ERRα promotes breast cancer cell dissemination to bone by increasing RANK expression in primary breast tumors.

Bone is the most common metastatic site for breast cancer. Estrogen-related-receptor alpha (ERRα) has been implicated in cancer cell invasiveness. Here, we established that ERRα promotes spontaneous metastatic dissemination of breast cancer cells from primary mammary tumors to the skeleton. We carried out cohort studies, pharmacological inhibition, gain-of-function analyses in vivo and cellular and molecular studies in vitro to identify new biomarkers in breast cancer metastases. Meta-analysis of human primary breast tumors revealed that high ERRα expression levels were associated with bone but not lung metastases. ERRα expression was also detected in circulating tumor cells from metastatic breast cancer patients. ERRα overexpression in murine 4T1 breast cancer cells promoted spontaneous bone micro-metastases formation when tumor cells were inoculated orthotopically, whereas lung metastases occurred irrespective of ERRα expression level. In vivo, Rank was identified as a target for ERRα. That was confirmed in vitro in Rankl stimulated tumor cell invasion, in mTOR/pS6K phosphorylation, by transactivation assay, ChIP and bioinformatics analyses. Moreover, pharmacological inhibition of ERRα reduced primary tumor growth, bone micro-metastases formation and Rank expression in vitro and in vivo. Transcriptomic studies and meta-analysis confirmed a positive association between metastases and ERRα/RANK in breast cancer patients and also revealed a positive correlation between ERRα and BRCA1mut carriers. Taken together, our results reveal a novel ERRα/RANK axis by which ERRα in primary breast cancer promotes early dissemination of cancer cells to bone. These findings suggest that ERRα may be a useful therapeutic target to prevent bone metastases.

Craniofacial and Dental Defects in the Col1a1Jrt/+ Mouse Model of Osteogenesis Imperfecta.

Certain mutations in the COL1A1 and COL1A2 genes produce clinical symptoms of both osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS) that include abnormal craniofacial growth, dental malocclusion, and dentinogenesis imperfecta. A mouse model (Col1a1(Jrt)/+) was recently developed that had a skeletal phenotype and other features consistent with moderate-to-severe OI and also with EDS. The craniofacial phenotype of 4- and 20-wk-old Col1a1(Jrt)/+ mice and wild-type littermates was assessed by micro-computed tomography (µCT) and morphometry. Teeth and the periodontal ligament compartment were analyzed by µCT, light microscopy/histomorphometry, and electron microscopy. Over time, at 20 wk, Col1a1(Jrt)/+ mice developed smaller heads, a shortened anterior cranial base, class III occlusion, and a mandibular side shift with shorter morphology in the masticatory region (maxilla and mandible). Col1a1(Jrt)/+ mice also had changes in the periodontal compartment and abnormalities in the dentin matrix and mineralization. These findings validate Col1a1(Jrt)/+ mice as a model for OI and EDS in humans.

Divergent regulation of the Osteopontin promoter by the estrogen receptor-related receptors is isoform- and cell context dependent.

We sought to determine whether the estrogen receptor-related receptor gamma (mEsrrg) regulated the Osteopontin (Opn) promoter through the same AP1/CAAT box element that we have previously described for mEsrra. In HeLa cells mEsrrg used an additional site present in the 5'UTR, while in ROS17/2.8 cells the AP1/CAAT site was not used, but a completely novel site surrounding the transcription start site was used. We also find that in ROS17/2.8 cells mEsrra repressed, while mEsrrg activated the Opn promoter. None of the sites identified conform to established Esrr response elements (ERREs). Additionally, the two reported mEsrrg protein isoforms showed differences in their activation potential. Mutations in the activation function 2 (AF2) of mEsrra, predicted to abolish activation, surprisingly turned mEsrra into a better activator. In contrast, similar AF2 mutations in Esrrg2 abolished its ability to activate the Opn promoter. Mutation of the DNA binding domain of mEsrra/g2 abolished transcriptional activity in HeLa and ROS17/2.8 cells. Our data indicate, first, that the two Esrr isoforms regulate Opn in a cell context-dependent manner. Second, they suggest that although the DNA binding domains of mEsrra and mEsrrg are 93% identical and required for regulation, the receptors bind to distinct Opn promoter elements, suggesting that the two isoforms may co-regulate Opn, and perhaps other genes, without competing for the same site in the promoter. Finally, the results suggest that each isoform interacts differently with co-activators and co-repressors, as highlighted by the AF2 mutation that turns mEsrra into a better activator but abolishes activity of Esrrg2.

