RESUMEN
Bone is a highly dynamic tissue undergoing continuous formation and resorption. Here, we investigated differential but complementary roles of hypoxia-inducible factor (HIF)-1α and HIF-2α in regulating bone remodeling. Using RNA-seq analysis, we identified that specific genes involved in regulating osteoblast differentiation were similarly but slightly differently governed by HIF-1α and HIF-2α. We found that increased HIF-1α expression inhibited osteoblast differentiation via inhibiting RUNX2 function by upregulation of Twist2, confirmed using Hif1a conditional knockout (KO) mouse. Ectopic expression of HIF-1α via adenovirus transduction resulted in the increased expression and activity of RANKL, while knockdown of Hif1a expression via siRNA or osteoblast-specific depletion of Hif1a in conditional KO mice had no discernible effect on osteoblast-mediated osteoclast activation. The unexpected outcome was elucidated by the upregulation of HIF-2α upon Hif1a overexpression, providing evidence that Hif2a is a transcriptional target of HIF-1α in regulating RANKL expression, verified through an experiment of HIF-2α knockdown after HIF-1α overexpression. The above results were validated in an ovariectomized- and aging-induced osteoporosis model using Hif1a conditional KO mice. Our findings conclude that HIF-1α plays an important role in regulating bone homeostasis by controlling osteoblast differentiation, and in influencing osteoclast formation through the regulation of RANKL secretion via HIF-2α modulation.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Homeostasis , Subunidad alfa del Factor 1 Inducible por Hipoxia , Ratones Noqueados , Osteoblastos , Animales , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ratones , Osteoblastos/metabolismo , Femenino , Huesos/metabolismo , Diferenciación Celular , Osteoclastos/metabolismo , Osteogénesis/genética , Ratones Endogámicos C57BL , Osteoporosis/genética , Osteoporosis/metabolismoRESUMEN
Oral diseases exhibit a significant association with metabolic syndrome, including dyslipidemia. However, direct evidence supporting this relationship is lacking, and the involvement of cholesterol metabolism in the pathogenesis of periodontitis (PD) has yet to be determined. In this study, we showed that high cholesterol caused periodontal inflammation in mice. Cholesterol homeostasis in human gingival fibroblasts was disrupted by enhanced uptake through C-X-C motif chemokine ligand 16 (CXCL16), upregulation of cholesterol hydroxylase (CH25H), and the production of 25-hydroxycholesterol (an oxysterol metabolite of CH25H). Retinoid-related orphan receptor α (RORα) mediated the transcriptional upregulation of inflammatory mediators; consequently, PD pathogenesis mechanisms, including alveolar bone loss, were stimulated. Our collective data provided direct evidence that hyperlipidemia is a risk factor for PD and supported that inhibition of the CXCL16-CH25H-RORα axis is a potential treatment mechanism for PD as a systemic disorder manifestation.
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Pérdida de Hueso Alveolar , Síndrome Metabólico , Periodontitis , Humanos , Ratones , Animales , Pérdida de Hueso Alveolar/etiología , Inflamación , HomeostasisRESUMEN
Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) interact closely with cancer cells to promote tumor development. Downregulation of SPIN90 in CAFs has been reported to facilitate breast cancer progression, but the underlying mechanism has not been elucidated. Here, we demonstrate that miR-130b-3p directly downregulates SPIN90 in stromal fibroblasts, leading to their differentiation into CAFs. As the decrease of SPIN90 in CAFs was shown to be more prominent in estrogen receptor (ER)-positive breast tumors in this study, miR-130b-3p was selected by bioinformatics analysis of data from patients with ER-positive breast cancer. Ectopic expression of miR-130b-3p in fibroblasts accelerated their differentiation to CAFs that promote cancer cell motility; this was associated with SPIN90 downregulation. We also found that miR-130b-3p was generated in luminal A-type cancer cells and activated fibroblasts after being secreted via exosomes from cancer cells. Finally, miR-130b-3p increased in SPIN90-downregulated tumor stroma of luminal A breast cancer patients and MCF7 cell-xenograft model mice. Our data demonstrate that miR-130b-3p is a key modulator that downregulates SPIN90 in breast CAFs. The inverse correlation between miR-130b-3p and SPIN90 in tumor stroma suggests that the miR-130b-3p/SPIN90 axis is clinically significant for CAF activation during breast cancer progression.
