RESUMO
Osteosarcoma (OS) is an aggressive bone cancer originating in the mesenchymal lineage. Prognosis for metastatic disease is poor, with a mortality rate of approximately 40%; OS is an aggressive disease for which new treatments are needed. All bone cells are sensitive to their mechanical/physical surroundings and changes in these surroundings can affect their behavior. However, it is not well understood how OS cells specifically respond to fluid movement, or substrate stiffness-two stimuli of relevance in the tumor microenvironment. We used cells from spontaneous OS tumors in a mouse engineered to have a bone-specific knockout of pRb-1 and p53 in the osteoblast lineage. We silenced Sox2 (which regulates YAP) and tested the effect of fluid flow shear stress (FFSS) and substrate stiffness on YAP expression/activity-which was significantly reduced by loss of Sox2, but that effect was reversed by FFSS but not by substrate stiffness. Osteogenic gene expression was also reduced in the absence of Sox2 but again this was reversed by FFSS and remained largely unaffected by substrate stiffness. Thus we described the effect of two distinct stimuli on the mechanosensory and osteogenic profiles of OS cells. Taken together, these data suggest that modulation of fluid movement through, or stiffness levels within, OS tumors could represent a novel consideration in the development of new treatments to prevent their progression.
RESUMO
Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family members generate phosphatidylinositol 4,5-bisphosphate (PIP2), a critical lipid regulator of diverse physiological processes. The PIP5K-dependent PIP2 generation can also act upstream of the oncogenic phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Many studies have demonstrated various mechanisms of spatiotemporal regulation of PIP5K catalytic activity. However, there are few studies on regulation of PIP5K protein stability. Here, we examined potential regulation of PIP5Kα, a PIP5K isoform, via ubiquitin-proteasome system, and its implication for breast cancer. Our results showed that the ubiquitin ligase NEDD4 (neural precursor cell expressed, developmentally down-regulated gene 4) mediated ubiquitination and proteasomal degradation of PIP5Kα, consequently reducing plasma membrane PIP2 level. NEDD4 interacted with the C-terminal region and ubiquitinated the N-terminal lysine 88 in PIP5Kα. In addition, PIP5Kα gene disruption inhibited epidermal growth factor (EGF)-induced Akt activation and caused significant proliferation defect in breast cancer cells. Notably, PIP5Kα K88R mutant that was resistant to NEDD4-mediated ubiquitination and degradation showed more potentiating effects on Akt activation by EGF and cell proliferation than wild-type PIP5Kα. Collectively, these results suggest that PIP5Kα is a novel degradative substrate of NEDD4 and that the PIP5Kα-dependent PIP2 pool contributing to breast cancer cell proliferation through PI3K/Akt activation is negatively controlled by NEDD4.
Assuntos
Membrana Celular/metabolismo , Regulação Neoplásica da Expressão Gênica , Ubiquitina-Proteína Ligases Nedd4/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Proliferação de Células , Fator de Crescimento Epidérmico/farmacologia , Feminino , Edição de Genes , Humanos , Mutação , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Ubiquitinação/efeitos dos fármacosRESUMO
The stem cell transcription factor Sox2 is highly expressed in many cancers where it is thought to mark cancer stem cells (CSCs). In osteosarcomas, the most common bone malignancy, high Sox2 expression marks and maintains a fraction of tumor-initiating cells that show all the properties of CSC. Knockdown of Sox2 expression abolishes tumorigenicity and suppresses the CSC phenotype. Here we show that, in a mouse model of osteosarcoma, osteoblast-specific Sox2 conditional knockout (CKO) causes a drastic reduction in the frequency and onset of tumors. The rare tumors detected in the Sox2 CKO animals were all Sox2 positive, indicating that they arose from cells that had escaped Sox2 deletion. Furthermore, Sox2 inactivation in cultured osteosarcoma cells by CRISPR/CAS technology leads to a loss of viability and proliferation of the entire cell population. Inactivation of the YAP gene, a major Hippo pathway effector which is a direct Sox2 target, causes similar results and YAP overexpression rescues cells from the lethality caused by Sox2 inactivation. These effects were osteosarcoma-specific, suggesting a mechanism of cell "addiction" to Sox2-initiated pathways. The requirement of Sox2 for osteosarcoma formation as well as for the survival of the tumor cells suggests that disruption of Sox2-initiated pathways could be an effective strategy for the treatment of osteosarcoma.
