High-dose vitamin D to attenuate bone loss in patients with prostate cancer on androgen deprivation therapy: A phase 2 RCT.

BACKGROUND: Androgen deprivation therapy (ADT) inhibits prostate cancer growth. However, ADT causes loss of bone mineral density (BMD) and an increase in fracture risk; effective interventions for ADT-induced bone loss are limited. METHODS: A phase 2 randomized controlled trial investigated the feasibility, safety, and preliminary efficacy of high-dose weekly vitamin D (HDVD, 50,000 IU/week) versus placebo for 24 weeks in patients with prostate cancer receiving ADT, with all subjects receiving 600 IU/day vitamin D and 1000 mg/day calcium. Participants were ≥60 years (mean years, 67.7), had a serum 25-hydroxyvitamin D level <32 ng/mL, and initiated ADT within the previous 6 months. At baseline and after intervention, dual-energy x-ray absorptiometry was used to assess BMD, and levels of bone cell, bone formation, and resorption were measured. RESULTS: The HDVD group (N = 29) lost 1.5% BMD at the total hip vs. 4.1% for the low-dose group (N = 30; p = .03) and 1.7% BMD at the femoral neck vs. 4.4% in the low-dose group (p = .06). Stratified analyses showed that, for those with baseline 25-hydroxyvitamin D level <27 ng/mL, the HDVD group lost 2.3% BMD at the total hip vs 7.1% for the low-dose group (p < .01). Those in the HDVD arm showed significant changes in parathyroid hormone (p < .01), osteoprotegerin (p < 0.01), N-terminal telopeptide of type 1 collagen (p < 0.01) and C-terminal telopeptide of type 1 collagen (p < 0.01). No difference in adverse events or toxicity was noted between the groups. CONCLUSIONS: HDVD supplementation significantly reduced hip and femoral neck BMD loss, especially for patients with low baseline serum 25-hydroxyvitamin D levels, although demonstrating safety and feasibility in prostate cancer patients on ADT.

Neonatal hyperoxia impairs adipogenesis of bone marrow-derived mesenchymal stem cells and fat accumulation in adult mice.

Preterm birth is a risk factor for growth failure and development of respiratory disease in children and young adults. Their early exposure to oxygen may contribute to lung disease because adult mice exposed to hyperoxia as neonates display reduced lung function, changes in the host response to respiratory viral infections, and develop pulmonary hypertension and heart failure that shortens their lifespan. Here, we provide new evidence that neonatal hyperoxia also impairs growth by inhibiting fat accumulation. Failure to accumulate fat may reflect a systemic defect in adipogenic potential of stem cells because bone marrow-derived mesenchymal cells (BMSCs) isolated from the mice grew slower and were more oxidized compared to controls. They also displayed reduced capacity to accumulate lipid and differentiate into adipocytes. BMSCs from adult mice exposed to neonatal hyperoxia express lower levels of peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor that drives adipocyte differentiation. The defect in adipogenesis was rescued by expressing PPARγ in these cells. These findings reveal early life exposure to high levels of oxygen may suppresses fat accumulation and impair adipogenic differentiation upstream of PPARγ signaling, thus potentially contributing to growth failure seen in people born preterm.

Secondary Fracture Prevention: Consensus Clinical Recommendations from a Multistakeholder Coalition.

