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1.
J Mol Cell Cardiol ; 140: 1-9, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32057736

RESUMO

Diabetes is an important risk factor for the development of cardiovascular disease including atherosclerosis and ischemic heart disease. Vascular complications including macro- and micro-vascular dysfunction are the leading causes of morbidity and mortality in diabetes. Disease mechanisms at present are unclear and no ideal therapies are available, which urgently calls for the identification of novel therapeutic targets/agents. An altered nucleotide- and nucleoside-mediated purinergic signaling has been implicated to cause diabetes-associated vascular dysfunction in major organs. Alteration of both purinergic P1 and P2 receptor sensitivity rather than the changes in receptor expression accounts for vascular dysfunction in diabetes. Activation of P2X7 receptors plays a crucial role in diabetes-induced retinal microvascular dysfunction. Recent findings have revealed that both ecto-nucleotidase CD39, a key enzyme hydrolyzing ATP, and CD73, an enzyme regulating adenosine turnover, are involved in the renal vascular injury in diabetes. Interestingly, erythrocyte dysfunction in diabetes by decreasing ATP release in response to physiological stimuli may serve as an important trigger to induce vascular dysfunction. Nucleot(s)ide-mediated purinergic activation also exerts long-term actions including inflammatory and atherogenic effects in hyperglycemic and diabetic conditions. This review highlights the current knowledge regarding the altered nucleot(s)ide-mediated purinergic signaling as an important disease mechanism for the diabetes-associated vascular complications. Better understanding the role of key receptor-mediated signaling in diabetes will provide more insights into their potential as targets for the treatment.

2.
Front Med ; 13(6): 639-645, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31468282

RESUMO

Esophageal cancer-related gene-4 (Ecrg4) is cloned from the normal epithelium of the esophagus. It is constitutively expressed in quiescent epithelial cells and downregulated during tumorigenesis, and Ecrg4 expression levels are inversely correlated with the malignant phenotype of tumor cells, validating that Ecrg4 is a real tumor suppressor gene. Unlike other tumor suppressor genes that usually encode membrane or intracellular proteins, Ecrg4 encodes a 148-amino acid pre-pro-peptide that is tethered on the cell surface in epithelial cells, specialized epithelial cells, and human leukocytes, where it can be processed tissue dependently into several small peptides upon cell activation. Ecrg4 is expressed in a wide variety of other cells/tissues, including cardiomyocytes and conduction system of the heart, the glomus cells of the carotid body, adrenal glands, choroid plexus, and leukocytes among others, where it exerts distinct functions, such as promoting/suppressing inflammation, inducing neuron senescence, stimulating the hypothalamus-pituitary-adrenal axis, maintaining the stemness of stem cells, participating in the rhythm and rate control of the heart, and possibly gauging the responsiveness of the cardiovascular system (CVS) to hypoxia, in addition to tumor suppression. Here, we briefly review the latest discoveries on Ecrg4 and its underlying molecular mechanisms as a tumor suppressor and focus on the emerging roles of Ecrg4 in the CVS.

3.
Gene ; 714: 143996, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31348980

RESUMO

The uniquely human α7-nAChR gene (CHRFAM7A) is evolved from the fusion of two partially duplicated genes, FAM7 and α7-nAChR gene (CHRNA7), and is inserted on same chromosome 15, 5' end of the CHRNA7 gene. Transcription of CHRFAM7A gene produces a 1256-bp open reading frame encoding dup-α7-nAChR, where a 27-aminoacid residues from FAM7 replaced the 146-aminoacid residues of the N-terminal extracellular ligand binding domain of α7-nAChR. In vitro, dup-α7-nAChR has been shown to form hetero-pentamer with α7-nAChR and dominant-negatively regulates the channel functions of α7-nAChR. However, the contribution of CHRFAM7A gene to the biology of α7-nAChR in the brain in vivo remains largely a matter of conjecture. CHRFAM7A transgenic mouse was created and differentially expressed proteins were profiled from the whole brain using iTRAQ-2D-LC-MS/MS proteomic technology. Proteins with a fold change of ≥1.2 or ≤0.83 and p < 0.05 were considered to be significant. Bioinformatics analysis showed that over-expression of the CHRFAM7A gene significantly modulated the proteins commonly involved in the signaling pathways of α7-nAChR-mediated neuropsychiatric disorders including Parkinson's disease, Alzheimer's disease, Huntington's disease, and alcoholism, suggesting that the CHRFAM7A gene contributes to the pathogenesis of neuropsychiatric disorders mostly likely through fine-tuning the functions of α7-nAChR in the brain.


