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

Open AccessPublished:November 03, 2021DOI:https://doi.org/10.1016/j.jbc.2021.101376
      Notch2tm1.1Ecan 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 Notch2tm1.1Ecan 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 Notch2tm1.1Ecan mice and the skeletal manifestations of Hajdu–Cheney syndrome, Hes1 was inactivated in Ctsk-expressing cells from Notch2tm1.1Ecan mice. Ctsk encodes the protease cathepsin K, which is expressed preferentially by osteoclasts. We found that the osteopenia of Notch2tm1.1Ecan 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 CtskCre/WT;Hes1Δ/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 Notch2tm1.1Ecan mice and is a novel determinant of osteoclast differentiation and function.

      Keywords

      Abbreviations:

      α-MEM (α-minimum essential medium), μCT (microcomputed tomography), Ad-Cre (adenoviruses carrying cytomegalovirus-Cre), Ad-GFP (adenoviruses carrying GFP), Blimp1 (B lymphocyte–induced maturation protein 1), BMM (bone marrow–derived macrophage), BV/TV (bone volume/total volume), cDNA (complementary DNA), FBS (fetal bovine serum), HCS (Hajdu–Cheney syndrome), HES1 (hairy and enhancer of split 1), IL (interleukin), IPA (ingenuity pathway analysis), M-CSF (macrophage colony-stimulating factor), NFATc1 (nuclear factor of activated T cells, cytoplasmic 1), RANKL (receptor activator of NF-κB ligand), SMI (structure model index), TNFα (tumor necrosis factor α), TRAP (tartrate resistant acid phosphatase)
      Osteoclasts are multinucleated giant cells that are responsible for bone resorption and essential to maintain bone homeostasis. Osteoclasts are derived from the differentiation and fusion of mononuclear cells of the myeloid lineage by the actions of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) (
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      Hajdu–Cheney syndrome (HCS) (Online Mendelian Inheritance in Man: 102500) is a rare and devastating disorder characterized by numerous skeletal manifestations, including craniofacial developmental defects, short stature, bone loss with fractures, and acroosteolysis associated with inflammation of the distal phalanges (
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      ). HCS is associated with mutations or short deletions in exon 34 of NOTCH2 upstream of the PEST domain, which is required for the ubiquitination and degradation of NOTCH2 (
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      • Simpson M.A.
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      • Stewart H.
      • Kim C.A.
      • et al.
      Mutations in NOTCH2 cause Hajdu-Cheney syndrome, a disorder of severe and progressive bone loss.
      ,
      • Zhao W.
      • Petit E.
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      • Robey P.G.
      • Seton M.
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      • Mannstadt M.
      Mutations in NOTCH2 in patients with Hajdu-Cheney syndrome.
      ). The HCS pathogenic variants lead to the premature termination of a protein product lacking sequences necessary for the proteasomal degradation of the NOTCH2 intracellular domain so that the protein is stable and a gain-of-NOTCH2 function ensues. Autosomal dominant inheritance as well as de novo heterozygous mutations have been reported (
      • Canalis E.
      Clinical and experimental aspects of notch receptor signaling: Hajdu-Cheney syndrome and related disorders.
      ,
      • Isidor B.
      • Lindenbaum P.
      • Pichon O.
      • Bezieau S.
      • Dina C.
      • Jacquemont S.
      • Martin-Coignard D.
      • Thauvin-Robinet C.
      • Le M.M.
      • Mandel J.L.
      • David A.
      • Faivre L.
      • Cormier-Daire V.
      • Redon R.
      • Le C.C.
      Truncating mutations in the last exon of NOTCH2 cause a rare skeletal disorder with osteoporosis.
      ,
      • Majewski J.
      • Schwartzentruber J.A.
      • Caqueret A.
      • Patry L.
      • Marcadier J.
      • Fryns J.P.
      • Boycott K.M.
      • Ste-Marie L.G.
      • McKiernan F.E.
      • Marik I.
      • Van E.H.
      • Michaud J.L.
      • Samuels M.E.
      Mutations in NOTCH2 in families with Hajdu-Cheney syndrome.
      ,
      • Simpson M.A.
      • Irving M.D.
      • Asilmaz E.
      • Gray M.J.
      • Dafou D.
      • Elmslie F.V.
      • Mansour S.
      • Holder S.E.
      • Brain C.E.
      • Burton B.K.
      • Kim K.H.
      • Pauli R.M.
      • Aftimos S.
      • Stewart H.
      • Kim C.A.
      • et al.
      Mutations in NOTCH2 cause Hajdu-Cheney syndrome, a disorder of severe and progressive bone loss.
      ,
      • Zhao W.
      • Petit E.
      • Gafni R.I.
      • Collins M.T.
      • Robey P.G.
      • Seton M.
      • Miller K.K.
      • Mannstadt M.
      Mutations in NOTCH2 in patients with Hajdu-Cheney syndrome.
      ).
      Our laboratory created a knock-in mouse model harboring a Notch26955C>T mutation reproducing HCS and termed Notch2tm1.1Ecan (also known as Notch2Q2319X) (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ,
      • Yu J.
      • Zanotti S.
      • Walia B.
      • Jellison E.
      • Sanjay A.
      • Canalis E.
      The Hajdu Cheney mutation is a determinant of B-cell allocation of the splenic marginal zone.
      ). The homozygous mutation is associated with craniofacial developmental abnormalities and is lethal, and heterozygous Notch2tm1.1Ecan mutant mice exhibit profound osteopenia and short limbs, reproducing functional outcomes of the human disease and establishing the first model for the study of HCS (
      • Canalis E.
      Clinical and experimental aspects of notch receptor signaling: Hajdu-Cheney syndrome and related disorders.
      ,
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ). Notch2tm1.1Ecan mice have increased bone resorption secondary to a direct effect of the gain-of-NOTCH2 function on osteoclastogenesis as well as the increased expression of RANKL by cells of the osteoblast lineage (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ). These are unique functional properties of NOTCH2, which are distinct from those reported for other Notch receptors (
      • Canalis E.
      Notch in skeletal physiology and disease.
      ,
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      The skeleton of lateral meningocele syndrome.
      ). Indeed, NOTCH1 inhibits osteoclastogenesis directly, and NOTCH3 is not expressed in the myeloid lineage; although, by inducing RANKL in cells of the osteoblast lineage, it enhances osteoclastogenesis indirectly (
      • Bai S.
      • Kopan R.
      • Zou W.
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      • Long F.
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      • Teitelbaum S.L.
      NOTCH1 regulates osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblast lineage cells.
      ,
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      • Hu X.
      • Ivashkiv L.B.
      TNF-induced osteoclastogenesis and inflammatory bone resorption are inhibited by transcription factor RBP-J.
      ,
      • Canalis E.
      • Yu J.
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      • Zanotti S.
      The lateral meningocele syndrome mutation causes marked osteopenia in mice.
      ). Low levels of NOTCH4 are expressed in the myeloid lineage, and it is not known to play a role in osteoblastogenesis (
      • Yu J.
      • Canalis E.
      Notch and the regulation of osteoclast differentiation and function.
      ).
      Cultures of bone marrow macrophages (BMMs) revealed that the expression of Hes1, a Notch target gene, is enhanced as cells mature as osteoclasts, and the increased expression is of greater magnitude in cultures from Notch2tm1.1Ecan mice (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ,
      • Yu J.
      • Canalis E.
      The Hajdu Cheney mutation sensitizes mice to the osteolytic actions of tumor necrosis factor alpha.
      ). Importantly, other Notch target genes, such as Hes3, Hes5, Hey1, Hey2, and HeyL, are either expressed at very low levels or not detected in BMMs from control or mutant mice. This observation suggests that hairy and enhancer of split 1 (HES1) may be an important regulator of osteoclastogenesis and is in part responsible for the HCS phenotype.
      HES1 is a transcriptional modulator that plays a role in the differentiation of embryonic stem and mesenchymal cells (
      • Kobayashi T.
      • Mizuno H.
      • Imayoshi I.
      • Furusawa C.
      • Shirahige K.
      • Kageyama R.
      The cyclic gene Hes1 contributes to diverse differentiation responses of embryonic stem cells.
      • Ross D.A.
      • Rao P.K.
      • Kadesch T.
      Dual roles for the Notch target gene Hes-1 in the differentiation of 3T3-L1 preadipocytes.
      ). Although HES1 is considered a transcriptional repressor, transcription factors can function as either positive or negative regulators of transcription in a cell context–dependent manner (
      • Alvarez M.
      • Rhodes S.J.
      • Bidwell J.P.
      Context-dependent transcription: All politics is local.
      ,
      • Yao J.
      • Lai E.
      • Stifani S.
      The winged-helix protein brain factor 1 interacts with groucho and hes proteins to repress transcription.
      ). In addition, calcium/calmodulin-dependent protein kinase 2 can convert HES1 from a repressor to an activator of transcription (
      • Sugita S.
      • Hosaka Y.
      • Okada K.
      • Mori D.
      • Yano F.
      • Kobayashi H.
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      • et al.
      Transcription factor Hes1 modulates osteoarthritis development in cooperation with calcium/calmodulin-dependent protein kinase 2.
      ,
      • Ju B.G.
      • Solum D.
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      • Lee K.J.
      • Rose D.W.
      • Glass C.K.
      • Rosenfeld M.G.
      Activating the PARP-1 sensor component of the groucho/TLE1 corepressor complex mediates a CaMKinase IIdelta-dependent neurogenic gene activation pathway.
      ). Misexpression of Hes1 in the osteoblast lineage has demonstrated a role as an inhibitor of osteoblast differentiation and function (
      • Zanotti S.
      • Smerdel-Ramoya A.
      • Canalis E.
      Hairy and enhancer of split (HES)1 is a determinant of bone mass.
      ). The role of HES1 in osteoclastogenesis is unknown.
      The intent of the present study was to determine whether HES1 was mechanistically relevant to the HCS phenotype and to define the function of HES1 in osteoclast differentiation in vitro and in vivo. For this purpose, Hes1 was induced or inactivated in Ctsk-expressing cells of the osteoclast lineage. To determine whether HES1 had a mechanistic role in the skeletal phenotype of HCS, Notch2tm1.1Ecan mice were studied in the context of the Hes1 inactivation in Ctsk-expressing cells. Skeletal phenotypes were determined by microcomputed tomography (μCT) and histomorphometry and cellular effects by the study of osteoclast differentiation and resorption activity in vitro.

