Prdm5 regulates collagen gene transcription by association with RNA polymerase II in developing bone.

PRDM family members are transcriptional regulators involved in tissue specific differentiation. PRDM5 has been reported to predominantly repress transcription, but a characterization of its molecular functions in a relevant biological context is lacking. We demonstrate here that Prdm5 is highly expressed in developing bones; and, by genome-wide mapping of Prdm5 occupancy in pre-osteoblastic cells, we uncover a novel and unique role for Prdm5 in targeting all mouse collagen genes as well as several SLRP proteoglycan genes. In particular, we show that Prdm5 controls both Collagen I transcription and fibrillogenesis by binding inside the Col1a1 gene body and maintaining RNA polymerase II occupancy. In vivo, Prdm5 loss results in delayed ossification involving a pronounced impairment in the assembly of fibrillar collagens. Collectively, our results define a novel role for Prdm5 in sustaining the transcriptional program necessary to the proper assembly of osteoblastic extracellular matrix.

Inhibition of p38alpha mitogen-activated protein kinase prevents the development of osteolytic bone disease, reduces tumor burden, and increases survival in murine models of multiple myeloma.

The bone microenvironment plays a critical role in supporting the growth and survival of multiple myeloma as well as in the development of osteolytic bone disease. Signaling through p38alpha mitogen-activated protein kinase (MAPK) mediates synthesis of multiple myeloma cell growth factors, and its inhibition reduces proliferation in vitro. However, it is unclear whether targeting p38alpha MAPK prevents multiple myeloma growth and the development of bone disease in vivo. In this study, we determined whether SCIO-469, a selective p38alpha MAPK inhibitor, inhibits multiple myeloma growth and prevents bone disease in the 5T2MM and 5T33MM models. SCIO-469 decreased constitutive p38alpha MAPK phosphorylation of both 5T2MM and 5T33MM cells in vitro. This was associated with decreased DNA synthesis and an induction of apoptosis when the cells were cultured with bone marrow stromal cells. Treatment of C57Bl/KaLwRij mice bearing 5T33MM cells with SCIO-469 inhibited p38alpha MAPK phosphorylation and was associated with a significant decrease in serum paraprotein, an almost complete reduction in tumor cells in the bone marrow, a decrease in angiogenesis, and a significant increase in disease-free survival. Injection of 5T2MM murine myeloma cells into C57Bl/KaLwRij mice resulted in myeloma bone disease characterized by increased osteoclast occupation of the bone surface, reduced cancellous bone, and the development of osteolytic bone lesions. Treatment of 5T2MM-injected mice with SCIO-469 reduced this development of bone disease. Together, these data show that targeting p38alpha MAPK with SCIO-469 decreases myeloma burden in vivo, in addition to preventing the development of myeloma bone disease.

Geranylgeranyl transferase type II inhibition prevents myeloma bone disease.

Geranylgeranyl transferase II (GGTase II) is an enzyme that plays a key role in the isoprenylation of proteins. 3-PEHPC, a novel GGTase II inhibitor, blocks bone resorption and induces myeloma cell apoptosis in vitro. Its effect on bone resorption and tumor growth in vivo is unknown. We investigated the effect of 3-PEHPC on tumor burden and bone disease in the 5T2MM model of multiple myeloma in vivo. 3-PEHPC significantly reduced osteoclast numbers and osteoclast surface. 3-PEHPC prevented the bone loss and the development of osteolytic bone lesions induced by 5T2MM myeloma cells. Treatment with 3-PEHPC also significantly reduced myeloma burden in bone. The magnitude of response was similar to that seen with the bisphosphonate, risedronate. These data show that targeting GGTase II with 3-PEHPC can prevent osteolytic bone disease and reduce tumor burden in vivo, and represents a novel approach to treating tumors that grow in bone.

Role of CCR1 and CCR5 in homing and growth of multiple myeloma and in the development of osteolytic lesions: a study in the 5TMM model.

Multiple myeloma (MM) is a plasma cell malignancy, characterized by the localization of the MM cells in the bone marrow (BM), where they proliferate and induce osteolysis. The MM cells first need to home or migrate to the BM to receive necessary survival signals. In this work, we studied the role of CCR1 and CCR5, two known chemokine receptors, in both chemotaxis and osteolysis in the experimental 5TMM mouse model. A CCR1-specific (BX471) and a CCR5-specific (TAK779) antagonist were used to identify the function of both receptors. We could detect by RT-PCR and flow cytometric analyses the expression of both CCR1 and CCR5 on the cells and their major ligand, macrophage inflammatory protein 1alpha (MIP1alpha) could be detected by ELISA. In vitro migration assays showed that MIP1alpha induced a 2-fold increase in migration of 5TMM cells, which could only be blocked by TAK779. In vivo homing kinetics showed a 30% inhibition in BM homing when 5TMM cells were pre-treated with TAK779. We found, in vitro, that both inhibitors were able to reduce osteoclastogenesis and osteoclastic resorption. In vivo end-term treatment of 5T2MM mice with BX471 resulted in a reduction of the osteolytic lesions by 40%; while TAK779 treatment led to a 20% decrease in lesions. Furthermore, assessment of the microvessel density demonstrated a role for both receptors in MM induced angiogenesis. These data demonstrate the differential role of CCR1 and CCR5 in MM chemotaxis and MM associated osteolysis and angiogenesis.

Radiographic classification of osteogenesis during bone distraction.

Successful limb lengthening requires serial radiological evaluation of the progression of healing of the regenerate bone. However, there is no radiographic classification system that shows how the regenerate should progress during treatment in adults. The study aimed to address this need.A series of radiographs were studied from 92 patients (125 segments) who had undergone bone lengthening. A radiographic classification of osteogenesis was developed based on callus shape and radiographic features that occur between osteotomy and fixator removal. This classification system used both shape and type of feature to condense and record the radiographic information, but type of feature alone was sufficient to predict outcome. The concurrence and reproducibility of the classification system was tested by inter- and intra-observer studies. The degree of consistent repetition and agreement between observers suggests that the classification system is reliable, reproducible, and therefore should be robust in use. This classification system provides an insight into osteogenesis; it allows the progress of the bone healing to be assessed against a successful pattern of healing. Hence, potential problems can be predicted and clinical changes made to improve outcome. The classification can be simplified to make it more appropriate for clinical use.

Effect of 900 MHz electromagnetic fields on nonthermal induction of heat-shock proteins in human leukocytes.

Despite many studies, the evidence as to whether radiofrequency fields are detrimental to health remains controversial, and the debate continues. Cells respond to some abnormal physiological conditions by producing cytoprotective heat-shock (or stress) proteins. The aim of this study was to determine whether exposure to mobile phone-type radiation causes a nonthermal stress response in human leukocytes. Human peripheral blood was sham-exposed or exposed to 900 MHz fields (continuous-wave or GSM-modulated signal) at three average specific absorption rates (0.4, 2.0 and 3.6 W/kg) for different durations (20 min, 1 h and 4 h) in a calibrated TEM cell placed in an incubator to give well-controlled atmospheric conditions at 37 degrees C and 95% air/5% CO(2). Positive (heat-stressed at 42 degrees C) and negative (kept at 37 degrees C) control groups were incubated simultaneously in the same incubator. Heat caused an increase in the number of cells expressing stress proteins (HSP70, HSP27), measured using flow cytometry, and this increase was dependent on time. However, no statistically significant difference was detected in the number of cells expressing stress proteins after RF-field exposure. These results suggest that mobile phone-type radiation is not a stressor of normal human lymphocytes and monocytes, in contrast to mild heating.

The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest.

Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology.

Effect of 50 Hz electromagnetic fields on the induction of heat-shock protein gene expression in human leukocytes.

Although evidence is controversial, exposure to environmental power-frequency magnetic fields is of public concern. Cells respond to some abnormal physiological conditions by producing cytoprotective heat-shock (or stress) proteins. In this study, we determined whether exposure to power-frequency magnetic fields in the range 0-100 microT rms either alone or concomitant with mild heating induced heat-shock protein gene expression in human leukocytes, and we compared this response to that induced by heat alone. Samples of human peripheral blood were simultaneously exposed to a range of magnetic-field amplitudes using a regimen that was designed to allow field effects to be distinguished from possible artifacts due to the position of the samples in the exposure system. Power-frequency magnetic-field exposure for 4 h at 37 degrees C had no detectable effect on expression of the genes encoding HSP27, HSP70A or HSP70B, as determined using reverse transcriptase-PCR, whereas 2 h at 42 degrees C elicited 10-, 5- and 12-fold increases, respectively, in the expression of these genes. Gene expression in cells exposed to power-frequency magnetic fields at 40 degrees C was not increased compared to cells incubated at 40 degrees C without field exposure. These findings and the extant literature suggest that power-frequency electromagnetic fields are not a universal stressor, in contrast to physical agents such as heat.

Knee joint reaction force during tibial diaphyseal lengthening: a study on a rabbit model.

The in vivo passive knee joint reaction force was measured in a rabbit model of tibial diaphyseal lengthening. This was based on the assumption that limb lengthening creates soft tissue tension that compresses the joint surface and generates the joint contact force. A measurement method was developed that involved the distraction of the joint and the determination of the distraction force that just separates the joint surfaces. Sixteen immature (mean+/-SD age=9+/-0.6 weeks) New Zealand White rabbits underwent 30% (left) tibial diaphyseal lengthening at a rate of two 0.4mm incremental lengthenings per day. The knee joint reaction force was measured at the end of lengthening (8 rabbits, mean+/-SD age=14+/-0.6 weeks) and five weeks after lengthening (8 rabbits, mean+/-SD age=19+/-0.7 weeks). An instrumented bilateral distractor and an extensometer were fixed cross the knee joint. The joint distraction force and distraction displacement were measured when the joint was distracted in steps and after the section of the Achilles tendon. The joint reaction force on the lengthened side was significantly higher than the control side at both time points (mean+/-SD 44.4+/-7.8 N v. 27.2+/-4.0 N at the end of lengthening, 44.3+/-S6.5 N v. 31.3+/-3.0 N at 5 weeks after lengthening). The contribution of the gastrocnemius to the joint reaction force on the lengthened side was also significantly higher than the control side at both time points (mean+/-SD 9.0+/-1.3N v. 2.8+/-0.8 N at the end of lengthening, 5.3+/-1.4N v. 2.7+/-0.5N at 5 weeks after lengthening). There were significant knee and ankle joint contractures at the end of lengthening, as evidenced by decreased range of motion (mean+/-SD 27+/-8 degrees and 36+/-13 degrees, respectively), which remained 5 weeks after lengthening (mean+/-SD 26+/-6 degrees and 35+/-8 degrees, respectively). The gastrocnemius contributed about 20% of the joint reaction force, indicating that changes in the other intra- and extra-articular structures due to joint contracture may be more important in generating the joint reaction force.

Distraction-resisting force during tibial diaphyseal lengthening and consolidation--a study on a rabbit model.

BACKGROUND: Distraction-resisting force is generated in the soft tissues and callus during limb lengthening. Monitoring this force may offer a method of studying the behaviour of soft tissue and detecting the distraction osteogenesis related problems, and help to prevent complications. Changes in the post distraction period have not been previously investigated and there are no reports on the contribution of gastrocnemius to the distraction-resisting force. METHODS: Sixteen immature New Zealand White rabbits underwent 30% (left) tibial diaphyseal lengthening at a rate of two 0.4 mm increments per day. Using an instrumented bilateral fixator, the passive distraction-resisting force and the contribution made by gastrocnemius were measured at the end of lengthening and 5 weeks after lengthening. FINDINGS: The distraction-resisting force at the end of lengthening (mean 44 N (SD 10)) was statistically higher (p < 0.01) than that five weeks after lengthening (mean 20 N (SD 8)), so was the contribution of the gastrocnemius to the force (mean 11 N (SD 5 N) or 25% (SD 7) at the end of lengthening and 3 N (SD 1) or 13% (SD 5.2) five weeks later). INTERPRETATION: The callus rather than the surrounding muscles generates most of the passive DRF and its share of the force increased during consolidation period.

The effect of tibial lengthening on immature articular cartilage of the ankle joint.

Is the articular cartilage of the immature ankle joint damaged by tibial lengthening? Sixteen immature rabbits underwent a 30% diaphyseal lengthening by tibial callotasis. Damage was assessed by scanning electronic microscopy and histomorphometry at the completion of distraction and after an additional 5 weeks. Despite joint contracture, little damage in the articular cartilage was observed in contrast to the knee joint. The findings show that the immature ankle joint is more resilient to stress than the knee and implies that reduced weight bearing and decreased joint movement alone are not sufficient to cause cartilage damage, at least in the ankle.

Alterations in the self-renewal and differentiation ability of bone marrow mesenchymal stem cells in a mouse model of rheumatoid arthritis.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease primarily involving the synovium. Evidence in recent years has suggested that the bone marrow (BM) may be involved, and may even be the initiating site of the disease. Abnormalities in haemopoietic stem cells' (HSC) survival, proliferation and aging have been described in patients affected by RA and ascribed to abnormal support by the BM microenvironment. Mesenchymal stem cells (MSC) and their progeny constitute important components of the BM niche. In this study we test the hypothesis that the onset of inflammatory arthritis is associated with altered self-renewal and differentiation of bone marrow MSC, which alters the composition of the BM microenvironment. METHODS: We have used Balb/C Interleukin-1 receptor antagonist knock-out mice, which spontaneously develop RA-like disease in 100% of mice by 20 weeks of age to determine the number of mesenchymal progenitors and their differentiated progeny before, at the start and with progression of the disease. RESULTS: We showed a decrease in the number of mesenchymal progenitors with adipogenic potential and decreased bone marrow adipogenesis before disease onset. This is associated with a decrease in osteoclastogenesis. Moreover, at the onset of disease a significant increase in all mesenchymal progenitors is observed together with a block in their differentiation to osteoblasts. This is associated with accelerated bone loss. CONCLUSIONS: Significant changes occur in the BM niche with the establishment and progression of RA-like disease. Those changes may be responsible for aspects of the disease, including the advance of osteoporosis. An understanding of the molecular mechanisms leading to those changes may lead to new strategies for therapeutic intervention.

Inhibiting activin-A signaling stimulates bone formation and prevents cancer-induced bone destruction in vivo.

Cancers that grow in bone, such as myeloma and breast cancer metastases, cause devastating osteolytic bone destruction. These cancers hijack bone remodeling by stimulating osteoclastic bone resorption and suppressing bone formation. Currently, treatment is targeted primarily at blocking bone resorption, but this approach has achieved only limited success. Stimulating osteoblastic bone formation to promote repair is a novel alternative approach. We show that a soluble activin receptor type IIA fusion protein (ActRIIA.muFc) stimulates osteoblastogenesis (p < .01), promotes bone formation (p < .01) and increases bone mass in vivo (p < .001). We show that the development of osteolytic bone lesions in mice bearing murine myeloma cells is caused by both increased resorption (p < .05) and suppression of bone formation (p < .01). ActRIIA.muFc treatment stimulates osteoblastogenesis (p < .01), prevents myeloma-induced suppression of bone formation (p < .05), blocks the development of osteolytic bone lesions (p < .05), and increases survival (p < .05). We also show, in a murine model of breast cancer bone metastasis, that ActRIIA.muFc again prevents bone destruction (p < .001) and inhibits bone metastases (p < .05). These findings show that stimulating osteoblastic bone formation with ActRIIA.muFc blocks the formation of osteolytic bone lesions and bone metastases in models of myeloma and breast cancer and paves the way for new approaches to treating this debilitating aspect of cancer.

Bortezomib alone or in combination with the histone deacetylase inhibitor JNJ-26481585: effect on myeloma bone disease in the 5T2MM murine model of myeloma.

The proteasome inhibitor bortezomib (Velcade) is currently approved as second-line treatment of multiple myeloma (MM). MM-related bone disease is one of the most debilitating complications of MM. Besides supportive care with biphosphonates, which have proven efficacy in reducing and delaying skeletal-related events, there is no specific treatment of lytic bone lesions. The present study investigated the effect of bortezomib alone or in combination with a hydroxamate-based histone deacetylase inhibitor, JNJ-26481585 on tumor burden, and MM bone disease in the 5T2MM model. Injection of 5T2MM cells into C57Bl/KaLwRij mice resulted in MM bone disease, characterized by an increase in the percentage osteoclasts, a decrease in osteoblasts, trabecular bone volume, trabecular number, and the development of bone lesions. Treatment of 5T2MM-bearing mice with bortezomib significantly reduced tumor burden, angiogenesis, and MM bone disease. More importantly, the combination of bortezomib with JNJ-26481585 resulted in a more pronounced reduction of osteoclasts and increase of osteoblasts, trabecular bone volume, and trabecular number compared with bortezomib as single agent. These data suggest that bortezomib has bone remodeling properties that can be improved in combination with low dose JNJ-26481585. The study indicates that this combination therapy could be a useful strategy for the treatment of MM patients, especially in those patients with skeletal complications.

Inhibiting Dickkopf-1 (Dkk1) removes suppression of bone formation and prevents the development of osteolytic bone disease in multiple myeloma.

