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The orphan nuclear estrogen receptor--related receptor [Alpha] (ERR[Alpha]), is expressed by many cell types, but is very highly expressed by osteoblastic cells in which it transactivates at least one osteoblast-associated gene, osteopontin. To study the putative involvement of ERR[Alpha] in bone, we first assessed its expression in rat calvaria (RC) in vivo and in RC cells in vitro. ERR[Alpha] mRNA and protein were expressed at all developmental stages from early osteoprogenitors to bone-forming osteoblasts, but protein was most abundant in mature cuboidal osteoblasts. To assess a functional role for ERR[Alpha] in osteoblast differentiation and bone formation, we blocked its expression by antisense oligonucleotides in either proliferating or differentiating RC cell cultures and found inhibition of cell growth and a proliferation-independent inhibition of differentiation. On the other hand, ERR[Alpha] overexpression in RC cells increased differentiation and maturation of progenitors to mature bone-forming cells. Our findings show that ERRet is highly expressed throughout the osteoblast developmental sequence and plays a physiological role in differentiation and bone formation at both proliferation and differentiation stages. In addition, we found that manipulation of receptor levels in the absence of known ligand is a fruitful approach for functional analysis of this orphan receptor and identification of potential target genes. Key words: osteoblasts * bone * nuclear receptor * estrogen related * differentiation

Les cancers pulmonaires non à petites cellules (CPNPC) sont fréquents et représentent la première cause de mortalité par cancer en France. L'immunothérapie par inhibiteurs de point de contrôle immunitaire (ICI) est une nouvelle arme thérapeutique très efficace, disponible en routine pour les patients en plus des doublets de chimiothérapies et des thérapeutiques ciblées. En contrepartie, les ICI déclenchent fréquemment des effets immuno-induits. Récemment, Ye et al. ont rapporté en épidémiologie que les patients sous ICI présentaient un risque accru de fractures bénignes. Les résultats préliminaires, chez la souris, semblent concordants. Notre étude vise à évaluer l'effet osseux des ICI, chez les patients atteints de CPNPC métastatique osseux traités par ICI en monothérapie en première ligne. Nous avons mis en place, entre un CHU et 2 Centres de lutte contre le cancer une étude rétrospective des fractures vertébrales (FV) survenant chez les patients adultes atteints d'un CPNPC métastatique osseux traités en première ligne par ICI en monothérapie. Les scanners à baseline et à 6 mois ont permis de comptabiliser l'existence de fractures bénignes entre T4 et L5. Les vertèbres métastatiques ont été exclues. Entre octobre 2016 et décembre 2020, 58 patients ont été identifiés et inclus, correspondants à 812 vertèbres situées entre T4 et L5. Soixante-dix vertèbres non analysables ou métastatiques ont été exclues. Les patients étaient majoritairement des hommes (75 %) de 65,8 ± 3,4 ans avec un IMC de 24,7 ± 1,2 kg/m2. Parmi eux, 83 % étaient fumeurs et 88 % avaient un bon score de Performans Status (≤ 1). Aucun patient n'avait été traité auparavant par anti-résorbeur osseux. 8,6 % avaient reçu une corticothérapie au long cours (> 3 mois) avant l'instauration de l'ICI. La prévalence des FV à baseline était de 21 % (12/58). Après 6 mois de traitement, 4 patients ont présenté une première FV, et 2, une nouvelle fracture, soit 10,3 % de FV incidentes à six mois portant à 16 (28 %) le nombre total de patients fracturés après 6 mois de traitement par ICI. Le nombre total de FV à 6 mois est de 24. Les fractures se localisaient principalement à la charnière thoracolombaire (38 % des cas). Les patients fracturés étaient significativement plus âgés (73,4 ans vs 64,2 ans, p = 0,01). Aucune différence significative n'a été observée pour le sexe, l'IMC, le tabagisme, la corticothérapie initiale ou un traitement osseux antérieur. De plus, au cours du suivi, 24 % des patients ont reçu un traitement antirésorbeur osseux après le début de l'ICI (13 denosumab et 1 bisphosphonate). Parmi les traités, 2 ont développé une FV incidente, contre 4 chez les non traités. Une différence numérique mais non significative a été observée dans l'utilisation de la corticothérapie après ICI (11,9 % vs 25 %). Les patients atteints de CPNPC métastatique osseux traités par ICI ont un risque élevé de FV à court terme. L'âge est le principal facteur de risque. Notre étude souligne aussi la faible prescription des antirésorbeurs osseux chez les patients métastatiques malgré les recommandations de l'ESMO 2020. La santé osseuse des patients oncologiques doit faire partie de leur prise en charge globale. [ABSTRACT FROM AUTHOR]

