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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.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.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).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 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-β signaling. As lead is concentrated in articular cartilage, we hypothesize that it can disrupt normal chondrocyte phenotype through suppression of TGF-β 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-β 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-β 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. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1760–1766, 2012

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.

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 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. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1145–1151, 2011

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 (β-TCP) scaffolds (hereafter PLA or PLA/βTCP, respectively), which were fabricated with low porosity (

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.

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.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.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%).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.

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 β-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 β-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 β-catenin signaling. In addition, β-catenin upregulation was also found in articular cartilage tissues in patients with OA. These findings indicate that β-catenin plays a central role in articular cartilage function and that activation of β-catenin signaling may represent a pathologic mechanism for OA development.

Osteoporosis, a skeletal disorder characterized by a reduction in bone strength, increases fracture risk. Primary osteoporosis is usually a result of reduced bone mineral density as a consequence of natural aging. Secondary osteoporosis is usually a result of a disease, such as cystic fibrosis, or medical treatment, such as corticosteroids or cancer treatment.Currently, ten million Americans are osteoporotic and an additional 34 million have the precursor condition, osteopenia. Osteoporosis leads to 1.5 million fractures and 500,000 hospitalizations annually. Osteoporosis-related fractures increase mortality and reduce quality of life. Calcitriol, the active form of vitamin D, regulates intestinal calcium absorption, among other actions. During the past four decades, many clinical trials investigating the effect of calcitriol on bone loss have been performed.We conducted a systematic qualitative review of clinical trials that assessed calcitriol for the treatment of osteoporosis and bone loss. In these clinical trials, calcitriol was used as a monotherapy and in combination with other therapeutic bone agents.Studies using calcitriol monotherapy, although not conclusive, found that calcitriol slowed the rate of bone loss in a variety of populations. Calcitriol in combination with other therapeutic bone agents was shown to have additional bone-preserving effects when compared to the use of therapeutic bone agents alone. A common side-effect of calcitriol therapy was hypercalcemia and hypercalciuria, but the degree of hypercalcemia was mild. Recent research found that intermittent dosing can reduce hypercalcemia rates. Calcitriol, alone or in combination with other agents, should be considered for the therapy of osteoporosis.

Type X collagen is produced by hypertrophic chondrocytes and serves as a highly specific marker for chondrocyte maturation. This study was designed to compare the expression of type II and type X collagen in growth plate sections and in distinct populations of chondrocytes in culture by in situ hybridization. Growth plate sections were treated with type II and type X collagen cDNA probes. Type II collagen mRNA was present throughout the growth plate but greatest in the lower proliferating and upper hypertrophic regions. In contrast, type X collagen was expressed only in the hypertrophic region. Northern analysis confirmed the specificity of the probe for type X collagen mRNA. Chick growth plate chondrocytes were separated by countercurrent centrifugal elutriation into five distinct populations and plated in serum-containing medium. These cultures were examined at varying times after plating for the expression of type II and type X collagen mRNA. At 3 h, type II collagen was present in the majority of the cells in all fractions, and approximately 15-20% of the cells expressed type X collagen mRNA. The cells expressing type X were from the hypertrophic region. At 24 h, however, nearly all cells in culture expressed type X mRNA, and there was a decrease in expression of type II collagen mRNA. Similar results were obtained in cultures in the absence of serum, and SDS-PAGE analysis of collagen synthesis confirmed the expression of type X collagen in all populations of fractionated cells at 24 h at the protein level. Type X collagen is an important marker through which cellular matruation can be evaluated in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The Runx family of transcription factors regulate cell growth and differentiation, and control the expression of target genes involved in cell fate decisions. We examined the role of the bone-related member of this family, Runx2, in regulating apoptosis via modulation of the Bcl2 family of genes in the osteosarcoma cell line Saos2. Our data demonstrate that Runx2 directly binds to two Runx-specific regulatory elements on the human bax promoter thereby inducing Bax expression. Furthermore, bone morphogenetic protein-induced or vector-mediated expression of Runx2 resulted in upregulation of Bax expression, and subsequent increased sensitivity of Saos2 cells to apoptosis. Finally, the observed upregulation of Bax expression and increased apoptosis were Runx2 dependent as Runx2 loss of function abrogated these effects. Our study provides the first evidence for Bax as a direct target of Runx2, suggesting that Runx2 may act as a proapoptotic factor in osteosarcoma cells.

Smurf2 is an E3 ubiquitin ligase that targets TGF-beta receptor activated Smad2 and Smad3 for the proteasome in primary articular chondrocytes, thus stimulating their hypertrophic differentiation. Comparatively, how Smurf2 functions in growth plate chondrocytes in a developing long bone is an open question. In this study, we measured the mRNA levels of endogenous Smurf2 and type X collagen in chick growth plate at different embryonic stages to monitor the correlation between the level of Smurf2 expression and chondrocyte maturational stage. We found that high levels of Smurf2 were associated with the differentiative and proliferative stages, while Smurf2 levels were thereafter decreased as the chondrocytes matured toward hypertrophy. In addition, we injected Smurf2-RCAS into chick wing buds at HH stage 20-23 and examined how the ectopic overexpression of Smurf2 in condensing chondrogenic mesenchyme affects the subsequent process of chondrocyte maturation and ossification during embryonic development. Histological analysis showed that overexpression of Smurf2 in a developing wing bud accelerated chondrocyte maturation and endochondral ossification, which may result from a decrease in TGF-beta signaling in the infected chondrocytes with Smurf2-RCAS.

Ectopic expression of Smurf2 in chondrocytes and perichondrial cells accelerated endochondral ossification by stimulating chondrocyte maturation and osteoblast development through upregulation of β-catenin in Col2a1-Smurf2 embryos. The mechanism underlying Smurf2-mediated morphological changes during embryonic development may provide new mechanistic insights and potential targets for prevention and treatment of human osteoarthritis. Introduction: Our recent finding that adult Col2a1-Smurf2 mice have an osteoarthritis-like phenotype in knee joints prompted us to examine the role of Smurf2 in the regulation of chondrocyte maturation and osteoblast differentiation during embryonic endochondral ossification. Materials and Methods: We analyzed gene expression and morphological changes in developing limbs by immunofluorescence, immunohistochemistry, Western blot, skeletal preparation, and histology. A series of markers for chondrocyte maturation and osteoblast differentiation in developing limbs were examined by in situ hybridization. Results: Ectopic overexpression of Smurf2 driven by the Col2a1 promoter was detected in chondrocytes and in the perichondrium/periosteum of 16.5 dpc transgenic limbs. Ectopic Smurf2 expression in cells of the chondrogenic lineage inhibited chondrocyte differentiation and stimulated maturation; ectopic Smurf2 in cells of the osteoblastic lineage stimulated osteoblast differentiation. Mechanistically, this could be caused by a dramatic increase in the expression of β-catenin protein levels in the chondrocytes and perichondrial/periosteal cells of the Col2a1-Smurf2 limbs. Conclusions: Ectopic expression of Smurf2 driven by the Col2a1 promoter accelerated the process of endochondral ossification including chondrocyte maturation and osteoblast differentiation through upregulation of β-catenin, suggesting a possible mechanism for development of osteoarthritis seen in these mice.