      Results

      Inactivation of Hes1 reverses the effect of the Hajdu–Cheney mutation on osteoclastogenesis

      To determine whether HES1 played a role in the enhanced osteoclastogenesis observed in Notch2tm1.1Ecan mice, osteoclast precursors from Notch2tm1.1Ecan;Hes1loxP/loxP and Hes1loxP/loxP littermate controls were transduced with adenoviruses carrying cytomegalovirus-Cre (Ad-Cre) or GFP (Ad-GFP) control vectors. Hes1 mRNA levels were increased in Notch2tm1.1Ecan cells and decreased significantly in Notch2tm1.1Ecan;Hes1Δ/Δ and Hes1Δ/Δ osteoclasts transduced with Ad-Cre compared with Notch2tm1.1Ecan;Hes1loxP/loxP and Hes1loxP/loxP cells transduced with Ad-GFP. Notch2 and Notch26955C>T mutant transcripts were not affected by the Hes1 inactivation (Fig. 1). Notch2tm1.1Ecan;Hes1loxP/loxP osteoclast precursors treated with RANKL exhibited an increase in osteoclast number compared with Hes1loxP/loxP cells. Osteoclast number was decreased significantly in Notch2tm1.1Ecan; Hes1Δ/Δ and Hes1Δ/Δ cells so that the Hes1 inactivation reversed the enhanced osteoclastogenesis observed in the context of the Notch2tm1.1Ecan mutation. In addition, basal levels of osteoclastogenesis were reduced in Hes1Δ/Δ cells, suggesting a role of HES1 in osteoclastogenesis under physiological conditions (Fig. 1).
      Figure thumbnail gr1
      Figure 1Hes1 inactivation reverses the effect of the Hajdu–Cheney mutation on osteoclastogenesis. BMMs derived from 2-month-old Notch2tm1.1Ecan;Hes1loxP/loxP and Hes1loxP/loxP littermate controls were cultured for 2 days with M-CSF at 30 ng/ml and RANKL at 10 ng/ml and transduced with adenoviruses carrying CMV-Cre (Ad-Cre) or adenoviruses carrying GFP (Ad-GFP) as control at MOI 100 and cultured for two additional days in the presence of M-CSF at 30 ng/ml and RANKL at 10 ng/ml until the formation of multinucleated TRAP-positive cells. A, total RNA was extracted, and gene expression was determined by quantitative RT–PCR. Data are expressed as Notch26955C>T, Notch2, and Hes1, corrected for Rpl38 copy number. B, representative images of TRAP-stained multinucleated cells are shown. The scale bars in the right corner represent 500 μm. C, TRAP-positive cells with more than three nuclei were considered osteoclasts and counted. Values are means ± SD; n = 4 technical replicates for WT (open circles) and Notch2tm1.1Ecan (closed circles) cells in the context of Hes1loxP/loxP (white bar) or Hes1Δ/Δ (gray bar) deleted alleles. Representative data are shown from two independent experiments. ∗Significantly different between Notch2tm1.1Ecan and control, p < 0.05. #Significantly different between Hes1Δ/Δ and Hes1loxP/loxP, p < 0.05. BMM, bone marrow–derived macrophage; M-CSF, macrophage colony-stimulating factor; MOI, multiplicity of infection; RANKL, receptor activator of NF-κB ligand; TRAP, tartrate resistant acid phosphatase.
      To determine whether the inactivation of Hes1 could reverse the osteopenia of the Notch2tm1.1Ecan mutation, CtskCre/WT;Hes1loxP/loxP mice were crossed with Notch2tm1.1Ecan;Hes1loxP/loxP mice to inactivate Hes1 in the context of the Notch2 mutation. The transcript levels of Hes1 were decreased in bone extracts from 2-month-old male CtskCre/WT;Hes1Δ/Δ and CtskCre/WT;Notch2tm1.1Ecan;Hes1Δ/Δ mice compared with control, whereas Notch2 WT and mutant (Notch26955C>T) mRNA levels were not affected (Fig. S1). Confirming prior observations, Notch2tm1.1Ecan mice displayed cancellous bone osteopenia associated with decreased connectivity and trabecular number (Fig. 2). The Hes1 inactivation by itself did not alter bone microarchitectural parameters in 2-month-old male mice compared with control sex-matched WT mice. The decreased cancellous bone volume/total volume (BV/TV) observed in Notch2tm1.1Ecan was significantly increased in the context of the Hes1 inactivation associated with increased connectivity and trabecular number so that the osteopenia of Notch2tm1.1Ecan mice was ameliorated in CtskCre/WT;Notch2tm1.1Ecan;Hes1Δ/Δ mice (Fig. 2). Cancellous bone histomorphometry confirmed previous work and demonstrated an increase in osteoclast number and bone resorption, without an effect on osteoblast number and bone formation, in Notch2tm1.1Ecan mice (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ). The increased osteoclast number and eroded surface found in Notch2tm1.1Ecan mice were decreased ∼50% in the context of the Hes1 inactivation, so that both parameters were no longer increased in CtskCre/WT;Notch2tm1.1Ecan;Hes1Δ/Δ male mice compared with Notch2tm1.1Ecan;HesloxP/loxP control mice (Table 1). These results indicate that HES1 is mechanistically relevant to the osteopenia of Notch2tm1.1Ecan mice although they suggest a minor role of HES1 in the bone architecture of male mice. The Hes1 deletion had only a modest effect on the cortical osteopenic phenotype (not shown) and did not affect the decrease in femoral length observed in Notch2tm1.1Ecan mice (Fig. S1).
      Figure thumbnail gr2
      Figure 2Hes1 inactivation in Ctsk-expressing cells ameliorates the osteopenia of the Hajdu–Cheney mutation. μCT was performed on 2-month-old WT (open circles) or Notch2tm1.1Ecan (closed circles) mice in a Hes1Δ/Δ (gray bar) or Hes1loxP/loxP (white bar) genetic background by crossing CtskCre/WT;Hes1loxP/loxP with Notch2tm1.1Ecan;Hes1loxP/loxP mice. A, representative images show osteopenic cancellous bone of the distal femur in Notch2tm1.1Ecan;Hes1loxP/loxP male mice and its amelioration by the Hes1 inactivation. The scale bar in the right corner represents 1 mm. B, parameters shown are bone volume/total volume (BV/TV, %); connectivity density (Conn.D, mm−3); structure model index (SMI); trabecular number (Tb.N, mm−1), trabecular separation (Tb.Sp, μm), and trabecular thickness (Tb.Th, μm). Values are means ± SD; n = 12 for control Hes1loxP/loxP and n = 6 for Notch2tm1.1Ecan;Hes1loxP/loxP; n = 10 for CtskCre/WT;Hes1Δ/Δ and n = 11 for CtskCre/WT;Notch2tm1.1Ecan;Hes1Δ/Δ. ∗Significantly different between Notch2tm1.1Ecan and control, p < 0.05. #Significantly different between Hes1Δ/Δ and Hes1loxP/loxP, p < 0.05. μCT, microcomputed tomography.
      Table 1Cancellous bone histomorphometry of 2-month-old Hes1loxP/loxP, Notch2tm1.1Ecan;Hes1loxP/loxP, CtskCre/WT;Hes1Δ/Δ and CtskCre/WT;Notch2tm1.1Ecan;Hes1Δ/Δ male mice
      Distal femur trabecular boneHes1loxP/loxPHes1Δ/Δ
      WTNotch2tm1.1EcanWTNotch2tm1.1Ecan
      n = 4
      BV/TV (%)32.1 ± 2.814.2 ± 3.6
      Significantly different between Notch2tm1.1Ecan and WT, p < 0.05.
      37.4 ± 1.924.9 ± 7.4
      Significantly different between Notch2tm1.1Ecan and WT, p < 0.05.
      ,
      Significantly different between Hes1Δ/Δ and Hes1loxP/loxP, p < 0.05.
      Trabecular separation (μm)117 ± 5236 ± 53
      Significantly different between Notch2tm1.1Ecan and WT, p < 0.05.
      94 ± 7130 ± 40
      Significantly different between Hes1Δ/Δ and Hes1loxP/loxP, p < 0.05.
      Trabecular number (1/mm)5.8 ± 0.13.9 ± 1.0
      Significantly different between Notch2tm1.1Ecan and WT, p < 0.05.
      6.7 ± 0.56.0 ± 1.2
      Significantly different between Hes1Δ/Δ and Hes1loxP/loxP, p < 0.05.
      Trabecular thickness (μm)55 ± 539 ± 4
      Significantly different between Notch2tm1.1Ecan and WT, p < 0.05.
      56 ± 541 ± 6
      Significantly different between Notch2tm1.1Ecan and WT, p < 0.05.
      Osteoblast surface/bone surface (%)14.7 ± 0.919 ± 1.313 ± 1.918 ± 4.3
      Osteoblasts/bone perimeter (1/mm)9.1 ± 0.911.3 ± 0.68.5 ± 1.010.1 ± 1.6
      Osteoclast surface/bone surface (%)10.4 ± 3.720.1 ± 3.4
      Significantly different between Notch2tm1.1Ecan and WT, p < 0.05.
      12.1 ± 2.29.1 ± 4.1
      Significantly different between Hes1Δ/Δ and Hes1loxP/loxP, p < 0.05.
      Osteoclasts/bone perimeter (1/mm)3.2 ± 1.36.1 ± 1.2
      Significantly different between Notch2tm1.1Ecan and WT, p < 0.05.
      3.6 ± 0.82.8 ± 1.7
      Significantly different between Hes1Δ/Δ and Hes1loxP/loxP, p < 0.05.
      Eroded surface/bone surface (%)10.3 ± 1.313.4 ± 2.2
      Significantly different between Notch2tm1.1Ecan and WT, p < 0.05.
      11.9 ± 0.87.1 ± 1.1
      Significantly different between Notch2tm1.1Ecan and WT, p < 0.05.
      ,
      Significantly different between Hes1Δ/Δ and Hes1loxP/loxP, p < 0.05.
      Mineral apposition rate (μm/day)3.3 ± 1.02.8 ± 0.22.6 ± 0.12.4 ± 0.2
      Mineralizing surface/bone surface (%)31.2 ± 2.827.7 ± 3.535.1 ± 4.432.3 ± 2.3
      Bone formation rate (μm3/μm2/day)1.0 ± 0.30.8 ± 0.10.9 ± 0.10.8 ± 0.1
      Bone histomorphometry was performed on distal femurs from 2-month-old Hes1loxP/loxP, Notch2tm1.1Ecan;Hes1loxP/loxP, CtskCre/WT;Hes1Δ/Δ, and CtskCre/WT;Notch2tm1.1Ecan;Hes1Δ/Δ male mice. Values are means ± SD.
      a Significantly different between Notch2tm1.1Ecan and WT, p < 0.05.
      b Significantly different between Hes1Δ/Δ and Hes1loxP/loxP, p < 0.05.

      HES1 is a determinant of osteoclastogenesis in vitro

      To ascertain the function of HES1 in cells of the osteoclast lineage, BMMs from either Hes1loxP/loxP or Rosa[STOP]Hes1 mice were isolated. BMMs were cultured in the presence of M-CSF and RANKL for 2 days and transduced with Ad-Cre to delete loxP flanked sequences or Ad-GFP as control. Excision of the STOP cassette in RosaHes1 cells resulted in a 20-fold induction of Hes1 mRNA and a 1.7-fold increase in osteoclastogenesis compared with Rosa[STOP]Hes1 cultures transduced with Ad-GFP (Fig. 3). Conversely, deletion of Hes1 resulted in a 50% reduction in Hes1 mRNA levels and a 50% decrease in osteoclast number compared with control cultures transduced with Ad-GFP (Fig. 3). The results demonstrate that HES1 is a determinant of osteoclast differentiation in vitro.
      Figure thumbnail gr3
      Figure 3HES1 is required for osteoclastogenesis in vitro. BMMs derived from 2-month-old RosaHes1 (A and B) or HesloxP/loxP mice (C and D) were cultured in the presence of M-CSF at 30 ng/ml and RANKL at 10 ng/ml for 2 days. Cells were transduced with Ad-Cre (closed circles), to recombine loxP flanked sequences, or Ad-GFP (open circles) as a control and then cultured for two additional days. A and C, representative images of TRAP-stained multinucleated cells are shown. The scale bar in the right corner represents 500 μm. B and D, Hes1 transcript levels were measured by quantitative RT–PCR in total RNA from osteoclasts. Transcript levels are reported as copy number corrected for Rpl38 (left). TRAP-positive cells with more than three nuclei were considered osteoclasts (right). Values are means ± SD; n = 3 or 4 technical replicates for control (open circles) and either Hes1Δ/Δ or recombined RosaHes1 (closed circles) cells. Representative data are shown from two independent experiments. ∗Significantly different between Hes1Δ/Δ and control, p < 0.05; or recombined RosaHes1 and control, p < 0.05. BMM, bone marrow–derived macrophage; HES1, hairy and enhancer of split 1; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of NF-κB ligand; TRAP, tartrate resistant acid phosphatase.