Assuntos
Camundongos Transgênicos/genética , Receptor Nicotínico de Acetilcolina alfa7/genética , Animais , Encéfalo/metabolismo , Cromatografia Líquida/métodos , Cromossomos Humanos Par 15/genética , Perfilação da Expressão Gênica/métodos , Genes Duplicados/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteômica/métodos , Transdução de Sinais/genética , Espectrometria de Massas em Tandem/métodos
4.
J Cell Mol Med ; 23(9): 6085-6097, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31270949

RESUMO

The surged systemic vascular inflammation after acute myocardial infarction (AMI) aggravates the atherosclerotic endothelial injury. To explore roles of miR-499 released from cardiomyocytes during AMI in endothelial injury. Using qPCR and ELISA, we discovered that patients with AMI had significantly increased plasma miR-499, which was directly correlated with serum thrombomodulin, a marker for endothelial injury. Plasma of AMI patients, when incubated with human umbilical vein endothelial cells (HUVECs), significantly increased the expression of endothelial injury markers, which could be abrogated by antagomiR-499. In vitro, neonatal rat cardiomyocytes subjected to hypoxia/reoxygenation (HX/R) released miR-499 that could be internalized into rat pulmonary microvascular endothelial cells (RPMECs), worsening the high glucose-induced injury. In silico analysis demonstrated that CHRNA7 encoding α7-nAchR is a target of miR-499, which was validated in cell lines expressing endogenous α7-nAchR. In high glucose-induced RPMECs injury model, miR-499 aggravated, whereas forced CHRNA7 expression ameliorated the injury. Moreover, the perfusate from Langendorff perfused rat heart subjected to HX/R contained higher level of miR-499 that significantly impaired the Bradykinin-mediated endothelium-dependent relaxation in both conduit and resistance arteries, which could be partially abrogated by antagomiR-499. Finally, the correlation between plasma miR-499 and endothelial injury was further confirmed in another cohort of AMI patients. We conclude that miR-499 released from injured cardiomyocytes contributes to the endothelial injury by targeting α7-nAchR. This study implies that miR-499 may serve as a potential target for the treatment of the surged vascular inflammation post-AMI.

5.
Cell Signal ; 62: 109327, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31152845

RESUMO

Esophageal cancer related gene-4 (ECRG4) inhibits the malignant phenotype of oral squamous cell carcinoma. However, the molecular mechanisms remain to be explored. Using the tongue carcinoma cell line, TCA8113 as a cell model, we showed that forced expression of ECRG4 down-regulated the expression of the BC200 long non-coding RNA (lncRNA) and matrix metalloproteinases (MMP-9 and MMP-13). Restoration of BC200 lncRNA rescued ECRG4-mediated down-regulation of MMP-9 and -13. Furthermore, over-expression of Ecrg4 inhibited cell proliferation and migration, which was abolished by forced expression of BC200 lncRNA in TCA8113 cells. Our results indicate that ECRG4 inhibits the malignant phenotype of TCA8113 cells most likely through suppression of BC200 lncRNA/MMPs signaling pathway, rationalizing that BC200 lncRNA may be a potential target for oral squamous cell carcinoma (OSCC) therapy.

6.
Cell Mol Life Sci ; 76(24): 5027-5039, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31190084

RESUMO

The embedding of small peptide ligands within large inactive pre-pro-precursor proteins encoded by orphan open reading frames (ORFs) makes them difficult to identify and study. To address this problem, we generated oligonucleotide (< 100-400 base pair) combinatorial libraries from either the epidermal growth factor (EGF) ORF that encodes the > 1200 amino acid EGF precursor protein or the orphan ECRG4 ORF, that encodes a 148 amino acid Esophageal Cancer Related Gene 4 (ECRG4), a putative cytokine precursor protein of up to eight ligands. After phage display and 3-4 rounds of biopanning for phage internalization into prostate cancer epithelial cells, sequencing identified the 53-amino acid EGF ligand encoded by the 5' region of the EGF ORF and three distinct domains within the primary sequence of ECRG4: its membrane targeting hydrophobic signal peptide, an unanticipated amino terminus domain at ECRG437-63 and a C-terminus ECRG4133-148 domain. Using HEK-blue cells transfected with the innate immunity receptor complex, we show that both ECRG437-63 and ECRG4133-148 enter cells by interaction with the TLR4 immune complex but neither stimulate NFkB. Taken together, the results help establish that phage display can be used to identify cryptic domains within ORFs of the human secretome and identify a novel TLR4-targeted internalization domain in the amino terminus of ECRG4 that may contribute to its effects on cell migration, immune cell activation and tumor suppression.