Osteoporosis-related fractures are undertreated, due in part to misinformation about recommended approaches to patient care and discrepancies among treatment guidelines. To help bridge this gap and improve patient outcomes, the American Society for Bone and Mineral Research assembled a multistakeholder coalition to develop clinical recommendations for the optimal prevention of secondary fractureamong people aged 65 years and older with a hip or vertebral fracture. The coalition developed 13 recommendations (7 primary and 6 secondary) strongly supported by the empirical literature. The coalition recommends increased communication with patients regarding fracture risk, mortality and morbidity outcomes, and fracture risk reduction. Risk assessment (including fall history) should occur at regular intervals with referral to physical and/or occupational therapy as appropriate. Oral, intravenous, andsubcutaneous pharmacotherapies are efficaciousandcanreduce risk of future fracture.Patientsneededucation,however, about thebenefitsandrisks of both treatment and not receiving treatment. Oral bisphosphonates alendronate and risedronate are first-line options and are generally well tolerated; otherwise, intravenous zoledronic acid and subcutaneous denosumab can be considered. Anabolic agents are expensive butmay be beneficial for selected patients at high risk.Optimal duration of pharmacotherapy is unknown but because the risk for second fractures is highest in the earlypost-fractureperiod,prompt treatment is recommended.Adequate dietary or supplemental vitaminDand calciumintake shouldbe assured. Individuals beingtreatedfor osteoporosis shouldbe reevaluated for fracture risk routinely, includingvia patienteducationabout osteoporosisandfracturesandmonitoringfor adverse treatment effects.Patients shouldbestronglyencouraged to avoid tobacco, consume alcohol inmoderation atmost, and engage in regular exercise and fall prevention strategies. Finally, referral to endocrinologists or other osteoporosis specialists may be warranted for individuals who experience repeated fracture or bone loss and those with complicating comorbidities (eg, hyperparathyroidism, chronic kidney disease).

Secondary Fracture Prevention: Consensus Clinical Recommendations from a Multistakeholder Coalition.

Osteoporosis-related fractures are undertreated, due in part to misinformation about recommended approaches to patient care and discrepancies among treatment guidelines. To help bridge this gap and improve patient outcomes, the American Society for Bone and Mineral Research assembled a multistakeholder coalition to develop clinical recommendations for the optimal prevention of secondary fracture among people aged 65 years and older with a hip or vertebral fracture. The coalition developed 13 recommendations (7 primary and 6 secondary) strongly supported by the empirical literature. The coalition recommends increased communication with patients regarding fracture risk, mortality and morbidity outcomes, and fracture risk reduction. Risk assessment (including fall history) should occur at regular intervals with referral to physical and/or occupational therapy as appropriate. Oral, intravenous, and subcutaneous pharmacotherapies are efficacious and can reduce risk of future fracture. Patients need education, however, about the benefits and risks of both treatment and not receiving treatment. Oral bisphosphonates alendronate and risedronate are first-line options and are generally well tolerated; otherwise, intravenous zoledronic acid and subcutaneous denosumab can be considered. Anabolic agents are expensive but may be beneficial for selected patients at high risk. Optimal duration of pharmacotherapy is unknown but because the risk for second fractures is highest in the early post-fracture period, prompt treatment is recommended. Adequate dietary or supplemental vitamin D and calcium intake should be assured. Individuals being treated for osteoporosis should be reevaluated for fracture risk routinely, including via patient education about osteoporosis and fractures and monitoring for adverse treatment effects. Patients should be strongly encouraged to avoid tobacco, consume alcohol in moderation at most, and engage in regular exercise and fall prevention strategies. Finally, referral to endocrinologists or other osteoporosis specialists may be warranted for individuals who experience repeated fracture or bone loss and those with complicating comorbidities (eg, hyperparathyroidism, chronic kidney disease). © 2019 American Society for Bone and Mineral Research.

The effects of high-dose calcitriol and individualized exercise on bone metabolism in breast cancer survivors on hormonal therapy: a phase II feasibility trial.

INTRODUCTION: Cancer treatment-induced bone loss (CTIBL) is a long-term side effect of breast cancer therapy. Both calcitriol and weight-bearing exercise improve bone metabolism for osteoporotic patients, but are unproven in a breast cancer population. We used a novel high-dose calcitriol regimen with an individualized exercise intervention to improve bone metabolism in breast cancer survivors. METHODS: We accrued 41 subjects to this open label, 2 × 2 factorial, randomized feasibility trial. Breast cancer survivors were randomized to receive the following: (1) calcitriol (45 micrograms/week), (2) individualized exercise with progressive walking and resistance training, (3) both, or (4) a daily multivitamin (control condition) for 12 weeks. Primary outcomes included changes in biomarkers of bone formation, bone resorption, and the bone remodeling index, a composite measure of bone formation and resorption. Safety measures included clinical and biochemical adverse events. A main effect analysis was used for these endpoints. RESULTS: Hypercalcemia was limited to three grade I cases with no grade ≥ 2 cases. Among exercisers, 100% engaged in the prescribed aerobic training and 44.4% engaged in the prescribed resistance training. Calcitriol significantly improved bone formation (Cohen's d = 0.64; p < 0.01), resulting in a non-significant increase in the bone remodeling index (Cohen's d = 0.21; p = 31). Exercise failed to improve any of the bone biomarkers. CONCLUSIONS: Both calcitriol and exercise were shown to be feasible and well tolerated. Calcitriol significantly improved bone formation, resulting in a net increase of bone metabolism. Compliance with the exercise intervention was sub-optimal, which may have led to a lack of effect of exercise on bone metabolism.

