RESUMO
BACKGROUND: Antigenic stimulation through cross-linking the IgE receptor and epithelial cell-derived cytokine IL-33 are potent stimuli of mast cell (MC) activation. Moreover, IL-33 primes a variety of cell types, including MCs to respond more vigorously to external stimuli. However, target genes induced by the combined IL-33 priming and antigenic stimulation have not been investigated in human skin mast cells (HSMCs) in a genome-wide manner. Furthermore, epigenetic changes induced by the combined IL-33 priming and antigenic stimulation have not been evaluated. RESULTS: We found that IL-33 priming of HSMCs enhanced their capacity to promote transcriptional synergy of the IL1B and CXCL8 genes by 16- and 3-fold, respectively, in response to combined IL-33 and antigen stimulation compared to without IL-33 priming. We identified the target genes in IL-33-primed HSMCs in response to the combined IL-33 and antigenic stimulation using RNA sequencing (RNA-seq). We found that the majority of genes synergistically upregulated in the IL-33-primed HSMCs in response to the combined IL-33 and antigenic stimulation were predominantly proinflammatory cytokine and chemokine genes. Moreover, the combined IL-33 priming and antigenic stimulation increase chromatin accessibility in the synergy target genes but not synergistically. Transcription factor binding motif analysis revealed more binding sites for NF-κB, AP-1, GABPA, and RAP1 in the induced or increased chromatin accessible regions of the synergy target genes. CONCLUSIONS: Our study demonstrates that IL-33 priming greatly potentiates MCs' ability to transcribe proinflammatory cytokine and chemokine genes in response to antigenic stimulation, shining light on how epithelial cell-derived cytokine IL-33 can cause exacerbation of skin MC-mediated allergic inflammation.
Assuntos
Citocinas , Mastócitos , Humanos , Citocinas/genética , Citocinas/metabolismo , Mastócitos/metabolismo , Interleucina-33/genética , Interleucina-33/metabolismo , Quimiocinas/genética , Cromatina/metabolismoRESUMO
Inflammation is pathogenic to skin diseases, including atopic dermatitis (AD) and eczema. Treatment for AD remains mostly symptomatic with newer but costly options, tainted with adverse side effects. There is an unmet need for safe therapeutic and preventative strategies for AD. Resveratrol (R) is a natural compound known for its anti-inflammatory properties. However, animal and human R studies have yielded contrasting results. Mast cells (MCs) are innate immune skin-resident cells that initiate the development of inflammation and progression to overt disease. R's effects on MCs are also controversial. Using a human-like mouse model of AD development consisting of a single topical application of antigen ovalbumin (O) for 7 days, we previously established that the activation of MCs by a bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) initiated substantial skin remodeling compared to controls. Here, we show that daily R application normalized O-mediated epidermal thickening, ameliorated cell infiltration, and inhibited skin MC activation and chemokine expression. We unraveled R's multiple mechanisms of action, including decreased activation of the S1P-producing enzyme, sphingosine kinase 1 (SphK1), and of transcription factors Signal Transducer and Activator of Transcription 3 (Stat3) and NF-κBp65, involved in chemokine production. Thus, R may be poised for protection against MC-driven pathogenic skin inflammation.
Assuntos
Dermatite Atópica , NF-kappa B , Animais , Humanos , Camundongos , Quimiocinas/metabolismo , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Mastócitos/metabolismo , NF-kappa B/metabolismo , Resveratrol/uso terapêutico , Esfingosina , Fator de Transcrição STAT3/metabolismoRESUMO
BACKGROUND: Mast cell activation is critical for the development of allergic diseases. Ligation of sialic acid-binding immunoglobin-like lectins (Siglecs), such as Siglec-6, -7, and -8 as well as CD33, have been shown to inhibit mast cell activation. Recent studies showed that human mast cells express Siglec-9, an inhibitory receptor also expressed by neutrophils, monocytes, macrophages, and dendritic cells. OBJECTIVE: We aimed to characterize Siglec-9 expression and function in human mast cells in vitro. METHODS: We assessed the expression of Siglec-9 and Siglec-9 ligands on human mast cell lines and human primary mast cells by real-time quantitative PCR, flow cytometry, and confocal microscopy. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt the SIGLEC9 gene. We evaluated Siglec-9 inhibitory activity on mast cell function by using native Siglec-9 ligands, glycophorin A (GlycA), and high-molecular-weight hyaluronic acid, a monoclonal antibody against Siglec-9, and coengagement of Siglec-9 with the high-affinity receptor for IgE (FcεRI). RESULTS: Human mast cells express Siglec-9 and Siglec-9 ligands. SIGLEC9 gene disruption resulted in increased expression of activation markers at baseline and increased responsiveness to IgE-dependent and IgE-independent stimulation. Pretreatment with GlycA or high-molecular-weight hyaluronic acid followed by IgE-dependent or -independent stimulation had an inhibitory effect on mast cell degranulation. Coengagement of Siglec-9 with FcεRI in human mast cells resulted in reduced degranulation, arachidonic acid production, and chemokine release. CONCLUSIONS: Siglec-9 and its ligands play an important role in limiting human mast cell activation in vitro.
