Bone health issues in breast cancer survivors: a Medicare Current Beneficiary Survey (MCBS) study.

PURPOSE: Breast cancer treatments (chemotherapy and hormone therapy) can cause a rapid loss in bone mineral density, leading to osteoporosis and fractures later in life. Fortunately, preventative measures (vitamin D, exercise, etc.) can delay bone loss if employed early enough. This study compares the prevalence of osteoporosis and osteoporosis-related discussions with physicians among female breast cancer survivors and females with no cancer history to determine if breast cancer patients are being correctly advised on their high risk of bone loss. METHODS: The 2003 Medicare Current Beneficiary Survey, a nationally representative sample of 550 women with a breast cancer history and 6,673 women with no cancer history aged ≥65, was used. The first set of dependent variables collected information on bone health (osteoporosis, falls, and fractures). The second set of dependent variables collected information on bone health discussions with their physician. Multivariate logistic regression models were used to evaluate whether breast cancer was independently associated with bone health issues. RESULTS: After adjustment for confounders, a breast cancer diagnosis was found to be associated with a higher prevalence of an osteoporosis diagnosis over their lifetime (adjusted odds ratio (OR(adj)) = 1.32, 95 % confidence interval (95 % CI) = 1.08-1.61) and falls in the previous year (OR(adj) = 1.23, 95 % CI = 1.01-1.51) compared to respondents without a cancer diagnosis. However, breast cancer respondents were not more likely than respondents without a cancer diagnosis to discuss osteoporosis with their physician (OR(adj) = 1.20, 95 % CI = 0.96-1.50) or be told they are at high risk for osteoporosis (OR(adj) = 1.41, 95 % CI = 0.95-2.10). CONCLUSIONS: A breast cancer diagnosis was associated with an increased prevalence of osteoporosis and falls. Nevertheless, breast cancer respondents were not more likely to discuss osteoporosis with their physician nor were they more likely to be considered high risk for osteoporosis. Increased dialogue between physician and breast cancer patient pertaining to bone loss is needed.

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.

Ski inhibits TGF-ß/phospho-Smad3 signaling and accelerates hypertrophic differentiation in chondrocytes.

Since transforming growing factor-ß (TGF-ß)/Smad signaling inhibits chondrocyte maturation, endogenous negative regulators of TGF-ß signaling are likely also important regulators of the chondrocyte differentiation process. One such negative regulator, Ski, is an oncoprotein that is known to inhibit TGF-ß/Smad3 signaling via its interaction with phospho-Smad3 and recruitment of histone deacetylases (HDACs) to the DNA binding complex. Based on this, we hypothesized that Ski inhibits TGF-ß signaling and accelerates maturation in chondrocytes via recruitment of HDACs to transcriptional complexes containing Smads. We tested this hypothesis in chick upper sternal chondrocytes (USCs), where gain and loss of Ski expression experiments were performed. Over-expression of Ski not only reversed the inhibitory effect of TGF-ß on the expression of hypertrophic marker genes such as type X collagen (colX) and osteocalcin, it induced these genes basally as well. Conversely, knockdown of Ski by RNA interference led to a reduction of colX and osteocalcin expression under basal conditions. Furthermore, Ski blocked TGF-ß induction of cyclinD1 and caused a basal up-regulation of Runx2, consistent with the observed acceleration of hypertrophy. Regarding mechanism, not only does Ski associate with phospho-Smad2 and 3, but its association with phospho-Smad3 is required for recruitment of HDAC4 and 5. Implicating this recruitment of HDACs in the phenotypic effects of Ski in chondrocytes, the HDAC inhibitor SAHA reversed the up-regulation of colX and osteocalcin in Ski over-expressing cells. These results suggest that inhibition of TGF-ß signaling by Ski, which involves its association with phospho-Smad3 and recruitment of HDAC4 and 5, leads to accelerated chondrocyte differentiation.

Targeting radioresistant osteosarcoma cells with parthenolide.

