Skeletal overexpression of gremlin impairs bone formation and causes osteopenia

Skeletal cells synthesize bone morphogenetic proteins (BMPs) and BMP antagonists. Gremlin, a BMP antagonist, is expressed in osteoblasts and opposes BMP effects on osteoblastic differentiation and function in vitro. However, its effects in vivo are not known. To investigate the actions of gremlin on bone remodeling in vivo, we generated transgenic mice overexpressing gremlin under the control of the osteocalcin promoter. Gremlin transgenics exhibited bone fractures and reduced bone mineral density by 20-30%, compared with controls. Static and dynamic histomorphometry of femurs revealed that gremlin overexpression caused reduced trabecular bone volume and the appearance of woven bone. Polarized light microscopy revealed disorganized collagen bundles at the endosteal cortical surface. Gremlin transgenic mice displayed a 70% decrease in the number of osteoblasts/trabecular area and reduced mineral apposition and bone formation rates. In vivo bromodeoxyuridine labeling and marrow stromal cell cultures demonstrated an inhibitory effect of gremlin on osteoblastic cell replication, but no change on apoptosis was detected. Marrow stromal cells from gremlin transgenics displayed a reduced response to BMP on phosphorylated mothers against decapentaplegic 1/5/8 phosphorylation and reduced free cytosolic beta-catenin levels. In conclusion, transgenic mice overexpressing gremlin in the bone microenvironment have decreased osteoblast number and function leading to osteopenia and spontaneous fractures.     

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.     

Conditional inactivation of noggin in the postnatal skeleton causes osteopenia

Noggin is an antagonist of bone morphogenetic proteins (BMP), and its overexpression causes suppressed osteoblastogenesis and osteopenia. Global inactivation of Noggin results in severe developmental defects and prenatal lethality, but the consequences of the conditional inactivation of Noggin on the postnatal skeleton are not known. To study the function of noggin in osteoblasts, we generated tissue-specific null Noggin mice by mating Noggin conditional mice, where the Noggin allele is flanked by loxP sequences, with mice expressing the Cre recombinase under the control of the osteocalcin promoter (Oc-Cre). Noggin conditional null mice exhibited decreased weight, shortened femoral length, and generalized osteopenia. Bone histomorphometric and microarchitectural analyses of distal femurs revealed decreased bone volume due to a reduced number of trabeculae in 1- and 3-month-old Noggin conditional null mice. Vertebral microarchitecture confirmed the osteopenia observed in Noggin conditional null mice. Osteoclast number was increased in 1-month-old male Noggin conditional null mice, and bone formation was increased in 3-month-old mice, but female mice did not exhibit increased bone remodeling. In conclusion, Noggin inactivation causes osteopenia, suggesting that BMP in excess have a detrimental effect on bone or that noggin has a BMP-independent role in skeletal homeostasis.     

A soluble bone morphogenetic protein type IA receptor increases bone mass and bone strength

Diseases such as osteoporosis are associated with reduced bone mass. Therapies to prevent bone loss exist, but there are few that stimulate bone formation and restore bone mass. Bone morphogenetic proteins (BMPs) are members of the TGFβ superfamily, which act as pleiotropic regulators of skeletal organogenesis and bone homeostasis. Ablation of the BMPR1A receptor in osteoblasts increases bone mass, suggesting that inhibition of BMPR1A signaling may have therapeutic benefit. The aim of this study was to determine the skeletal effects of systemic administration of a soluble BMPR1A fusion protein (mBMPR1A-mFc) in vivo. mBMPR1A-mFc was shown to bind BMP2/4 specifically and with high affinity and prevent downstream signaling. mBMPR1A-mFc treatment of immature and mature mice increased bone mineral density, cortical thickness, trabecular bone volume, thickness and number, and decreased trabecular separation. The increase in bone mass was due to an early increase in osteoblast number and bone formation rate, mediated by a suppression of Dickkopf-1 expression. This was followed by a decrease in osteoclast number and eroded surface, which was associated with a decrease in receptor activator of NF-κB ligand (RANKL) production, an increase in osteoprotegerin expression, and a decrease in serum tartrate-resistant acid phosphatase (TRAP5b) concentration. mBMPR1A treatment also increased bone mass and strength in mice with bone loss due to estrogen deficiency. In conclusion, mBMPR1A-mFc stimulates osteoblastic bone formation and decreases bone resorption, which leads to an increase in bone mass, and offers a promising unique alternative for the treatment of bone-related disorders.     

