Weirong Xing

Ephrin B1 Regulates Bone Marrow Stromal Cell Differentiation and Bone Formation by Influencing TAZ Transactivation via Complex Formation with NHERF1

ABSTRACT Mutations of ephrin B1 in humans result in craniofrontonasal syndrome. Because little is known of the role and mechanism of action of ephrin B1 in bone, we examined the function of osteoblast-produced ephrin B1 in vivo and identified the molecular mechanism by which ephrin B1 reverse signaling regulates bone formation. Targeted deletion of the ephrin B1 gene in type 1α2 collagen-producing cells resulted in severe calvarial defects, decreased bone size, bone mineral density, and trabecular bone volume, caused by impairment in osterix expression and osteoblast differentiation. Coimmunoprecipitation of the TAZ complex with TAZ-specific antibody revealed a protein complex containing ephrin B1, PTPN13, NHERF1, and TAZ in bone marrow stromal (BMS) cells. Activation of ephrin B1 reverse signaling with soluble EphB2-Fc led to a time-dependent increase in TAZ dephosphorylation and shuttling from cytoplasm to nucleus. Treatment of BMS cells with exogenous EphB2-Fc resulted in a 4-fold increase in osterix expression as determined by Western blotting. Disruption of TAZ expression using specific lentivirus small hairpin RNA (shRNA) decreased TAZ mRNA by 80% and ephrin B1 reverse signaling-mediated increases in osterix mRNA by 75%. Knockdown of NHERF1 expression reduced basal levels of osterix expression by 90% and abolished ephrin B1-mediated induction of osterix expression. We conclude that locally produced ephrin B1 mediates its effects on osteoblast differentiation by a novel molecular mechanism in which activation of reverse signaling leads to dephosphorylation of TAZ and subsequent release of TAZ from the ephrin B1/NHERF1/TAZ complex to translocate to the nucleus to induce expression of the osterix gene and perhaps other osteoblast differentiation genes. Our findings provide strong evidence that ephrin B1 reverse signaling in osteoblasts is critical for BMS cell differentiation and bone formation.

Nuclear Factor-E2-related Factor-1 Mediates Ascorbic Acid Induction of Osterix Expression via Interaction with Antioxidant-Responsive Element in Bone Cells

We recently found that deletion of the gulonolactone oxidase gene, which is involved in the synthesis of ascorbic acid (AA), was responsible for the fracture phenotype in spontaneous fracture mice. To explore the molecular mechanisms by which AA regulates osteoblast differentiation, we examined the effect of AA on osterix expression via Nrf1 (NF-E2-related factor-1) binding to antioxidant-responsive element (ARE) in bone marrow stromal (BMS) cells. AA treatment caused a 6-fold increase in osterix expression in mutant BMS cells at 24 h, which was unaffected by pretreatment with protein synthesis inhibitor. Sequence analyses of mouse osterix promoter revealed a putative ARE located at -1762 to -1733 upstream of the transcription start site to which Nrf potentially binds. A gel mobility shift assay revealed that nuclear proteins from AA-treated BMS cells bound to radiolabeled ARE much more strongly than nuclear extracts from AA-untreated cells. A chromatin immunoprecipitation assay with Nrf1 antibody confirmed the interaction of Nrf1 with the mouse osterix promoter. A reporter assay demonstrated that the promoter activity of mouse osterix containing an ARE was stimulated 4-fold by a 48-h treatment with AA in spontaneous fracture BMS cells. Treatment of mutant BMS cells with AA resulted in a 3.9-fold increase in the nuclear accumulation of Nrf1. Transfection of mutant BMS cells with Nrf1 small interfering RNA decreased Nrf1 protein by 4.5-fold, blocked AA induction of osterix expression, and impaired BMS cell differentiation. Our data provided the first experimental evidence that AA modulated osterix expression via a novel mechanism involving Nrf1 nuclear translocation and Nrf1 binding to ARE to activate genes critical for cell differentiation.