Deficiency in acellular cementum and periodontal attachment in bsp null mice.

Bone sialoprotein (BSP) is an extracellular matrix protein found in mineralized tissues of the skeleton and dentition. BSP is multifunctional, affecting cell attachment and signaling through an RGD integrin-binding region, and acting as a positive regulator for mineral precipitation by nucleating hydroxyapatite crystals. BSP is present in cementum, the hard tissue covering the tooth root that anchors periodontal ligament (PDL) attachment. To test our hypothesis that BSP plays an important role in cementogenesis, we analyzed tooth development in a Bsp null ((-/-)) mouse model. Developmental analysis by histology, histochemistry, and SEM revealed a significant reduction in acellular cementum formation on Bsp (-/-) mouse molar and incisor roots, and the cementum deposited appeared hypomineralized. Structural defects in cementum-PDL interfaces in Bsp (-/-) mice caused PDL detachment, likely contributing to the high incidence of incisor malocclusion. Loss of BSP caused progressively disorganized PDL and significantly increased epithelial down-growth with aging. Bsp (-/-) mice displayed extensive root and alveolar bone resorption, mediated by increased RANKL and the presence of osteoclasts. Results collected here suggest that BSP plays a non-redundant role in acellular cementum formation, likely involved in initiating mineralization on the root surface. Through its importance to cementum integrity, BSP is essential for periodontal function.

Estrogen receptor-related receptor-alpha (ERR-alpha) is dysregulated in inflammatory arthritis.

OBJECTIVES: Subchondral bone loss is a characteristic feature of inflammatory arthritis. Recently, estrogen receptor-related receptor-alpha (ERR-alpha), an orphan nuclear receptor, has been found to be involved in activation of macrophages. We hypothesized that ERR-alpha which is expressed and also functional in articular chondrocytes, osteoblasts and osteoclasts, may be involved in rodent models of inflammatory arthritis. METHODS: Erosive arthritis was induced in DBA/1 mice by injection of type II collagen in Freund's complete adjuvant. RNA was isolated from the bone and joints and expression of ERR-alpha and cartilage (GDF5 and Col2a1) and bone [bone sialoprotein (BSP) and osteocalcin (OCN)] markers was analysed by semi-quantitative PCR. RESULTS: We report for the first time that the expression of ERR-alpha is dysregulated in bones and joints in a mouse model of inflammatory arthritis. Specifically, we show that ERR-alpha expression is down-regulated early in bone and later in joints of mice with type II CIA. Concomitantly, temporal changes were observed in GDF-5 and Col2a1 expression in joints following both initial injection and booster injection of type II collagen. Similarly, down-regulation of ERR-alpha mRNA expression in subchondral bone in mice with induced joint inflammation was also paralleled by down-regulation of markers of bone formation (BSP, OCN). CONCLUSIONS: These data suggest that dysregulation of ERR-alpha expression may precede and contribute to the destruction of cartilage and bone accompanying inflammatory arthritis.

The orphan nuclear estrogen receptor-related receptor-alpha regulates cartilage formation in vitro: implication of Sox9.

We report for the first time the expression of estrogen receptor-related receptor (ERR)-alpha in fetal and adult rat chondrocytes in growth plate and articular cartilage and the rat chondrogenic cell line C5.18 cells in vitro. ERRalpha mRNA and protein were expressed from proliferating chondrocyte to mature chondrocyte stages. We show that overexpressing ERRalpha in C5.18 cell cultures induces an increase in Sry-type high-mobility-group box transcription factor (Sox)-9 expression, a master gene in cartilage formation. In parallel, we report Sox9 promoter regulation by ERRalpha in C5.18 cells. To assess a functional role for ERRalpha in chondrogenesis, its expression was blocked by antisense oligonucleotides in C5.18 cell cultures, and this led to inhibition of cartilage formation associated with down-regulation of Sox9 and Indian hedgehog expression and maturation of proliferating chondrocytes into hypertrophic chondrocytes in vitro. Together these results implicate ERRalpha in the formation and maintenance of cartilage and also suggest that agonists and antagonists of ERRalpha may be useful as therapeutic agents in a wide variety of diseases affecting cartilage and joints.

