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

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