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Alzheimer's disease (AD) is the most common form of dementia in the elderly population, but its underlying cause has not been fully elucidated. Recent studies have shown that microRNAs (miRNAs) play important roles in regulating the expression levels of genes associated with AD development. In this study, we analyzed miRNAs in plasma and cerebrospinal fluid (CSF) from AD patients and cognitively normal (including amyloid positive) individuals. miR-1273g-3p was identified as an AD-associated miRNA and found to be elevated in the CSF of early-stage AD patients. The overexpression of miR-1273g-3p enhanced amyloid beta (Aß) production by inducing oxidative stress and mitochondrial impairments in AD model cell lines. A biotin-streptavidin pull-down assay demonstrated that miR-1273g-3p primarily interacts with mitochondrial genes, and that their expression is downregulated by miR-1273g-3p. In particular, the miR-1273g-3p-target gene TIMM13 showed reduced expression in brain tissues from human AD patients. These results suggest that miR-1273g-3p expression in an early stage of AD notably contributes to Aß production and mitochondrial impairments. Thus, miR-1273g-3p might be a biomarker for early diagnosis of AD and a potential therapeutic target to prevent AD progression.
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Enfermedad de Alzheimer/genética , Regulación de la Expresión Génica , Genes Mitocondriales , MicroARNs/genética , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/líquido cefalorraquídeo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo/genética , Femenino , Hipocampo/patología , Humanos , Masculino , MicroARNs/sangre , MicroARNs/líquido cefalorraquídeo , MicroARNs/metabolismo , Mitocondrias/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/metabolismo , Modelos Biológicos , Estrés Oxidativo/genética , Regulación hacia Arriba/genéticaRESUMEN
Osteoarthritis (OA) is a degenerative disorder that can result in the loss of articular cartilage. No effective treatment against OA is currently available. Thus, interest in natural health products to relieve OA symptoms is increasing. However, their qualities such as efficacy, toxicity, and mechanism are poorly understood. In this study, we determined the efficacy of avenanthramide (Avn)-C extracted from oats as a promising candidate to prevent OA progression and its mechanism of action to prevent the expression of matrix-metalloproteinases (MMPs) in OA pathogenesis. Interleukin-1 beta (IL-1ß), a proinflammatory cytokine as a main causing factor of cartilage destruction, was used to induce OAlike condition of chondrocytes in vitro. Avn-C restrained IL-1ß- mediated expression and activity of MMPs, such as MMP-3, -12, and -13 in mouse articular chondrocytes. Moreover, Avn-C alleviated cartilage destruction in experimental OA mouse model induced by destabilization of the medial meniscus (DMM) surgery. However, Avn-C did not affect the expression of inflammatory mediators (Ptgs2 and Nos) or anabolic factors (Col2a1, Aggrecan, and Sox9), although expression levels of these genes were upregulated or downregulated by IL-1ß, respectively. The inhibition of MMP expression by Avn-C in articular chondrocytes was mediated by p38 kinase and c-Jun N-terminal kinase (JNK) signaling, but not by ERK or NF-κB. Interestingly, Avn-C added with SB203580 and SP600125 as specific inhibitors of p38 kinase and JNK, respectively, enhanced its inhibitory effect on the expression of MMPs in IL-1ß treated chondrocytes. Taken together, these results suggest that Avn-C is an effective candidate to prevent OA progression and a natural health product to relieve OA pathogenesis. [BMB Reports 2021; 54(10): 528-533].
