Stefano Zanotti

Notch Inhibits Osteoblast Differentiation and Causes Osteopenia

Notch receptors are determinants of cell fate decisions. To define the role of Notch in the adult skeleton, we created transgenic mice overexpressing the Notch intracellular domain (NICD) under the control of the type I collagen promoter. First-generation transgenics were small and osteopenic. Bone histomorphometry revealed that NICD caused a decrease in bone volume, secondary to a reduction in trabecular number; osteoblast and osteoclast number were decreased. Low fertility of founder mice and lethality of young pups did not allow the complete establishment of transgenic lines. To characterize the effect of Notch overexpression in vitro, NICD was induced in osteoblasts and stromal cells from Rosa(notch) mice, in which a STOP cassette flanked by lox(P) sites is upstream of NICD, by transduction with an adenoviral vector expressing Cre recombinase (Cre) under the control of the cytomegalovirus (CMV) promoter (Ad-CMV-Cre). NICD impaired osteoblastogenesis and inhibited Wnt/beta-catenin signaling. To determine the effects of notch1 deletion in vivo, mice in which notch1 was flanked by lox(P) sequences (notch1(loxP/loxP)) were mated with mice expressing Cre recombinase under the control of the osteocalcin promoter. Conditional null notch1 mice had no obvious skeletal phenotype, possibly because of rescue by notch2; however, 1-month-old females exhibited a modest increase in osteoclast surface and eroded surface. Osteoblasts from notch1(loxP/loxP) mice, transduced with Ad-CMV-Cre and transfected with Notch2 small interfering RNA, displayed increased alkaline phosphatase activity. In conclusion, Notch signaling in osteoblasts causes osteopenia and impairs osteo-blastogenesis by inhibiting the Wnt/beta-catenin pathway.

Notch and the Skeleton

ABSTRACT Notch receptors are transmembrane receptors that regulate cell fate decisions. There are four Notch receptors in mammals. Upon binding to members of the Delta and Jagged family of transmembrane proteins, Notch is cleaved and the Notch intracellular domain (NICD) is released. NICD then translocates to the nucleus, where it associates with the CBF-1, Suppressor of Hairless, and Lag-2 (CSL) and Mastermind-Like (MAML) proteins. This complex activates the transcription of Notch target genes, such as Hairy Enhancer of Split (Hes) and Hes-related with YRPF motif (Hey). Notch signaling is critical for the regulation of mesenchymal stem cell differentiation. Misexpression of Notch in skeletal tissue indicates a role as an inhibitor of skeletal development and postnatal bone formation. Overexpression of Notch inhibits endochondral bone formation and osteoblastic differentiation, causing severe osteopenia. Conditional inactivation of Notch in the skeleton causes an increase in cancellous bone volume and enhanced osteoblastic differentiation. Notch ligands are expressed in the hematopoietic stem cell niche and are critical for the regulation of hematopoietic stem cell self-renewal. Dysregulation of Notch signaling is the underlying cause of diseases affecting the skeletal tissue, including Alagille syndrome, spondylocostal dysostosis, and possibly, osteosarcoma.

Conditional deletion of gremlin causes a transient increase in bone formation and bone mass

Gremlin is a glycoprotein that binds bone morphogenetic proteins (BMPs) 2, 4, and 7, antagonizing their actions. Gremlin opposes BMP effects on osteoblastic differentiation and function in vitro and in vivo, and its overexpression causes osteopenia. To define the function of gremlin in the skeleton, we generated gremlin 1 (grem1) conditional null mice by mating mice where grem1 was flanked by loxP sequences with mice expressing the Cre recombinase under the control of the osteocalcin promoter. grem1 null male mice displayed increased trabecular bone volume due to enhanced osteoblastic activity, because mineral apposition and bone formation rates were increased. Osteoblast number and bone resorption were not altered. Marrow stromal cells from grem1 conditional null mice expressed higher levels of alkaline phosphatase activity. Gremlin down-regulation by RNA interference in ST-2 stromal and MC3T3 osteoblastic cells increased the BMP-2 stimulatory effect on alkaline phosphatase activity, on Smad 1/5/8 phosphorylation, and on the transactivation of the BMP/Smad reporter construct 12×SBE-Oc-pGL3. Gremlin down-regulation also enhanced osteocalcin and Runx-2 expression, Wnt 3a signaling, and activity in ST-2 cells. In conclusion, deletion of grem1 in the bone microenvironment results in sensitization of BMP signaling and activity and enhanced bone formation in vivo.