Estrogen receptor-related receptor alpha: a mediator of estrogen response in bone.

Estrogen receptor-related receptor-alpha (ERRalpha) is an orphan nuclear receptor with sequence homology to the estrogen receptors, ERalpha/beta, but it does not bind estrogen. However, several recent studies suggest that ERRalpha not only plays a functional role in osteoblasts but also impinges on the estrogen axis in bone, as it does in at least certain other estrogen target tissues. We summarize here data on ERRalpha and its cellular and molecular modes of action that have broad implications for considering the potential role of this orphan receptor as a new therapeutic target in osteopenic disorders such as osteoporosis as well as other estrogen-responsive conditions.

Expression of leukemia inhibitory factor (LIF)/interleukin-6 family cytokines and receptors during in vitro osteogenesis: differential regulation by dexamethasone and LIF.

The leukemia inhibitory factor/interleukin-6 (LIF/IL-6) family of cytokines is known to play a major role in bone physiology. Although much work has focused on the regulation of bone resorption by IL-6 and related cytokines, their effects on osteoblast development and bone formation have not been as well studied. Previously, we reported that LIF inhibits, in a non-IL-6-dependent manner, osteoblast differentiation and bone nodule formation in the rat calvaria (RC) model, an effect that is antagonized by dexamethasone (Dex). The culture time-sensitive window suggested that LIF targets late preosteoblasts or early osteoblasts, and that this stage-specific effect coincided with a period of low endogenous production of LIF and IL-6. To detect potential crosstalk between members of this family, we have extended these observations by assessing the expression levels of other LIF/IL-6 cytokines (CNTF, OSM, IL-11, CT-1) and their receptors in the same RC cell model treated with or without LIF or Dex. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that IL-11 and its receptor, CNTF and its receptor, LIFR, and gp130 were constitutively expressed throughout the culture period. Expression of CT-1 and OSM increased with culture time - that is, with osteoblast differentiation - whereas the specific receptor for OSM (OSMR) was highly expressed at early timepoints and either plateaued or decreased thereafter. Continuous treatment with Dex (10(-8) mol/L) inhibited the endogenous production of IL-6, LIF, OSM, IL-11R, and OSMR, but had no detectable effect on the expression of IL-11, CT-1, CNTF, CNTFR, LIFR, or gp130. Finally, treatment with exogenously added LIF stimulated IL-6, LIF, LIFR, and OSMR, but had no other detectable effects. These data indicate that multiple members of the LIF/IL-6 family and their receptors are expressed in RC cell cultures, and are differentially regulated by Dex and LIF, suggesting that these cytokines play a complex and interdependent role, further modulated by glucocorticoid levels, in osteoprogenitor differentiation and bone nodule formation.

Biphasic effects of leukemia inhibitory factor on osteoblastic differentiation.

Leukemia inhibitory factor (LIF) is a cytokine produced by multiple cell types including osteoblasts and which is active on bone metabolism. We have previously shown that in a bone nodule forming in vitro model of osteogenesis, the fetal rat calvaria (RC) cell model, LIF inhibits osteoblast differentiation, acting on late osteoprogenitors and/or early osteoblasts. These results are in contrast to in vivo experiments, in which LIF has been found to increase bone formation. To resolve this discrepancy, we have tested the effect of LIF on rat bone marrow (RBM) stromal cell cultures, an in vitro model encompassing earlier osteoprogenitor stages. LIF inhibited cell growth in early, proliferating RBM cultures, but increased the culture saturation density. The effect of LIF on bone nodule formation in this model was cell density dependent and biphasic. Continuous treatment with LIF reduced the number of bone nodules present in confluent, more mature cultures, and the inhibitory effect was strongest when cells were plated at higher cell density than lower. In contrast, during the early stages of RBM culture, nodule numbers were higher in LIF-treated dishes than in controls, and this effect was greater in lower density cultures. Acute LIF treatment restricted to early time points increased the final number of bone nodules formed in mature RBM cell cultures, but not in RC cell cultures. Our results indicate that LIF exerts complex, stage-specific effects on osteoprogenitor recruitment, differentiation, and bone formation, and that the effects are cell nonautonomous, in the rat bone marrow stromal cell model. J. Cell. Biochem. Suppl. 36: 63-70, 2001.