Assuntos
Ácido Hialurônico , Mastócitos , Humanos , Ligantes , Ácido Hialurônico/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Imunoglobulina E/metabolismoRESUMO
Mas-related G protein-coupled receptor-X2 (MRGPRX2) expressed on mast cells (MCs) contributes to hypersensitivity reactions to cationic US-Food and Drug Administration (FDA) approved drugs such as the neuromuscular blocking agent, rocuronium. In addition, activation of MRGPRX2 by the neuropeptide substance P (SP) and the pro-adrenomedullin peptide (PAMP-12) is associated with a variety of cutaneous conditions such as neurogenic inflammation, pain, atopic dermatitis, urticaria, and itch. Thus, small molecules aimed at blocking MRGPRX2 constitute potential options for modulating IgE-independent MC-mediated disorders. Two inverse MRGPRX2 agonists, named C9 and C9-6, have recently been identified, which inhibit basal G protein activation and agonist-induced calcium mobilization in transfected HEK293 cells. Substance P serves as a balanced agonist for MRGPRX2 whereby it activates both G protein-mediated degranulation and ß-arrestin-mediated receptor internalization. The purpose of this study was to determine if C9 blocks MRGPRX2's G protein and ß-arrestin-mediated signaling and to determine its specificity. We found that C9, but not its inactive analog C7, inhibited degranulation in RBL-2H3 cells stably expressing MRGPRX2 in response to SP, PAMP-12 and rocuronium with an IC50 value of ~300 nM. C9 also inhibited degranulation as measured by cell surface expression of CD63, CD107a and ß-hexosaminidase release in LAD2 cells and human skin-derived MCs in response to SP but not the anaphylatoxin, C3a or FcϵRI-aggregation. Furthermore, C9 inhibited ß-arrestin recruitment and MRGPRX2 internalization in response to SP and PAMP-12. We found that a G protein-coupling defective missense MRGPRX2 variant (V282M) displays constitutive activity for ß-arrestin recruitment, and that this response was significantly inhibited by C9. Rocuronium, SP and PAMP-12 caused degranulation in mouse peritoneal MCs and these responses were abolished in the absence of MrgprB2 or cells treated with pertussis toxin but C9 had no effect. These findings suggest that C9 could provide an important framework for developing novel therapeutic approaches for the treatment of IgE-independent MC-mediated drug hypersensitivity and cutaneous disorders.
Assuntos
Hipersensibilidade a Drogas , Neuropeptídeos , Camundongos , Animais , Humanos , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Degranulação Celular , Adrenomedulina/metabolismo , Receptores de IgE/metabolismo , Substância P/farmacologia , Cálcio/metabolismo , Rocurônio , Toxina Pertussis/farmacologia , Células HEK293 , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Mastócitos/metabolismo , Neuropeptídeos/metabolismo , Hipersensibilidade a Drogas/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , beta-Arrestinas/metabolismo , beta-Arrestinas/farmacologia , Anafilatoxinas/metabolismo , Imunoglobulina E/metabolismoRESUMO
While marine natural products have been investigated for anticancer drug discovery, they are barely screened against rare cancers. Thus, in our effort to discover potential drug leads against the rare cancer pseudomyxoma peritonei (PMP), which currently lacks effective drug treatments, we screened extracts of marine actinomycete bacteria against the PMP cell line ABX023-1. This effort led to the isolation of nine rearranged angucyclines from Streptomyces sp. CNZ-748, including five new analogues, namely, grincamycins P-T (1-5). The chemical structures of these compounds were unambiguously established based on spectroscopic and chemical analyses. Particularly, grincamycin R (3) possesses an S-containing α-l-methylthio-aculose residue, which was discovered in nature for the first time. All of the isolated compounds were evaluated against four PMP cell lines and some exhibited low micromolar inhibitory activities. To identify a candidate biosynthetic gene cluster (BGC) encoding the grincamycins, we sequenced the genome of the producing strain, Streptomyces sp. CNZ-748, and compared the BGCs detected with those linked to the production of angucyclines with different aglycon structures.