Multivalent Presentation of Peptide Targeting Groups Alters Polymer Biodistribution to Target Tissues.

Drug delivery to bone is challenging, whereby drug distribution is commonly <1% of injected dose, despite development of several bone-targeted drug delivery systems specific to hydroxyapatite. These bone-targeted drug delivery systems still suffer from poor target cell localization within bone, as at any given time overall bone volume is far greater than acutely remodeling bone volume, which harbors relevant cell targets (osteoclasts or osteoblasts). Thus, there exists a need to target bone-acting drugs specifically to sites of bone remodeling. To address this need, this study synthesized oligo(ethylene glycol) copolymers based on a peptide with high affinity to tartrate-resistant acid phosphatase (TRAP), an enzyme deposited by osteoclasts during the bone resorption phase of bone remodeling, which provides greater specificity relevant for bone cell drugging. Gradient and random peptide orientations, as well as polymer molecular weights, were investigated. TRAP-targeted, high molecular weight (Mn) random copolymers exhibited superior accumulation in remodeling bone, where fracture accumulation was observed for at least 1 week and accounted for 14% of tissue distribution. Intermediate and low Mn random copolymer accumulation was lower, indicating residence time depends on Mn. High Mn gradient polymers were cleared, with only 2% persisting at fractures after 1 week, suggesting TRAP binding depends on peptide density. Peptide density and Mn are easily modified in this versatile targeting platform, which can be applied to a range of bone drug delivery applications.

Fracture-Targeted Delivery of ß-Catenin Agonists via Peptide-Functionalized Nanoparticles Augments Fracture Healing.

Despite several decades of progress, bone-specific drug delivery is still a major challenge. Current bone-acting drugs require high-dose systemic administration which decreases therapeutic efficacy and increases off-target tissue effects. Here, a bone-targeted nanoparticle (NP) delivery system for a ß-catenin agonist, 3-amino-6-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-N-(pyridin-3-yl)pyrazine-2-carboxamide, a glycogen synthase kinase 3 beta (GSK-3ß) inhibitor, was developed to enhance fracture healing. The GSK-3ß inhibitor loading capacity was found to be 15 wt % within highly stable poly(styrene-alt-maleic anhydride)-b-poly(styrene) NPs, resulting in ∼50 nm particles with ∼ -30 mV surface charge. A peptide with high affinity for tartrate-resistant acid phosphatase (TRAP), a protein deposited by osteoclasts on bone resorptive surfaces, was introduced to the NP corona to achieve preferential delivery to fractured bone. Targeted NPs showed improved pharmacokinetic profiles with greater accumulation at fractured bone, accompanied by significant uptake in regenerative cell types (mesenchymal stem cells (MSCs) and osteoblasts). MSCs treated with drug-loaded NPs in vitro exhibited 2-fold greater ß-catenin signaling than free drug that was sustained for 5 days. To verify similar activity in vivo, TOPGAL reporter mice bearing fractures were treated with targeted GSK-3ß inhibitor-loaded NPs. Robust ß-galactosidase activity was observed in fracture callus and periosteum treated with targeted carriers versus controls, indicating potent ß-catenin activation during the healing process. Enhanced bone formation and microarchitecture were observed in mice treated with GSK-3ß inhibitor delivered via TRAP-binding peptide-targeted NPs. Specifically, increased bone bridging, ∼4-fold greater torsional rigidity, and greater volumes of newly deposited bone were observed 28 days after treatment, indicating expedited fracture healing.

Sclerostin activity plays a key role in the negative effect of glucocorticoid signaling on osteoblast function in mice.

