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Antisense oligonucleotides targeting Notch2 ameliorate the osteopenic phenotype in a mouse model of Hajdu-Cheney syndrome

Open AccessPublished:January 28, 2020DOI:https://doi.org/10.1074/jbc.RA119.011440
      Notch receptors play critical roles in cell-fate decisions and in the regulation of skeletal development and bone remodeling. Gain–of–function NOTCH2 mutations can cause Hajdu-Cheney syndrome, an untreatable disease characterized by osteoporosis and fractures, craniofacial developmental abnormalities, and acro-osteolysis. We have previously created a mouse model harboring a point 6955C→T mutation in the Notch2 locus upstream of the PEST domain, and we termed this model Notch2tm1.1Ecan. Heterozygous Notch2tm1.1Ecan mutant mice exhibit severe cancellous and cortical bone osteopenia due to increased bone resorption. In this work, we demonstrate that the subcutaneous administration of Notch2 antisense oligonucleotides (ASO) down-regulates Notch2 and the Notch target genes Hes-related family basic helix–loop–helix transcription factor with YRPW motif 1 (Hey1), Hey2, and HeyL in skeletal tissue from Notch2tm1.1Ecan mice. Results of microcomputed tomography experiments indicated that the administration of Notch2 ASOs ameliorates the cancellous osteopenia of Notch2tm1.1Ecan mice, and bone histomorphometry analysis revealed decreased osteoclast numbers in Notch2 ASO-treated Notch2tm1.1Ecan mice. Notch2 ASOs decreased the induction of mRNA levels of TNF superfamily member 11 (Tnfsf11, encoding the osteoclastogenic protein RANKL) in cultured osteoblasts and osteocytes from Notch2tm1.1Ecan mice. Bone marrow-derived macrophage cultures from the Notch2tm1.1Ecan mice displayed enhanced osteoclastogenesis, which was suppressed by Notch2 ASOs. In conclusion, Notch2tm1.1Ecan mice exhibit cancellous bone osteopenia that can be ameliorated by systemic administration of Notch2 ASOs.

      Introduction

      Notch receptors are four single-pass transmembrane proteins that play a critical function in cell fate determination (
      • Siebel C.
      • Lendahl U.
      Notch signaling in development, tissue homeostasis, and disease.
      ,
      • Zanotti S.
      • Canalis E.
      Notch signaling and the skeleton.
      ). Notch1–3 and low levels of Notch4 transcripts are detected in bone cells, where they play a key role in osteoblast and osteoclast differentiation and function (
      • Canalis E.
      Notch in skeletal physiology and disease.
      ). Notch receptors are activated following interactions with ligands of the Jagged and Delta-like families, and JAGGED1 is the prevalent ligand expressed by skeletal cells (
      • Canalis E.
      Notch in skeletal physiology and disease.
      ,
      • Zanotti S.
      • Canalis E.
      Parathyroid hormone inhibits Notch signaling in osteoblasts and osteocytes.
      ). Interactions of NOTCH with its ligands lead to the proteolytic cleavage of the NOTCH protein and to the release of the NOTCH intracellular domain (NICD)
      The abbreviations used are: NICD
      NOTCH intracellular domain
      ASO
      antisense oligonucleotide
      BMM
      bone marrow macrophage
      BV/TV
      bone volume/tissue volume
      CTX
      carboxyl-terminal collagen cross-link
      Ctrl
      control
      DMEM
      Dulbecco's modified Eagle's medium
      FBS
      fetal bovine serum
      HCS
      Hajdu-Cheney syndrome
      HES
      Hairy Enhancer of Split
      M-CSF
      macrophage–colony-stimulating factor
      MAML
      mastermind
      μCT
      microcomputed tomography
      N2ICD
      NOTCH2 intracellular domain
      NRR
      negative regulatory region
      PEST
      proline (P), glutamic acid (E), serine (S), and threonine (T)
      qRT
      quantitative reverse transcription
      RANKL
      receptor activator of nuclear factor κB ligand
      SMI
      structure model index
      TRAP
      tartrate-resistant acid phosphatase.
      (
      • Zanotti S.
      • Canalis E.
      Notch and the skeleton.
      ,
      • Schroeter E.H.
      • Kisslinger J.A.
      • Kopan R.
      Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain.
      ). The NICD is translocated into the nucleus where it forms a complex with recombination signal-binding protein for Ig of κ (RBPJκ) and mastermind (MAML) to induce the transcription of target genes, including those encoding Hairy Enhancer of Split (HES)-1, -5, and -7 and HES-related with YRPW motif (HEY)-1, -2, and -L (
      • Kovall R.A.
      More complicated than it looks: assembly of Notch pathway transcription complexes.
      ,
      • Nam Y.
      • Sliz P.
      • Song L.
      • Aster J.C.
      • Blacklow S.C.
      Structural basis for cooperativity in recruitment of MAML coactivators to Notch transcription complexes.
      ,
      • Wilson J.J.
      • Kovall R.A.
      Crystal structure of the CSL–Notch–mastermind ternary complex bound to DNA.
      ).
      Although activation of NOTCH1, -2, and -3 in the skeleton results in osteopenia, the mechanisms responsible for the bone loss are distinct (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu-Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ,
      • 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.
      ,
      • Fukushima H.
      • Nakao A.
      • Okamoto F.
      • Shin M.
      • Kajiya H.
      • Sakano S.
      • Bigas A.
      • Jimi E.
      • Okabe K.
      The association of Notch2 and NF-κB accelerates RANKL-induced osteoclastogenesis.
      ,
      • Canalis E.
      • Yu J.
      • Schilling L.
      • Yee S.P.
      • Zanotti S.
      The lateral meningocele syndrome mutation causes marked osteopenia in mice.
      ,
      • Canalis E.
      • Parker K.
      • Feng J.Q.
      • Zanotti S.
      Osteoblast lineage-specific effects of Notch activation in the skeleton.
      ). NOTCH2 has unique properties and impairs osteoblast maturation and induces osteoclastogenesis by acting directly on cells of the myeloid lineage and by inducing receptor activator of NF-κB ligand (RANKL) in 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.
      ,
      • Fukushima H.
      • Nakao A.
      • Okamoto F.
      • Shin M.
      • Kajiya H.
      • Sakano S.
      • Bigas A.
      • Jimi E.
      • Okabe K.
      The association of Notch2 and NF-κB accelerates RANKL-induced osteoclastogenesis.
      ,
      • Yu J.
      • Canalis E.
      The Hajdu-Cheney mutation sensitizes mice to the osteolytic actions of tumor necrosis factor α.
      ).
      Hajdu-Cheney syndrome (HCS) is a dominant inherited disease characterized by craniofacial developmental abnormalities, acro-osteolysis, generalized osteoporosis with fractures and neurological complications (
      • Cheney W.D.
      Acro-osteolysis.
      ,
      • Hajdu N.
      • Kauntze R.
      Cranio-skeletal dysplasia.
      ,
      • Canalis E.
      Clinical and experimental aspects of notch receptor signaling: Hajdu-Cheney syndrome and related disorders.
      ). HCS is associated with point mutations in, or short deletions of, exon 34 of NOTCH2 that lead to the creation of a stop codon upstream of the proline (P), glutamic acid (E), serine (S), and threonine (T) (PEST) domain (
      • Gray M.J.
      • Kim C.A.
      • Bertola D.R.
      • Arantes P.R.
      • Stewart H.
      • Simpson M.A.
      • Irving M.D.
      • Robertson S.P.
      Serpentine fibula polycystic kidney syndrome is part of the phenotypic spectrum of Hajdu-Cheney syndrome.
      ,
      • Isidor B.
      • Lindenbaum P.
      • Pichon O.
      • Bézieau S.
      • Dina C.
      • Jacquemont S.
      • Martin-Coignard D.
      • Thauvin-Robinet C.
      • Le Merrer M.
      • Mandel J.L.
      • David A.
      • Faivre L.
      • Cormier-Daire V.
      • Redon R.
      • Le Caignec 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 Esch H.
      • FORGE Canada Consortium
      • 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.
      ). The PEST domain is required for the ubiquitination and degradation of NOTCH2. As a consequence, the mutations result in the translation of a stable truncated protein product and a gain–of–NOTCH2 function. Iliac crest bone biopsies obtained from subjects afflicted by HCS have demonstrated the presence of osteopenia, increased bone resorption, and trabecularization of cortical bone (
      • Udell J.
      • Schumacher Jr, H.R.
      • Kaplan F.
      • Fallon M.D.
      Idiopathic familial acro-osteolysis: histomorphometric study of bone and literature review of the Hajdu-Cheney syndrome.
      ,
      • Blumenauer B.T.
      • Cranney A.B.
      • Goldstein R.
      Acro-osteolysis and osteoporosis as manifestations of the Hajdu-Cheney syndrome.
      ,
      • 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.
      • Högler W.
      Bone structural characteristics and response to bisphosphonate treatment in children with Hajdu-Cheney syndrome.
      ).
      To gain an understanding of the HCS skeletal phenotype and the mechanisms involved, we introduced a Notch2 mutation (6955C→T) in the mouse genome to reproduce a mutation (6949C→T) found in a subject with HCS (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu-Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ,
      • Isidor B.
      • Lindenbaum P.
      • Pichon O.
      • Bézieau S.
      • Dina C.
      • Jacquemont S.
      • Martin-Coignard D.
      • Thauvin-Robinet C.
      • Le Merrer M.
      • Mandel J.L.
      • David A.
      • Faivre L.
      • Cormier-Daire V.
      • Redon R.
      • Le Caignec C.
      Truncating mutations in the last exon of NOTCH2 cause a rare skeletal disorder with osteoporosis.
      ,
      • 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.
      ). The mutation creates a stop codon in exon 34 leading to the translation of a truncated NOTCH2 protein of 2318 amino acids (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu-Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ). The mouse line, termed Notch2tm1.1Ecan, exhibits NOTCH2 gain–of–function, and homozygous mice display craniofacial developmental abnormalities and newborn lethality. Heterozygous Notch2tm1.1Ecan mice have cancellous and cortical bone osteopenia due to enhanced bone resorption. This is secondary to an increase in the number of osteoclasts due to enhanced expression of RANKL by cells of the osteoblast lineage as well as due to direct effects of NOTCH2 on osteoclastogenesis (
      • 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 α.
      ). The discovery of the mechanisms responsible for the bone loss provided clues to offer improved treatments to individuals with HCS, such as the use of the RANKL antibody denosumab (
      • Adami G.
      • Rossini M.
      • Gatti D.
      • Orsolini G.
      • Idolazzi L.
      • Viapiana O.
      • Scarpa A.
      • Canalis E.
      Hajdu-Cheney syndrome; report of a novel NOTCH2 mutation and treatment with denosumab.
      ). However, none of the available interventions offers the opportunity to correct the mechanisms responsible for the disease.
      Approaches to down-regulate Notch signaling include the use of biochemical inhibitors of Notch activation, thapsigargin, antibodies to nicastrin, which forms part of the γ-secretase complex, or to Notch receptors or their ligands, and stapled peptides that prevent the assembly of a NICD/RBPJκ/MAML ternary complex (
      • Ryeom S.W.
      The cautionary tale of side effects of chronic Notch1 inhibition.
      ,
      • De Strooper B.
      • Annaert W.
      • Cupers P.
      • Saftig P.
      • Craessaerts K.
      • Mumm J.S.
      • Schroeter E.H.
      • Schrijvers V.
      • Wolfe M.S.
      • Ray W.J.
      • Goate A.
      • Kopan R.
      A presenilin-1-dependent γ-secretase-like protease mediates release of Notch intracellular domain.
      ,
      • Duggan S.P.
      • McCarthy J.V.
      Beyond γ-secretase activity: the multifunctional nature of presenilins in cell signalling pathways.
      ,
      • Ilagan M.X.
      • Kopan R.
      Selective blockade of transport via SERCA inhibition: the answer for oncogenic forms of Notch?.
      ,
      • Moellering R.E.
      • Cornejo M.
      • Davis T.N.
      • Del Bianco C.
      • Aster J.C.
      • Blacklow S.C.
      • Kung A.L.
      • Gilliland D.G.
      • Verdine G.L.
      • Bradner J.E.
      Direct inhibition of the NOTCH transcription factor complex.
      ). A limitation of these approaches is that either they are not specific inhibitors of Notch signaling or they prevent the indiscriminate activation of all Notch receptors, leading to a generalized Notch activation knockdown and side effects. Anti-Notch NRR antibodies have been effective at preventing the activation of specific Notch receptors (
      • Wu Y.
      • Cain-Hom C.
      • Choy L.
      • Hagenbeek T.J.
      • de Leon G.P.
      • Chen Y.
      • Finkle D.
      • Venook R.
      • Wu X.
      • Ridgway J.
      • Schahin-Reed D.
      • Dow G.J.
      • Shelton A.
      • Stawicki S.
      • Watts R.J.
      • et al.
      Therapeutic antibody targeting of individual Notch receptors.
      ,
      • Canalis E.
      • Sanjay A.
      • Yu J.
      • Zanotti S.
      An antibody to Notch2 reverses the osteopenic phenotype of Hajdu-Cheney mutant male mice.
      ,
      • Yu J.
      • Siebel C.W.
      • Schilling L.
      • Canalis E.
      An antibody to Notch3 reverses the skeletal phenotype of lateral meningocele syndrome in male mice.
      ). However, the pronounced down-regulation of Notch activation may result in gastrointestinal toxicity.
      Antisense oligonucleotides (ASOs) are single-stranded synthetic nucleic acids that bind target mRNA by Watson-Crick pairing resulting in mRNA degradation by RNase H (
      • Bennett C.F.
      • Baker B.F.
      • Pham N.
      • Swayze E.
      • Geary R.S.
      Pharmacology of antisense drugs.
      ,
      • Cerritelli S.M.
      • Crouch R.J.
      Ribonuclease H: the enzymes in eukaryotes.
      ). The administration of ASOs has emerged as a novel therapeutic approach to down-regulate WT and mutant transcripts, and it has been successful in the silencing of mutant genes in the central and peripheral nervous system, retina, and liver (
      • Murray S.F.
      • Jazayeri A.
      • Matthes M.T.
      • Yasumura D.
      • Yang H.
      • Peralta R.
      • Watt A.
      • Freier S.
      • Hung G.
      • Adamson P.S.
      • Guo S.
      • Monia B.P.
      • LaVail M.M.
      • McCaleb M.L.
      Allele-specific inhibition of rhodopsin with an antisense oligonucleotide slows photoreceptor cell degeneration.
      ,
      • Shy M.E.
      Antisense oligonucleotides offer hope to patients with Charcot-Marie-Tooth disease type 1A.
      ,
      • Carroll J.B.
      • Warby S.C.
      • Southwell A.L.
      • Doty C.N.
      • Greenlee S.
      • Skotte N.
      • Hung G.
      • Bennett C.F.
      • Freier S.M.
      • Hayden M.R.
      Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene/allele-specific silencing of mutant huntingtin.
      ,
      • Limmroth V.
      • Barkhof F.
      • Desem N.
      • Diamond M.P.
      • Tachas G.
      • ATL1102 Study Group
      CD49d antisense drug ATL1102 reduces disease activity in patients with relapsing-remitting MS.
      ,
      • McCampbell A.
      • Cole T.
      • Wegener A.J.
      • Tomassy G.S.
      • Setnicka A.
      • Farley B.J.
      • Schoch K.M.
      • Hoye M.L.
      • Shabsovich M.
      • Sun L.
      • Luo Y.
      • Zhang M.
      • Comfort N.
      • Wang B.
      • Amacker J.
      • et al.
      Antisense oligonucleotides extend survival and reverse decrement in muscle response in ALS models.
      ,
      • Zhao H.T.
      • Damle S.
      • Ikeda-Lee K.
      • Kuntz S.
      • Li J.
      • Mohan A.
      • Kim A.
      • Hung G.
      • Scheideler M.A.
      • Scherer S.S.
      • Svaren J.
      • Swayze E.E.
      • Kordasiewicz H.B.
      PMP22 antisense oligonucleotides reverse Charcot-Marie-Tooth disease type 1A features in rodent models.
      ,
      • Zhu C.
      • Kim K.
      • Wang X.
      • Bartolome A.
      • Salomao M.
      • Dongiovanni P.
      • Meroni M.
      • Graham M.J.
      • Yates K.P.
      • Diehl A.M.
      • Schwabe R.F.
      • Tabas I.
      • Valenti L.
      • Lavine J.E.
      • Pajvani U.B.
      Hepatocyte Notch activation induces liver fibrosis in nonalcoholic steatohepatitis.
      ,
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ). ASOs have been used to down-regulate specific genes in the skeleton, although information about their possible use as a therapeutic intervention in genetic disorders of the skeleton is limited (
      • Wang F.S.
      • Wu R.W.
      • Ko J.Y.
      • Tai M.H.
      • Ke H.C.
      • Yeh D.W.
      • Wu S.L.
      • Chen M.W.
      Heat shock protein 60 protects skeletal tissue against glucocorticoid-induced bone mass loss by regulating osteoblast survival.
      ,
      • Wang F.S.
      • Ko J.Y.
      • Lin C.L.
      • Wu H.L.
      • Ke H.J.
      • Tai P.J.
      Knocking down dickkopf-1 alleviates estrogen deficiency induction of bone loss. A histomorphological study in ovariectomized rats.
      ).
      The purpose of this work was to answer the question whether the phenotype of the Notch2tm1.1Ecan mouse model could be ameliorated or reversed by down-regulating Notch2 expression with Notch2-specific ASOs. To this end, heterozygous Notch2tm1.1Ecan and control littermate mice were treated with second generation phosphorothioate-modified ASOs targeting Notch2 and characterized by bone microarchitectural analysis. The direct effects of the Notch2 ASO on osteoblast, osteocyte, and osteoclast cultures from control and experimental mice also were tested.