Assuntos
Imunidade Inata/genética , Neoplasias da Próstata/genética , Receptor 4 Toll-Like/genética , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Técnicas de Visualização da Superfície Celular , Genes Supressores de Tumor , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Masculino , NF-kappa B/genética , Oligonucleotídeos/genética , Fases de Leitura Aberta/genética , Neoplasias da Próstata/patologia , Domínios Proteicos/genética , Transfecção
7.
Cancer Gene Ther ; 25(9-10): 248-259, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29983418

RESUMO

Esophageal cancer related gene-4 (Ecrg4) has been shown to be a tumor suppressor in many organs. Exosomes are naturally secreted nanosized particles that carry signal molecules including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and messenger RNAs (mRNAs) among others. Upon internalization, exosomes unload their cargos that in turn modulate the biology of the recipient cells. Mounting evidence has shown that exosomal miRNAs are functional. However, reports that exosomes carry functional mRNAs remain scarce. We found that serum exosomes contain ECRG4 open reading frame. To simulate serum exosomal ECRG4, stable cell line expressing ECRG4 was created, from which exosomes were isolated and characterized, and the internalization and the resulting biological effects of exosomal ECRG4 were evaluated. Results showed that serum exosomes contain higher levels of ECRG4 mRNA in healthy individuals than their cancer counterparts. Exosomal ECRG4 can be internalized and unload the encapsulated ECRG4 into recipient cells, which subsequently suppressed cell proliferation in vitro, and inhibited tumor growth in a xenograft mouse model. Mechanistically, ECRG4-containing exosomes, when internalized, suppressed the expression of genes commonly implicated in inflammation, cell proliferation, and angiogenesis. Given that exosome is an ideal vehicle for therapeutics delivery and that ECRG4 is a tumor suppressor gene, the exosomal ECRG4 can be exploited as a formulation for cancer gene therapy.


Assuntos
Proliferação de Células , Exossomos/metabolismo , Genes Supressores de Tumor , Proteínas de Neoplasias/sangue , Neoplasias , Neovascularização Patológica/sangue , RNA Mensageiro/sangue , RNA Neoplásico/sangue , Células A549 , Animais , Exossomos/patologia , Feminino , Células HEK293 , Humanos , Inflamação/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/sangue , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/patologia , Proteínas Supressoras de Tumor
8.
Gene ; 642: 26-31, 2018 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-29126922

RESUMO

Epidemiogical evidence has shown that the incidence of atrial fibrillation in tumor patients is higher than non-tumor patients and general population. The potential risk factors predisposing tumor patients to atrial fibrillation include advanced age, comorbidities, direct anatomic local occupying effect of tumors in the heart or adjacent organs, paraneoplastic manifestations of some tumors, tumor-induced dys-regulation of metabolism, radio-, bio- and chemo-therapeutics, disturbance of autonomous nerve system because of physical pain and psychological sufferings, chronic inflammation typical of most tumors, and surgical interventions among others. However, whether tumor suppressor genes commonly mutated or dys-regulated in tumor play any roles in the pathogenesis of atrial fibrillation remain largely unexplored. Tumor suppressor genes or genes possessing tumor suppressing function have been reported to be constitutively expressed in quiescent heart, and mutations, small nucleotide polymorphisms, or disturbed expression of tumor suppressor genes has been implicated in the pathogenesis of atrial fibrillation. Here, we provide a state-of-the-art overview of the unrecognized roles of tumor suppressor genes in the pathogenesis of atrial fibrillation, focusing mainly on the two well-characterized tumor suppressor genes, zinc finger homeobox protein-3 and esophageal cancer related gene-4.


Assuntos
Fibrilação Atrial/genética , Proteínas de Homeodomínio/genética , Proteínas de Neoplasias/genética , Neoplasias/complicações , Fibrilação Atrial/epidemiologia , Fibrilação Atrial/etiologia , Feminino , Regulação da Expressão Gênica , Predisposição Genética para Doença , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Mutação , Miocárdio/metabolismo , Miocárdio/patologia , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Polimorfismo Genético , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
9.
Gene ; 636: 103-111, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-28870864

