RESUMO
Alterations in bone remodeling are a major public health issue, as therapeutic options for widespread bone disorders such as osteoporosis and tumor-induced osteolysis are still limited. Therefore, a detailed understanding of the regulatory mechanism governing bone cell differentiation in health and disease are of utmost clinical importance. Here we report a novel function of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a member of the immunoglobulin superfamily involved in inflammation and tumorigenesis, in the physiologic regulation of bone remodeling. Assessing the expression of all members of the murine Ceacam family in bone tissue and marrow, we found CEACAM1 and CEACAM10 to be differentially expressed in both bone-forming osteoblasts and bone-resorbing osteoclasts. While Ceacam10-deficient mice displayed no alteration in structural bone parameters, static histomorphometry demonstrated a reduced trabecular bone mass in mice lacking CEACAM1. Furthermore, cellular and dynamic histomorphometry revealed an increased osteoclast formation in Ceacam1-deficient mice, while osteoblast parameters and the bone formation rate remained unchanged. In line with these findings, we detected accelerated osteoclastogenesis in Ceacam1-deficient bone marrow cells, while osteoblast differentiation, as determined by mineralization and alkaline phosphatase assays, was not affected. Therefore, our results provide in vivo and in vitro evidence for a physiologic role of CEACAM1 in the regulation of osteoclastogenesis.
Assuntos
Moléculas de Adesão Celular/deficiência , Osteoclastos/citologia , Animais , Antígenos CD/genética , Medula Óssea/metabolismo , Remodelação Óssea , Moléculas de Adesão Celular/genética , Linhagem Celular , Regulação da Expressão Gênica , Camundongos , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese , FenótipoRESUMO
The hormone calcitonin (CT) is primarily known for its pharmacologic action as an inhibitor of bone resorption, yet CT-deficient mice display increased bone formation. These findings raised the question about the underlying cellular and molecular mechanism of CT action. Here we show that either ubiquitous or osteoclast-specific inactivation of the murine CT receptor (CTR) causes increased bone formation. CT negatively regulates the osteoclast expression of Spns2 gene, which encodes a transporter for the signalling lipid sphingosine 1-phosphate (S1P). CTR-deficient mice show increased S1P levels, and their skeletal phenotype is normalized by deletion of the S1P receptor S1P3. Finally, pharmacologic treatment with the nonselective S1P receptor agonist FTY720 causes increased bone formation in wild-type, but not in S1P3-deficient mice. This study redefines the role of CT in skeletal biology, confirms that S1P acts as an osteoanabolic molecule in vivo and provides evidence for a pharmacologically exploitable crosstalk between osteoclasts and osteoblasts.
Assuntos
Calcitonina/metabolismo , Lisofosfolipídeos/metabolismo , Osteoclastos/citologia , Osteogênese , Esfingosina/análogos & derivados , Alelos , Animais , Osso e Ossos/metabolismo , Colagenases/metabolismo , Cruzamentos Genéticos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/citologia , Osteoporose/fisiopatologia , Fenótipo , Porosidade , Receptores da Calcitonina/metabolismo , Transdução de Sinais , Esfingosina/metabolismoRESUMO
Stable Fos expression in the osteoblast lineage results in the development of osteosarcomas (OS) in mice, yet the underlying mechanisms are poorly understood. Using a genetic system in which Fos expression can be induced in osteoblasts in a doxycycline-dependent manner and through subsequent RNA sequencing and gene set enrichment analysis, we were able to identify novel transcriptional targets of Fos in osteoblasts. These included a distinct activation of cellular response toward replication stress (RS), exemplified by a Fos-dependent induction of the RS-suppressing Chk1 kinase. Importantly, Fos expression protects osteoblasts from RS and DNA damage likely through upregulation of Chk1 and facilitates transformation by Ras/E1A oncogenes. These data reveal a novel function of Fos in safeguarding genome stability during replication, which is particularly relevant in conditions of oncogene-induced S-phase entry.