The orphan nuclear estrogen receptor-related receptor alpha (ERRalpha) is expressed throughout osteoblast differentiation and regulates bone formation in vitro.

The orphan nuclear estrogen receptor-related receptor alpha (ERRalpha), 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 ERRalpha in bone, we first assessed its expression in rat calvaria (RC) in vivo and in RC cells in vitro. ERRalpha 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 ERRalpha 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, ERRalpha overexpression in RC cells increased differentiation and maturation of progenitors to mature bone-forming cells. Our findings show that ERRalpha 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.

Developmental expression and tissue distribution of Phex protein: effect of the Hyp mutation and relationship to bone markers.

Mutations in PHEX, a phosphate-regulating gene with homology to endopeptidases on the X chromosome, are responsible for X-linked hypophosphatemia (XLH). The murine Hyp homologue has the phenotypic features of XLH and harbors a large deletion in the 3' region of the Phex gene. We characterized the developmental expression and tissue distribution of Phex protein, using a monoclonal antibody against human PHEX, examined the effect of the Hyp mutation on Phex expression, and compared neprilysin (NEP), osteocalcin, and parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor gene expression in bone of normal and Hyp mice. Phex encodes a 100- to 105-kDa glycoprotein, which is present in bones and teeth of normal mice but not Hyp animals. These results were confirmed by in situ hybridization (ISH) and ribonuclease protection assay. Phex protein expression in femur and calvaria decreases with age, suggesting a correlation between Phex expression and bone formation. Immunohistochemical studies detected Phex protein in osteoblasts, osteocytes, and odontoblasts, but not in osteoblast precursors. In contrast to Phex, the abundance of NEP messenger RNA (mRNA) and protein is not significantly altered in Hyp bone. Similarly, osteocalcin and PTH/PTHrP receptor gene expression are not compromised in bone of Hyp mice. Our results are consistent with the hypothesis that loss of Phex function affects the mineralizing activity of osteoblasts rather than their differentiation.

Osteoprotegerin and its ligand: A new paradigm for regulation of osteoclastogenesis and bone resorption.

In just 3 years, striking new advances have been made in understanding the molecular mechanisms that govern the crosstalk between osteoblasts/stromal cells and hemopoietic osteoclast precursor cells that leads to osteoclastogenesis. Led first by the discovery of osteoprotegerin (OPG), a naturally occurring protein with potent osteoclastogenesis inhibitory activity, rapid progress was made to the isolation of RANKL, a transmembrane ligand expressed on osteoblasts/stromal cells, that binds to RANK, a transmembrane receptor on hemopoietic osteoclast precursor cells. The interaction of RANK and RANKL initiates a signaling and gene expression cascade that results in differentiation and maturation of osteoclast precursor cells to active osteoclasts capable of resorbing bone. Osteoprotegerin acts as a decoy receptor; it binds to RANKL and blocks its interaction with RANK, thus inhibiting osteoclast development. Many of the calciotropic hormones and cytokines, including vitamin D3, parathyroid hormone, prostaglandin E2 and interleukin-11, appear to stimulate osteoclastogenesis through the dual action of inhibiting production of OPG and stimulating production of RANKL. Estrogen, on the other hand, appears to inhibit production of RANKL and RANKL-stimulated osteoclastogenesis. Recently, the results of the first clinical trial with OPG supported its potential as a therapeutic agent for osteoporosis. The new understanding provided by the RANK/RANKL/OPG paradigm for both differentiation and activation of osteoclasts has had tremendous impact on the field of bone biology and has opened new avenues for development of possible treatments of diseases characterized by excessive bone resorption.

Osteoprotegerin and its ligand: a new paradigm for regulation of osteoclastogenesis and bone resorption.