Bone loss associated with estrogen deficiency indicates a fundamental role of these hormones in skeletal growth and bone remodeling. In the last decades, growing recent evidence demonstrated that estrogens can also affect the immune compartment of the bone. In this review, we summarize the impacts of estrogens on bone immune cells and their consequences on bone homeostasis, metastasis settlement into the bone and tumor progression. We also addressed the role of an orphan nuclear receptor ERRalpha ("Estrogen-receptor Related Receptor alpha") on macrophages and T lymphocytes, and as an immunomodulator in bone metastases. Hence, this review links estrogens to bone immune cells in osteo-oncology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Marie and Bonnelye.)

Skeletal metastases are frequent complications of many cancers, causing bone complications (fractures, bone pain, disability) that negatively affect the patient's quality of life. Here, we first discuss the burden of skeletal complications in cancer bone metastasis. We then describe the pathophysiology of bone metastasis. Bone metastasis is a multistage process: long before the development of clinically detectable metastases, circulating tumor cells settle and enter a dormant state in normal vascular and endosteal niches present in the bone marrow, which provide immediate attachment and shelter, and only become active years later as they proliferate and alter the functions of bone-resorbing (osteoclasts) and bone-forming (osteoblasts) cells, promoting skeletal destruction. The molecular mechanisms involved in mediating each of these steps are described, and we also explain how tumor cells interact with a myriad of interconnected cell populations in the bone marrow, including a rich vascular network, immune cells, adipocytes, and nerves. We discuss metabolic programs that tumor cells could engage with to specifically grow in bone. We also describe the progress and future directions of existing bone-targeted agents and report emerging therapies that have arisen from recent advances in our understanding of the pathophysiology of bone metastases. Finally, we discuss the value of bone turnover biomarkers in detection and monitoring of progression and therapeutic effects in patients with bone metastasis.

The current paradigm of osteoblast fate is that the majority undergo apoptosis, while some further differentiate into osteocytes and others flatten and cover bone surfaces as bone lining cells. Osteoblasts have been described to exhibit heterogeneous expression of a variety of osteoblast markers at both transcriptional and protein levels. To explore further this heterogeneity and its biological significance, Venus-positive (Venus + ) cells expressing the fluorescent protein Venus under the control of the 2.3-kb Col1a1 promoter were isolated from newborn mouse calvariae and subjected to single-cell RNA sequencing. Functional annotation of the genes expressed in 272 Venus + single cells indicated that Venus + cells are osteoblasts that can be categorized into four clusters. Of these, three clusters (clusters 1 to 3) exhibited similarities in their expression of osteoblast markers, while one (cluster 4) was distinctly different. We identified a total of 1920 cluster-specific genes and pseudotime ordering analyses based on established concepts and known markers showed that clusters 1 to 3 captured osteoblasts at different maturational stages. Analysis of gene co-expression networks showed that genes involved in protein synthesis and protein trafficking between endoplasmic reticulum (ER) and Golgi are active in these clusters. However, the cells in these clusters were also defined by extensive heterogeneity of gene expression, independently of maturational stage. Cells of cluster 4 expressed Cd34 and Cxcl12 with relatively lower levels of osteoblast markers, suggesting that this cell type differs from actively bone-forming osteoblasts and retain or reacquire progenitor properties. Based on expression and machine learning analyses of the transcriptomes of individual osteoblasts, we also identified genes that may be useful as new markers of osteoblast maturational stages. Taken together, our data show much more extensive heterogeneity of osteoblasts than previously documented, with gene profiles supporting diversity of osteoblast functional activities and developmental fates. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research., (© 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.)

