RESUMEN
BACKGROUND: Osteoblasts and adipocytes share a common mesenchymal stem cell origin. Therefore, it has been suggested that the accumulation of marrow adipocytes observed in bone loss is caused by a shift in the commitment of mesenchymal stem cells from the osteogenic pathway to the adipogenic pathway. Supporting this hypothesis the competition between adipogenic and osteogenic lineages was widely demonstrated on partially homogeneous cell populations. However, some data from mouse models showed the existence of an independent relationship between bone mineral content and bone marrow adiposity. Therefore, the combination of adipogenesis and osteogenesis in primary culture would be helpful to determine if this competition would be observed on a whole bone marrow stromal cell population in a culture medium allowing both lineages. In this aim, mouse bone marrow stromal cells were cultured in a standard osteogenic medium added with different concentrations of Dexamethasone, known to be an important regulator of mesenchymal progenitor cell differentiation. RESULTS: Gene expression of osteoblast and adipocyte markers, biochemical and physical analyses demonstrated the presence of both cell types when Dexamethasone was used at 100 nM. Overall, our data showed that in this co-differentiation medium both differentiation lineages were enhanced compared to classical adipogenic or osteogenic culture medium. This suggests that in this model, adipocyte phenotype does not seem to increase at the expense of the osteoblast lineage. CONCLUSION: This model appears to be a promising tool to study osteoblast and adipocyte differentiation capabilities and the interactions between these two processes.
Asunto(s)
Adipocitos/citología , Antiinflamatorios/farmacología , Diferenciación Celular/efectos de los fármacos , Dexametasona/farmacología , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Adipocitos/metabolismo , Adipogénesis/efectos de los fármacos , Animales , Células de la Médula Ósea/citología , Linaje de la Célula , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Medios de Cultivo Condicionados/farmacología , Células Madre Mesenquimatosas/metabolismo , Ratones , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogénesis/efectos de los fármacosRESUMEN
BACKGROUND: In osteoporosis, bone loss is accompanied by increased marrow adiposity. Given their proximity in the bone marrow and their shared origin, a dialogue between adipocytes and osteoblasts could be a factor in the competition between human Mesenchymal Stem Cells (hMSC) differentiation routes, leading to adipocyte differentiation at the expense of osteoblast differentiation. The adipocyte/osteoblast balance is highly regulated at the level of gene transcription. In our work, we focused on PPARgamma, CEBPalpha and CEBPdelta, as these transcription factors are seen as master regulators of adipogenesis and expressed precociously, and on leptin and adiponectin, considered as adipocyte marker genes. In 2010, our group has demonstrated, thanks to a coculture model, that in the presence of hMSC-derived adipocytes (hMSC-Adi), hMSC-derived osteoblasts (hMSC-Ost) express lesser amounts of osteogenic markers but exhibit the expression of typical adipogenic genes. Nevertheless, the mechanisms underlying this modulation of gene expression are not clarified. Recently, adipocytes were described as releasing extracellular vesicles (EVs), containing and transferring adipocyte specific transcripts, like PPARgamma, leptin and adiponectin. Here, we investigated whether EVs could be the way in which adipocytes transfer adipogenic RNAs in our coculture model. RESULTS: We observed in hMSC-Ost incubated in hAdi-CM an increase in the adipogenic PPARγ, leptin, CEBPα and CEBPδ transcripts as well as the anti-osteoblastic miR-138, miR30c, miR125a, miR-125b, miR-31 miRNAs, probably implicated in the observed osteocalcin (OC) and osteopontin (OP) expression decrease. Moreover, EVs were isolated from conditioned media collected from cultures of hMSC at different stages of adipocyte differentiation and these specific adipogenic transcripts were detected inside. Finally, thanks to interspecies conditioned media exposition, we could highlight for the first time a horizontal transfer of adipogenic transcripts from medullary adipocytes to osteoblasts. CONCLUSIONS: Here, we have shown, for the first time, RNA transfer between hMSC-derived adipocytes and osteoblasts through EVs. Additional studies are needed to clarify if this mechanism has a role in the adipocytic switch driven on osteoblasts by adipocytes inside bone marrow and if EVs could be a target component to regulate the competition between osteoblasts and adipocytes in the prevention or in the therapy of osteoporosis and other osteopenia.