Osteosarcoma is a devastating tumor of bone, primarily affecting adolescents. Osteosarcoma tumors are notoriously radioresistant. Radioresistant cancers, including osteosarcoma, typically exhibit a considerable potential for relapse and development of metastases following treatment. Relapse and metastatic potential can, in part, be due to a specific radioresistant subpopulation of cells with stem-like characteristics, cancer stem cells, which maintain the capacity to regenerate entire tumors. In the current study, we have investigated whether in vitro treatments with parthenolide, a naturally occurring small molecule that interferes with NF-κB signaling and has various other effects, will re-sensitize cancer stem cells and the entire cell population to radiotherapy in osteosarcoma. Our results indicate that parthenolide and ionizing radiation synergistically induce cell death in LM7 osteosarcoma cells. Importantly, the combination treatment results in a significant reduction in the viability of both the overall population of osteosarcoma cells and the cancer stem cell subpopulation. This effect is dependent on the ability of parthenolide to induce oxidative stress. Therefore, as a supplement to current multimodal therapy, parthenolide may sensitize osteosarcoma tumors to radiation and greatly reduce the prevalence of relapse and metastatic progression.

High-fat diet accelerates progression of osteoarthritis after meniscal/ligamentous injury.

INTRODUCTION: Increasing obesity and type 2 diabetes, in part due to the high-fat (HF) Western diet, parallels an increased incidence of osteoarthritis (OA). This study was undertaken to establish a causal relation between the HF diet and accelerated OA progression in a mouse model and to determine the relative roles of weight gain and metabolic dysregulation in this progression. METHODS: Five-week-old C57BL/6 mice were placed on HF (60% kcal) or low-fat (lean, 10% kcal) diets for 8 or 12 weeks before transecting the medial collateral ligament and excising a segment of the medial meniscus of the knee to initiate OA. One group was switched from lean to HF diet at the time of surgery. RESULTS: Body weight of mice on the HF diet peaked at 45.9 ± 2.1 g compared with 29.9 ± 1.8 g for lean diets, with only those on the HF becoming diabetic. Severity of OA was greater in HF mice, evidenced by the Osteoarthritis Research Society International (OARSI) histopathology initiative scoring method for mice and articular cartilage thickness and area. To assess the importance of weight gain, short- and long-term HF diets were compared with the lean diet. Short- and long-term HF groups outweighed lean controls by 6.2 g and 20.5 g, respectively. Both HF groups became diabetic, and OA progression, evidenced by increased OARSI score, decreased cartilage thickness, and increased osteophyte diameter, was comparably accelerated relative to those of lean controls. CONCLUSIONS: These results demonstrate that the HF diet accelerates progression of OA in a type 2 diabetic mouse model without correlation to weight gain, suggesting that metabolic dysregulation is a comorbid factor in OA-related cartilage degeneration.

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.

Establishment of an index with increased sensitivity for assessing murine arthritis.

The goals of our study were to establish quantitative outcomes for assessing murine knee arthritis and develop an Arthritis Index that incorporates multiple outcomes into a single calculation that provides enhanced sensitivity. Using an accepted model of meniscal/ligamentous injury (MLI)-induced osteoarthritis (OA), we assessed mouse knee arthritis using several approaches. Histology-based methods were performed to visualize joint tissues including articular cartilage and subchondral bone. Accepted histologic scoring methods and histomorphometry were performed to grade cartilage degeneration and determine articular cartilage area, respectively. MicroCT was used to visualize and quantify the bony structures of the joint including osteophytes and joint bone volume. A statistical algorithm was then developed that combined histologic scores and cartilage areas into a single Arthritis Index. MLI induced progressive, OA-like articular cartilage degeneration characterized by increasing (worsening) histologic score and decreasing cartilage area. MicroCT revealed osteophytes and increased joint bone volume between the femoral and tibial physes following MLI. Lastly, an Arthritis Index calculation was established, which incorporated histologic scoring and cartilage area. The Arthritis Index provided enhanced quantitative sensitivity in assessing the level of joint degeneration compared to either histologic scoring or cartilage area determination alone; when using the Index, between 29% and 43% fewer samples are needed to establish statistical significance in studies of murine arthritis. Arthritis in the mouse knee can be quantitatively assessed by histologic scoring, measuring cartilage area, and determining joint bone volume. Enhanced sensitivity can be achieved by performing the Arthritis Index calculation, a novel method for quantitatively assessing mouse knee arthritis.