      Results

      Effect of Notch2 ASOs on Notch2 expression and signaling in vivo

      In initial experiments, we tested whether mouse Notch2 ASOs down-regulated Notch2 mRNA in vivo in tissues where Notch2 is expressed and is known to have a function (
      • 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.
      ,
      • Saito T.
      • Chiba S.
      • Ichikawa M.
      • Kunisato A.
      • Asai T.
      • Shimizu K.
      • Yamaguchi T.
      • Yamamoto G.
      • Seo S.
      • Kumano K.
      • Nakagami-Yamaguchi E.
      • Hamada Y.
      • Aizawa S.
      • Hirai H.
      Notch2 is preferentially expressed in mature B cells and indispensable for marginal zone B lineage development.
      ,
      • Sparks E.E.
      • Huppert K.A.
      • Brown M.A.
      • Washington M.K.
      • Huppert S.S.
      Notch signaling regulates formation of the three-dimensional architecture of intrahepatic bile ducts in mice.
      ,
      • McCright B.
      • Gao X.
      • Shen L.
      • Lozier J.
      • Lan Y.
      • Maguire M.
      • Herzlinger D.
      • Weinmaster G.
      • Jiang R.
      • Gridley T.
      Defects in development of the kidney, heart and eye vasculature in mice homozygous for a hypomorphic Notch2 mutation.
      ,
      • Liu Z.
      • Chen S.
      • Boyle S.
      • Zhu Y.
      • Zhang A.
      • Piwnica-Worms D.R.
      • Ilagan M.X.
      • Kopan R.
      The extracellular domain of Notch2 increases its cell-surface abundance and ligand responsiveness during kidney development.
      ). The subcutaneous administration of ASOs targeting murine Notch2 to C57BL/6 WT mice at a dose of 50 mg/kg caused an ˜40–50% down-regulation of Notch2 mRNA 40 h later in the spleen, kidney, and femur and an 80% reduction of Notch2 transcripts in the liver (Fig. 1). In a subsequent experiment, Notch2 ASOs, administered subcutaneously to WT C57BL/6 mice at 50 mg/kg, down-regulated Notch2 mRNA in femur by ˜40% 48–96 h after the administration of the ASO.
      Figure thumbnail gr1
      Figure 1Effect of control (open circles) or Notch2 ASOs (closed circles) administered subcutaneously at a dose of 50 mg/kg to C57BL/6 mice on Notch2 mRNA. Panel A, Notch2 copy number corrected for Rpl38 was determined 40 h later in femur, spleen, kidney, and liver; panel B, Notch2 copy number was determined 6–96 h later in femur, and values are shown as relative expression corrected for Rpl38 and normalized to a control value of 1. Panel A, individual values are shown, and bars and ranges represent means ± S.D.; n = 3, except for 6-h data in panel B, where n = 2 of biological replicates. *, significantly different between Notch2 and control ASO, p < 0.05.
      There was evidence of enhanced Notch signaling in skeletal tissue from Notch2tm1.1Ecan mice, and the Notch target genes Hey1, Hey2, and HeyL were induced in bone extracts from mutant mice in relationship to control littermates (Fig. 2). The subcutaneous administration of mouse Notch2 ASOs decreased the expression of Notch2 and Notch26955C→T mutant mRNA. Notch2 ASOs also decreased the Notch target genes Hey1 and Hey2 in bone extracts from WT mice and Hey1, Hey2, and HeyL in extracts from Notch2tm1.1Ecan mice demonstrating a suppressive effect of Notch2 ASOs on Notch signaling in the skeleton. As a result, the mRNA levels of Hey1, Hey2, and HeyL in tibiae from Notch2tm1.1Ecan mice treated with Notch2 ASOs approached the levels found in tibiae from WT mice treated with control ASOs. A modest induction of Tnfsf11 (encoding RANKL, p > 0.05) was observed in tibiae from Notch2tm1.1Ecan mice, and this was reduced by Notch2 ASOs.
      Figure thumbnail gr2
      Figure 2Gene expression analysis of tibiae from 2-month-old male Notch2tm1.1Ecan mutant mice (closed circles) and sex-matched littermate controls (open circles) treated with Notch2 ASO or control ASO (Ctrl), both at 50 mg/kg subcutaneously once a week for 4 weeks prior to sacrifice. Transcript levels for Notch2, Notch26955C→T, Hey1, Hey2, HeyL, and Tnfsf11 are expressed as relative expression corrected for Rpl38 and normalized to a control value of 1. Individual values are shown, and bars and ranges represent means ± S.D.; n = 9–13 of biological replicates. *, significantly different between Notch2tm1.1Ecan mutant and WT mouse, p < 0.05; #, significantly different between Notch2 ASO and control ASO, p < 0.05.