The clinician-scientist faces many career barriers that changes in medicine over the past two decades have accentuated. Political, socioeconomic, and cultural changes in medicine have increased financial pressures on physicians and hospitals, negatively impacting the ability of clinicians to pursue research activities. Examples of institutional barriers include pressure on physicians to increase clinical productivity to help offset rising costs and declining reimbursements, inadequate resources and structures to protect research time, a lack of consideration of the importance of spatial colocalization of interacting researchers and clinicians, insufficient focus on integrating clinician-scientists into research teams, and inadequate accessibility of administrative research infrastructure. Lack of mentorship and role models in orthopaedics also contributes to the barriers confronting the clinician-scientist. A number of steps could be taken by institutions to positively influence orthopaedic clinician-scientist career development. Creation of research teams in which orthopaedic clinician-scientists can collaborate effectively with other researchers is a critical step, as is providing protected time free of clinical responsibilities to be devoted to research. Increased attention to physical co-localization of the research activities of clinicians and scientists, and provision of research infrastructure at the department level are additional approaches that can be taken to ameliorate the institutional barriers to the orthopaedic clinician-scientist.

To elucidate the possible effect of NFkappaB on radioresistance, we used the osteosarcoma cell line Saos2, stably expressing the NFkappaB constitutive inhibitor, mIkappaB (Saos2-mIkappaB) or stably transfected with the empty vector (Saos2-EV). Ionizing radiation induced "intrinsic" apoptosis in Saos2-mIkappaB cells but not in Saos2-EV control cells, with intact NFkappaB activity. We find as expected, that this NFkappaB activity was enhanced following irradiation in the Saos2-EV control cells. On the other hand, inhibition of NFkappaB signaling in Saos2-mIkappaB cells led to the upregulation of the pro-apoptotic systems, such as Bax protein and c-Jun N-terminal Kinase (JNK)/c-Jun/AP1 signaling. Inhibition of NFkappaB resulted in decreased expression of the DNA damage protein GADD45beta, a known inhibitor of JNK. Subsequently, JNK activation of c-Jun/AP-1 proteins increased radiation-induced apoptosis in these mutants. Radiation-induced apoptosis in Saos2-mIkappaB cells was inhibited by the JNK specific inhibitor SP600125 as well as by Bcl-2 over-expression. Furthermore, release of cytochrome-c from mitochondria was increased and caspase-9 and -3 were activated following irradiation in Saos2-mIkappaB cells. Antisense inhibition of GADD45beta in Saos2-EV cells significantly enhanced apoptosis following irradiation. Our results demonstrate that radioresistance of Saos2 osteosarcoma cells is due to NFkappaB-mediated inhibition of JNK. Our study brings new insight into the mechanisms underlying radiation-induced apoptosis of osteosarcoma, and may lead to development of new therapeutic strategies against osteosarcoma.

Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF-beta signaling factors, but BMP-related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3-/- chondrocytes. BMP signaling is increased in TGF-beta deficiency and is required for accelerated chondrocyte maturation.Disruption of TGF-beta signaling results in accelerated chondrocyte maturation and leads to postnatal dwarfism and premature osteoarthritis. The mechanisms involved in this process were studied using in vitro murine chondrocyte cultures.Primary chondrocytes were isolated from the sterna of neonatal wildtype and Smad3-/- mice. Expressions of maturational markers, as well as genes involved in TGF-beta and BMP signaling were examined. Chondrocytes were treated with TGF-beta and BMP-2, and effects on maturation-related genes and BMP/TGF-beta responsive reporters were examined. Recombinant noggin or retroviral vectors expressing Smad2 or Smad3 were added to the cultures.Expression of colX and other maturational markers was markedly increased in Smad3-/- chondrocytes. Smad3-/- chondrocytes lacked compensatory increases in Smad2, Smad4, TGFRII, Sno, or Smurf2 and had reduced expression of TGF-beta1 and TGFRI. In contrast, Smad1, Smad5, BMP2, and BMP6 expression was increased, suggesting a shift from TGF-beta toward BMP signaling. In Smad3-/- chondrocytes, alternative TGF-beta signaling pathways remained responsive, as shown by luciferase assays. These non-Smad3-dependent TGF-beta pathways reduced colX expression and alkaline phosphatase activity in TGF-beta-treated Smad3-/- cultures, but only partially. In contrast, Smad3-/- chondrocytes were more responsive to BMP-2 treatment and had increased colX expression, phosphoSmads 1, 5, and 8 levels, and luciferase reporter activity. Overexpression of both Smad2 and Smad3 blocked spontaneous maturation in Smad3-deficient chondrocytes. Maturation was also abrogated by the addition of noggin, an extracellular BMP inhibitor.These findings show a key role for BMP signaling during the chondrocyte maturation, occurring with loss of TGF-beta signaling with important implications for osteoarthritis and cartilage diseases.

The principle function of articular cartilage is to provide a low friction load-bearing surface that facilitates free movement of joints. Maintenance of this surface depends on the maturational arrest of chondrocytes before terminal hypertrophic differentiation occurs [Exp. Cell Res. 216 (1995) 191; Osteoarthritis Cartilage 7 (1999) 389; J. Cell Biol. 139 (1997) 541; J. Cell Biol. 145 (1999) 783]. In contrast to endochondral ossification which involves a programmed process of chondrocyte maturation culminating in terminal hypertrophy and mineralization [Nat. Genet. 9 (1995) 15], articular chondrocytes (ACs) are constrained from completing the maturational program as evidenced by a lack of type X collagen (colX) and alkaline phosphatase expression [Arthritis Res. 3 (2001) 107; Biochem. J. 362 (2002) 473]. Also, ACs are not responsive to factors that impact the maturational process, including bone morphogenetic protein-2 (BMP-2), a potent stimulator of chondrocyte maturation [J. Orthop. Res. 14 (1996) 937]. Factors that constrain AC maturation are only relieved under unique circumstances such as in osteoarthritis (OA), where proliferation and an increase in the expression of hypertrophic hallmarks indicates that the cells have differentiated into a mature phenotype [Calcif. Tissue Int. 63 (2000) 230]. OA may thus involve the functional loss of mechanisms that arrest articular cartilage differentiation. Responsiveness to various growth or systemic factors translates into activation or repression of specific genes through transcriptional mediators. Understanding the downstream mechanisms involved in this process is of paramount importance. Thus, unraveling the molecular interplay between various factors that regulate chondrocyte maturation during OA occurrence and progression is the main focus of ongoing efforts.

Fallopian tube carcinoma is a rare gynecologic tumor that has metastasized to bone in only one documented case. This case report is of a 56-year-old woman with a primary fallopian tube cancer metastasizing to her right femur and rib cage. This patient was referred to our orthopaedic department from her gynecologic oncologist. A total abdominal hysterectomy and bilateral salpingectomy and oophorectomy revealed fallopian tube cancer. After surgery, the patient had progressive right thigh pain with activity. Radiographs of the femur showed a lytic lesion in the right proximal femur diaphysis with erosion of the medial cortex. We did an open biopsy and curettage of the lesion and intramedullary rod placement. The biopsy confirmed a metastatic lesion arising from the primary fallopian tube cancer. Currently, the patient is recovering and is receiving radiation and chemotherapy. In this case report, we discuss the presentation and treatment course of the patient and summarize the available literature on fallopian tube carcinomas.