Assuntos
Replicação do DNA/fisiologia , Proteínas Oncogênicas v-fos/metabolismo , Osteoblastos/metabolismo , Proteínas Quinases/metabolismo , Animais , Células Cultivadas , Quinase 1 do Ponto de Checagem , Dano ao DNA/fisiologia , Técnicas de Inativação de Genes , Camundongos , Proteínas Oncogênicas v-fos/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Although Wnt signaling is considered a key regulatory pathway for bone formation, inactivation of ß-catenin in osteoblasts does not affect their activity but rather causes increased osteoclastogenesis due to insufficient production of osteoprotegerin (Opg). By monitoring the expression pattern of all known genes encoding Wnt receptors in mouse tissues and bone cells we identified Frizzled 8 (Fzd8) as a candidate regulator of bone remodeling. Fzd8-deficient mice displayed osteopenia with normal bone formation and increased osteoclastogenesis, but this phenotype was not associated with impaired Wnt signaling or Opg production by osteoblasts. The deduced direct negative influence of canonical Wnt signaling on osteoclastogenesis was confirmed in vitro and through the generation of mice lacking ß-catenin in the osteoclast lineage. Here, we observed increased bone resorption despite normal Opg production and a resistance to the anti-osteoclastogenic effect of Wnt3a. These results demonstrate that Fzd8 and ß-catenin negatively regulate osteoclast differentiation independent of osteoblasts and that canonical Wnt signaling controls bone resorption by two different mechanisms.
Assuntos
Osteoclastos/citologia , Osteoprotegerina/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Reabsorção Óssea , Diferenciação Celular , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/metabolismo , Osteogênese/fisiologia , Osteoprotegerina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Receptores Wnt/genética , Receptores Wnt/metabolismo , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo , Proteína Wnt3A/fisiologia , beta Catenina/genética , beta Catenina/metabolismo , beta Catenina/fisiologiaRESUMO
X-linked hypophosphatemic rickets (XLH) is a genetic disorder caused by mutational inactivation of the PHEX gene, encoding a transmembrane endopeptidase expressed in osteoblasts. Since several experiments involving Phex-deficient Hyp mice have demonstrated that an increased expression of Fgf23 in osteoblasts is causative for the renal phosphate loss characteristic of XLH, we performed genome-wide expression analysis to compare differentiated osteoblasts from wildtype and Hyp mice. Here we did not only observe the expected increase of Fgf23 expression in the latter ones, but also a differential expression of genes that are either induced by or involved in retinoic acid signaling, which led us to analyze whether dietary retinol deprivation would influence the phenotype of Hyp mice. Unexpectedly, feeding a retinol-free diet resulted in a partial rescue of the growth plate and bone mineralization defects in 6 weeks old Hyp mice. When we fed the same diet for 24 weeks the amount of non-mineralized bone matrix (osteoid) was reduced by more than 70%, although phosphate homeostasis was unaffected. In contrast, a dietary normalization of serum phosphate levels in Hyp mice reduced the osteoid amount by less than 30%, thereby demonstrating a previously unknown impact of retinol on the cell-autonomous mineralization defect of Phex-deficient osteoblasts.