Assuntos
Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Proliferação de Células/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Fatores de Transcrição SOXB1/genética , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Camundongos , Camundongos Knockout , Células-Tronco Neoplásicas/patologia , Transdução de Sinais/genéticaRESUMO
The transcription factor (TF) yes-associated protein 1 (YAP1) is a major effector of the tumor suppressive Hippo signaling pathway and is also necessary to maintain pluripotency in embryonic stem cells. Elevated levels of YAP1 expression antagonize the tumor suppressive effects of the Hippo pathway that normally represses YAP1 function. High YAP1 expression is observed in several types of human cancers and is particularly prominent in cancer stem cells (CSCs). The stem cell TF Sox2, which marks and maintains CSCs in osteosarcomas (OSs), promotes YAP1 expression by binding to an intronic enhancer element and YAP1 expression is also crucial for the maintainance of OS stem cells. To further understand the regulation of YAP1 expression in OSs, we subjected the YAP1 intronic enhancer to scanning mutagenesis to identify all DNA cis-elements critical for enhancer function. Through this approach, we identified two novel TFs, GA binding protein (GABP) and myeloid zinc finger 1 (MZF1), which are essential for basal YAP1 transcription. These factors are highly expressed in OSs and bind to distinct sites in the YAP1 enhancer. Depletion of either factor leads to drastically reduced YAP1 expression and thus a reversal of stem cell properties. We also found that YAP1 can regulate the expression of Sox2 by binding to two distinct DNA binding sites upstream and downstream of the Sox2 gene. Thus, Sox2 and YAP1 reinforce each others expression to maintain stemness and tumorigenicity in OSs, but the activity of MZF1 and GABP is essential for YAP1 transcription. Stem Cells 2017;35:2340-2350.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Linhagem Celular Tumoral , Humanos , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
Osteosarcomas are malignant tumors of bone, most commonly seen in children and adolescents. Despite advances in modern medicine, the poor survival rate of metastatic osteosarcoma has not improved in two decades. In the present study we have investigated the effect of Riluzole on a human and mouse metastatic osteosarcoma cells. We show that LM7 cells secrete glutamate in the media and that mGluR5 receptors are required for the proliferation of LM7 cells. Riluzole, which is known to inhibit glutamate release, inhibits proliferation, induces apoptosis and prevents migration of LM7 cells. This is also seen with Fenobam, a specific blocker of mGluR5. We also show that Riluzole alters the phosphorylation status of AKT/P70 S6 kinase, ERK1/2 and JNK1/2. Thus Riluzole is an effective drug to inhibit proliferation and survival of osteosarcoma cells and has therapeutic potential for the treatment of osteosarcoma exhibiting autocrine glutamate signaling.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Osteossarcoma/tratamento farmacológico , Receptor de Glutamato Metabotrópico 5/metabolismo , Riluzol/farmacologia , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Humanos , Camundongos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
Osteosarcoma (OS) is a highly aggressive pediatric bone cancer in which most tumor cells remain immature and fail to differentiate into bone-forming osteoblasts. However, OS cells readily respond to adipogenic stimuli suggesting they retain mesenchymal stem cell-like properties. Here we demonstrate that nuclear receptor PPARγ agonists such as the anti-diabetic, thiazolidinedione (TZD) drugs induce growth arrest and cause adipogenic differentiation in human, mouse and canine OS cells as well as in tumors in mice. Gene expression analysis reveals that TZDs induce lipid metabolism pathways while suppressing targets of the Hippo-YAP pathway, Wnt signaling and cancer-related proliferation pathways. Significantly, TZD action appears to be restricted to the high Sox2 expressing cancer stem cell population and is dependent on PPARγ expression. TZDs also affect growth and cell fate by causing the cytoplasmic sequestration of the transcription factors SOX2 and YAP that are required for tumorigenicity. Finally, we identify a TZD-regulated gene signature based on Wnt/Hippo target genes and PPARγ that predicts patient outcomes. Together, this work highlights a novel connection between PPARγ agonist in inducing adipogenesis and mimicking the tumor suppressive hippo pathway. It also illustrates the potential of drug repurposing for TZD-based differentiation therapy for osteosarcoma.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Osteossarcoma/metabolismo , PPAR gama/agonistas , PPAR gama/metabolismo , Fosfoproteínas/metabolismo , Adipócitos/citologia , Adipogenia , Animais , Ciclo Celular , Proteínas de Ciclo Celular , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Cães , Via de Sinalização Hippo , Humanos , Metabolismo dos Lipídeos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Osteossarcoma/genética , Proteínas Serina-Treonina Quinases/metabolismo , Rosiglitazona , Transdução de Sinais , Tiazolidinedionas/química , Fatores de Transcrição , Proteínas Wnt/metabolismo , Proteínas de Sinalização YAPRESUMO