Bone metastasis remains the most frequent and the deadliest complication of prostate cancer (PCa). Mechanisms leading to the homing of tumor cells to bone remain poorly characterized. Role of chemokines in providing navigational cues to migrating cancer cells bearing specific receptors is well established. Bone is an adipocyte-rich organ since 50 to 70% of the adult bone marrow (BM) volume comprise bone marrow adipocytes (BM-Ads), which are likely to produce chemokines within the bone microenvironment. Using in vitro migration assays, we demonstrated that soluble factors released by human primary BM-Ads are able to support the directed migration of PCa cells in a CCR3-dependent manner. In addition, we showed that CCL7, a chemokine previously involved in the CCR3-dependent migration of PCa cells outside of the prostate gland, is released by human BM-Ads. These effects are amplified by obesity and ageing, two clinical conditions known to promote aggressive and metastatic PCa. In human tumors, we found an enrichment of CCR3 in bone metastasis vs. primary tumors at mRNA levels using Oncomine microarray database. In addition, immunohistochemistry experiments demonstrated overexpression of CCR3 in bone versus visceral metastases. These results underline the potential importance of BM-Ads in the bone metastatic process and imply a CCR3/CCL7 axis whose pharmacological interest needs to be evaluated.

Bone metastasis remains a major cause of mortality and morbidity in breast cancer. Therefore, there is an urgent need to better select high-risk patients in order to adapt patient's treatment and prevent bone recurrence. Here, we found that integrin alpha5 (ITGA5) was highly expressed in bone metastases, compared to lung, liver, or brain metastases. High ITGA5 expression in primary tumors correlated with the presence of disseminated tumor cells in bone marrow aspirates from early stage breast cancer patients (n = 268; p = 0.039). ITGA5 was also predictive of poor bone metastasis-free survival in two separate clinical data sets (n = 855, HR = 1.36, p = 0.018 and n = 427, HR = 1.62, p = 0.024). This prognostic value remained significant in multivariate analysis (p = 0.028). Experimentally, ITGA5 silencing impaired tumor cell adhesion to fibronectin, migration, and survival. ITGA5 silencing also reduced tumor cell colonization of the bone marrow and formation of osteolytic lesions in vivo. Conversely, ITGA5 overexpression promoted bone metastasis. Pharmacological inhibition of ITGA5 with humanized monoclonal antibody M200 (volociximab) recapitulated inhibitory effects of ITGA5 silencing on tumor cell functions in vitro and tumor cell colonization of the bone marrow in vivo. M200 also markedly reduced tumor outgrowth in experimental models of bone metastasis or tumorigenesis, and blunted cancer-associated bone destruction. ITGA5 was not only expressed by tumor cells but also osteoclasts. In this respect, M200 decreased human osteoclast-mediated bone resorption in vitro. Overall, this study identifies ITGA5 as a mediator of breast-to-bone metastasis and raises the possibility that volociximab/M200 could be repurposed for the treatment of ITGA5-positive breast cancer patients with bone metastases.

Bone is the most common metastatic site for breast cancer. Although the estrogen-related receptor alpha (ERRα) has been implicated in breast cancer cell dissemination to the bone from the primary tumor, its role after tumor cell anchorage in the bone microenvironment remains elusive. Here, we reveal that ERRα inhibits the progression of bone metastases of breast cancer cells by increasing the immune activity of the bone microenvironment. Overexpression of ERRα in breast cancer bone metastases induced expression of chemokines CCL17 and CCL20 and repressed production of TGFβ3. Subsequently, CD8 + T lymphocytes recruited to bone metastases escaped TGFβ signaling control and were endowed with exacerbated cytotoxic features, resulting in significant reduction in metastases. The clinical relevance of our findings in mice was confirmed in over 240 patients with breast cancer. Thus, this study reveals that ERRα regulates immune properties in the bone microenvironment that contributes to decreasing metastatic growth. SIGNIFICANCE: This study places ERRα at the interplay between the immune response and bone metastases of breast cancer, highlighting a potential target for intervention in advanced disease., (©2020 American Association for Cancer Research.)

Communication between osteoblasts and osteoclasts plays a key role in bone metabolism. We describe here an unexpected role for matrix vesicles (MVs), which bud from bone-forming osteoblasts and have a well-established role in initiation of bone mineralization, in osteoclastogenesis. We show that the MV cargo miR-125b accumulates in the bone matrix, with increased accumulation in transgenic (Tg) mice overexpressing miR-125b in osteoblasts. Bone formation and osteoblasts in Tg mice are normal, but the number of bone-resorbing osteoclasts is reduced, leading to higher trabecular bone mass. miR-125b in the bone matrix targets and degrades Prdm1, a transcriptional repressor of anti-osteoclastogenic factors, in osteoclast precursors. Overexpressing miR-125b in osteoblasts abrogates bone loss in different mouse models. Our results show that the MV cargo miR-125b is a regulatory element of osteoblast-osteoclast communication, and that bone matrix provides extracellular storage of miR-125b that is functionally active in bone resorption.