Asunto(s)
Adipocitos/citología , Células de la Médula Ósea/citología , ARN Mensajero/metabolismo , Vesículas Secretoras/metabolismo , Adipogénesis/efectos de los fármacos , Animales , Proteína delta de Unión al Potenciador CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Humanos , Leptina/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , MicroARNs/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteopontina/metabolismo , PPAR gamma/antagonistas & inhibidores , PPAR gamma/genética , PPAR gamma/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
K+ channels are key molecules in the progression of several cancer types and are considered to be potential targets for cancer therapy. In this study, we investigated the intermediate- conductance Ca2+-activated K+ channels (hKCa3.1) expression in both breast carcinoma (BC) specimens and human breast cancer epithelial primary cell cultures (hBCE) using immuno-histochemistry (60 samples), quantitative Real-Time RT-PCR (30 samples) and Western blot assay (30 samples). We also looked at whether or not the expression of these channels is correlated with breast carcinomas grade tumours and metastasis status. Furthermore, we characterized the hKCa3.1 channel activity in hBCE cells by using the Whole Cell Patch Clamp Technique. We found that hKCa3.1 transcripts and proteins were expressed in both BC samples and hBCE cells. Clinicopathologic evaluation indicated a significant correlation between hKCa3.1-expression and tumour grade. hKCa3.1 mRNA and protein were more highly expressed in grade III tumours than in both grades I and II. However, the hKCa3.1 expression-increase according to grade was only observed in tumours with negative metastasis status. Moreover, the hKCa3.1 channels expressed in hBCE cells are functional. This was attested by patch-clamp recordings showing typical hKCa3.1-mediated currents in these cells. In conclusion, these data suggest that hKCa3.1 might contribute to breast tumour-progression and can serve as a useful prognostic marker for breast cancer.
Asunto(s)
Adenocarcinoma/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/secundario , Biomarcadores de Tumor/genética , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Distribución de Chi-Cuadrado , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Potenciales de la Membrana , Estadificación de Neoplasias , Técnicas de Placa-Clamp , Potasio/metabolismo , Pronóstico , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
BACKGROUND: DNA methylation is an important mechanism for gene silencing and has already been described for several genes in breast cancer. A previous immunohistochemistry study demonstrated a decrease of K(v)1.3 potassium channel expression in breast adenocarcinoma compared to normal breast tissue. METHODS: Methyl-specific PCR (MSP), immunohistochemistry and RNA extraction were performed on breast adenocarcinoma. MSP and DNA extraction were also performed on one breast carcinoma cell line and on primary culture normal cells. RESULTS: DNA methylation of K(v)1.3 gene promoter was observed in 42.3% of samples (22/52). The methylated status was associated with poorly differentiated tumors (p=0.04) and younger patients (p=0.048). Decreased K(v)1.3 expression was observed in grade III tumors, at both the mRNA and protein levels, while methylation increased in grade III tumors. Finally, K(v)1.3 gene promoter was methylated in the MCF-7 breast carcinoma cell line while promoter methylation was absent in primary culture of normal breast cells (HMEpC). CONCLUSION: We report, for the first time, the methylation of the K(v)1.3 gene promoter in breast adenocarcinoma. Our data suggest that DNA methylation is responsible for a decrease of K(v)1.3 gene expression in breast adenocarcinoma and is associated with poorly differentiated tumors and younger patients.
