RESUMO
Purpose: Erianin has been reported to inhibit tumor activity by suppressing the expression of integrins. It is hypothesized that erianin can inhibit retinal neovascularization in collagen by suppressing the expression of integrins. With an aim to test this hypothesis, the regulation of erianin on collagen-mediated retinal angiogenesis via the Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing protein kinase 1 (ROCK1) signaling pathway induced by α2 and ß1 integrin-collagen interactions was investigated. Methods: The effects of erianin on human retinal vascular endothelial cells (HRVECs) were assessed in vitro using a hypoxia model in a three-dimensional cell culture induced by cobalt (II) chloride (CoCl2). A hypoxia-induced retinopathy model in adult zebrafish and zebrafish embryos was established to assess the antiangiogenic effect of erianin with and without vitreous collagen in vivo. The expression of α2 and ß1 integrin and RhoA/ROCK1 pathway in HRVECs and zebrafish retinas were analyzed. Results: In vitro, collagen improved the angiogenic potential of HRVECs, including migration, adhesion, and tube formation, in a three-dimensional cell culture model. Erianin suppressed the angiogenic processes of the CoCl2-induced hypoxia HRVEC model in a concentration-dependent manner. In vivo, erianin reduced retinal angiogenesis in the hypoxia-induced retinopathy model in adult and embryo zebrafish. Erianin inhibited the expression of α2 and ß1 integrin and RhoA/ROCK1 in a hypoxia-induced model in vitro in three-dimensional cell culture and in vivo in adult zebrafish. Conclusions: Collagen-mediated retinal angiogenesis may be regulated by erianin via the RhoA/ROCK1 signaling pathway induced by α2 and ß1 integrin-collagen interactions. These findings suggest that erianin has the therapeutic potential on intraocular collagen-mediated retinal angiogenesis.
Assuntos
Bibenzilas/farmacologia , Regulação da Expressão Gênica , Integrina alfa1/genética , Integrina beta1/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Fenol/farmacologia , Neovascularização Retiniana/genética , Proteínas de Peixe-Zebra/genética , Quinases Associadas a rho/genética , Animais , Movimento Celular , Células Cultivadas , Colágeno/metabolismo , Modelos Animais de Doenças , Integrina alfa1/biossíntese , Integrina beta1/biossíntese , Proteínas Monoméricas de Ligação ao GTP/biossíntese , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/biossíntese , Quinases Associadas a rho/biossínteseRESUMO
Background: Cell therapy of diabetes aims at restoring the physiological control of blood glucose by transplantation of functional pancreatic islet cells. A potentially unlimited source of cells for such transplantations would be islet cells derived from an in vitro differentiation of human pluripotent stem cells (hESC/hiPSC). The islet-like clusters (ILC) produced by the known differentiation protocols contain various cell populations. Among these, the ß-cells that express both insulin and the transcription factor Nkx6.1 seem to be the most efficient to restore normoglycemia in diabetes animal models. Our aim was to find markers allowing selection of these efficient cells. Methods: Functional Cell-Capture Screening (FCCS) was used to identify markers that preferentially capture the cells expressing both insulin and Nkx6.1, from hESC-derived ILC cells. In order to test whether selection for such markers could improve cell therapy in diabetic mouse models, we used ILC produced from a clinical-grade line of hESC by a refined differentiation protocol adapted to up-scalable bioreactors. Re-aggregated MACS sorted cells were encapsulated in microspheres made of alginate modified to reduce foreign body reaction. Implantation was done intraperitoneally in STZ-treated C57BL/6 immuno-competent mice. Results: CD49A (integrin alpha1) was identified by FCCS as a marker for cells that express insulin (or C-peptide) as well as Nkx6.1 in ILC derived by hESC differentiation. The ILC fraction enriched in CD49A + cells rapidly reduced glycemia when implanted in diabetic mice, whereas mice receiving the CD49A depleted population remained highly diabetic. CD49A-enriched ILC cells also produced higher levels of human C-peptide in the blood of transplanted mice. However, the difference between CD49A-enriched and total ILC cells remained small. Another marker, CD26 (DPP4), was identified by FCCS as binding insulin-expressing cells which are Nkx6.1 negative. Depletion of CD26 + cells followed by enrichment for CD49A + cells increased insulin+/Nkx6.1+ cells fraction to ~70%. The CD26 - /CD49A + enriched ILC exhibited improved function over non-sorted ILC or CD49A + cells in diabetic mice and maintain prolonged blood C-peptide levels. Conclusions: Refining the composition of ILC differentiated from hPSC by negative selection to remove cells expressing CD26 and positive selection for CD49A expressing cells could enable more effective cell therapy of diabetes.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Dipeptidil Peptidase 4/biossíntese , Integrina alfa1/biossíntese , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Peptídeo C/biossíntese , Diferenciação Celular , Separação Celular , Proteínas de Homeodomínio/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Endogâmicos C57BL , MicroesferasRESUMO
