Deena Durant

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.

Platelet-derived Growth Factor Induces Interleukin-6 Transcription in Osteoblasts through the Activator Protein-1 Complex and Activating Transcription Factor-2

Platelet-derived growth factor (PDGF) BB, a mitogen that stimulates bone resorption, increases the expression of interleukin-6 (IL-6), a cytokine that induces osteoclast recruitment. The mechanisms involved in IL-6 induction by PDGF BB are poorly understood. We examined the effect of PDGF BB on IL-6 expression in cultures of osteoblasts from fetal rat calvariae (Ob cells). PDGF BB increased IL-6 mRNA and heterogeneous nuclear RNA levels, the rate of transcription, and the activity of base pairs (bp) −2906 to +20 IL-6 promoter fragments transiently transfected into Ob cells. Deletion analysis revealed two responsive regions, one containing an activator protein-1 (AP-1) site located between bp −276 and −257, and a second, less well defined, downstream of −257. Targeted mutations of a cyclic AMP-responsive element (CRE), and nuclear factor-IL-6 and nuclear factor-κB binding sites in a bp −257 to +20 IL-6 construct that was transfected into Ob cells, revealed that the CRE also contributed to IL-6 promoter induction by PDGF BB. Electrophoretic mobility shift assay revealed AP-1 and CRE nuclear protein complexes that were enhanced by PDGF BB. Supershift assays revealed binding of Jun and Fos to AP-1 and CRE sequences and binding of activating transcription factor-2 to CRE. In conclusion, PDGF BB induces IL-6 transcription in osteoblasts by regulating nuclear proteins of the AP-1 complex and activating transcription factor-2.

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.

Cortisol regulates the expression of Notch in osteoblasts

Glucocorticoids have important effects on osteoblastic replication, differentiation, and function, and the Notch family of receptors is considered to play a role in osteoblastic cell differentiation. We postulated that cortisol could regulate Notch and Notch ligand expression in osteoblastic cells, providing an additional mechanism by which glucocorticoids could regulate osteoblastic differentiation. We examined the expression and regulation of Notch1, 2, 3, and 4 and their ligands Jagged 1 and 2 and Delta 1 and 3 by cortisol in cultures of osteoblastic MC3T3‐E1 cells. Cortisol caused a time‐dependent increase in Notch1 and 2 mRNA levels in MC3T3 cells. Notch3 and 4 were not detected in the presence or absence of cortisol. MC3T3 cells expressed Delta 1 and Jagged 1 but not Jagged 2 or Delta 3 mRNAs, and cortisol did not have a substantial effect on the expression of any of these ligands. Cortisol increased the rate of Notch1 and 2 transcription and, in transcriptionally arrested cells, did not modify the decay of the transcripts, indicating a transcriptional level of control. In conclusion, cortisol stimulates Notch1 and 2 transcription in osteoblasts. Since Notch signaling appears to play a negative role in osteoblastic differentiation, its increased expression could be relevant to the actions of cortisol in bone. J. Cell. Biochem. 85: 252–258, 2002.

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.

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.

Platelet-derived growth factor induces collagenase 3 transcription in osteoblasts through the activator protein 1 complex.

Platelet‐derived growth factor (PDGF) BB is a mitogen that stimulates bone resorption and increases collagenase 3 transcription in osteoblasts, although the mechanisms involved are as yet unknown. We examined the effect of PDGF BB on collagenase 3 transcription in cultures of osteoblasts from fetal rat calvariae (Ob cells). PDGF BB increased the activity of collagenase 3 promoter fragments transiently transfected into Ob cells. Deletion analysis of the collagenase promoter revealed three regions that impaired the induction of collagenase 3 by PDGF BB. A construct spanning base pair −53 to +28 collagenase 3 sequences, in relation to the start site of transcription +1, was fully responsive to PDGF BB and was studied in detail. Targeted mutations of an AP‐1 site in this fragment decreased basal collagenase promoter activity and the responsiveness to PDGF BB, whereas mutations of Stat3 and Ets binding sites did not alter the response to PDGF. Electrophoretic mobility shift assay, using nuclear extracts from control and treated cells, revealed AP‐1 nuclear protein complexes that were enhanced in extracts from PDGF BB–treated Ob cells. Supershift assays revealed that antibodies to c‐Fos, Fos B, Fra‐2, c‐Jun, Jun B, and Jun D shifted the binding of nuclear extracts from cells treated with PDGF BB to AP‐1 sequences. In conclusion, PDGF BB induces collagenase 3 transcription in osteoblasts by regulating nuclear proteins interacting with AP‐1 sequences. J. Cell. Physiol. 184:326–333, 2000.