Ovarian cancer presents as an aggressive, advanced stage cancer with widespread metastases that depend primarily on multicellular spheroids in the peritoneal fluid. To identify new druggable pathways related to metastatic progression and spheroid formation, we integrated microRNA and mRNA sequencing data from 293 tumors from The Cancer Genome Atlas (TCGA) ovarian cancer cohort. We identified miR-509-3p as a clinically significant microRNA that is more abundant in patients with favorable survival in both the TCGA cohort (P = 2.3E-3), and, by in situ hybridization (ISH), in an independent cohort of 157 tumors (P < 1.0E-3). We found that miR-509-3p attenuated migration and disrupted multi-cellular spheroids in HEYA8, OVCAR8, SKOV3, OVCAR3, OVCAR4 and OVCAR5 cell lines. Consistent with disrupted spheroid formation, in TCGA data miR-509-3p's most strongly anti-correlated predicted targets were enriched in components of the extracellular matrix (ECM). We validated the Hippo pathway effector YAP1 as a direct miR-509-3p target. We showed that siRNA to YAP1 replicated 90% of miR-509-3p-mediated migration attenuation in OVCAR8, which contained high levels of YAP1 protein, but not in the other cell lines, in which levels of this protein were moderate to low. Our data suggest that the miR-509-3p/YAP1 axis may be a new druggable target in cancers with high YAP1, and we propose that therapeutically targeting the miR-509-3p/YAP1/ECM axis may disrupt early steps in multi-cellular spheroid formation, and so inhibit metastasis in epithelial ovarian cancer and potentially in other cancers.
Assuntos
Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Biomarcadores Tumorais/análise , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/mortalidade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/mortalidade , Fosfoproteínas/biossíntese , Esferoides Celulares/patologia , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
The molecular basis by which receptor tyrosine kinases (RTKs) recruit and phosphorylate Src Homology 2 (SH2) domain-containing substrates has remained elusive. We used X-ray crystallography, NMR spectroscopy, and cell-based assays to demonstrate that recruitment and phosphorylation of Phospholipase Cγ (PLCγ), a prototypical SH2 containing substrate, by FGF receptors (FGFR) entails formation of an allosteric 2:1 FGFR-PLCγ complex. We show that the engagement of pTyr-binding pocket of the cSH2 domain of PLCγ by the phosphorylated tail of an FGFR kinase induces a conformational change at the region past the cSH2 core domain encompassing Tyr-771 and Tyr-783 to facilitate the binding/phosphorylation of these tyrosines by another FGFR kinase in trans. Our data overturn the current paradigm that recruitment and phosphorylation of substrates are carried out by the same RTK monomer in cis and disclose an obligatory role for receptor dimerization in substrate phosphorylation in addition to its canonical role in kinase activation.
Assuntos
Fosfolipase C gama/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos , Ressonância Magnética Nuclear Biomolecular , Fosfatidilinositóis/metabolismo , Fosfolipase C gama/química , Fosfolipase C gama/genética , Fosforilação , Ligação Proteica , Conformação Proteica , Transporte Proteico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Relação Estrutura-Atividade , Transfecção , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Domínios de Homologia de srcRESUMO
The repressive Hippo pathway has a profound tumour suppressive role in cancer by restraining the growth-promoting function of the transcriptional coactivator, YAP. We previously showed that the stem cell transcription factor Sox2 maintains cancer stem cells (CSCs) in osteosarcomas. We now report that in these tumours, Sox2 antagonizes the Hippo pathway by direct repression of two Hippo activators, Nf2 (Merlin) and WWC1 (Kibra), leading to exaggerated YAP function. Repression of Nf2, WWC1 and high YAP expression marks the CSC fraction of the tumor population, while the more differentiated fraction has high Nf2, high WWC1 and reduced YAP expression. YAP depletion sharply reduces CSCs and tumorigenicity of osteosarcomas. Thus, Sox2 interferes with the tumour-suppressive Hippo pathway to maintain CSCs in osteosarcomas. This Sox2-Hippo axis is conserved in other Sox2-dependent cancers such as glioblastomas. Disruption of YAP transcriptional activity could be a therapeutic strategy for Sox2-dependent tumours.