Lead exposure continues to be a significant public health problem. In addition to acute toxicity, Pb has an extremely long half-life in bone. Individuals with past exposure develop increased blood Pb levels during periods of high bone turnover or resorption. Pb is known to affect osteoblasts, osteoclasts, and chondrocytes and has been associated with osteoporosis. However, its effects on skeletal repair have not been studied. We exposed C57/B6 mice to various concentrations of Pb acetate in their drinking water to achieve environmentally relevant blood Pb levels, measured by atomic absorption. After exposure for 6 weeks, each mouse underwent closed tibia fracture. Radiographs were followed and histologic analysis was performed at 7, 14, and 21 days. In mice exposed to low Pb concentrations, fracture healing was characterized by a delay in bridging cartilage formation, decreased collagen type II and type X expression at 7 days, a 5-fold increase in cartilage formation at day 14 associated with delayed maturation and calcification, and a persistence of cartilage at day 21. Fibrous nonunions at 21 days were prevalent in mice receiving very high Pb exposures. Pb significantly inhibited ex vivo bone nodule formation but had no effect on osteoclasts isolated from Pb-exposed animals. No significant effects on osteoclast number or activity were observed. We conclude that Pb delays fracture healing at environmentally relevant doses and induces fibrous nonunions at higher doses by inhibiting the progression of endochondral ossification.

Treatment of various types of cells with the mitochondrial ATP-sensitive K+ channel opener, diazoxide, preconditions cells to subsequent injuries and inhibits apoptosis. The mechanism of such preconditioning is not well understood. We have studied the effect of diazoxide pretreatment on mitochondrial morphology and function in HL60 cells and on susceptibility of these cells to apoptosis. We have found that diazoxide pretreatment inhibited etoposide-induced apoptosis and mitochondrial dysfunction. Diazoxide induced moderate mitochondrial swelling and increase in the cytosolic fraction of mitochondrial intermembrane proteins including cytochrome c without any significant effect on the oxidative phosphorylation function or membrane potential. Possibly as an adaptive response, total protein and mRNA levels of cytochrome c and of the anti-apoptotic Bcl-2 family member, Bcl-xl, increased. These effects coincided with activation of the transcription factors cAMP-response element-binding protein (CREB) and NFkappaB. The gene encoding cytochrome c carries the cAMP-response element (CRE), and the gene encoding Bcl-xl carries both the CRE and NFkappaB response elements. The inability of etoposide to trigger apoptosis in preconditioned cells was most likely because of prosurvival signaling by CREB and NFkappaB, which included up-regulation of cytochrome c and Bcl-xl. All described effects were reversed by a specific mitochondrial ATP-sensitive K+ channel inhibitor, 5-hydroxydecanoate, proving the specificity of the action of diazoxide. Preconditioning was also reversed by a specific NFkappaB inhibitor, SN50, proving the importance of this transcription factor for the phenomenon of preconditioning. CREB and NFkappaB were activated most likely in response to an observed elevation in cytosolic calcium following diazoxide treatment. We, therefore, conclude that diazoxide-mediated preconditioning against apoptosis involves activation of the pro-survival transcription factors CREB and NFkappaB.

Prostaglandins are ubiquitous metabolites of arachidonic acid, and cyclooxygenase inhibitors prevent their production and secretion. Animals with loss of cyclooxygenase-2 function have reduced reparative bone formation, but the role of prostaglandins during endochondral bone formation is not defined. The role of PGE2 as a regulator of chondrocyte differentiation in chick growth plate chondrocytes (GPCs) was examined. While PGE2, PGD2, PGF2alpha, and PGJ2 all inhibited colX expression, approximately 80% at 10(-6) M, PGE2 was the most potent activator of cAMP response element (CRE)-mediated transcription. PGE2 dose-dependently inhibited the expression of the differentiation-related genes, colX, VEGF, MMP-13, and alkaline phosphatase gene, and enzyme activity with significant effects at concentrations as low as 10(-10) M. PGE2 induced cyclic AMP response element binding protein (CREB) phosphorylation and increased c-Fos protein levels by 5 min, and activated transcription at CRE-Luc, AP-1-Luc, and c-Fos promoter constructs. The protein kinase A (PKA) inhibitor, H-89, completely blocked PGE2-mediated induction of CRE-Luc and c-Fos promoter-Luc promoters, and partially inhibited induction of AP-1-Luc, while the protein kinase C (PKC) inhibitor Go-6976 partially inhibited all three promoters, demonstrating substantial cross-talk between these signaling pathways. PGE2 inhibition of colX gene expression was dependent upon both PKA and PKC signaling. These observations demonstrate potent prostaglandin regulatory effects on chondrocyte maturation and show a role for both PKA and PKC signaling in PGE2 regulatory events.

Osteoporosis is a decrease in bone mineral density (BMD) that predisposes individuals to fractures. Although an elderly affliction, a predisposition may develop during adolescence if a sufficient peak BMD is not achieved. Rat studies have found that lead exposure is associated with decreased BMD. However, human studies are limited. We hypothesized that the BMD of children with high lead exposure would be lower than the BMD of children with low lead exposure. We collected data on 35 subjects; 16 had low cumulative lead exposure (mean, 6.5 microg/dL), and 19 had high exposure (mean, 23.6 micro g/dL). All were African American; there was no difference between the groups by sex, age, body mass index, socioeconomic status, physical activity, or calcium intake. Significant differences in BMD between low and high cumulative lead exposure were noted in the head (1.589 vs. 1.721 g/cm2), third lumbar vertebra (0.761 vs. 0.819 g/cm2), and fourth lumbar vertebra (0.712 vs. 0.789 g/cm2). Contrary to our hypothesis, subjects with high lead exposure had a significantly higher BMD than did subjects with low lead exposure. This may reflect a true phenomenon because lead exposure has been reported to accelerate bony maturation by inhibiting the effects of parathyroid hormone-related peptide. Accelerated maturation of bone may ultimately result in a lower peak BMD being achieved in young adulthood, thus predisposing to osteoporosis in later life. Future studies need to investigate this proposed model.

In vitro models of endochondral bone formation using both primary and immortalized cells have provided insight regarding factors and signaling pathways involved in chondrocyte maturation and endochondral bone formation. However, primary murine cell culture models of chondrocyte differentiation have not been established but have enormous potential due to the possible use of cells from transgenic and knockout animals. Here, we show that stage E11.5 embryonic murine limb bud mesenchymal stem cells in micromass cell culture progress through the stages of chondrogenesis, chondrocyte hypertrophy, terminal differentiation, and matrix calcification. This cell culture system recapitulated the sequential expression of genes that characterize chondrocyte differentiation, including Sox9, col2, colX, MMP13, VEGF, and osteocalcin. TGF-beta treatment for up to 21 days markedly delayed the rate of chondrocyte maturation and inhibited matrix calcification and its related gene expression. In TGF-beta-treated cultures, the hypertrophic and terminal differentiation markers colX, VEGF, MMP13, and osteocalcin were reduced or absent. PGE2 had minimal effects on chondrocyte hypertrophy but delayed terminal differentiation and matrix calcification. Thus, primary murine mesenchymal cells sequentially differentiate through the various stages of chondrocyte maturation and establish a model whereby the role of specific signaling molecules can be examined in cells derived from transgenic or knockout mice.