Assuntos
Antraquinonas/farmacologia , Antineoplásicos/farmacologia , Pseudomixoma Peritoneal/tratamento farmacológico , Streptomyces/química , Antraquinonas/isolamento & purificação , Antineoplásicos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , California , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Sedimentos Geológicos/microbiologia , Humanos , Estrutura Molecular , Família Multigênica , Streptomyces/genéticaRESUMO
Mast cells (MCs) are tissue resident immune cells that play important roles in the pathogenesis of allergic disorders. These responses are mediated via the cross-linking of cell surface high affinity IgE receptor (FcϵRI) by antigen resulting in calcium (Ca2+) mobilization, followed by degranulation and release of proinflammatory mediators. In addition to FcϵRI, cutaneous MCs express Mas-related G protein-coupled receptor X2 (MRGPRX2; mouse ortholog MrgprB2). Activation of MRGPRX2/B2 by the neuropeptide substance P (SP) is implicated in neurogenic inflammation, chronic urticaria, mastocytosis and atopic dermatitis. Although Ca2+ entry is required for MRGPRX2/B2-mediated MC responses, the possibility that calcium release-activated calcium (CRAC/Orai) channels participate in these responses has not been tested. Lentiviral shRNA-mediated silencing of Orai1, Orai2 or Orai3 in a human MC line (LAD2 cells) resulted in partial inhibition of SP-induced Ca2+ mobilization, degranulation and cytokine/chemokine generation (TNF-α, IL-8, and CCL-3). Synta66, which blocks homo and hetero-dimerization of Orai channels, caused a more robust inhibition of SP-induced responses than knockdown of individual Orai channels. Synta66 also blocked SP-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation and abrogated cytokine/chemokine production. It also inhibited SP-induced Ca2+ mobilization and degranulation in primary human skin MCs and mouse peritoneal MCs. Furthermore, Synta66 attenuated both SP-induced cutaneous vascular permeability and leukocyte recruitment in mouse peritoneum. These findings demonstrate that Orai channels contribute to MRGPRX2/B2-mediated MC activation and suggest that their inhibition could provide a novel approach for the modulation of SP-induced MC/MRGPRX2-mediated disorders.
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Ativados pela Liberação de Cálcio/antagonistas & inibidores , Canais de Cálcio Ativados pela Liberação de Cálcio/genética , Sinalização do Cálcio , Permeabilidade Capilar/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Knockout , Família MultigênicaRESUMO
microRNA-155 (miR155) is pro-atherogenic; however, its role in vascular calcification is unknown. In this study, we aim to examine whether miR155 regulates vascular calcification and to understand the underlying mechanism. Quantitative real-time PCR showed that miR155 is highly expressed in human calcific carotid tissue and positively correlated with the expression of osteogenic genes. Wound-healing assay and TUNEL staining showed deletion of miR155 inhibited vascular smooth muscle cell (VSMC) migration and apoptosis. miR155 deficiency attenuated calcification of cultured mouse VSMCs and aortic rings induced by calcification medium, whereas miR155 overexpression promoted VSMC calcification. Compared with wild-type mice, miR155-/- mice showed significant resistance to vitamin D3 induced vascular calcification. Protein analysis showed that miR155 deficiency alleviated the reduction of Rictor, increased phosphorylation of Akt at S473 and accelerated phosphorylation and degradation of FOXO3a in cultured VSMCs and in the aortas of vitamin D3-treated mice. A PI3K inhibitor that suppresses Akt phosphorylation increased, whereas a pan-caspase inhibitor that suppresses apoptosis reduced VSMC calcification; and both inhibitors diminished the protective effects of miR155 deficiency on VSMC calcification. In conclusion, miR155 deficiency attenuates vascular calcification by increasing Akt phosphorylation and FOXO3a degradation, and thus reducing VSMC apoptosis induced by calcification medium.