Asunto(s)
Adenocarcinoma/genética , Neoplasias de la Mama/genética , Metilación de ADN , Canal de Potasio Kv1.3/genética , Regiones Promotoras Genéticas , Adenocarcinoma/patología , Adulto , Anciano , Neoplasias de la Mama/patología , Femenino , Humanos , Canal de Potasio Kv1.3/metabolismo , Persona de Mediana Edad , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
Because transient receptor potential (TRP) channels have been implicated in tumor progression, we have investigated the potential role of TRPM7 channel in breast cancer cell proliferation. Under whole cell patch clamp, a Mg(2+)-inhibited cationic (MIC) current was observed in MCF-7 cells. This current was characterized by an inward current and a strong outward rectifying current that were both inhibited in a concentration-dependent manner by the presence of intracellular Mg(2+) or Mg(2+)-ATP. The inward current was reduced by La(3+), and the outward current was sensitive to 2-aminoethoxydiphenyl borate (2-APB), spermine, La(3+), and flufenamic acid. Importantly, a similar MIC current was also recorded in the primary culture of human breast cancerous epithelial cells (hBCE). Moreover, TRPM7 transcripts were found in both hBCE and MCF-7 cells. In MCF-7 cells, the MIC current was inhibited by TRPM7 small interfering RNA. Interestingly, we found that cell proliferation and intracellular Ca(2+) concentration were also reduced by TRPM7 silencing in MCF-7 cells. TRPM7 channels were also found in both human breast cancer and healthy tissues. Importantly, TRPM7 channel was overexpressed in grade III breast cancer samples associated with important Ki67 or tumor size. Our findings strongly suggest that TRPM7 is involved in the proliferative potentiality of breast cancer cells, probably by regulating Ca(2+) influx.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Regulación Neoplásica de la Expresión Génica/fisiología , Canales Catiónicos TRPM/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Magnesio/metabolismo , Manganeso , Potenciales de la Membrana/fisiología , Proteínas Serina-Treonina Quinasas , Canales Catiónicos TRPM/genética , Técnicas de Cultivo de TejidosRESUMEN
BACKGROUND: TRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7), a human breast cancer epithelial primary culture (hBCE) and in normal and tumour breast tissues. METHODS: Molecular (Western blot and RT-PCR), and immunohistochemical techniques were used to investigate TRPC6 expression. To investigate the channel activity in both MCF-7 cells and hBCE we used electrophysiological technique (whole cell patch clamp configuration). RESULTS: A non selective cationic current was activated by the oleoyl-2-acetyl-sn-glycerol (OAG) in both hBCE and MCF-7 cells. OAG-inward current was inhibited by 2-APB, SK&F 96365 and La3+. TRPC6, but not TRPM7, was expressed both in hBCE and in MCF-7 cells. TRPC3 was only expressed in hBCE. Clinically, TRPC6 mRNA and protein were elevated in breast carcinoma specimens in comparison to normal breast tissue. Furthermore, we found that the overexpression of TRPC6 protein levels were not correlated with tumour grades, estrogen receptor expression or lymph node positive tumours. CONCLUSION: Our results indicate that TRPC6 channels are strongly expressed and functional in breast cancer epithelial cells. Moreover, the overexpression of these channels appears without any correlation with tumour grade, ER expression and lymph node metastasis. Our findings support the idea that TRPC6 may have a role in breast carcinogenesis.
Asunto(s)
Neoplasias de la Mama/metabolismo , Glándulas Mamarias Humanas/metabolismo , Neoplasias Glandulares y Epiteliales/metabolismo , Canales Catiónicos TRPC/biosíntesis , Western Blotting , Neoplasias de la Mama/patología , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Femenino , Humanos , Inmunohistoquímica , Glándulas Mamarias Humanas/citología , Estadificación de Neoplasias , Neoplasias Glandulares y Epiteliales/patología , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales Catiónicos TRPC/fisiología , Canal Catiónico TRPC6RESUMEN
In this work, we investigated the effect of inorganic phosphate (Pi) on the differentiation of monocyte/macrophage precursors into an "osteoclastic" phenotype, and we delineated the molecular mechanisms which could be involved in this phenomenon. This was achieved by stimulating human peripheral blood monocytic cells and RAW 264.7 monocyte-macrophage precursor cells to differentiate into osteoclast-like cells in the presence of receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). RANKL has been previously reported to stimulate the signaling kinases ERK 1/2, p38, Akt, JNK, and the DNA-binding activity of the transcription factors AP-1 and NF-kappaB. Increase in extracellular Pi concentration (1.5-4.5 mM) dose-dependently inhibits both osteoclastic differentiation and bone resorption activity induced by RANKL and M-CSF. Pi was found to specifically inhibit the RANKL-induced JNK and Akt activation, while RANKL-induced p38 and ERK 1/2 phosphorylation were not significantly affected. Moreover, we found that Pi significantly reduced the RANKL-stimulated DNA-binding activity of NF-kappaB. The effects of Pi on osteoclast differentiation and DNA-binding activity of NF-kappaB were prevented by Foscarnet, a sodium-phosphate cotransport inhibitor, suggesting that the effects of Pi occur subsequently to its intake. These results demonstrate that Pi downregulates the differentiation of osteoclasts via a negative feedback exerted on RANK-RANKL signaling.