      Effect of Notch2 ASOs on general characteristics, femoral microarchitecture, and histomorphometry of Notch2tm1.1Ecan mice

      Heterozygous Notch2tm1.1Ecan mutant male mice were compared with WT sex-matched littermate mice in a C57BL/6 genetic background because the skeletal phenotype was similar in both sexes and the homozygous mutation of Notch2tm1.1Ecan results in perinatal lethality. Confirming prior results, Notch2tm1.1Ecan heterozygous mice had ˜10% less weight than littermate controls, and their femoral length was slightly shorter than that of controls (Fig. 3) (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu-Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ). Following the administration of mouse Notch2 ASOs, control and Notch2tm1.1Ecan experimental mice appeared healthy, although a 6% decrease in weight was noted in WT mice treated with Notch2 ASOs when compared with control ASOs. Femoral length was not affected by Notch2 ASOs in either control or Notch2tm1.1Ecan mice (Fig. 3).
      Figure thumbnail gr3
      Figure 3Body weight and femoral length of 2-month-old male Notch2tm1.1Ecan mutant mice (closed circles) and littermate WT controls (open circles) treated with Notch2 ASO or control ASO (Ctrl) at 50 mg/kg subcutaneously, once a week for 4 weeks. Individual values are shown, and bars and ranges represent means ± S.D.; n = 12–15 of biological replicates. Numerical values express data normalized to a control value of 100%. *, significantly different between Notch2tm1.1Ecan mutant and control mice, p < 0.05; #, significantly different between Notch2 ASO and control ASO, p < 0.05.
      Validating previous observations, μCT of the distal femur revealed that 2-month-old Notch2tm1.1Ecan mutant male mice had a significant decrease in trabecular bone volume/total volume (BV/TV) associated with reduced connectivity and a higher structure model index (SMI) (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu-Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ). Trabecular number and thickness were both reduced in Notch2tm1.1Ecan mice, contributing to the decrease in BV/TV (Fig. 4). The subcutaneous administration of mouse Notch2 ASOs once a week at 50 mg/kg for 4 weeks did not change microarchitectural parameters of femoral bone in WT mice. In contrast, Notch2tm1.1Ecan mice receiving Notch2 ASOs had a BV/TV that was 30% greater than in mutant mice receiving control ASOs. As a consequence, BV/TV in Notch2tm1.1Ecan mice was reduced by 28% when compared with control WT mice, whereas Notch2tm1.1Ecan treated with control ASOs exhibited a 45% reduction in BV/TV compared with WT littermate controls (Fig. 4). The partial restoration of BV/TV by Notch2 ASOs was associated with a significant increase in trabecular number. Notch2tm1.1Ecan mice presented with cortical osteopenia and cortical bone was thin, and bone area and cortical thickness were reduced (Table 1). The cortical osteopenia was not affected by Notch2 ASOs, so the cortical bone area and thickness in Notch2tm1.1Ecan mice treated with Notch2 ASOs were not different from values obtained in mutant mice treated with control ASOs.
      Figure thumbnail gr4
      Figure 4Cancellous bone microarchitecture assessed by μCT of the distal femur from 2-month-old Notch2tm1.1Ecan mutant male mice (closed circles) and sex-matched littermate controls (open circles) treated with Notch2 ASO (n = 15 for WT, n = 14 for Notch2tm1.1Ecan) or control ASO (Ctrl; n = 15 for WT, n = 12 for Notch2tm1.1Ecan), both at 50 mg/kg subcutaneously, once a week for 4 weeks prior to sacrifice. Parameters shown are as follows: bone volume/tissue volume (BV/TV); trabecular separation (Tb.Sp); number (Tb.N) and thickness (Tb.Th); connectivity density (Conn.D); structure model index (SMI); and density of material expressed as milligrams of hydroxyapatite (HA) per cm3. Individual values are shown, and bars and ranges represent means ± S.D. of biological replicates. *, significantly different between Notch2tm1.1Ecan and control, p < 0.05; #, significantly different between Notch2 and control ASO, p < 0.05. Representative images show cancellous bone osteopenia in Notch2tm1.1Ecan mutant mice and its amelioration by Notch2 ASOs. Scale bars in the right corner represent 100 μm.
      Table 1Femoral cortical microarchitecture assessed by μCT of Notch2tm1.1Ecan male mice and littermate sex-matched controls treated with Notch2 ASOs
      Control ASONotch2 ASO
      Wildtype, n = 8Notch2tm1.1Ecan, n = 7Wildtype, n = 8Notch2tm1.1Ecan, n = 9
      Femoral midshaft cortical bone
      Bone volume/total volume (%)91.0 ± 1.087.4 ± 1.8
      Data are significantly different between Notch2tm1.1Ecan and wild type littermate mice, p < 0.05.
      90.7 ± 1.588.4 ± 0.9
      Data are significantly different between Notch2tm1.1Ecan and wild type littermate mice, p < 0.05.
      Porosity (%)9.0 ± 1.012.6 ± 1.8
      Data are significantly different between Notch2tm1.1Ecan and wild type littermate mice, p < 0.05.
      9.3 ± 1.511.6 ± 0.9
      Data are significantly different between Notch2tm1.1Ecan and wild type littermate mice, p < 0.05.
      Cortical thickness (μm)162 ± 10118 ± 17
      Data are significantly different between Notch2tm1.1Ecan and wild type littermate mice, p < 0.05.
      161 ± 14124 ± 9
      Data are significantly different between Notch2tm1.1Ecan and wild type littermate mice, p < 0.05.
      Total area (mm2)2.0 ± 0.21.8 ± 0.1
      Data are significantly different between Notch2tm1.1Ecan and wild type littermate mice, p < 0.05.
      2.0 ± 0.21.9 ± 0.1
      Bone area (mm2)0.9 ± 0.10.7 ± 0.1
      Data are significantly different between Notch2tm1.1Ecan and wild type littermate mice, p < 0.05.
      0.9 ± 0.10.7 ± 0.1
      Data are significantly different between Notch2tm1.1Ecan and wild type littermate mice, p < 0.05.
      Periosteal perimeter (mm)5.1 ± 0.24.8 ± 0.2
      Data are significantly different between Notch2tm1.1Ecan and wild type littermate mice, p < 0.05.
      5.0 ± 0.34.8 ± 0.1
      Endocortical perimeter (mm)3.8 ± 0.23.8 ± 0.13.8 ± 0.23.9 ± 0.1
      Density of material (mg HA/ml)1126 ± 221093 ± 36
      Data are significantly different between Notch2tm1.1Ecan and wild type littermate mice, p < 0.05.
      1114 ± 291095 ± 14
      a Data are significantly different between Notch2tm1.1Ecan and wild type littermate mice, p < 0.05.
      Cancellous bone histomorphometric analysis revealed that osteoclast number was increased in Notch2tm1.1Ecan mice; Notch2 ASOs did not change osteoclast number in WT mice, but significantly reduced the osteoclast number in Notch2tm1.1Ecan mice so that the osteoclast number was not different between Notch2tm1.1Ecan mice treated with Notch2 ASOs and control littermate WT mice (Table 2). Confirming prior observations, osteoblast number was not different between control and Notch2tm1.1Ecan mice. Accordingly, dynamic parameters of bone formation were not different between WT and mutant mice and were not affected by Notch2 ASOs. In accordance with the cellular phenotype of Notch2tm1.1Ecan mice, fasting serum levels of carboxyl-terminal collagen cross-links (CTX) were increased from (means ± S.D.; n = 5–6) control 34.6 ± 2.4 to 49.2 ± 8.9 ng/ml (p < 0.05) in Notch2tm1.1Ecan mice treated with control ASOs. Notch2 ASOs reduced the serum levels of CTX in both WT mice to 24.1 ± 9.7 ng/ml (p < 0.052) and Notch2tm1.1Ecan mice to 23.2 ± 3.9 ng/ml (p < 0.05) demonstrating a normalization of bone resorption in experimental mice.
      Table 2Cancellous bone histological parameters of 2-month-old Notch2tm1.1Ecan mice and littermate sex-matched controls treated with Notch2 ASOs
      Control ASONotch2 ASO
      Wildtype, n = 11Notch2tm1.1Ecan, n = 10Wildtype, n = 13Notch2tm1.1Ecan, n = 12
      Osteoblast surface/bone surface (%)12.6 ± 4.514.2 ± 5.79.5 ± 4.110.2 ± 4.4
      Osteoblasts/bone perimeter (1/mm)9.7 ± 3.411.1 ± 4.47.6 ± 3.38.3 ± 3.3
      Osteoclast surface/bone surface (%)6.9 ± 2.010.8 ± 2.4
      Data are significantly different between Notch2tm1.1Ecan and wildtype controls, p < 0.05.
      6.4 ± 2.37.8 ± 2.7
      Data are significantly different between Notch2 ASO and control ASO, p < 0.05.
      Osteoclasts/bone perimeter (1/mm)2.7 ± 0.64.2 ± 1.0
      Data are significantly different between Notch2tm1.1Ecan and wildtype controls, p < 0.05.
      2.5 ± 0.93.1 ± 1.0
      Data are significantly different between Notch2 ASO and control ASO, p < 0.05.
      Eroded surface/bone surface (%)3.0 ± 0.63.9 ± 2.02.6 ± 1.22.8 ± 1.2
      Mineral apposition rate (μm/day)1.2 ± 0.31.6 ± 0.71.3 ± 0.31.5 ± 0.4
      Mineralizing surface/bone surface (%)3.2 ± 1.12.1 ± 1.12.8 ± 1.52.0 ± 1.2
      Bone formation rate (μm3/μm2/day)0.03 ± 0.010.03 ± 0.020.04 ± 0.020.03 ± 0.02
      a Data are significantly different between Notch2tm1.1Ecan and wildtype controls, p < 0.05.
      b Data are significantly different between Notch2 ASO and control ASO, p < 0.05.

      Effect of Notch2 ASOs on Notch2 expression and signaling in osteoblast and osteocyte cell cultures