The effect of various vitamin D supplementation regimens in breast cancer patients.

Vitamin D deficiency in the patients treated for breast cancer is associated with numerous adverse effects (bone loss, arthralgia, and falls). The first aim of this study was to assess vitamin D status, determined by 25-OH vitamin D levels, among women diagnosed with breast cancer according to demographic/clinical variables and bone mineral density (BMD). The second aim of this study was to evaluate the effect of daily low-dose and weekly high-dose vitamin D supplementation on 25-OH vitamin D levels. This retrospective study included 224 women diagnosed with stage 0-III breast cancer who received treatment at the James P. Wilmot Cancer Center at the University of Rochester Medical Center. Total 25-OH vitamin D levels (D(2) + D(3)) were determined at baseline for all participants. Vitamin D deficiency was defined as a 25-OH vitamin D level < 20 ng/ml, insufficiency as 20-31 ng/ml, and sufficiency as ≥32 ng/ml. BMD was assessed during the period between 3 months before and 6 months following the baseline vitamin D assessment. Based on the participants' baseline levels, they received either no supplementation, low-dose supplementation (1,000 IU/day), or high-dose supplementation (≥50,000 IU/week), and 25-OH vitamin D was reassessed in the following 8-16 weeks. Approximately 66.5% had deficient/insufficient vitamin D levels at baseline. Deficiency/insufficiency was more common among non-Caucasians, women with later-stage disease, and those who had previously received radiation therapy (P < 0.05). Breast cancer patients with deficient/insufficient 25-OH vitamin D levels had significantly lower lumbar BMD (P = 0.03). Compared to the no-supplementation group, weekly high-dose supplementation significantly increased 25-OH vitamin D levels, while daily low-dose supplementation did not significantly increase levels. Vitamin D deficiency and insufficiency were common among women with breast cancer and associated with reduced BMD in the spine. Clinicians should carefully consider vitamin D supplementation regimens when treating vitamin D deficiency/insufficiency in breast cancer patients.

The orally bioavailable met inhibitor PF-2341066 inhibits osteosarcoma growth and osteolysis/matrix production in a xenograft model.

Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. Ninety percent of patients who present with metastatic and 30% to 40% of patients with nonmetastatic disease experience relapse, creating an urgent need for novel therapeutic strategies. The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), are important for mitosis, motility, and cell survival. Upregulation of Met/HGF signaling via receptor overexpression, amplification, or mutation drives the proliferation, invasiveness, and metastasis of a variety of cancer cells, including OS, prompting the development of Met/HGF inhibitors. OS cells depend on Met overexpression because introduction of dominant-negative Met inhibits in vivo tumorigenicity. Despite the importance of Met/HGF signaling in the development and maintenance of OS, the potential efficacy of pharmacologic Met inhibition in OS has been addressed only in in vitro studies. PF-2341066 is an orally bioavailable, selective ATP-competitive Met inhibitor that showed promising results recently in a phase I clinical trial in non-small cell lung cancer (NSCLC) patients. We tested the ability of PF-2341066 to inhibit malignant properties of osteosarcoma cells in vitro and orthotopic xenograft growth in vivo. In vitro, PF-2341066 inhibited osteosarcoma behavior associated with primary tumor growth (eg, proliferation and survival) as well as metastasis (eg, invasion and clonogenicity). In nude mice treated with PF-2341066 via oral gavage, the growth and associated osteolysis and extracortical bone matrix formation of osteosarcoma xenografts were inhibited by PF-2341066. PF-2341066 may represent an effective new systemic therapy for localized and potentially disseminated osteosarcoma.