RESUMO

The human cytokine precursor ECRG4 has been associated with multiple physiological, developmental and pathophysiological processes involving cell proliferation, cell migration, innate immunity, inflammation, cancer progression and metastases. Although down-regulation of ECRG4 gene expression has been largely attributed to hypermethylation of CpG islands in the 5'untranslated region of the ECRG4 promoter, the mechanisms that underlie the dynamics of its regulation have never been systematically described. Here we show that the ECRG4 gene is widely expressed in human tissues and report that its core promoter lies between the -780 to +420 base pairs relative to the ATG start codon of the ECRG4 open reading frame. This sequence, which contains several CpG islands, also includes multiple overlapping Sp1 consensus binding sequences and a putative binding site for NF-kB activation. 5'RACE of mRNA derived from human leukocytes shows that ECRG4 transcription initiates from the guanidine at -11 from the initiation ATG of the ECRG4 open reading frame. While there is no canonical TATA- or CAAT-boxes proximal to this translational initiation site, there is a distal TATA-sequence in the 5'UTR. This region was identified as the sequence targeted by hypermethylation because in vitro methylation of plasmids encoding the ECRG4 promoter abolish promoter activity and the treatment of Jurkat cells (which naturally express ECRG4) with the methylation inhibitor 5-AzaC, increases endogenous ECRG4 expression. Because ChIP assays show that Sp1 binds the ECRG4 promoter, that forced Sp1 expression trans-activates the ECRG4 promoter and Sp1 inhibition with mithramycin inhibits ECRG4 expression, we conclude that the dynamic positive and negative regulatory elements controlling ECRG4 expression include a counter regulation between promoter methylation and Sp1 activation.


Assuntos
Metilação de DNA , Regulação da Expressão Gênica , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Regiões 5' não Traduzidas , Células Cultivadas , Clonagem Molecular , Regulação para Baixo , Humanos , Células Jurkat , Leucócitos/metabolismo , Proteínas de Neoplasias/metabolismo , Distribuição Tecidual , Sítio de Iniciação de Transcrição , Ativação Transcricional , Proteínas Supressoras de Tumor
10.
Sci Rep ; 7(1): 2717, 2017 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-28578429

RESUMO

Epidemiological studies have shown a strong correlation between tumor and AF. However, the molecular link between tumor and AF remains unknown. ECRG4, a tumor suppressor gene that is expressed in the A-V node and in sporadic ventricular myocytes, inhibits tumorigenesis and monitors tissue homeostasis by functioning as a 'sentinel' molecule gauging inflammatory and cell proliferative responses. To explore the potential physiological function of Ecrg4 in heart, we evaluated its distribution in heart, analyzed its expression in patients with persistent AF and in a canine AF model, and dissected the molecular events downstream of Ecrg4. The results showed that the level of Ecrg4 expression is homogenously high in atria and the conduction systems and in sporadic ventricular myocytes. Importantly, the expression of Ecrg4 was significantly decreased in atrial appendages of AF patients than patients with SR. Moreover, in rapid pacing canine AF models, the expression of ECRG4 in atria was significantly decreased compared to that of the controls. Mechanistically, knockdown ECRG4 in atrial myocytes significantly shortened the APDs, inhibited the expression of Gja1, and activated pro-inflammatory cascades and genes involved in cardiac remodeling. These results suggest that Ecrg4 may play a critical role in the pathogenesis of AF.


Assuntos
Fibrilação Atrial/genética , Predisposição Genética para Doença , Proteínas de Neoplasias/genética , Potenciais de Ação , Adulto , Animais , Apêndice Atrial/metabolismo , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Remodelamento Atrial , Cães , Eletrocardiografia , Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Átrios do Coração/metabolismo , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Proteínas de Neoplasias/metabolismo , Ratos , Proteínas Supressoras de Tumor
11.
Wound Repair Regen ; 24(6): 1004-1014, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27663454

RESUMO

Mice engrafted with human CD34+ hematopoietic stem and progenitor cells (CD34+ -HSPCs) have been used to study human infection, diabetes, sepsis, and burn, suggesting that they could be highly amenable to characterizing the human inflammatory response to injury. To this end, human leukocytes infiltrating subcutaneous implants of polyvinyl alcohol (PVA) sponges were analyzed in immunodeficient NSG mice reconstituted with CD34+ -HSPCs. It was reported that human CD45+ (hCD45+ ) leukocytes were present in PVA sponges 3 and 7 days postimplantation and could be localized within the sponges by immunohistochemistry. The different CD45+ subtypes were characterized by flow cytometry and the profile of human cytokines they secreted into PVA wound fluid was assessed using a human-specific multiplex bead analyses of human IL-12p70, TNFα, IL-10, IL-6, IL1ß, and IL-8. This enabled tracking the functional contributions of HLA-DR+ , CD33+ , CD19+ , CD62L+ , CD11b+ , or CX3CR1+ hCD45+ infiltrating inflammatory leukocytes. PCR of cDNA prepared from these cells enabled the assessment and differentiation of human, mouse, and uniquely human genes. These findings support the hypothesis that mice engrafted with CD34+ -HSPCs can be deployed as precision avatars to study the human inflammatory response to injury.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Inflamação/imunologia , Inflamação/patologia , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/patologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Células Supressoras Mieloides/metabolismo , Transdução de Sinais , Cicatrização/imunologia , Ferimentos e Lesões/imunologia , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologia
12.
BBA Clin ; 5: 66-71, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27051591

RESUMO

BACKGROUND: The α7-subunit of the α7-nicotinic acetylcholine receptor (α7-nAChR) is an obligatory intermediate for the anti-inflammatory effects of the vagus nerve. But in humans, there exists a second gene called CHRFAM7A that encodes a dominant negative α7-nAChR inhibitor. Here, we investigated whether their expression was altered in inflammatory bowel disease (IBD) and colon cancer. METHODS: Quantitative RT-PCR measured gene expression of human α7-nAChR gene (CHRNA7), CHRFAM7A, TBC3D1, and actin in biopsies of normal large and small intestine, and compared to their expression in biopsies of ulcerative colitis, Crohn's disease, and colon cancer. RESULTS: qRT-PCR showed that CHRFAM7A and CHRNA7 gene expression was significantly (p < .02) up-regulated in IBD (N = 64). Gene expression was unchanged in colon cancer. Further analyses revealed that there were differences in ulcerative colitis and Crohn's Disease. Colon biopsies of ulcerative colitis (N = 33) confirmed increased expression of CHRFAM7A and decreased in CHRNA7 expression (p < 0.001). Biopsies of Crohn's disease (N = 31), however, showed only small changes in CHRFAM7A expression (p < 0.04) and no change in CHRNA7. When segregated by tissue source, both CHRFAM7A up-regulation (p < 0.02) and CHRNA7 down-regulation (p < 0.001) were measured in colon, but not in small intestine. CONCLUSION: The human-specific CHRFAM7A gene is up-regulated, and its target, CHRNA7, down-regulated, in IBD. Differences between ulcerative colitis and Crohn's disease tie to location of disease. SIGNIFICANCE: The appearance of IBD in modern humans may be consequent to the emergence of CHRFAM7A, a human-specific α7-nAChR antagonist. CHRFAM7A could present a new, unrecognized target for development of IBD therapeutics.

13.
J Pharmacol Sci ; 132(4): 235-243, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27107824

RESUMO

Acehytisine, a multi-ion channel blocker, can markedly inhibit INa, ICa, IKur, If at various concentrations and effectively terminate and prevent atrial fibrillation (AF) in patients and animal models, but the molecular mechanism underlying its blockage remains elusive. In this study, we investigated the effects of acehytisine on action potentials and sodium channels of atrial and ventricular myocytes isolated from rabbit, using whole-cell recording system. We found that acehytisine exerted stronger blocking effects on sodium channels in atria than in ventricles, especially at depolarization (IC50: 48.48 ± 7.75 µmol/L in atria vs. 560.17 ± 63.98 µmol/L in ventricles). It also significantly shifted steady state inactivation curves toward negative potentials in atrial myocytes, without affecting the recovery kinetics from inactivation of sodium channels in the same cells. In addition, acehytisine inhibited INa in a use-dependent manner and regulated slow inactivation kinetics by different gating configurations. These findings indicate that acehytisine selectively blocks atrial sodium channels and possesses affinity to sodium channel in certain states, which provides additional evidence for the anti-AF of acehytisine.


Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Fibrilação Atrial/tratamento farmacológico , Fibrilação Atrial/metabolismo , Átrios do Coração/citologia , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Técnicas de Patch-Clamp , Coelhos
14.
Ann Surg ; 263(1): 199-204, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25575256