Stress during prenatal development is correlated with detrimental cognitive and behavioral outcomes in offspring. However, the long-term impact of prenatal stress (PS) and disrupted glucocorticoid signaling on bone mass and strength is not understood. In contrast, the detrimental effect of lead (Pb) on skeletal health is well documented. As stress and Pb act on common biological targets via glucocorticoid signaling pathways and co-occur in the environment, this study first sought to assess the combined effect of stress and Pb on bone quality in association with alterations in glucocorticoid signaling. Bone parameters were evaluated using microCT, histomorphometry, and strength determination in 8-month-old male mouse offspring subjected to PS on gestational days 16 and 17, lifetime Pb exposure (100 p.p.m. Pb in drinking water), or to both. Pb reduced trabecular bone mass and, when combined with PS, Pb unmasked an exaggerated decrement in bone mass and tensile strength. Next, to characterize a mechanism of glucocorticoid effect on bone, prednisolone was implanted subcutaneously (controlled-release pellet, 5 mg·kg-1 per day) in 5-month-old mice that decreased osteoblastic activity and increased sclerostin and leptin levels. Furthermore, the synthetic glucocorticoid dexamethasone alters the anabolic Wnt signaling pathway. The Wnt pathway inhibitor sclerostin has several glucocorticoid response elements, and dexamethasone administration to osteoblastic cells induces sclerostin expression. Dexamethasone treatment of isolated bone marrow cells decreased bone nodule formation, whereas removal of sclerostin protected against this decrement in mineralization. Collectively, these findings suggest that bone loss associated with steroid-induced osteoporosis is a consequence of sclerostin-mediated restriction of Wnt signaling, which may mechanistically facilitate glucocorticoid toxicity in bone.

Elevated Lifetime Lead Exposure Impedes Osteoclast Activity and Produces an Increase in Bone Mass in Adolescent Mice.

The heavy metal lead (Pb) has a deleterious effect on skeletal health. Because bone mass is maintained through a balance of bone formation and resorption, it is important to understand the effect of Pb levels on osteoblastic and osteoclastic activity. Pb exposure is associated with low bone mass in animal models and human populations; however, the correlation between Pb dosing and corresponding bone mass has been poorly explored. Thus, mice were exposed to increasing Pb and at higher levels (500 ppm), there was unexpectedly an increase in femur-tibial bone mass by 3 months of age. This is contrary to several studies alluded to earlier. Increased bone volume (BV) was accompanied by a significant increase in cortical thickness of the femur and trabecular bone that extended beyond the epiphyseal area into the marrow cavity. Subsequent evaluations revealed an increase in osteoclast numbers with high Pb exposure, but a deficiency in osteoclastic activity. These findings were substantiated by observed increases in levels of the resorption-altering hormones calcitonin and estrogen. In addition we found that pro-osteoclastic nuclear factor-kappa beta (NF-κB) pathway activity was dose dependently elevated with Pb, both in vivo and in vitro. However, the ability of osteoclasts to resorb bone was depressed in the presence of Pb in media and within test bone wafers. These findings indicate that exposure to high Pb levels disrupts early life bone accrual that may involve a disruption of osteoclast activity. This study accentuates the dose dependent variation in Pb exposure and consequent effects on skeletal health.

Heavy Metal Ion Regulation of Gene Expression: MECHANISMS BY WHICH LEAD INHIBITS OSTEOBLASTIC BONE-FORMING ACTIVITY THROUGH MODULATION OF THE Wnt/ß-CATENIN SIGNALING PATHWAY.