Prostate cancers have a strong propensity to metastasize to bone and promote osteoblastic lesions. TMPRSS2:ERG is the most frequent gene rearrangement identified in prostate cancer, but whether it is involved in prostate cancer bone metastases is largely unknown. We exploited an intratibial metastasis model to address this issue and we found that ectopic expression of the TMPRSS2:ERG fusion enhances the ability of prostate cancer cell lines to induce osteoblastic lesions by stimulating bone formation and inhibiting the osteolytic response. In line with these in vivo results, we demonstrate that the TMPRSS2:ERG fusion protein increases the expression of osteoblastic markers, including Collagen Type I Alpha 1 Chain and Alkaline Phosphatase, as well as Endothelin-1, a protein with a documented role in osteoblastic bone lesion formation. Moreover, we determined that the TMPRSS2:ERG fusion protein is bound to the regulatory regions of these genes in prostate cancer cell lines, and we report that the expression levels of these osteoblastic markers are correlated with the expression of the TMPRSS2:ERG fusion in patient metastasis samples. Taken together, our results reveal that the TMPRSS2:ERG gene fusion is involved in osteoblastic lesion formation induced by prostate cancer cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Periostin is an extracellular matrix protein that actively contributes to tumor progression and metastasis. Here, we hypothesized that it could be a marker of bone metastasis formation. To address this question, we used two polyclonal antibodies directed against the whole molecule or its C-terminal domain to explore the expression of intact and truncated forms of periostin in the serum and tissues (lung, heart, bone) of wild-type and periostin-deficient mice. In normal bones, periostin was expressed in the periosteum and specific periostin proteolytic fragments were found in bones, but not in soft tissues. In animals bearing osteolytic lesions caused by 4T1 cells, C-terminal intact periostin (iPTN) expression disappeared at the invasive front of skeletal tumors where bone-resorbing osteoclasts were present. In vitro, we found that periostin was a substrate for osteoclast-derived cathepsin K, generating proteolytic fragments that were not recognized by anti-periostin antibodies directed against iPTN. In vivo, using an in-house sandwich immunoassay aimed at detecting iPTN only, we observed a noticeable reduction of serum periostin levels (- 26%; P < 0.002) in animals bearing osteolytic lesions caused by 4T1 cells. On the contrary, this decrease was not observed in women with breast cancer and bone metastases when periostin was measured with a human assay detecting total periostin. Collectively, these data showed that mouse periostin was degraded at the bone metastatic sites, potentially by cathepsin K, and that the specific measurement of iPTN in serum should assist in detecting bone metastasis formation in breast cancer.

