RESUMEN
A cell-free approach utilizing the paracrine effects of mesenchymal stromal cells is receiving attention in regenerative medicine. In the present study, we evaluated the effects of a conditioned medium of amniotic fluid-derived stromal cells (AFSC-CM) on bone metabolism. In mice, intraperitoneal injections of AFSC-CM increased bone mass and enhanced bone turnover. The precursor populations of myeloid and mesenchymal lineages, as well as endothelial cells in bone marrow, were also augmented by AFSC-CM administration. In an in vitro culture experiment, AFSC-CM increased osteoclast differentiation of bone marrow-derived macrophages, but had no significant effect on the osteogenic differentiation of preosteoblasts. However, AFSC-CM administration dramatically accelerated the migration and tube formation of endothelial cells, and a cytokine array showed that AFSC-CM contained many angiogenic factors. These results indicate that AFSC-CM exerts a bone anabolic effect by changing the bone marrow microenvironment, including angiogenesis and precursor expansion. Therefore, ameliorating marrow angiogenesis is a potential therapeutic strategy for bone regeneration, for which AFSCs can be a good cellular source.
Asunto(s)
Anabolizantes , Células Madre Mesenquimatosas , Líquido Amniótico , Anabolizantes/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Medios de Cultivo Condicionados/metabolismo , Medios de Cultivo Condicionados/farmacología , Citocinas/metabolismo , Células Endoteliales , Ratones , OsteogénesisRESUMEN
Background: Metformin has antitumoral actions in human cancers, including the thyroid, while its effects on metastatic lesions are unclear. Patients with bone metastasis (BM) from thyroid cancers have poor survival. Because metformin inhibits the activation of osteoclasts, which has essential roles in BM, the aim of this study was to investigate the therapeutic effects of metformin on thyroid cancer BM and osteoclast activation in the bone microenvironment. Methods: The anaplastic thyroid cancer (ATC) cell lines FRO and SW1736 were used to test the antitumoral effect of metformin in vitro and in vivo. A murine model of BM was established by intratibial injection of cancer cells. To mimic the BM microenvironment, osteoblasts were treated with conditioned media from the FRO (FRO-CM) and SW1736 (SW1736-CM) cells. Thyroid cancer patients with or without BM were recruited, and the serum receptor activator of nuclear factor kappa-B ligand (RANKL) levels was measured. Results: Metformin treatment significantly reduced the viabilities of the FRO and SW1736 cells in vitro and the tumor growth of SW1736 in vivo. In the murine model of BM, metformin delayed tumor growth in the bone and decreased the numbers of tartrate-resistant acid phosphatase-positive osteoclasts on the bone surface with reduced RANKL in the bone marrow. Furthermore, FRO- or SW1736-CM significantly increased the osteoblastic RANKL productions and activated osteoclast differentiation in whole marrow cultures, which were blocked by metformin treatment. Among 67 thyroid cancer patients, the serum RANKL levels were significantly increased in BM patients compared with patients with lung-only metastasis or no distant metastasis. In addition, the interleukin-6 superfamily in the FRO- or SW1736-CM stimulated STAT3 phosphorylation, which was inhibited by gp130 blocking. Metformin treatment decreased the FRO- or SW1736-CM-induced STAT3 phosphorylation by AMPK phosphorylation. Metformin also inhibited the FRO- or SW1736-CM-induced osteoclastic differentiation of bone marrow-derived monocyte/macrophage by RANK/c-Fos/NFATC1 signaling. Conclusions: In the microenvironment of BM, metformin effectively reduced ATC tumor growth by inhibiting cancer cell viability, blocking cancer cell-induced osteoblastic RANKL production, which further activated osteoclastogenesis, and directly reduced osteoclast differentiation. These multifactorial actions of metformin suggest that it has potential therapeutic effects in thyroid cancer BM.