RESUMO

OBJECTIVE: To develop an animal model of injury that more closely represents the human inflammatory cell response to injury. BACKGROUND: Because the mouse inflammatory response to burn injury cannot account for the contribution of human-specific genes, animal models are needed to more closely recapitulate the human inflammatory response and improve the translational impact of injury research. To this end, we hypothesized that the human inflammatory cell response to injury could be selectively assessed after severe burn injury using humanized mice. METHODS: NOD-Scid-IL2Rγ null mice were transplanted with human hematopoietic CD34+ progenitor cells; their engraftment confirmed and then subjected to 30% total body surface area steam burn injury. Blood, bone marrow, and lung tissue were collected 4 hours after injury and human inflammatory cell mobilization analyzed using flow cytometry and immunohistochemistry. RESULTS: Burn injury caused mobilization of human inflammatory cells into the systemic circulation. Next, burn injury was accompanied by evidence of histologic lung injury and concomitant mobilization of human CD45+ immune cells into the lung that were associated with increased trafficking of human CD11b+ myeloid cells. CONCLUSIONS: These experiments are the first to demonstrate the suitability of humanized mice for injury research. They offer the possibility to address very specific research questions that are not amenable to traditional mouse models of injury, for example, the emerging role of certain human-specific genes that are either unrepresented or totally absent, from the mouse genome.


Assuntos
Queimaduras/imunologia , Modelos Animais de Doenças , Transplante de Células-Tronco Hematopoéticas , Animais , Humanos , Camundongos
15.
Sci Rep ; 5: 12410, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26174737

RESUMO

Copper, a strictly regulated trace element, is essential for many physiological processes including angiogenesis. Dysregulated angiogenesis has been associated with increased copper in tumors, and thus copper chelators have been used to inhibit tumor angiogenesis. However, it remains unclear whether copper has any effect on epithelial-mesenchymal transition (EMT). Using CoCl2-induced EMT of human breast carcinoma MCF-7 cells, we found that TEPA, a copper chelator, inhibited EMT-like cell morphology and cytoskeleton arrangement triggered by CoCl2; decreased the expression of vimentin and fibronectin, markers typical of EMT; inhibited HIF-1 activation and HIF1-α accumulation in nuclear; and down-regulated the expression of hypoxia-associated transcription factors, Snail and Twist1. Moreover, knockdown copper transport protein, Ctr1, also inhibited CoCl2-induced EMT and reversed the mesenchymal phenotype. In EMT6 xenograft mouse models, TEPA administration inhibited the tumor growth and increased mice survival. Immunohistochemical analysis of the xenograft further demonstrated that TEPA administration significantly inhibited tumor angiogenesis, down-regulated hypoxia-induced transcription factors, Snail and Twist1, leading to decreased transactivation of EMT-associated marker genes, vimentin and fibronectin. These results indicate that TEPA inhibits CoCl2-induced EMT most likely via HIF1-α-Snail/Twist signaling pathway, and copper depletion may be exploited as a therapeutic for breast cancer.


Assuntos
Cobre/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Animais , Antineoplásicos Alquilantes/uso terapêutico , Antineoplásicos Alquilantes/toxicidade , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Polaridade Celular , Cobalto/química , Cobalto/farmacologia , Regulação para Baixo/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Imuno-Histoquímica , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Nucleares/antagonistas & inibidores , Fenótipo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição da Família Snail , Fatores de Transcrição/antagonistas & inibidores , Ativação Transcricional , Transplante Heterólogo , Trietilenofosforamida/uso terapêutico , Trietilenofosforamida/toxicidade , Proteína 1 Relacionada a Twist/antagonistas & inibidores , Vimentina/genética , Vimentina/metabolismo
16.
Mol Med ; 21: 323-36, 2015 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-25860877

RESUMO

The human genome contains a variant form of the α7-nicotinic acetylcholine receptor (α7nAChR) gene that is uniquely human. This CHRFAM7A gene arose during human speciation and recent data suggests that its expression alters ligand tropism of the normally homopentameric human α7-AChR ligand-gated cell surface ion channel that is found on the surface of many different cell types. To understand its possible significance in regulating inflammation in humans, we investigated its expression in normal human leukocytes and leukocyte cell lines, compared CHRFAM7A expression to that of the CHRNA7 gene, mapped its promoter and characterized the effects of stable CHRFAM7A overexpression. We report here that CHRFAM7A is highly expressed in human leukocytes but that the levels of both CHRFAM7A and CHRNA7 mRNAs were independent and varied widely. To this end, mapping of the CHRFAM7A promoter in its 5'-untranslated region (UTR) identified a unique 1-kb sequence that independently regulates CHRFAM7A gene expression. Because overexpression of CHRFAM7A in THP1 cells altered the cell phenotype and modified the expression of genes associated with focal adhesion (for example, FAK, P13K, Akt, rho, GEF, Elk1, CycD), leukocyte transepithelial migration (Nox, ITG, MMPs, PKC) and cancer (kit, kitL, ras, cFos cyclinD1, Frizzled and GPCR), we conclude that CHRFAM7A is biologically active. Most surprisingly however, stable CHRFAM7A overexpression in THP1 cells upregulated CHRNA7, which, in turn, led to increased binding of the specific α7nAChR ligand, bungarotoxin, on the THP1 cell surface. Taken together, these data confirm the close association between CHRFAM7A and CHRNA7 expression, establish a biological consequence to CHRFAM7A expression in human leukocytes and support the possibility that this human-specific gene might contribute to, and/or gauge, a human-specific response to inflammation.