miRNAs are master regulators of gene expression that play key roles in cancer metastasis. During bone metastasis, metastatic tumor cells must rewire their biology and express genes that are normally expressed by bone cells (a process called osteomimicry), which endow tumor cells with full competence for outgrowth in the bone marrow. Here, we establish miR-30 family members miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e as suppressors of breast cancer bone metastasis that regulate multiple pathways, including osteomimicry. Low expression of miR-30 in primary tumors from patients with breast cancer were associated with poor relapse-free survival. In addition, estrogen receptor (ER)-negative/progesterone receptor (PR)-negative breast cancer cells expressed lower miR-30 levels than their ER/PR-positive counterparts. Overexpression of miR-30 in ER/PR-negative breast cancer cells resulted in the reduction of bone metastasis burden in vivo In vitro , miR-30 did not affect tumor cell proliferation, but did inhibit tumor cell invasion. Furthermore, overexpression of miR-30 restored bone homeostasis by reversing the effects of tumor cell-conditioned medium on osteoclastogenesis and osteoblastogenesis. A number of genes associated with osteoclastogenesis stimulation ( IL8, IL11 ), osteoblastogenesis inhibition ( DKK-1 ), tumor cell osteomimicry ( RUNX2, CDH11 ), and invasiveness ( CTGF, ITGA5, ITGB3 ) were identified as targets for repression by miR-30. Among these genes, silencing CDH11 or ITGA5 in ER-/PR-negative breast cancer cells recapitulated inhibitory effects of miR-30 on skeletal tumor burden in vivo Overall, our findings provide evidence that miR-30 family members employ multiple mechanisms to impede breast cancer bone metastasis and may represent attractive targets for therapeutic intervention. Significance: These findings suggest miR-30 family members may serve as an effective means to therapeutically attenuate metastasis in triple-negative breast cancer. Cancer Res; 78(18); 5259-73. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Introduction: Pierson syndrome is caused by a mutation of LAMB2, encoding for laminin β2. Clinical phenotype is variable but usually associates congenital nephrotic syndrome (CNS) and ocular abnormalities. Neuromuscular impairment has also been described., Methods: We report on a 15-year old girl, suffering from Pierson Syndrome, who developed severe bone deformations during puberty. This patient initially displayed CNS and microcoria, leading to the clinical diagnosis of Pierson syndrome. Genetic analysis revealed a truncating mutation and a splice site mutation of LAMB2. The patient received a renal transplantation (R-Tx) at the age of 3. After R-Tx, renal evolution was simple, the patient receiving low-dose corticosteroids, tacrolimus and mycophenolate mofetil. At the age of 12, bone deformations progressively appeared. At the time of bone impairment, renal function was subnormal (glomerular filtration rate using iohexol clearance 50mL/min per 1.73m 2 ), and parameters of calcium/phosphate metabolism were normal (calcium 2.45mmol/L, phosphorus 1.30mmol/L, PTH 81ng/L, ALP 334U/L, 25OH-D 73nmol/L). Radiographs showed major deformations such as scoliosis, genu varum and diffuse epiphyseal abnormalities. A high resolution scanner (HR-pQCT) was performed, demonstrating a bone of "normal low" quantity and quality; major radial and cubital deformations were observed. Stainings of laminin β2 were performed on bone and renal samples from the patient and healthy controls: as expected, laminin β2 was expressed in the control kidney but not in the patient's renal tissue, and a similar pattern was observed in bone., Conclusion: This is the first case of skeletal impairment ever described in Pierson syndrome. Integrin α3β1, receptor for laminin β2, are found in podocytes and osteoblasts, and the observation of both the presence of laminin β2 staining in healthy bone and its absence in the patient's bone raises the question of a potential role of laminin β2 in bone physiology., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Lysyl oxidase (LOX) is a secreted copper-dependent amine oxidase whose primary function is to drive collagen crosslinking and extracellular matrix stiffness. LOX in colorectal cancer synergizes with hypoxia-inducible factor-1 (HIF-1) to promote tumor progression. Here we investigated whether LOX/HIF1 endows colorectal cancer cells with full competence for aggressive colonization in bone. We show that a high LOX expression in primary tumors from patients with colorectal cancer was associated with poor clinical outcome, irrespective of HIF-1 In addition, LOX was expressed by tumor cells in the bone marrow from colorectal cancer patients with bone metastases. In vivo experimental studies show that LOX overexpression in colorectal cancer cells or systemic delivery of the conditioned medium from LOX-overexpressing colorectal cancer cells promoted tumor cell dissemination in the bone marrow and enhanced osteolytic lesion formation, irrespective of HIF-1 Conversely, silencing or pharmacologic inhibition of LOX activity blocked dissemination of colorectal cancer cells in the bone marrow and tumor-driven osteolytic lesion formation. In vitro, tumor-secreted LOX supported the attachment and survival of colorectal cancer cells to and in the bone matrix, and inhibited osteoblast differentiation. LOX overexpression in colorectal cancer cells also induced a robust production of IL6. In turn, both LOX and IL6 were acting in concert to promote RANKL-dependent osteoclast differentiation, thereby creating an imbalance between bone resorption and bone formation. Collectively, our findings show that LOX supports colorectal cancer cell dissemination in the bone marrow and they reveal a novel mechanism through which LOX-driven IL6 production by colorectal cancer cells impairs bone homeostasis. Cancer Res; 77(2); 268-78. ©2016 AACR., (©2016 American Association for Cancer Research.)

Bone metastases are one of the main complications of prostate cancer and they are incurable. We investigated whether and how estrogen receptor-related receptor alpha (ERRα) is involved in bone tumor progression associated with advanced prostate cancer. By meta-analysis, we first found that ERRα expression is correlated with castration-resistant prostate cancer (CRPC), the hallmark of progressive disease. We then analyzed tumor cell progression and the associated signaling pathways in gain-of-function/loss-of-function CRPC models in vivo and in vitro. Increased levels of ERRα in tumor cells led to rapid tumor progression, with both bone destruction and formation, and direct impacts on osteoclasts and osteoblasts. VEGF-A, WNT5A and TGFβ1 were upregulated by ERRα in tumor cells and all of these factors also significantly and positively correlated withERRα expression in CRPC patient specimens. Finally, high levels of ERRα in tumor cells stimulated the pro-metastatic factor periostin expression in the stroma, suggesting that ERRα regulates the tumor stromal cell microenvironment to enhance tumor progression. Taken together, our data demonstrate that ERRα is a regulator of CRPC cell progression in bone. Therefore, inhibiting ERRα may constitute a new therapeutic strategy for prostate cancer skeletal-related events.

