks of age) with no alteration in bone resorption. Collectively, these data raise the possibility that Bmpr2 mutant mice may serve as a novel model for elucidating mechanisms that regulate osteoblast activity in aging mice. We sought to identify the gene signature associated with elevated osteoblast activity using genome-wide transcriptome profiling of marrow-free humerii from control and Bmpr2 mutant mice. Applying stringent criteria comparing individual transcripts to eight well-accepted housekeeping genes (Ppib, Gapdh, Hprt, Tbp, Ppia, GusB, Prkg1, and Ywhaz), and contrasting the results at 35 weeks of age to the transcriptome profile at 55 weeks of age, we constructed a Venn diagram sorting the genes into 15 distinct zones. Bioinformatic analyses on this refined gene set indicates that elevated bone formation rate in Bmpr2 mutant mice correlates with enrichment for genes containing binding sites for transcription factors associated with skeletal homeostasis. Further, several genes corresponding with osteoblast differentiation and activity are up-regulated in Bmpr2 mutant mice at 35 weeks of age

Comparative Genomics Identifies the Mouse BMP3 Promoter and an Upstream Evolutionary Conserved Region (ECR) in Mammals.

The Bone Morphogenetic Protein (BMP) pathway is a multi-member signaling cascade whose basic components are found in all animals. One member, BMP3, which arose more recently in evolution and is found only in deuterostomes, serves a unique role as an antagonist to both the canonical BMP and Activin pathways. However, the mechanisms that control BMP3 expression, and the cis-regulatory regions mediating this regulation, remain poorly defined. With this in mind, we sought to identify the Bmp3 promoter in mouse (M. musculus) through functional and comparative genomic analyses. We found that the minimal promoter required for expression in resides within 0.8 kb upstream of Bmp3 in a region that is highly conserved with rat (R. norvegicus). We also found that an upstream region abutting the minimal promoter acts as a repressor of the minimal promoter in HEK293T cells and osteoblasts. Strikingly, a portion of this region is conserved among all available eutherian mammal genomes (47/47), but not in any non-eutherian animal (0/136). We also identified multiple conserved transcription factor binding sites in the Bmp3 upstream ECR, suggesting that this region may preserve common cis-regulatory elements that govern Bmp3 expression across eutherian mammals. Since dysregulation of BMP signaling appears to play a role in human health and disease, our findings may have application in the development of novel therapeutics aimed at modulating BMP signaling in humans

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

Elucidating the Molecular Signatures Associated with Elevated Bone Formation Rate

Osteoporosis is a disease of decreased bone density that occurs when bone resorption exceeds bone formation, thereby placing individuals at greater risk of fracture and disability. We previously reported that deletion of the Bmpr2 gene in embryonic skeletal progenitor cells causes substantially elevated bone density in young adulthood and reduced age-related decline in bone density, likely due to elevated bone formation rate. Thus, these mice may serve as a novel model in which to explore the mechanisms regulating bone formation in the aging skeleton. Here, we performed transcriptome profiling and identified a concise gene signature associated with elevated bone formation rate in Bmpr2 mutant mice, with 120 transcripts up-regulated and 131 transcripts down-regulated. Candidate-driven qRT-PCR provided secondary confirmation of this dataset. Notably, only 8 of these differentially-expressed transcripts have been previously implicated in bone physiology (Pak4, Rpl38, B2m, Fgf1, Nmu, Phospho1, Smpd3 and Inbe), thus representing potentially novel regulators of osteoblast function in the aging skeleton. Additionally, we sought to examine the cell communication events that are associated with elevated bone formation rate. Using protein samples from control and mutant mice, we took advantage of recent advancements in high-throughput phospho-profiling antibody arrays, which allow simultaneous detection of \u3e1,300 targets using very small quantities of protein. These results indicate that the phosphorylation status of at least 86 signaling effectors is differentially regulated in Bmpr2 mutant mice as compared to control littermates, including numerous proteins known to regulate osteoblast differentiation and/or activity. Collectively, our work highlights novel factors associated with elevated bone formation rate and may identify new opportunities for treating low bone density in humans

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

Abnormal Trafficking of Endogenously Expressed BMPR2 Mutant Allelic Products in Patients with Heritable Pulmonary Arterial Hypertension

More than 200 heterozygous mutations in the type 2 BMP receptor gene, BMPR2, have been identified in patients with Heritable Pulmonary Arterial Hypertension (HPAH). More severe clinical outcomes occur in patients with BMPR2 mutations by-passing nonsense-mediated mRNA decay (NMD negative mutations). These comprise 40% of HPAH mutations and are predicted to express BMPR2 mutant products. However expression of endogenous NMD negative BMPR2 mutant products and their effect on protein trafficking and signaling function have never been described. Here, we characterize the expression and trafficking of an HPAH-associated NMD negative BMPR2 mutation that results in an in-frame deletion of BMPR2 EXON2 (BMPR2ΔEx2) in HPAH patient-derived lymphocytes and in pulmonary endothelial cells (PECs) from mice carrying the same in-frame deletion of Exon 2 (Bmpr2 (ΔEx2/+) mice). The endogenous BMPR2ΔEx2 mutant product does not reach the cell surface and is retained in the endoplasmic reticulum. Moreover, chemical chaperones 4-PBA and TUDCA partially restore cell surface expression of Bmpr2ΔEx2 in PECs, suggesting that the mutant product is mis-folded. We also show that PECs from Bmpr2 (ΔEx2/+) mice have defects in the BMP-induced Smad1/5/8 and Id1 signaling axis, and that addition of chemical chaperones restores expression of the Smad1/5/8 target Id1. These data indicate that the endogenous NMD negative BMPRΔEx2 mutant product is expressed but has a folding defect resulting in ER retention. Partial correction of this folding defect and restoration of defective BMP signaling using chemical chaperones suggests that protein-folding agents could be used therapeutically in patients with these NMD negative BMPR2 mutations

BMPR-II is Dispensable for Formation of the Limb Skeleton.

Initiation of BMP signaling is dependent upon activation of Type I BMP receptor by constitutively active Type II BMP receptor. Three Type II BMP receptors have been identified; Acvr2a and Acvr2b serve as receptors for BMPs and for activin-like ligands whereas BMPR-II functions only as a BMP receptor. As BMP signaling is required for endochondral ossification and loss of either Acvr2a or Acvr2b is not associated with deficits in limb development, we hypothesized that BMPR-II would be essential for BMP signaling during skeletogenesis. We removed BMPR-II from early limb mesoderm by crossing BMPR-II floxed mice with those carrying the Prx1-Cre transgene. Mice lacking limb expression of BMPR-II have normal skeletons that could not be distinguished from control littermates. From these data, we conclude that BMPR-II is not required for endochondral ossification in the limb where loss of BMPR-II may be compensated by BMP utilization of Acvr2a and Acvr2b