Hairy and enhancer of split 1 is a primary effector of NOTCH2 signaling and induces osteoclast differentiation and...

Notch2^tm1.1Ecan mice, which harbor a mutation replicating that found in Hajdu-Cheney syndrome, exhibit marked osteopenia because of increased osteoclast number and bone resorption. Hairy and enhancer of split 1 (HES1) is a Notch target gene and a transcriptional modulator that determines osteoclast cell fate decisions. Transcript levels of Hes1 increase in Notch2^tm1.1Ecan bone marrow-derived macrophages (BMMs) as they mature into osteoclasts, suggesting a role in osteoclastogenesis. To determine whether HES1 is responsible for the phenotype of Notch2^tm1.1Ecan mice and the skeletal manifestations of Hajdu-Cheney syndrome, Hes1 was inactivated in Ctsk-expressing cells from Notch2^tm1.1Ecan mice. Ctsk encodes the protease cathepsin K, which is expressed preferentially by osteoclasts. We found that the osteopenia of Notch2^tm1.1Ecan mice was ameliorated, and the enhanced osteoclastogenesis was reversed in the context of the Hes1 inactivation. Microcomputed tomography revealed that the downregulation of Hes1 in Ctsk-expressing cells led to increased bone volume/total volume in female mice. In addition, cultures of BMMs from Ctsk^Cre/WT;Hes1^Δ/Δ mice displayed a decrease in osteoclast number and size and decreased bone-resorbing capacity. Moreover, activation of HES1 in Ctsk-expressing cells led to osteopenia and enhanced osteoclast number, size, and bone resorptive capacity in BMM cultures. Osteoclast phenotypes and RNA-Seq of cells in which HES1 was activated revealed that HES1 modulates cell-cell fusion and bone-resorbing capacity by supporting sealing zone formation. In conclusion, we demonstrate that HES1 is mechanistically relevant to the skeletal manifestation of Notch2^tm1.1Ecan mice and is a novel determinant of osteoclast differentiation and function.