Transgenic mice expressing selected insulin-like growth factor-binding protein-5 fragments do not exhibit enhanced bone formation.

Skeletal cells synthesize insulin like growth factors (IGF) and six IGF binding proteins (IGFBP). IGFBP-5 and fragments were reported to stimulate bone cell growth and parameters of osteoblastic function. We investigated the effects of IGFBP-5 1-162 and 1-193 on bone remodeling in transgenic mice overexpressing these fragments under the control of the osteocalcin promoter. Transgenic mice had normal appearance, weight, and bone mineral density. Static and dynamic histomorphometry revealed that transgenic mice overexpressing IGFBP-5 1-162 or 1-193 had normal trabecular bone volume, osteoblast and osteoclast number, and normal bone formation rate. MC3T3 cells transduced with retroviral vectors overexpressing IGFBP-5 1-235, 1-193, and 1-162 fragments displayed normal cell growth and maturation, and failed to enhance the expression of alkaline phosphatase, osteocalcin, and type I collagen mRNA when compared to cells transduced with vector alone. In conclusion, transgenic mice expressing IGFBP-5 1-162 and 1-193 in the bone microenvironment do not exhibit an obvious skeletal phenotype.     

Role of BMPs and Smads during endochondral bone formation

Biochemical research has revealed that BMP signals are mainly mediated through ligand binding to the receptors followed by activation of Smad proteins. BMP signaling is subjected to regulation at extracellular level by noggin and at intracellular level by inhibitory-Smads. Analysis of transgenic mice in which BMP signals are activated or inactivated has been clarifying a role of BMP signaling during endochondral bone formation. BMPs promote chondrocyte proliferation and hypertrophy, whereas Smad signaling regulates chondrocyte hypertrophy. Proper regulation of these signals is necessary for normal bone formation.     

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.     

Noggin haploinsufficiency differentially affects tissue responses in destructive and remodeling arthritis

OBJECTIVE: The balance between destruction and homeostatic or reparative responses determines the outcome of arthritis. Increasing evidence suggests a role for signaling pathways, essential for development and growth, in the maintenance of tissue homeostasis and attempts at repair. Inappropriate activation of such pathways may also have a role in disease progression. We undertook this study to determine the effect of shifting the balance in bone morphogenetic protein (BMP) signaling in different mouse models of arthritis. METHODS: Endogenous levels of noggin, a BMP antagonist, were reduced using heterozygous noggin(+/LacZ) mice in a model of inflammation-driven destruction (methylated bovine serum albumin [mBSA]-induced monarthritis), a model of systemic autoimmune arthritis (collagen-induced arthritis [CIA]), and a model of joint ankylosis (spontaneous arthritis in DBA/1 mice). In addition, we studied BMP inactivation by adenoviral noggin overexpression in destructive arthritis. Cartilage damage and activation of BMP signaling were studied by digital image analysis using Safranin O sulfated glycosaminoglycan staining and immunohistochemistry for phosphorylated Smads (Smads 1, 5, and 8), respectively. RESULTS: Noggin haploinsufficiency provided protection for articular cartilage against destruction in mBSA-induced arthritis. Antagonist overexpression rendered cartilage more vulnerable in this model. Noggin gene transfer in knees affected by CIA also enhanced cartilage damage. Haploinsufficiency did not affect CIA, but noggin(+/LacZ) mice had an increased number of CD4-positive cells with normal immune responses. In noggin(+/LacZ) DBA/1 mice with spontaneous arthritis, we observed delayed progression from cartilage to bone formation. CONCLUSION: Tight spatiotemporal control of BMP signaling appears to be critical in the response of joint tissues in models of arthritis.     