      Inactivation of Hes1 in osteoclasts of female mice increases BV in vivo

      To confirm a role of HES1 in osteoclastogenesis and bone homeostasis, Hes1 was inactivated in vivo in Ctsk-expressing cells. For this purpose, CtskCre/WT;Hes1loxP/loxP mice were crossed with Hes1loxP/loxP mice to generate CtskCre/WT;Hes1Δ/Δ and littermate Hes1loxP/loxP controls. CtskCre/WT;Hes1Δ/Δ appeared healthy, and their weight and femoral length were not different from littermate Hes1loxP/loxP mice (Fig. S2). CtskCre-mediated recombination was documented in genomic DNA from tibiae of CtskCre/WT;Hes1Δ/Δ mice with a consequent decrease in Hes1 mRNA. Confirming the results observed in the context of the Notch2tm1.1Ecan mutant mice, inactivation of Hes1 in 2- or 4-month-old male mice did not result in an obvious skeletal phenotype, although trabecular number and connectivity were modestly increased (Table 2). In contrast, 2-month and particularly 4-month-old female mice harboring the inactivation of Hes1 exhibited a significant increase in femoral BV/TV (Table 2 and Fig. 4). Femoral μCT of 4-month-old female CtskCre/WT;Hes1Δ/Δ mice revealed an 85% increase in BV/TV associated with an increase in trabecular number and connectivity density and a decrease in structure model index (SMI) compared with controls. Bone histomorphometry of 4-month-old CtskCre/WT;Hes1Δ/Δ female mice demonstrated an ∼50% decrease in osteoclast number and ∼35% decrease in eroded surface, compared with littermate controls, confirming that HES1 is required for osteoclast differentiation and function in vivo (Table 3 and Fig. 5). Osteoblast number and bone formation were not affected by the Hes1 deletion.
      Table 2Femoral microarchitecture assessed by μCT of 2- and 4-month-old CtskCre/WT;Hes1Δ/Δ mice and sex-matched littermate controls
      Two months oldMalesFemales
      ControlHes1Δ/ΔControlHes1Δ/Δ
      n = 3n = 6n = 6–7n = 14
      Distal femur trabecular bone
       BV/TV (%)19.3 ± 3.820.4 ± 3.310.4 ± 1.413.0 ± 1.0
      Significantly different from control, p < 0.05.
       Trabecular separation (μm)150 ± 9140 ± 8206 ± 11180 ± 10
      Significantly different from control, p < 0.05.
       Trabecular number (1/mm)6.6 ± 0.67.3 ± 0.3
      Significantly different from control, p < 0.05.
      5.0 ± 0.35.6 ± 0.3
      Significantly different from control, p < 0.05.
       Trabecular thickness (μm)44 ± 642 ± 239 ± 239 ± 3
       Connectivity density (1/mm3)391 ± 34474 ± 35
      Significantly different from control, p < 0.05.
      220 ± 37322 ± 40
      Significantly different from control, p < 0.05.
       Structure model index1.7 ± 0.31.6 ± 0.42.3 ± 0.32.2 ± 0.2
       Density of material (mg HA/cm3)849 ± 28797 ± 50814 ± 56801 ± 55
      Femoral midshaft cortical bone
       BV/TV (%)88.6 ± 1.488.0 ± 0.488.5 ± 0.888.1 ± 1.2
       Porosity (%)11.4 ± 1.412.0 ± 0.411.5 ± 0.811.9 ± 1.2
       Cortical thickness (μm)147 ± 13144 ± 5141 ± 7144 ± 12
       Total area (mm2)2.0 ± 0.12.0 ± 0.21.8 ± 0.21.8 ± 0.1
       Bone area (mm2)0.9 ± 0.10.8 ± 0.10.7 ± 0.00.7 ± 0.1
       Periosteal perimeter (mm)5.1 ± 0.25.0 ± 0.24.8 ± 0.04.7 ± 0.2
       Endocortical perimeter (mm)3.8 ± 0.13.8 ± 0.23.7 ± 0.13.6 ± 0.1
       Density of material (mg HA/cm3)1071 ± 451040 ± 131079 ± 421062 ± 42
      Four months oldMalesFemales
      ControlHes1Δ/ΔControlHes1Δ/Δ
      n = 6n = 11–12n = 6n = 10
      Distal femur trabecular bone
       BV/TV (%)15.8 ± 3.017.9 ± 1.74.6 ± 1.08.5 ± 2.8
      Significantly different from control, p < 0.05.
       Trabecular separation (μm)192 ± 15174 ± 10
      Significantly different from control, p < 0.05.
      302 ± 27255 ± 17
      Significantly different from control, p < 0.05.
       Trabecular number (1/mm)5.2 ± 0.55.7 ± 0.33.3 ± 0.34.0 ± 0.3
      Significantly different from control, p < 0.05.
       Trabecular thickness (μm)44 ± 242 ± 437 ± 340 ± 7
       Connectivity density (1/mm3)212 ± 44271 ± 40
      Significantly different from control, p < 0.05.
      82 ± 23158 ± 44
      Significantly different from control, p < 0.05.
       Structure model index1.4 ± 0.41.2 ± 0.22.8 ± 0.12.2 ± 0.3
      Significantly different from control, p < 0.05.
       Density of material (mg HA/cm3)903 ± 26908 ± 24902 ± 27896 ± 28
      Femoral midshaft cortical bone
       BV/TV (%)91.7 ± 2.090.0 ± 2.692.0 ± 0.491.8 ± 0.6
       Porosity (%)8.3 ± 2.010.0 ± 2.68.0 ± 0.48.2 ± 0.6
       Cortical thickness (μm)182 ± 13174 ± 10186 ± 6184 ± 7
       Total area (mm2)2.1 ± 0.22.2 ± 0.21.7 ± 0.11.8 ± 0.1
      Significantly different from control, p < 0.05.
       Bone area (mm2)1.0 ± 0.11.0 ± 0.10.8 ± 0.00.9 ± 0.0
      Significantly different from control, p < 0.05.
       Periosteal perimeter (mm)5.2 ± 0.25.3 ± 0.24.6 ± 0.14.8 ± 0.1
      Significantly different from control, p < 0.05.
       Endocortical perimeter (mm)3.7 ± 0.23.8 ± 0.23.3 ± 0.23.4 ± 0.1
       Density of material (mg HA/cm3)1168 ± 251166 ± 191190 ± 371193 ± 22
      μCT was performed on distal femurs for trabecular bone and midshaft for cortical bone. Values are means ± SD.
      a Significantly different from control, p < 0.05.
      Figure thumbnail gr4
      Figure 4Inactivation of Hes1 in Ctsk-expressing cells increases bone volume in female mice. Representative microcomputed tomography image of femurs from 2- (A) and 4-month-old (B) male and female CtskCre/WT;Hes1Δ/Δ mice and Hes1loxP/loxP sex-matched control littermates. The scale bar in the right corner represents 1 mm.
      Table 3Cancellous bone histomorphometry of 4-month-old CtskCre/WT;Hes1Δ/Δ female mice and sex-matched littermate controls
      Distal femur trabecular boneControlHes1Δ/Δ
      n = 4–5n = 6–8
      BV/TV (%)9.3 ± 2.313.6 ± 2.2
      Significantly different from control, p < 0.05.
      Trabecular separation (μm)317 ± 69229 ± 38
      Significantly different from control, p < 0.05.
      Trabecular number (1/mm)3.0 ± 0.73.9 ± 0.5
      Significantly different from control, p < 0.05.
      Trabecular thickness (μm)31 ± 3.234 ± 4.0
      Osteoblast surface/bone surface (%)15.9 ± 2.215.3 ± 2.0
      Osteoblasts/bone perimeter (1/mm)12.4 ± 2.612.6 ± 1.6
      Osteoclast surface/bone surface (%)24.4 ± 8.112.6 ± 3.5
      Significantly different from control, p < 0.05.
      Osteoclasts/bone perimeter (1/mm)8.3 ± 2.14.7 ± 1.1
      Significantly different from control, p < 0.05.
      Eroded surface/bone surface (%)16.5 ± 6.510.6 ± 2.4
      Significantly different from control, p < 0.05.
      Mineral apposition rate (μm/day)1.7 ± 0.11.8 ± 0.4
      Mineralizing surface/bone surface (%)31.1 ± 5.132.8 ± 2.5
      Bone formation rate (μm3/μm2/day)0.5 ± 0.10.6 ± 0.1
      Bone histomorphometry was performed on distal femurs from 4-month-old CtskCre/WT;Hes1Δ/Δ female mice and sex-matched littermate controls. Values are means ± SD.
      a Significantly different from control, p < 0.05.
      Figure thumbnail gr5
      Figure 5Inactivation of Hes1 in Ctsk-expressing cells decreases osteoclast number and bone resorption in vivo. Representative static (upper panels) and dynamic (lower panels) cancellous bone histomorphometry of the distal femur from 4-month-old CtskCre/WT;Hes1Δ/Δ female mice and Hes1loxP/loxP sex-matched control littermates. The scale bar in the right corner represents 50 μm.

      Inactivation of Hes1 decreases osteoclast differentiation in vitro

      To confirm that the phenotype of CtskCre/WT;Hes1Δ/Δ mice was due to a decrease in osteoclast differentiation, BMMs derived from CtskCre/WT;Hes1Δ/Δ and control littermates were cultured in the presence of M-CSF and RANKL. CtskCre/WT;Hes1Δ/Δ cultures revealed a 42% decrease in osteoclast number when compared with cells from littermate controls (Fig. 6). The number of osteoclasts with high number of nuclei was decreased in CtskCre/WT;Hes1Δ/Δ cultures compared with controls, indicating that the size of osteoclasts was reduced because of a decrease in the fusion capacity of CtskCre/WT;Hes1Δ/Δ cells. Mature osteoclasts have a distinct cytoskeletal structure, namely the sealing zone, a circular actin-rich structure formed by podosomes in a cluster to create a ring that is tightly adherent to the bone matrix for efficient bone resorption (
      • Takito J.
      • Inoue S.
      • Nakamura M.
      The sealing zone in osteoclasts: A self-organized structure on the bone.
      ). Phalloidin staining of osteoclasts from CtskCre/WT;Hes1Δ/Δ mice cultured on bone slices revealed smaller sealing zones than controls and a ∼30% decrease in the perimeter of the sealing zone (Fig. 6). CtskCre/WT;Hes1Δ/Δ osteoclasts also exhibited a ∼60% decrease in total bone resorption area, indicating a decrease in osteoclast resorptive activity (Fig. 6).
      Figure thumbnail gr6
      Figure 6Number, size, and resorptive capacity are decreased in CtskCre/WT;Hes1Δ/Δ osteoclasts. BMMs derived from 2-month-old CtskCre/WT;Hes1Δ/Δ (closed circles) mice and control littermates (open circles) were cultured for 4 days in the presence of M-CSF at 30 ng/ml and of RANKL at 10 ng/ml in cell culture–coated plates (A and B) or bone discs (CF). A, representative images of TRAP-stained multinucleated cells in cell culture–coated plates are shown. The scale bar in the right corner represents 500 μm. B, Hes1 transcript levels were measured by quantitative RT–PCR in total RNA from osteoclasts. Transcript levels are reported as copy number corrected for Rpl38 (left). TRAP-positive cells with more than three nuclei were considered osteoclasts and counted (middle). TRAP-positive cells with differential counting of nuclei/osteoclast are shown (right). C, representative images of Alexa Fluor 594 phalloidin-stained multinucleated cells on bone discs are shown. The scale bar in the right corner represents 100 μm. D, the perimeter of sealing zones was measured in n = 145 osteoclasts from control and n = 139 osteoclasts from CtskCre/WT;Hes1Δ/Δ cultures. E, representative images of toluidine blue–stained resorption pits. The scale bar in the right corner represents 200 μm. F, the total resorption pit area was measured (%). Values are means ± SD; n = 3 or 4 biological replicates for control and CtskCre/WT;Hes1Δ/Δ. ∗Significantly different between CtskCre/WT;Hes1Δ/Δ and control, p < 0.05. BMM, bone marrow–derived macrophage; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of NF-κB ligand; TRAP, tartrate resistant acid phosphatase.