Assuntos
Calcificação Fisiológica , Raquitismo Hipofosfatêmico Familiar/fisiopatologia , Doenças Genéticas Ligadas ao Cromossomo X , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Vitamina A/antagonistas & inibidores , Animais , Sequência de Bases , Primers do DNA , Dieta , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Camundongos , Reação em Cadeia da Polimerase , Vitamina A/administração & dosagemRESUMO
WNT5A has recently been implicated in inflammatory processes, but its role as a bone marrow stromal cell (BMSC)-derived mediator of joint inflammation in arthritis is unclear. Here, we investigated whether inflammatory stimuli induce WNT5A in BMSC to control inflammatory responses. WNT5A levels were determined in human BMSC after stimulation with lipopolysaccharide (LPS) or tumor necrosis factor α (TNF-α,) and in synovial cells and tissue of patients with rheumatoid arthritis (RA) and human TNF-α transgenic (hTNFtg) mice. A microarray analysis of WNT5A-treated murine osteoblasts was performed using Affymetrix gene chips. The regulation of cytokine/chemokine expression was confirmed by qPCR, ELISA, and Luminex technology in BMSC after stimulation with WNT5A or WNT5A knockdown. Relevant signaling pathways were identified using specific inhibitors. Migration of MACS-purified T lymphocytes and monocytes was assessed using the FluoroBlok system. WNT5A expression was increased threefold in BMSC after stimulation with LPS or TNF-α. Synovial fibroblasts from patients with RA showed a twofold increase of WNT5A expression compared with control cells, and its expression was highly induced in the synovial tissue of patients with RA and hTNFtg mice. Microarray analysis of WNT5A-treated osteoblasts identified cytokines and chemokines as targets. The induction of IL-1ß, IL-6, CCL2, CCL5, CXCL1, and CXCL5 by WNT5A was confirmed in BMSC and depended on the activation of the NF-κB, mitogen-activated protein (MAPK), and Akt pathways. Accordingly, knockdown of WNT5A markedly reduced the basal and LPS-induced cytokine/chemokine production. Finally, migration of monocytes and T cells toward the supernatant of WNT5A-treated BMSC was increased by 25% and 20%, respectively. This study underlines the critical role of BMSC-derived WNT5A in the regulation of inflammatory processes and suggests its participation in the pathogenesis of RA.
Assuntos
Células da Medula Óssea/metabolismo , Quimiocinas/biossíntese , Citocinas/biossíntese , Mediadores da Inflamação/fisiologia , Proteínas Proto-Oncogênicas/biossíntese , Células Estromais/metabolismo , Proteínas Wnt/biossíntese , Adulto , Animais , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Células da Medula Óssea/efeitos dos fármacos , Movimento Celular , Separação Celular , Células Cultivadas , Feminino , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Células Estromais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Proteína Wnt-5aRESUMO
Interleukin-33 (IL-33) is the most recently identified member of the IL-1 family of cytokines, which is primarily known for its proinflammatory functions. We have previously reported that IL-33 is expressed by bone-forming osteoblasts, and that administration of recombinant IL-33 to bone marrow cultures inhibits their differentiation into bone-resorbing osteoclasts. Likewise, while the inhibitory effect of IL-33 on osteoclast differentiation was fully abolished in cultures lacking the IL-33 receptor ST2, mice lacking ST2 displayed low bone mass caused by increased osteoclastogenesis. Although these data suggested a physiological role of IL-33 as an inhibitor of bone resorption, direct in vivo evidence supporting such a function was still missing. Here we describe the generation and bone histomorphometric analysis of a transgenic mouse model (Col1a1-Il33) over-expressing IL-33 specifically in osteoblasts. While we did not observe differences in osteoblast number and bone formation between wildtype and Col1a1-Il33 mice, the number of osteoclasts was significantly reduced compared to wildtype littermates in two independent transgenic lines. Since we did not observe quantitative differences in the populations of eosinophils, neutrophils, basophils or M2-macrophages from the bone marrow of wildtype and Col1a1-Il33 mice, our data demonstrate that an inhibition of osteoclastogenesis is one of the major physiological functions of IL-33, at least in mice.