Maintenance of the articular surface depends on the function of articular chondrocytes (ACs) which produce matrix and are constrained from undergoing the maturation program seen in growth plate chondrocytes. Only during pathologic conditions, such as in osteoarthritis, are maturational constraints lost causing recapitulation of the process that occurs during endochondral ossification. With the aim of establishing a model to identify regulatory mechanisms that suppress AC hypertrophy, we examined the capability of 5-azacytidine (Aza) to have an impact on the maturational program of these cells. Primary ACs do not spontaneously express markers of maturation and are refractory to treatment by factors that normally regulate chondrocyte maturation. However, following exposure to Aza, ACs (i) were induced to express type X collagen (colX), Indian hedgehog, and alkaline phosphatase and (ii) showed altered colX and AP expression in response to bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), and parathyroid hormone-related protein (PTHrP). Since Aza unmasked responsiveness of ACs to BMP-2 and TGF-beta, we examined the effect of Aza treatment on signaling via these pathways by assessing the expression of the TGF-beta Smads (2 and 3), the BMP-2 Smads (1 and 5), and the Smad2 and 3-degrading ubiquitin E3 ligase Smurf2. Aza-treated ACs displayed less Smad2 and 3 and increased Smad1, 5, and Smurf2 protein and showed a loss of TGF-beta signaling on the P3TP-luciferase reporter. Suggesting that Aza-induction of Smurf2 may be responsible for the loss of Smad2 and 3 protein via this pathway, immunoprecipitation and metabolic labeling experiments confirmed that Aza accelerated the ubiquitination and degradation of these targets. Overall, Aza-treated ACs represent a novel model for the study of mechanisms that regulate maturational potential of articular cartilage, with the data suggesting that maturation of these cells may be due to up-regulation of Smad1 and 5 coupled with a Smurf2-dependent degradation of Smad2 and 3 and loss of TGF-beta signaling.

Aseptic loosening of orthopaedic implants is precipitated by wear debris-induced osteolysis. Central to this process are the pro-inflammatory mediators that are produced in response to wear by the fibroblastic cells, which comprise the majority of periprosthetic membranes. Since this pro-inflammatory cascade is mediated by a plethora of factors with redundant functions, it is imperative to establish a hierarchy. Two well-known fibroblast derived pro-inflammatory factors that stimulate wear debris-induced osteoclastic resorption are prostaglandin E2 (PGE2) and IL-6. However, their relationship to each other in this process is poorly defined. Here we show immunohistochemistry of retrieval membranes indicating that COX-2 is the principal cyclooxygenase responsible for PGE2 production in fibroblasts around failed implants. We also performed in vitro experiments with fibroblasts derived from wild-type (WT), COX-1 (-/-) and COX-2 (-/-) mice, which demonstrated that COX-2 is required for Ti wear debris-induced PGE2 production. Interestingly, COX-2 was also required for IL-6 production in these assays, which could be rescued by the addition of exogenous PGE2 (10(-6) M). Pharmacology studies that utilized the COX-1 selective inhibitor SC 560, the COX-2 selective inhibitor celecoxib, and the nonselective COX inhibitor indomethacin confirmed these results. Taken together, these results indicate that selective inhibition of prostaglandin signaling could favorably impact aseptic loosening beyond its direct effects on PGE2 synthesis, in that it inhibits downstream pro-inflammatory/pro-osteoclastic cytokine production.

Articular cartilage is a complex tissue maintained by chondrocytes, which undergo metabolic changes as a result of aging, disease, and injury. These changes may hinder tissue maintenance and repair, resulting in accelerated loss of articular surface and leading to end-stage arthritis. Researchers are investigating both normal and pathologic cellular and molecular processes as well as the development of chondroprotective agents to improve the metabolic function of articular cartilage. Current research is helping to clarify the mechanisms by which a variety of agents, such as glucosamine, chondroitin sulfate, hyaluronic acid, green tea, glucocorticoids, and nonsteroidal anti-inflammatory drugs, can modify the symptoms and course of osteoarthritis. Also under investigation are methods of stimulating repair or replacing damaged cartilage, such as matrix metalloproteinase inhibitors, gene therapy, growth factors, cytokine inhibitors, and artificial cartilage substitutes. Tissue engineering, the combining of artificial matrices with cells and growth factors or genes, offers great potential for improving patient care.

Growth plate chondrocytes integrate multiple signals during normal development. The type I BMP receptor ALK2 is expressed in cartilage and expression of constitutively active (CA) ALK2 and other activated type I BMP receptors results in maturation-independent expression of Ihh in chondrocytes in vitro and in vivo. The findings suggest that BMP signaling modulates the Ihh/PTHrP signaling pathway that regulates the rate of chondrocyte differentiation. Introduction: Bone morphogenetic proteins (BMPs) have an important role in vertebrate limb development. The expression of the BMP type I receptors BMPR-IA (ALK3) and BMPR-IB (ALK6) have been more completely characterized in skeletal development than ALK2. Methods: ALK2 expression was examined in vitro in isolated chick chondrocytes and osteoblasts and in vivo in the developing chick limb bud. The effect of overexpression of CA ALK2 and the other type I BMP receptors on the expression of genes involved in chondrocyte maturation was determined. Results: ALK2 was expressed in isolated chick osteoblasts and chondrocytes and specifically mediated BMP signaling. In the developing chick limb bud, ALK2 was highly expressed in mesenchymal soft tissues. In skeletal elements, expression was higher in less mature chondrocytes than in chondrocytes undergoing terminal differenti- ation. CA ALK2 misexpression in vitro enhanced chondrocyte maturation and induced Ihh. Surprisingly, although parathyroid hormone-related peptide (PTHrP) strongly inhibited CA ALK2 mediated chondrocyte differentiation, Ihh expression was minimally decreased. CA ALK2 viral infection in stage 19 -23 limbs resulted in cartilage expansion with joint fusion. Enhanced periarticular expression of PTHrP and delayed maturation of the cartilage elements were observed. In the cartilage element, CA ALK2 misexpression precisely colocalized with the expression with Ihh. These findings were most evident in partially infected limbs where normal morphology was maintained. In contrast, BMP-6 had a normal pattern of differentiation-related expression. CA BMPR-IA and CA BMPR-IB overexpression similarly induced Ihh and PTHrP. Conclusions: The findings show that BMP signaling induces Ihh. Although the colocalization of the activated type I receptors and Ihh suggests a direct BMP-mediated signaling event, other indirect mechanisms may also be involved. Thus, while BMPs act directly on chondrocytes to induce maturation, this effect is counterbalanced in vivo by induction of the Ihh/PTHrP signaling loop. The findings suggest that BMPs are integrated into the Ihh/PTHrP signaling loop and that a fine balance of BMP signaling is essential for normal chondrocyte maturation and skeletal development. J Bone Miner Res 2003;18:1593-1604

