RESUMO
Six GATA transcription factors play important roles in eukaryotic development. Among these, GATA2, an essential factor for the hematopoietic cell lineage, exhibits low expression in human gastric tissues, whereas GATA6, which is crucial for gastrointestinal development and differentiation, is frequently amplified and/or overexpressed in human gastric cancer. Interestingly, we found that GATA6 was overexpressed in human gastric cancer cells only when GATA2 expression was completely absent, thereby showing an inverse correlation between GATA2 and GATA6. In gastric cancer cells that express high GATA6 levels, a GATA2 CpG island is hypermethylated, repressing expression in these cells. In contrast, GATA6 expression is undetectable in GATA2-overexpressing gastric cancer cells, which lack GATA2 DNA methylation. Furthermore, PRC2 complex-mediated transcriptional silencing of GATA6 was observed in the GATA2-overexpressing cells. We also show that the GATA2 and PRC2 complexes are enriched within the GATA6 locus, and that the recruitment of the PRC2 complex is impaired by disrupting GATA2 expression, resulting in GATA6 upregulation. In addition, ectopic GATA2 expression significantly downregulates GATA6 expression, suggesting GATA2 directly represses GATA6. Furthermore, GATA6 downregulation showed antitumor activity by inducing growth arrest. Finally, we show that aberrant GATA2 methylation occurs early during the multistep process of gastric carcinogenesis regardless of Helicobacter pylori infection. Taken together, GATA2 dysregulation by epigenetic modification is associated with unfavorable phenotypes in human gastric cancer cells by allowing GATA6 expression.
Assuntos
Biomarcadores Tumorais/metabolismo , Metilação de DNA , Epigênese Genética , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA6/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Gástricas/patologia , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Fator de Transcrição GATA2/metabolismo , Fator de Transcrição GATA6/genética , Humanos , Prognóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Células Tumorais CultivadasRESUMO
The CCCTC-binding factor (CTCF)/cohesin complex regulates gene transcription via high-order chromatin organization of the genome. De novo methylation of CpG islands in the promoter region is an epigenetic hallmark of gene silencing in cancer. Although the CTCF/cohesin complex preferentially targets hypomethylated DNA, it remains unclear whether the CTCF/cohesin-mediated high-order chromatin structure is affected by DNA methylation during tumorigenesis. We found that DNA methylation downregulates the expression of prostaglandin-endoperoxide synthase 2 (PTGS2), which is an inducible, rate-limiting enzyme for prostaglandin synthesis, by disrupting CTCF/cohesin-mediated chromatin looping. We show that the CTCF/cohesin complex is enriched near a CpG island associated with PTGS2 and that the PTGS2 locus forms chromatin loops through methylation-sensitive binding of the CTCF/cohesin complex. DNA methylation abolishes the association of the CTCF/cohesin complex with the PTGS2 CpG island. Disruption of chromatin looping by DNA methylation abrogates the enrichment of transcriptional components, such as positive elongation factor b, at the transcriptional start site of the PTGS2 locus. These alterations result in the downregulation of PTGS2. Our results provide evidence that CTCF/cohesin-mediated chromatin looping of the PTGS2 locus is dynamically influenced by the DNA methylation status.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Ciclo-Oxigenase 2/biossíntese , Metilação de DNA , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas Repressoras/metabolismo , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/patologia , Proteínas Cromossômicas não Histona/genética , Ilhas de CpG , Ciclo-Oxigenase 2/genética , Humanos , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteínas Repressoras/genética , CoesinasRESUMO
Mesenchymal stem cells (MSCs) possess immunomodulatory activities, including suppression of T- and B-cell activation. However, their effects on atopic dermatitis (AD) have not yet been studied. Using an ovalbumin-induced AD mouse model, we investigated whether MSCs can be used as therapeutics in AD. We isolated both allogeneic and syngeneic clonal MSCs (cMSCs) from mouse bone marrow according to the subfractionation culturing method. Our cMSCs suppressed both T- and B-cell activation. T-cell proliferation and cytokine production, including interferon (IFN)-γ and interleukin (IL)-4, were suppressed by inhibition of transcription factors, such as T-bet, GATA-3, and c-Maf. Those transcription factors were nitric oxide dependent. Immunoglobulin E (IgE) suppression occurred through downregulation of AID and BLIMP-1, important regulators for isotype class switch and B-cell differentiation. The cMSCs were injected intravenously into ovalbumin-induced AD mouse model, and the therapeutic effects were analyzed. Injection of both allogeneic and syngeneic cMSCs in an AD mouse model inhibited cell infiltration in skin lesions and decreased the serum level of IgE. IL-4 expression was also suppressed by cMSCs in both the lymph node and skin. The cMSCs migrated to skin lesions and draining lymph nodes. Taken together, these data demonstrated that cMSCs, which suppressed T- and B-cell functions, can be used for the treatment of AD in mice.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos , Dermatite Atópica/terapia , Transplante de Células-Tronco Mesenquimais , Animais , Linfócitos B/imunologia , Citocinas/imunologia , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/imunologia , Modelos Animais de Doenças , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Ovalbumina/efeitos adversos , Linfócitos T/imunologiaRESUMO
Mesenchymal stem cells (MSCs) have immunomodulatory functions such as the suppression of T and B cells. MSCs suppress immunoglobulin (Ig) production by B cells via cell-cell contact as well as via secretion of soluble factors. Our study showed that the conditioned medium (CM) of MSCs infected with a mycoplasma strain, Mycoplasma arginini, has marked inhibitory effects on Ig production by lipopolysaccharide/interleukin-4-induced B cells compared with mycoplasma-free MSC-CM. We analyzed mycoplasma-infected MSC-CM by fast protein liquid chromatography and liquid chromatography to screen the molecules responsible for Ig inhibition. Complement C3 (C3) was the most critical molecule among the candidates identified. C3 was shown to be involved in the suppression of the Ig production of B cells. C3 was secreted by mycoplasma-infected MSCs, but not by mycoplasma-free MSCs or B cells. It was able to directly inhibit Ig production by B cells. In the presence of a C3 inhibitor, Ig inhibition by MSC-CM was abrogated. This inhibitory effect was concomitant with the downregulation of B-cell-induced maturation protein-1, which is a regulator of the differentiation of antibody-secreting plasma cells. These results suggest that C3 secreted from mycoplasma-infected MSCs has an important role in the immunomodulatory functions of MSCs. However, its role in vivo needs to be explored.