      Induction of HES1 in osteoclasts causes osteopenia

      To determine the effect of the HES1 induction on osteoclastogenesis in vivo, homozygous Rosa[STOP]Hes1 mice were crossed with CtskCre/WT mice for the creation of CtskCre/WT;RosaHes1 experimental mice and Rosa[STOP]Hes1 littermate controls. CtskCre/WT;RosaHes1 mice appeared healthy, and their weight was not different from that of littermate controls (Fig. S3). CtskCre-mediated recombination was demonstrated in genomic DNA from tibiae of CtskCre/WT;RosaHes1 mice, and Hes1 mRNA levels were increased in bone extracts from CtskCre/WT;RosaHes1 mice.
      Femoral architecture of 10-week-old male and female CtskCre/WT;RosaHes1 mice revealed a 30% decrease in BV/TV associated with a decrease in connectivity and an increase in SMI in CtskCre/WT;RosaHes1 mice that reached statistical significance in female but not in male mice (Table 4). Bone histomorphometry of 10-week-old female CtskCre/WT;RosaHes1 mice demonstrated a 1.7-fold increase in osteoclast surface and number, and approximately twofold increase in eroded surface, when compared with littermate controls, confirming that HES1 increases osteoclast differentiation and function in vivo (Table 5 and Fig. 7).
      Table 4Femoral microarchitecture assessed by μCT of 10-week-old CtskCre/WT;RosaHes1 mice and sex-matched littermate controls
      μCT parametersMalesFemales
      ControlRosaHes1ControlRosaHes1
      n = 7n = 9n = 6n = 6
      Distal femur trabecular bone
       BV/TV (%)12.5 ± 2.610.5 ± 4.14.7 ± 0.73.3 ± 0.6
      Significantly different from control, p < 0.05.
       Trabecular separation (μm)206 ± 26225 ± 25313 ± 33346 ± 54
       Trabecular number (1/mm)4.9 ± 0.64.5 ± 0.53.3 ± 0.33.0 ± 0.4
       Trabecular thickness (μm)44 ± 542 ± 639 ± 339 ± 4
       Connectivity density (1/mm3)280 ± 82214 ± 8392 ± 1946 ± 18
      Significantly different from control, p < 0.05.
       Structure model index2.0 ± 0.32.1 ± 0.52.7 ± 0.23.0 ± 0.3
      Significantly different from control, p < 0.05.
       Density of material (mg HA/cm3)980 ± 10973 ± 9.7995 ± 9993 ± 16
      Femoral midshaft cortical bone
       BV/TV (%)89.4 ± 0.188.8 ± 0.887.3 ± 0.887.8 ± 1.1
       Porosity (%)10.6 ± 0.111.2 ± 0.812.7 ± 0.812.2 ± 1.1
       Cortical thickness (μm)191 ± 14186 ± 17152 ± 10159 ± 17
       Total area (mm2)2.3 ± 0.22.2 ± 0.31.8 ± 0.11.7 ± 0.1
       Bone area (mm2)1.1 ± 0.11.1 ± 0.20.8 ± 0.10.8 ± 0.1
       Periosteal perimeter (mm)5.4 ± 0.35.2 ± 0.44.7 ± 0.24.6 ± 0.2
       Endocortical perimeter (mm)3.9 ± 0.23.7 ± 0.23.5 ± 0.23.4 ± 0.1
       Density of material (mg HA/cm3)1181 ± 121187 ± 181195 ± 141195 ± 16
      μCT was performed on distal femurs for trabecular bone and midshaft for cortical bone. Values are means ± SD.
      a Significantly different from control, p < 0.05.
      Table 5Cancellous bone histomorphometry of 10-week-old CtskCre/WT;RosaHes1 female mice and sex-matched littermate controls
      Distal femur trabecular boneControlRosaHes1
      n = 3–4n = 3–5
      BV/TV (%)11.2 ± 1.48.2 ± 1.6
      Significantly different from control, p < 0.05.
      Trabecular separation (μm)286 ± 42383 ± 60
      Significantly different from control, p < 0.05.
      Trabecular number (1/mm)3.1 ± 0.42.4 ± 0.4
      Significantly different from control, p < 0.05.
      Trabecular thickness (μm)35 ± 3.034 ± 2.9
      Osteoblast surface/bone surface (%)17.2 ± 5.616.9 ± 3.8
      Osteoblasts/bone perimeter (1/mm)11.3 ± 3.112.4 ± 1.5
      Osteoclast surface/bone surface (%)10.8 ± 1.718.7 ± 1.4
      Significantly different from control, p < 0.05.
      Osteoclasts/bone perimeter (1/mm)4.1 ± 0.66.8 ± 0.8
      Significantly different from control, p < 0.05.
      Eroded surface/bone surface (%)12.3 ± 2.823.1 ± 4.3
      Significantly different from control, p < 0.05.
      Mineral apposition rate (μm/day)1.7 ± 0.31.4 ± 0.7
      Mineralizing surface/bone surface (%)17.7 ± 7.718.7 ± 2.8
      Bone formation rate (μm3/μm2/day)0.3 ± 0.20.3 ± 0.1
      Bone histomorphometry was performed on distal femurs from 10-week-old CtskCre/WT;RosaHes1 female mice and sex-matched littermate controls. Values are means ± SD.
      a Significantly different from control, p < 0.05.
      Figure thumbnail gr7
      Figure 7Activation of HES1 in Ctsk-expressing cells increases osteoclast number and bone resorption in vivo. Representative static (upper panels) and dynamic (lower panels) cancellous bone histomorphometry of the distal femur from 10-week-old CtskCre/WT;RosaHes1 female mice and sex-matched littermate controls. The scale bar in the right corner represents 50 μm. HES1, hairy and enhancer of split 1.

      Induction of HES1 enhances osteoclast differentiation in vitro

      To verify that the phenotype of CtskCre/WT;RosaHes1 mice was due to a direct effect in cells of the osteoclast lineage, BMMs from CtskCre/WT;RosaHes1 and control littermates were cultured in the presence of M-CSF and RANKL. BMMs from CtskCre/WT;RosaHes1 mice exhibited a 4.5-fold increase in osteoclast number in comparison to cells from littermate controls (Fig. 8). In addition, osteoclasts with a high number of nuclei were significantly increased in CtskCre/WT;RosaHes1 cultures compared with controls, indicating that the size of osteoclasts was larger because of highly activated fusion in CtskCre/WT;RosaHes1 cells. Phalloidin staining of osteoclasts from CtskCre/WT;RosaHes1 mice cultured on bone slices confirmed larger cells with sealing zones that were 25% larger than in cells from control littermates (Fig. 8). Accordingly, CtskCre/WT;RosaHes1 osteoclasts exhibited a sixfold increase in total resorption pit area (Fig. 8), indicating enhanced bone resorptive capacity in CtskCre/WT;RosaHes1 osteoclasts.
      Figure thumbnail gr8
      Figure 8Number, size, and resorptive capacity are increased in CtskCre/WT;RosaHes1 osteoclasts. BMMs derived from 10-week-old CtskCre/WT;RosaHes1 (closed circles) mice and control littermates (open circles) were cultured for 4 days in the presence of M-CSF at 30 ng/ml and of RANKL at 10 ng/ml in cell culture–coated plates (A and B) or bone discs (CF). A, representative images of TRAP-stained multinucleated cells in cell culture–coated plates are shown. The scale bar in the right corner represents 500 μm. B, Hes1 transcript levels were measured by quantitative RT–PCR in total RNA from osteoclasts. Transcript levels are reported as copy number corrected for Rpl38 (left). TRAP-positive cells with more than three nuclei were considered osteoclasts and counted (middle). TRAP-positive cells with differential counting of nuclei/osteoclast are shown (right). C, representative images of Alexa Fluor 594 phalloidin-stained multinucleated cells on bone discs are shown. The scale bar in the right corner represents 100 μm. D, the perimeter of sealing zones was measured in n = 68 osteoclasts from control and in n = 131 osteoclasts from CtskCre/WT;RosaHes1 cultures. E, representative images of toluidine blue–stained resorption pits. The scale bar in the right corner represents 200 μm. F, the total resorption pit area was measured (%). Values are means ± SD; n = 3 biological replicates for control and CtskCre/WT;RosaHes1. ∗Significantly different between CtskCre/WT;RosaHes1 and control, p < 0.05. BMM, bone marrow–derived macrophage; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of NF-κB ligand; TRAP, tartrate resistant acid phosphatase.