We previously reported reciprocal regulation of extracellular matrix degrading enzymes and their endogenous inhibitors by NFkappaB. As such, dominant negative inhibition of NFkappaB in a murine lung alveolar carcinoma cell, Line 1, afforded a decrease in malignant proclivity [Cancer Res. 60(23) (2000) 6557-6562]. Contrasting the gene expression profile of malignant Line 1 tumor cells (WT-Line 1) and their non-malignant counterparts transduced with a dominant negative inhibitor of NFkappaB (mIkappaB-Line 1), we observed upregulated retinoic acid receptors (RARs) and the cAMP response element modulator (CREM), in mIkappaB-Line 1 tumor cells, and utilized heterologous promoter-reporter constructs to confirm enhanced responsiveness. We translate these findings by inducing retinoid and cAMP transcriptional programs in WT-Line 1 tumor cells with pharmacologic doses of all-trans retinoic acid (at-RA) and pentoxyfilline (PTX), respectively, and demonstrate suppression of NFkappaB activity, tumor cell derived matrix metalloprotease 9 activity, tumor cell invasiveness in vitro and spontaneous metastasis in vivo. Our results are consistent with the putative role of retinoids and cAMP in the malignant reversion of tumor cells and illustrative of the binary nature of transcriptional programs that modulate malignant progression.

The formation of new bone during the process of bone remodeling occurs almost exclusively at sites of prior bone resorption. In an attempt to discover what regulatory pathways are utilized by osteoblasts to effect this site-specific formation event we probed components of an active bone resorption surface with an osteoblast phage expression library. In these experiments primary cultures of rat osteoblasts were used to construct a phage display library in T7 phage. Tartrate-resistant acid phosphatase (type V) (TRAP) was used as the bait in a biopanning procedure. 40 phage clones with very high affinity for TRAP were sequenced, and of the clones with multiple consensus sequences we identified a regulatory protein that modulates osteoblast differentiation. This protein is the TGFbeta receptor-interacting protein (TRIP-1). Our data demonstrate that TRAP activation of TRIP-1 evokes a TGFbeta-like differentiation process. Specifically, TRIP-1 activation increases the activity and expression of osteoblast alkaline phosphatase, osteoprotegerin, collagen, and Runx2. Moreover, we show that TRAP interacts with TRIP intracellularly, that activation of the TGFbeta type II receptor by TRIP-1 occurs in the presence of TRAP and that the differentiation process is mediated through the Smad2/3 pathway. A final experiment demonstrates that osteoblasts, when cultured in osteoclast lacunae containing TRAP, rapidly and specifically differentiate into a mature bone-forming phenotype. We hypothesize that binding to TRAP may be one mechanism by which the full osteoblast phenotype is expressed during the process of bone remodeling.

Parathyroid hormone-related peptide is one of the most important regulators of chondrocyte proliferation. Although cartilaginous neoplasms express different collagens, including Types II and X, the pathogenesis of these tumors has not been elucidated. The current study examined the hypothesis that parathyroid hormone-related peptide is expressed in cartilaginous neoplasms and its level of expression may correlate with the proliferative rate of cartilaginous neoplasms with higher levels in more malignant tumors and lower levels in benign lesions. Two hundred thirty-four biopsy and resection specimens of benign and malignant cartilage tumors from 179 patients were retrieved from surgical pathology archival material and analyzed immunohistochemically using an antibody to human parathyroid hormone-related peptide. Most cartilaginous neoplasms had some level of expression of parathyroid hormone-related peptide, and tumors with a more proliferative phenotype had higher levels of parathyroid hormone-related peptide. Although benign lesions such as enchondromas and osteochondromas had low levels of parathyroid hormone-related peptide, malignant neoplasms such as extraskeletal myxoid chondrosarcomas, dedifferentiated chondrosarcomas, and mesenchymal chondrosarcomas expressed high levels of parathyroid hormone-related peptide. Parathyroid hormone-related peptide expression correlated with grade of malignancy in chondrosarcoma. Although there were highly significant differences between Grade I chondrosarcoma versus Grade II and Grade III lesions, the difference between Grade II and Grade III chondrosarcomas approached significance. Parathyroid hormone-related peptide may represent a new tumor marker with potential diagnostic use in classifying cartilaginous neoplasms.

Regulation of phenotype in chick tibial growth plate chondrocytes (GPCs) by parathyroid hormone-related peptide (PTHrP) is facilitated via signaling through three pathways: protein kinase A (PKA), protein kinase C (PKC) and inositol-1,4,5-trisphosphate-induced Ca 2+ transients. To establish the underlying signaling specificity for PTHrP-regulation of chondrocyte maturation, we examined the separate involvement of each of these three pathways in the PTHrP regulation of key hallmarks of GPC phenotype: stimulation of proliferation and proteoglycan synthesis and reduction of alkaline phosphatase activity and type X collagen expression. Mimicking the PTHrP stimulation either of PKC with 1-oleoyl 2-acetyl glycerol or of a Ca 2+ pulse with 65 mM KCl did not lead to PTHrP-like effects on any of the four markers examined. Also, inhibition of PKC with myr- Ψ PKC or blockade of Ca 2+ signals with an intracellular chelator did not inhibit PTHrP action. However, PKA activation with dibutyryl cAMP mimicked PTHrP and blockade of PTHrP stimulation of PKA with H-89 inhibited the regulatory action of the factor. These data demonstrate that although activation of PKC or Ca 2+ signals is not required, the cylic AMP-dependent A kinase is required for PTHrP to regulate key hallmarks of GPC phenotype.

The skeletal system is an important target for lead toxicity. One of the impacts of lead in the skeleton, the inhibition of axial bone development, is likely due to its effect on the normal progression of chondrocyte maturation that is central to the process of endochondral ossification. Since little is known about the effect of lead on chondrocyte function/maturation, its impact on (1) growth factor-induced proliferation, (2) expression of maturation-specific markers type X collagen and BMP-6, and (3) the activity of AP-1 and NF-kappaB was examined in chick growth plate and sternal chondrocyte models. Exposure to lead alone (1-30 microM) resulted in a dose-dependent inhibition of thymidine incorporation in growth plate chondrocytes. Lead also blunted the stimulation of thymidine incorporation by parathyroid hormone-related peptide (PTHrP) and transforming growth factor-beta1 (TGF-beta1), two critical regulators of chondrocyte maturation. Lead (1 and 10 microM), TGF-beta1 (3 ng/ml) and PTHrP (10(-7) M) all significantly inhibited the expression of type X collagen, a marker of chondrocyte terminal differentiation. However, when in combination, lead completely reversed the inhibition of type X collagen by PTHrP and TGF-beta1. The effect of lead on BMP-6. an inducer of terminal differentiation. was also examined. Independently, lead and TGF-beta1 were without effect on BMP-6 expression, but PTHrP significantly suppressed it. Comparatively, lead did not alter PTHrP-mediated suppression of BMP-6, but in combination with TGF-beta1. BMP-6 expression was increased 3-fold. To determine if lead effects on signaling might play a role in facilitating these events, the impact of lead on NF-kappaB and AP-1 signaling was assessed using luciferase reporter constructs in sternal chondrocytes. Lead had no effect on the AP-1 reporter, but it dose-dependently inhibited the NF-kappaB reporter. PTHrP, which signals through AP-1, did not activate the NF-kappaB reporter and did not affect inhibition of this reporter by lead. In contrast, PTHrP activation of the AP-1 reporter was dose-dependently enhanced by lead. These findings, which establish that chondrocytes are important targets for lead toxicity, suggest that the effects of lead on bone growth are derived from its impact on the modulation of chondrocyte maturation by growth factors and second messenger signaling responses.