Asunto(s)
Neoplasias Óseas/secundario , Hipoglucemiantes/farmacología , Metformina/farmacología , Osteoblastos/efectos de los fármacos , Carcinoma Anaplásico de Tiroides/secundario , Neoplasias de la Tiroides/patología , Microambiente Tumoral/efectos de los fármacos , Animales , Médula Ósea/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Medios de Cultivo Condicionados , Técnicas In Vitro , Ratones , Ligando RANK/efectos de los fármacos , Ligando RANK/metabolismoRESUMEN
Osteoporosis is a systemic skeletal disorder characterized by reduced bone mineral density (BMD) and increased risk of fracture. We studied the effects of transplantation of mesenchymal stem cells (MSCs) overexpressing receptor activator of nuclear factor-kappaB (RANK)-Fc and CXC chemokine receptor-4 (CXCR4) using retrovirus on ovariectomy (OVX)-induced bone loss in mice. Ten-week-old adult female C57BL/6 mice were divided into six groups as follows: Sham-operated mice treated with phosphate-buffered saline (PBS) (Sham-op + PBS); OVX mice intravenously transplanted with syngeneic MSCs overexpressing RANK-Fc-DsRED and CXCR4-GFP (RANK-Fc + CXCR4); RANK-Fc-DsRED and GFP (RANK-Fc + GFP); CXCR4-GFP and DsRED (CXCR4 + RED); DsRED and GFP (RED + GFP); or treated with PBS only (OVX + PBS). Measurement of BMD showed that introduction of RANK-Fc resulted in significant protection against OVX-induced bone loss compared to treatment with PBS (-0.1% versus -6.2%, P < 0.05) at 8 weeks after cell infusion. CXCR4 + RED group also significantly prevented bone loss compared to OVX + PBS group (2.7% versus -6.2%, P < 0.05). Notably, the effect of RANK-Fc + CXCR4 was greater than that of RANK-Fc + GFP (4.4% versus -0.1%, P < 0.05) while it was not significantly different from that in CXCR4 + RFP group (4.4% versus 2.7%, P = 0.055) at 8 weeks. Transplantation of MSCs with control virus (RED + GFP group) also resulted in amelioration of bone loss compared to OVX + PBS group (-1.7% versus -6.2%, P < 0.05). Fluorescence-activated cell sorting (FACS) and real-time quantitative PCR (qPCR) analysis for GFP from bone tissue revealed enhanced cell trafficking to bone by co-overexpression of CXCR4. In conclusion, we have demonstrated that intravenous transplantation of syngeneic MSCs overexpressing CXCR4 could promote increased in vivo cell trafficking to bone in OVX mice, which could in itself protect against bone loss but also enhance the therapeutic effects of RANK-Fc.
Asunto(s)
Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Osteoporosis/prevención & control , Receptores CXCR4/fisiología , Proteínas Recombinantes de Fusión/fisiología , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Expresión Génica , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos BALB C , Ovariectomía , Receptores CXCR4/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Current diagnosis of bone metastasis (BM) in breast cancer relies on structural changes of bone that occur only in the advanced stage. A sensitive biomarker for detecting early progression of bone metastasis is urgently required. We performed clinical and preclinical studies to investigate diagnostic value of circulating osteocalcin-positive cells (cOC) in breast cancer bone metastasis. Metastatic breast cancer patients (n = 92) with or without bone metastasis (ie, BM+ or BM- ) were enrolled, and cOC were measured at enrollment. Patients were followed up for bone metastasis progression for 18 months. BM+ patients (n = 59) were divided into progressive (PD) or stable disease (SD) groups, based on imaging studies at the end of the 18-month study. The PD group had higher baseline cOC compared with the SD group. Furthermore, higher cOC resulted in reduced BM progression-free survival. Three patients in the BM- group (n = 33) developed new BM during the 18-month study, and these patients had a higher level of baseline cOC compared with the remaining BM- patients. In murine preclinical studies, cOC increased at early time points when micro-metastases were evident only by histology but undetectable by bioluminescence imaging. Also, cOC levels predicted the progression of BM and correlated significantly with BM tumor burden. cOC increased in the early phase of breast cancer BM and can predict BM progression, supporting cOC as a potential novel biomarker. © 2020 American Society for Bone and Mineral Research.