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Condrocitos/metabolismo , Osteoartritis/tratamiento farmacológico , ortoaminobenzoatos/farmacología , Animales , Avena/metabolismo , Condrocitos/efectos de los fármacos , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Interleucina-1beta/efectos de los fármacos , Interleucina-1beta/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Metaloproteinasas de la Matriz/efectos de los fármacos , Metaloproteinasas de la Matriz/genética , Ratones , FN-kappa B/metabolismo , Osteoartritis/patología , Extractos Vegetales/farmacología , Cultivo Primario de Células , Transducción de Señal/efectos de los fármacos , ortoaminobenzoatos/metabolismoRESUMEN
Aging is associated with cellular senescence followed by bone loss leading to bone fragility in humans. However, the regulators associated with cellular senescence in aged bones need to be identified. Hypoxia-inducible factor (HIF)-2α regulates bone remodeling via the differentiation of osteoblasts and osteoclasts. Here, we report that HIF-2α expression was highly upregulated in aged bones. HIF-2α depletion in male mice reversed age-induced bone loss, as evidenced by an increase in the number of osteoblasts and a decrease in the number of osteoclasts. In an in vitro model of doxorubicin-mediated senescence, the expression of Hif-2α and p21, a senescence marker gene, was enhanced, and osteoblastic differentiation of primary mouse calvarial preosteoblast cells was inhibited. Inhibition of senescence-induced upregulation of HIF-2α expression during matrix maturation, but not during the proliferation stage of osteoblast differentiation, reversed the age-related decrease in Runx2 and Ocn expression. However, HIF-2α knockdown did not affect p21 expression or senescence progression, indicating that HIF-2α expression upregulation in senescent osteoblasts may be a result of aging rather than a cause of cellular senescence. Osteoclasts are known to induce a senescent phenotype during in vitro osteoclastogenesis. Consistent with increased HIF-2α expression, the expression of p16 and p21 was upregulated during osteoclastogenesis of bone marrow macrophages. ChIP following overexpression or knockdown of HIF-2α using adenovirus revealed that p16 and p21 are direct targets of HIF-2α in osteoclasts. Osteoblast-specific (Hif-2αfl/fl;Col1a1-Cre) or osteoclast-specific (Hif-2αfl/fl;Ctsk-Cre) conditional knockout of HIF-2α in male mice reversed age-related bone loss. Collectively, our results suggest that HIF-2α acts as a senescence-related intrinsic factor in age-related dysfunction of bone homeostasis.
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Envejecimiento/genética , Envejecimiento/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Susceptibilidad a Enfermedades , Osteoporosis/etiología , Osteoporosis/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores , Densidad Ósea , Remodelación Ósea , Huesos/diagnóstico por imagen , Huesos/metabolismo , Huesos/patología , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Genotipo , Humanos , Masculino , Ratones , Ratones Noqueados , Osteoblastos/metabolismo , Osteoporosis/diagnóstico por imagen , Osteoporosis/patología , Microtomografía por Rayos XRESUMEN
Cancer-associated fibroblasts (CAFs) in the tumor microenvironment play major roles in supporting cancer progression. A previous report showed that SPIN90 downregulation is correlated with CAF activation and that SPIN90-deficient CAFs promote breast cancer progression. However, the mechanisms that mediate cancer-stroma interaction and how such interactions regulate cancer progression are not well understood. Here, we show that extra domain A (EDA)-containing fibronectin (FN), FN(+)EDA, produced by mouse embryonic fibroblasts (MEFs) derived from Spin90-knockout (KO) mice increases their own myofibroblast differentiation, which facilitates breast cancer progression. Increased FN(+)EDA in Spin90-KO MEFs promoted fibril formation in the extracellular matrix (ECM) and specifically interacted with integrin α4ß1 as the mediating receptor. Moreover, FN(+)EDA expression by Spin90-KO MEFs increased proliferation, migration, and invasion