Exposure to lead (Pb) from environmental sources remains an overlooked and serious public health risk. Starting in childhood, Pb in the skeleton can disrupt epiphyseal plate function, constrain the growth of long bones, and prevent attainment of a high peak bone mass, all of which will increase susceptibility to osteoporosis later in life. We hypothesize that the effects of Pb on bone mass, in part, come from depression of Wnt/ß-catenin signaling, a critical anabolic pathway for osteoblastic bone formation. In this study, we show that depression of Wnt signaling by Pb is due to increased sclerostin levels in vitro and in vivo. Downstream activation of the ß-catenin pathway using a pharmacological inhibitor of GSK-3ß ameliorates the Pb inhibition of Wnt signaling activity in the TOPGAL reporter mouse. The effect of Pb was determined to be dependent on sclerostin expression through use of the SOST gene knock-out mice, which are resistant to Pb-induced trabecular bone loss and maintain their mechanical bone strength. Moreover, isolated bone marrow cells from the sclerostin null mice show improved bone formation potential even after exposure to Pb. Also, our data suggest that the TGFß canonical signaling pathway is the mechanism by which Pb controls sclerostin production. Taken together these results support our hypothesis that the osteoporotic-like phenotype observed after Pb exposure is, in part, regulated through modulation of the Wnt/ß-catenin pathway.

Effects of Combined Exposure to Lead and High-Fat Diet on Bone Quality in Juvenile Male Mice.

BACKGROUND: Lead (Pb) exposure and obesity are co-occurring risk factors for decreased bone mass in the young, particularly in low socioeconomic communities. OBJECTIVES: The goal of this study was to determine whether the comorbidities of Pb exposure and high-fat diet-induced obesity amplify skeletal deficits independently associated with each of these risk factors, and to explore associated mechanisms of the observed deficiencies. METHODS: Five-week-old male C57BL/6J mice were placed on low-fat (10% kcal, LFD) or high-fat (60% kcal, HFD) diets for 12 weeks. Mice were exposed to lifetime Pb (50 ppm) through drinking water. RESULTS: HFD was associated with increased body mass and glucose intolerance. Both HFD and Pb increased fasting glucose and serum leptin levels. Pb and HFD each reduced trabecular bone quality and together had a further detrimental effect on these bone parameters. Mechanical bone properties of strength were depressed in Pb-exposed bones, but HFD had no significant effect. Both Pb and HFD altered progenitor cell differentiation, promoting osteoclastogenesis and increasing adipogenesis while suppressing osteoblastogenesis. In support of this lineage shift being mediated through altered Wnt signaling, Pb and non-esterified fatty acids in MC3T3 cells increased in vitro PPAR-γ activity and inhibited ß-catenin activity. Combining Pb and non-esterified fatty acids enhanced these effects. CONCLUSIONS: Pb and HFD produced selective deficits in bone accrual that were associated with alterations in progenitor cell activity that may involve reduced Wnt signaling. This study emphasizes the need to assess toxicants together with other risk factors relevant to human health and disease.

Inhibition of beta-catenin signaling by Pb leads to incomplete fracture healing.

There is strong evidence in the clinical literature to suggest that elevated lead (Pb) exposure impairs fracture healing. Since Pb has been demonstrated to inhibit bone formation, and Wnt signaling is an important anabolic pathway in chondrocyte maturation and endochondral ossification, we investigated the impact of Wnt therapy on Pb-exposed mice undergoing bone repair in a mouse tibial fracture model. We established that tibial fracture calluses from Pb-treated mice were smaller and contained less mineralized tissue than vehicle controls. This resulted in the persistence of immature cartilage in the callus and decreased ß-catenin levels. Reduction of ß-catenin protein was concurrent with systemic elevation of LRP5/6 antagonists DKK1 and sclerostin in Pb-exposed mice throughout fracture healing. ß-catenin stimulation by the GSK3 inhibitor BIO reversed these molecular changes and restored the amount of mineralized callus. Overall, Pb is identified as a potent inhibitor of endochondral ossification in vivo with correlated effects on bone healing with noted deficits in ß-catenin signaling, suggesting the Wnt/ß-catenin as a pivotal pathway in the influence of Pb on fracture repair.

Heavy metal lead exposure, osteoporotic-like phenotype in an animal model, and depression of Wnt signaling.