Assuntos
Interleucinas/fisiologia , Osteoblastos/fisiologia , Animais , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Interleucina-33 , Interleucinas/genética , Camundongos , Camundongos Transgênicos , Osteoblastos/citologia , TransgenesRESUMO
Skeletal metastases are a frequent complication of prostate, breast and lung cancer, and the interactions of tumor cells with bone-forming osteoblasts and bone-resorbing osteoclasts have been suggested to play critical roles in disease progression. We have previously shown that treatment of primary murine osteoblasts with conditioned medium of the human osteolytic prostate cancer cell line PC-3 results in a rapid induction of chemokine expression, thereby providing further evidence for a molecular crosstalk between bone and tumor cells. The aim of our current study was to identify PC-3-derived molecules mediating this effect. Using Affymetrix Gene Chip hybridization followed by qRT-PCR we were able to confirm that the expression of chemokine-encoding genes is markedly induced in human primary osteoblasts following incubation with PC-3-conditioned medium. Since this induction was significantly affected upon alteration of p65-levels in PC-3 cells, we performed a second genome-wide expression analysis to identify p65-regulated cytokines, which were then tested for their ability to induce chemokine expression. Here we observed that interleukin-1ß (IL-1B) did not only increase the expression of chemokines in osteoblasts, but also the phosphorylation of p65 and thereby its own expression. Since immunohistochemistry on bone biopsy sections from prostate cancer metastases demonstrated IL-1B expression in both, tumor cells and osteoblasts, our data suggest that IL-1B is one of the relevant cytokines involved in the skeletal complications of cancer metastases.
Assuntos
Neoplasias Ósseas/metabolismo , Quimiocinas/metabolismo , Interleucina-1beta/metabolismo , Osteoblastos/metabolismo , Comunicação Parácrina , Neoplasias da Próstata/metabolismo , Fator de Transcrição RelA/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Quimiocinas/genética , Meios de Cultivo Condicionados/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Imuno-Histoquímica , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/imunologia , Fosforilação , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição RelA/genética , Transfecção , Regulação para CimaRESUMO
Although Wnt signaling in osteoblasts is of critical importance for the regulation of bone remodeling, it is not yet known which specific Wnt receptors of the Frizzled family are functionally relevant in this process. In this paper, we show that Fzd9 is induced upon osteoblast differentiation and that Fzd9(-/-) mice display low bone mass caused by impaired bone formation. Our analysis of Fzd9(-/-) primary osteoblasts demonstrated defects in matrix mineralization in spite of normal expression of established differentiation markers. In contrast, we observed a reduced expression of chemokines and interferon-regulated genes in Fzd9(-/-) osteoblasts. We also identified the ubiquitin-like modifier Isg15 as one potential downstream mediator of Fzd9 in these cells. Importantly, our molecular analysis further revealed that canonical Wnt signaling is not impaired in the absence of Fzd9, thus explaining the absence of a bone resorption phenotype. Collectively, our results reveal a previously unknown function of Fzd9 in osteoblasts, a finding that may have therapeutic implications for bone loss disorders.
Assuntos
Receptores de Neurotransmissores/metabolismo , Proteínas Wnt/metabolismo , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Quimiocinas/metabolismo , Receptores Frizzled , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese , Receptores de Neurotransmissores/genética , Transdução de Sinais/fisiologia , Distribuição Tecidual , Proteínas Wnt/genéticaRESUMO
Since the hematopoetic system is located within the bone marrow, it is not surprising that recent evidence has demonstrated the existence of molecular interactions between bone and immune cells. While interleukin 1 (IL-1) and IL-18, two cytokines of the IL-1 family, have been shown to regulate differentiation and activity of bone cells, the role of IL-33, another IL-1 family member, has not been addressed yet. Since we observed that the expression of IL-33 increases during osteoblast differentiation, we analyzed its possible influence on bone formation and observed that IL-33 did not affect matrix mineralization but enhanced the expression of Tnfsf11, the gene encoding RANKL. This finding led us to analyze the skeletal phenotype of Il1rl1-deficient mice, which lack the IL-33 receptor ST2. Unexpectedly, these mice displayed normal bone formation but increased bone resorption, thereby resulting in low trabecular bone mass. Since this finding suggested a negative influence of IL-33 on osteoclastogenesis, we next analyzed osteoclast differentiation from bone marrow precursor cells and observed that IL-33 completely abolished the generation of TRACP(+) multinucleated osteoclasts, even in the presence of RANKL and macrophage colony-stimulating factor (M-CSF). Although our molecular studies revealed that IL-33 treatment of bone marrow cells caused a shift toward other hematopoetic lineages, we further observed a direct negative influence of IL-33 on the osteoclastogenic differentiation of RAW264.7 macrophages, where IL-33 repressed the expression of Nfatc1, which encodes one of the key transciption factors of osteoclast differentiation. Taken together, these findings have uncovered a previously unknown function of IL-33 as an inhibitor of bone resorption.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Interleucinas/fisiologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Fosfatase Ácida/metabolismo , Animais , Basófilos/citologia , Basófilos/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Reabsorção Óssea/metabolismo , Osso e Ossos/patologia , Calcitriol/farmacologia , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Eosinófilos/citologia , Eosinófilos/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/farmacologia , Isoenzimas/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoclastos/patologia , Ligante RANK/genética , Ligante RANK/farmacologia , Receptores de Interleucina/genética , Células Estromais/citologia , Células Estromais/fisiologia , Fosfatase Ácida Resistente a TartaratoRESUMO
Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders.