Assuntos
Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Células-Tronco Neoplásicas/metabolismo , Osteossarcoma/genética , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXB1/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Via de Sinalização Hippo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Osteossarcoma/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
Macrophages adopt an alternatively activated phenotype (AAMs) when activated by the interleukin-4receptor(R)α. AAMs can be derived either from proliferation of tissue resident macrophages or recruited inflammatory monocytes, but it is not known whether these different sources generate AAMs that are phenotypically and functionally distinct. By transcriptional profiling analysis, we show here that, although both monocyte and tissue-derived AAMs expressed high levels of Arg1, Chi3l3, and Retnla, only monocyte-derived AAMs up-regulated Raldh2 and PD-L2. Monocyte-derived AAMs were also CX3CR1-green fluorescent protein (GFP)(high) and expressed CD206, whereas tissue-derived AAMs were CX3CR1-GFP and CD206 negative. Monocyte-derived AAMs had high levels of aldehyde dehydrogenase activity and promoted the differentiation of FoxP3(+) cells from naïve CD4(+) cells via production of retinoic acid. In contrast, tissue-derived AAMs expressed high levels of uncoupling protein 1. Hence monocyte-derived AAM have properties associated with immune regulation, and the different physiological properties associated with AAM function may depend on the distinct lineage of these cells.
Assuntos
Perfilação da Expressão Gênica , Ativação de Macrófagos , Macrófagos/imunologia , Monócitos/imunologia , Animais , Antígenos CD4/análise , Proliferação de Células , Células Cultivadas , Fatores de Transcrição Forkhead/análise , Expressão Gênica , Canais Iônicos/análise , Canais Iônicos/genética , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/genética , Monócitos/citologia , Monócitos/metabolismo , Proteína Desacopladora 1RESUMO
The osteoblastic and adipocytic lineages arise from mesenchymal stem cells (MSCs), but few regulators of self-renewal and early cell-fate decisions are known. Here, we show that the Hippo pathway effector YAP1 is a direct target of SOX2 and can compensate for the self-renewal defect caused by SOX2 inactivation in osteoprogenitors and MSCs. Osteogenesis is blocked by high SOX2 or YAP1, accelerated by depletion of either one, and the inhibition of osteogenesis by SOX2 requires YAP1. SOX2 favors adipogenesis and induces PPARγ, but adipogenesis can only occur with moderate levels of YAP1. YAP1 induction by SOX2 is restrained in adipogenesis, and both YAP1 overexpression and depletion inhibit the process. YAP1 binds ß-catenin and directly induces the Wnt antagonist Dkk1 to dampen pro-osteogenic Wnt signals. We demonstrate a Hippo-independent regulation of YAP1 by SOX2 that cooperatively antagonizes Wnt/ß-catenin signals and regulates PPARγ to determine osteogenic or adipocytic fates.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adipócitos/citologia , Células-Tronco Mesenquimais/citologia , Osteócitos/citologia , Fosfoproteínas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adipócitos/metabolismo , Adipogenia , Animais , Proteínas de Ciclo Celular , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Linhagem da Célula , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Osteócitos/metabolismo , Osteogênese , Fosfoproteínas/genética , Fatores de Transcrição SOXB1/genética , Transdução de Sinais , Transfecção , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Proteínas de Sinalização YAP , beta Catenina/metabolismoRESUMO
Osteosarcoma is an aggressive pediatric tumor of growing bones that, despite surgery and chemotherapy, is prone to relapse. These mesenchymal tumors are derived from progenitor cells in the osteoblast lineage that have accumulated mutations to escape cell cycle checkpoints leading to excessive proliferation and defects in their ability to differentiate appropriately into mature bone-forming osteoblasts. Like other malignant tumors, osteosarcoma is often heterogeneous, consisting of phenotypically distinct cells with features of different stages of differentiation. The cancer stem cell hypothesis posits that tumors are maintained by stem cells and it is the incomplete eradication of a refractory population of tumor-initiating stem cells that accounts for drug resistance and tumor relapse. In this review we present our current knowledge about the biology of osteosarcoma stem cells from mouse and human tumors, highlighting new insights and unresolved issues in the identification of this elusive population. We focus on factors and pathways that are implicated in maintaining such cells, and differences from paradigms of epithelial cancers. Targeting of the cancer stem cells in osteosarcoma is a promising avenue to explore to develop new therapies for this devastating childhood cancer.