of breast cancer cells. Irigenin, a specific inhibitor of the interaction between integrin α4ß1 and FN(+)EDA, significantly blocked the effects of FN(+)EDA, such as fibril formation by Spin90-KO MEFs and proliferation, migration, and invasion of breast cancer cells. In orthotopic breast cancer mouse models, irigenin injection remarkably reduced tumor growth and lung metastases. It was supported by that FN(+)EDA in assembled fibrils was accumulated in cancer stroma of human breast cancer patients in which SPIN90 expression was downregulated. Our data suggest that SPIN90 downregulation increases FN(+)EDA and promotes ECM stiffening in breast cancer stroma through an assembly of long FN(+)EDA-rich fibrils; moreover, engagement of the Integrin α4ß1 receptor facilitates breast cancer progression. Inhibitory effects of irigenin on tumor growth and metastasis suggest the potential of this agent as an anticancer therapeutic.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/metabolismo , Fibronectinas/metabolismo , Proteínas Musculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células Cultivadas , Femenino , Fibronectinas/genética , Eliminación de Gen , Humanos , Neoplasias Mamarias Animales , Ratones , Ratones Endogámicos C57BL , Proteínas Musculares/genética , Neoplasias Experimentales , Proteínas del Tejido Nervioso/genética , Regulación hacia ArribaRESUMEN
During ligand-mediated receptor endocytosis, the small GTPase Rab5 functions in vesicle fusion and trafficking. Rab5 activation is known to require interactions with its guanine nucleotide-exchange factors (GEFs); however, the mechanism regulating Rab5 interactions with GEFs remains unclear. Here, we show that the SH3-adapter protein SPIN90 participates in the activation of Rab5 through the recruitment of both Rab5 and its GEF, Gapex5, to endosomal membranes during epidermal growth factor (EGF)-mediated endocytosis. SPIN90 strongly interacts with the inactive Rab5/GDI2 complex through its C-terminus. In response to EGF signaling, extracellular signal-regulated kinase (ERK)-mediated phosphorylation of SPIN90 at Thr-242 enables SPIN90 to bind Gapex5 through its N-terminal SH3 domain. Gapex5 is a determinant of Rab5 membrane targeting, while SPIN90 mediates the interaction between Gapex5 and Rab5 in a phosphorylation-dependent manner. Collectively, our findings suggest that SPIN90, as an adaptor protein, simultaneously binds inactive Rab5 and Gapex5, thereby altering their spatial proximity and facilitating Rab5 activation.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Endocitosis/fisiología , Endosomas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Musculares/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rab5/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Endocitosis/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Musculares/genética , Fosforilación , Unión Proteica , Proteínas de Unión al GTP rab5/genética , Dominios Homologos srcRESUMEN
Pathological bone loss is caused by an imbalance between bone formation and resorption. The bone microenvironments are hypoxic, and hypoxia-inducible factor (HIF) is known to play notable roles in bone remodeling. However, the relevant functions of HIF-2α are not well understood. Here, we have shown that HIF-2α deficiency in mice enhances bone mass through its effects on the differentiation of osteoblasts and osteoclasts. In vitro analyses revealed that HIF-2α inhibits osteoblast differentiation by targeting Twist2 and stimulates RANKL-induced osteoclastogenesis via regulation of Traf6. In addition, HIF-2α appears to contribute to the crosstalk between osteoblasts and osteoclasts by directly targeting RANKL in osteoprogenitor cells. Experiments performed with osteoblast- and osteoclast-specific conditional knockout mice supported a role of HIF-2α in this crosstalk. HIF-2α deficiency alleviated ovariectomy-induced bone loss in mice, and specific inhibition of HIF-2α with ZINC04179524 significantly blocked RANKL-mediated osteoclastogenesis. Collectively, our results suggest that HIF-2α functions as a catabolic regulator in bone remodeling, which is critical for the maintenance of bone homeostasis.