Genetic evidence that thyroid hormone is indispensable for prepubertal insulin-like growth factor–I expression and bone acquisition in mice

Understanding how bone growth is regulated by hormonal and mechanical factors during early growth periods is important for optimizing the attainment of peak bone mass to prevent or postpone the occurrence of fragility fractures later in life. Using genetic mouse models that are deficient in thyroid hormone (TH) (Tshr−/− and Duox2−/−), growth hormone (GH) (Ghrhrlit/lit), or both (Tshr−/−; Ghrhrlit/lit), we demonstrate that there is an important period prior to puberty when the effects of GH are surprisingly small and TH plays a critical role in the regulation of skeletal growth. Daily administration of T3/T4 during days 5 to 14, the time when serum levels of T3 increase rapidly in mice, rescued the skeletal deficit in TH‐deficient mice but not in mice lacking both TH and GH. However, treatment of double‐mutant mice with both GH and T3/T4 rescued the bone density deficit. Increased body fat in the TH‐deficient as well as TH/GH double‐mutant mice was rescued by T3/T4 treatment during days 5 to 14. In vitro studies in osteoblasts revealed that T3 in the presence of TH receptor (TR) α1 bound to a TH response element in intron 1 of the IGF‐I gene to stimulate transcription. In vivo studies using TRα and TRβ knockout mice revealed evidence for differential regulation of insulin‐like growth factor (IGF)‐I expression by the two receptors. Furthermore, blockade of IGF‐I action partially inhibited the biological effects of TH, thus suggesting that both IGF‐I–dependent and IGF‐I–independent mechanisms contribute to TH effects on prepubertal bone acquisition.

Targeted disruption of nuclear factor erythroid-derived 2-like 1 in osteoblasts reduces bone size and bone formation in mice

Previous in vitro studies found that nuclear factor erythroid-derived 2-like 1 (NFE2L1) was involved in mediating ascorbic acid-induced osterix expression and osteoblast differentiation via binding to the antioxidant response element of the osterix promoter. To test the role of NFE2L1 in regulating bone formation in vivo, we disrupted NFE2L1 specifically in osteoblasts. Mice expressing Cre under the control of Col1alpha2 promoter were crossed with NFE2L1 loxP mice to generate Cre+ knockout (KO) and Cre- wild-type (WT) mice. Skeletal measurements by DEXA revealed 8-10% and 9-11% reduction in total body BMC and bone area in the KO mice from 3 to 8 wk of age. Peripheral quantitative computed tomography analyses found both periosteal and endosteal circumferences were reduced by 6% at the middiaphysis of the femurs from 8 wk old KO mice. Histomorphometric analyses revealed reduced bone formation was a cause for reduced bone size in the KO mice. Microcomputed tomography analysis of the metaphysis of the femur revealed that trabecular bone volume/total volume, and trabecular numbers were decreased by 30 and 53% in the NFE2L1 KO mice. Expression of osterix was decreased by 57% in the bones of NFE2L1 KO mice. In vitro nodule assay demonstrated that mineralized nodule area was reduced by 68% in the cultures of bone marrow stromal cells from NFE2L1 KO mice. Treatment of primary osteoblasts with ascorbic acid increased osterix expression by fourfold, whereas loss of NFE2L1 in osteoblasts diminished ascorbic acid stimulation of osterix expression by 50%. Our data provide the first in vivo experimental evidence that NFE2L1 produced by osteoblasts is involved in regulating osterix expression, osteoblast differentiation, and bone formation.

Glutaredoxin 5 regulates osteoblast apoptosis by protecting against oxidative stress

There is now increasing evidence which suggests an important role for reactive oxygen species (ROS) in the pathogenesis of osteoporosis. However, little is known on the molecular components of the oxidative stress pathway or their functions in bone. In this study, we evaluated the role and mechanism of action of glutaredoxin (Grx) 5, a protein that is highly expressed in bone. Osteoblasts were transfected with Grx5 siRNA and treated with hydrogen peroxide (H(2)O(2)). Grx5 siRNA treatment increased apoptosis while Grx5 overexpression protected MC3T3-E1 cells against H(2)O(2) induced apoptosis and ROS formation. Grx5 deficiency results in impaired biogenesis of Fe-S cluster in yeast. Accordingly, activity of mitochondrial aconitase, whose activity is dependent on Fe-S cluster, decreased in Grx5 siRNA treated cells. Since reduced formation of Fe-S cluster would lead to increased level of free iron, a competitive inhibitor of manganese superoxide dismutase (MnSOD), we measured MnSOD activity in Grx5 deficient osteoblasts and found MnSOD activity was significantly reduced. The consequence of long term inhibition of Grx5 on osteoblast apoptosis was evaluated using lentiviral shRNA technology. Grx5 shRNA cells exhibited higher caspase activity and cardiolipin oxidation in the presence of H(2)O(2). MnSOD activity was rescued by the addition of MnCl(2) to Grx5 shRNA osteoblasts in the presence of H(2)O(2). Our findings are consistent with the hypothesis that Grx5 is an important determinant of osteoblast apoptosis and acts via a molecular pathway that involves regulation of ROS production, cardiolipin oxidation, caspase activity, Fe-S cluster formation, and MnSOD activity.