Osteoblastic expansion induced by parathyroid hormone receptor signaling in murine osteocytes is not sufficient to increase hematopoietic stem cells

Microenvironmental expansion of hematopoietic stem cells (HSCs) is induced by treatment with parathyroid hormone (PTH) or activation of the PTH receptor (PTH1R) in osteoblastic cells; however, the osteoblastic subset mediating this action of PTH is unknown. Osteocytes are terminally differentiated osteoblasts embedded in mineralized bone matrix but are connected with the BM. Activation of PTH1R in osteocytes increases osteoblastic number and bone mass. To establish whether osteocyte-mediated PTH1R signaling expands HSCs, we studied mice expressing a constitutively active PTH1R in osteocytes (TG mice). Osteoblasts, osteoclasts, and trabecular bone were increased in TG mice without changes in BM phenotypic HSCs or HSC function. TG mice had progressively increased trabecular bone but decreased HSC function. In severely affected TG mice, phenotypic HSCs were decreased in the BM but increased in the spleen. TG osteocytes had no increase in signals associated with microenvironmental HSC support, and the spindle-shaped osteoblastic cells that increased with PTH treatment were not present in TG bones. These findings demonstrate that activation of PTH1R signaling in osteocytes does not expand BM HSCs, which are instead decreased in TG mice. Therefore, osteocytes do not mediate the HSC expansion induced by PTH1R signaling. Further, osteoblastic expansion is not sufficient to increase HSCs.     

Extracellular matrix-associated bone morphogenetic proteins are essential for differentiation of murine osteoblastic cells in vitro

Osteoblastic differentiation is an essential part of bone formation that compensates resorbed bone matrix to maintain its structural integrity. Cells in an osteoblast lineage develop differentiated phenotypes during a long-term culture in vitro. However, intrinsic mechanisms whereby these cells differentiate into mature osteoblasts are yet unclear. Bone morphogenetic proteins (BMPs) stimulate osteoblastic differentiation and bone formation. We demonstrate that mouse osteoblastic MC3T3-E 1 cells constitutively expressed messenger RNAs (mRNAs) for BMP-2 and BMP-4 and accumulated BMPs in collagen-rich extracellular matrices. BMPs associated with the extracellular matrices were involved in the induction of osteoblastic differentiation of nonosteogenic mesenchymal cells as well as cells in the osteoblast lineage. MC3T3-E1 cells constitutively expressed type IA and type II BMP receptors. When a kinase-deficient type IA BMP receptor was stably transfected to MC3T3-E 1 cells to obliterate BMP-2/4 signaling, these cells not only failed to respond to exogenous BMP-2 but lost their capability of differentiation into osteoblasts that form mineralized nodules. These observations strongly suggest that endogenous BMP-2/4 accumulated in extracellular matrices are essential for the osteoblastic differentiation of cells in the osteoblast lineage. Therefore, the regulatory mechanism of BMP-2/4 actions in osteoblastic cells is a principal issue to be elucidated for better understanding of pathogenesis of bone losing diseases such as osteoporosis.     