Assuntos
MicroRNAs/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Fosfatidilinositol 3-Quinases/genética , Fosforilação/genética , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/imunologia , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Indole-3-carbinol (I3C) and other aryl hydrocarbon receptor agonists are known to modulate the immune system and ameliorate various inflammatory and autoimmune diseases in animal models, including colitis induced by dextran sulfate sodium (DSS). MicroRNAs (miRNAs) are also gaining traction as potential therapeutic agents or diagnostic elements. Enterohepatic Helicobacter (EHH) species are associated with an increased risk of inflammatory bowel disease, but little is known about how these species affect the immune system or response to treatment. AIM: To determine whether infection with an EHH species alters the response to I3C and how the immune and miRNA responses of an EHH species compare with responses to DSS and inflammatory bowel disease. METHODS: We infected C57BL/6 mice with Helicobacter muridarum (H. muridarum), with and without DSS and I3C treatment. Pathological responses were evaluated by histological examination, symptom scores, and cytokine responses. MiRNAs analysis was performed on mesenteric lymph nodes to further evaluate the regional immune response. RESULTS: H. muridarum infection alone caused colonic inflammation and upregulated proinflammatory, macrophage-associated cytokines in the colon similar to changes seen in DSS-treated mice. Further upregulation occurred upon treatment with DSS. H. muridarum infection caused broad changes in mesenteric lymph node miRNA expression, but colitis-associated miRNAs were regulated similarly in H. muridarum-infected and uninfected, DSS-treated mice. In spite of causing colitis exacerbation, H. muridarum infection did not prevent disease amelioration by I3C. I3C normalized both macrophage- and T cell-associated cytokines. CONCLUSION: Thus, I3C may be useful for inflammatory bowel disease patients regardless of EHH infection. The miRNA changes associated with I3C treatment are likely the result of, rather than the cause of immune response changes.
Assuntos
Colite , MicroRNAs , Animais , Colite/induzido quimicamente , Colite/genética , Colo , Citocinas , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Helicobacter , Humanos , Indóis , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genéticaRESUMO
A Gram-stain-negative, microaerophilic, non-motile, rod-shaped bacterium strain designated PMP191FT, was isolated from a human peritoneal tumour. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the organism formed a lineage within the family Chitinophagaceae that was distinct from members of the genus Pseudoflavitalea (95.1-95.2â% sequence similarity) and Pseudobacter ginsenosidimutans (94.4â% sequence similarity). The average nucleotide identity values between strain PMP191FT and Pseudoflavitalea rhizosphaerae T16R-265T and Pseudobacter ginsenosidimutans Gsoil 221T was 68.9 and 62.3% respectively. The only respiratory quinone of strain PMP191FT was MK-7 and the major fatty acids were iso-C15â:â0, iso-C15â:â1 G and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The polar lipids consisted of phosphatidylethanolamine and some unidentified amino and glycolipids. The G+C content of strain PMP191FT calculated from the genome sequence was 43.4 mol%. Based on phylogenetic, phenotypic and chemotaxonomic evidence, strain PMP191FT represents a novel species and genus for which the name Parapseudoflavitalea muciniphila gen. nov., sp. nov. is proposed. The type strain is PMP191FT (=DSM 104999T=ATCC BAA-2857T = CCUG 72691T). The phylogenetic analyses also revealed that Pseudobacter ginsenosidimutans shared over 98â% sequence similarly to members of the genus Pseudoflavitalea. However, the average nucleotide identity value between Pseudoflavitalea rhizosphaerae T16R-265T, the type species of the genus and Pseudobacter ginsenosidimutans Gsoil 221T was 86.8â%. Therefore, we also propose that Pseudobacter ginsenosidimutans be reclassified as Pseudoflavitalea ginsenosidimutans comb. nov.