      Mouse Notch2 ASOs added to the culture medium of osteoblast-enriched cells from WT C57BL/6 mice at 1–20 μm decreased Notch2 mRNA by ˜40 to ˜80% 72 h after ASO addition without evidence of cellular toxicity or changes in cell replication (Fig. 5). The effect of the Notch2 ASO was specific for Notch2 mRNA because, at a concentration as high as 20 μm, it did not decrease the expression of Notch1, -3, or -4 mRNA. The NOTCH2 intracellular domain (N2ICD), representative of NOTCH2 cleavage and signal activation, was increased in Notch2tm1.1Ecan osteoblasts, and the truncated form of NOTCH2, lacking the PEST domain (N2ICDΔPEST), was detected only in Notch2tm1.1Ecan cells. Therefore, the total levels of N2ICDs, intact and truncated, were ˜2-fold greater in Notch2tm1.1Ecan cells than in control cells (Fig. 5). Notch2 ASOs decreased the total levels of N2ICD in WT and Notch2tm1.1Ecan cells demonstrating a suppression of NOTCH2 activation. Notch26955C→T transcripts were present in cells from Notch2tm1.1Ecan mutant mice but not in control cultures, and Hey1 and Hey2 transcripts were increased in Notch2tm1.1Ecan osteoblasts confirming that Notch signaling was activated (Fig. 6). In accordance with prior observations, tumor necrosis factor superfamily member 11 (Tnfsf11), encoding RANKL, was induced, whereas Bglap, encoding osteocalcin, was not changed in Notch2tm1.1Ecan osteoblasts (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu-Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ). Notch2 ASOs decreased Notch2 mRNA in WT and mutant cells and Notch26955C→T mRNA in osteoblasts from Notch2tm1.1Ecan mice. In addition, Notch2 ASOs decreased Hey1, Hey2, and Tnfsf11 mRNA in cells from Notch2tm1.1Ecan mice without an effect on Bglap expression (Fig. 6).
      Figure thumbnail gr5
      Figure 5Effect of control (open circles) or Notch2 ASOs (closed circles) on Notch2 mRNA and NOTCH2 protein expression in calvarial osteoblast-enriched cells. Panel A, Notch2 mRNA levels were obtained 72 h after the addition of Notch2 (closed circles) or control ASO (open circles) at 1–20 μm to cells from WT C57BL/6 mice. Values are means ± S.D.; n = 3 technical replicates. Panel B, Notch1–4 mRNA levels were obtained 72 h after the addition of Notch2 (closed circles) or control ASO (open circles) at 20 μm to cells from WT C57BL/6 mice. Panels A and B, transcript levels are expressed as relative number following correction for Rpl38. Panels C and D, osteoblasts from WT (control) or Notch2tm1.1Ecan mutant littermates were cultured with Notch2 or control ASOs at 20 μm for 72 h and analyzed for Notch2 mRNA expression (copy number corrected for Rpl38) (left) and for NOTCH2 by immunoblotting (right). The band intensity was quantified by Image LabTM software (version 5.2.1), and the numerical ratio of NOTCH2/β-actin, N2ICD (including N2ICDΔPEST)/β-actin is shown below each blot with WT cells cultured with control ASO normalized to 1. Panels B and C, individual values are shown, and bars and ranges represent means ± S.D. of technical replicates; n = 3 for B and n = 4 for C. Panels A and B, *, significantly different between Notch2 ASO and control ASO, p < 0.05. Panel C, *, significantly different between Notch2tm1.1Ecan and control mice, p < 0.05; #, significantly different between Notch2 and control ASO, p < 0.05.
      Figure thumbnail gr6
      Figure 6Gene expression analysis of calvarial osteoblast-enriched cells from Notch2tm1.1Ecan mutant mice (closed circles) and littermate controls (open circles). Notch2, Notch26955C→T, Hey1, Hey2, HeyL, Tnfsf11, and Bglap mRNA levels were obtained 72 h after the addition of Notch2 or control ASO (Ctrl) at 20 μm. Transcript levels are expressed as copy number corrected for Rpl38. Individual values are shown, and bars and ranges represent means ± S.D. of technical replicates; n = 4. *, significantly different between Notch2tm1.1Ecan and control osteoblasts, p < 0.05; #, significantly different between Notch2 ASO and control ASO, p < 0.05.
      Notch26955C→T mRNA was present in osteocyte-enriched cultures from Notch2tm1.1Ecan mice and not in control cultures, and Hey2 and Tnfsf11 were significantly increased in Notch2tm1.1Ecan cells (Fig. 7). Notch2 ASOs suppressed Notch2 mRNA in WT and mutant cells and Notch26955C→T mRNA levels in cells from Notch2tm1.1Ecan mice and suppressed Hey2 and Tnfsf11 in Notch2tm1.1Ecan cells to levels that were similar to those found in WT cells treated with control ASOs.
      Figure thumbnail gr7
      Figure 7Gene expression analysis of osteocyte-enriched preparations from 2-month-old Notch2tm1.1Ecan mutant mice (closed circles) and littermate controls (open circles) cultured for 72 h in the presence of Notch2 ASO or control ASO (Ctrl), both at 20 μm. Transcript levels for Notch2, Notch26955C→T, Hey2, and Tnfsf11 are expressed as copy number corrected for Rpl38. Individual values are shown, and bars and ranges represent means ± S.D.; n = 3–4 of biological replicates. *, significantly different between Notch2tm1.1Ecan and control osteocytes, p < 0.05; #, significantly different between Notch2 ASO and control ASO, p < 0.05.

      Effect of Notch2 ASOs on Notch2 expression and activity in BMM cultures and osteoclast formation

      Notch2 ASOs were added to either BMM cultures at the initiation of the culture period or following the addition of RANKL for 2 days to determine their effect in cells of the myeloid lineage and in osteoclast precursors. Mouse Notch2 ASOs at 1 and 5 μm suppressed Notch2 mRNA levels in BMMs from WT C57BL/6 mice by 85–95% and in osteoclast precursors by 70–85% without evidence of cellular toxicity and without altering cell proliferation (Fig. 8). Confirming results in osteoblast cultures, the N2ICD was slightly increased in Notch2tm1.1Ecan osteoclasts, and the truncated form of NOTCH2 lacking the PEST domain (N2ICDΔPEST) was detected only in Notch2tm1.1Ecan cells. Consequently, the total levels of N2ICD, intact and truncated, were ˜2-fold greater in Notch2tm1.1Ecan cells than in control cells (Fig. 8). Notch2 ASOs decreased the total levels of N2ICD in WT and Notch2tm1.1Ecan cells demonstrating a suppression of NOTCH2 activation.
      Figure thumbnail gr8
      Figure 8Effect of control (open circles) or Notch2 ASOs (closed circles) on Notch2 mRNA and NOTCH2 protein expression in the osteoclast lineage. Panel A, BMM (left panel) and osteoclast precursors (OCP) (right panel) from WT C57BL/6 mice were cultured. BMMs were cultured for 72 h in the presence of Notch2 or control ASOs at 1 or 5 μm (left panel) in the presence of M-CSF at 30 ng/ml or cultured in the presence of M-CSF at 30 ng/ml and then seeded in the presence of M-CSF at 30 ng/ml and RANKL at 10 ng/ml with Notch2 or control ASOs at 1 or 5 μm (right panel). Notch2 mRNA levels are expressed as copy number corrected for Rpl38. Individual values are shown, and bars and ranges represent means ± S.D. for three biological replicates. *, significantly different between Notch2 ASO and control ASO, p < 0.05. Panel B, BMMs from WT (control) or Notch2tm1.1Ecan mutant littermates were cultured in M-CSF at 30 ng/ml and then seeded in the presence of M-CSF at 30 ng/ml and RANKL at 10 ng/ml with Notch2 or control ASOs at 1 μm for 48 h and analyzed for NOTCH2 by immunoblotting. The band intensity was quantified by Image LabTM software (version 5.2.1) and the numerical ratio of NOTCH2/β-actin, N2ICD (including N2ICDΔPEST)/β-actin is shown below each blot with WT cells cultured with control ASO normalized to 1.
      There was a significant increase in osteoclast formation in BMMs from Notch2tm1.1Ecan mice cultured in the presence of M-CSF and RANKL (Fig. 9). The increased osteoclastogenesis was prevented by the addition of Notch2 ASOs to BMM cultures at 1 μm so that the osteoclastogenic potential of Notch2tm1.1Ecan cells cultured with Notch2 ASOs was no longer different from that of control cells. The decrease in osteoclastogenesis by Notch2 ASOs in Notch2tm1.1Ecan cells was associated with a concomitant decrease in Notch2 WT and Notch26955C→T mutant transcripts.
      Figure thumbnail gr9
      Figure 9Osteoclast formation and gene expression analysis of BMM from Notch2tm1.1Ecan mutant (closed circles) and WT littermate controls (open circles) cultured for 72 h in the presence of M-CSF at 30 ng/ml and then seeded in the presence of M-CSF at 30 ng/ml and RANKL at 10 ng/ml and either Notch2 or control ASO (Ctrl) at 1 μm. Panel A, representative images of TRAP-stained multinucleated cells and data expressed as number of TRAP-positive multinucleated cells. The scale bars in the right corner represent 500 μm. Panel B, Notch2 and Notch26955C→T mRNA levels were obtained 96 h after the addition of Notch2 or control ASO (Ctrl) at 1 μm. mRNA levels are expressed as copy number corrected for Rpl38 copy number. Individual values are shown, and bars and ranges represent means ± S.D.; n = 4 of technical replicates of one representative out of three experiments. *, significantly different between Notch2tm1.1Ecan mutant and control cells; #, significantly different between Notch2 ASO and control ASO.