Asunto(s)
Neoplasias Óseas , Neoplasias de la Mama , Osteocalcina/análisis , Animales , Neoplasias Óseas/diagnóstico por imagen , Neoplasias Óseas/secundario , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Humanos , Ratones , Carga TumoralRESUMEN
PURPOSE: Thyroid-stimulating hormone (TSH) suppression is widely used to treat well-differentiated thyroid cancer, whereas its role in poorly differentiated thyroid cancer (PDTC) is undetermined. Besides thyrocytes, TSH also binds to stromal cells, comprising tumor microenvironments. This study aimed to investigate the effects of TSH on tumor microenvironments in PDTC. EXPERIMENTAL DESIGN: An ectopic tumor model using PDTC cells (BHP10-3SCp and FRO), which exhibit TSH/cAMP-independent cell growth, was treated with TSH. IHC was performed using tissue microarrays from 13 PDTCs. RESULTS: TSH treatment significantly enhanced tumor growth of PDTCs with increased vascularity but not that of breast cancer cells, suggesting this effect is unique to thyroid cancer cells, not stromal cells. TSH significantly upregulated VEGF-A and CXCL8 expressions in BHP10-3SCp cells via AKT and ERK signaling, resulting in higher concentrations of VEGF-A and CXCL8 in conditioned medium of TSH-treated BHP10-3SCp cells (TSH-CM) compared with controls. TSH-CM treatment enhanced tube formation potentials of endothelial cells, and blocking VEGF and/or CXCL8 reduced them. Blocking VEGF and/or CXCL8 also reduced TSH-dependent tumor growth with reduced tumor vasculature in vivo. TSH-treated tumors showed increased macrophage densities, and macrophage inhibition reduced TSH-dependent tumor growth in vivo. In human PDTCs, preoperative TSH levels were positively associated with VEGF-A and tumor size, and the expression of VEGF-A was positively correlated with CD31, CD163, and CXCL8, and their clinical poor prognosis. CONCLUSIONS: Aberrant TSH receptor signaling modulates tumor angiogenesis by stimulating VEGF-A and CXCL8 secretion from PDTC cells and enhances tumor growth; thus, TSH suppression is beneficial for treating PDTCs.
Asunto(s)
Interleucina-8/genética , Neovascularización Patológica/genética , Receptores de Tirotropina/genética , Neoplasias de la Tiroides/genética , Factor A de Crecimiento Endotelial Vascular/genética , Inhibidores de la Angiogénesis/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Receptores de Tirotropina/antagonistas & inhibidores , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/patología , Tirotropina/genética , Tirotropina/metabolismo , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although various chemokines have pro-tumorigenic actions in cancers, the effects of CXCL16 remain controversial. The aim of this study was to investigate the molecular characteristics of CXCL16-expressing papillary thyroid cancers (PTCs). CXCL16 expressions were significantly higher in PTCs than benign or normal thyroid tissues. In the TCGA dataset for PTCs, a higher CXCL16 expression was associated with M2 macrophage- and angiogenesis-related genes and poor prognostic factors including a higher TNM staging and the BRAFV600E mutation. PTCs with a higher expression of 3-gene panel including CXCL16, AHNAK2, and THBS2 showed poor recurrence-free survivals than that of the lower expression group. Next, shCXCL16 was introduced into BHP10-3SCp cells to deplete the endogenous CXCL16, and then, the cells were subcutaneously injected to athymic mice. Tumors from the BHP10-3SCpshCXCL16 exhibited a delayed tumor growth with decreased numbers of ERG+ endothelial cells and F4/80+ macrophages than those from the BHP10-3SCpcontrol. CXCL16-related genes including AHNAK2 and THBS2 were downregulated in the tumors from the BHP10-3SCpshCXCL16 compared with that from the BHP10-3SCpcontrol. In conclusion, a higher CXCL16 expression was associated with macrophage- and angiogenesis-related genes and aggressive phenotypes in PTC. Targeting CXCL16 may be a good therapeutic strategy for advanced thyroid cancer.
