Osmotic stress suppresses osteogenic differentiation by inhibiting nuclear translocation of YAP via perinuclear actin accumulation

Hyperglycemia is a well-recognized cause of osteoblast dysfunction. Recent evidence, however, indicates that elevated extracellular osmolarity associated with hyperglycemia may independently impair osteogenic differentiation. However, the mechanisms underlying these effects remain poorly understood. In this study, we examined how osmotic stress influences osteoblast differentiation, with a focus on actin cytoskeletal remodeling and subcellular localization of the Yes-associated protein (YAP), a mechanosensitive transcriptional coactivator involved in osteogenesis. Using MC3T3-E1 pre-osteoblasts, we found that osteogenic induction enhanced cell proliferation, migration, nuclear deformation, and nuclear translocation of YAP, accompanied by upregulated expression of genes encoding osteogenic markers. In contrast, treatment with either glucose or mannitol, used to isolate the osmotic component of hyperglycemia, preserved nuclear morphology, decreased nuclear localization of YAP, and led to perinuclear actin accumulation, as confirmed by radial profile analysis of actin distribution. These effects were accompanied by downregulation of target genes of YAP and reduction in alkaline phosphatase (ALP)-positive cells. Similar effects observed following treatments with both glucose and mannitol suggest that the impairment arises primarily from osmotic stress rather than from glucose-specific metabolic signaling. Notably, pharmacological inhibition of Rho-associated kinase using Y-27632 attenuated perinuclear actin accumulation, restored nuclear translocation of YAP, and rescued the expression of YAP-dependent osteogenic genes under osmotic conditions. Y-27632 also increased the number of ALP-positive cells after treatment with both glucose and mannitol. These findings underscore cytoskeletal remodeling as a central regulator of YAP activity and osteogenesis under osmotic stress, and propose potential therapeutic targets for skeletal fragility in diabetes.