The transcription factor TWIST1 induces epithelial-mesenchymal transition and/or escape to the oncogenic-induced failsafe program, facilitating the intravasation of breast cancer cells in the systemic circulation and their dissemination to the lungs. Its involvement in breast cancer bone metastasis is unknown. To address this question, human osteotropic MDA-MB-231/B02 breast cancer cells were stably transfected with a Tet-inducible vector encoding for TWIST1, whose expression was specifically repressed in the presence of doxycycline (dox). The intra-arterial inoculation of transfectants expressing TWIST1 in immunodeficient mice substantially increased the extent of osteolytic lesions in these animals, being 50% larger than that of animals bearing mock-transfected tumors, as determined by radiography. This difference was accompanied by a sharp reduction of the bone volume (indicating a higher bone destruction) and a twofold increase in the tumor volume compared with mice bearing mock-transfected tumors, as determined by histomorphometry. Importantly, the suppression of TWIST1 expression in MDA-MB-231/B02 cells in the presence of dox abolished the stimulatory effect of TWIST1 on bone metastasis formation in vivo. Additionally, examination of the bone marrow from untreated and dox-treated animals on day 7 after tumor cell inoculation, at which time there was no evidence of radiographic osteolytic lesions, revealed that the number of tumor cell colonies that were recovered from the bone marrow of untreated mice was dramatically increased compared with that of dox-fed animals. In vitro, TWIST1 expression promoted tumor cell invasion and enhanced microRNA 10b (miR-10b) expression, a proinvasive factor, but was dispensable for growth of tumor cells. In vivo, the repression of miR-10b substantially decreased the presence of TWIST1-expressing breast cancer cells in the bone marrow. Overall, these results establish that TWIST1 facilitates breast cancer bone metastasis formation through a mechanism dependent of miR-10b, which leads to increase tumor burden and bone destruction., (© 2014 American Society for Bone and Mineral Research.)

Adult Ibsp-knockout mice (BSP-/-) display shorter stature, lower bone turnover and higher trabecular bone mass than wild type, the latter resulting from impaired bone resorption. Unexpectedly, BSP knockout also affects reproductive behavior, as female mice do not construct a proper "nest" for their offsprings. Multiple crossing experiments nonetheless indicated that the shorter stature and lower weight of BSP-/- mice, since birth and throughout life, as well as their shorter femur and tibia bones are independent of the genotype of the mothers, and thus reflect genetic inheritance. In BSP-/- newborns, µCT analysis revealed a delay in membranous primary ossification, with wider cranial sutures, as well as thinner femoral cortical bone and lower tissue mineral density, reflected in lower expression of bone formation markers. However, trabecular bone volume and osteoclast parameters of long bones do not differ between genotypes. Three weeks after birth, osteoclast number and surface drop in the mutants, concomitant with trabecular bone accumulation. The growth plates present a thinner hypertrophic zone in newborns with lower whole bone expression of IGF-1 and higher IHH in 6 days old BSP-/- mice. At 3 weeks the proliferating zone is thinner and the hypertrophic zone thicker in BSP-/- than in BSP+/+ mice of either sex, maybe reflecting a combination of lower chondrocyte proliferation and impaired cartilage resorption. Six days old BSP-/- mice display lower osteoblast marker expression but higher MEPE and higher osteopontin(Opn)/Runx2 ratio. Serum Opn is higher in mutants at day 6 and in adults. Thus, lack of BSP alters long bone growth and membranous/cortical primary bone formation and mineralization. Endochondral development is however normal in mutant mice and the accumulation of trabecular bone observed in adults develops progressively in the weeks following birth. Compensatory high Opn may allow normal endochondral development in BSP-/- mice, while impairing primary mineralization.