There is a temporal coupling between the processes of bone resorption and bone formation in normal skeletal remodeling. That is, osteoblastic activity usually follows episodes of osteoclastic activity. However, what has not been universally appreciated is that there also is a spatial coupling between these processes. Bone formation only occurs in the immediate vicinity of the resorptive event. In this study, we describe a phage display technique that has been used to identify the mechanisms by which osteoblasts recognize components of the prior resorbed lacunar surface. Using a type V tartrate-resistant acid phosphatase (TRAP) as the bait and a random peptide M13 phage display library as the probe, we have identified specific sequences that show a very high affinity for TRAP. One of these peptides, designated clone 5, has a subnanomolar Kd for TRAP, interacts with TRAP in a Far-Western assay, binds exclusively to TRAP within osteoclast lacunae, is present in osteoblasts, and can effectively block osteoblast binding to resorption surfaces. The clone 5 peptide shows a high homology to glypican 4 (GPC4), a proteoglycan attachment receptor found in a number of cell types.

Aminobisphosphonates have shown significant antitumor activity in vitro and in vivo with selective pharmacodistribution to bone, and an established role in the treatment of malignant bone disease. Given that the mode of action of aminobisphosphonates involves decreasing the prenylation of the Rho family of proteins, through decreasing the availability of prenyl groups (farnesyl and geranylgeranyl isoprenoids), the authors sought the inhibition of Rho protein prenylation at two points, by using an aminobiphosphonate (alendronate) in conjunction with a prenyl transferase inhibitor (R115777, a specific farnesyl transferase inhibitor with limited effects in geranylgeranyl transferase). The authors show synergistic inhibition of the prenylation dependent membrane association and migratory function of Rho proteins, translating into a suppressive effect on in vitro tumor cell invasiveness and in vivo metastasis. The findings support the use of aminobisphosphonates in conjunction with farnesyl transferase inhibitors in the prevention of metastatic progression and suggest that metastatic progression is a valid end point in assessing the antitumor activity of farnesyl transferase inhibitors.

Background and Objectives Osteolytic bone tumors produce intercellular signaling proteins that regulate bone remodeling by altering the rates of osteoclast and osteoblast differentiation and activity. This report examines osteolytic bone tumor expression of receptor activator of nuclear factor B-ligand (RANK-L), a cytokine that is arguably the most critical regulator of osteoclast differentiation and activation. Methods This prospective immunohistochemical study examined RANK-L expression in frozen tissues from sixteen surgical specimens of patients who underwent surgery for the treatment of osteolytic bone tumors between 1999 and 2000. Results RANK-L was positive in 13 of the 16 cases. Primary benign bone tumors, primary malignant bone tumors, and metastasis to bone were positive for RANK-L. Conclusions The cells in some, but not all, osteolytic tumors produce the cytokine RANK-L. Further study is necessary to determine in which specific tumors RANK-L is the cytokine responsible for increased osteoclastic activity, and to develop possible therapeutic use of RANK-L antagonists such as osteoprotegerin (OPG). J. Surg. Oncol. 2002;79:174–179. © 2002 Wiley–Liss, Inc.

Prosthesis failure due to wear debris-induced osteolysis remains a major clinical problem and the greatest limitation for total joint arthroplasty. Based on our knowledge of osteoclast involvement in this process and the requirements of receptor activator of NF-kappaB (RANK) signaling in osteoclastogenesis and bone resorption, we investigated the efficacy of RANK blockade in preventing and ameliorating titanium (Ti)-induced osteolysis in a mouse calvaria model. Compared with placebo controls we found that all doses of RANK:Fc above 1 mg/kg intraperitoneally (ip) per 48 h significantly inhibited osteoclastogenesis and bone resorption in response to Ti implanted locally. Complete inhibition occurred at 10 mg/kg ip per 48 h, yielding results that were statistically equivalent to data obtained with Ti-treated RANK-/- mice. We also evaluated the effects of a single injection of RANK:Fc on day 5 on established osteolysis and found that Ti-treated were still depleted for multinucleated tartrate-resistant acid phosphatase-positive (TRAP+) cells 16 days later. More importantly, this osteoclast depletion did not affect bone formation because the bone lost from the osteolysis on day 5 was restored by day 21. An assessment of the quantity and quality of the newly formed bone in these calvariae by calcein labeling and infrared (IR) microscopy, respectively, showed no significant negative effect of RANK:Fc treatment. These studies indicate that osteoclast depletion via RANK blockade is an effective method to prevent and reverse wear debris-induced osteolysis without jeopardizing osteogenesis.

Bone morphogenetic proteins (BMPs) are ubiquitous regulators of cellular growth and differentiation. A variety of processes modulate BMP activity, including negative regulation by several distinct binding proteins. One such BMP antagonist chordin has a role in axis determination and neural induction in the early embryo. In this study, a role for chordin during endochondral ossification has been investigated. During limb development, Chordin expression was detected only at the distal ends of the skeletal elements. In cultured embryonic sternal chondrocytes, Chordin expression was related inversely to the stages of maturation. Further, treating cultured chondrocytes with chordin interfered with maturation induced by treatment with BMP-2. These results suggest that chordin may negatively regulate chondrocyte maturation and limb growth in vivo. To address this hypothesis, chordin protein was expressed ectopically in Hamburger-Hamilton (HH) stage 25-27 embryonic chick limbs. The phenotypic changes and alteration of gene expression in treated limbs revealed that overexpression of chordin protein delayed chondrocyte maturation in developing skeletal elements. In summary, these findings strongly support a role for chordin as a negative regulator of endochondral ossification.