Assuntos
Bacteroidetes/classificação , Neoplasias Peritoneais/microbiologia , Filogenia , Técnicas de Tipagem Bacteriana , Bacteroidetes/isolamento & purificação , Baltimore , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Glicolipídeos/química , Humanos , Fosfatidiletanolaminas/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMO
Tumor microenvironment (TME) contains a variety of infiltrating immune cells. Among them, tumor-associated macrophages (TAMs) and their alternative activation contribute greatly to the progression of tumors. The mechanisms governing macrophage polarization in the TME are unclear. Here, we show that in TAMs or macrophages under tumor-conditioned medium treatment, the expression of transcription factor EB (TFEB) is reduced and more of the TFEB protein is in an inactive cytosolic form. Transforming growth factor (TGF)-ß is identified as a main driving force for the reduced TFEB expression and activity in TAMs via activating ERK signaling. TFEB interference in macrophages significantly enhanced their alternative activation, with reduced expression of MHC-II and co-stimulatory molecule CD80, decreased ability to activate T cells, and increased ability to attract tumor cells. When co-inoculated with tumor cells, macrophages with TFEB knockdown significantly enhanced tumor growth with increased infiltration of M2-like macrophages, reduced infiltration of CD8+ T cells, and enhanced angiogenesis in the tumors. Mechanistic studies revealed that TFEB downregulation resulted in macrophage M2 polarization through reducing SOCS3 production and enhancing STAT3 activation. We further demonstrate that the activation of TFEB by hydroxypropyl-ß-cyclodextrin in macrophages suppressed their M2 polarization and tumor-promoting capacity, and that macrophage-specific TFEB overexpression inhibited breast tumor growth in mice. Therefore, our data suggest that TFEB plays critical roles in macrophage polarization, and the downregulation of TFEB expression and activation is an integral part of tumor-induced immune editing in the TME. This study provides a rationale for a new cancer treatment strategy by modulating macrophage polarization through activating TFEB.
RESUMO
Macrophages are pleiotropic cells capable of performing a broad spectrum of functions. Macrophage phenotypes are classified along a continuum between the extremes of proinflammatory M1 macrophages and anti-inflammatory M2 macrophages. The seemingly opposing functions of M1 and M2 macrophages must be tightly regulated for an effective and proper response to foreign molecules or damaged tissue. Excessive activation of either M1 or M2 macrophages contributes to the pathology of many diseases. Emodin is a Chinese herb-derived compound and has shown potential to inhibit inflammation in various settings. In this study, we tested the ability of emodin to modulate the macrophage response to both M1 and M2 stimuli. Primary mouse macrophages were stimulated with LPS/IFNγ or IL4 with or without emodin, and the effects of emodin on gene transcription, cell signaling pathways, and histone modifications were examined by a variety of approaches, including microarray, quantitative real-time PCR, Western blotting, chromatin immunoprecipitation, and functional assays. We found that emodin bidirectionally tunes the induction of LPS/IFNγ- and IL4-responsive genes through inhibiting NFκB/IRF5/STAT1 signaling and IRF4/STAT6 signaling, respectively. Thereby, emodin modulates macrophage phagocytosis, migration, and NO production. Furthermore, emodin inhibited the removal of H3K27 trimethylation (H3K27m3) marks and the addition of H3K27 acetylation (H3K27ac) marks on genes required for M1 or M2 polarization of macrophages. In conclusion, our data suggest that emodin is uniquely able to suppress the excessive response of macrophages to both M1 and M2 stimuli and therefore has the potential to restore macrophage homeostasis in various pathologies.
Assuntos
Polaridade Celular/efeitos dos fármacos , Emodina/farmacologia , Memória Imunológica/efeitos dos fármacos , Inflamação/genética , Inflamação/imunologia , Macrófagos/imunologia , Animais , Células Cultivadas , Epigenômica , Humanos , Inflamação/tratamento farmacológico , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/imunologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Overexpression of HOXB7 in breast cancer cells induces an epithelial-mesenchymal transition and promotes tumor progression and lung metastasis. However, the underlying mechanisms for HOXB7-induced aggressive phenotypes in breast cancer remain largely unknown. Here, we report that phosphorylation of SMAD3 was detected in a higher percentage in primary mammary tumor tissues from double-transgenic MMTV-Hoxb7/Her2 mice than tumors from single-transgenic Her2/neu mice, suggesting activation of TGFß/SMAD3 signaling by HOXB7 in breast tumor tissues. As predicted, TGFß2 was high in four MMTV-Hoxb7/Her2 transgenic mouse tumor cell lines and two breast cancer cell lines transfected with HOXB7, whereas TGFß2 was low in HOXB7-depleted cells. HOXB7 directly bound to and activated the TGFß2 promoter in luciferase and chromatin immunoprecipitation assays. Increased migration and invasion as a result of HOXB7 overexpression in breast cancer cells were reversed by knockdown of TGFß2 or pharmacologic inhibition of TGFß signaling. Furthermore, knockdown of TGFß2 in HOXB7-overexpressing MDA-MB-231 breast cancer cells dramatically inhibited metastasis to the lung. Interestingly, HOXB7 overexpression also induced tumor-associated macrophage (TAM) recruitment and acquisition of an M2 tumor-promoting phenotype. TGFß2 mediated HOXB7-induced activation of macrophages, suggesting that TAMs may contribute to HOXB7-promoted tumor metastasis. Providing clinical relevance to these findings, by real-time PCR analysis, there was a strong correlation between HOXB7 and TGFß2 expression in primary breast carcinomas. Taken together, our results suggest that HOXB7 promotes tumor progression in a cell-autonomous and non-cell-autonomous manner through activation of the TGFß signaling pathway.
Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Proteínas de Homeodomínio/biossíntese , Fator de Crescimento Transformador beta2/biossíntese , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Transgênicos , Receptor ErbB-2/genética , Transdução de Sinais/genética , Proteína Smad3/biossínteseRESUMO
STARD6 is a member of the StAR-related lipid transfer (START) domain family of proteins whose function thus far remains obscure. While it recently was shown to facilitate steroidogenesis in a cell-free setting, it has not been localized to steroidogenic cells of normal reproductive tissues. In a recent microarray study, we detected STARD6 mRNA in cultured porcine ovarian granulosa cells which are steroidogenic. In the present study, we examined regulation of STARD6 mRNA in porcine granulosa cultures, and found that it was not regulated by cyclic AMP, but it was reduced by combined knockdown of the transcription factors GATA4 and GATA6. We detected both STARD6 mRNA and protein in fresh granulosa cells and whole antral follicles and different stage corpora lutea of pig. The highest levels were discovered in the mid-luteal phase corpus luteum. Immunolocalization within ovarian tissues indicated robust STARD6 immunoreactivity in steroidogenic cells of the corpus luteum. Relatively lesser amounts of STARD6 signal were found in granulosa cells, theca cells, and oocytes. To test the ability of STARD6 to facilitate de novo steroidogenesis, non-steroidogenic COS-1 cells were co-transfected with components of the P450 cholesterol side-chain cleavage system, enabling them to make pregnenolone, and STARD6. STARD6 increased pregnenolone production by two- to three-fold over the empty vector control. In summary, STARD6 is found in the pig ovary, exhibits the strongest expression in highly steroidogenic luteal cells, and significantly enhances pregnenolone production in transfected COS cells independent of cyclic AMP treatment. Collectively, these findings indicate that STARD6 may contribute to steroidogenesis in ovarian cells, but also suggests other cellular functions that require cholesterol trafficking.
Assuntos
Proteínas de Membrana Transportadoras/fisiologia , Ovário/metabolismo , Suínos/metabolismo , Animais , Células COS , Chlorocebus aethiops , Corpo Lúteo/metabolismo , AMP Cíclico/metabolismo , Ciclo Estral/metabolismo , Feminino , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA6/genética , Técnicas de Silenciamento de Genes , Células da Granulosa/metabolismo , Imuno-Histoquímica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Folículo Ovariano/metabolismo , Interferência de RNA , RNA MensageiroRESUMO
OBJECTIVE: microRNA-155 (miR155) plays a critical role in immunity and macrophage inflammation. We aim to investigate the role of miR155 in atherogenesis. APPROACH AND RESULTS: Quantitative real-time polymerase chain reaction showed that miR155 was expressed in mouse and human atherosclerotic lesions. miR155 expression in macrophages was correlated positively with proinflammatory cytokine expression. Lentivirus-mediated overexpression of miR155 in macrophages enhanced their inflammatory response to lipopolysaccharide through targeting suppressor of cytokine signaling-1 and impaired cholesterol efflux from acetylated low-density lipoprotein-loaded macrophages, whereas deficiency of miR155 blunted macrophage inflammatory responses and enhanced cholesterol efflux possibly via enhancing lipid loading-induced macrophage autophagy. We next examined the atherogenesis in apolipoprotein E-deficient (apoE(-/-)) and miR155(-/-)/apoE(-/-) (double knockout) mice fed a Western diet. Compared with apoE(-/-) mice, the double knockout mice developed less atherosclerosis lesion in aortic root, with reduced neutral lipid content and macrophages. Flow cytometric analysis showed that there were increased number of regulatory T cells and reduced numbers of Th17 cells and CD11b+/Ly6C(high) cells in the spleen of double knockout mice. Peritoneal macrophages from the double knockout mice had significantly reduced proinflammatory cytokine expression and secretion both in the absence and presence of lipopolysaccharide stimulation. To determine whether miR155 in leukocytes contributes to atherosclerosis, we performed a bone marrow transplantation study. Deficiency of miR155 in bone marrow-derived cells suppressed atherogenesis in apoE(-/-) mice, demonstrating that hematopoietic cell-derived miR155 plays a critical role. CONCLUSIONS: miR155 deficiency attenuates atherogenesis in apoE(-/-) mice by reducing inflammatory responses of macrophages, enhancing macrophage cholesterol efflux and resulting in an antiatherogenic leukocyte profile. Targeting miR155 may be a promising strategy to halt atherogenesis.
Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/prevenção & controle , Inflamação/prevenção & controle , Macrófagos/metabolismo , MicroRNAs/metabolismo , Animais , Antígenos Ly/metabolismo , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/metabolismo , Biomarcadores/metabolismo , Transplante de Medula Óssea , Antígeno CD11b/metabolismo , Células Cultivadas , Colesterol/metabolismo , Citocinas/metabolismo , Dieta Aterogênica , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Fatores de Tempo , Receptor 4 Toll-Like/metabolismo , TransfecçãoRESUMO
BACKGROUND: Since a disproportionate amount of medical education still occurs in hospitals, there are concerns that medical school graduates are not fully prepared to deliver efficient and effective care in ambulatory settings to increasingly complex patients. AIMS: To understand the current extent of scholarship in this area. METHOD: A scoping review was conducted by searching electronic databases and grey literature sources for articles published between 2001 and 2011 that identified key challenges and models of practice for undergraduate teaching of ambulatory care. Relevant articles were charted and assigned key descriptors, which were mapped onto Canadian recommendations for the future of undergraduate medical education. RESULTS: Most of the relevant articles originated in the United States, Australia, or the United Kingdom. Recommendations related to faculty development, learning contexts and addressing community needs had numerous areas of scholarly activity while scholarly activity was lacking for recommendations related to inter-professional practice, the use of technology, preventive medicine, and medical leadership. CONCLUSIONS: Systems should be established to support education and research collaboration between medical schools to develop best practices and build capacity for change. This method of scoping the field can be applied using best practices and recommendations in other countries.
Assuntos
Assistência Ambulatorial , Educação de Graduação em Medicina , Canadá , Competência Clínica , Humanos , Modelos Organizacionais , Ensino/métodosRESUMO
Current theories of skill learning suggest that novices learn optimally in a simplified environment. This information can be incorporated in simulator designs. Our purpose was to assess whether basic visuospatial training is beneficial for performance on an arthroscopy model. One group of trainees practiced three visuomotor tasks while the other group was not given this opportunity. Both groups then performed three different surgical tasks on a simulated arthroscopy model. Practice with the visuomotor tasks enhanced performance on two of the tasks on the arthroscopy model. The basic navigational skills learned through practice transferred to the performance of arthroscopic surgery tasks and these skills should be included in the design of a comprehensive arthroscopy simulator.
Assuntos
Artroscopia/educação , Simulação por Computador , Laparoscopia/educação , Humanos , Estudantes de Medicina , Análise e Desempenho de TarefasRESUMO
PCSK9 (proprotein convertase subtilisin/kexin type 9) has emerged as a novel therapeutic target for hypercholesterolemia due to its LDL receptor (LDLR)-reducing activity. Although its structure has been solved, the lack of a detailed understanding of the structure-function relation hinders efforts to develop small molecule inhibitors. In this study, we used mutagenesis and transfection approaches to investigate the roles of the prodomain (PD) and the C-terminal domain (CD) and its modules (CM1-3) in the secretion and function of PCSK9. Deletion of PD residues 31-40, 41-50, or 51-60 did not affect the self-cleavage, secretion, or LDLR-degrading activity of PCSK9, whereas deletion of residues 61-70 abolished all of these functions. Deletion of the entire CD protein did not impair PCSK9 self-cleavage or secretion but completely abolished LDLR-degrading activity. Deletion of any one or two of the CD modules did not affect self-cleavage but influenced secretion and LDLR-reducing activity. Furthermore, in cotransfection experiments, a secretion-defective PD deletion mutant (ΔPD) was efficiently secreted in the presence of CD deletion mutants. This was due to the transfer of PD from the cotransfected CD mutants to the ΔPD mutant. Finally, we found that a discrete CD protein fragment competed with full-length PCSK9 for binding to LDLR in vitro and attenuated PCSK9-mediated hypercholesterolemia in mice. These results show a previously unrecognized domain interaction as a critical determinant in PCSK9 secretion and function. This knowledge should fuel efforts to develop novel approaches to PCSK9 inhibition.