Assuntos
Neoplasias Ósseas/patologia , Células-Tronco Neoplásicas/patologia , Osteoblastos/patologia , Osteossarcoma/patologia , Animais , Neoplasias Ósseas/genética , Transformação Celular Neoplásica/genética , Criança , Humanos , Camundongos , Modelos Genéticos , Células-Tronco Neoplásicas/metabolismo , Osteoblastos/metabolismo , Osteossarcoma/genéticaRESUMO
BACKGROUND: Supernumerary teeth are often observed in patients suffering from cleidocranial dysplasia due to a mutation in Runx2 that results in haploinsufficiency. However, the underlying molecular mechanisms are poorly defined. In this study, we assessed the roles of Runx2 and its functional antagonist Twist1 in regulating fibroblast growth factor (FGF) signaling using in vitro biochemical approaches. RESULTS: We showed that Twist1 stimulated Fgfr2 and Fgf10 expression in a mesenchymal cell line and that it formed heterodimers with ubiquitously expressed E12 (together with E47 encoded by E2A gene) and upregulated Fgfr2 and Fgf10 promoter activities in a dental mesenchyme-derived cell line. We further demonstrated that the bHLH domain of Twist1 was essential for its synergistic activation of Fgfr2 promoter with E12 and that the binding of E12 stabilized Twist1 by preventing it from undergoing lysosomal degradation. Although Runx2 had no apparent effects on Fgfr2 and Fgf10 promoter activities, it inhibited the stimulatory activity of Twist1 on Fgfr2 promoter. CONCLUSIONS: These findings suggest that Runx2 haploinsufficiency might result in excessive unbound Twist1 that can freely bind to E12 and enhance FGF signaling, thereby promoting the formation of extra teeth.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Animais , Western Blotting , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Eletroforese em Gel de Poliacrilamida , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Imunoprecipitação , Camundongos , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteína 1 Relacionada a Twist/genéticaRESUMO
The transcription factor Sox2 is a key player in the maintenance of pluripotency and "stemness." We have previously shown that Sox2 maintains self-renewal in the osteoblast lineage while inhibiting differentiation (U. Basu-Roy et al., Cell Death Differ. 17:1345-1353, 2010; A. Mansukhani, D. Ambrosetti, G. Holmes, L. Cornivelli, and C. Basilico, J. Cell Biol. 168:1065-1076, 2005). Sox2 also interferes with Wnt signaling by binding ß-catenin, a central mediator of the Wnt pathway. Here we show that these multiple functions of Sox2 are encoded in distinct domains. The self-renewal function of Sox2 is dependent on its transcriptional activity and requires both its DNA-binding and C-terminal activation regions, while only the third C-terminal transactivation (TA) region is required for binding ß-catenin and interfering with Wnt-induced transcription. The results of gene expression analysis upon Sox2 deletion strongly support the notion that Sox2 maintains stemness. We show also that Sox2 suppresses differentiation by attenuating Wnt signaling by posttranscriptional and transcriptional mechanisms and that adenomatous polyposis coli (APC) and GSK3ß, which are negative regulators of the Wnt pathway, are direct Sox2 targets in osteoblasts. Several genes, such as the FoxP1 and BMI-1 genes, that are associated with stemness are downregulated upon Sox2 inactivation. Constitutive expression of the Polycomb complex member BMI-1 can bypass the Sox2 requirement for self-renewal but does not affect differentiation. Our results establish a connection between Sox2 and BMI-1 in maintaining self-renewal and identify BMI-1 as a key mediator of Sox2 function.