      Mechanisms of HES1 action on osteoclastogenesis

      To understand the molecular mechanisms associated with the effect of HES1 on osteoclast differentiation, total RNA from CtskCre/WT;RosaHes1 and control osteoclasts was examined by RNA-Seq analysis. Ingenuity pathway analysis (IPA) revealed that ∼200 genes associated with cellular functions, including movement, spreading, cell–cell contact, and organization of the cytoskeleton, were upregulated in cells from CtskCre/WT;RosaHes1 mice (Fig. S4). Known fusion markers of osteoclastogenesis, such as Ocstamp, Dcstamp, and Atp6v0d2, were significantly increased in CtskCre/WT;RosaHes1 osteoclasts compared with controls (Fig. 9) (
      • Lee S.H.
      • Rho J.
      • Jeong D.
      • Sul J.Y.
      • Kim T.
      • Kim N.
      • Kang J.S.
      • Miyamoto T.
      • Suda T.
      • Lee S.K.
      • Pignolo R.J.
      • Koczon-Jaremko B.
      • Lorenzo J.
      • Choi Y.
      v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation.
      ,
      • Kim K.
      • Lee S.H.
      • Ha Kim J.
      • Choi Y.
      • Kim N.
      NFATc1 induces osteoclast fusion via up-regulation of Atp6v0d2 and the dendritic cell-specific transmembrane protein (DC-STAMP).
      ,
      • Kukita T.
      • Wada N.
      • Kukita A.
      • Kakimoto T.
      • Sandra F.
      • Toh K.
      • Nagata K.
      • Iijima T.
      • Horiuchi M.
      • Matsusaki H.
      • Hieshima K.
      • Yoshie O.
      • Nomiyama H.
      RANKL-induced DC-STAMP is essential for osteoclastogenesis.
      ,
      • Yagi M.
      • Miyamoto T.
      • Sawatani Y.
      • Iwamoto K.
      • Hosogane N.
      • Fujita N.
      • Morita K.
      • Ninomiya K.
      • Suzuki T.
      • Miyamoto K.
      • Oike Y.
      • Takeya M.
      • Toyama Y.
      • Suda T.
      DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells.
      ,
      • Yang M.
      • Birnbaum M.J.
      • MacKay C.A.
      • Mason-Savas A.
      • Thompson B.
      • Odgren P.R.
      Osteoclast stimulatory transmembrane protein (OC-STAMP), a novel protein induced by RANKL that promotes osteoclast differentiation.
      ,
      • Takagi T.
      • Inoue H.
      • Takahashi N.
      • Katsumata-Tsuboi R.
      • Uehara M.
      Sulforaphene attenuates multinucleation of pre-osteoclasts by suppressing expression of cell-cell fusion-associated genes DC-STAMP, OC-STAMP, and Atp6v0d2.
      ). Tetraspanins, a family of 32 distinct members, are known to affect cell–cell fusion, motility, and sealing zone formation (
      • Hemler M.E.
      Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain.
      ,
      • Ishii M.
      • Saeki Y.
      Osteoclast cell fusion: Mechanisms and molecules.
      ,
      • Kwon J.O.
      • Lee Y.D.
      • Kim H.
      • Kim M.K.
      • Song M.K.
      • Lee Z.H.
      • Kim H.H.
      Tetraspanin 7 regulates sealing zone formation and the bone-resorbing activity of osteoclasts.
      ). Among the tetraspanins, the transcripts of Cd9, Cd63, Cd82, Tspan5, Tspan7, and Tspan10 were expressed in osteoclasts and significantly increased in CtskCre/WT;RosaHes1 osteoclasts. Analysis of altered canonical pathway in CtskCre/WT;RosaHes1 osteoclasts by IPA revealed upregulation of integrin signaling in CtskCre/WT;RosaHes1 osteoclasts (Fig. S5). Osteoclasts express αvβ3 integrins, and they play a role in the adhesion of osteoclasts to bone matrix, cytoskeletal organization, and sealing zone formation (
      • Takito J.
      • Inoue S.
      • Nakamura M.
      The sealing zone in osteoclasts: A self-organized structure on the bone.
      ,
      • Nakamura I.
      • Duong L.T.
      • Rodan S.B.
      • Rodan G.A.
      Involvement of alpha(v)beta3 integrins in osteoclast function.
      ,
      • Spinardi L.
      • Marchisio P.C.
      Podosomes as smart regulators of cellular adhesion.
      ). Expression of genes associated with integrin signaling, including itgb3 (integrin β3), Src, Syk, Rac2, Vav3, Vcl and Dock5, was upregulated in CtskCre/WT;RosaHes1 osteoclasts (Fig. 9). The transcriptional repressors of Nfatc1, including Bcl6, Mafb, Id1, and Irf8, were decreased, and Nfatc1 was increased in CtskCre/WT;RosaHes1 osteoclasts; B lymphocyte–induced maturation protein 1 (Blimp1) was not affected (
      • Zhao B.
      • Ivashkiv L.B.
      Negative regulation of osteoclastogenesis and bone resorption by cytokines and transcriptional repressors.
      ,
      • Lee J.
      • Kim K.
      • Kim J.H.
      • Jin H.M.
      • Choi H.K.
      • Lee S.H.
      • Kook H.
      • Kim K.K.
      • Yokota Y.
      • Lee S.Y.
      • Choi Y.
      • Kim N.
      Id helix-loop-helix proteins negatively regulate TRANCE-mediated osteoclast differentiation.
      ,
      • Nishikawa K.
      • Nakashima T.
      • Hayashi M.
      • Fukunaga T.
      • Kato S.
      • Kodama T.
      • Takahashi S.
      • Calame K.
      • Takayanagi H.
      Blimp1-mediated repression of negative regulators is required for osteoclast differentiation.
      ,
      • Zhao B.
      • Takami M.
      • Yamada A.
      • Wang X.
      • Koga T.
      • Hu X.
      • Tamura T.
      • Ozato K.
      • Choi Y.
      • Ivashkiv L.B.
      • Takayanagi H.
      • Kamijo R.
      Interferon regulatory factor-8 regulates bone metabolism by suppressing osteoclastogenesis.
      ). Interleukin (IL) 1β, known to induce osteoclast differentiation in physiological conditions and following inflammation, and its receptor Il1r1, were markedly upregulated in CtskCre/WT;RosaHes1 osteoclasts (Fig. 9) (
      • Shiratori T.
      • Kyumoto-Nakamura Y.
      • Kukita A.
      • Uehara N.
      • Zhang J.
      • Koda K.
      • Kamiya M.
      • Badawy T.
      • Tomoda E.
      • Xu X.
      • Yamaza T.
      • Urano Y.
      • Koyano K.
      • Kukita T.
      IL-1beta induces pathologically activated osteoclasts bearing extremely high levels of resorbing activity: A possible pathological subpopulation of osteoclasts, accompanied by suppressed expression of Kindlin-3 and Talin-1.
      ,
      • Lee Y.M.
      • Fujikado N.
      • Manaka H.
      • Yasuda H.
      • Iwakura Y.
      IL-1 plays an important role in the bone metabolism under physiological conditions.
      ,
      • Kim J.H.
      • Jin H.M.
      • Kim K.
      • Song I.
      • Youn B.U.
      • Matsuo K.
      • Kim N.
      The mechanism of osteoclast differentiation induced by IL-1.
      ). The levels of other osteoclastogenic markers, such as Oscar, Calcr, Car2, and Acp5, also were increased in CtskCre/WT;RosaHes1 osteoclasts. The mRNA expression of Bcl6, Mafb, Nfatc1, Atp6v0d2, Ocstamp, and Acp5 was demonstrated by quantitative RT–PCR (qRT–PCR) (Fig. 9). In accordance with these results, NFATc1 protein levels were increased in CtskCre/WT;RosaHes1 osteoclasts (Fig. 9). HES1 protein levels were increased in differentiated osteoclasts, and the increase was greater in CtskCre/WT;RosaHes1 cells.
      Figure thumbnail gr9
      Figure 9Expression of osteoclastogenic genes is increased in CtskCre/WT;RosaHes1 osteoclasts. BMMs derived from 10-week-old CtskCre/WT;RosaHes1 mice and control littermates were cultured for 4 days in the presence of M-CSF at 30 ng/ml and of RANKL at 10 ng/ml. Cells were collected for total RNA and protein extraction. A, RNA was analyzed by RNA-Seq. The bars indicate Log2 fold changes (p < 0.05) of gene expression between control and CtskCre/WT;RosaHes1 osteoclasts; n = 3 control and CtskCre/WT;RosaHes1 biological replicates. B, Bcl6, Mafb, Nfatc1, Atp6vOd2, Ocstamp, and Acp mRNA levels were measured by quantitative RT–PCR and reported as copy number corrected for Rpl38 mRNA levels. Values are means ± SD; n = 3 control and CtskCre/WT;RosaHes1 biological replicates. C, representative data of protein levels of NFATc1 and HES1. About 40 μg of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NFATc1 and HES1 levels were detected using anti-NFATc1 and anti-HES1 antibodies, respectively. β-Actin served as a loading control in the same blot. The band intensity was quantified by Image Lab software (version 5.2.1), and the numerical ratio of NFATc1/β-actin and HES1/β-actin is shown under each blot. Control ratios at the initiation of the culture in the presence of RANKL (day 0) are normalized to 1. ∗Significantly different between CtskCre/WT;RosaHes1 and control, p < 0.05. BMM, bone marrow–derived macrophage; HES1, hairy and enhancer of split 1; M-CSF, macrophage colony-stimulating factor; NFATc1, nuclear factor of activated T cells, cytoplasmic 1; RANKL, receptor activator of NF-κB ligand.