BACKGROUND: Exposure to lead (Pb) from environmental and industrial sources remains an overlooked serious public health risk. Elucidating the effect of Pb on bone cell function is therefore critical for understanding its risk associated with diseases of low bone mass. OBJECTIVES: We tested the hypothesis that Pb negatively affects bone mass. We also assessed the underlying mechanisms of Pb on bone signaling pathways. METHODS: We used a model of low-level Pb exposure in a rodent beginning before conception and continuing over 18 months. We characterized the effect of Pb on bone quality using dual-energy X-ray absorptiometry (DXA), micro-computed tomography, Raman spectroscopy, and histology. We assessed the effect of Pb on bone and adipocyte formation by mineral deposition, lipid droplet formation, and Western blot and RNA analysis. RESULTS: Pb-exposed animals had decreased bone mass that resulted in bones that were more susceptible to fracture. Pb decreased osteoblastic cell number leading to a depression of bone formation. Accompanying this, Pb exposure elevated sclerostin protein levels in the skeleton, and correspondingly reduced levels of ß-catenin and Runx2 in stromal precursor cells. Pb also increased skeletal expression of peroxisome proliferator-activated receptor-γ (PPAR-γ). These results indicate a shift in mesenchymal differentiation wherein Pb promoted enhanced adipogenesis and decreased osteoblastogenesis. Substantial differences in bone marrow composition were observed, highlighted by an increase in adipocytes. CONCLUSIONS: The disruption Pb has on bone mass and bone homeostasis is principally explained by inhibition of the Wnt/ß-catenin pathway, which may provide a molecular basis for novel therapeutic strategies to combat Pb-induced bone pathologies.

Influence of upper extremity assistance on lower extremity force application symmetry in individuals post-hip fracture during the sit-to-stand task.

STUDY DESIGN: Controlled laboratory study using a cross-sectional design. OBJECTIVES: To compare lower extremity force applications during a sit-to-stand (STS) task with and without upper extremity assistance in older individuals post-hip fracture to those of age-matched controls. BACKGROUND: A recent study documented the dependence on upper extremity assistance and the uninvolved lower limb during an STS task in individuals post-hip fracture. This study extends this work by examining the effect of upper extremity assistance on symmetry of lower extremity force applications. METHODS: Twenty-eight community-dwelling elderly subjects, 14 who had recovered from a hip fracture and 14 controls, participated in the study. All participants were independent ambulators. Four force plates were used to determine lower extremity force applications during an STS task with and without upper extremity assistance. The summed vertical ground reaction forces (vGRFs) of both limbs were used to determine STS phases (preparation/rising). The lower extremity force applications were assessed statistically using analysis of variance models. RESULTS: During the preparation phase, side-to-side symmetry of the rate of force development was significantly lower for the hip fracture group for both STS tasks (P<.001). During the rising phase, the vGRF impulse of the involved limb was significantly lower for the hip fracture group for both STS tasks (P = .045). The vGRF impulse for the uninvolved limb was significantly increased when participants with hip fracture did not use upper extremity assistance compared to elderly controls (P = .002). This resulted in a significantly lower vGRF symmetry for the hip fracture group during both STS tasks (P<.001). CONCLUSION: Participants with hip fracture who were discharged from rehabilitative care demonstrated decreased side-to-side symmetry of lower extremity loading during an STS task, irrespective of whether upper extremity assistance was provided. These findings suggest that learned motor control strategies may influence movement patterns post-hip fracture.

Lead induces an osteoarthritis-like phenotype in articular chondrocytes through disruption of TGF-ß signaling.

Lead remains a significant environmental toxin, and we believe we may have identified a novel target of lead toxicity in articular chondrocytes. These cells are responsible for the maintenance of joint matrix, and do so under the regulation of TGF-ß signaling. As lead is concentrated in articular cartilage, we hypothesize that it can disrupt normal chondrocyte phenotype through suppression of TGF-ß signaling. These experiments examine the effects of lead exposure in vivo and in vitro at biologically relevant levels, from 1 nM to 10 µM on viability, collagen levels, matrix degrading enzyme activity, TGF-ß signaling, and articular surface morphology. Our results indicate that viability was unchanged at levels ≤100 µM Pb, but low and high level lead in vivo exposure resulted in fibrillation and degeneration of the articular surface. Lead treatment also decreased levels of type II collagen and increased type X collagen, in vivo and in vitro. Additionally, MMP13 activity increased in a dose-dependent manner. Active caspase 3 and 8 were dose-dependently elevated, and treatment with 10 µM Pb resulted in increases of 30% and 500%, respectively. Increasing lead treatment resulted in a corresponding reduction in TGF-ß reporter activity, with a 95% reduction at 10µM. Levels of phosphoSmad2 and 3 were suppressed in vitro and in vivo and lead dose-dependently increased Smurf2. These changes closely parallel those seen in osteoarthritis. Over time this phenotypic shift could compromise maintenance of the joint matrix.

