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

ABSTRACT: 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.

SUBMITTER: Yu J

PROVIDER: S-EPMC8633688 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Peroxisome proliferator-activated receptor gamma (PPAR?) is a master regulator for white adipocyte differentiation and lipid storage. The increased level of hepatic PPAR?2 isoform reprograms liver for lipid storage and causes abnormal fat accumulation in certain pathophysiologic conditions. The current study aimed to investigate a role of transcriptional repressor hairy and enhancer of split 6 (HES6) in the regulation of Pparg2 expression and hepatic steatosis induced by diet. Liver-specific overexpression of Hes6 using adenovirus reduced Pparg2 messenger RNA levels by 90% and hepatic triglyceride accumulation by 22% compared to the levels in mice injected with an adenoviral empty vector with Western diet feeding. In sharp contrast, silencing Hes6 gene expression using short hairpin RNA increased hepatic lipid accumulation and Pparg2 messenger RNA levels by 70% and 4-fold, respectively. To locate hepatocyte nuclear factor 4 alpha (HNF4?) binding site(s), through which repressional activity of HES6 is mediated, a 2.5-kb Pparg2 promoter-driven luciferase reporter was constructed for transient transfection assays. Subsequently, chromatin immunoprecipitation and electrophoretic mobility shift assays were performed. An HNF4? binding consensus sequence was identified at 903 base pairs upstream from the transcription start site of Pparg2. Deletion or point mutation of the sequence in a luciferase reporter containing the Pparg2 promoter abolished HNF4?-mediated activation in HeLa cells. Chromatin immunoprecipitation and electrophoretic mobility shift assays further confirmed direct recruitment and binding of HNF4? to the site. Gene expression analysis with liver samples from subjects with nonalcoholic steatohepatitis suggested that the axis of the Hes6-Hnf4a-Pparg2 transcriptional cascade is also responsible for hepatic fat accumulation in humans. Conclusion: HES6 represses Pparg2 gene expression, thereby preventing hepatic lipid accumulation induced by chronic Western diet feeding or pathophysiologic conditions. (Hepatology Communications 2017;1:1085-1098).

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

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