Bone morphogenetic protein 2 is expressed by, and acts upon, mature epithelial cells in the colon

BACKGROUND AND AIMS: The recent findings of bone morphogenetic protein (BMP) receptor Ia mutations in juvenile polyposis and frequent Smad4 mutations in colon cancer suggest a role for BMPs in the colonic epithelium and colon cancer. We investigated the role of BMP2 in the colon. METHODS: We assessed BMP receptor expression in cell lines using the reverse-transcribed polymerase chain reaction and immunoblotting. We investigated the effect of BMP2 on cell lines using the MTT assay and by immunoblotting for markers of differentiation, proliferation, and apoptosis. We assessed the expression of BMP2, its receptors, and signal transduction elements in mouse and human colon tissue using immunohistochemistry. We also investigated the effect of the BMP antagonist noggin in vivo in mice by assessing colon tissue with immunohistochemistry and immunoblotting. Finally, we investigated the expression of BMP2 in microadenomas from familial adenomatous polyposis patients. RESULTS: BMP receptors (BMPR) Ia, BMPR Ib, and BMPR II are all expressed in colonic epithelial cell lines. BMP2 inhibits colonic epithelial cell growth in vitro, promoting apoptosis and differentiation and inhibiting proliferation. BMP2, BMPRIa, BMPRIb, BMPRII, phosphorylated Smad1, and Smad4 are expressed predominantly in mature colonocytes at the epithelial surface in normal adult human and mouse colon. Noggin inhibits apoptosis and proliferation in mouse colonic epithelium in vivo. BMP2 expression is lost in the microadenomas of familial adenomatous polyposis patients. CONCLUSIONS: These data suggest that BMP2 acts as a tumor suppressor promoting apoptosis in mature colonic epithelial cells.     

Constitutive activity of the osteoblast Ca2+-sensing receptor promotes loss of cancellous bone

Changes in extracellular [Ca2+] modulate the function of bone cells in vitro via the extracellular Ca2+-sensing receptor (CaR). Within bone microenvironments, resorption increases extracellular [Ca2+] locally. To determine whether enhanced CaR signaling could modulate remodeling and thereby bone mass in vivo, we generated transgenic mice with a constitutively active mutant CaR (Act-CaR) targeted to their mature osteoblasts by the 3.5 kb osteocalcin promoter. Longitudinal microcomputed tomography of cancellous bone revealed reduced bone volume and density, accompanied by a diminished trabecular network, in the Act-CaR mice. The bone loss was secondary to an increased number and activity of osteoclasts, demonstrated by histomorphometry of secondary spongiosa. Histomorphometry, conversely, indicates that bone formation rates were unchanged in the transgenic mice. Constitutive signaling of the CaR in mature osteoblasts resulted in increased expression of RANK-L (receptor activator of nuclear factor-kappaB ligand), the major stimulator of osteoclast differentiation and activation, which is the likely underlying mechanism for the bone loss. The phenotype of Act-CaR mice is not attributable to systemic changes in serum [Ca2+] or PTH levels. We provide the first in vivo evidence that increased signaling by the CaR in mature osteoblasts can enhance bone resorption and further propose that fluctuations in the [Ca2+] within the bone microenvironment may modulate remodeling via the CaR.     

Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A

During bone formation and fracture healing there is a cross-talk between endothelial cells and osteoblasts. We previously showed that vascular endothelial growth factor A (VEGF-A) might be an important factor in this cross-talk, as osteoblast-like cells produce this angiogenic factor in a differentiation-dependent manner. Moreover, exogenously added VEGF-A enhances osteoblast differentiation. In the present study we investigated, given the coupling between angiogenesis and bone formation, whether bone morphogenetic proteins (BMPs) stimulate osteoblastogenesis and angiogenesis through the production of VEGF-A. For this we used the murine preosteoblast-like cell line KS483, which forms mineralized nodules in vitro, and an angiogenesis assay comprising 17-d-old fetal mouse bone explants that have the ability to form tube-like structures in vitro. Treatment of KS483 cells with BMP-2, -4, and -6 enhanced nodule formation, osteocalcin mRNA expression, and subsequent mineralization after 18 d of culture. This was accompanied by a dose-dependent increase in VEGF-A protein levels throughout the culture period. BMP-induced osteoblast differentiation, however, was independent of VEGF-A, as blocking VEGF-A activity by a VEGF-A antibody or a VEGF receptor 2 tyrosine kinase inhibitor did not affect BMP-induced mineralization. To investigate whether BMPs stimulate angiogenesis through VEGF-A, BMPs were assayed for their angiogenic activity. Treatment of bone explants with BMPs enhanced angiogenesis. This was inhibited by soluble BMP receptor 1A or noggin. In the presence of a VEGF-A antibody, both unstimulated and BMP-stimulated angiogenesis were arrested. Conditioned media of KS483 cells treated with BMPs also induced a strong angiogenic response, which was blocked by antimouse VEGF-A but not by noggin. These effects were specific for BMPs, as TGF beta inhibited osteoblast differentiation and angiogenesis while stimulating VEGF-A production. These findings indicate that BMPs stimulate angiogenesis through the production of VEGF-A by osteoblasts. In conclusion, VEGF-A produced by osteoblasts in response to BMPs is not involved in osteoblast differentiation, but couples angiogenesis to bone formation.     

