The RhoGAP Myo9b Promotes Bone Growth by Mediating Osteoblastic Responsiveness to IGF-1

McMichael B., Jeong Y., Auerbach J., Han C., Sedlar R., Shettigar V., Bähler M., Agarwal S., Kim D., Lee B.

Abstract

The Ras homolog A (RhoA) subfamily of Rho guanosine triphosphatases (GTPases) regulates actin-based cellular functions in bone such as differentiation, migration, and mechanotransduction. Polymorphisms or genetic ablation of RHOA and some of its regulatory guanine exchange factors (GEFs) have been linked to poor bone health in humans and mice, but the effects of RhoA-specific GTPase-activating proteins (GAPs) on bone quality have not yet been identified. Therefore, we examined the consequences of RhoGAP Myo9b gene knockout on bone growth, phenotype, and cellular activity. Male and female mice lacking both alleles demonstrated growth retardation and decreased bone formation rates during early puberty. These mice had smaller, weaker bones by 4 weeks of age, but only female KOs had altered cellular numbers, with fewer osteoblasts and more osteoclasts. By 12 weeks of age, bone quality in KOs worsened. In contrast, 4-week-old heterozygotes demonstrated bone defects that resolved by 12 weeks of age. Throughout, Myo9b ablation affected females more than males. Osteoclast activity appeared unaffected. In primary osteogenic cells, Myo9b was distributed in stress fibers and focal adhesions, and its absence resulted in poor spreading and eventual detachment from culture dishes. Similarly, MC3T3-E1 preosteoblasts with transiently suppressed Myo9b levels spread poorly and contained decreased numbers of focal adhesions. These cells also demonstrated reduced ability to undergo IGF-1–induced spreading or chemotaxis toward IGF-1, though responses to PDGF and BMP-2 were unaffected. IGF-1 receptor (IGF1R) activation was normal in cells with diminished Myo9b levels, but the activated receptor was redistributed from stress fibers and focal adhesions into nuclei, potentially affecting receptor accessibility and gene expression. These results demonstrate that Myo9b regulates a subset of RhoA-activated processes necessary for IGF-1 responsiveness in osteogenic cells, and is critical for normal bone formation in growing mice. © 2017 American Society for Bone and Mineral Research.