Tissue-resident memory T (Trm) cells form a heterogeneous population that provides localized protection against pathogens. Here, we identify CD49a as a marker that differentiates CD8+ Trm cells on a compartmental and functional basis. In human skin epithelia, CD8+CD49a+ Trm cells produced interferon-γ, whereas CD8+CD49a- Trm cells produced interleukin-17 (IL-17). In addition, CD8+CD49a+ Trm cells from healthy skin rapidly induced the expression of the effector molecules perforin and granzyme B when stimulated with IL-15, thereby promoting a strong cytotoxic response. In skin from patients with vitiligo, where melanocytes are eradicated locally, CD8+CD49a+ Trm cells that constitutively expressed perforin and granzyme B accumulated both in the epidermis and dermis. Conversely, CD8+CD49a- Trm cells from psoriasis lesions predominantly generated IL-17 responses that promote local inflammation in this skin disease. Overall, CD49a expression delineates CD8+ Trm cell specialization in human epithelial barriers and correlates with the effector cell balance found in distinct inflammatory skin diseases.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica/imunologia , Integrina alfa1/imunologia , Pele/imunologia , Subpopulações de Linfócitos T/imunologia , Separação Celular , Citometria de Fluxo , Humanos , Memória Imunológica/imunologia , Integrina alfa1/biossíntese , Ativação Linfocitária/imunologia , Microscopia Confocal , Psoríase/imunologia , Vitiligo/imunologiaRESUMO
Neuromedin U (NMU) was originally named based on its strong uterine contractile activity, but little is known regarding its signaling/functions in utero. We identified that NMU and one of its receptors, NMUR2, are not only present in normal uterine endometrium but also co-expressed in endometrial cancer tissues, where the NMU level is correlated with the malignant grades and survival of patients. Cell-based assays further confirmed that NMU signaling can promote cell motility and proliferation of endometrial cancer cells derived from grade II tumors. Activation of NMU pathway in these endometrial cancer cells is required in order to sustain expression of various adhesion molecules, such as CD44 and integrin alpha1, as well as production of their corresponding extracellular matrix ligands, hyaluronan and collagen IV; it also increased the activity of SRC and its downstream proteins RHOA and RAC1. Thus, it is concluded that NMU pathway positively controls the adhesion signaling-SRC-Rho GTPase axis in the tested endometrial cancer cells and that changes in cell motility and proliferation can occur when there is manipulation of NMU signaling in these cells either in vitro or in vivo. Intriguingly, this novel mechanism also explains how NMU signaling promotes the EGFR-driven and TGFß receptor-driven mesenchymal transitions. Through the above axis, NMU signaling not only can promote malignancy of the tested endometrial cancer cells directly, but also helps these cells to become more sensitive to niche growth factors in their microenvironment.
Assuntos
Transformação Celular Neoplásica/patologia , Neoplasias do Endométrio/patologia , Endométrio/patologia , Neuropeptídeos/metabolismo , Transdução de Sinais/fisiologia , Animais , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Colágeno/biossíntese , Progressão da Doença , Endométrio/metabolismo , Feminino , Humanos , Receptores de Hialuronatos/biossíntese , Ácido Hialurônico/biossíntese , Integrina alfa1/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos Nus , Pessoa de Meia-Idade , Receptores de Neurotransmissores/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismoRESUMO
A cell line was established from ventral prostate (VP) tumors of one-year-old Hi-Myc mice. These cells, called HMVP2 cells, are LinnegSca-1highCD49fhigh with high CD44 and CD29 expression and express CK14, Sca-1 and CD49f (but not CK8), suggesting basal-epithelial characteristics. Furthermore, HMVP2 cells form spheroids and both the cells and spheroids produce tumors in syngeneic mice. After four days of culture, HMVP2 spheroids underwent a gradual transition from LinnegSca-1highCD49fhigh expression to LinnegSca-1lowCD49flow while a subpopulation of the cells retained the original LinnegSca-1highCD49fhigh expression pattern. Additional cell subpopulations expressing Lin positive markers were also present suggesting further differentiation of HMVP2 spheroids. Two additional highly tumorigenic cell lines (HMVP2A1 and HMVP2A2) were isolated from HMVP2 cells after subsequent tumor formation in FVB/N mice. Concurrently, we also established cell lines from the VP of 6 months old Hi-Myc mice (named as HMVP1) and FVB/N mice (called NMVP) having less aggressive growth properties compared to the other three cell lines. AR expression was reduced in HMVP2 cells compared to NMVP and HMVP1 cells and almost absent in HMVP2A1 and HMVP2A2 cells. These cell lines will provide valuable tools for further mechanistic studies as well as preclinical studies to evaluate preventive and/or therapeutic agents for prostate cancer.