      Discussion

      Findings from this work confirm that a mouse model replicating a mutation found in HCS displays femoral cancellous and cortical bone osteopenia. The osteopenic phenotype is manifested early in life in mice of both sexes; and in this study, we elected to treat 1-month-old male mice with Notch2 ASOs in an attempt to ameliorate the osteopenic femoral phenotype of 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.
      ). Because only male mice were treated, one needs to be cautious and not to extrapolate the results to female mice. Phenotypic alterations of experimental and control mice were assessed by μCT, and analyses required the ex vivo exam of bone following the sacrifice of mice. Consequently, the same animal could not be analyzed before and after the administration of Notch2 ASOs. Another limitation of the work is the fact that all the analyses were performed in femoral bone because the osteopenia of Notch2tm1.1Ecan mice was established at this skeletal site (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu-Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ). Although Notch2 ASOs down-regulated Notch2 WT and mutant transcripts in femoral bone, it was not determined whether the same effect occurs at other skeletal, possibly less vascularized, sites. The Notch2 ASO utilized is specific to Notch2 so that the results obtained should not be attributed to the down-regulation of other Notch receptors.
      The phenotype of the Notch2tm1.1Ecan mutant mouse recapitulates aspects of HCS, including osteopenia, short limbs, and in the homozygous state of craniofacial abnormalities, including micrognathia and early-lethality (
      • 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 α.
      ).
      E. Canalis, unpublished observations.
      However, neither Notch2tm1.1Ecan nor an alternate murine model of HCS manifest acro-osteolysis (
      • Vollersen N.
      • Hermans-Borgmeyer I.
      • Cornils K.
      • Fehse B.
      • Rolvien T.
      • Triviai I.
      • Jeschke A.
      • Oheim R.
      • Amling M.
      • Schinke T.
      • Yorgan T.A.
      High bone turnover in mice carrying a pathogenic Notch2 mutation causing Hajdu-Cheney syndrome.
      ). In this work, we confirm that Notch2 has unique actions on trabecular bone physiology and induces osteoclastogenesis by increasing the expression of RANKL by cells of the osteoblast lineage and by inducing the differentiation of cells of the myeloid lineage toward mature osteoclasts (
      • Yu J.
      • Canalis E.
      The Hajdu-Cheney mutation sensitizes mice to the osteolytic actions of tumor necrosis factor α.
      ). Notch2 ASOs decreased both effects in vitro and decreased serum levels of CTX, a marker of bone resorption, so that CTX levels in Notch2tm1.1Ecan ASO-treated mice were not different from those of WT mice. These effects would explain the amelioration of the osteopenia observed in Notch2tm1.1Ecan mice.
      Notch2 ASOs down-regulated Notch2 and Notch26955C→T transcripts and decreased the enhanced Notch signaling found in Notch2tm1.1Ecan cells as well as in bone extracts without an effect on basal levels of Notch activation. Only Notch2tm1.1Ecan mutant cells synthesized the truncated form of the N2ICD (N2ICDΔPEST) and the intact N2ICD. The summation of the intact and truncated forms of N2ICD resulted in an ˜2-fold greater expression of N2ICD in Notch2tm1.1Ecan mutants than in control cells, and this was suppressed by Notch2 ASOs confirming the down-regulation of Notch2 signaling. The N2ICDΔPEST is more stable than WT N2ICD because it is resistant to ubiquitin-mediated degradation, explaining the gain–of–NOTCH2 function and the induction of Notch target genes in Notch2tm1.1Ecan cells. Notch2tm1.1Ecan mice do not exhibit an increase in osteoblast number or a bone-forming response to the increase in bone resorption, indicating a possible negative regulation of osteoblastogenesis or osteoblast function by the Notch2 mutation. However, in this study, we confirm that osteoblast gene markers, such as Bglap (osteocalcin), are not affected in cells from Notch2tm1.1Ecan mice. The inactivation of Notch2 in cells of the osteoblastic lineage causes an increase in the osteogenic potential of these cells suggesting an inhibitory role of Notch signaling in osteoblastogenesis (
      • Yorgan T.
      • Vollersen N.
      • Riedel C.
      • Jeschke A.
      • Peters S.
      • Busse B.
      • Amling M.
      • Schinke T.
      Osteoblast-specific Notch2 inactivation causes increased trabecular bone mass at specific sites of the appendicular skeleton.
      ,
      • Zanotti S.
      • Smerdel-Ramoya A.
      • Stadmeyer L.
      • Durant D.
      • Radtke F.
      • Canalis E.
      Notch inhibits osteoblast differentiation and causes osteopenia.
      ,
      • Deregowski V.
      • Gazzerro E.
      • Priest L.
      • Rydziel S.
      • Canalis E.
      Notch 1 overexpression inhibits osteoblastogenesis by suppressing Wnt/β-catenin but not bone morphogenetic protein signaling.
      ).
      Although approaches to down-regulate Notch signaling are various, they are often not specific to this signaling pathway or to a specific Notch receptor. A recent alternative has been the use of antibodies to the negative regulatory region (NRR) of specific Notch receptors that prevent the exposure of the NRR to the γ-secretase complex and thus the activation of Notch (
      • Wu Y.
      • Cain-Hom C.
      • Choy L.
      • Hagenbeek T.J.
      • de Leon G.P.
      • Chen Y.
      • Finkle D.
      • Venook R.
      • Wu X.
      • Ridgway J.
      • Schahin-Reed D.
      • Dow G.J.
      • Shelton A.
      • Stawicki S.
      • Watts R.J.
      • et al.
      Therapeutic antibody targeting of individual Notch receptors.
      ,
      • Canalis E.
      • Sanjay A.
      • Yu J.
      • Zanotti S.
      An antibody to Notch2 reverses the osteopenic phenotype of Hajdu-Cheney mutant male mice.
      ,
      • Yu J.
      • Siebel C.W.
      • Schilling L.
      • Canalis E.
      An antibody to Notch3 reverses the skeletal phenotype of lateral meningocele syndrome in male mice.
      ). Recently, we demonstrated that anti-Notch2 NRR antibodies reverse the skeletal phenotype of Notch2tm1.1Ecan mice, and anti-Notch3 NRR antibodies reverse the skeletal phenotype of Notch3tm1.1Ecan mice, a model of lateral meningocele syndrome (
      • Canalis E.
      • Sanjay A.
      • Yu J.
      • Zanotti S.
      An antibody to Notch2 reverses the osteopenic phenotype of Hajdu-Cheney mutant male mice.
      ,
      • Yu J.
      • Siebel C.W.
      • Schilling L.
      • Canalis E.
      An antibody to Notch3 reverses the skeletal phenotype of lateral meningocele syndrome in male mice.
      ). Although anti-Notch NRR antibodies are specific, the significant down-regulation of the Notch receptor throughout the organism may lead to potential side effects, such as gastrointestinal toxicity. In this study, we demonstrate that down-regulation of Notch expression by specific Notch ASOs is a suitable alternative to decrease Notch activation in conditions of Notch gain–of–function. Although the effect of Notch2 ASOs was less pronounced than the one reported with anti-Notch2 NRR antibodies, Notch2 ASOs were effective at ameliorating the skeletal phenotype of Notch2tm1.1Ecan mice and appeared to be well-tolerated by this experimental model of HCS.
      Although attempts have been made to transport ASOs to bone, complex delivery systems were necessary, and the technology has not been applied to the correction of gene mutations in the skeleton (
      • Zhang G.
      • Guo B.
      • Wu H.
      • Tang T.
      • Zhang B.T.
      • Zheng L.
      • He Y.
      • Yang Z.
      • Pan X.
      • Chow H.
      • To K.
      • Li Y.
      • Li D.
      • Wang X.
      • Wang Y.
      • et al.
      A delivery system targeting bone formation surfaces to facilitate RNAi-based anabolic therapy.
      ). In this study, we used a practical systemic approach to down-regulate Notch2 in skeletal and nonskeletal tissue. We demonstrate that a second generation phosphorothioate-modified murine Notch2 ASO down-regulated Notch2 in tissues where the gene is expressed and has a function, including bone. The decrease in Notch2 in a mouse model of Notch2 gain–of–function was associated with a concomitant decrease in Notch target gene expression in skeletal cells documenting a tempering effect on Notch activation. As a consequence, a recovery of bone mass was observed. Although this was not complete, a significant effect on BV/TV was achieved with amelioration of the Notch2tm1.1Ecan skeletal phenotype.
      In conclusion, Notch2 ASOs down-regulate Notch2 expression and signal activation, decrease RANKL and osteoclastogenesis in a model of HCS, and consequently ameliorate its osteopenic phenotype. The down-regulation of NOTCH2 may offer a potential therapeutic opportunity for subjects with HCS in the future.

      Experimental procedures

      Notch2 antisense oligonucleotides

      ASOs targeting Notch2 mRNA were designed in silico by scanning through the sequence of murine Notch2 pre-mRNA. The entire Notch2 pre-mRNA sequence was covered for potential 16-mer oligonucleotides complementary to the pre-mRNA. Sequence motifs that were intrinsically problematic because of unfavorable hybridization properties, such as polyG stretches, or potential toxicity due to immunogenic responses were avoided. Notch2 ASOs were tested for activity in vitro for down-regulation of Notch2 mRNA in HEPA 1–6 cells at Ionis Pharmaceuticals (Carlsbad, CA), and 14 ASOs targeting Notch2 mRNA were screened for activity and toxicity in vivo at the Korea Institute of Toxicology (Daejeon, Korea). To this end, 7-week-old BALB/c male mice were administered ASOs at a dose of 50 mg/kg once a week by subcutaneous injection for a total of 3.5 weeks (four doses). Body weights were measured weekly, and mice were euthanized 48 h after the last dose of ASO. Liver, kidney, and spleen were weighed, normalized to body weight, and compared with organs from control mice. Blood was obtained by cardiac puncture, and plasma was collected for the measurement of alanine aminotransferase, aspartate aminotransferase, total bilirubin, albumin, and blood urea nitrogen. Total RNA was extracted from liver samples to determine Notch2 mRNA levels corrected for cyclophilin A expression. Based on the information obtained, ASOs found to down-regulate Notch2 liver mRNA by more than 75% compared with a control mismatched ASO without toxicity in vivo were selected. Procedures were performed at and approved by the Animal Care and Use Committee of the Korea Institute of Toxicology. For this study, mouse Notch2 ASO Ionis 977472 of sequence GTTATATAATCTTCCA and control mismatched ASO Ionis 549144 of sequence GGCCAATACGCCGTCA were selected.

      Notch2tm1.1Ecan mutant mice

      A mouse model of HCS, termed Notch2tm1.1Ecan, harboring a 6955C→T substitution in exon 34 of Notch2 was previously reported and validated (
      • 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 were backcrossed into a C57BL/6J background for eight or more generations, and genotyping was conducted in tail DNA extracts by PCR using forward primer Nch2Lox gtF 5′-CCCTTCTCTCTGTGCGGTAG-3′ and reverse primer Nch2Lox gtR 5′-CTCAGAGCCAAAGCCTCACTG-3′. In this study, 1-month-old mice heterozygous for the Notch26955C→T allele and control mice were obtained by crossing heterozygous mutants with WT mice to assess the impact of Notch2 ASOs on the Notch2tm1.1Ecan skeletal phenotype. One-month-old male Notch2tm1.1Ecan heterozygous mutant and control sex-matched littermate mice were treated with Notch2 ASO (Ionis 977472) or control ASO (Ionis 549144) that was suspended in PBS and administered subcutaneously at a dose of 50 mg/kg once a week for 4 consecutive weeks. Mice were euthanized at 2 months of age. Studies were approved by the Institutional Animal Care and Use Committee of UConn Health.

      μCT

      Bone microarchitecture of femurs from experimental and control mice was determined using a μCT (μCT 40; Scanco Medical AG, Bassersdorf, Switzerland), 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.
      • Müller 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 kilovoltage peaks, intensity of 145 μA, and integration time of 200 ms. A total of 100 slices at midshaft and 160 slices at the distal metaphysis were acquired at an isotropic voxel size of 216 μm3, with a slice thickness of 6 μm, and then chosen for analysis. Trabecular bone volume fraction and microarchitecture were evaluated starting ˜1.0-mm proximal from the femoral condyles. Contours were manually drawn a few voxels away from the endocortical boundary every 10 slices to define the region of interest for analysis. The remaining slice contours were iterated automatically. Trabecular regions were assessed for total volume, bone volume, bone volume fraction (bone volume/total volume), trabecular thickness, trabecular number, trabecular separation, connectivity density, and SMI, using a Gaussian filter (σ = 0.8), and a threshold of 240 per mil eq to 355.5 mg/cm3 hydroxyapatite (
      • Bouxsein M.L.
      • Boyd S.K.
      • Christiansen B.A.
      • Guldberg R.E.
      • Jepsen K.J.
      • Müller 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 femoral cortical bone, contours were iterated across 100 slices along the cortical shell of the femoral midshaft, excluding the marrow cavity. Analysis of bone volume/total volume, porosity, cortical thickness, total cross-sectional and cortical bone area, periosteal perimeter, endosteal perimeter, and material density were performed using a Gaussian filter (σ = 0.8, support = 1), and a threshold of 400 per mil eq to 704.7 mg/cm3 hydroxyapatite.

      Bone histomorphometric analysis

      Static cancellous bone histomorphometry was carried out on experimental and control mice. The 5-μm longitudinal sections of undecalcified femurs embedded in methyl methacrylate were cut on a microtome (Microm, Richards-Allan Scientific, Kalamazoo, MI) and stained with 0.1% toluidine blue. Static and dynamic parameters of bone formation and resorption were measured in a defined area between 360 and 2160 μm from the growth plate, using an OsteoMeasure morphometry system (OsteoMetrics, Atlanta, GA). Stained sections were used to measure osteoblast and osteoclast number and eroded surface. Mineralizing surface per bone surface and the 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. The terminology and units used are those recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (
      • 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.
      ,
      • Parfitt A.M.
      • Drezner M.K.
      • Glorieux F.H.
      • Kanis J.A.
      • Malluche H.
      • Meunier P.J.
      • Ott S.M.
      • Recker R.R.
      Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee.
      ).

      Osteoblast-enriched cell cultures

      The parietal bones of 3–5-day-old control and Notch2tm1.1Ecan mutant mice were exposed to Liberase TL 1.2 units/ml (Sigma) for 20 min at 37 °C, and cells were extracted in five consecutive reactions (
      • Yesil P.
      • Michel M.
      • Chwalek K.
      • Pedack S.
      • Jany C.
      • Ludwig B.
      • Bornstein S.R.
      • Lammert E.
      A new collagenase blend increases the number of islets isolated from mouse pancreas.
      ). Cells from the last three digestions were pooled and seeded at a density of 10 × 104 cells/cm2, as described previously (
      • McCarthy T.L.
      • Centrella M.
      • Canalis E.
      Further biochemical and molecular characterization of primary rat parietal bone cell cultures.
      ). Osteoblast-enriched cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with nonessential amino acids (both from Life Technologies, Inc.) and 10% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, GA) in a humidified 5% CO2 incubator at 37 °C. Confluent osteoblast-enriched cells were exposed to DMEM supplemented with 10% heat-inactivated FBS, 100 μg/ml ascorbic acid, and 5 mm β-glycerophosphate (both from Sigma) in the presence of Notch2 ASO or control ASO at various doses as indicated in figure legends.