Bone density changes after radiation for extremity sarcomas: exploring the etiology of pathologic fractures.

PURPOSE: The incidental irradiation (RT) of adjacent bone that takes place during treatment of soft tissue extremity sarcomas is generally presumed to "weaken" the bone by decreasing its density, which subsequently increases the risk for pathologic fracture. This investigation intended to assess the relative effects on bone density of both RT and diminished mechanical loading secondary to tumor-induced and therapy-induced functional extremity impairment. METHODS AND MATERIALS: 19 patients treated with surgical excision and RT for soft tissue extremity sarcomas had bone density measured using dual energy X-ray absorptiometry at four sites: the irradiated (A) and contralateral (B) bone, and an uninvolved bone (C) in the treated extremity and its contralateral counterpart (D). Analysis included (1) [A-B], (2) [C-D], (3) [(A-B), - (C-D)], and (4) [(A-B)/B - (C-D)/D]. RESULTS: The mean bone density for all irradiated sites was increased 0.08 ± 0.22 g/cm(2) (variance) compared to the contralateral unirradiated side when corrected for weight-bearing effects (3). An average increase in bone density of 9 ± 22% (p = 0.08) was also seen when the differences were divided by individual control densities to normalize variation in density of different anatomic sites (4). CONCLUSIONS: RT does not routinely decrease bone density when corrected for weight bearing or mechanical effects. The pathogenesis for the known increased risk of pathologic fracture in irradiated bones is likely multifactorial, including possible alterations in bone remodeling that can result in stable, or even increased, bone density. Further clinical and basic studies are needed to confirm our unexpected findings.

Teriparatide therapy and beta-tricalcium phosphate enhance scaffold reconstruction of mouse femoral defects.

To investigate the efficacy of endocrine parathyroid hormone treatment on tissue-engineered bone regeneration, massive femoral defects in C57Bl/6 mice were reconstructed with either 100:0 or 85:15 poly-lactic acid (PLA)/beta-tricalcium phosphate (ß-TCP) scaffolds (hereafter PLA or PLA/ßTCP, respectively), which were fabricated with low porosity (<30%) to improve their structural rigidity. Experimental mice were treated starting at 1 week postop with daily subcutaneous injections of 40 µg/kg teriparatide until sacrifice at 9 weeks, whereas control mice underwent the same procedure but were injected with sterile saline. Bone regeneration was assessed longitudinally using planar X-ray and quantitative microcomputed tomography, and the reconstructed femurs were evaluated at 9 weeks either histologically or biomechanically to determine their torsional strength and rigidity. Teriparatide treatment increased bone volume and bone mineral content significantly at 6 weeks and led to enhanced trabeculated bone callus formation that appeared to surround and integrate with the scaffold, thereby establishing union by bridging bone regeneration across the segmental defect in 30% of the reconstructed femurs, regardless of the scaffold type. However, the bone volume and mineral content in the PLA reconstructed femurs treated with teriparatide was reduced at 9 weeks to control levels, but remained significantly increased in the PLA/ßTCP scaffolds. Further, bridged teriparatide-treated femurs demonstrated a prototypical brittle bone torsion behavior, and were significantly stronger and stiffer than control specimens or treated specimens that failed to form bridging bone union. Taken together, these observations suggest that intermittent, systemic parathyroid hormone treatment can enhance bone regeneration in scaffold-reconstructed femoral defects, which can be further enhanced by mineralized (ßTCP) particles within the scaffold.

The histone deacetylase inhibitor vorinostat selectively sensitizes fibrosarcoma cells to chemotherapy.