Assuntos
Linhagem Celular Tumoral , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/patologia , Animais , Antígenos Ly/biossíntese , Biomarcadores Tumorais/metabolismo , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Integrina alfa1/biossíntese , Masculino , Proteínas de Membrana/biossíntese , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND: There is increasing evidence to suggest that pericytes play a crucial role in regulating the remodeling state of blood vessels. As cerebral pericytes are embedded within the extracellular matrix (ECM) of the vascular basal lamina, it is important to understand how individual ECM components influence pericyte remodeling behavior, and how cytokines regulate these events. METHODS: The influence of different vascular ECM substrates on cerebral pericyte behavior was examined in assays of cell adhesion, migration, and proliferation. Pericyte expression of integrin receptors was examined by flow cytometry. The influence of cytokines on pericyte functions and integrin expression was also examined, and the role of specific integrins in mediating these effects was defined by function-blocking antibodies. Expression of pericyte integrins within remodeling cerebral blood vessels was analyzed using dual immunofluorescence (IF) of brain sections derived from the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). RESULTS: Fibronectin and collagen I promoted pericyte proliferation and migration, but heparan sulfate proteoglycan (HSPG) had an inhibitory influence on pericyte behavior. Flow cytometry showed that cerebral pericytes express high levels of α5 integrin, and lower levels of α1, α2, and α6 integrins. The pro-inflammatory cytokine tumor necrosis factor (TNF)-α strongly promoted pericyte proliferation and migration, and concomitantly induced a switch in pericyte integrins, from α1 to α2 integrin, the opposite to the switch seen when pericytes differentiated. Inhibition studies showed that α2 integrin mediates pericyte adhesion to collagens, and significantly, function blockade of α2 integrin abrogated the pro-modeling influence of TNF-α. Dual-IF on brain tissue with the pericyte marker NG2 showed that while α1 integrin was expressed by pericytes in both stable and remodeling vessels, pericyte expression of α2 integrin was strongly induced in remodeling vessels in EAE brain. CONCLUSIONS: Our results suggest a model in which ECM constituents exert an important influence on pericyte remodeling status. In this model, HSPG restricts pericyte remodeling in stable vessels, but during inflammation, TNF-α triggers a switch in pericyte integrins from α1 to α2, thereby stimulating pericyte proliferation and migration on collagen. These results thus define a fundamental molecular mechanism in which TNF-α stimulates pericyte remodeling in an α2 integrin-dependent manner.
Assuntos
Cérebro/citologia , Cérebro/metabolismo , Integrina alfa1/biossíntese , Integrina alfa2/biossíntese , Pericitos/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Células Cultivadas , Cérebro/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Pericitos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Tissue factor pathway inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI-2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI-2 is frequently down-regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI-2 down-regulation in the National Cancer Institute (NCI)-H460 non-small cell lung cancer cell line using specific micro interfering micro-interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI-2 down-regulation enhanced cell adhesion to collagen IV and laminin via an increase in α(1) integrin on cell surface, and increased MMP expression (mainly MMP-1 and -3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI-2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP-1, -3 and -7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Neoplasias Pulmonares/fisiopatologia , Metaloproteinases da Matriz/metabolismo , Invasividade Neoplásica , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Colágeno Tipo IV/metabolismo , Regulação para Baixo , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina alfa1/biossíntese , Laminina/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Metaloproteinases da Matriz/biossíntese , