Shin-Tai Chen

Insulin-like growth factor-binding protein 5 (IGFBP-5) interacts with a four and a half LIM protein 2 (FHL2)

Recent studies using insulin-like growth factor I (IGF-I) knockout mice demonstrate that IGF-binding protein (IGFBP)-5, an important bone formation regulator, itself is a growth factor with cellular effects not dependent on IGFs. Because IGFBP-5 contains a nuclear localization sequence that mediates transport of IGFBP-5 into the nucleus, we propose that IGFBP-5 interacts with nuclear proteins to affect transcription of genes involved in bone formation. We therefore undertook studies to identify proteins that bind to IGFBP-5 using IGFBP-5 as bait in a yeast two-hybrid screen of a U2 human osteosarcoma cDNA library. Five related clones that interacted strongly with the bait corresponded to the FHL2 gene, which contains four and a half LIM domains. Co-immunoprecipitation studies with lysates from U2 cells overexpressing FHL2 and IGFBP-5 confirmed that interaction between IGFBP-5 and FHL2 occurs in whole cells.In vitro interaction studies revealed that purified FHL2 interacted with IGFBP-5 but not with IGFBP-3, -4, or -6. Northern blot analysis showed that FHL2 was strongly expressed in human osteoblasts. Nuclear localization of both FHL2 and IGFBP-5 was evident from Western immunoblot analysis and immunofluorescence. The role of FHL2 as an intracellular mediator of the effects of IGFBP-5 and other osteoregulatory agents in osteoblasts will need to be verified in future studies.

Ex vivo gene therapy with stromal cells transduced with a retroviral vector containing the BMP4 gene completely heals critical size calvarial defect in rats

In order to develop a successful gene therapy system for the healing of bone defects, we developed a murine leukemia virus (MLV)-based retroviral system expressing the human bone morphogenetic protein (BMP) 4 transgene with high transduction efficiency. The bone formation potential of BMP4 transduced cells was tested by embedding 2.5 × 106 transduced stromal cells in a gelatin matrix that was then placed in a critical size defect in calvariae of syngenic rats. Gelatin matrix without cells or with untransduced stromal cells were the two control groups. The defect area was completely filled with new bone in experimental rats after 4 weeks, while limited bone formation occurred in either control group. Bone mineral density (BMD) of the defect in the gene therapy group was 67.8 ± 5.7 mg/cm2 (mean ± s.d., n = 4), which was 119 ± 10% of the control BMD of bone surrounding the defect (57.2 ± 1.5 mg/cm2). In contrast, BMD of rats implanted with untransduced stromal cells was five-fold lower (13.8 ± 7.4 mg/cm2, P < 0.001). Time course studies revealed that there was a linear increase in BMD between 2–4 weeks after inoculation of the critical size defect with 2.5 × 106 implanted BMP4 cells. In conclusion, the retroviral-based BMP4 gene therapy system that we have developed has the potential for regeneration of large skeletal defects.

In vivo bone formation in fracture repair induced by direct retroviral-based gene therapy with bone morphogenetic protein-4

This study sought to develop an in vivo gene therapy to accelerate the repair of bone fractures. In vivo administration of an engineered viral vector to promote fracture healing represents a potential high-efficacy, low-risk procedure. We selected a murine leukemia virus (MLV)-based retroviral vector, because this vector would be expected to target transgene expression to the proliferating periosteal cells arising shortly after bone fracture. This vector transduced a hybrid gene that consisted of a bone morphogenetic protein (BMP)-4 transgene with the BMP-2 secretory signal to enhance the secretion of mature BMP-4. The MLV vector expressing this BMP-2/4 hybrid gene or beta-galactosidase control gene was administered at the lateral side of the fracture periosteum at 1 day after fracture in the rat femoral fracture model. X-ray examination by radiograph and peripheral quantitative computed tomography at 7, 14, and 28 days after fracture revealed a highly significant enhancement of fracture tissue size in the MLV-BMP-2/4-treated fractures compared to the control fractures. The tissue was extensively ossified at 14 and 28 days, and the newly formed bone exhibited normal bone histology. This tissue also exhibited strong immunohistochemical staining of BMP-4. Additional control and MLV-BMP-2/4-treated animals each were monitored for 70 days to determine the fate of the markedly enhanced fracture callus. Radiographs showed that the hard callus had been remodeled and substantial healing at the fracture site had occurred, suggesting that the union of the bone at the fracture site was at least as high in the BMP-4-treated bone as in the control bone. There was no evidence of viral vector infection of extraskeletal tissues, suggesting that this in vivo gene therapy for fracture repair is safe. In summary, we have demonstrated for the first time that a MLV-based retroviral vector is a safe and effective means of introducing a transgene to a fracture site and that this procedure caused an enormous augmentation of fracture bone formation.

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.

Local ex vivo gene therapy with bone marrow stromal cells expressing human BMP4 promotes endosteal bone formation in mice.

Bone loss in osteoporosis is caused by an imbalance between resorption and formation on endosteal surfaces of trabecular and cortical bone. We investigated the feasibility of increasing endosteal bone formation in mice by ex vivo gene therapy with bone marrow stromal cells (MSCs) transduced with a MLV‐based retroviral vector to express human bone morphogenetic protein 4 (BMP4).

Retroviral‐based gene therapy with cyclooxygenase‐2 promotes the union of bony callus tissues and accelerates fracture healing in the rat

An in vivo gene therapy strategy was developed to accelerate bone fracture repair.

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.