Misexpression of CCAAT/enhancer binding protein beta causes osteopenia

CCAAT/enhancer binding proteins (C/EBPs) are expressed by osteoblasts and adipocytes during differentiation. C/EBP beta is critical for adipogenesis; however, its role in osteoblastogenesis is unclear, and its function in the postnatal skeleton is not known. To study C/EBP beta in osteoblasts in vivo, we created transgenic mice expressing full length C/EBP beta under the control of a 3.8 kb fragment of the human osteocalcin promoter. Two transgenic lines were established in a friend leukemia virus strain B genetic background, and compared with wild type littermate controls. Both C/EBP beta transgenic lines exhibited osteopenia, with a 30% decrease in bone volume, due to a decrease in trabecular number. The number of osteoblasts and osteoclasts per bone perimeter was not changed. Bone marrow stromal cells from C/EBP beta transgenics showed reduced mineralization, and reduced alkaline phosphatase mRNA levels. Calvarial osteoblasts from C/EBP beta transgenics displayed reduced alkaline phosphatase activity. To determine the consequences of the Cebpb deletion in vivo, the phenotype of Cebpb null mice was compared with that of wild type controls of identical genetic composition. Cebpb null mice exhibited reduced weight, body fat, and bone mineral density, and decreased bone volume, due to a decrease in trabecular number. The number of osteoblasts and osteoclasts per bone perimeter was not changed. C/EBP beta downregulation by RNA interference in calvarial osteoblasts had no effect on osteoblast differentiation/function. The phenotype of the Cebpb inactivation may be secondary to systemic indirect effects, and to direct effects of C/EBP beta in osteoblasts. In conclusion, C/EBP beta plays a role in mesenchymal cell differentiation and its misexpression in vivo causes osteopenia.

Nephroblastoma Overexpressed (Nov) Inactivation Sensitizes Osteoblasts to Bone Morphogenetic Protein-2, But Nov Is Dispensable for Skeletal Homeostasis

Overexpression of nephroblastoma overexpressed (Nov), a member of the Cyr 61, connective tissue growth factor, Nov family of proteins, inhibits osteoblastogenesis and causes osteopenia. The consequences of Nov inactivation on osteoblastogenesis and the postnatal skeleton are not known. To study the function of Nov, we inactivated Nov by homologous recombination. Nov null mice were maintained in a C57BL/6 genetic background after the removal of the neomycin selection cassette and compared with wild-type controls of identical genetic composition. Nov null mice were identified by genotyping and absent Nov mRNA in calvarial extracts and osteoblast cultures. Nov null mice did not exhibit developmental skeletal abnormalities or postnatal changes in weight, femoral length, body fat, or bone mineral density and appeared normal. Bone volume and trabecular number were decreased only in 1-month-old female mice. In older mice, after 7 months of age, osteoblast surface and bone formation were increased in females, and osteoclast and eroded surfaces were increased in male Nov null mice. Calvarial osteoblasts from Nov null mice displayed enhanced alkaline phosphatase activity, alkaline phosphatase mRNA, and transactivation of a bone morphogenetic protein (BMP)/phosphorylated mothers against decapentaplegic reporter construct in response to BMP-2. Similar results were obtained after the down-regulation of Nov by RNA interference in ST-2 stromal and MC3T3 cells. Osteoclast number was increased in marrow stromal cell cultures from Nov null mice. Surface plasmon resonance demonstrated direct interactions between Nov and BMP-2. In conclusion, Nov sensitizes osteoblasts to BMP-2, but Nov is dispensable for the maintenance of bone mass.