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