Osteoblast lineage-specific effects of notch activation in the skeleton.

Transgenic overexpression of the Notch1 intracellular domain inhibits osteoblast differentiation and causes osteopenia, and inactivation of Notch1 and Notch2 increases bone volume transiently and induces osteoblastic differentiation. However, the biology of Notch is cell-context-dependent, and consequences of Notch activation in cells of the osteoblastic lineage at various stages of differentiation and in osteocytes have not been defined. For this purpose, Rosa(Notch) mice, where a loxP-flanked STOP cassette placed between the Rosa26 promoter and the NICD coding sequence, were crossed with transgenics expressing the Cre recombinase under the control of the Osterix (Osx), Osteocalcin (Oc), Collagen 1a1 (Col2.3), or Dentin matrix protein1 (Dmp1) promoters. At 1 month, Osx-Cre;Rosa(Notch) and Oc-Cre;Rosa(Notch) mice exhibited osteopenia due to impaired bone formation. In contrast, Col2.3-Cre;Rosa(Notch) and Dmp1-Cre;Rosa(Notch) exhibited increased femoral trabecular bone volume due to a decrease in osteoclast number and eroded surface. In the four lines studied, cortical bone was either not present, was porous, or had the appearance of trabecular bone. Oc-Cre;Rosa(Notch) and Col2.3-Cre;Rosa(Notch) mice exhibited early lethality so that their adult phenotype was not established. At 3 months, Osx-Cre;Rosa(Notch) and Dmp1-Cre;Rosa(Notch) mice displayed increased bone volume, and increased osteoblasts although calcein-demeclocycline labels were diffuse and fragmented, indicating abnormal bone formation. In conclusion, Notch effects in the skeleton are cell-context-dependent. When expressed in immature osteoblasts, Notch arrests their differentiation, causing osteopenia, and when expressed in osteocytes, it causes an initial suppression of bone resorption and increased bone volume, a phenotype that evolves as the mice mature.

Nephroblastoma overexpressed (NOV) inhibits osteoblastogenesis and causes osteopenia

Nephroblastoma overexpressed (Nov), a member of the Cyr 61, connective tissue growth factor, Nov (CCN) family of proteins, is expressed by osteoblasts, but its function in cells of the osteoblastic lineage is not known. We investigated the effects of Nov overexpression by transducing murine ST-2 stromal and MC3T3 osteoblastic cells with a retroviral vector where Nov is under the control of the cytomegalovirus promoter. We also examined the skeletal phenotype of transgenic mice expressing Nov under the control of the human osteocalcin promoter. Overexpression of Nov in ST-2 cells inhibited the appearance of mineralized nodules and decreased alkaline phosphatase activity and osteocalcin mRNA levels. Nov overexpression inhibited the effect of bone morphogenetic protein (BMP)-2 on the phosphorylation of Smad 1/5/8; on the transactivation of 12xSBE-Oc-pGL3, a BMP/Smad signaling reporter construct, and of Wnt 3 on cytoplasmic β-catenin levels; and on the transactivation of the Wnt/β-catenin signaling reporter construct 16xTCF-Luc. Nov overexpression did not activate Notch or transforming growth factor β signaling. Glutathione S-transferase pulldown assays demonstrated direct Nov-BMP interactions. Nov transgenic mice exhibited osteopenia. In conclusion, Nov binds BMP-2 and antagonizes BMP-2 and Wnt activity, and its overexpression inhibits osteoblastogenesis and causes osteopenia.