Soft tissue sarcoma (STS) is a rare malignancy that is generally resistant to chemotherapy. We investigated the ability of the histone deacetylase inhibitor vorinostat to sensitize STS cells versus normal fibroblasts to chemotherapy. Fibrosarcoma, leiomyosarcoma, and liposarcoma cells and normal fibroblasts were treated with vorinostat to determine effects on proliferation and basal apoptosis as measured by total cell number and cleaved caspase 3 staining. Effects on histone deacetylases (HDAC) activity were confirmed by Western blotting for acetylated histone H3. A clinically relevant dose of vorinostat that had no effect on basal apoptosis was selected to examine altered sensitivity to doxorubicin. The effects of vorinostat, doxorubicin, or the combination on fibrosarcoma growth in vivo were determined in a xenograft model. Tumor volume was measured biweekly and HDAC activity and cell death were assessed by immunohistochemical analysis of acetylated histone H3, cleaved caspase 3, and TUNEL staining. Vorinostat inhibited proliferation and induced histone acetylation without affecting basal apoptosis levels. Combined treatment with vorinostat and doxorubicin synergistically induced apoptosis in vitro in fibrosarcoma but not leiomyosarcoma, liposarcoma, or normal fibroblasts. In nude mice, the combination of vorinostat and doxorubicin inhibited fibrosarcoma xenograft growth further than either agent alone. Cell death, as measured by cleaved caspase 3 and TUNEL staining, was greatest in xenografts from mice treated with vorinostat and doxorubicin. Vorinostat inhibits growth and induces chemosensitivity in fibrosarcoma cells in vitro and in vivo, suggesting that the combination of vorinostat and chemotherapy may represent a novel treatment option for this STS subtype. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:623-632, 2011.

Effects of a structured weight-bearing exercise program on bone metabolism among breast cancer survivors: a feasibility trial.

PURPOSE: Treatments for breast cancer, specifically hormonal therapy, accelerate bone loss (BL) among breast cancer survivors, leading to osteoporosis and an increase in fracture risk. Tai Chi Chuan (TCC) is a moderate form of weight-bearing exercise, equivalent to walking, and it has been shown to improve aerobic capacity and strength among breast cancer survivors and might also be effective in slowing bone loss in breast cancer survivors. This pilot study compared the influence of TCC with that of standard support therapy (ST; exercise control) on BL biomarkers among breast cancer survivors. PATIENTS AND METHODS: Randomly assigned breast cancer survivors (N = 16; median age, 53 years; < 30 months after treatment) completed 12 weeks (3 times per week, 60 minutes per session) of TCC or ST. Serum levels of N-telopeptides of type I collagen (NTx), a marker of bone resorption, and bone-specific alkaline phosphatase (BSAP), a marker of bone formation, were determined according to enzyme-linked immunosorbent assay at baseline and after the intervention. RESULTS: Using analysis of covariance, survivors in the TCC group experienced a greater increase in levels of bone formation (BSAP [microg/L]: before, 8.3; after, 10.2; change, 1.9 microg/L and 22.4%), compared with survivors in ST (BSAP [microg/L]: before, 7.6; after, 8.1; change, 0.5 microg/L [6.3%]). Survivors in the TCC group also experienced a significant decrease in bone resorption (NTx [nanomoles bone collagen equivalent; nmBCE]: before, 17.6; after, 11.1; change, -6.5 nmBCE; -36.9%), whereas women in the ST group did not (NTx [nmBCE]: before, 20.8; after, 18.8; change, -2.0 nmBCE; -9.6%). CONCLUSION: This pilot study suggests that weight-bearing exercise exerts positive effects on BL, through increased bone formation and decreased bone resorption. Further examinations of the influence of TCC on bone health are warranted.

Beta-catenin, cartilage, and osteoarthritis.