Assuntos
Leucócitos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Processamento Alternativo , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Regiões Promotoras Genéticas , Isoformas de Proteínas , RNA Mensageiro/genética , Transcrição Genética
17.
FASEB J ; 29(6): 2292-302, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25681457

RESUMO

The human genome contains a unique, distinct, and human-specific α7-nicotinic acetylcholine receptor (α7nAChR) gene [CHRNA7 (gene-encoding α7-nicotinic acetylcholine receptor)] called CHRFAM7A (gene-encoding dup-α7-nicotinic acetylcholine receptor) on a locus of chromosome 15 associated with mental illness, including schizophrenia. Located 5' upstream from the "wild-type" CHRNA7 gene that is found in other vertebrates, we demonstrate CHRFAM7A expression in a broad range of epithelial cells and sequenced the CHRFAM7A transcript found in normal human fetal small intestine epithelial (FHs) cells to prove its identity. We then compared its expression to CHRNA7 in 11 gut epithelial cell lines, showed that there is a differential response to LPS when compared to CHRNA7, and characterized the CHRFAM7A promoter. We report that both CHRFAM7A and CHRNA7 gene expression are widely distributed in human epithelial cell lines but that the levels of CHRFAM7A gene expression vary up to 5000-fold between different gut epithelial cells. A 3-hour treatment of epithelial cells with 100 ng/ml LPS increased CHRFAM7A gene expression by almost 1000-fold but had little effect on CHRNA7 gene expression. Mapping the regulatory elements responsible for CHRFAM7A gene expression identifies a 1 kb sequence in the UTR of the CHRFAM7A gene that is modulated by LPS. Taken together, these data establish the presence, identity, and differential regulation of the human-specific CHRFAM7A gene in human gut epithelial cells. In light of the fact that CHRFAM7A expression is reported to modulate ligand binding to, and alter the activity of, the wild-type α7nAChR ligand-gated pentameric ion channel, the findings point to the existence of a species-specific α7nAChR response that might regulate gut epithelial function in a human-specific fashion.


Assuntos
Células Epiteliais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/genética , Regiões 5' não Traduzidas/genética , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células HCT116 , Células HT29 , Humanos , Immunoblotting , Mucosa Intestinal/metabolismo , Intestinos/citologia , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor Nicotínico de Acetilcolina alfa7/metabolismo
18.
J Leukoc Biol ; 97(2): 247-57, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25473097

RESUMO

Conventional wisdom presumes that the α7nAChR product of CHRNA7 expression mediates the ability of the vagus nerve to regulate the inflammatory response to injury and infection. Yet, 15 years ago, a 2nd structurally distinct and human-specific α7nAChR gene was discovered that has largely escaped attention of the inflammation research community. The gene, originally called dupα7nAChR but now known as CHRFAM7A, has been studied exhaustively in psychiatric research because of its association with mental illness. However, dupα7nAChR/CHRFAM7A expression is relatively low in human brain but elevated in human leukocytes. Furthermore, α7nAChR research in human tissues has been confounded by cross-reacting antibodies and nonspecific oligonucleotide primers that crossreact in immunoblotting, immunohistochemistry, and RT-PCR. Yet, 3 independent reports show the human-specific CHRFAM7A changes cell responsiveness to the canonical α7nAChR/CHRNA7 ion-gated channel. Because of its potential for the injury research community, its possible significance to human leukocyte biology, and its relevance to human inflammation, we review the discovery and structure of the dupα7nAChR/CHRFAM7A gene, the distribution of its mRNA, and its biologic activities and then discuss its possible role(s) in specifying human inflammation and injury. In light of emerging concepts that point to a role for human-specific genes in complex human disease, the existence of a human-specific α7nAChR regulating inflammatory responses in injury underscores the need for caution in extrapolating findings in the α7nAChR literature to man. To this end, we discuss the translational implications of a uniquely human α7nAChR-like gene on new drug target discovery and therapeutics development for injury, infection, and inflammation.


