Mechanically regulated proteostasis by Hsp27 governs osteogenesis of mesenchymal stromal cells on soft matrices.

Matrix stiffness is a critical biophysical cue that governs mesenchymal stromal cell (MSC) fate, yet the molecular mechanisms by which soft extracellular environments impair osteogenesis remain poorly defined. In this study, we utilized a stiffness-tunable in vitro model to dissect focal adhesion (FA)-mediated mechanotransduction and identified the small heat shock protein Hsp27 as a mechanosensitive regulator that selectively localizes to the FAs of MSCs on soft matrices. Proteomic profiling revealed that soft substrates reprogram FA composition to recruit chaperones and ubiquitin-proteasome system (UPS) components, with Hsp27 acting as a central effector. Mechanistically, Hsp27 facilitates the localized, proteasome-mediated degradation of the small GTPase Rac1, thereby attenuating lamellipodia formation, cytoskeletal tension, and pro-osteogenic signaling. Genetic deletion or pharmacological inhibition of Hsp27 successfully restored FA maturation, actin stress fiber architecture, and intracellular traction forces, effectively rescuing osteogenic differentiation on compliant substrates. In a murine model of age-related osteoporosis, we observed that femoral bone tissues exhibit hallmarks of matrix softening, including collagen fiber misalignment and increased marrow adiposity, accompanied by Hsp27-mediated Rac1 depletion. These findings establish Hsp27 as a mechanically responsive regulator of FA proteostasis and identify a stiffness-sensitive degradation pathway as a potential therapeutic target to restore bone formation in mechanically compromised environments.