In just 3 years, striking new advances have been made in understanding the molecular mechanisms that govern the crosstalk between osteoblasts/stromal cells and hematopoietic osteoclast precursor cells that leads to osteoclastogenesis. Led first by the discovery of osteoprotegerin (OPG), a naturally occurring protein with potent osteoclastogenesis inhibitory activity, rapid progress was made to the isolation of RANKL, a transmembrane ligand expressed on osteoblasts/stromal cells that binds to RANK, a transmembrane receptor on hematopoietic osteoclast precursor cells. The interaction of RANK and RANKL initiates a signaling and gene expression cascade that results in differentiation and maturation of osteoclast precursor cells to active osteoclasts capable of resorbing bone. OPG acts as a decoy receptor, binding to RANKL and blocking its interaction with RANK, inhibiting osteoclast development. Many of the calciotropic hormones and cytokines, including 1,25(OH)2D3, PTH, PGE2 and IL-11, appear to act through a dual capacity to inhibit production of OPG and stimulate production of RANKL. Estrogen, on the other hand, appears to inhibit production of RANKL and RANKL-stimulated osteoclastogenesis. Recently, the results of the first clinical trial with OPG supported its potential as a therapeutic agent for diseases such as osteoporosis. The new understanding provided by the RANK/RANKL/OPG paradigm for both differentiation of osteoclasts and their activation has had tremendous impact on the field and opened new avenues for development of possible treatments of diseases characterized by excessive bone resorption.

Aluminum accelerates osteoblastic differentiation but is cytotoxic in long-term rat calvaria cell cultures.

We have examined the effects of aluminum (Al) on osteoprogenitor proliferation and differentiation, cell survival, and bone formation in long-term rat calvaria (RC) cell cultures. RC cells were grown in alpha minimal essential medium containing 10% fetal bovine serum, 50 microg/ml ascorbic acid, and 10 mM beta-glycerophosphate with or without Al added to final concentrations of 1 microM-1 mM. Al caused a dose-dependent increase in the number of bone nodules present at early times (day 11) but had no significant effect on nodule numbers at later times (day 17). Time course experiments showed that Al increased nodule number beginning from day 7. Alkaline phosphatase activity, assessed at four stages during the differentiation sequence of RC cell cultures (from 4 to 13 days) was stimulated by Al at all times. However, Al decreased colony formation, inhibited cell growth in late log phase, and decreased saturation density of the treated cultures. Al concentrations of 30 microM and above resulted in degeneration of the cell layer and an increasing fibrillar appearance of the matrix present in between or adjacent to nodules when cultures were maintained for more than 15 days. The presence of Al significantly decreased the viability of cells obtained from 13-17 days cultures, as determined by plating efficiency and trypan blue exclusion. We frequently observed cellular toxicity (in 8 of 10 experiments) in cultures containing 300 microM Al, and by days 17-19, cells, nodules, and matrix were disintegrating in these cultures. We conclude that Al accelerates the rate of osteoprogenitor cell differentiation and the formation of bone nodules while concomitantly inhibiting nodule mineralization. However, concentrations that accelerate differentiation appear to be cytotoxic in long-term cultures.

cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes.

A cDNA fingerprinting strategy was developed to identify genes based on their differential expression pattern during osteoblast development. Preliminary biological and molecular staging of cDNA pools prepared by global amplification PCR allowed discrim-inating choices to be made in selection of expressed sequence tags (ESTs) to be isolated. Sequencing of selected ESTs confirmed that both known and novel genes can be isolated from any developmental stage of interest, e.g. from primitive progenitors, intermediate precursors or mature osteoblasts. EST expression provides insight into possible interrelated physiological functions and putative interacting molecules during differentiation. This method offers a functional genomics approach to isolate differentiation stage-specific genes in samples as small as a single cell.

Osteoprogenitor cell frequency in rat bone marrow stromal populations: role for heterotypic cell-cell interactions in osteoblast differentiation.