The early cellular events during the development of osteoarthritis (OA) are accelerated articular chondrocyte maturation and extracellular matrix degradation, which are usually seen in the weight-bearing region of articular cartilage. The results of our recent studies from transgenic OA mouse models indicate that upregulation of beta-catenin signaling in articular chondrocytes is most likely responsible for the conversion of normal articular chondrocytes into maturing (arthritic) chondrocytes, which is associated with activation of chondrocyte maturational genes and matrix degradation. Conditional activation of the beta-catenin gene in articular chondrocytes leads to an OA-like phenotype. Overexpression of Smurf2, an E3 ubiquitin ligase, also induces an OA-like phenotype through upregulation of beta-catenin signaling. In addition, beta-catenin upregulation was also found in articular cartilage tissues in patients with OA. These findings indicate that beta-catenin plays a central role in articular cartilage function and that activation of beta-catenin signaling may represent a pathologic mechanism for OA development.

Smurf2 induces degradation of GSK-3beta and upregulates beta-catenin in chondrocytes: a potential mechanism for Smurf2-induced degeneration of articular cartilage.

We have previously demonstrated that Smurf2 is highly expressed in human osteoarthritis (OA) tissue, and overexpression of Smurf2 under the control of the type II collagen promoter (Col2a1) induces an OA-like phenotype in aged Col2a1-Smurf2 transgenic mice, suggesting that Smurf2 is located upstream of a signal cascade which initiates OA development. However, the factors downstream of Smurf2 in this signal cascade and how Smurf2-induced OA is initiated are largely unknown. In this study, we further characterized the phenotypic changes in Col2a1-Smurf2 transgenic and WT articular cartilage from the postnatal stage to adulthood. We found that the articular cartilage degeneration occurring at the cartilage surface in 6 month-old Col2a1-Smurf2 transgenic mice progressed from an expanded hypertrophic domain in the basal layer of the deep articular cartilage at 2.5 weeks of age, which may lead to an accelerated calcification and ectopic ossification of this region at 1 month of age, and aggregation and maturation of articular chondrocytes in the middle and deep zones at 2 months and 4.5 months of age, respectively. Furthermore, we discovered that ectopically expressed Smurf2 interacted with GSK-3beta and induced its ubiquitination and subsequent proteasomal degradation, and hence upregulated beta-catenin in Col2a1-Smurf2 transgenic chondrocytes ex vivo. It is therefore likely that Smurf2-mediated upregulation of beta-catenin through induction of proteasomal degradation of GSK-beta in chondrocytes may activate articular chondrocyte maturation and associated alteration of gene expression, the early events of OA.

Activation of beta-catenin signaling in articular chondrocytes leads to osteoarthritis-like phenotype in adult beta-catenin conditional activation mice.

Osteoarthritis (OA) is a degenerative joint disease, and the mechanism of its pathogenesis is poorly understood. Recent human genetic association studies showed that mutations in the Frzb gene predispose patients to OA, suggesting that the Wnt/beta-catenin signaling may be the key pathway to the development of OA. However, direct genetic evidence for beta-catenin in this disease has not been reported. Because tissue-specific activation of the beta-catenin gene (targeted by Col2a1-Cre) is embryonic lethal, we specifically activated the beta-catenin gene in articular chondrocytes in adult mice by generating beta-catenin conditional activation (cAct) mice through breeding of beta-catenin(fx(Ex3)/fx(Ex3)) mice with Col2a1-CreER(T2) transgenic mice. Deletion of exon 3 of the beta-catenin gene results in the production of a stabilized fusion beta-catenin protein that is resistant to phosphorylation by GSK-3beta. In this study, tamoxifen was administered to the 3- and 6-mo-old Col2a1-CreER(T2);beta-catenin(fx(Ex3)/wt) mice, and tissues were harvested for histologic analysis 2 mo after tamoxifen induction. Overexpression of beta-catenin protein was detected by immunostaining in articular cartilage tissues of beta-catenin cAct mice. In 5-mo-old beta-catenin cAct mice, reduction of Safranin O and Alcian blue staining in articular cartilage tissue and reduced articular cartilage area were observed. In 8-mo-old beta-catenin cAct mice, cell cloning, surface fibrillation, vertical clefting, and chondrophyte/osteophyte formation were observed. Complete loss of articular cartilage layers and the formation of new woven bone in the subchondral bone area were also found in beta-catenin cAct mice. Expression of chondrocyte marker genes, such as aggrecan, Mmp-9, Mmp-13, Alp, Oc, and colX, was significantly increased (3- to 6-fold) in articular chondrocytes derived from beta-catenin cAct mice. Bmp2 but not Bmp4 expression was also significantly upregulated (6-fold increase) in these cells. In addition, we also observed overexpression of beta-catenin protein in the knee joint samples from patients with OA. These findings indicate that activation of beta-catenin signaling in articular chondrocytes in adult mice leads to the premature chondrocyte differentiation and the development of an OA-like phenotype. This study provides direct and definitive evidence about the role of beta-catenin in the development of OA.