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Calcium and calcium-binding proteins play crucial roles in the regulation of actin dynamics, which contributes to cancer cell migration and invasion. In this chapter, we have focused on a three-dimensional imaging method to explore the pathophysiological function of EF-hand domain-containing protein D2 (EFHD2), a novel actin-binding protein. To overcome the limitations of two-dimensional imaging on substrate-coated cover glass for examination of invasive protrusions of cancer cells, we suggest three-dimensional reconstruction from optical z-sections of cells cultured on substrate-impregnated membrane filters of Transwell.
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Proteínas de Unión al Calcio/metabolismo , Técnicas de Cultivo de Célula/métodos , Imagenología Tridimensional/métodos , Melanoma/metabolismo , Podosomas/metabolismo , Actinas/metabolismo , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Línea Celular Tumoral , Movimiento Celular , Humanos , Ratones , Microscopía ConfocalRESUMEN
Alterations in mechanical properties in the extracellular matrix are modulated by myofibroblasts and are required for progressive fibrotic diseases. Recently, we reported that fibroblasts depleted of SPIN90 showed enhanced differentiation into myofibroblasts via increased acetylation of microtubules in the soft matrix; the mechanisms of the underlying signaling network, however, remain unclear. In this study, we determine the effect of depletion of SPIN90 on FAK/ROCK signaling modules. Transcriptome analysis of Spin90 KO mouse embryonic fibroblasts (MEF) and fibroblasts activated by TGF-ß revealed that Postn is the most significantly upregulated gene. Knockdown of Postn by small interfering RNA suppressed cell adhesion and myofibroblastic differentiation and downregulated FAK activity in Spin90 KO MEF. Our results indicate that SPIN90 depletion activates FAK/ROCK signaling, induced by Postn expression, which is critical for myofibroblastic differentiation on soft matrices mimicking the mechanical environment of a normal tissue.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Moléculas de Adhesión Celular/metabolismo , Regulación hacia Abajo/genética , Fibroblastos/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal , Quinasas Asociadas a rho/metabolismo , Animales , Diferenciación Celular , Adhesiones Focales/metabolismo , Ratones Noqueados , Miofibroblastos/metabolismoRESUMEN
The importance of actin-binding proteins (ABPs) in the regulation of synapse morphology and plasticity has been well established. SH3 protein interacting with Nck, 90 kDa (SPIN90), an Nck-interacting protein highly expressed in synapses, is essential for actin remodeling and dendritic spine morphology. Synaptic targeting of SPIN90 to spine heads or dendritic shafts depends on its phosphorylation state, leading to blockage of cofilin-mediated actin depolymerization and spine shrinkage. However, the physiological role of SPIN90 in long-term plasticity, learning and memory are largely unknown. In this study, we demonstrate that Spin90-knockout (KO) mice exhibit substantial deficits in synaptic plasticity and behavioral flexibility. We found that loss of SPIN90 disrupted dendritic spine density in CA1 neurons of the hippocampus and significantly impaired long-term depression (LTD), leaving basal synaptic transmission and long-term potentiation (LTP) intact. These impairments were due in part to deficits in AMPA receptor endocytosis and its pre-requisites, GluA1 dephosphorylation and postsynaptic density (PSD) 95 phosphorylation, but also by an intrinsic activation of Akt-GSK3ß signaling as a result of Spin90-KO. In accordance with these defects, mice lacking SPIN90 were found to carry significant deficits in object-recognition and behavioral flexibility, while learning ability was largely unaffected. Collectively, these findings demonstrate a novel modulatory role for SPIN90 in hippocampal LTD and behavioral flexibility.