Asunto(s)
Quimiocina CXCL16/genética , Macrófagos/inmunología , Neovascularización Patológica/patología , Cáncer Papilar Tiroideo/patología , Animales , Carcinogénesis/patología , Línea Celular Tumoral , Movimiento Celular/fisiología , Quimiocina CXCL16/metabolismo , Proteínas del Citoesqueleto/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Pronóstico , Proteínas Proto-Oncogénicas B-raf/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Trombospondinas/metabolismo , Cáncer Papilar Tiroideo/mortalidad , Glándula Tiroides/patología , Microambiente Tumoral/fisiologíaRESUMEN
Secreted frizzled-related proteins (sFRPs) are modulators of Wnt signaling. This study was undertaken for definitive assessment of contribution of different sFRPs in osteoblastic differentiation of mesenchymal progenitor cells and apoptosis of osteoblasts. Treatment of C3H10T1/2 cells with sFRP-2 at concentrations of 10, 50, and 100nM and sFRP-4 at low concentrations (5nM) significantly increased Wnt-3A-induced alkaline phosphatase (ALP) activities, whereas sFRP-1 or 3 did not. Retroviral transduction of the sFRP-2 but not other sFRPs also significantly enhanced ALP activity induced by beta-glycerophosphate and ascorbic acid. Furthermore, transfection of all the sFRP expression vectors significantly increased beta-catenin/TCF reporter activity and the effects were most prominent with sFRP-2 and -4. In osteoblast apoptosis assay, only sFRP-3 increased etoposide-induced apoptosis in MC3T3-E1 mouse osteoblasts. In conclusion, we found that different repertoires of sFRPs exert differential effects on osteoblastic differentiation of mouse mesenchymal cells and cellular apoptosis of mouse osteoblasts in vitro.
Asunto(s)
Glicoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Osteoblastos/citología , Osteoblastos/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Animales , Apoptosis/fisiología , Diferenciación Celular/fisiología , Línea Celular , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C3HRESUMEN
Although previous studies have reported the effects of extensive subculturing on proliferation rates and osteogenic potential of human mesenchymal stem cells (hMSCs), the results remain controversial. The aim of our study was to characterize the proliferation and osteogenic potential of hMSCs during serial subculture, and also to identify proteins that are differentially regulated in hMSCs during serial subculture and osteogenic differentiation using proteome analysis. Here we show that the proliferation and osteogenic capacity of hMSCs decrease during serial subculturing. Several proteins were shown to be differentially regulated during serial subculture; among these the expression of T-complex protein 1 alpha subunit (TCP-1alpha), a protein known to be associated with cell proliferation, cell cycle, morphological changes, and apoptosis, gradually decreased during serial subculture. Among proteins that were differentially regulated during osteogenic differentiation, chloride intracellular channel 1 (CLIC1) was downregulated only during the early passages eukaryotic translation elongation factor, and acidic ribosomal phosphoprotein P0 was downregulated during the middle passages, while annexin V, LIM, and SH3 domain protein 1 (LASP-1), and 14-3-3 protein gamma (YWHAG) were upregulated during the later passage. These studies suggest that differentially regulated passage-specific proteins may play a role in the decrease of osteogenic differentiation potential under serial subculturing.