Induction of an osteoarthritis-like phenotype and degradation of phosphorylated Smad3 by Smurf2 in transgenic mice.

OBJECTIVE: To determine whether Smurf2, an E3 ubiquitin ligase known to inhibit transforming growth factor beta (TGFbeta) signaling, is expressed in human osteoarthritic (OA) cartilage and can initiate OA in mice. METHODS: Human OA cartilage was obtained from patients undergoing knee arthroplasty. Samples were graded histologically using the Mankin scale and were examined immunohistochemically for Smurf2 expression. A transgene driven by the collagen 2alpha1 promoter was used to overexpress Smurf2 in mice. Smurf2 overexpression in mouse sternal chondrocytes was confirmed by reverse transcription-polymerase chain reaction and Western blotting. Changes in articular cartilage area, chondrocyte number, and chondrocyte diameter were assessed histomorphometrically using OsteoMeasure software. Alterations in type X collagen and matrix metalloproteinase 13 (MMP-13) in articular chondrocytes were examined by in situ hybridization and immunohistochemistry, respectively. Joint bone phenotypes were evaluated by microfocal computed tomography. The effects of Smurf2 overexpression on TGFbeta signaling were examined using a luciferase-based reporter and immunoprecipitation/Western blotting. RESULTS: Human OA cartilage strongly expressed Smurf2 as compared with nonarthritic human cartilage. By 8 months of age, Smurf2-transgenic mice exhibited decreased articular cartilage area, fibrillation, clefting, eburnation, subchondral sclerosis, and osteophytes. Increased expression of type X collagen and MMP-13 were also detected in articular cartilage from transgenic mice. Transgenic sternal chondrocytes showed reduced TGFbeta signaling as well as decreased expression and increased ubiquitination of pSmad3. CONCLUSION: Smurf2 is up-regulated during OA in humans, and Smurf2-transgenic mice spontaneously develop an OA-like phenotype that correlates with decreased TGFbeta signaling and increased pSmad3 degradation. Overall, these results suggest a role of Smurf2 in the pathogenesis of OA.

Erythropoietin accelerates functional recovery after peripheral nerve injury.