Lysophosphatidic acid (LPA) is a natural bioactive lipid that acts through six different G protein-coupled receptors (LPA1-6) with pleiotropic activities on multiple cell types. We have previously demonstrated that LPA is necessary for successful in vitro osteoclastogenesis of bone marrow cells. Bone cells controlling bone remodeling (i.e. osteoblasts, osteoclasts, and osteocytes) express LPA1, but delineating the role of this receptor in bone remodeling is still pending. Despite Lpar1(-/-) mice displaying a low bone mass phenotype, we demonstrated that bone marrow cell-induced osteoclastogenesis was reduced in Lpar1(-/-) mice but not in Lpar2(-/-) and Lpar3(-/-) animals. Expression of LPA1 was up-regulated during osteoclastogenesis, and LPA1 antagonists (Ki16425, Debio0719, and VPC12249) inhibited osteoclast differentiation. Blocking LPA1 activity with Ki16425 inhibited expression of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and dendritic cell-specific transmembrane protein and interfered with the fusion but not the proliferation of osteoclast precursors. Similar to wild type osteoclasts treated with Ki16425, mature Lpar1(-/-) osteoclasts had reduced podosome belt and sealing zone resulting in reduced mineralized matrix resorption. Additionally, LPA1 expression markedly increased in the bone of ovariectomized mice, which was blocked by bisphosphonate treatment. Conversely, systemic treatment with Debio0719 prevented ovariectomy-induced cancellous bone loss. Moreover, intravital multiphoton microscopy revealed that Debio0719 reduced the retention of CX3CR1-EGFP(+) osteoclast precursors in bone by increasing their mobility in the bone marrow cavity. Overall, our results demonstrate that LPA1 is essential for in vitro and in vivo osteoclast activities. Therefore, LPA1 emerges as a new target for the treatment of diseases associated with excess bone loss.

Background: Up to 80% of patients dying from prostate carcinoma have developed bone metastases that are incurable. Castration is commonly used to treat prostate cancer. Although the disease initially responds to androgen blockade strategies, it often becomes castration-resistant (CRPC for Castration Resistant Prostate Cancer). Most of the murine models of mixed lesions derived from prostate cancer cells are androgen sensitive. Thus, we established a new model of CRPC (androgen receptor (AR) negative) that causes mixed lesions in bone., Methods: PC3 and its derived new cell clone PC3c cells were directly injected into the tibiae of SCID male mice. Tumor growth was analyzed by radiography and histology. Direct effects of conditioned medium of both cell lines were tested on osteoclasts, osteoblasts and osteocytes., Results: We found that PC3c cells induced mixed lesions 10 weeks after intratibial injection. In vitro, PC3c conditioned medium was able to stimulate tartrate resistant acid phosphatase (TRAP)-positive osteoclasts. Osteoprotegerin (OPG) and endothelin-1 (ET1) were highly expressed by PC3c while dikkopf-1 (DKK1) expression was decreased. Finally, PC3c highly expressed bone associated markers osteopontin (OPN), Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP) and produced mineralized matrix in vitro in osteogenic conditions., Conclusions: We have established a new CRPC cell line as a useful system for modeling human metastatic prostate cancer which presents the mixed phenotype of bone metastases that is commonly observed in prostate cancer patients with advanced disease. This model will help to understand androgen-independent mechanisms involved in the progression of prostate cancer in bone and provides a preclinical model for testing the effects of new treatments for bone metastases.

Estrogen receptor-related receptor alpha (ERRα) is an orphan nuclear receptor with sequence homology to the estrogen receptors, ERα/β, but it does not bind estrogen. ERRα not only plays a functional role in osteoblasts but also in osteoclasts and chondrocytes. In addition, the ERRs, including ERRα, can be activated by coactivators such as peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC1α and β) and are implicated in adipogenesis, fatty acid oxidation, and oxidative stress defense, suggesting that ERRα-through its activity in bone resorption and adipogenesis--may regulate the insulin and leptin pathways and contribute to aging-related changes in bone and cartilage. In this review, we discuss data on ERRα and its cellular and molecular modes of action, which have broad implications for considering the potential role of this orphan receptor in cartilage and bone endocrine function, on whole-organism physiology, and in the bone aging process., (Copyright © 2013 American Society for Bone and Mineral Research.)