Multiple myeloma (MM) is associated with the development of osteolytic bone disease, mediated by increased osteoclastic bone resorption and impaired osteoblastic bone formation. Dickkopf-1 (Dkk1), a soluble inhibitor of wingless/int (Wnt) signaling and osteoblastogenesis, is elevated in patients with MM and correlates with osteolytic bone disease. In this study, we investigated the effect of inhibiting Dkk1 on the development of osteolytic lesions in the 5T2MM murine model of myeloma. We showed that Dkk1 is expressed by murine 5T2MM myeloma cells. Injection of 5T2MM cells into C57BL/KaLwRij mice resulted in the development of osteolytic bone lesions (p < 0.05), mediated by increased osteoclast numbers (p < 0.001) and a decrease in osteoblast numbers (p < 0.001) and mineralizing surface (p < 0.05). Mice bearing 5T2MM cells were treated with an anti-Dkk1 antibody (BHQ880, 10 mg/kg, IV, twice weekly for 4 wk) from time of paraprotein detection. Anti-Dkk1 treatment prevented 5T2MM-induced suppression of osteoblast numbers (p < 0.001) and surface (p < 0.001). Treatment increased mineralizing surface by 28% and bone formation rate by 25%; however, there was no change in mineral apposition rate. Inhibiting Dkk1 had no effect on osteoclast numbers. muCT analysis showed that anti-Dkk1 treatment significantly protected against 5T2MM-induced trabecular bone loss (p < 0.05) and reduced the development of osteolytic bone lesions (p < 0.05). Treatment had no significant effect on tumor burden. These data suggest that inhibiting Dkk1 prevents the suppression of bone formation and in doing so is effective in preventing the development of osteolytic bone disease in myeloma, offering an effective therapeutic approach to treating this clinically important aspect of myeloma.

The effect of tibial diaphyseal lengthening on the longitudinal growth of the tibia.

Limb lengthening by tibial callotasis is usually performed in the metaphysis but may cause growth inhibition. Is diaphyseal lengthening more advantageous? Sixteen immature rabbits underwent 30% diaphyseal lengthening by tibial callotasis. The tibial length was measured on radiographs at the end of the distraction period and after an additional 5 weeks. The proximal and distal growth plates were assessed histomorphometrically. Osteotomy stimulated tibial elongation; however, combined with diaphyseal lengthening the stimulation was suppressed resulting in longitudinal growth that matched the control side. In longer lengthenings of limbs diaphyseal callotasis may be more advantageous than metaphyseal by not inhibiting longitudinal growth.

Targeting the IGF-1R using picropodophyllin in the therapeutical 5T2MM mouse model of multiple myeloma: beneficial effects on tumor growth, angiogenesis, bone disease and survival.

During the last decade, a central role for insulin-like growth factor 1 (IGF-1) in the pathophysiology of multiple myeloma (MM) has been well established. IGF-I provided by the tumor-microenvironment interaction may directly and indirectly facilitate the migration, survival and expansion of the MM cells in the bone marrow (BM). The inhibition of the IGF-1R-mediated signaling pathway has recently been suggested to be a possible new therapeutic principle in MM. Using the mouse 5T2MM model, we now demonstrate that targeting the IGF-1R using picropodophyllin (PPP) in a therapeutical setting not only has strong antitumor activity on the established MM tumor but also influences the BM microenvironment by inhibiting angiogenesis and bone disease, having a profound effect on the survival of the mice. At therapeutically achievable concentrations of PPP, the average survival was 180 days for the PPP-treated mice as compared to 100 days for vehicle-treated mice. PPP used as single drug treatment in the 5T2MM model resulted in a decrease of tumor burden by 65% while the paraprotein concentrations were reduced by 75%. This decrease was associated with a significant inhibition of tumor-associated angiogenesis and osteolysis. The present studies on the biological effects of PPP in the 5T2MM model constitute an important experimental platform for future therapeutic implementation.

Effect of diaphyseal lengthening of tibia on knee joint reactive force / 中国矫形外科杂志

[Objective]To measure the in vivo knee joint reaction force in a rabbit model of tibial diaphyseal lengthening.[Method]Sixteen immature (8 weeks old) New Zealand White rabbits were underwent 30% (left) tibial diaphyseal lengthening at a rate of two 0.4 mm incremental lengthenings per day.The knee joint reaction force was measured at the end of lengthening (8 rabbits,13 weeks old) and 5 weeks later (8 rabbits,18 weeks old).An instrumented bilateral distractor and an extensometer were fixed cross the knee joint.The joint distraction force and distraction displacement were measured immediately after each incremental distraction of the joint.[Result]The joint reaction force on the lengthened side was significantly higher than that of the control side at both time points (44.4?7.8 N v.27.2?4.0 N at 13 weeks of age,44.3?6.5 N v.31.3?3.0 N at 18 weeks of age).[Conclusion]The knee joint reactive force could be increased in 30% tibial disphyseal lengthening which potentially could lead to the risk of damage to artic ular cartilage.