      Discussion

      The present work uncovers a new function of HES1 on osteoclast differentiation and bone remodeling. The deletion of Hes1 in Ctsk-expressing cells decreased the osteoclastogenic potential of preosteoclasts, whereas its induction enhanced osteoclastogenesis. Osteoclast phenotypes and RNA-Seq analysis revealed that HES1 regulates cell–cell fusion and the formation of the sealing zone. The gene subsets of fusion markers, integrin signaling, and structural proteins for sealing zone formation were significantly upregulated in osteoclasts overexpressing HES1. These results indicate that HES1 has a direct role in osteoclast differentiation and function. Our study also reveals that the expression of Nfatc1 and that of inhibitors of osteoclastogenesis acting as transcriptional brakes of Nfatc1, such as Irf8, Bcl6, Mafb, and Id1, were regulated by HES1. It is possible that HES1 interacts with transcriptional repressors of osteoclastogenesis in a manner analogous to BLIMP1, although the expression of Blimp1 was not affected by HES1 (
      • Nishikawa K.
      • Nakashima T.
      • Hayashi M.
      • Fukunaga T.
      • Kato S.
      • Kodama T.
      • Takahashi S.
      • Calame K.
      • Takayanagi H.
      Blimp1-mediated repression of negative regulators is required for osteoclast differentiation.
      ,
      • Shin B.
      • Yu J.
      • Park E.S.
      • Choi S.
      • Yu J.
      • Hwang J.M.
      • Yun H.
      • Chung Y.H.
      • Hong K.S.
      • Choi J.S.
      • Takami M.
      • Rho J.
      Secretion of a truncated osteopetrosis-associated transmembrane protein 1 (OSTM1) mutant inhibits osteoclastogenesis through down-regulation of the B lymphocyte-induced maturation protein 1 (BLIMP1)-nuclear factor of activated T cells c1 (NFATc1) axis.
      ). It is probable that HES1 acts as a transcriptional repressor of inhibitors of osteoclastogenesis and as a consequence causes enhanced Nfatc1 expression. Under selected cellular conditions, HES1 can act as a transcriptional activator so that one cannot exclude a direct effect of HES1 on the transcriptional activation of Nfatc1 (
      • Sugita S.
      • Hosaka Y.
      • Okada K.
      • Mori D.
      • Yano F.
      • Kobayashi H.
      • Taniguchi Y.
      • Mori Y.
      • Okuma T.
      • Chang S.H.
      • Kawata M.
      • Taketomi S.
      • Chikuda H.
      • Akiyama H.
      • Kageyama R.
      • et al.
      Transcription factor Hes1 modulates osteoarthritis development in cooperation with calcium/calmodulin-dependent protein kinase 2.
      ). In accordance with our observations, γ-secretase inhibitors, known to prevent Notch activation, were found to inhibit osteoclast cell fusion and the formation of the podosomal actin belt structure by suppressing HES1/mitogen-activated protein kinase/AKT-mediated induction of NFATc1 in vitro (
      • Chen X.
      • Chen X.
      • Zhou Z.
      • Qin A.
      • Wang Y.
      • Fan B.
      • Xu W.
      • Zhang S.
      LY411575, a potent gamma-secretase inhibitor, suppresses osteoclastogenesis in vitro and LPS-induced calvarial osteolysis in vivo.
      ). However, it is important to note that γ-secretase inhibitors can target many substrates, and their effect is not specific to Notch signaling (
      • Golde T.E.
      • Koo E.H.
      • Felsenstein K.M.
      • Osborne B.A.
      • Miele L.
      gamma-Secretase inhibitors and modulators.
      ,
      • Duggan S.P.
      • McCarthy J.V.
      Beyond gamma-secretase activity: The multifunctional nature of presenilins in cell signalling pathways.
      ).
      Although HES1 had a pronounced effect on osteoclast differentiation and function in vitro, this effect was restricted to female mice in vivo. The Hes1 inactivation caused an 85% increase in BV in mature female mice, and the induction of HES1 in Ctsk-expressing cells caused an osteopenic phenotype. The inactivation of Hes1 in male mice did not result in a prominent skeletal phenotype; however, it opposed the osteopenic and resorptive phenotype of Notch2tm1.1Ecan mice harboring an HCS mutation causing a gain-of-NOTCH2 function. The absence of a phenotype in male mice facilitated the interpretation of the rescue of the Notch2tm1.1Ecan phenotype by the Hes1 deletion. Since female Hes1-inactivated mice had an increase and Notch2tm1.1Ecan a decrease in BV, one would expect Notch2tm1.1Ecan;Hes1Δ/Δ female mice to have an intermediate BV. So that an increase in the BV of Notch2tm1.1Ecan would not necessarily represent a rescue of the osteopenic phenotype and that HES1 was a mediator of NOTCH2. It is not readily apparent why the Hes1 inactivation caused a phenotype in female but not in male mice, and the observation stresses the importance of examining phenotypes in mice of different sexes independently (
      • Zanotti S.
      • Kalajzic I.
      • Aguila H.L.
      • Canalis E.
      Sex and genetic factors determine osteoblastic differentiation potential of murine bone marrow stromal cells.
      ,
      • Mun S.H.
      • Jastrzebski S.
      • Kalinowski J.
      • Zeng S.
      • Oh B.
      • Bae S.
      • Eugenia G.
      • Khan N.M.
      • Drissi H.
      • Zhou P.
      • Shin B.
      • Lee S.K.
      • Lorenzo J.
      • Park-Min K.H.
      Sexual dimorphism in differentiating osteoclast precursors demonstrates enhanced inflammatory pathway activation in female cells.
      ). It is not unusual to observe sex-specific phenotypes in genetically engineered mice (
      • Li S.
      • He T.
      • Wu D.
      • Zhang L.
      • Chen R.
      • Liu B.
      • Yuan J.
      • Tickner J.
      • Qin A.
      • Xu J.
      • Rong L.
      Conditional knockout of PKC-delta in osteoclasts favors bone mass accrual in males due to decreased osteoclast function.
      ,
      • Csete D.
      • Simon E.
      • Alatshan A.
      • Aradi P.
      • Dobo-Nagy C.
      • Jakus Z.
      • Benko S.
      • Gyori D.S.
      • Mocsai A.
      Hematopoietic or osteoclast-specific deletion of Syk leads to increased bone mass in experimental mice.
      ,
      • Yang Y.
      • Chung M.R.
      • Zhou S.
      • Gong X.
      • Xu H.
      • Hong Y.
      • Jin A.
      • Huang X.
      • Zou W.
      • Dai Q.
      • Jiang L.
      STAT3 controls osteoclast differentiation and bone homeostasis by regulating NFATc1 transcription.
      ). Possible explanations for the prevalence of a phenotype in female mice include genetic influences, a loss of the inhibitory actions of estrogens on osteoclastogenesis in the context of the Hes1 inactivation as well as the earlier NF-κB–NFATc1 activation and osteoclastogenesis that occurs in female mice (
      • Mun S.H.
      • Jastrzebski S.
      • Kalinowski J.
      • Zeng S.
      • Oh B.
      • Bae S.
      • Eugenia G.
      • Khan N.M.
      • Drissi H.
      • Zhou P.
      • Shin B.
      • Lee S.K.
      • Lorenzo J.
      • Park-Min K.H.
      Sexual dimorphism in differentiating osteoclast precursors demonstrates enhanced inflammatory pathway activation in female cells.
      ,
      • Nakamura T.
      • Imai Y.
      • Matsumoto T.
      • Sato S.
      • Takeuchi K.
      • Igarashi K.
      • Harada Y.
      • Azuma Y.
      • Krust A.
      • Yamamoto Y.
      • Nishina H.
      • Takeda S.
      • Takayanagi H.
      • Metzger D.
      • Kanno J.
      • et al.
      Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts.
      ,
      • Martin-Millan M.
      • Almeida M.
      • Ambrogini E.
      • Han L.
      • Zhao H.
      • Weinstein R.S.
      • Jilka R.L.
      • O'Brien C.A.
      • Manolagas S.C.
      The estrogen receptor-alpha in osteoclasts mediates the protective effects of estrogens on cancellous but not cortical bone.
      ).
      In previous work, we demonstrated that HES1 is induced as osteoclasts mature, particularly in the context of the Notch2tm1.1Ecan mutation (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ). A plausible explanation for the modest skeletal phenotype of the Hes1 inactivation in male mice is that under basal conditions HES1 levels are low and play a modest role in skeletal physiology, and only following Notch activation, HES1 plays a significant role in bone homeostasis. This explanation is substantiated by the amelioration of the Notch2tm1.1Ecan osteopenic phenotype following the Hes1 inactivation. The Notch2tm1.1Ecan phenotype was not fully reversed, and this is explained by the effects of NOTCH2 enhancing RANKL expression by cells of the osteoblast lineage since these are independent of the induction of HES1 in the myeloid lineage (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ,
      • Yu J.
      • Canalis E.
      Notch and the regulation of osteoclast differentiation and function.
      ). Other Notch target genes, such as Hey1, Hey2, and HeyL, are not expressed in cells of the myeloid lineage and as a consequence could not be responsible for the stimulatory effects of NOTCH2 on osteoclastogenesis (
      • Canalis E.
      Notch in skeletal physiology and disease.
      ). HES3 and HES5 could compensate for the effects of HES1, but their expression in osteoclasts is low and their role in osteoclastogenesis is unknown (
      • Kageyama R.
      • Ohtsuka T.
      • Kobayashi T.
      Roles of Hes genes in neural development.
      ). Whereas, HES1 mediates direct effects of NOTCH2 on osteoclastogenesis, it is not likely to mediate the effects of NOTCH1, known to inhibit and not enhance osteoclast maturation, or NOTCH3, since this Notch receptor is not expressed in the myeloid lineage and its effects on osteoclastogenesis are indirect (
      • Bai S.
      • Kopan R.
      • Zou W.
      • Hilton M.J.
      • Ong C.T.
      • Long F.
      • Ross F.P.
      • Teitelbaum S.L.
      NOTCH1 regulates osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblast lineage cells.
      ,
      • Canalis E.
      • Yu J.
      • Schilling L.
      • Yee S.P.
      • Zanotti S.
      The lateral meningocele syndrome mutation causes marked osteopenia in mice.
      ). NOTCH4 is expressed at low levels in the myeloid lineage and not known to play a role in osteoclast differentiation (
      • Canalis E.
      Notch in skeletal physiology and disease.
      ,
      • Yu J.
      • Canalis E.
      Notch and the regulation of osteoclast differentiation and function.
      ).
      In the present work, we confirm that Notch2tm1.1Ecan mice are osteopenic because of direct effects of NOTCH2 in cells of the myeloid lineage. The stimulatory effect of NOTCH2 on osteoclastogenesis has been attributed to direct interactions of the NOTCH2 intracellular domain with NF-κB in the context of Nfatc1 regulatory regions and increased Nfatc1 transcription (
      • Fukushima H.
      • Nakao A.
      • Okamoto F.
      • Shin M.
      • Kajiya H.
      • Sakano S.
      • Bigas A.
      • Jimi E.
      • Okabe K.
      The association of Notch2 and NF-kappaB accelerates RANKL-induced osteoclastogenesis.
      ). However, recent work from our laboratory has demonstrated that NOTCH2 has NF-κB–independent effects on tumor necrosis factor α (TNFα)–induced osteoclastogenesis, and some of these effects are secondary to the activation of AKT and Il1b expression (
      • Yu J.
      • Canalis E.
      The Hajdu Cheney mutation sensitizes mice to the osteolytic actions of tumor necrosis factor alpha.
      ,
      • Lee S.E.
      • Woo K.M.
      • Kim S.Y.
      • Kim H.M.
      • Kwack K.
      • Lee Z.H.
      • Kim H.H.
      The phosphatidylinositol 3-kinase, p38, and extracellular signal-regulated kinase pathways are involved in osteoclast differentiation.
      ). The present work demonstrates that the direct effects of NOTCH2 on osteoclastogenesis are HES1 dependent confirming previous work from this laboratory revealing that the enhancement of the osteolytic actions of TNFα by the Notch2tm1.1Ecan mutation depend on the induction of HES1 (
      • Yu J.
      • Canalis E.
      The Hajdu Cheney mutation sensitizes mice to the osteolytic actions of tumor necrosis factor alpha.
      ).
      HES1 is known to inhibit phosphatase and tensin homolog and as a consequence enhance phosphoinositide 3-kinase–AKT signaling (
      • Wong G.W.
      • Knowles G.C.
      • Mak T.W.
      • Ferrando A.A.
      • Zuniga-Pflucker J.C.
      HES1 opposes a PTEN-dependent check on survival, differentiation, and proliferation of TCRbeta-selected mouse thymocytes.
      ). AKT signaling is required for cell–cell fusion during osteoclast differentiation, and inhibitors of AKT lead to a decrease in Dcstamp transcripts and osteoclast size (
      • Tiedemann K.
      • Le Nihouannen D.
      • Fong J.E.
      • Hussein O.
      • Barralet J.E.
      • Komarova S.V.
      Regulation of osteoclast growth and fusion by mTOR/raptor and mTOR/rictor/Akt.
      ). However, the levels of phosphatase and tensin homolog transcripts and the phosphorylation of AKT were not different between CtskCre/WT;RosaHes1 osteoclasts and controls (data not shown). Although RANKL and TNFα share and activate similar downstream molecules, mechanisms triggering osteoclastogenesis are different in part because Nfatc1 and Dcstamp levels are not changed in conditions of proinflammatory cytokine-induced osteoclastogenesis (
      • Hotokezaka H.
      • Sakai E.
      • Ohara N.
      • Hotokezaka Y.
      • Gonzales C.
      • Matsuo K.
      • Fujimura Y.
      • Yoshida N.
      • Nakayama K.
      Molecular analysis of RANKL-independent cell fusion of osteoclast-like cells induced by TNF-alpha, lipopolysaccharide, or peptidoglycan.
      ,
      • Xing L.
      • Xiu Y.
      • Boyce B.F.
      Osteoclast fusion and regulation by RANKL-dependent and independent factors.
      ). Hes1 inactivation decreases Il1b in TNFα-induced Notch2tm1.1Ecan osteoclasts, and the present work confirms that Il1b and Il1r1 transcripts are increased in HES1-overexpressing osteoclasts (
      • Yu J.
      • Canalis E.
      The Hajdu Cheney mutation sensitizes mice to the osteolytic actions of tumor necrosis factor alpha.
      ). IL1β induces pathologically activated osteoclasts bearing a high level of bone-resorbing activity and may be mechanistically relevant to the actions of HES1 in osteoclasts (
      • Shiratori T.
      • Kyumoto-Nakamura Y.
      • Kukita A.
      • Uehara N.
      • Zhang J.
      • Koda K.
      • Kamiya M.
      • Badawy T.
      • Tomoda E.
      • Xu X.
      • Yamaza T.
      • Urano Y.
      • Koyano K.
      • Kukita T.
      IL-1beta induces pathologically activated osteoclasts bearing extremely high levels of resorbing activity: A possible pathological subpopulation of osteoclasts, accompanied by suppressed expression of Kindlin-3 and Talin-1.
      ).
      The phenotype of Notch2tm1.1Ecan mice as well as the osteopenia of humans harboring HCS pathogenic variants is secondary to an increase in bone resorption with no evidence of impaired bone formation (
      • Sakka S.
      • Gafni R.I.
      • Davies J.H.
      • Clarke B.
      • Tebben P.
      • Samuels M.
      • Saraff V.
      • Klaushofer K.
      • Fratzl-Zelman N.
      • Roschger P.
      • Rauch F.
      • Hogler W.
      Bone structural characteristics and response to bisphosphonate treatment in children with Hajdu-Cheney syndrome.
      ,
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ,
      • Canalis E.
      Notch in skeletal physiology and disease.
      ). The direct effects of NOTCH2 in the myeloid lineage appear mediated by Notch target gene Hes1. This is further substantiated by the fact that other target genes, such as Hey1, Hey2, and HeyL, are not expressed by cells of the osteoclast lineage; therefore, these cannot mediate the effects of NOTCH2 in this cell population.
      A limitation of the present work is the use of a CtskCre mouse model to deliver Cre recombinase since the expression of Ctsk is not exclusive to osteoclasts and Ctsk is also detected in alternate skeletal and nonskeletal cells (
      • Nakamura T.
      • Imai Y.
      • Matsumoto T.
      • Sato S.
      • Takeuchi K.
      • Igarashi K.
      • Harada Y.
      • Azuma Y.
      • Krust A.
      • Yamamoto Y.
      • Nishina H.
      • Takeda S.
      • Takayanagi H.
      • Metzger D.
      • Kanno J.
      • et al.
      Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts.
      ,
      • Jacome-Galarza C.
      • Soung do Y.
      • Adapala N.S.
      • Pickarski M.
      • Sanjay A.
      • Duong L.T.
      • Lorenzo J.A.
      • Drissi H.
      Altered hematopoietic stem cell and osteoclast precursor frequency in cathepsin K null mice.
      ,
      • Chiu W.S.
      • McManus J.F.
      • Notini A.J.
      • Cassady A.I.
      • Zajac J.D.
      • Davey R.A.
      Transgenic mice that express Cre recombinase in osteoclasts.
      ,
      • Winkeler C.L.
      • Kladney R.D.
      • Maggi Jr., L.B.
      • Weber J.D.
      Cathepsin K-Cre causes unexpected germline deletion of genes in mice.
      ). Although one cannot fully exclude effects outside the osteoclast lineage, it is reasonable to believe that the effects observed in the present work are secondary to the misexpression of Hes1 in osteoclasts since cultures of BMMs from CtskCre/WT;RosaHes1 and CtskCreWT;Hes1Δ/Δ mice revealed profound effects on osteoclast differentiation. Moreover, the activation and inactivation of Hes1 in BMM cultures using adenoviruses to deliver Cre demonstrated a direct effect of HES1 in the osteoclast lineage.
      In conclusion, HES1 plays a critical role in osteoclastogenesis and bone resorption and is mechanistically relevant to the skeletal phenotype of an experimental model of HCS.

      Experimental procedures

      Genetically modified mice

      Notch2tm1.1Ecan mice harboring a 6955C>T substitution in the Notch2 locus have been characterized in previous studies and were backcrossed into a C57BL/6 background for eight and more generations (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ,
      • Yu J.
      • Zanotti S.
      • Walia B.
      • Jellison E.
      • Sanjay A.
      • Canalis E.
      The Hajdu Cheney mutation is a determinant of B-cell allocation of the splenic marginal zone.
      ,
      • Canalis E.
      • Sanjay A.
      • Yu J.
      • Zanotti S.
      An antibody to Notch2 reverses the osteopenic phenotype of Hajdu-Cheney mutant male mice.
      ). Hes1loxP/loxP (Hes1<tm1Imayo) mice, where loxP sequences are knocked into the first intron and downstream of the 3′ UTR of Hes1 alleles, were obtained from RIKEN (RBRC06047; Wako Saitama) in a C57BL/6 background (
      • Imayoshi I.
      • Shimogori T.
      • Ohtsuka T.
      • Kageyama R.
      Hes genes and neurogenin regulate non-neural versus neural fate specification in the dorsal telencephalic midline.
      ). Rosa[STOP]Hes1 (Gt(ROSA)26Sor<tm1(Hes1.EGFP)Imayo>) were obtained from RIKEN (RBRC06002) in an ICR background (
      • Kobayashi T.
      • Mizuno H.
      • Imayoshi I.
      • Furusawa C.
      • Shirahige K.
      • Kageyama R.
      The cyclic gene Hes1 contributes to diverse differentiation responses of embryonic stem cells.
      ). In Rosa[STOP]Hes1 mice, Hes1 coding sequences are cloned into the Rosa26 locus downstream of a Neo-STOP cassette flanked by loxP sequences, so that HES1-IRES-GFP is expressed following the excision of the cassette by Cre recombination. To induce or delete Hes1 in differentiated cells of the osteoclast lineage, mice harboring sequences coding for the Cre recombinase knocked-in into the Ctsk locus (CtskCre) were used in a C57BL/6 background (
      • Nakamura T.
      • Imai Y.
      • Matsumoto T.
      • Sato S.
      • Takeuchi K.
      • Igarashi K.
      • Harada Y.
      • Azuma Y.
      • Krust A.
      • Yamamoto Y.
      • Nishina H.
      • Takeda S.
      • Takayanagi H.
      • Metzger D.
      • Kanno J.
      • et al.
      Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts.
      ,
      • Jacome-Galarza C.
      • Soung do Y.
      • Adapala N.S.
      • Pickarski M.
      • Sanjay A.
      • Duong L.T.
      • Lorenzo J.A.
      • Drissi H.
      Altered hematopoietic stem cell and osteoclast precursor frequency in cathepsin K null mice.
      ). Genotyping was conducted in tail DNA extracts by PCR using specific primers from Integrated DNA Technologies (IDT) (Table S1).
      For the deletion of Hes1, Hes1loxP alleles were introduced into CtskCre mice to create CtskCre/WT;Hes1loxP/loxP mice, and these were crossed with Hes1loxP/loxP to generate Hes1Δ/Δ-deleted and Hes1loxP/loxP control littermates. For the induction of HES1, CtskCre/WT mice were crossed with homozygous Rosa[STOP]Hes1 mice to generate ∼50% Hes1-induced and ∼50% Rosa[STOP]Hes1 control littermates. For the deletion of Hes1 in the context of the Notch2tm1.1Ecan mutation, CtskCre/WT;Hes1loxP/loxP mice were crossed with Notch2tm1.1Ecan;Hes1loxP/loxP mice to create Notch2tm1.1Ecan;Hes1Δ/Δ and Notch2tm1.1Ecan;Hes1loxP/loxP controls. Recombination of loxP flanked sequences was documented in extracts from tibiae using specific primers (Table S1). All animal experiments were approved by the Institutional Animal Care and Use Committee of UConn Health.

      BMM cultures, osteoclast formation, and adenovirus-Cre-mediated recombination

      To obtain BMMs, the marrow from experimental and control sex-matched littermate mice was removed by flushing with a 26-gauge needle, and erythrocytes were lyzed in 150 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA (pH 7.4), as described previously (
      • Canalis E.
      • Sanjay A.
      • Yu J.
      • Zanotti S.
      An antibody to Notch2 reverses the osteopenic phenotype of Hajdu-Cheney mutant male mice.
      ). Cells were centrifuged, and the sediment suspended in α-minimum essential medium (α-MEM) in the presence of 10% fetal bovine serum (FBS; both from Thermo Fisher Scientific) and recombinant human M-CSF at 30 ng/ml. M-CSF complementary DNA (cDNA) and expression vector were obtained from D. Fremont, and M-CSF was purified as previously reported (
      • Lee S.H.
      • Rho J.
      • Jeong D.
      • Sul J.Y.
      • Kim T.
      • Kim N.
      • Kang J.S.
      • Miyamoto T.
      • Suda T.
      • Lee S.K.
      • Pignolo R.J.
      • Koczon-Jaremko B.
      • Lorenzo J.
      • Choi Y.
      v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation.
      ). Cells were seeded on uncoated plastic petri dishes at a density of 300,000 cells/cm2 and cultured for 3 days. For osteoclast formation, cells were collected following treatment with 0.25% trypsin/EDTA for 5 min and seeded on tissue culture plates at a density of 62,500 cells/cm2 in α-MEM with 10% FBS, M-CSF at 30 ng/ml, and recombinant murine RANKL at 10 ng/ml. Tnfsf11, encoding RANKL, cDNA expression vector was obtained from M. Glogauer, and glutathione-S-transferase–tagged RANKL was expressed and purified as described (
      • Wang Y.
      • Lebowitz D.
      • Sun C.
      • Thang H.
      • Grynpas M.D.
      • Glogauer M.
      Identifying the relative contributions of Rac1 and Rac2 to osteoclastogenesis.
      ). Cultures were carried out until multinucleated tartrate resistant acid phosphatase (TRAP)–positive cells were formed. TRAP enzyme histochemistry was conducted using a commercial kit (Sigma–Aldrich), in accordance with the manufacturer's instructions. TRAP-positive cells containing more than three nuclei were considered osteoclasts.
      For actin structure staining and bone resorption assay of osteoclasts in vitro, BMMs were seeded at a density of 62,500 cells/cm2 on bovine cortical bone slices and cultured in α-MEM with 10% FBS, M-CSF at 30 ng/ml, and RANKL at 10 ng/ml. To visualize the sealing zone of osteoclasts on bone slices, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.3% Triton X-100 for 5 min. To block nonspecific background staining, cells on bone discs were incubated with 2% bovine serum albumin for 1 h and stained with Alexa Fluor 594 Phalloidin (Thermo Fisher Scientific) at a 1:40 dilution for 20 min. The sealing zone was viewed on a Leica fluorescence microscope (model DMI6000B), and collected images were processed using the Leica Application Suite X 1.5.1.1387 (Leica Microsystems). After visualizing the sealing zone, cells were stained for TRAP to assess cellular morphology. To visualize bone resorption pits, bone slices were sonicated to remove osteoclasts and stained with 1% toluidine blue in 1% sodium borate. To assess the ability of osteoclasts to resorb bone, the total resorption area/total bone area was measured on images acquired with an Olympus DP72 camera using cellSens Dimension software, version 1.6 (Olympus Corporation). The total resorption area/total bone area was corrected for the total number of TRAP-positive multinucleated cells (
      • Canalis E.
      • Sanjay A.
      • Yu J.
      • Zanotti S.
      An antibody to Notch2 reverses the osteopenic phenotype of Hajdu-Cheney mutant male mice.
      ).
      To inactivate or induce Hes1 in osteoclast precursors in vitro, BMMs from homozygous Hes1loxP/loxP or Rosa[STOP]Hes1 mice were cultured in the presence of M-CSF at 30 ng/ml and RANKL at 10 ng/ml for 2 days, prior to being transduced with Ad-Cre or CMV-GFP (Ad-GFP [Vector Biolabs]) as control, at multiplicity of infection of 100 and cultured with M-CSF and RANKL for two additional days until the formation of multinucleated TRAP-positive cells. To inactivate Hes1 in the context of the Notch2tm1.1Ecan mutation, Hes1loxP/loxP alleles were introduced into Notch2tm1.1Ecan mice to create Notch2tm1.1Ecan;Hes1loxP/loxP mice, and BMMs were cultured and transduced with Ad-Cre or Ad-GFP.

      qRT–PCR

      Total RNA was extracted from osteoclasts with the RNeasy Mini kit (Qiagen) and homogenized bones with the RNeasy Micro kit (Qiagen), in accordance with the manufacturer's instructions. The integrity of the RNA extracted from bones was assessed by microfluidic electrophoresis on an Experion system (Bio-Rad), and RNA with a quality indicator number equal to or higher than 7.0 was used for subsequent analysis. Equal amounts of RNA were reverse transcribed using the iScript RT-PCR kit (Bio-Rad) and amplified in the presence of specific primers (all from IDT; Table S2) with the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) at 60 °C for 40 cycles. Transcript copy number was estimated by comparison with a serial dilution of cDNA for Acp5 and Notch2 (all from Thermo Fisher Scientific), Hes1 (American Type Culture Collection), Nfatc1 (Addgene; plasmid 11793; created by A. Rao), Bcl6, Mafb, Atp6vOd2, and Ocstamp (all from Dharmacon).
      The level of Notch26955C>T mutant transcript was measured as described previously (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ). Total RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase in the presence of reverse primers for Notch2 (5′-GGATCTGGTACATAGAG-3′) and Rpl38 (Table S2). Notch2 cDNA was amplified by qPCR in the presence of TaqMan gene expression assay mix, including specific primers (5′-CATCGTGACTTTCCA-3′ and 5′-GGATCTGGTACATAGAG-3′) and a 6-carboxyfluorexcein-labeled DNA probe of sequence 5′-CATTGCCTAGGCAGC-3′ covalently attached to a 3′-minor groove binder quencher (Thermo Fisher Scientific), and SsoAdvanced Universal Probes Supermix (Bio-Rad) at 60 °C for 45 cycles (
      • Kutyavin I.V.
      • Afonina I.A.
      • Mills A.
      • Gorn V.V.
      • Lukhtanov E.A.
      • Belousov E.S.
      • Singer M.J.
      • Walburger D.K.
      • Lokhov S.G.
      • Gall A.A.
      • Dempcy R.
      • Reed M.W.
      • Meyer R.B.
      • Hedgpeth J.
      3'-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures.
      ). Notch26955C>T transcript copy number was estimated by comparison with a serial dilution of a synthetic DNA fragment (IDT) containing ∼200 bp surrounding the 6955C>T mutation in the Notch2 locus and cloned into pcDNA3.1(−) (Thermo Fisher Scientific) by isothermal single reaction assembly using commercially available reagents (New England Biolabs) (
      • Gibson D.G.
      • Young L.
      • Chuang R.Y.
      • Venter J.C.
      • Hutchison 3rd, C.A.
      • Smith H.O.
      Enzymatic assembly of DNA molecules up to several hundred kilobases.
      ).
      Amplification reactions were conducted in CFX96 qRT–PCR detection systems (Bio-Rad), and fluorescence was monitored at the end of the elongation step during every PCR cycle. Data are expressed as copy number corrected for Rpl38 expression estimated by comparison with a serial dilution of cDNA for Rpl38 (American Type Culture Collection) (
      • Kouadjo K.E.
      • Nishida Y.
      • Cadrin-Girard J.F.
      • Yoshioka M.
      • St-Amand J.
      Housekeeping and tissue-specific genes in mouse tissues.
      ).

      Illumina transcriptome library preparation and sequencing

      Total RNA was quantified, and purity ratios were determined for each sample using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). To assess RNA quality, total RNA was analyzed on the Agilent TapeStation 4200 (Agilent Technologies) using the RNA High Sensitivity assay. Ribosomal integrity numbers were recorded for each sample. Only samples with ribosomal integrity number values above 9.0 were used for library preparation.
      Total RNA samples were prepared for mRNA-Seq using the Illumina TruSeq Stranded mRNA Sample Preparation kit following the manufacturer's protocol (Illumina). Libraries were validated for length and adapter dimer removal using the Agilent TapeStation 4200 D1000 High Sensitivity assay (Agilent Technologies), and then they were quantified and normalized using the dsDNA High Sensitivity Assay for Qubit 3.0 (Thermo Fisher Scientific). Sample libraries were prepared for Illumina sequencing by denaturing and diluting the libraries per manufacturer's protocol (Illumina). All samples were pooled into one sequencing pool, equally normalized, and run as one sample pool across the Illumina NextSeq 500 using version 2.5 chemistry. Target read depth was achieved for each sample with paired end 75 bp reads. Raw reads were trimmed with Sickle (version 1.33), with a quality threshold of 30 and length threshold of 45, following that the trimmed reads were mapped to Homo Sapiens genome (GRCh38 ensembl release 99) with HISAT2 (version 2.1.0) (
      • Kim D.
      • Langmead B.
      • Salzberg S.L.
      HISAT: A fast spliced aligner with low memory requirements.
      ). The resulting SAM files were then converted into BAM format using samtools (version 1.9) (
      • Li H.
      • Handsaker B.
      • Wysoker A.
      • Fennell T.
      • Ruan J.
      • Homer N.
      • Marth G.
      • Abecasis G.
      • Durbin R.
      • Genome Project Data Processing Subgroup
      The sequence alignment/map format and SAMtools.
      ), and the PCR duplicates were removed using PICARD (http://broadinstitute.github.io/picard/). The counts were generated against the features with HTSeq-count (
      • Anders S.
      • Pyl P.T.
      • Huber W.
      HTSeq--a Python framework to work with high-throughput sequencing data.
      ). The differential expression of genes between conditions was evaluated using DESeq2 (
      • Love M.I.
      • Huber W.
      • Anders S.
      Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
      ). Covariates were introduced in the DESeq2 analysis to increase the accuracy of results, and genes showing less than ten counts across the compared samples were excluded from the analysis. Genes with a false discovery rate <0.05 were considered significant and used in the downstream analysis. The processed RNA-Seq results were further analyzed by using IPA (Qiagen).

      Immunoblotting

      Cells from control and experimental mice were extracted in buffer containing 25 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 1 mM EDTA, 0.5% Triton X-100, 1 mM sodium orthovanadate, 10 mM NaF, 1 mM phenyl methyl sulfonyl fluoride, and a protease inhibitor cocktail (all from Sigma–Aldrich). Total cell lysates (40 μg of total protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 8 or 12% polyacrylamide gels and transferred to Immobilon-P membranes (Millipore). The blots were probed with anti-HES1 (11988) and β-actin (3700) antibodies from Cell Signaling Technology or anti-NFATc1 antibody (556602) from BD Biosciences. The blots were exposed to anti-rabbit, antirat, or antimouse IgG conjugated to horseradish peroxidase (Sigma–Aldrich) and incubated with a chemiluminescence detection reagent (Bio-Rad). Chemiluminescence was detected by ChemiDoc XSR+ molecular imager (Bio-Rad) with Image Lab software (version 5.2.1), and the amount of protein present in individual bands was quantified (
      • Yu J.
      • Canalis E.
      The Hajdu Cheney mutation sensitizes mice to the osteolytic actions of tumor necrosis factor alpha.
      ).

      μCT

      Femoral microarchitecture was determined using a μCT instrument (Scanco μCT 40; Scanco Medical AG), which was calibrated periodically using a phantom provided by the manufacturer (
      • Bouxsein M.L.
      • Boyd S.K.
      • Christiansen B.A.
      • Guldberg R.E.
      • Jepsen K.J.
      • Muller R.
      Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.
      ,
      • Glatt V.
      • Canalis E.
      • Stadmeyer L.
      • Bouxsein M.L.
      Age-related changes in trabecular architecture differ in female and male C57BL/6J mice.
      ). Femurs were scanned in 70% ethanol at high resolution, energy level of 55 kVp, intensity of 145 μA, and integration time of 200 ms. Evaluation of skeletal microarchitecture was started 1.0 mm proximal from the condyles of the distal femur. A total of 160 consecutive 6 μm thick slices were acquired at an isotropic voxel dimension of 216 μm3 and selected for analysis. Contours were drawn manually every ten slices a few voxels away from the endocortical boundary to define the region of analysis. The remaining slice contours were iterated automatically. BV/TV, trabecular separation, number and thickness, connectivity density, SMI, and material density were measured in trabecular regions using a Gaussian filter (σ = 0.8) (
      • Bouxsein M.L.
      • Boyd S.K.
      • Christiansen B.A.
      • Guldberg R.E.
      • Jepsen K.J.
      • Muller R.
      Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.
      ,
      • Glatt V.
      • Canalis E.
      • Stadmeyer L.
      • Bouxsein M.L.
      Age-related changes in trabecular architecture differ in female and male C57BL/6J mice.
      ). For analysis of cortical bone, contours were iterated across 100 slices along the cortical shell of the femoral midshaft, excluding the marrow cavity. Analyses of BV/TV, cortical thickness, periosteal perimeter, endosteal perimeter, total cross-sectional area, and cortical bone area were conducted using a Gaussian filter (σ = 0.8, support = 1).

      Bone histomorphometry

      Bone histomorphometry was carried out in CtskCre/WT;Notch2tm1.1Ecan;Hes1Δ/Δ, CtskCre/WT;Hes1Δ/Δ, and CtskCre/WT;RosaHes1 mice, and sex-matched controls were injected with calcein 20 mg/kg and demeclocycline 50 mg/kg at a 5 or 7 days of interval and sacrificed 2 days after demeclocycline administration. For static cancellous bone histomorphometry and to assess for the presence of TRAP-positive multinucleated cells, bones were decalcified in 14% EDTA for 14 days and embedded in paraffin, and 7 μm sections were stained for the presence of TRAP and counterstained with hematoxylin and analyzed at a 100× magnification using OsteoMeasureXP software (Osteometrics). Stained sections were used to draw bone tissue and measure trabecular separation, number and thickness, and eroded surface, as well as to count osteoblast and osteoclast number. To assess dynamic parameters of bone histomorphometry, undecalcified femurs were embedded in methyl methacrylate, and 5 μm sections were cut using Microm microtome (Richards-Allan Scientific). Mineralizing surface per bone surface and mineral apposition rate were measured on unstained sections visualized under UV light and a triple diamidino-2-phenylindole/fluorescein/Texas red set long-pass filter, and bone formation rate was calculated (
      • Dempster D.W.
      • Compston J.E.
      • Drezner M.K.
      • Glorieux F.H.
      • Kanis J.A.
      • Malluche H.
      • Meunier P.J.
      • Ott S.M.
      • Recker R.R.
      • Parfitt A.M.
      Standardized nomenclature, symbols, and units for bone histomorphometry: A 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee.
      ).

      Statistics

      Data are expressed as means ± SD and presented as biological replicates except for experiments where BMMs were transduced with adenoviruses or cells were extracted for immunoblotting, and these are presented as technical replicates representative of two or more experiments. Statistical differences were determined by Student's t test or two-way analysis of variance with Tukey analysis for multiple comparisons, respectively.

      Data availability

      Data not shown will be shared upon request to Ernesto Canalis at [email protected]

      Supporting information

      This article contains supporting information.

      Conflict of interest

      The authors declare no conflicts of interest with the contents of this article.

      Acknowledgments

      The authors thank R. Kageyama for Hes1loxP/loxP and RosaHes1 mice, obtained through RIKEN, D. Fremont for M-CSF cDNA, M. Glogauer for Tnfsf11 cDNA, A. Rao for Nfatc1 cDNA, Lydia Schlaefke for technical assistance, and Mary Yurczak for secretarial support.

      Author contributions

      J. Y. and E. C. conceptualization; J. Y. validation; J. Y. formal analysis; J. Y., L. S., and T. E. investigation; J. Y. and E. C. writing–original draft; J. Y. and E. C. writing–review and editing; J. Y. visualization; E. C. project administration; E. C. funding acquisition.

      Funding and additional information

      This work was supported by grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health grant AR078149 (to E. C.) and AR072987 (to E. C.) and from the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health DK045227 (to E. C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

      Supporting information

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