Assuntos
Proteínas Nucleares/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Via de Sinalização Wnt/genética , Polipose Adenomatosa do Colo/metabolismo , Animais , Ciclo Celular/genética , Linhagem Celular , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Forkhead/biossíntese , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Camundongos , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Complexo Repressor Polycomb 1 , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Deleção de Sequência , Transdução de Sinais , Crânio , Transcrição Gênica , Ativação Transcricional , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
Activating mutations of FGFRs1-3 cause craniosynostosis (CS), the premature fusion of cranial bones, in man and mouse. The mechanisms by which such mutations lead to CS have been variously ascribed to increased osteoblast proliferation, differentiation, and apoptosis, but it is not always clear how these disturbances relate to the process of suture fusion. We have reassessed coronal suture fusion in an Apert Fgfr2 (S252W) mouse model. We find that the critical event of CS is the early loss of basal sutural mesenchyme as the osteogenic fronts, expressing activated Fgfr2, unite to form a contiguous skeletogenic membrane. A mild increase in osteoprogenitor proliferation precedes but does not accompany this event, and apoptosis is insignificant. On the other hand, the more apical coronal suture initially forms appropriately but then undergoes fusion, albeit at a slower rate, accompanied by a significant decrease in osteoprogenitor proliferation, and increased osteoblast maturation. Apoptosis now accompanies fusion, but is restricted to bone fronts in contact with one another. We correlated these in vivo observations with the intrinsic effects of the activated Fgfr2 S252W mutation in primary osteoblasts in culture, which show an increased capacity for both proliferation and differentiation. Our studies suggest that the major determinant of Fgfr2-induced craniosynostosis is the failure to respond to signals that would halt the recruitment or the advancement of osteoprogenitor cells at the sites where sutures should normally form.
Assuntos
Apoptose/fisiologia , Craniossinostoses/embriologia , Osteoblastos/patologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Células-Tronco/patologia , Acrocefalossindactilia/embriologia , Acrocefalossindactilia/genética , Acrocefalossindactilia/patologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Craniossinostoses/genética , Craniossinostoses/patologia , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Camundongos Mutantes , Osteoblastos/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Células-Tronco/fisiologiaRESUMO
Fibroblast growth factor receptor (FGFR) is expressed in a variety of cells and is involved in their proliferation/migration/survival. To elucidate FGFR-mediated specific action of vascular endothelial cells (ECs) on myocardial ischemia, we generated endothelium-targeted transgenic mice overexpressing constitutively active FGFR2 using Tie2 promoter (FGFR2-Tg). Infarct size, vessel formation and blood perfusion were significantly improved 28 days after myocardial infarction (MI) in FGFR2-Tg, compared with wild-type mice. Aortic ECs isolated from FGFR-Tg showed a marked increase in migratory capacity and tube formation. These in vitro angiogenic activities were blocked by PI3-kinase inhibitor. Whereas, parameters obtained from echocardiography were already improved at three days after MI. Cardiomyocyte apoptosis at the ischemic border zone was decreased in FGFR2-Tg (32.1%, p < 0.05) and cardiac mRNA expression of FGF2 (basic FGF) was also up-regulated (142%, p < 0.05) at 3 days after MI. 1% oxygen-mediated apoptosis was significantly inhibited in FGFR2-Tg-ECs and this inhibition was abolished by PI3-kinase inhibitor. FGFR2-Tg-ECs exposed to 1% oxygen exhibited enhanced phosphorylation of 416-Tyr-Src, 473-Ser-Akt, and HIF1alpha accumulation. The production of FGF2 was enhanced 2.1-fold in FGFR-Tg-ECs under 1% oxygen via the Src/Akt/HIF1alpha pathway, which induced the peri-vessel migration of vascular smooth muscle cells (VSMCs) and anti-apoptotic effects on VSMCs and cardiomyocytes. FGF receptor signaling in ECs promoted migration, survival and autocrine production of FGF2, leading to reduced infarct size, which is associated with anti-apoptotic action in the early stage and with enhanced angiogenesis in the late stage after MI.