Asunto(s)
Células Madre Mesenquimatosas/metabolismo , Osteogénesis/fisiología , Adolescente , Adulto , Biomarcadores/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Chaperonina con TCP-1 , Chaperoninas , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Regulación hacia Abajo , Femenino , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Histocitoquímica , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Persona de Mediana Edad , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteoma , Proteómica , ARN Mensajero/metabolismoRESUMEN
Macrophages in tumor microenvironment have pivotal roles in tumor growth, metastasis, and angiogenesis. We investigated the interacting mechanism of macrophage actions in human papillary thyroid cancer (PTC). Co-cultures of macrophage/PTC significantly increased the cancer cell migration potentials, compared with the PTC culture alone. Treatment of conditioned medium (CM) of macrophage/PTC co-cultures enhanced cell invasions in 3D invasion assay. Cytokine array analysis demonstrated that CM of macrophage/PTC co-cultures contained a high level of CXCL16, while it was not found in CM of PTC culture alone. Treatment with CXCL16 enhanced the cell migration potentials in PTC cells, and blocking CXCL16 signaling using anti-CXCL16 antibody or metalloproteinase inhibitor (TAPI2) attenuated macrophage-mediated enhancement of PTC cell migration potentials. In PTC cells, CXCL16 treatment or co-cultures with macrophages increased Akt phosphorylation, and these macrophage-dependent increases of Akt phosphorylation was inhibited by anti-CXCL16 antibody. Moreover, Akt inhibitor attenuated macrophage-mediated increases of PTC cell migration potential. In macrophages, treatment of macrophage/PTC co-cultured CMs up-regulated CD163, Il10, and CD206, which were attenuated by anti-CXCL16 antibody treatment. Finally, CXCR6 and CXCL16 expressions were evaluated by immunohistochemical staining with a thyroid tissue microarray including 136 PTC. CXCR6 expressions showed positive correlation with the density of CD163(+) macrophages and associated with lymph node metastasis. In conclusion, CXCL16 signaling partly mediated macrophage actions on PTC tumor cell invasion and also changed the macrophage phenotypes into M2-macrophages in PTC tumor microenvironment. These data suggested that CXCL16 signaling, a bidirectional player in macrophage-associated tumor microenvironment, might be a potential therapeutic target of human PTC.
Asunto(s)
Carcinoma Papilar/metabolismo , Quimiocinas CXC/metabolismo , Macrófagos/metabolismo , Invasividad Neoplásica/patología , Receptores Depuradores/metabolismo , Transducción de Señal/fisiología , Neoplasias de la Tiroides/metabolismo , Carcinoma Papilar/patología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Quimiocina CXCL16 , Quimiocinas CXC/farmacología , Humanos , Macrófagos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Transducción de Señal/efectos de los fármacos , Neoplasias de la Tiroides/patologíaRESUMEN
Chondrogenesis involves the recruitment of mesenchymal cells to differentiate into chondroblasts, and also the cells must synthesize a cartilage-specific extracellular matrix. There were two representative culture systems that promoted the chondrogenic differentiation of human mesenchymal stem cells. These systems were adaptations of the "pellet" culture system, which was originally described as a method for preventing the phenotypic modulation of chondrocytes, and the "alginate bead" culture system, which was used to maintain encapsulated cells at their differentiated phenotype over time, and also it was used to maintain the cells' proteoglycan synthesis at a rate similar to that of primary chondrocytes. We performed test on the differences of phenotypic characterization with the two methods of differentiating human mesenchymal stem cells into chondrocytes. The typical gene for articular cartilage, collagen type II, was more strongly expressed in the "alginate bead" system than in the "pellet" culture system, in addition, specific gene for hypertrophic cartilage, collagen type X, was more rapidly expressed in the "pellet" system than in "alginate bead" culture system. Therefore, the "alginate bead" culture system is a more phenotypical, practical and appropriate system to differentiate human mesenchymal stem cells into articular chondrocytes than the "pellet" culture system.