Assuntos
Proteínas de Membrana/fisiologia , Osteogênese , Transdução de Sinais , Proteínas Wnt/antagonistas & inibidores , Animais , Remodelação Óssea , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Osteoblastos/patologia , Osteoporose/etiologiaRESUMO
Prostate cancer primarily metastasizes to bone, and the interaction of cancer cells with bone cells results in a local activation of bone formation and/or bone resorption. Since the cellular and molecular mechanisms underlying the development of these tumor-induced osteoblastic or osteolytic lesions are still poorly understood, we have compared the effects of two prostate cancer cell lines, osteoblastic MDA-PCa-2b cells and osteolytic PC-3 cells, on bone-forming osteoblasts. Using Affymetrix Gene Chip hybridization followed by qRT-PCR confirmation we were able to identify specific genes, including Smpd3 and Dmp1, whose expression is significantly reduced upon treatment with PC-3-conditioned medium. Moreover, we observed that PC-3-conditioned medium led to a marked induction of several cytokine genes, including Cxcl5, Cxcl12 and Tnfsf11, the latter one encoding for the osteoclast differentiation factor Rankl. Likewise, when we analyzed the effects of MDA-PCa-2b- and PC-3-conditioned medium on signal transduction in osteoblasts we did not only observe opposite effects on the canonical Wnt signalling pathway, but also a specific induction of Erk and Stat phosphorylation by PC-3-conditioned medium. Most importantly, the induction of Cxcl5, Cxcl12 and Tnfsf11 in osteoblasts by PC-3-conditioned medium was abrogated by the Stat3/5 inhibitor piceatannol, whereas the selective blockade of Stat1 and Erk activation had no effect. Together with the finding, that activated Stat3 in osteoblasts was detectable in bone biopsies from patients with osteolytic metastases, our data suggest that the Stat3/5-dependent activation of cytokine expression in osteoblasts may have a significant impact on cancer cell migration and proliferation, but also on osteoclast activation.
Assuntos
Citocinas/genética , Osteoblastos/metabolismo , Osteogênese , Osteólise/patologia , Neoplasias da Próstata/patologia , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiocinas/genética , Quimiocinas/metabolismo , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteólise/complicações , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/complicações , Neoplasias da Próstata/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/metabolismoRESUMO
The availability of Rahman, and the virulent HM-1:IMSS strain of E. histolytica, provides a powerful tool for identifying virulence factors of E. histolytica. Here we report an attempt to identify potential virulence factors of E. histolytica by comparing the transcriptome of E. histolytica HM-1:IMSS and E. histolytica Rahman. With phenotypically defined strains, we compared the transcriptome of Rahman and HM-1:IMSS using a custom 70mer oligonucleotide based microarray that has essentially full representation of the E. histolytica HM-1:IMSS genome. We find extensive differences between the two strains, including distinct patterns of gene expression of cysteine proteinases, AIG family members, and lectin light chains.