Sequencing reads were mapped by the STAR aligner98 to the mm9 reference genome using Ensembl annotation. remain unknown. Here, we report that class IIa histone deacetylases (HDAC4 and HDAC5) are required for loading-induced suppression and bone formation. FFSS signaling drives class IIa HDAC nuclear translocation through a signaling pathway involving direct HDAC5 tyrosine 642 phosphorylation by focal adhesion kinase (FAK), a HDAC5 post-translational modification that controls its subcellular localization. Osteocyte cell adhesion supports FAK tyrosine phosphorylation, and FFSS triggers FAK dephosphorylation. Pharmacologic FAK catalytic inhibition reduces mRNA expression in vitro and in vivo. These studies demonstrate a role for HDAC5 as a transducer of matrix-derived cues to regulate cell type-specific gene expression. gene) are both central regulators of bone remodeling. Osteocyte-derived RANKL is a crucial osteoclastogenic factor6, and the target of the osteoporosis drug denosumab7. Sclerostin is a canonical WNT pathway inhibitor that blocks osteoblast activity stimulated by WNTs8. Romosozumab, a neutralizing sclerostin antibody, is now approved for osteoporosis treatment9,10. expression by osteocytes is mechanically regulated, with sclerostin levels increasing with unloading11 and decreasing with skeletal loading12. Necrostatin-1 Osteocytic downregulation is important for loading-induced bone formation13, and upregulation contributes to immobilization-induced bone loss14,15. While it is clear that modulating expression is an important strategy used by osteocytes to link mechanical cues to bone formation, the intracellular signaling pathways through which this occurs are largely unknown. Like mechanical loading, parathyroid hormone (PTH) stimulates bone formation, in part, by reducing sclerostin levels16,17. expression is positively regulated by the transcription factor MEF2C, which binds to a?+?45?kB downstream enhancer site18,19 that is absent in Rabbit Polyclonal to TCF7L1 high bone-mass patients with Van Buchem disease20. In many biologic systems, class IIa histone Necrostatin-1 deacetylases are potent inhibitors of MEF2-driven gene expression21. Class IIa HDACs are uniquely endowed with long N-terminal extensions that confer responsiveness to external signals and allow inhibitory binding to MEF2 family transcription factors22. HDAC4 and HDAC5 inhibit MEF2-driven osteocytic expression23. Moreover, PTH signaling drives HDAC4/5 translocation from the cytosol to the nucleus via a cAMP-dependent pathway involving inhibition of salt-inducible kinases24. Despite these advances, whether class IIa HDACs participate in osteocyte mechanotransduction and loading-induced suppression is currently unknown. It is generally accepted that osteocytes sense mechanical cues by changes in fluid-flow shear stress (FFSS) across their dendritic processes25,26. Skeletal loading induced during functional activity primarily places long bones in bending27, which due to heterogeneous strain distribution within a given cross-section facilitates interstitial fluid flow within the lacunarCcanalicular system28,29. This interstitial FFSS produces focal strains at attachment sites surrounding osteocyte cell processes30. Integrin V/?3 heterodimers have been proposed to play a key role in osteocyte/matrix interaction and mechanotransduction31C33. Multiple membrane proximal signaling mechanisms have been described Necrostatin-1 downstream of FFSS across dendritic processes. These include outside-in integrin signaling, ATP release34, local calcium fluxes35, TRPV4-mediated microtubule reorganization and ROS generation36, plasma membrane disruptions37, and effects on connexin hemichannels38. However, precise links between these proximal signaling steps and suppression remain to be determined. Here, we report that FFSS triggers class IIa HDAC nuclear translocation in osteocytes, and that HDAC4/5 are required for loading-induced bone formation in vivo. While class IIa HDACs are involved in both PTH and FFSS-mediated suppression, these two external cues utilize distinct upstream signaling mechanisms to drive HDAC4/5 nuclear translocation. In osteocytes, constitutive cell/matrix interactions lead to basal activation of focal adhesion kinase (FAK) through outside-in integrin signaling39 for review of integrin-mediated signaling). FAK is known to play crucial roles in mechanotransduction in many tissue types40C43, although links between FAK and class IIa HDACs have not been described. Here, we show that FAK regulates class IIa HDAC subcellular localization by direct HDAC5 tyrosine 642 phosphorylation. FFSS inhibits FAK activity, a step that is required for FFSS-induced suppression. Moreover, many of the transcriptomic effects of FFSS are mimicked by small molecule FAK inhibitors, and by RGD peptides that block integrin/matrix adhesion. Finally, pharmacologic FAK inhibitors can suppress expression in vivo, indicating the therapeutic potential of this FAK/class IIa HDAC/signaling axis. Results Mechanosensitive class IIa HDACs are required for loading-induced bone formation We previously demonstrated that parathyroid hormone (PTH) signaling promotes the dephosphorylation and nuclear translocation of HDAC4 and HDAC5 in osteocytes, and that HDAC4/5 are required for PTH-induced suppression of expression in vitro and in vivo24. Mechanical.