Asunto(s)
Diferenciación Celular , Condrogénesis , Células Madre Mesenquimatosas/citología , Adulto , Alginatos , Colágeno/genética , Ácido Glucurónico , Ácidos Hexurónicos , Humanos , Inmunohistoquímica , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Coloración y EtiquetadoRESUMEN
Metformin, an anti-diabetic drug used in type 2 diabetes treatment, is reported to have oncopreventive or therapeutic roles in several human cancers. The present study investigated the therapeutic potential of physiologic dose of metformin in PTC. Metformin inhibited PTC cell viability and increased cell apoptosis in various doses (0.5-20mM) in BCPAP and BHP10-3SC cells. Western blot analysis demonstrated that the p-AMPK/AMPK ratio increased with increased metformin treatment. The ectopic tumor experiment was performed using BHP10-3SC cells and athymic nude mice. Oral metformin treatment via drinking water significantly delayed tumor growth in both tumor development model and established tumor models. Necrotic area in tumors significantly increased with metformin treatment. Western blot analysis revealed an increase in p-AMPK/AMPK ratio and suppressions of mTOR and Akt expressions in metformin-treated mice compared to the results in mock-treated control mice. Our results indicate that a physiologic dose of metformin has anti-tumorigenic effects that result from activation of AMPK signaling and inhibition of Akt signaling.
Asunto(s)
Carcinoma/tratamiento farmacológico , Metformina/uso terapéutico , Neoplasias de la Tiroides/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Western Blotting , Carcinoma Papilar , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Metformina/farmacología , Ratones , Ratones Desnudos , Transducción de Señal/efectos de los fármacos , Cáncer Papilar TiroideoRESUMEN
BACKGROUND: Aberrant activation of the Wnt/ß-catenin pathway is a common pathogenesis of various human cancers. We investigated the role of the Wnt inhibitor, Dkk-1, in papillary thyroid cancer (PTC). METHODS: Immunohistochemical ß-catenin staining was performed in tissue microarray containing 148 PTCs and five normal thyroid tissues. In vivo effects of Dkk-1 were explored using ectopic tumors with BHP10-3SC cells. RESULTS: In 27 PTC patients, 60% of patients showed ß-catenin up-regulation and Dkk-1 down-regulation in tumor vs normal tissues. Tissue microarray analysis showed that 14 of 148 PTC samples exhibited cytoplasmic-dominant ß-catenin expression compared to membranous-dominant expression in normal tissues. Aberrant ß-catenin expression was significantly correlated with higher rates of the loss of membranous E-cadherin expression and poor disease-free survival than that in the normal membranous expression group over a median follow-up period of 14 years. Implantation of Dkk-1-overexpressing BHP10-3SC cells revealed delayed tumor growth, resulting from the rescue of membranous ß-catenin and E-cadherin expressions. Furthermore, tissue microarray analysis demonstrated that BRAF(WT) patients had higher rates of aberrant expressions of ß-catenin and E-cadherin than BRAF(V600E) patients. Indeed, the inhibitory effects of Dkk-1 on cell survival were more sensitive in BRAF(WT) (BHP10-3SC and TPC-1) than in BRAF(V600E) (SNU-790 and BCPAP) cells. Overexpression of BRAF(V600E) in normal thyroid epithelial (H tori) cells also reduced the effects of Dkk-1 on cell survival. CONCLUSION: A subset of PTC patients showed aberrant expression of ß-catenin/E-cadherin signaling and poor disease-free survival. Dkk-1 might have a therapeutic role, particularly in BRAF(WT) patients.