Background: Pentoxifylline (Trental) is a methylxanthine-derivative drug that has been used for more than twenty years in the treatment of peripheral vascular disease. Pentoxifylline is also a potent inhibitor of tumor necrosis factor-alpha (TNF-α) secretion, both in vitro and in vivo, and has demonstrated efficacy in the treatment of certain animal and human inflammatory diseases. Pentoxifylline has a potential therapeutic role in the treatment of aseptic loosening of total joint replacement components because it inhibits TNF-α secretion by particle-stimulated human peripheral blood monocytes. The purpose of our study was to determine whether the particle-stimulated secretion of TNF-α by peripheral blood monocytes was inhibited in volunteers who had received pentoxifylline orally. Methods: Human peripheral blood monocytes were harvested from eight healthy volunteers and were exposed to three different concentrations of titanium particles or to 500 ng/mL of lipopolysaccharide as a positive control. The same volunteers were then given pentoxifylline (400 mg, five times per day) for seven days. Their peripheral blood monocytes were again isolated and exposed to experimental conditions, and the TNF-α levels were measured. Results: The peripheral blood monocytes from all eight volunteers showed a significant reduction in TNF-α release following oral treatment with pentoxifylline. This reduction was observed at exposures of 107 and 106 titanium particles/mL and in the lipopolysaccharide-treated group, but not at 105 particles/mL. Conclusions: To our knowledge, this is the first study to demonstrate the ability of an oral drug to decrease the release of TNF-α from human peripheral blood monocytes exposed ex vivo to particle debris. TNF-α is involved in the pathogenesis of osteolysis and subsequent loosening of total joint arthroplasty components. The ability to suppress the release of TNF-α in patients with a total joint replacement may help to control osteolysis and to reduce the development of aseptic loosening. This effect could increase implant longevity and decrease the need for revision arthroplasty.

Aseptic loosening is a major complication of prosthetic joint surgery and is manifested as chronic inflammation, pain, and osteolysis at the bone implant interface. The osteolysis is believed to be driven by a host inflammatory response to wear debris generated from the implant. In our current study, we use a selective inhibitor (celecoxib) of cyclo-oxygenase 2 (COX-2) and mice that lack either COX-1 (COX-1-/-) or COX-2 (COX-2-/-) to show that COX-2, but not COX-1, plays an important role in wear debris-induced osteolysis. Titanium (Ti) wear debris was implanted surgically onto the calvaria of the mice. An intense inflammatory reaction and extensive bone resorption, which closely resembles that observed in patients with aseptic loosening, developed within 10 days of implantation in wild-type and COX-1-/- mice. COX-2 and prostaglandin E2 (PGE2) production increased in the calvaria and inflammatory tissue overlying it after Ti implantation. Celecoxib (25 mg/kg per day) significantly reduced the inflammation, the local PGE2 production, and osteolysis. In comparison with wild-type and COX-1-/- mice, COX-2-/- mice implanted with Ti had a significantly reduced calvarial bone resorption response, independent of the inflammatory response, and significantly fewer osteoclasts were formed from cultures of their bone marrow cells. These results provide direct evidence that COX-2 is an important mediator of wear debris-induced osteolysis and suggests that COX-2 inhibitors are potential therapeutic agents for the prevention of wear debris-induced osteolysis.

Transforming growth factor-b (TGF-b) is a multifunctional regulator of a variety of cellular functions, including proliferation, differentiation, matrix synthesis, and apoptosis. In growth plate chondrocytes, TGF-b slows the rate of maturation. Because the current paradigm of TGF-b signaling involves Smad proteins as downstream regulators of target genes, we have characterized their role as mediators of TGF-b effects on chondrocyte maturation. Both Smad2 and 3 translocated to the nucleus upon TGF-b1 signaling, but not upon BMP-2 signaling. Cotransfection experiments using the TGF-b responsive and Smad3 sensitive p3TP-Lux luciferase reporter demonstrated that wild-type Smad3 potentiated, whereas dominant negative Smad3 inhibited TGF-b1 induced luciferase activity. To confirm the role of Smad2 and 3 as essential mediators of TGF-b1 effects on chondrocyte maturation, we overexpressed both wild-type and dominant negative Smad2 and 3 in virally infected chondrocyte cultures. Overexpression of both wildtype Smad2 and 3 potentiated the inhibitory effect of TGF-b on chondrocyte maturation, as determined by colx and alkaline phosphatase activity, whereas dominant negative Smad2 and 3 blocked these effects. Wild-type and dominant negative forms of Smad3 had more pronounced effects than Smad2. Our results define Smad2 and 3 as key mediators of the inhibitory effect of TGF-b1 signaling on chondrocyte maturation. (Endocrinology 141: 4728 ‐ 4735, 2000)

Although PTHrP has been identified as a key regulator of chondrocyte differentiation in the growth plate, the factors directly regulating PTHrP expression have not been identified. Furthermore, while cells from the epiphysis are considered the physiologic source of PTHrP, the relative expression of PTHrP in epiphyseal and growth plate chondrocytes has not been defined. PTHrP expression was examined in chondrocytes isolated from 3- to 5-week-old chick long bones. The expression of PTHrP mRNA was 10-fold higher in epiphyseal chondrocytes compared to cells from the growth plate. Growth plate chondrocytes were isolated into populations with distinct maturational characteristics by countercurrent centrifugal elutriation and analyzed for PTHrP expression. The expression was highest in the least mature cells and progressively declined with the onset of maturation. The regulation of PTHrP expression was further examined in epiphyseal chondrocytes. Both TGF-beta1 and cis-retinoic acid stimulation markedly increased PTHrP mRNA levels, while BMP-2 and PTHrP stimulation decreased the expression of this transcript. The effects of TGF-beta1 (8.9-fold stimulation) and TGF-beta3 (9.2-fold) were slightly greater than the effects of TGF-beta2 (4.9-fold). The effect of TGF-beta was dose-dependent and increases could be detected after 68 h of treatment. To analyze the paracrine effect of epiphyseal and growth plate chondrocytes on each other, these cells were placed in coculture and the mRNA from each of the populations was harvested separately after 24 h. Following coculture the PTHrP mRNA levels increased in the epiphyseal cells while the expression of type X collagen and Indian hedgehog transcripts decreased in growth plate chondrocytes. The results demonstrate potentially important paracrine interactions between these cell populations, possibly mediated by TGF-beta and PTHrP.