Notch Signaling in Osteocytes Differentially Regulates Cancellous and Cortical Bone Remodeling

Background: Notch signaling regulates skeletal development and remodeling. Results: Activation of Notch preferentially in osteocytes induces osteoprotegerin and Wnt signaling, decreases cancellous bone remodeling, and increases cortical bone formation; as a result, Notch prevents immobilization-induced osteopenia. Conclusion: In osteocytes, Notch decreases cancellous bone remodeling and enhances cortical bone formation. Significance: Notch has distinct actions in osteocytes leading to a marked increase in bone mass. Notch receptors play a role in skeletal development and homeostasis, and Notch activation in undifferentiated and mature osteoblasts causes osteopenia. In contrast, Notch activation in osteocytes increases bone mass, but the mechanisms involved and exact functions of Notch are not known. In this study, Notch1 and -2 were inactivated preferentially in osteocytes by mating Notch1/2 conditional mice, where Notch alleles are flanked by loxP sequences, with transgenics expressing Cre directed by the Dmp1 (dentin matrix protein 1) promoter. Notch1/2 conditional null male and female mice exhibited an increase in trabecular bone volume due to an increase in osteoblasts and decrease in osteoclasts. In male null mice, this was followed by an increase in osteoclast number and normalization of bone volume. To activate Notch preferentially in osteocytes, Dmp1-Cre transgenics were crossed with RosaNotch mice, where a loxP-flanked STOP cassette is placed between the Rosa26 promoter and Notch1 intracellular domain sequences. Dmp1-Cre+/−;RosaNotch mice exhibited an increase in trabecular bone volume due to decreased bone resorption and an increase in cortical bone due to increased bone formation. Biomechanical and chemical properties were not affected. Osteoprotegerin mRNA was increased, sclerostin and dickkopf1 mRNA were decreased, and Wnt signaling was enhanced in Dmp1-Cre+/−;RosaNotch femurs. Botulinum toxin A-induced muscle paralysis caused pronounced osteopenia in control mice, but bone mass was preserved in mice harboring the Notch activation in osteocytes. In conclusion, Notch plays a unique role in osteocytes, up-regulates osteoprotegerin and Wnt signaling, and differentially regulates trabecular and cortical bone homeostasis.

Notch Regulation of Bone Development and Remodeling and Related Skeletal Disorders

Notch signaling mediates cell-to-cell interactions that are critical for embryonic development and tissue renewal. In the canonical signaling pathway, the Notch receptor is cleaved following ligand binding, resulting in the release and nuclear translocation of the Notch intracellular domain (NICD). NICD induces gene expression by forming a ternary complex with the DNA binding protein CBF1/Rbp-Jk, Suppressor of Hairless, Lag1, and Mastermind-Like (Maml). Hairy Enhancer of Split (Hes) and Hes related with YRPW motif (Hey) are classic Notch targets. Notch canonical signaling plays a central role in skeletal development and bone remodeling by suppressing the differentiation of skeletal cells. The skeletal phenotype of mice misexpressing Hes1 phenocopies partially the effects of Notch misexpression, suggesting that Hey proteins mediate most of the skeletal effects of Notch. Dysregulation of Notch signaling is associated with diseases affecting human skeletal development, such as Alagille syndrome, brachydactyly and spondylocostal dysostosis. Somatic mutations in Notch receptors and ligands are found in tumors of the skeletal system. Overexpression of NOTCH1 is associated with osteosarcoma, and overexpression of NOTCH3 or JAGGED1 in breast cancer cells favors the formation of osteolytic bone metastasis. Activating mutations in NOTCH2 cause Hajdu-Cheney syndrome, which is characterized by skeletal defects and fractures, and JAG1 polymorphisms, are associated with variations in bone mineral density. In conclusion, Notch is a regulator of skeletal development and bone remodeling, and abnormal Notch signaling is associated with developmental and postnatal skeletal disorders.