BACKGROUND: Erythropoietin is a naturally occurring hormone with multiple effects on a number of different cell types. Recent data have suggested neuroprotective and perhaps even neurotrophic roles for erythropoietin. We hypothesized that these functional effects could be demonstrable in standard models of peripheral nerve injury. METHODS: Experiments were undertaken to evaluate the effect of erythropoietin on the previously reported standard course of healing of sciatic injuries in mice. The injury groups included mice that were subjected to (1) sham surgery, (2) a calibrated sciatic crush injury, (3) transection of the sciatic nerve followed by epineural repair, or (4) a transection followed by burial of the proximal stump in the adjacent muscle tissue (neurectomy). Either erythropoietin or saline solution was administered to the mice in each of these experimental groups twenty-four hours preinjury, immediately after surgical creation of the injury, twenty-four hours postinjury, or one week postinjury. All mice were evaluated on the basis of the published model for recovery of sciatic nerve motor function by measuring footprint parameters at specific times after the injury. Immunohistochemistry was also performed to assess the erythropoietin-receptor expression profile at the site of injury. RESULTS: In general, the mice treated with erythropoietin recovered sciatic nerve motor function significantly faster than did the untreated controls. This conclusion was based on a sciatic function index that was 60% better in the erythropoietin-treated mice at seven days postinjury (p < 0.05). Although the group that had been given the erythropoietin immediately postinjury showed the best enhancement of recovery, the timing of the administration of the drug was not critical. Histological analysis demonstrated enhanced erythropoietin-receptor positivity in the nerves that recovered fastest, suggesting that accelerated healing correlates with expression of the receptor in nerve tissue. CONCLUSIONS: Erythropoietin treatment of an acute sciatic nerve crush injury leads to an effect consistent with functional neuroprotection. This protective effect may have clinical relevance, especially since it was detectable even when erythropoietin had been administered up to one week after injury.

Quantification of massive allograft healing with dynamic contrast enhanced-MRI and cone beam-CT: a pilot study.

Although massive allografts are widely used for reconstruction of critical defects in long bones caused by tumor or trauma, many will have inadequate long-term outcomes. Toward a tissue engineering solution to this problem, we developed experimental stem cell and gene therapy adjuvants that induce angiogenesis, osteogenesis, and remodeling of the structural allografts. We present data from pilot studies to show the utility of dynamic contrast enhanced MRI (DCE-MRI) to quantify vascularity after femoral osteotomy in a canine femur model and cone beam CT (CB-CT) to quantify bone volume in a patient after composite prosthetic-allograft reconstructive surgery. The results demonstrate our ability to suppress the artifacts generated by the metal implants required to secure massive allografts such that precise quantification of cortical bone revascularization (>10-fold increase at 3 weeks postoperatively) and new bone formation (accurate to about 193 mum(3)) around the graft can be performed longitudinally via DCE-MRI and CB-CT, respectively.

Inhibition of beta-catenin signaling in articular chondrocytes results in articular cartilage destruction.

OBJECTIVE: Osteoarthritis is a degenerative joint disease whose molecular mechanism is currently unknown. Wnt/beta-catenin signaling has been demonstrated to play a critical role in the development and function of articular chondrocytes. To determine the role of beta-catenin signaling in articular chondrocyte function, we generated Col2a1-ICAT-transgenic mice to inhibit beta-catenin signaling in chondrocytes. METHODS: The expression of the ICAT transgene was determined by immunostaining and Western blot analysis. Histologic analyses were performed to determine changes in articular cartilage structure and morphology. Cell apoptosis was determined by TUNEL staining and the immunostaining of cleaved caspase 3 and poly(ADP-ribose) polymerase (PARP) proteins. Expression of Bcl-2, Bcl-x(L), and Bax proteins and caspase 9 and caspase 3/7 activities were examined in primary sternal chondrocytes isolated from 3-day-old neonatal Col2a1-ICAT-transgenic mice and their wild-type littermates and in primary chicken and porcine articular chondrocytes. RESULTS: Expression of the ICAT transgene was detected in articular chondrocytes of the transgenic mice. Associated with this, age-dependent articular cartilage destruction was observed in Col2a1-ICAT-transgenic mice. A significant increase in cell apoptosis in articular chondrocytes was identified by TUNEL staining and the immunostaining of cleaved caspase 3 and PARP proteins in these transgenic mice. Consistent with this, Bcl-2 and Bcl-x(L) expression were decreased and caspase 9 and caspase 3/7 activity were increased, suggesting that increased cell apoptosis may contribute significantly to the articular cartilage destruction observed in Col2a1-ICAT-transgenic mice. CONCLUSION: Inhibition of beta-catenin signaling in articular chondrocytes causes increased cell apoptosis and articular cartilage destruction in Col2a1-ICAT- transgenic mice.