Assuntos
Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Animais , Western Blotting , Linhagem Celular , Células Hep G2 , Humanos , Camundongos , Mutagênese , Mutação , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/genética , Estrutura Terciária de Proteína/genética , Serina Endopeptidases/genéticaRESUMO
Polybrominated diphenyl ethers (PBDEs) are flame retardants that have been widely used in manufacturing. They are major household and environmental contaminants that bioaccumulate. Humans are exposed primarily through dust inhalation and dietary ingestion of animal products. In animal studies, high doses of penta-brominated diphenyl ethers (penta-BDEs) in the mg/kg body weight (BW) range negatively impact brain development, behavior, memory, circulating thyroid hormone concentrations, the reproductive system and bone development. We investigated the effects of ingestion of a relatively low dose of the penta-BDE mixture DE-71 by pregnant and lactating rats on reproductive and thyroid parameters of the F1 offspring. F0 mothers received 60 µg/kg BW of DE-71 or vehicle daily by gavage from Day 1.5 of pregnancy through lactation (except the day of parturition). F1 pups were sacrificed at 21 d of age or outbred at approximately 80 d of age. Bred F1 females were sacrificed at Day 14.5 of pregnancy or at five months of age. Bred F1 males were sacrificed at five months of age. DE-71 treatment of the mothers affected the F1 females as evidenced by lower body weights at 80 d and five months of age, elevated serum T3 and T4 concentrations at Day 14.5 of pregnancy and increased thyroid gland weight and ovarian osteopontin mRNA at five months of age. Perinatal DE-71 exposure also increased testicular osteopontin mRNA in 21-day-old F1 males. Utilizing a granulosa cell in vitro model, we demonstrated that DE-71 activated the rat osteopontin gene promoter. Our results are the first to demonstrate that PBDEs increase rodent circulating T3 and T4 concentrations and gonadal osteopontin mRNA, and activate the osteopontin gene promoter. These changes may have clinical implications as others have shown associations between human exposure to PBDEs and subclinical hyperthyroidism, and overexpression of ovarian osteopontin has been associated with ovarian cancer.
Assuntos
Expressão Gênica/efeitos dos fármacos , Éteres Difenil Halogenados/farmacologia , Exposição Materna , Osteopontina/genética , Hormônios Tireóideos/sangue , Animais , Sequência de Bases , Primers do DNA , Relação Dose-Resposta a Droga , Feminino , Imuno-Histoquímica , Masculino , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Radioimunoensaio , RatosRESUMO
GATA4 and GATA6 are zinc-finger transcription factors that regulate specific genes involved in steroidogenesis. Using RNA interference (RNAi)-mediated reduction of GATA4 and/or GATA6 with microarray analysis, we aimed to identify novel GATA target genes in luteinizing porcine granulosa cells under vehicle- and cAMP-treated conditions. Microarray analysis identified IGF1 mRNA to be cAMP- and GATA-responsive, and real-time PCR demonstrated that the cAMP-induced increase in IGF1 mRNA was reduced under conditions of GATA6 depletion and GATA4 plus GATA6 depletion, but not GATA4 depletion. Insulin-like growth factor 1 protein levels in media were also decreased by GATA6 or GATA4 plus GATA6 reduction. IGFBP2 and IGFBP4 mRNAs were increased and IGFBP5 mRNA decreased with vehicle and cAMP treatment under GATA4 plus GATA6 RNAi conditions. GATA6 reduction alone increased basal IGFBP4 and decreased IGFBP5 with both vehicle and cAMP, and GATA4 reduction alone lowered cAMP IGFBP5 levels with cAMP. No changes in IGFBP3 mRNA were observed with GATA reduction relative to the control RNAi condition. Levels of insulin-like growth factor binding proteins 2-5 in media as assessed by Western ligand blotting were not altered by GATA reduction. Electromobility gel shift assays with two GATA-containing oligonucleotides of the IGF1 5'-regulatory region showed GATA4 and GATA6 could bind the more proximal GATA-B site. These studies indicate that although GATA4 and GATA6 can bind the porcine IGF1 5'-region, GATA6 is functionally most important for cAMP-stimulated mRNA levels. Using microarray analysis, we identified other mRNAs that were altered by GATA-reduced conditions, including ALDH1, DIO2, and EDNRB. Our findings further support GATA as a coordinator of endocrine/paracrine/autocrine signals in the ovary.