Due to irreversible joint destruction caused by the various arthritides, more than 400,000 total joint arthroplasties are performed each year in the United States. As many as 20% of these require revision surgery because of aseptic loosening. The current paradigm to explain aseptic loosening is that wear debris generated from the prosthesis stimulates the release of proinflammatory cytokines (i.e., tumor necrosis factor-alpha and interleukins 1 and 6) following phagocytosis by resident macrophages. These cytokines, in turn, initiate an inflammatory response, with the development of an erosive pannus that stimulates bone resorption by osteoclasts. In support of this model, we have previously shown that human monocytes produce large quantities of tumor necrosis factor-alpha in response to titanium particles in vitro. In the current study, we characterized the role of tumor necrosis factor-alpha/nuclear transcription factor-kappaB signaling in the proinflammatory response to titanium particles in vitro and in vivo. Using the mouse macrophage cell line J774, we showed that these cells produce an amount of tumor necrosis factor-alpha in response to titanium particles similar to that produced by human peripheral blood monocytes. The production of tumor necrosis factor-alpha was preceded by a drop in cellular levels of inhibitory factor-kappaBalpha protein and translocation of p50/p65 nuclear transcription factor-KB to the nucleus 30 minutes after stimulation. Levels of tumor necrosis factor-alpha and inhibitory factor-kappaBalpha mRNA increased 30 minutes after stimulation, consistent with the activation of nuclear transcription factor-kappaB. Interleukin-6 mRNA was first seen 4 hours after the addition of the titanium particles, indicating that the production of this cytokine is secondary to the immediate nuclear transcription factor-kappaB response. To test the relevance of tumor necrosis factor-alpha/nuclear transcription factor-kappaB signaling in response to titanium particles in vivo, we adopted an animal model in which the particles were surgically implanted on the calvaria of mice. The animals displayed a dramatic histological response to the debris, with the formation of fibrous tissue and extensive bone resorption after only 1 week. With use of immunohistochemistry and tartrate-resistant acid phosphatase staining, tumor necrosis factor-alpha and osteoclasts were readily detected at the site of inflammation and bone resorption in the calvaria of the treated mice. By testing mice that genetically over-produce tumor necrosis factor-alpha (hTNFalpha-Tg), those defective in tumor necrosis factor-alpha signaling (TNF-RI-/-), and those that are nuclear transcription factor-kappaB1-deficient (NFkappaB1-/-), we evaluated the importance of tumor necrosis factor-alpha/nuclear transcription factor-kappaB signaling in the biological processes responsible for aseptic loosening. The hTNFalpha-Tg mice had a grossly exaggerated response, the TNF-RI(-/-) mice showed little evidence of inflammation or bone resorption, and the nuclear transcription factor-kappaB1(-/-) mice had an inflammatory response without bone resorption. On the basis of these results, we propose a model for periprosthetic osteolysis in which wear debris particles are phagocytosed by macrophages, resulting in the activation of nuclear transcription factor-kappaB and the production of tumor necrosis factor-alpha. Tumor necrosis factor-alpha directly induces fibroblast proliferation and tissue fibrosis and recruits or activates, or both, osteoclasts to resorb adjacent bone.

Multiple inositol polyphosphate phosphatase (Minpp1) metabolizes inositol 1,3,4,5,6-pentakisphosphate (InsP(5)) and inositol hexakisphosphate (InsP(6)) with high affinity in vitro. However, Minpp1 is compartmentalized in the endoplasmic reticulum (ER) lumen, where access of enzyme to these predominantly cytosolic substrates in vivo has not previously been demonstrated. To gain insight into the physiological activity of Minpp1, Minpp1-deficient mice were generated by homologous recombination. Tissue extracts from Minpp1-deficient mice lacked detectable Minpp1 mRNA expression and Minpp1 enzyme activity. Unexpectedly, Minpp1-deficient mice were viable, fertile, and without obvious defects. Although Minpp1 expression is upregulated during chondrocyte hypertrophy, normal chondrocyte differentiation and bone development were observed in Minpp1-deficient mice. Biochemical analyses demonstrate that InsP(5) and InsP(6) are in vivo substrates for ER-based Minpp1, as levels of these polyphosphates in Minpp1-deficient embryonic fibroblasts were 30 to 45% higher than in wild-type cells. This increase was reversed by reintroducing exogenous Minpp1 into the ER. Thus, ER-based Minpp1 plays a significant role in the maintenance of steady-state levels of InsP(5) and InsP(6). These polyphosphates could be reduced below their natural levels by aberrant expression in the cytosol of a truncated Minpp1 lacking its ER-targeting N terminus. This was accompanied by slowed cellular proliferation, indicating that maintenance of cellular InsP(5) and InsP(6) is essential to normal cell growth. Yet, depletion of cellular inositol polyphosphates during erythropoiesis emerges as an additional physiological activity of Minpp1; loss of this enzyme activity in erythrocytes from Minpp1-deficient mice was accompanied by upregulation of a novel, substitutive inositol polyphosphate phosphatase.

Multiple inositol polyphosphate phosphatase is the only enzyme known to hydrolyze the abundant metabolites inositol pentakisphosphate and inositol hexakisphosphate. We have previously demonstrated that the chick homolog of multiple inositol polyphosphate phosphatase, designated HiPER1, has a role in growth plate chondrocyte differentiation. The relationship of these enzymes to intracellular signaling is obscure, and as part of our investigation we have examined the murine ((MMU)Minpp1) and human ((HSA)MINPP1) homologs. Northern blot analysis demonstrated expression of ((MMU)Minpp1 in a variety of mouse tissues, comparable to the expression of other mammalian homologs, but less restricted than the expression of HiPER1 in chick. A purified (MMU)Minpp1 fusion protein cleaved phosphate from inositol (1,3,4,5)-tetrakisphosphate and para-nitrophenyl phosphate. When the presumptive active site histidine was altered to alanine by site-directed mutagenesis, enzyme activity was abolished, confirming the classification of (MMU)Minpp1 as a histidine phosphatase. The amino acid sequences of the murine and human MINPP proteins share80% identity with the rat enzyme and56% identity with HiPER1, with conservation of the C-terminal consensus sequence that retains proteins in the endoplasmic reticulum. The intron/exon structure of the mammalian (MMU)Minpp1 and (HSA)MINPP1 genes is also conserved compared to the chick HiPER1 gene. Sequence analysis of plant and fruit fly MINPP homologs supports the hypothesis that the MINPP enzymes constitute a distinct evolutionary group within the histidine phosphatase family. We have mapped (HSA)MINPP1 to human chromosome 10q23 by fluorescence in situ hybridization, YAC screening, and radiation hybrid mapping. This assignment places (HSA)MINPP1 in a region of chromosome 10 that is frequently mutated in human cancers and places (HSA)MINPP1 proximal to the tumor suppressor PTEN, which maps to 10q23.3. Using a radiation hybrid panel, we localized (MMU)Minpp1 to a region of mouse chromosome 19 that includes the murine homolog of Pten. The evolutionary conservation of this novel enzyme within the inositol polyphosphate pathway suggests a significant role for multiple inositol polyphosphate phosphatase throughout higher eukaryotes.

The anti-inflammatory mediator interleukin-10 was investigated as a potential inhibitor of pro-inflammatory cytokine release in human peripheral blood monocytes activated with titanium particles. It inhibited the secretion of both tumor necrosis factor-alpha and interleukin-6 in a dose-dependent manner, with complete inhibition observed at 2 ng/ml. Co-culture experiments were performed to determine whether this cytokine may have functional importance as an inhibitor of the inflammatory response. When unstimulated lymphocytes and monocytes were co-cultured with titanium-stimulated monocytes, they significantly suppressed the secretion of both interleukin-6 and tumor necrosis factor-alpha. The inhibitory effect of these co-cultured cells could be partially blocked with the addition of an interleukin-10 neutralizing antibody. Interleukin-10 levels were measured in monocyte cultures treated with titanium particles as well as in fresh monocyte cultures treated with conditioned medium from titanium-stimulated monocytes. The latter experiments demonstrated marked stimulation of interleukin-10 secretion in conditioned medium-treated cultures, an effect that was related to the presence of tumor necrosis factor-alpha in the conditioned medium. The addition of titanium to conditioned medium-treated cultures markedly reduced the secretion of interleukin-10, suggesting that the most responsive cells are unstimulated monocytes exposed to agents released from activated monocytes. Altogether, the expression and responsiveness to interleukin-10 suggest a potential role for anti-inflammatory cytokines in regulation of the inflammatory response to wear debris.