1,25-Dihydroxyvitamin D3 suppresses TLR8 expression and TLR8-mediated inflammatory responses in monocytes in vitro and experimental autoimmune encephalomyelitis in vivo.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) suppresses autoimmunity and inflammation; however, the mechanism of its action has not been fully understood. We sought in this study to determine whether the anti-immune/anti-inflammatory action of 1,25(OH)2D3 is in part mediated through an interplay between 1,25(OH)2D3 and toll-like receptor (TLR)7/8 signaling. 1,25(OH)2D3 treatment prior to and/or following experimental autoimmune encephalomyelitis (EAE) induction effectively reduced inflammatory cytokine expression in the spinal cord and ameliorated EAE. These effects were accompanied with a reduction in expression of several TLRs with the most profound effect observed for TLR8. The expression of TLR8 adaptor protein MyD88 was also significantly reduced by 1,25(OH)2D3. To determine the molecular mechanism by which 1,25(OH)2D3 suppresses EAE induction of TLR8 and inflammatory cytokine expression, we evaluated whether 1,25(OH)2D3 can directly inhibit TLR8 signaling and the resulting inflammatory responses in human THP-1 monocytes. 1,25(OH)2D3 treatment not only significantly reduced TLR8 expression but also the expression or activity of MyD88, IRF-4, IRF-7 and NF-kB in monocytes challenged with TLR8 ligands. TLR8 promoter-luciferase reporter assays indicated that 1,25(OH)2D3 decreases TLR8 mRNA level in part via inhibiting TLR8 gene transcription activity. As a result of inhibition on TLR8 signaling cascade at various stages, 1,25(OH)2D3 significantly diminished the TLR8 target gene expression (TNF-α and IL-1β). In summary, our novel findings suggest that TLR8 is a new target of 1,25(OH)2D3 and may mediate the anti-inflammatory action of 1,25(OH)2D3. Our findings also point to a destructive role of TLR8 in EAE and shed lights on pathogenesis of multiple sclerosis.

Targeted Disruption of Ephrin B1 in Cells of Myeloid Lineage Increases Osteoclast Differentiation and Bone Resorption in Mice

Disruption of ephrin B1 in collagen I producing cells in mice results in severe skull defects and reduced bone formation. Because ephrin B1 is also expressed during osteoclast differentiation and because little is known on the role of ephrin B1 reverse signaling in bone resorption, we examined the bone phenotypes in ephrin B1 conditional knockout mice, and studied the function of ephrin B1 reverse signaling on osteoclast differentiation and resorptive activity. Targeted deletion of ephrin B1 gene in myeloid lineage cells resulted in reduced trabecular bone volume, trabecular number and trabecular thickness caused by increased TRAP positive osteoclasts and bone resorption. Histomorphometric analyses found bone formation parameters were not changed in ephrin B1 knockout mice. Treatment of wild-type precursors with clustered soluble EphB2-Fc inhibited RANKL induced formation of multinucleated osteoclasts, and bone resorption pits. The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation. Similarly, activation of ephrin B1 reverse signaling by EphB2-Fc treatment led to inhibition of TRAP, cathepsin K and NFATc1 mRNA expression in osteoclasts derived from wild-type mice but not conditional knockout mice. Immunoprecipitation with NHERF1 antibody revealed ephrin B1 interacted with NHERF1 in differentiated osteoclasts. Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin. We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.

Epiphyseal Chondrocyte Secondary Ossification Centers Require Thyroid Hormone Activation of Indian Hedgehog and Osterix Signaling