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Periodontal disease is one of the most prevalent chronic disorders worldwide. It is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss. Here, we focused on the role of adipokines, which are locally expressed by periodontal tissues, in the regulation of catabolic gene expression leading to periodontal inflammation. The expression of the nicotinamide phosphoribosyltransferase (NAMPT) adipokine was dramatically increased in inflamed human and mouse gingival tissues. NAMPT expression was also increased in lipopolysaccharide- and proinflammatory cytokine-stimulated primary cultured human gingival fibroblasts (GF). Adenovirus-mediated NAMPT (Ad-Nampt) overexpression upregulated the expression and activity of COX-2, MMP1 and MMP3 in human GF. The upregulation of IL-1ß- or Ad-Nampt-induced catabolic factors was significantly abrogated by the intracellular NAMPT (iNAMPT) inhibitor, FK866 or by the sirtuin (SIRT) inhibitor, nicotinamide (NIC). Recombinant NAMPT protein or extracellular NAMPT (eNAMPT) inhibition using a blocking antibody did not alter NAMPT target gene expression levels. Moreover, intragingival Ad-Nampt injection mediated periodontitis-like phenotypes including alveolar bone loss in mice. SIRT2, a part of the SIRT family, was positively associated with NAMPT actions in human GF. Furthermore, in vivo inhibition of the NAMPT-NAD+-SIRT axis by NIC injection in mice ameliorated the periodontal inflammation and alveolar bone erosion caused by intragingival injection of Ad-Nampt. Our findings indicate that NAMPT is highly upregulated in human GF, while its enzymatic activity acts as a crucial mediator of periodontal inflammation and alveolar bone destruction via regulation of COX-2, MMP1, and MMP3 levels.
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Ciclooxigenasa 2/genética , Citocinas/metabolismo , Regulación de la Expresión Génica , Expresión Génica , Encía/patología , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/genética , Nicotinamida Fosforribosiltransferasa/metabolismo , Periodontitis/genética , Adipoquinas/metabolismo , Adulto , Pérdida de Hueso Alveolar/metabolismo , Animales , Citocinas/genética , Modelos Animales de Enfermedad , Femenino , Fibroblastos/metabolismo , Humanos , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Morfolinas/farmacología , Niacinamida/farmacología , Nicotinamida Fosforribosiltransferasa/genética , Piperazinas/farmacología , Cultivo Primario de Células , Sirtuina 2/genética , Sirtuina 2/metabolismoRESUMEN
Biomechanical remodeling of stroma by cancer-associated fibroblasts (CAF) in early stages of cancer is critical for cancer progression, and mechanical cues such as extracellular matrix stiffness control cell differentiation and malignant progression. However, the mechanism by which CAF activation occurs in low stiffness stroma in early stages of cancer is unclear. Here, we investigated the molecular mechanism underlying CAF regulation by SPIN90 and microtubule acetylation under conditions of mechanically soft matrices corresponding to normal stromal rigidity. SPIN90 was downregulated in breast cancer stroma but not tumor, and this low stromal expression correlated with decreased survival in breast cancer patients. Spin90 deficiency facilitated recruitment of mDia2 and APC complex to microtubules, resulting in increased microtubule acetylation. This increased acetylation promoted nuclear localization of YAP, which upregulated expression of myofibroblast marker genes on soft matrices. Spin90 depletion enhanced tumor progression, and blockade of microtubule acetylation in CAF significantly inhibited tumor growth in mice. Together, our data demonstrate that loss of SPIN90-mediated microtubule acetylation is a key step in CAF activation in low stiffness stroma. Moreover, correlation among these factors in human breast cancer tissue supports the clinical relevance of SPIN90 and microtubule acetylation in tumor development. Cancer Res; 77(17); 4710-22. ©2017 AACR.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Neoplasias de la Mama/patología , Fibroblastos/patología , Microtúbulos/patología , Proteínas Musculares/metabolismo , Proteínas del Tejido Nervioso/fisiología , Células del Estroma/patología , Acetilación , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Diferenciación Celular , Progresión de la Enfermedad , Femenino , Fibroblastos/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Microtúbulos/metabolismo , Proteínas Musculares/genética , Miofibroblastos/metabolismo , Miofibroblastos/patología , Fosfoproteínas/metabolismo , Células del Estroma/metabolismo , Factores de Transcripción , Células Tumorales Cultivadas , Microambiente Tumoral , Proteínas Señalizadoras YAPRESUMEN
MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression by suppressing translation or facilitating mRNA decay. Differential expression of miRNAs is involved in the pathogenesis of several diseases including cancer. Here, we investigated the role of-miR-24-3p as a downregulated miRNA in metastatic cancer. miR-24-3p was decreased in metastatic cancer and lower expression of miR-24-3p was related to poor survival of cancer patients. Consistently, ectopic expression of miR-24-3p suppressed the cell migration, invasion, and proliferation of MCF7, Hep3B, B16F10, SK-Hep1, and PC-3 cells by directly targeting p130Cas. Stable expression of p130Cas restored miR-24-3p-mediated inhibition of cell migration and invasion. These results suggest that miR-24-3p functions as a tumor suppressor and the miR-24-3p/p130Cas axis is a novel factor of cancer progression by regulating cell migration and invasion.
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Proteína Sustrato Asociada a CrK/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Interferencia de ARN , Regiones no Traducidas 3' , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Ratones , Metástasis de la Neoplasia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/mortalidad , Transcriptoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Rheumatoid arthritis (RA) and osteoarthritis (OA), two common types of arthritis, affect the joints mainly by targeting the synovium and cartilage. Increasing evidence indicates that a significant network connects synovitis and cartilage destruction during the progression of arthritis. We recently demonstrated that hypoxia-inducible factor (HIF)-2α causes RA and OA by regulating the expression of catabolic factors in fibroblast-like synoviocytes (FLS) or chondrocytes. To address the reciprocal influences of HIF-2α on FLS and chondrocytes, we applied an in vitro co-culture system using a transwell apparatus. When co-cultured with HIF-2α-overexpressing chondrocytes, FLS exhibited increased expression of matrix metalloproteinases and inflammatory mediators, similar to the effects induced by tumor-necrosis factor (TNF)-α treatment of FLS. Moreover, chondrocytes co-cultured with HIF-2α-overexpressing FLS exhibited upregulation of Mmp3 and Mmp13, which is similar to the effects induced by interleukin (IL)-6 treatment of chondrocytes. We confirmed these differential HIF-2α-induced effects via distinct secretory mediators using Il6-knockout cells and a TNF-α-blocking antibody. The FLS-co-culture-induced gene expression changes in chondrocytes were significantly abrogated by IL-6 deficiency, whereas TNF-α neutralization blocked the alterations in gene expression associated with co-culture of FLS with chondrocytes. Our results further suggested that the observed changes might reflect the HIF-2α-induced upregulation of specific receptors for TNF-α (in FLS) and IL-6 (in chondrocytes). This study broadens our understanding of the possible regulatory mechanisms underlying the crosstalk between the synovium and cartilage in the presence of HIF-2α, and may suggest potential new anti-arthritis therapies.