Asunto(s)
Carcinoma Papilar/metabolismo , Carcinoma/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias de la Tiroides/metabolismo , Vía de Señalización Wnt/fisiología , Adulto , Anciano , Animales , Cadherinas/metabolismo , Carcinoma/genética , Carcinoma Papilar/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células Epiteliales/citología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones Desnudos , Persona de Mediana Edad , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Cáncer Papilar Tiroideo , Glándula Tiroides/citología , Neoplasias de la Tiroides/genética , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/metabolismoRESUMEN
Osteoporosis is a skeletal disorder characterized by reduced bone mineral density (BMD) and increased risk of fracture. We studied the effects of cell therapy of human adipose tissue-derived stromal cell (ADSC) on ovariectomy-induced bone loss in T cell deficient nude mice. Twelve-week-old female nude mice underwent ovariectomy and were treated with ADSC, estrogen, or phosphate buffered saline (PBS). Whole body BMD revealed that treatment of ADSC was more protective against ovariectomy-induced attenuation in bone mass gain compared with PBS control after cell therapy (8.4±1.1 vs. 2.4%±1.4%, p<0.05 at 4 weeks, 13.7±1.3 vs. 7.7%±1.8%, p<0.05 at 8 weeks) and this effect was comparable to that of estrogen. µCT analysis revealed that the effect of ADSCs was specific to trabecular bone. Serum osteocalcin levels were increased 4 weeks after ovariectomy and treatment with ADSCs (76.4±11.6 ng/mL) increased osteocalcin to a greater extent when compared with estrogen (63.1±6.7 ng/mL, p<0.05) or PBS treatment (58.0±9.2 ng/mL, p<0.05). Flow cytometry analysis for PKH26-labeled ADSCs and quantitative real-time PCR analysis for human ß-globin from bone revealed that transplanted ADSCs were trafficking in bone 48 h after injection and subsequently disappeared. There was no evidence of long-term engraftment of infused ADSCs in bone. In vitro, treatment with ADSC-conditioned medium enhanced osteogenic differentiation in stromal cells and preosteoblasts. These results suggest that cell therapy of ADSCs protects against ovariectomy-induced bone loss in nude mice in a paracrine manner.
Asunto(s)
Tejido Adiposo/citología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Osteoporosis/prevención & control , Ovariectomía/efectos adversos , Células del Estroma/citología , Animales , Densidad Ósea/fisiología , Femenino , Humanos , Ratones , Ratones Desnudos , Osteoporosis/etiología , Células del Estroma/fisiologíaRESUMEN
PPARγ has critical role in the differentiation of mesenchymal stem cells into adipocytes while suppressing osteoblastic differentiation. We generated transgenic mice that overexpress PPARγ specifically in osteoblasts under the control of a 2.3-kb procollagen type 1 promoter (Col.1-PPARγ). Bone mineral density (BMD) of 6- to 14-week-old Col.1 - PPARγ male mice was 8% to 10% lower than that of their wild-type littermates, whereas no difference was noticed in Col.1-PPARγ female mice. Col.1-PPARγ male mice exhibited decreased bone volume (45%), trabecular thickness (23%), and trabecular number (27%), with a reciprocal increase in trabecular spacing (51%). Dynamic histomorphometric analysis also revealed that bone-formation rate (42%) and mineral apposition rate (32%) were suppressed significantly in Col.1-PPARγ male mice compared with their wild-type littermates. Interestingly, osteoclast number and surface also were decreased by 40% and 58%, respectively, in Col.1-PPARγ male mice. In vitro whole-marrow culture for osteoclastogenesis also showed a significant decrease in osteoclast formation (approximately 35%) with the cells from Col.1-PPARγ male mice, and OPG/RANKL ratio was reduced in stromal cells from Col.1-PPARγ male mice. Although there was no significant difference in BMD in Col.1-PPARγ female mice up to 30 weeks, bone loss was accelerated after ovariectomy compared with wild-type female mice (-3.9% versus -6.8% at 12 weeks after ovariectomy, p < .01), indicating that the effects of PPARγ overexpression becomes more evident in an estrogen-deprived state in female mice. In conclusion, in vivo osteoblast-specific overexpression of PPARγ negatively regulates bone mass in male mice and accelerates estrogen-deficiency-related bone loss in female mice.