Connective Tissue Growth Factor Enhances Osteoblastogenesis in Vitro

Connective tissue growth factor (CTGF), a member of the CCN family of proteins, is expressed by osteoblasts, but its function in cells of the osteoblastic lineage has not been established. We investigated the effects of CTGF overexpression by transducing murine ST-2 stromal cells with a retroviral vector, where CTGF is under the control of the cytomegalovirus promoter. Overexpression of CTGF in ST-2 cells increased alkaline phosphatase activity, osteocalcin and alkaline phosphatase mRNA levels, and mineralized nodule formation. CTGF overexpression decreased the effect of bone morphogenetic protein-2 on Smad 1/5/8 phosphorylation and of Wnt 3 on cytosolic β-catenin, indicating that the stimulatory effect on osteoblastogenesis was unrelated to BMP and Wnt signaling. CTGF overexpression suppressed Notch signaling and induced the transcription of hairy and E (spl)-1 (HES)-1, by Notch-independent mechanisms. CTGF induced nuclear factor of activated T cells (NFAT) transactivation by a calcineurin-dependent mechanism. Down-regulation of CTGF enhanced Notch signaling and decreased HES-1 transcription and NFAT transactivation. Similar effects were observed following forced CTGF overexpression, the addition of CTGF protein, or the transduction of ST-2 cells with a retroviral vector expressing HES-1. In conclusion, CTGF enhances osteoblastogenesis, possibly by inhibiting Notch signaling and inducing HES-1 transcription and NFAT transactivation.

Skeletal Overexpression of Connective Tissue Growth Factor Impairs Bone Formation and Causes Osteopenia

Connective tissue growth factor (CTGF), a member of the CCN family of proteins, is expressed in skeletal cells, and the ctgf null mutation leads to neonatal lethality due to defects in skeletal development. To define the function of CTGF in the postnatal skeleton, we created transgenic mice overexpressing CTGF under the control of the human osteocalcin promoter. CTGF transgenic female and male mice exhibited a significant decrease in bone mineral density, compared with wild-type littermate controls. Bone histomorphometry revealed that CTGF overexpression caused decreased trabecular bone volume due to impaired osteoblastic activity because mineral apposition and bone formation rates were decreased. Osteoblast and osteoclast number and bone resorption were not altered. Calvarial osteoblasts and stromal cells from CTGF transgenics displayed decreased alkaline phosphatase and osteocalcin mRNA levels and reduced bone morphogenetic protein (BMP) signaling mothers against decapentaplegic, Wnt/beta-catenin, and IGF-I/Akt signaling. In conclusion, CTGF overexpression in vivo causes osteopenia, secondary to decreased bone formation, possibly by antagonizing BMP, Wnt, and IGF-I signaling and activity.

Mechanisms in Endocrinology: Notch signaling in skeletal health and disease

Notch receptors are single-pass transmembrane proteins that determine cell fate. Upon Notch ligand interactions, proteolytic cleavages release the Notch intracellular domain, which translocates to the nucleus to regulate the transcription of target genes, including Hairy enhancer of split (Hes) and Hes related to YRPW motif (Hey). Notch is critical for skeletal development and activity of skeletal cells, and dysregulation of Notch signaling is associated with human diseases affecting the skeleton. Inherited or sporadic mutations in components of the Notch signaling pathway are associated with spondylocostal dysostosis, spondylothoracic dysostosis and recessive brachydactyly, diseases characterized by skeletal patterning defects. Inactivating mutations of the Notch ligand JAG1 or of NOTCH2 are associated with Alagille syndrome, and activating mutations in NOTCH2 are associated with Hajdu-Cheney syndrome (HCS). Individuals affected by HCS exhibit osteolysis in distal phalanges and osteoporosis. NOTCH is activated in selected tumors, such as osteosarcoma, and in breast cancer cells that form osteolytic bone metastases. In conclusion, Notch regulates skeletal development and bone remodeling, and gain- or loss-of-function mutations of Notch signaling result in important skeletal diseases.