T-Box 3 Negatively Regulates Osteoblast Differentiation by Inhibiting Expression of Osterix and Runx2

T‐box (Tbx)3, a known transcriptional repressor, is a member of a family of transcription factors, which contain a highly homologous DNA binding domain known as the Tbx domain. Based on the knowledge that mutation of the Tbx3 gene results in limb malformation, Tbx3 regulates osteoblast proliferation and its expression increases during osteoblast differentiation, we predicted that Tbx3 is an important regulator of osteoblast cell functions. In this study, we evaluated the consequence of transgenic overexpression of Tbx3 on osteoblast differentiation. Retroviral overexpression increased Tbx3 expression >100‐fold at the mRNA and protein level. Overexpression of Tbx3 blocked mineralized nodule formation (28 ± 8 vs. 7 ± 1%) in MC3T3‐E1 cells. In support of these data, alkaline phosphatase (ALP) activity was reduced 33–70% (P < 0.05) in both MC3T3‐E1 cells and primary calvaria osteoblasts overexpressing Tbx3. In contrast, Tbx3 overexpression did not alter ALP activity in bone marrow stromal cells. Tbx3 overexpression blocked the increase in expression of key osteoblast marker genes, ALP, bone sialoprotein, and osteocalcin that occurs during normal osteoblast differentiation, but had little or no effect on expression of proliferation genes p53 and Myc. In addition, Tbx3 overexpression abolished increased osterix and runx2 expression observed during normal osteoblast differentiation, but the change in Msx1 and Msx2 expression over time was similar between control and Tbx3 overexpressing cells. Interestingly, osterix and runx2, but not Msx1 and Msx2, contain Tbx binding site in the regulatory region. Based on these data and our previous findings, we conclude that Tbx3 promotes proliferation and suppresses differentiation of osteoblasts and may be involved in regulating expression of key transcription factors involved in osteoblast differentiation. J. Cell. Biochem. 106: 482–490, 2009.

Potential involvement of the interaction between insulin-like growth factor binding protein (IGFBP)-6 and LIM mineralization protein (LMP)-1 in regulating osteoblast differentiation.

Insulin‐like growth factor binding protein (IGFBP)‐6 has been reported to inhibit differentiation of myoblasts and osteoblasts. In the current study, we explored the mechanisms underlying IGFBP‐6 effects on osteoblast differentiation. During MC3T3‐E1 osteoblast differentiation, we found that IGFBP‐6 protein was down‐regulated. Overexpression of IGFBP‐6 in MC3T3‐E1 and human bone cells inhibited nodule formation, osteocalcin mRNA expression and ALP activity. Furthermore, accumulation of IGFBP‐6 in the culture media was not required for any of these effects suggesting that IGFBP‐6 suppressed osteoblast differentiation by an intracellular mechanism. A yeast two‐hybrid screen of an osteosarcoma library was conducted to identify intracellular binding partners to account for IGFBP‐6 inhibitory effects on osteoblast differentiation. LIM mineralizing protein (LMP‐1) was identified as a high affinity IGFBP‐6 binding partner. Physical interaction between IGFBP‐6 and LMP‐1 was confirmed by co‐immunoprecipitation. Fluorescent protein fusion constructs for LMP‐1 and IGFBP‐6 were transiently transfected into osteoblasts to provide evidence of subcellular locations for each protein. Coexpression of LMP‐1‐GFP and IGFBP‐6‐RFP resulted in overlapping subcellular localization of LMP‐1 and IGFBP‐6. To determine if there was a functional association of IGFBP‐6 and LMP‐1 as well as a physical association, we studied the effect of IGFBP‐6, LMP‐1 and their combination on type I procollagen promoter activity. LMP‐1 increased promoter activity while IGFBP‐6 reduced promoter activity, and coexpression of LMP‐1 with IGFBP‐6 abrogated IGFBP‐6 suppression. These studies provide evidence that overexpression of IGFBP‐6 suppresses human and murine osteoblast differentiation, that IGFBP‐6 and LMP‐1 physically interact, and supports the conclusion that this interaction may be functionally relevant. J. Cell. Biochem. 104: 1890–1905, 2008.

Sca-1+ Hematopoietic Cell–based Gene Therapy with a Modified FGF-2 Increased Endosteal/Trabecular Bone Formation in Mice

This study assessed the feasibility of using an ex vivo stem cell antigen-1-positive (Sca-1+) cell-based systemic fibroblast growth factor-2 (FGF-2) gene therapy to promote endosteal bone formation. Sca-1+ cells were used because of their ability to home to, and engraft into, the bone marrow cavity. The human FGF-2 gene was modified to increase protein secretion and stability by adding the bone morphogeneic protein (BMP)-2/4 hybrid signal sequence and by mutating two key cysteines. Retro-orbital injection of Sca-1+ cells transduced with a Moloney leukemia virus (MLV)-based vector expressing the modified FGF-2 gene into sub-lethally irradiated W41/W41 recipient mice resulted in long-term engraftment, more than 100-fold elevation in serum FGF-2 level, increased serum bone-formation markers, and massive endosteal bone formation. In recipient mice showing very high serum FGF-2 levels (>2,000 pg/ml), this enhanced endosteal bone formation was so robust that the marrow space was filled with bony tissues and insufficient calcium was available for the mineralization of all the newly formed bone, which led to secondary hyperparathyroidism and osteomalacia. These adverse effects appeared to be dose related. In conclusion, this study provided compelling test-of-principle evidence for the feasibility of using an Sca-1+ cell-based ex vivo systemic FGF-2 gene therapy strategy to promote endosteal bone formation.