TRIP-1: a regulator of osteoblast function.

Transforming growth factor ß (TGFß) receptor interacting protein-1 (TRIP-1) is an intracellular protein expressed in osteoblasts with high affinity for type 5b tartrate resistant acid phosphatase (TRAP). It is suggested that through this interaction, TRIP-1 serves as a positive regulator of TGFß signaling and osteoblast differentiation during bone remodeling. We show here that TRIP-1 is abundant in osteoblasts in vivo and in vitro. TRIP-1 mRNA and protein expression were increased at early stages and decreased at later stages during osteoblast differentiation, suggesting a predominant role during early maturation. To investigate a role during bone remodeling, primary osteoblasts were treated with different hormones and factors that are known to affect remodeling. TRIP-1 levels were decreased with dexamethasone and increased with vitamin D(3) , dihydrotestosterone (DHT), TGFß1, and bone morphogenic protein 2 (BMP-2). Treatment with parathyroid hormone (PTH) and ß-estradiol did not affect TRIP-1 levels. Transfected small interfering RNA (siRNA) against TRIP-1 inhibited osteoblast differentiation as characterized by a decrease in alkaline phosphatase staining and enzyme activity, and decrease in the expression of collagen I, alkaline phosphatase, Runx2, osteopontin, and osteocalcin. The proliferation of osteoblasts was also affected by TRIP-1 siRNA. This particular effect was defined by decreased cell number, marked reduction of cyclin D1, a 38% decrease of cells in S phase (p < 0.001) and a 97% increase of cells in the G2/M phase (p < 0.01) of the cell cycle. However, TRIP-1 siRNA did not induce an effect in apoptosis. Using a TGFß luciferase reporter we found that knocking down TRIP-1 decreased the activation of TGFß signaling by 40% percent (p < 0.001). In conclusion, our characterization of TRIP-1 in osteoblasts provides the first evidence of its key role as a positive regulator of osteoblast function.

Teriparatide as a chondroregenerative therapy for injury-induced osteoarthritis.

There is no disease-modifying therapy for osteoarthritis, a degenerative joint disease that is projected to afflict more than 67 million individuals in the United States alone by 2030. Because disease pathogenesis is associated with inappropriate articular chondrocyte maturation resembling that seen during normal endochondral ossification, pathways that govern the maturation of articular chondrocytes are candidate therapeutic targets. It is well established that parathyroid hormone (PTH) acting via the type 1 PTH receptor induces matrix synthesis and suppresses maturation of chondrocytes. We report that the PTH receptor is up-regulated in articular chondrocytes after meniscal injury and in osteoarthritis in humans and in a mouse model of injury-induced knee osteoarthritis. To test whether recombinant human PTH(1-34) (teriparatide) would inhibit aberrant chondrocyte maturation and associated articular cartilage degeneration, we administered systemic teriparatide (Forteo), a Food and Drug Administration-approved treatment for osteoporosis, either immediately after or 8 weeks after meniscal/ligamentous injury in mice. Knee joints were harvested at 4, 8, or 12 weeks after injury to examine the effects of teriparatide on cartilage degeneration and articular chondrocyte maturation. Microcomputed tomography revealed increased bone volume within joints from teriparatide-treated mice compared to saline-treated control animals. Immediate systemic administration of teriparatide increased proteoglycan content and inhibited articular cartilage degeneration, whereas delayed treatment beginning 8 weeks after injury induced a regenerative effect. The chondroprotective and chondroregenerative effects of teriparatide correlated with decreased expression of type X collagen, RUNX2 (runt-related transcription factor 2), matrix metalloproteinase 13, and the carboxyl-terminal aggrecan cleavage product NITEGE. These preclinical findings provide proof of concept that Forteo may be useful for decelerating cartilage degeneration and inducing matrix regeneration in patients with osteoarthritis.