Assuntos
Endotélio Vascular/enzimologia , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/prevenção & controle , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Apoptose , Comunicação Autócrina , Movimento Celular , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Endotélio Vascular/patologia , Ativação Enzimática , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/citologia , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , Miócitos de Músculo Liso/patologia , Neovascularização Fisiológica , Especificidade de Órgãos , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor TIE-2/genética , Quinases da Família src/metabolismoRESUMO
Fibroblast growth factor (FGF) and Wnt signals are both critical for proper bone development. We previously reported that the expression of activating FGF receptor mutations in osteoblasts downregulated the expression of many genes reported as targets of Wnt signaling, suggesting an antagonistic effect between Wnt signaling, which promotes osteoblast differentiation and function, and FGF signaling, which inhibits these processes. To analyze the effect of FGF on Wnt signaling in osteoblasts, we created reporter cell lines where a Wnt-responsive promoter drives luciferase expression and showed that Wnt3a-induced luciferase expression was specifically inhibited by FGF treatment. FGF specifically prevented the formation of a Wnt-induced transcriptional complex of TCF1 and -4 with beta-catenin on DNA. FGF did not significantly affect the activation of beta-catenin, although it reduced both the expression of TCF/LEF factors and their induction by Wnt. Microarray analysis using osteoblasts treated with Wnt3a and FGF alone or in combination showed that about 70% of the genes induced by Wnt3a were downregulated by combined FGF treatment. These included novel and previously identified Wnt target genes and genes involved in osteoblast differentiation. Furthermore, FGF alone could downregulate the expression of four Fzd Wnt receptor genes. Our results show that FGF antagonizes Wnt signaling by inhibiting Wnt-induced transcription and suggest that multiple mechanisms, including downregulation of TCFs and Wnt receptors, contribute to this effect.
Assuntos
Fator 1 de Crescimento de Fibroblastos/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Proteínas Wnt/farmacologia , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Proteínas HMGB/metabolismo , Humanos , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXB1 , Fatores de Tempo , Fatores de Transcrição/metabolismo , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/metabolismoRESUMO
Skeletal development requires the correct balance of osteoblast proliferation, survival, and differentiation which is modulated by a network of signaling pathways and transcription factors. We have examined the role of the AKT (PKB), and ERK1/2 signaling pathways in the osteoblast response to FGFs, which inhibit differentiation, and to IGF-1 and Wnt signaling, which promote it. Using osteoblastic cell lines as well as primary calvarial osteoblasts, we show that ERK1/2 and AKT have distinct effects in FGF-induced osteoblast proliferation and differentiation. ERK1/2 is a primary mediator of FGF-induced proliferation, but also contributes to osteoblast differentiation, while AKT is important for osteoblast survival. Signaling by IGF-1, that promotes osteoblast differentiation, strongly activates AKT and weakly ERK1/2, while the opposite results are obtained with FGF, which inhibits differentiation. By introducing a constitutively active form of AKT, we found that increased AKT activity drives osteoblasts to differentiation. Increasing the AKT signal in osteoblasts that harbor FGFR2 activating mutations, found in Crouzon (342Y) and Apert (S22W) syndromes, is also able to drive differentiation in these cells, that normally fail to differentiate. Wnt signals, that promotes differentiation, also induce AKT phosphorylation, and cells expressing active AKT have increased levels of stabilized beta-catenin, a central molecule in Wnt signaling. Our results indicate that the relative strengths of ERK and AKT signaling pathways determine whether osteoblasts are driven into proliferation or differentiation, and that the effects of AKT may be due, in part, to synergy with the Wnt pathway as well as with the Runx2 transcription factor.
Assuntos
Osteoblastos/citologia , Transdução de Sinais/fisiologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Ativação Enzimática , Fator 1 de Crescimento de Fibroblastos/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação , Osteoblastos/fisiologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
Enhanced mesenchymal expression of FGF10 led to the formation of multifocal PIN or prostate cancer. Inhibition of epithelial FGFR1 signaling using DN FGFR1 led to reversal of the cancer phenotype. A subset of the FGF10-induced carcinoma was serially transplantable. Paracrine FGF10 led to an increase in epithelial androgen receptor and synergized with cell-autonomous activated AKT. Our observations indicate that stromal FGF10 expression may facilitate the multifocal histology observed in prostate adenocarcinoma and suggest the FGF10/FGFR1 axis as a potential therapeutic target in treating hormone-sensitive or refractory prostate cancer. We also show that transient exposure to a paracrine growth factor may be sufficient for the initiation of oncogenic transformation.