Assuntos
Duplicação Gênica/imunologia , Ativação do Canal Iônico/imunologia , Leucócitos/imunologia , Receptor Nicotínico de Acetilcolina alfa7/imunologia , Desenho de Drogas , Regulação da Expressão Gênica/imunologia , Humanos , /genética , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Leucócitos/patologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Receptor Nicotínico de Acetilcolina alfa7/genética
19.
Exp Lung Res ; 41(3): 162-72, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25513848

RESUMO

PURPOSE: The human c2orf40 gene encodes a candidate tumor suppressor called Esophageal Cancer-Related Gene-4 (ECRG4) that is a cytokine-like epigenetically-regulated protein that is characteristically downregulated in cancer, injury, inflammation, and infection. Here, we asked whether ECRG4 gene expression is detectable in lung epithelial cells and if its expression changes with inflammation, infection, and/or protective preconditioning. MATERIALS AND METHODS: We used immunoblotting, PCR, and quantitative PCR to measure ECRG4 and either inhalation anesthesia preconditioning, lipopolysaccharide injection, or laparotomy to modulate lung inflammation. RESULTS: Immunoblotting establishes the presence of the full-length 14 kDa ECRG4 peptide in mouse lung. Immunohistochemistry localizes ECRG4 to type l alveolar epithelial cells. Basal ECRG4 mRNA is greater than TNF-α, IL-1ß, and IL-6 but following inflammatory lung injury, TNF-α, IL-1ß, IL-6, and IL-10 are upregulated while ECRG4 gene expression is decreased. Similar findings are observed after an intravenous administration of lipopolysaccharide. In contrast, lung preconditioning with isoflurane anesthesia increases lung ECRG4 gene expression. Over-expression of ECRG4 in human lung epithelial cells in vitro decreases cell proliferation implying that a loss of ECRG4 in vivo would be permissive to cell growth. CONCLUSIONS: This study supports the hypothesis that ECRG4 acts as a sentinel growth inhibitor in lung alveolar epithelial cells. Its downregulation by injury, infection, and inflammation and upregulation by preconditioning supports a role for ECRG4 in regulating the alveolar epithelium response to injury and inflammation. By extension, the findings support a functional consequence to its inhibition by promoter hypermethylation (i.e. lung cancer) and suggest potential benefits to its upregulation.


Assuntos
Lesão Pulmonar/genética , Proteínas de Neoplasias/genética , Pneumonia/genética , Animais , Proliferação de Células/genética , Regulação para Baixo/genética , Células Epiteliais/metabolismo , Feminino , Genes Supressores de Tumor , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Pulmão/metabolismo , Lesão Pulmonar/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Pneumonia/metabolismo , Regiões Promotoras Genéticas/genética , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/genética
20.
Inflamm Res ; 64(2): 107-18, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25511108

RESUMO

OBJECTIVE AND DESIGN: The human c2orf40 gene encodes a tumor suppressor gene called esophageal cancer-related gene-4 (ECRG4) with pro- and anti-inflammatory activities that depend on cell surface processing. Here, we investigated its physical and functional association with the innate immunity receptor complex. METHODS: Interactions between ECRG4 and the innate immunity receptor complex were assessed by flow cytometry, immunohistochemistry, confocal microscopy, and co-immunoprecipitation. Phage display was used for ligand targeting to cells that overexpress the TLR4-MD2-CD14. RESULTS: Immunoprecipitation and immunohistochemical studies demonstrate a physical interaction between ECRG4 and TLR4-MD2-CD14 on human granulocytes. Flow cytometry shows ECRG4 on the cell surface of a subset of CD14(+) and CD16(+) leukocytes. In a cohort of trauma patients, the C-terminal 16 amino acid domain of ECRG4 (ECRG4(133-148)) appears to be processed and shed, presumably at a thrombin-like consensus sequence. Phage targeting this putative ligand shows that this peptide sequence internalizes into cells through the TLR4/CD14/MD2 complex, but modulates inflammation through non-canonical, NFκB signal transduction. CONCLUSIONS: ECRG4 is present on the surface of human monocytes and granulocytes. Its interaction with the human innate immunity receptor complex supports a role for cell surface activation of ECRG4 during inflammation and implicates this receptor in its mechanism of action.


Assuntos
Granulócitos/imunologia , Monócitos/imunologia , Proteínas de Neoplasias/imunologia , Adulto , Feminino , Células HEK293 , Humanos , Imunidade Inata , Receptores de Lipopolissacarídeos/imunologia , Antígeno 96 de Linfócito/imunologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Receptor 4 Toll-Like/imunologia , Proteínas Supressoras de Tumor , Adulto Jovem
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