Loading-induced bone formation requires sclerostin (SOST) down-regulation in osteocytes. Class IIa HDACs move from the cytoplasm to the nucleus to suppress SOST in response to PTH. We recently reported that HDAC4 and 5 in osteocytes are required for loading-induced SOST suppression and periosteal bone formation in vivo.
To understand the upstream mechanisms responsible for Fluid Flow Shear Stress(FFSS)-induced HDAC4/5-dependent SOST regulation, we first performed RNA-seq and upstream pathway analyses in FFSS-treated Ocy454 osteocytic cells. This analysis suggested the integrin-FAK axis to be a candidate pathway responsible for coordinate regulation of FFSS-dependent genes. Therefore, we used CRISPR/Cas9 to generate integrin subunit and FAK mutant Ocy454 cells. Only FAK-deficient (KO) clones show low basal SOST expression, and fail to further suppress SOST in response to FFSS. Notably, FAK is required for 94.8% of FFSS-regulated gene expression changes, as assessed by RNA-seq in WT and FAK-KO Ocy454 cells. Small molecule FAK inhibitors block FFSS-induced SOST inhibition and HDAC4/5 nuclear translocation by FFSS. We next explored the effects of FFSS on FAK activity in osteocytes. Surprisingly, FFSS rapidly reduced FAK Y397 auto-phosphorylation and subsequent phosphorylation of the FAK substrate paxillin. Treatment (in the absence of FFSS) with FAK inhibitors, including VS6063, reduced SOST expression both in vitro and in vivo (0.00154±0.00025 vs 0.00086±0.00016 relative to actin, p=0.0346, n=8) (see Figure). We next tested the role of tonic cell/matrix interactions (which are disrupted by FFSS) in driving constitutive FAK activity in Ocy454 cells. The integrin inhibitor cilengitide reduced FAK Y397 tyrosine phosphorylation, and reduced expression of SOST and other FFSS regulated genes. SOST reduction by both integrin and FAK inhibitors did not occur in HDAC4/5 KO cells.
Finally, we tested the link between FAK, a tyrosine kinase, and class IIa HDACs tyrosine phosphorylation. HDAC4/5 tyrosine phosphorylation was reduced by FFSS in cells, and recombinant HDAC5 is directly phosphorylated by FAK at Y642.
In conclusions, our in vitro and in vivo results indicate that class IIa HDACs are vital regulators of osteocyte mechanotransduction downstream of the integrin-FAK signaling pathway. These data suggest that FAK-dependent tyrosine phosphorylation represents a novel mechanism used to regulate class IIa HDAC in response to dynamic changes in cell/matrix interactions.