Glucocorticoids, notably dexamethasone (Dex), have been reported to be a requirement for osteoprogenitor cell differentiation in young adult rat bone marrow stromal cell populations. We have reinvestigated the requirement for Dex and analyzed the frequency of osteoprogenitor cells present. Stromal cells were grown as primary or first subcultures in the presence or absence of Dex and their expression of osteogenic markers (alkaline phosphatase activity, hormone responsiveness, and matrix molecules, including type I collagen, osteopontin, bone sialoprotein, and osteocalcin), as well as their functional capacity to differentiate to form a mineralized bone nodule, were assessed. Dex increased, but was not an absolute requirement for, the expression of osteogenic markers. Bone nodule formation was plating cell density dependent and occurred under all combinations of treatment with or without Dex but was maximal when Dex was present in both the primary and secondary cultures. Dex increased CFU-F by approximately 2-fold, but increased CFU-O (osteoprogenitor cells; bone nodule forming cells) by 5- to 50-fold depending on the cell density and duration of treatment. Neither CFU-F nor CFU-O expression followed a linear relationship in limiting dilution analysis until very high cell densities were reached, suggesting cooperativity of cell types within the population and a multitarget phenomenon leading to osteoprogenitor differentiation. When a large number of nonadherent bone marrow cells or their conditioned medium was added to the stromal cells, osteoprogenitors comprised approximately 1/100 of plated adherent cells and their expression followed a linear, single-hit relationship. By contrast, rat skin fibroblasts or their conditioned medium totally inhibited bone nodule formation. These data support the hypothesis that in marrow stroma, as in other bone cell populations such as those from calvaria, there are at least two classes of osteoprogenitor cells: those differentiating in the absence of added glucocorticoid and those requiring glucocorticoid to differentiate, that more than one cell type is limiting for stromal osteoprogenitor differentiation suggesting a role for heterotypic cell-cell interactions in osteogenesis in this tissue, and that Dex may be acting directly and/or indirectly through accessory cells in the bone marrow to alter osteoprogenitor cell expression.

Cloning of a 2.5 kb murine bone sialoprotein promoter fragment and functional analysis of putative Osf2 binding sites.

Bone sialoprotein (BSP) is an extracellular matrix protein that is intimately associated with the process of biomineralization. Osf2, a member of the Cbf/runt family of transcription factors, is required for the development of osteoblasts in vivo and has been reported to stimulate the transcription of BSP when overexpressed in mesenchymal cell lines. To investigate the role of Osf2 in BSP expression, we cloned a 2.5 kb fragment of a 5' untranscribed sequence from the murine BSP gene and evaluated it for putative Osf2 binding sites. This promoter, which was able to direct 5- to 10-fold higher levels of luciferase reporter expression in osteoblastic cells than in nonbone cell lines, contains two consensus core binding sites for members of the Cbf/runt family. One, at -61 relative to the start of transcription, is within a region having 75% overall sequence identity with the rat and human BSP promoters. The other is located at -1335, outside this highly conserved region. Neither site is completely conserved in the rat or human sequences. Only the -1335 site was able to bind a protein in nuclear extracts of osteoblastic cells, and this protein was identified as Osf2. Despite this in vitro binding ability, we detected no significant enhancer activity in the -1335 element when placed in front of a minimal osteocalcin promoter driving a luciferase reporter gene in osteoblastic cells nor any loss in transcriptional activity of a 5' promoter deletion which eliminated this element as compared with the full-length 2.5 kb promoter. These results suggest that Osf2 binding to the BSP promoter is not essential for its osteoblast-selective expression.

LIF, but not IL-6, regulates osteoprogenitor differentiation in rat calvaria cell cultures: modulation by dexamethasone.

Cytokines of the interleukin 6 (IL-6) subfamily are a group of factors produced by osteoblasts and acting through the same transducing element, membrane protein gp130. We have previously shown that exogenous (added to the culture medium) leukemia inhibitory factor (LIF) inhibits bone nodule formation and expression of osteoblast-associated genes in fetal rat calvaria (RC) cell cultures and that dexamethasone (Dex) increases the ID50 of LIF. To investigate the respective roles of IL-6-related cytokines and receptors in osteprogenitor differentiation, and their regulatory interplay with Dex, we used reverse transcribed polymerase chain reaction, bioassay, and blocking antibody techniques to assess the time courses of LIF, IL-6, LIF transmembrane receptor, IL-6 receptor, and gp130 expression in RC cell cultures grown with and without Dex. The levels of the mRNAs for IL-6, LIF, and gp130 decreased concomitantly with the formation of bone nodules. Dex treatment, which stimulates bone nodule formation, reduced the expression of LIF and IL-6 mRNAs and IL-6 bioactivity in the culture medium. LIF treatment strongly stimulated the expression of IL-6. Incubation with anti-LIF antibodies increased the number of nodules, while an antibody blocking IL-6 activity had little or no effect on nodule numbers and did not antagonize the action of exogenous LIF, indicating that IL-6 does not mediate the action of LIF in this system. Moreover, although exogenously added IL-6 was active in the cultures as noted by a reduction of nodule mineralization, it had no effect on nodule numbers, i.e., on osteoprogenitor differentiation, in the presence or absence of Dex. In conclusion, IL-6, LIF, and their receptors are expressed throughout the time-course of osteogenesis in RC cell cultures. However, only LIF, but not IL-6, appears to play a significant role in autocrine regulation of osteoblastic differentiation in this system. The antagonist action of Dex on the effects of exogenously added LIF, as well as the bone-promoting action of Dex in RC cell cultures, could be exerted partly through the down-regulation of the expression of endogenous LIF.