Thyroid hormones (THs) are known to regulate endochondral ossification during skeletal development via acting directly in chondrocytes and osteoblasts. In this study, we focused on TH effects on the secondary ossification center (SOC) because the time of appearance of SOCs in several species coincides with the time when peak levels of TH are attained. Accordingly, micro–computed tomography (µCT) evaluation of femurs and tibias at day 21 in TH‐deficient and control mice revealed that endochondral ossification of SOCs is severely compromised owing to TH deficiency and that TH treatment for 10 days completely rescued this phenotype. Staining of cartilage and bone in the epiphysis revealed that whereas all of the cartilage is converted into bone in the prepubertal control mice, this conversion failed to occur in the TH‐deficient mice. Immunohistochemistry studies revealed that TH treatment of thyroid stimulating hormone receptor mutant (Tshr−/−) mice induced expression of Indian hedgehog (Ihh) and Osx in type 2 collagen (Col2)‐expressing chondrocytes in the SOC at day 7, which subsequently differentiate into type 10 collagen (Col10)/osteocalcin‐expressing chondro/osteoblasts at day 10. Consistent with these data, treatment of tibia cultures from 3‐day‐old mice with 10 ng/mL TH increased expression of Osx, Col10, alkaline phosphatase (ALP), and osteocalcin in the epiphysis by sixfold to 60‐fold. Furthermore, knockdown of the TH‐induced increase in Osx expression using lentiviral small hairpin RNA (shRNA) significantly blocked TH‐induced ALP and osteocalcin expression in chondrocytes. Treatment of chondrogenic cells with an Ihh inhibitor abolished chondro/osteoblast differentiation and SOC formation. Our findings indicate that TH regulates the SOC initiation and progression via differentiating chondrocytes into bone matrix–producing osteoblasts by stimulating Ihh and Osx expression in chondrocytes.

Inactivation of insulin-like-growth factors diminished the anabolic effects of pregnancy-associated plasma protein-A (PAPP-A) on bone in mice

In vivo studies have provided ubiquitous evidence that pregnancy-associated plasma protein-A (PAPP-A) functions as a potent anabolic factor. While some evidence supports the prediction that increasing IGF bioavailability contributes to the anabolic effects of PAPP-A, definitive evidence has been lacking. This important issue has been addressed in this study using a unique mouse model in which PAPP-A was overexpressed in bone either alone or together with a protease-resistant IGFBP-4 analog (PRBP-4) which serves as an IGF inhibitor. PAPP-A transgenic mice exhibited a 25% increase in skull bone mineral density (BMD) whereas PRBP-4 transgenic mice showed a 20-25% decrease in this parameter at an age of 3months. Femur/tibia size-related parameters were significantly increased in PAPP-A transgenic mice but decreased in PRBP-4 transgenic mice. This data clearly demonstrates that PAPP-A transgenic mice exhibit opposite phenotypes in both flat bone and long bone compared to PRBP-4 transgenic mice which have reduced IGF bioavailability in bone. Importantly, PRBP-4 and PRBP-4/PAPP-A double transgenic mice shared essentially identical phenotypes in both flat and long bones. Calvarial thickness, skull BMD and long bone parameters were reduced to similar degrees in PRBP-4 and PRBP-4/PAPP-A transgenic mice relative to wild-type littermates. Our findings provide compelling evidence that PAPP-A increases bone formation primarily by increasing IGF bioavailability and that other alternative pathways may play a negligible role in mediating the anabolic effect of PAPPA in bone. This clear definition of PAPP-As mechanism of action is critical for future translational studies on the therapeutic application of PAPP-A.

Claudin 18 is a novel negative regulator of bone resorption and osteoclast differentiation

Claudin 18 (Cldn‐18) belongs to a large family of transmembrane proteins that are important components of tight junction strands. Although several claudin members are expressed in bone, the functional role for any claudin member in bone is unknown. Here we demonstrate that disruption of Cldn‐18 in mice markedly decreased total body bone mineral density, trabecular bone volume, and cortical thickness in Cldn‐18−/− mice. Histomorphometric studies revealed that bone resorption parameters were increased significantly in Cldn‐18−/− mice without changes in bone formation. Serum levels of tartrate‐resistant acid phosphatase 5b (TRAP5b) and mRNA expression levels of osteoclast specific markers and signaling molecules were also increased. Loss of Cldn‐18 further exacerbated calcium deficiency induced bone loss by influencing bone resorption, thereby resulting in mechanically weaker bone. In vitro studies with bone marrow macrophages revealed Cldn‐18 disruption markedly enhanced receptor activator of NF‐κB ligand (RANKL)‐induced osteoclast differentiation but not macrophage colony‐stimulating factor (MCSF)‐induced bone marrow macrophage (BMM) proliferation. Consistent with a direct role for Cldn‐18 in regulating osteoclast differentiation, overexpression of wild type but not PDZ binding motif deleted Cldn‐18 inhibited RANKL‐induced osteoclast differentiation. Furthermore, our findings indicate that Cldn‐18 interacts with Zonula occludens 2 (ZO‐2) to modulate RANKL signaling in osteoclasts. In conclusion, we demonstrate that Cldn‐18 is a novel negative regulator of bone resorption and osteoclast differentiation.