      Osteocyte-enriched cultures

      Femurs from 6- to 7-week-old WT or Notch2tm1.1Ecan mice were collected after sacrifice; the surrounding tissues were dissected; the proximal epiphysis was excised; and the bone marrow was removed by centrifugation. The distal epiphysis was removed, and to release the endosteal and periosteal cellular layers, the femoral fragments were sequentially exposed for 20-min periods to type II collagenase pretreated with 17 μg/ml Nα-tosyl-l-lysine chloromethyl ketone hydrochloride and 5 mm EDTA (Life Technologies, Inc.) at 37 °C, as described previously (
      • Zanotti S.
      • Canalis E.
      Parathyroid hormone inhibits Notch signaling in osteoblasts and osteocytes.
      ,
      • Halleux C.
      • Kramer I.
      • Allard C.
      • Kneissel M.
      Isolation of mouse osteocytes using cell fractionation for gene expression analysis.
      ). Osteocyte-enriched bone fragments were obtained and cultured individually in DMEM supplemented with nonessential amino acids, 100 μg/ml ascorbic acid, and heat-inactivated 10% FBS for 72 h in a humidified 5% CO2 incubator at 37 °C in the presence of control or Notch2 ASOs, as indicated in figure legends (
      • Zanotti S.
      • Canalis E.
      Parathyroid hormone inhibits Notch signaling in osteoblasts and osteocytes.
      ,
      • Canalis E.
      • Schilling L.
      • Zanotti S.
      Effects of sex and Notch signaling on the osteocyte cell pool.
      ).

      Bone marrow-derived macrophage (BMM) cultures and osteoclast formation

      To obtain BMMs, bone marrow cells were isolated from long bones by flushing the marrow with a 26-gauge needle. Red blood cells were lysed in lysis buffer containing 150 mm NH4Cl, 10 mm KHCO3, and 0.1 mm EDTA (pH 7.4). The cell suspension was centrifuged, and the pellet was suspended in α-minimum essential medium (Life Technologies, Inc.) containing 10% heat-inactivated FBS and recombinant human macrophage colony-stimulating factor (M-CSF) at 30 ng/ml. M-CSF complementary DNA (cDNA) and expression vector were obtained from D. Fremont (St. Louis, MO), and M-CSF was purified as reported previously (
      • 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 at a density of 3 × 105 cells/cm2 on uncoated Petri dishes and cultured for 3 days.
      For osteoclast formation, cells were collected following treatment with 0.05% trypsin/EDTA and seeded at a density of 4.7 × 104 cells/cm2 on tissue culture plates in the presence of M-CSF at 30 ng/ml and murine RANKL at 10 ng/ml until the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells. RANKL cDNA and expression vector were obtained from M. Glogauer (Toronto, Ontario, Canada), and GSH 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.
      ). TRAP enzyme histochemistry was conducted using a commercial kit (Sigma), in accordance with manufacturer’s instructions. TRAP-positive cells containing more than three nuclei were considered osteoclasts. Cultures were carried out in the presence of Notch2 or control ASO at various doses as indicated in the figure legends.

      Cell proliferation assay

      Cell replication was determined using the Cell Counting Kit-8. In this kit, the tetrazolium salt WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenhyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) produces a formazan dye, measured at an absorbance of 450 nm, upon reduction by cellular dehydrogenases. The assay quantifies viable cells and was used in accordance with the manufacturer's instructions (Dojindo Molecular Technologies, Rockville, MD).

      Quantitative reverse transcription (qRT)-PCR

      Total RNA was extracted from either cultured cells or tibiae following the removal of the bone marrow by centrifugation, and mRNA levels were determined by qRT-PCR (
      • Nazarenko I.
      • Pires R.
      • Lowe B.
      • Obaidy M.
      • Rashtchian A.
      Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes.
      ,
      • Nazarenko I.
      • Lowe B.
      • Darfler M.
      • Ikonomi P.
      • Schuster D.
      • Rashtchian A.
      Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore.
      ). For this purpose, equal amounts of RNA were reverse-transcribed using the iScript RT-PCR kit (Bio-Rad), according to the manufacturer’s instructions, and were amplified in the presence of specific primers (Table 3, all primers from Integrated DNA Technologies (IDT), Coralville, IA), and iQ SYBR Green Supermix (Bio-Rad), at 60 °C for 35 cycles. Transcript copy number was estimated by comparison with a serial dilution of cDNA for Bglap (from J. Lian, Burlington, VT), Hey1 and Hey2 (both from T. Iso, Gunma, Japan), HeyL (from D. Srivastava, San Francisco, CA), Notch2 (from Thermo Fisher Scientific), Notch1 (from J. S. Nye, Cambridge, MA), Notch4 (from Y. Shirayoshi, Tottori, Japan), or Tnfsf11 (from Source BioScience, Nottingham, UK) (
      • Iso T.
      • Sartorelli V.
      • Chung G.
      • Shichinohe T.
      • Kedes L.
      • Hamamori Y.
      HERP, a new primary target of Notch regulated by ligand binding.
      ,
      • Nakagawa O.
      • Nakagawa M.
      • Richardson J.A.
      • Olson E.N.
      • Srivastava D.
      HRT1, HRT2, and HRT3: a new subclass of bHLH transcription factors marking specific cardiac, somitic, and pharyngeal arch segments.
      ,
      • Wong B.R.
      • Rho J.
      • Arron J.
      • Robinson E.
      • Orlinick J.
      • Chao M.
      • Kalachikov S.
      • Cayani E.
      • Bartlett 3rd, F.S
      • Frankel W.N.
      • Lee S.Y.
      • Choi Y.
      TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells.
      ,
      • Nye J.S.
      • Kopan R.
      • Axel R.
      An activated Notch suppresses neurogenesis and myogenesis but not gliogenesis in mammalian cells.
      ,
      • Shirayoshi Y.
      • Yuasa Y.
      • Suzuki T.
      • Sugaya K.
      • Kawase E.
      • Ikemura T.
      • Nakatsuji N.
      Proto-oncogene of int-3, a mouse Notch homologue, is expressed in endothelial cells during early embryogenesis.
      ,
      • Lian J.
      • Stewart C.
      • Puchacz E.
      • Mackowiak S.
      • Shalhoub V.
      • Collart D.
      • Zambetti G.
      • Stein G.
      Structure of the rat osteocalcin gene and regulation of vitamin D-dependent expression.
      ). Notch3 copy number was estimated by comparison with a serial dilution of an ˜100-base pair synthetic DNA template (IDT) cloned into pcDNA3.1 (Thermo Fischer Scientific) by isothermal single reaction assembly using commercially available reagents (New England Biolabs, Ipswich, MA) (
      • 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.
      ).
      Table 3Primers used for qRT-PCR determinations
      GeneStrandSequenceGenBankTM accession no.
      BglapForward5′-GACTCCGGCGCTACCTTGGGTAAG-3′NM_001037939
      Reverse5′-CCCAGCACAACTCCTCCCTA-3′
      Hey1Forward5′-ATCTCAACAACTACGCATCCCAGC-3′NM_010423
      Reverse5′-GTGTGGGTGATGTCCGAAGG-3′
      Hey2Forward5′-AGCGAGAACAATTACCCTGGGCAC-3′NM_013904
      Reverse5′-GGTAGTTGTCGGTGAATTGGACCT-3′
      HeyLForward5′-CAGTAGCCTTTCTGAATTGCGAC-3′NM_013905
      Reverse5′-AGCTTGGAGGAGCCCTGTTTC-3′
      Notch1Forward5′-GTCCCACCCATGACCACTACCCAGTTC-3′NM_008714
      Reverse5′-GGGTGTTGTCCACAGGTGA-3′
      Notch2Forward5′-TGACGTTGATGAGTGTATCTCCAAGCC-3′NM_010928
      Reverse5′-GTAGCTGCCCTGAGTGTTGTGG-3′
      Notch3Forward5′-CCGATTCTCCTGTCGTTGTCTCC-3′NM_008716
      Reverse5′-TGAACACAGGGCCTGCTGAC-3′
      Notch4Forward5′-CCAGCAGACAGACTACGGTGGAC-3′NM_010929
      Reverse5′-GCAGCCAGCATCAAAGGTGT-3′
      Rpl38Forward5′-AGAACAAGGATAATGTGAAGTTCAAGGTTC-3′NM_001048057; NM_001048058; NM_023372
      Reverse5′-CTGCTTCAGCTTCTCTGCCTTT-3′
      Tnfsf11Forward5′-TATAGAATCCTGAGACTCCATGAAAAC-3′NM_011613
      Reverse5′-CCCTGAAAGGCTTGTTTCATCC-3′
      To measure levels of the Notch26955C→T mutant transcript, total RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase in accordance with the manufacturer’s instructions (Life Technologies, Inc.) in the presence of reverse primers for Notch2 and of reverse primers for ribosomal protein L38 (Rpl38) (Table 3). Notch2 cDNA was amplified by PCR in the presence of specific primers, a 6-carboxyfluorescein-labeled DNA probe of sequence 5′-CATTGCCTAGGCAGC-3′ covalently bound to a 3′-minor groove binder quencher (Life Technologies, Inc.), and SsoAdvanced Universal Probes Supermix (Bio-Rad) at 60 °C for 45 cycles (
      • Canalis E.
      • Schilling L.
      • Yee S.P.
      • Lee S.K.
      • Zanotti S.
      Hajdu-Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
      ,
      • 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 it was cloned into pcDNA3.1(−) (Life Technologies, Inc.) by isothermal single-reaction assembly using commercially-available reagents (New England Biolabs, Ipswich, MA) (
      • 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 a CFX96 qRT-PCR detection system (Bio-Rad), and fluorescence was monitored during every PCR cycle at the annealing step. Data are expressed as copy number corrected for Rpl38 copy number, as estimated by comparison with a serial dilution of Rpl38 cDNA (from ATCC) (
      • Kouadjo K.E.
      • Nishida Y.
      • Cadrin-Girard J.F.
      • Yoshioka M.
      • St-Amand J.
      Housekeeping and tissue-specific genes in mouse tissues.
      ). In selected experiments, control data were normalized to one following correction for Rpl38 expression.

      Immunoblotting

      Pre-osteoclasts or osteoblasts from control or Notch2tm1.1Ecan 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 phenylmethylsulfonyl fluoride, and protease inhibitor mixture (all from Sigma). Quantified total cell lysates (35 μg of total protein) were separated by SDS-PAGE in 8% polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Billerica, MA). The blots were probed with anti-NOTCH2 (C651.6DbHN) antibodies (Developmental Studies Hybridoma Bank (DSHB C651.6DbHN, University of Iowa, Iowa City)) and β-actin (3700) antibodies (Cell Signaling Technology, Danvers, MA) and exposed to anti-rabbit IgG and anti-rat IgG conjugated to horseradish peroxidase (Sigma) and incubated with a chemiluminescence detection reagent (Bio-Rad). Chemiluminescence was detected by ChemiDocTM XSR+ molecular imager (Bio-Rad) with Image LabTM software (version 5.2.1), and the amount of protein in individual bands was quantified (
      • Yu J.
      • Canalis E.
      The Hajdu-Cheney mutation sensitizes mice to the osteolytic actions of tumor necrosis factor α.
      ).

      Serum carboxyl-terminal collagen cross-links assay

      Serum samples from control and experimental mice were obtained after an overnight fast. CTX levels were measured using an ELISA kit according to manufacturer's instructions (Immunodiagnostic Systems, Gaithersburg, MD).

      Statistics

      Data are expressed as means ± S.D. Statistical differences were determined by analysis of variance with Holm-Sidak’s post hoc analysis for pairwise or multiple comparisons.

      Author contributions

      E. C. conceptualization; E. C. and J. Y. formal analysis; E. C. supervision; E. C. funding acquisition; E. C. writing-original draft; E. C., T. R. G., M. C., L. S., and J. Y. writing-review and editing; T. R. G. and M. C. resources; L. S. and J. Y. investigation.

      Acknowledgments

      We thank D. Srivastava for HeyL cDNA; D. Fremont for M-CSF cDNA; M. Glogauer for RANKL cDNA; T. Iso for Hey1 and Hey2 cDNA; J. Lian for Bglap cDNA; J. S. Nye for Notch1 cDNA; Y. Shirayoshi for Notch4 cDNA; T. Eller for technical assistance; and Mary Yurczak for secretarial support.

      References

        • Siebel C.
        • Lendahl U.
        Notch signaling in development, tissue homeostasis, and disease.
        Physiol. Rev. 2017; 97 (28794168): 1235-1294
        • Zanotti S.
        • Canalis E.
        Notch signaling and the skeleton.
        Endocr. Rev. 2016; 37 (27074349): 223-253
        • Canalis E.
        Notch in skeletal physiology and disease.
        Osteoporos. Int. 2018; 29 (30194467): 2611-2621
        • Zanotti S.
        • Canalis E.
        Parathyroid hormone inhibits Notch signaling in osteoblasts and osteocytes.
        Bone. 2017; 103 (28676438): 159-167
        • Zanotti S.
        • Canalis E.
        Notch and the skeleton.
        Mol. Cell. Biol. 2010; 30 (19995916): 886-896
        • Schroeter E.H.
        • Kisslinger J.A.
        • Kopan R.
        Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain.
        Nature. 1998; 393 (9620803): 382-386
        • Kovall R.A.
        More complicated than it looks: assembly of Notch pathway transcription complexes.
        Oncogene. 2008; 27 (18758478): 5099-5109
        • Nam Y.
        • Sliz P.
        • Song L.
        • Aster J.C.
        • Blacklow S.C.
        Structural basis for cooperativity in recruitment of MAML coactivators to Notch transcription complexes.
        Cell. 2006; 124 (16530044): 973-983
        • Wilson J.J.
        • Kovall R.A.
        Crystal structure of the CSL–Notch–mastermind ternary complex bound to DNA.
        Cell. 2006; 124 (16530045): 985-996
        • Canalis E.
        • Schilling L.
        • Yee S.P.
        • Lee S.K.
        • Zanotti S.
        Hajdu-Cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis and bone resorption.
        J. Biol. Chem. 2016; 291 (26627824): 1538-1551
        • 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.
        J. Biol. Chem. 2008; 283 (18156632): 6509-6518
        • Fukushima H.
        • Nakao A.
        • Okamoto F.
        • Shin M.
        • Kajiya H.
        • Sakano S.
        • Bigas A.
        • Jimi E.
        • Okabe K.
        The association of Notch2 and NF-κB accelerates RANKL-induced osteoclastogenesis.
        Mol. Cell. Biol. 2008; 28 (18710934): 6402-6412
        • Canalis E.
        • Yu J.
        • Schilling L.
        • Yee S.P.
        • Zanotti S.
        The lateral meningocele syndrome mutation causes marked osteopenia in mice.
        J. Biol. Chem. 2018; 293 (30042232): 14165-14177
        • Canalis E.
        • Parker K.
        • Feng J.Q.
        • Zanotti S.
        Osteoblast lineage-specific effects of Notch activation in the skeleton.
        Endocrinology. 2013; 154 (23275471): 623-634
        • Yu J.
        • Canalis E.
        The Hajdu-Cheney mutation sensitizes mice to the osteolytic actions of tumor necrosis factor α.
        J. Biol. Chem. 2019; 294 (31371452): 14203-14214
        • Cheney W.D.
        Acro-osteolysis.
        Am. J. Roentgenol. Radium. Ther. Nucl. Med. 1965; 94 (14303950): 595-607
        • Hajdu N.
        • Kauntze R.
        Cranio-skeletal dysplasia.
        Br. J. Radiol. 1948; 21 (18918373): 42-48
        • Canalis E.
        Clinical and experimental aspects of notch receptor signaling: Hajdu-Cheney syndrome and related disorders.
        Metabolism. 2018; 80 (28941602): 48-56
        • Gray M.J.
        • Kim C.A.
        • Bertola D.R.
        • Arantes P.R.
        • Stewart H.
        • Simpson M.A.
        • Irving M.D.
        • Robertson S.P.
        Serpentine fibula polycystic kidney syndrome is part of the phenotypic spectrum of Hajdu-Cheney syndrome.
        Eur. J. Hum. Genet. 2012; 20 (21712856): 122-124
        • Isidor B.
        • Lindenbaum P.
        • Pichon O.
        • Bézieau S.
        • Dina C.
        • Jacquemont S.
        • Martin-Coignard D.
        • Thauvin-Robinet C.
        • Le Merrer M.
        • Mandel J.L.
        • David A.
        • Faivre L.
        • Cormier-Daire V.
        • Redon R.
        • Le Caignec C.
        Truncating mutations in the last exon of NOTCH2 cause a rare skeletal disorder with osteoporosis.
        Nat. Genet. 2011; 43 (21378989): 306-308
        • 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 Esch H.
        • FORGE Canada Consortium
        • Michaud J.L.
        • Samuels M.E.
        Mutations in NOTCH2 in families with Hajdu-Cheney syndrome.
        Hum. Mutat. 2011; 32 (21681853): 1114-1117
        • 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.
        Nat. Genet. 2011; 43 (21378985): 303-305
        • 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.
        Osteoporos. Int. 2013; 24 (23389697): 2275-2281
        • Udell J.
        • Schumacher Jr, H.R.
        • Kaplan F.
        • Fallon M.D.
        Idiopathic familial acro-osteolysis: histomorphometric study of bone and literature review of the Hajdu-Cheney syndrome.
        Arthritis Rheum. 1986; 29 (3527178): 1032-1038
        • Blumenauer B.T.
        • Cranney A.B.
        • Goldstein R.
        Acro-osteolysis and osteoporosis as manifestations of the Hajdu-Cheney syndrome.
        Clin. Exp. Rheumatol. 2002; 20 (12175120): 574-575
        • 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.
        • Högler W.
        Bone structural characteristics and response to bisphosphonate treatment in children with Hajdu-Cheney syndrome.
        J. Clin. Endocrinol. Metab. 2017; 102 (28938420): 4163-4172
        • Adami G.
        • Rossini M.
        • Gatti D.
        • Orsolini G.
        • Idolazzi L.
        • Viapiana O.
        • Scarpa A.
        • Canalis E.
        Hajdu-Cheney syndrome; report of a novel NOTCH2 mutation and treatment with denosumab.
        Bone. 2016; 92 (27592446): 150-156
        • Ryeom S.W.
        The cautionary tale of side effects of chronic Notch1 inhibition.
        J. Clin. Invest. 2011; 121 (21266769): 508-509
        • De Strooper B.
        • Annaert W.
        • Cupers P.
        • Saftig P.
        • Craessaerts K.
        • Mumm J.S.
        • Schroeter E.H.
        • Schrijvers V.
        • Wolfe M.S.
        • Ray W.J.
        • Goate A.
        • Kopan R.
        A presenilin-1-dependent γ-secretase-like protease mediates release of Notch intracellular domain.
        Nature. 1999; 398 (10206645): 518-522
        • Duggan S.P.
        • McCarthy J.V.
        Beyond γ-secretase activity: the multifunctional nature of presenilins in cell signalling pathways.
        Cell. Signal. 2016; 28 (26498858): 1-11
        • Ilagan M.X.
        • Kopan R.
        Selective blockade of transport via SERCA inhibition: the answer for oncogenic forms of Notch?.
        Cancer Cell. 2013; 23 (23518343): 267-269
        • Moellering R.E.
        • Cornejo M.
        • Davis T.N.
        • Del Bianco C.
        • Aster J.C.
        • Blacklow S.C.
        • Kung A.L.
        • Gilliland D.G.
        • Verdine G.L.
        • Bradner J.E.
        Direct inhibition of the NOTCH transcription factor complex.
        Nature. 2009; 462 (19907488): 182-188
        • Wu Y.
        • Cain-Hom C.
        • Choy L.
        • Hagenbeek T.J.
        • de Leon G.P.
        • Chen Y.
        • Finkle D.
        • Venook R.
        • Wu X.
        • Ridgway J.
        • Schahin-Reed D.
        • Dow G.J.
        • Shelton A.
        • Stawicki S.
        • Watts R.J.
        • et al.
        Therapeutic antibody targeting of individual Notch receptors.
        Nature. 2010; 464 (20393564): 1052-1057
        • Canalis E.
        • Sanjay A.
        • Yu J.
        • Zanotti S.
        An antibody to Notch2 reverses the osteopenic phenotype of Hajdu-Cheney mutant male mice.
        Endocrinology. 2017; 158 (28323963): 730-742
        • Yu J.
        • Siebel C.W.
        • Schilling L.
        • Canalis E.
        An antibody to Notch3 reverses the skeletal phenotype of lateral meningocele syndrome in male mice.
        J. Cell. Physiol. 2020; 235 (31188489): 210-220
        • Bennett C.F.
        • Baker B.F.
        • Pham N.
        • Swayze E.
        • Geary R.S.
        Pharmacology of antisense drugs.
        Annu. Rev. Pharmacol. Toxicol. 2017; 57 (27732800): 81-105
        • Cerritelli S.M.
        • Crouch R.J.
        Ribonuclease H: the enzymes in eukaryotes.
        FEBS J. 2009; 276 (19228196): 1494-1505
        • Murray S.F.
        • Jazayeri A.
        • Matthes M.T.
        • Yasumura D.
        • Yang H.
        • Peralta R.
        • Watt A.
        • Freier S.
        • Hung G.
        • Adamson P.S.
        • Guo S.
        • Monia B.P.
        • LaVail M.M.
        • McCaleb M.L.
        Allele-specific inhibition of rhodopsin with an antisense oligonucleotide slows photoreceptor cell degeneration.
        Invest. Ophthalmol. Vis. Sci. 2015; 56 (26436889): 6362-6375
        • Shy M.E.
        Antisense oligonucleotides offer hope to patients with Charcot-Marie-Tooth disease type 1A.
        J. Clin. Invest. 2018; 128 (29199996): 110-112
        • Carroll J.B.
        • Warby S.C.
        • Southwell A.L.
        • Doty C.N.
        • Greenlee S.
        • Skotte N.
        • Hung G.
        • Bennett C.F.
        • Freier S.M.
        • Hayden M.R.
        Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene/allele-specific silencing of mutant huntingtin.
        Mol. Ther. 2011; 19 (21971427): 2178-2185
        • Limmroth V.
        • Barkhof F.
        • Desem N.
        • Diamond M.P.
        • Tachas G.
        • ATL1102 Study Group
        CD49d antisense drug ATL1102 reduces disease activity in patients with relapsing-remitting MS.
        Neurology. 2014; 83 (25239835): 1780-1788
        • McCampbell A.
        • Cole T.
        • Wegener A.J.
        • Tomassy G.S.
        • Setnicka A.
        • Farley B.J.
        • Schoch K.M.
        • Hoye M.L.
        • Shabsovich M.
        • Sun L.
        • Luo Y.
        • Zhang M.
        • Comfort N.
        • Wang B.
        • Amacker J.
        • et al.
        Antisense oligonucleotides extend survival and reverse decrement in muscle response in ALS models.
        J. Clin. Invest. 2018; 128 (30010620): 3558-3567
        • Zhao H.T.
        • Damle S.
        • Ikeda-Lee K.
        • Kuntz S.
        • Li J.
        • Mohan A.
        • Kim A.
        • Hung G.
        • Scheideler M.A.
        • Scherer S.S.
        • Svaren J.
        • Swayze E.E.
        • Kordasiewicz H.B.
        PMP22 antisense oligonucleotides reverse Charcot-Marie-Tooth disease type 1A features in rodent models.
        J. Clin. Invest. 2018; 128 (29202483): 359-368
        • Zhu C.
        • Kim K.
        • Wang X.
        • Bartolome A.
        • Salomao M.
        • Dongiovanni P.
        • Meroni M.
        • Graham M.J.
        • Yates K.P.
        • Diehl A.M.
        • Schwabe R.F.
        • Tabas I.
        • Valenti L.
        • Lavine J.E.
        • Pajvani U.B.
        Hepatocyte Notch activation induces liver fibrosis in nonalcoholic steatohepatitis.
        Sci. Transl. Med. 2018; 10 (30463916): eaat0344
        • Crooke S.T.
        • Witztum J.L.
        • Bennett C.F.
        • Baker B.F.
        RNA-targeted therapeutics.
        Cell Metab. 2018; 27 (29617640): 714-739
        • Wang F.S.
        • Wu R.W.
        • Ko J.Y.
        • Tai M.H.
        • Ke H.C.
        • Yeh D.W.
        • Wu S.L.
        • Chen M.W.
        Heat shock protein 60 protects skeletal tissue against glucocorticoid-induced bone mass loss by regulating osteoblast survival.
        Bone. 2011; 49 (21854881): 1080-1089
        • Wang F.S.
        • Ko J.Y.
        • Lin C.L.
        • Wu H.L.
        • Ke H.J.
        • Tai P.J.
        Knocking down dickkopf-1 alleviates estrogen deficiency induction of bone loss. A histomorphological study in ovariectomized rats.
        Bone. 2007; 40 (17055793): 485-492
        • 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.
        Am. J. Pathol. 2018; 188 (29037852): 149-159
        • Saito T.
        • Chiba S.
        • Ichikawa M.
        • Kunisato A.
        • Asai T.
        • Shimizu K.
        • Yamaguchi T.
        • Yamamoto G.
        • Seo S.
        • Kumano K.
        • Nakagami-Yamaguchi E.
        • Hamada Y.
        • Aizawa S.
        • Hirai H.
        Notch2 is preferentially expressed in mature B cells and indispensable for marginal zone B lineage development.
        Immunity. 2003; 18 (12753744): 675-685
        • Sparks E.E.
        • Huppert K.A.
        • Brown M.A.
        • Washington M.K.
        • Huppert S.S.
        Notch signaling regulates formation of the three-dimensional architecture of intrahepatic bile ducts in mice.
        Hepatology. 2010; 51 (20069650): 1391-1400
        • McCright B.
        • Gao X.
        • Shen L.
        • Lozier J.
        • Lan Y.
        • Maguire M.
        • Herzlinger D.
        • Weinmaster G.
        • Jiang R.
        • Gridley T.
        Defects in development of the kidney, heart and eye vasculature in mice homozygous for a hypomorphic Notch2 mutation.
        Development. 2001; 128 (11171333): 491-502
        • Liu Z.
        • Chen S.
        • Boyle S.
        • Zhu Y.
        • Zhang A.
        • Piwnica-Worms D.R.
        • Ilagan M.X.
        • Kopan R.
        The extracellular domain of Notch2 increases its cell-surface abundance and ligand responsiveness during kidney development.
        Dev. Cell. 2013; 25 (23806616): 585-598
        • Vollersen N.
        • Hermans-Borgmeyer I.
        • Cornils K.
        • Fehse B.
        • Rolvien T.
        • Triviai I.
        • Jeschke A.
        • Oheim R.
        • Amling M.
        • Schinke T.
        • Yorgan T.A.
        High bone turnover in mice carrying a pathogenic Notch2 mutation causing Hajdu-Cheney syndrome.
        J. Bone Miner. Res. 2018; 33 (28856714): 70-83
        • Yorgan T.
        • Vollersen N.
        • Riedel C.
        • Jeschke A.
        • Peters S.
        • Busse B.
        • Amling M.
        • Schinke T.
        Osteoblast-specific Notch2 inactivation causes increased trabecular bone mass at specific sites of the appendicular skeleton.
        Bone. 2016; 87 (27102824): 136-146
        • Zanotti S.
        • Smerdel-Ramoya A.
        • Stadmeyer L.
        • Durant D.
        • Radtke F.
        • Canalis E.
        Notch inhibits osteoblast differentiation and causes osteopenia.
        Endocrinology. 2008; 149 (18420737): 3890-3899
        • Deregowski V.
        • Gazzerro E.
        • Priest L.
        • Rydziel S.
        • Canalis E.
        Notch 1 overexpression inhibits osteoblastogenesis by suppressing Wnt/β-catenin but not bone morphogenetic protein signaling.
        J. Biol. Chem. 2006; 281 (16407293): 6203-6210
        • Zhang G.
        • Guo B.
        • Wu H.
        • Tang T.
        • Zhang B.T.
        • Zheng L.
        • He Y.
        • Yang Z.
        • Pan X.
        • Chow H.
        • To K.
        • Li Y.
        • Li D.
        • Wang X.
        • Wang Y.
        • et al.
        A delivery system targeting bone formation surfaces to facilitate RNAi-based anabolic therapy.
        Nat. Med. 2012; 18 (22286306): 307-314
        • Bouxsein M.L.
        • Boyd S.K.
        • Christiansen B.A.
        • Guldberg R.E.
        • Jepsen K.J.
        • Müller R.
        Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.
        J. Bone Miner. Res. 2010; 25 (20533309): 1468-1486
        • Glatt V.
        • Canalis E.
        • Stadmeyer L.
        • Bouxsein M.L.
        Age-related changes in trabecular architecture differ in female and male C57BL/6J mice.
        J. Bone Miner. Res. 2007; 22 (17488199): 1197-1207
        • 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.
        J. Bone Miner. Res. 2013; 28 (23197339): 2-17
        • Parfitt A.M.
        • Drezner M.K.
        • Glorieux F.H.
        • Kanis J.A.
        • Malluche H.
        • Meunier P.J.
        • Ott S.M.
        • Recker R.R.
        Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee.
        J. Bone Miner. Res. 1987; 2 (3455637): 595-610
        • Yesil P.
        • Michel M.
        • Chwalek K.
        • Pedack S.
        • Jany C.
        • Ludwig B.
        • Bornstein S.R.
        • Lammert E.
        A new collagenase blend increases the number of islets isolated from mouse pancreas.
        Islets. 2009; 1 (21099271): 185-190
        • McCarthy T.L.
        • Centrella M.
        • Canalis E.
        Further biochemical and molecular characterization of primary rat parietal bone cell cultures.
        J. Bone Miner. Res. 1988; 3 (3265577): 401-408
        • Halleux C.
        • Kramer I.
        • Allard C.
        • Kneissel M.
        Isolation of mouse osteocytes using cell fractionation for gene expression analysis.
        Methods Mol. Biol. 2012; 816 (22130922): 55-66
        • Canalis E.
        • Schilling L.
        • Zanotti S.
        Effects of sex and Notch signaling on the osteocyte cell pool.
        J. Cell. Physiol. 2017; 232 (27192486): 363-370
        • 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.
        Nat. Med. 2006; 12 (17128270): 1403-1409
        • Wang Y.
        • Lebowitz D.
        • Sun C.
        • Thang H.
        • Grynpas M.D.
        • Glogauer M.
        Identifying the relative contributions of Rac1 and Rac2 to osteoclastogenesis.
        J. Bone Miner. Res. 2008; 23 (17922611): 260-270
        • Nazarenko I.
        • Pires R.
        • Lowe B.
        • Obaidy M.
        • Rashtchian A.
        Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes.
        Nucleic Acids Res. 2002; 30 (11972350): 2089-2195
        • Nazarenko I.
        • Lowe B.
        • Darfler M.
        • Ikonomi P.
        • Schuster D.
        • Rashtchian A.
        Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore.
        Nucleic Acids Res. 2002; 30 (11972352): e37
        • Iso T.
        • Sartorelli V.
        • Chung G.
        • Shichinohe T.
        • Kedes L.
        • Hamamori Y.
        HERP, a new primary target of Notch regulated by ligand binding.
        Mol. Cell. Biol. 2001; 21 (11486044): 6071-6079
        • Nakagawa O.
        • Nakagawa M.
        • Richardson J.A.
        • Olson E.N.
        • Srivastava D.
        HRT1, HRT2, and HRT3: a new subclass of bHLH transcription factors marking specific cardiac, somitic, and pharyngeal arch segments.
        Dev. Biol. 1999; 216 (10588864): 72-84
        • Wong B.R.
        • Rho J.
        • Arron J.
        • Robinson E.
        • Orlinick J.
        • Chao M.
        • Kalachikov S.
        • Cayani E.
        • Bartlett 3rd, F.S
        • Frankel W.N.
        • Lee S.Y.
        • Choi Y.
        TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells.
        J. Biol. Chem. 1997; 272 (9312132): 25190-25194
        • Nye J.S.
        • Kopan R.
        • Axel R.
        An activated Notch suppresses neurogenesis and myogenesis but not gliogenesis in mammalian cells.
        Development. 1994; 120 (7956822): 2421-2430
        • Shirayoshi Y.
        • Yuasa Y.
        • Suzuki T.
        • Sugaya K.
        • Kawase E.
        • Ikemura T.
        • Nakatsuji N.
        Proto-oncogene of int-3, a mouse Notch homologue, is expressed in endothelial cells during early embryogenesis.
        Genes Cells. 1997; 2 (9189758): 213-224
        • Lian J.
        • Stewart C.
        • Puchacz E.
        • Mackowiak S.
        • Shalhoub V.
        • Collart D.
        • Zambetti G.
        • Stein G.
        Structure of the rat osteocalcin gene and regulation of vitamin D-dependent expression.
        Proc. Natl. Acad. Sci. U.S.A. 1989; 86 (2784002): 1143-1147
        • 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.
        Nat. Methods. 2009; 6 (19363495): 343-345
        • 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.
        Nucleic Acids Res. 2000; 28 (10606668): 655-661
        • Kouadjo K.E.
        • Nishida Y.
        • Cadrin-Girard J.F.
        • Yoshioka M.
        • St-Amand J.
        Housekeeping and tissue-specific genes in mouse tissues.
        BMC Genomics. 2007; 8 (17519037): 127