Tob deficiency superenhances osteoblastic activity after ovariectomy to block estrogen deficiency-induced osteoporosis

Tob (transducer of erbB2) is a member of antiproliferative family proteins and acts as a bone morphogenic protein inhibitor as well as a suppressor of proliferation in T cells, which have been implicated in postmenopausal bone loss. To determine the effect of Tob deficiency on estrogen deficiency-induced bone loss, we analyzed bone metabolism after ovariectomy or sham operation in Tob-deficient mice. Ovariectomy in WT mice decreased trabecular bone volume and bone mineral density (BMD) as expected. In Tob-deficient mice, ovariectomy reduced bone volume and BMD. However, even after ovariectomy, both trabecular bone volume and BMD levels in Tob-deficient bone were comparable to those in sham-operated WT bones. Bone formation parameters (mineral apposition rate and bone formation rate) in the ovariectomized Tob-deficient mice were significantly higher than those in the ovariectomized WT mice. In contrast, the ovariectomy-induced increase in the bone resorption parameters, osteoclast surface, and osteoclast number was similar between Tob-deficient mice and WT mice. Furthermore, in ex vivo nodule formation assay, ovariectomy-induced enhancement of nodule formation was significantly higher in the bone marrow cells from Tob-deficient mice than in the bone marrow cells from ovariectomized WT mice. Both Tob and estrogen signalings converge at bone morphogenic protein activation of alkaline phosphatase and GCCG-reporter gene expression in osteoblasts, revealing interaction between the two signals. These data indicate that Tob deficiency prevents ovariectomy-induced bone loss through the superenhancement of osteoblastic activities in bone and that this results in further augmentation in the bone formation rate and the mineral apposition rate after ovariectomy in vivo.     

Targeted overexpression of insulin-like growth factor I to osteoblasts of transgenic mice: increased trabecular bone volume without increased osteoblast proliferation

Insulin-like growth factor I (IGF-I) is an important growth factor for bone, yet the mechanisms that mediate its anabolic activity in the skeleton are poorly understood. To examine the effects of locally produced IGF-I in bone in vivo, we targeted expression IGF-I to osteoblasts of transgenic mice using a human osteocalcin promoter. The IGF-I transgene was expressed in bone osteoblasts in OC-IGF-I transgenic mice at high levels in the absence of any change in serum IGF-I levels, or of total body growth. Bone formation rate at the distal femur in 3-week-old OC-IGF-I transgenic mice was approximately twice that of controls. By 6 weeks, bone mineral density as measured by dual energy x-ray, and quantitative computed tomography was significantly greater in OC-IGF-I transgenic mice compared with controls. Histomorphometric measurements revealed a marked (30%) increase femoral cancellous bone volume in the OC-IGF-I transgenic mice, but no change in the total number of osteoblasts or osteoclasts. Transgenic mice also demonstrated an increase in the osteocyte lacunea occupancy, suggesting that IGF-I may extend the osteocyte life span. We conclude that IGF-I produced locally in bone osteoblasts exerts its anabolic effect primarily by increasing the activity of resident osteoblasts.