Bone Morphogenetic Protein-Based Therapeutic Approaches.

Bone morphogenetic proteins (BMPs) constitute the largest subdivision of the transforming growth factor (TGF)-β family of ligands and exert most of their effects through the canonical effectors Smad1, 5, and 8. Appropriate regulation of BMP signaling is critical for the development and homeostasis of numerous human organ systems. Aberrations in BMP pathways or their regulation are increasingly associated with diverse human pathologies, and there is an urgent and growing need to develop effective approaches to modulate BMP signaling in the clinic. In this review, we provide a wide perspective on diseases and/or conditions associated with dysregulated BMP signal transduction, outline the current strategies available to modulate BMP pathways, highlight emerging second-generation technologies, and postulate prospective avenues for future investigation

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

On the Emerging Role of the Taste Receptor Type 1 (T1R) Family of Nutrient-Sensors in the Musculoskeletal System.

The special sense of taste guides and guards food intake and is essential for body maintenance. Salty and sour tastes are sensed via ion channels or gated ion channels while G protein-coupled receptors (GPCRs) of the taste receptor type 1 (T1R) family sense sweet and umami tastes and GPCRs of the taste receptor type 2 (T2R) family sense bitter tastes. T1R and T2R receptors share similar downstream signaling pathways that result in the stimulation of phospholipase-C-β2. The T1R family includes three members that form heterodimeric complexes to recognize either amino acids or sweet molecules such as glucose. Although these functions were originally described in gustatory tissue, T1R family members are expressed in numerous non-gustatory tissues and are now viewed as nutrient sensors that play important roles in monitoring global glucose and amino acid status. Here, we highlight emerging evidence detailing the function of T1R family members in the musculoskeletal system and review these findings in the context of the musculoskeletal diseases sarcopenia and osteoporosis, which are major public health problems among the elderly that affect locomotion, activities of daily living, and quality of life. These studies raise the possibility that T1R family member function may be modulated for therapeutic benefit