Asunto(s)
Resorción Ósea/metabolismo , Resorción Ósea/patología , Huesos/patología , Osteoblastos/metabolismo , Ovariectomía , PPAR gamma/metabolismo , Animales , Western Blotting , Peso Corporal , Densidad Ósea , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Resorción Ósea/fisiopatología , Huesos/metabolismo , Huesos/fisiopatología , Extractos Celulares , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Tamaño de los Órganos , Osteogénesis , Fenotipo , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
Secreted frizzled-related protein-4 (sFRP4) is a member of secreted modulators of Wnt signaling pathways and has been recognized to play important role in the pathogenesis of oncogenic osteomalacia as a potential phosphatonin. To investigate the role of sFRP4 in bone biology and phosphorus homeostasis in postnatal life, we generated transgenic mice that overexpress sFRP4 under the control of the serum amyloid P promoter (SAP-sFRP4), which drives transgene expression postnatally. Serum phosphorus level and urinary phosphorus excretion were slightly lower and higher, respectively, in SAP-sFRP4 compared to wild-type (WT) littermate, but the difference did not reach statistical significance. However, renal Na(+/-)/Pi co-transporter (Npt) 2a and 1alpha-hydroxylase gene expression were up-regulated in SAP-sFRP4 mice. In addition, the level of serum 1,25-dihydroxyvitamin D(3) was higher in SAP-sFRP4 mice. At 5 weeks of age, bone mineral density (BMD) in SAP-sFRP4 was similar to that in WT. However, with advancing age, SAP-sFRP4 mice gained less BMD so that areal BMD of SAP-sFRP4 mice was significantly lower compared to WT at 15 weeks of age. Histomorphometric analysis of proximal tibia showed that trabecular bone volume (BV/TV) and thickness (Tb.Th) were significantly lower in SAP-sFRP4 mice. There was no evidence of osteomalacia in histological analysis. Our data do not support the role of sFRP4 per se as a phosphatonin but suggest that sFRP4 negatively regulates bone formation without disrupting phosphorus homeostasis.
Asunto(s)
Huesos/citología , Homeostasis , Fosfatos/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/metabolismo , Componente Amiloide P Sérico/genética , Animales , Animales Recién Nacidos , Peso Corporal/fisiología , Densidad Ósea/fisiología , Huesos/metabolismo , Diferenciación Celular , Separación Celular , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Tamaño de los Órganos , Osteoblastos/citología , Fosfatos/sangre , Fosfatos/orina , Proteínas Proto-Oncogénicas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cráneo/citologíaRESUMEN
Wnt inhibitory factor (WIF)-1 belongs to the members of secreted modulators of Wnt proteins. Secreted frizzled-related proteins (sFRPs), another member of Wnt modulators, have been shown to play differential roles in Wnt signaling depending on the subtypes and cell models. This study was undertaken to investigate the functional role of WIF-1 in osteoblastic differentiation of mouse mesenchymal C3H10T1/2 cells. C3H10T1/2 cells express endogenous WIF-1 and its expression level decreases during osteoblastogenesis. Treatment of C3H10T1/2 cells with WIF-1 significantly reduced alkaline phosphatase (ALP) activities induced by either osteogenic medium (OM, ascorbic acid and beta-glycerophosphate) or Wnt-3a conditioned medium (CM) in a dose-dependent manner. In contrast, the expression level of endogenous WIF-1 increased during adipogenesis and WIF-1 treatment resulted in increased adipogenesis. C3H10T1/2 cells transduced with WIF-1 retrovirus also exhibited reduced ALP activity and decreased mRNA expression of Runx2, collagen type 1, ALP and osteocalcin during osteoblastic differentiation compared to empty virus-transduced cells. Moreover, treatment with WIF-1 dose-dependently attenuates beta-catenin/T-cell factor (TCF) transcriptional activity in this cell line. Finally, knockdown of WIF-1 in C3H10T1/2 cells by RNA interference leads to increase in ALP activities. Collectively, these results indicate that WIF-1 plays as a negative regulator of osteoblastic differentiation in mouse mesenchymal C3H10T1/2 cells in vitro.