Osteogenic protein-1 up-regulation of the collagen X promoter activity is mediated by a MEF-2-like sequence and requires an adjacent AP-1 sequence.

Bone morphogenetic proteins induce chondrogenesis and osteogenesis in vivo. To investigate molecular mechanisms involved in chondrocyte induction, we examined the effect of osteogenic protein (OP)-1/bone morphogenetic protein-7 on the collagen X promoter. In rat calvaria-derived chondrogenic C5.18 cells, OP-1 up-regulates collagen X mRNA levels and its promoter activity in a cell type- specific manner. Deletion analysis localizes the OP-1 response region to 33 bp (-310/-278), which confers OP-1 responsiveness to both the minimal homologous and heterologous Rous sarcoma virus promoter. Transforming growth factor-beta2 or activin, which up-regulates the expression of a transforming growth factor-beta-inducible p3TP-Lux construct, has little effect on collagen X mRNA and on this 33-bp region. Mutational analysis shows that both an AP-1 like sequence (-294/-285, TGAATCATCA) and an A/T-rich myocyte enhancer factor (MEF)-2 like sequence (-310/-298, TTAAAAATAAAAA) in the 33-bp region are necessary for the OP-1 effect. Gel shift assays show interaction of distinct nuclear proteins from C5.18 cells with the AP-1-like and the MEF-2-like sequences. OP-1 rapidly induces nuclear protein interaction with the MEF-2-like sequence but not with the AP-1 like sequence. MEF-2-like binding activity induced by OP-1 is distinct from the MEF-2 family proteins present in C2C12 myoblasts, in which OP-1 does not induce collagen X mRNA or up-regulate its promoter activity. In conclusion, we identified a specific response region for OP-1 in the mouse collagen X promoter. Mutational and gel shift analyses suggest that OP-1 induces nuclear protein interaction with an A/T-rich MEF-2 like sequence, distinct from the MEF-2 present in myoblasts, and up-regulates collagen X promoter activity, which also requires an AP-1 like sequence.

The ERR-1 orphan receptor is a transcriptional activator expressed during bone development.

We studied the expression of estrogen-related receptor ERR-1 during mouse embryonic development. ERR-1 mRNA is present in bones formed by both the endochondral and intramembranous routes, and the onset of its expression coincides with bone formation. By RT-PCR experiments, we found that ERR-1, but not the related receptor ERR-2, is expressed in osteoblastic osteosarcoma cell lines as well as in primary osteoblastic cell populations derived from normal human bone. By gel shift analysis we found that ERR-1 binds as a monomer specifically to the SFRE sequence (SF-1-responsive-element; TCAAGGTCA). Mutation analysis revealed that both the core AGGTCA motif and the TCA 5'-extension are required for efficient ERR-1 binding. In transient transfection assays, ERR-1 acts as a potent transactivator through the SFRE sequence. This effect is cell-specific since ERR-1 activates transcription in the rat osteosarcoma cell line ROS 17.2/8 as well as in HeLa, NB-E, and FREJ4 cells but not in COS1 and HepG2 cells. Notably, the osteopontin (a protein expressed by osteoblasts and released in the bone matrix) gene promoter is a target for ERR-1 transcriptional regulation. Our findings suggest a role for ERR-1 in bone development and metabolism.