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Artritis/inmunología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Condrocitos/patología , Fibroblastos/patología , Interleucina-6/inmunología , Membrana Sinovial/patología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Artritis/genética , Artritis/patología , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células Cultivadas , Condrocitos/inmunología , Condrocitos/metabolismo , Técnicas de Cocultivo , Fibroblastos/inmunología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Interleucina-6/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoartritis/genética , Osteoartritis/inmunología , Osteoartritis/patología , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismo , Factor de Necrosis Tumoral alfa/genética , Regulación hacia ArribaRESUMEN
INTRODUCTION: Pannus formation and resulting cartilage destruction during rheumatoid arthritis (RA) depends on the migration of synoviocytes to cartilage tissue. Here, we focused on the role of hypoxia-inducible factor (HIF)-2α-induced chemokines by chondrocytes in the regulation of fibroblast-like synoviocyte (FLS) migration into the cartilage-pannus interface and cartilage erosion. METHODS: Collagen-induced arthritis (CIA), K/BxN serum transfer, and tumor necrosis factor-α transgenic mice were used as experimental RA models. Expression patterns of HIF-2α and chemokines were determined via immunostaining, Western blotting and RT-PCR. FLS motility was evaluated using transwell migration and invasion assays. The specific role of HIF-2α was determined via local deletion of HIF-2α in joint tissues or using conditional knockout (KO) mice. Cartilage destruction, synovitis and pannus formation were assessed via histological analysis. RESULTS: HIF-2α and various chemokines were markedly upregulated in degenerating cartilage and pannus of RA joints. HIF-2α induced chemokine expression by chondrocytes in both primary culture and cartilage tissue. HIF-2α -induced chemokines by chondrocytes regulated the migration and invasion of FLS. Local deletion of HIF-2α in joint tissues inhibited pannus formation adjacent to cartilage tissue and cartilage destruction caused by K/BxN serum transfer. Furthermore, conditional knockout of HIF-2α in cartilage blocked pannus formation in adjacent cartilage but not bone tissue, along with inhibition of cartilage erosion caused by K/BxN serum transfer. CONCLUSION: Our findings suggest that chemokines induced by IL-1ß or HIF-2α in chondrocytes regulate pannus expansion by stimulating FLS migration and invasion, leading to cartilage erosion during RA pathogenesis.
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Artritis Experimental/patología , Artritis Reumatoide/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Cartílago Articular/patología , Condrocitos/inmunología , Fibroblastos/metabolismo , Animales , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Western Blotting , Cartílago Articular/inmunología , Cartílago Articular/metabolismo , Movimiento Celular/inmunología , Quimiocinas/inmunología , Condrocitos/metabolismo , Inmunoprecipitación de Cromatina , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
In response to brain injury, microglia rapidly extend processes that isolate lesion sites and protect the brain from further injury. Here we report that microglia carrying a pathogenic mutation in the Parkinson's disease (PD)-associated gene, G2019S-LRRK2 (GS-Tg microglia), show retarded ADP-induced motility and delayed isolation of injury, compared with non-Tg microglia. Conversely, LRRK2 knockdown microglia are highly motile compared with control cells. In our functional assays, LRRK2 binds to focal adhesion kinase (FAK) and phosphorylates its Thr-X-Arg/Lys (TXR/K) motif(s), eventually attenuating FAK activity marked by decreased pY397 phosphorylation (pY397). GS-LRRK2 decreases the levels of pY397 in the brain, microglia and HEK cells. In addition, treatment with an inhibitor of LRRK2 kinase restores pY397 levels, decreased pTXR levels and rescued motility of GS-Tg microglia. These results collectively suggest that G2019S mutation of LRRK2 may contribute to the development of PD by inhibiting microglial response to brain injury.
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Lesiones Encefálicas , Movimiento Celular/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Microglía/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Cicatrización de Heridas/genética , Animales , Western Blotting , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Inmunoprecipitación , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Ratones , Ratones Transgénicos , Mutación , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , RatasRESUMEN
Ectopic expression of Swiprosin-1, an actin-binding protein (also known as EF hand domain containing 2; EFHD2), enhanced motile protrusions associated with actin, such as lamellipodia and membrane ruffles. Swiprosin-1 levels were increased in various human cancer tissues, particularly at highly invasive stages of malignant melanoma. Expression of Swiprosin-1 was correlated with that of epidermal growth factor receptor (EGFR) and induced by EGF. In a mouse metastasis model, Swiprosin-1 overexpression induced pulmonary metastasis whereas its knockdown led to marked inhibition of metastasis of highly invasive melanoma cells. Swiprosin-1 at the lamellipodia and membrane ruffles controlled the direction of cell protrusion and enhanced migration velocity through activating the Rho family of small GTPases, including Rac1, Cdc42 and RhoA. Our collective findings support the potential utility of Swiprosin-1 as a therapeutic target to prevent cancer invasion and metastasis.