Purpose: Nitrogen-containing bisphosphonates (N-BP) such as zoledronate and risedronate exhibit antitumor effects. They block the activity of farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway, leading to intracellular accumulation of mevalonate metabolites (IPP/ApppI), which are recognized as tumor phosphoantigens by Vγ9Vδ2 T cells. However, mechanisms responsible for Vγ9Vδ2 T-cell recognition of N-BP-treated tumors producing IPP/ApppI remain unclear., Experimental Design: The effects of N-BPs on Vγ9Vδ2 T-cell expansion and anticancer activity were evaluated in vitro and in animal models of human breast cancers. The modalities of recognition of breast tumors by Vγ9Vδ2 T cells in N-BP-treated animals were also examined., Results: We found a strong correlation between Vγ9Vδ2 T-cell anticancer activity and intracellular accumulation of IPP/ApppI in risedronate-treated breast cancer cells in vitro. In addition, following risedronate treatment of immunodeficient mice bearing human breast tumors, human Vγ9Vδ2 T cells infiltrated and inhibited growth of tumors that produced high IPP/ApppI levels but not those expressing low IPP/ApppI levels. The combination of doxorubicin with a N-BP improved, however, Vγ9Vδ2 T-cell cytotoxicity against breast tumors expressing low IPP/ApppI levels. Moreover, Vγ9Vδ2 T-cell cytotoxicity in mice treated with risedronate or zoledronate did not only depend on IPP/ApppI accumulation in tumors but also on expression of tumor cell surface receptor intercellular adhesion molecule-1 (ICAM-1), which triggered the recognition of N-BP-treated breast cancer cells by Vγ9Vδ2 T cells in vivo., Conclusion: These findings suggest that N-BPs can have an adjuvant role in cancer therapy by activating Vγ9Vδ2 T-cell cytotoxicity in patients with breast cancer that produces high IPP/ApppI levels after N-BP treatment., (©2012 AACR.)

Bone metastasis is a complication occurring in up to 70% of advanced breast cancer patients. The estrogen receptor-related receptor alpha (ERRα) has been implicated in breast cancer and bone development, prompting us to examine whether ERRα may function in promoting the osteolytic growth of breast cancer cells in bone. In a mouse xenograft model of metastatic human breast cancer, overexpression of wild-type ERRα reduced metastasis, whereas overexpression of a dominant negative mutant promoted metastasis. Osteoclasts were directly affected and ERRα upregulated the osteoclastogenesis inhibitor, osteoprotegerin (OPG), providing a direct mechanistic basis for understanding how ERRα reduced breast cancer cell growth in bone. In contrast, ERRα overexpression increased breast cancer cell growth in the mammary gland. ERRα-overexpressing primary tumors were highly vascularized, consistent with an observed upregulation of angiogenic growth factor, the VEGF. In support of these findings, we documented that elevated expression of ERRα mRNA in breast carcinomas was associated with high expression of OPG and VEGF and with disease progression. In conclusion, our results show that ERRα plays a dual role in breast cancer progression in promoting the local growth of tumor cells, but decreasing metastatic growth of osteolytic lesions in bone., (©2011 AACR.)

Objective: We reported previously that the orphan nuclear receptor, estrogen receptor-related receptor α (ERRα), is expressed in articular chondrocytes and is dysregulated in a mouse model of inflammatory arthritis. The aim of this study, therefore, was to determine whether ERRα is also dysregulated in patients with osteoarthritis (OA)., Methods: ERRα messenger RNA (mRNA) and protein were quantified in normal and OA cartilage samples and in OA chondrocytes in vitro, with and without short-term treatment with a variety of OA-associated factors and signaling pathway agonists and inhibitors., Results: ERRα expression was lower in OA than in normal articular cartilage. Interleukin-1β (IL-1β) markedly up-regulated ERRα expression in OA chondrocytes in vitro, and agonist or inhibitor treatment indicated that the up-regulation was dependent on cyclooxygenase 2 (COX-2; NS398), prostaglandin E(2), cAMP (8-bromo-cAMP), and protein kinase A (PKA; KT5720). Treatment with the ERRα inverse agonist XCT790 decreased the expression of SOX9 and the up-regulation of ERRα by IL-1β, suggesting autoregulation of ERRα in the IL-1β pathway. Matrix metalloproteinase 13 (MMP-13) expression was also decreased by treatment with XCT790 plus IL-1β versus IL-1β alone, and the down-regulation of MMP-13 mRNA and protein observed with XCT790 alone suggests that the up-regulation of MMP-13 by IL-1β is ERRα-dependent., Conclusion: We report the first evidence that ERRα expression is regulated by IL-1β in COX-2-, cAMP-, and PKA-dependent pathways in OA chondrocytes. We confirmed that SOX9 is an ERRα target gene in human, as in rodent, chondrocytes and identified MMP-13 as a potential new target gene, which suggests that ERRα may both respond to the healing signal and contribute to extracellular degradation in OA cartilage., (Copyright © 2011 by the American College of Rheumatology.)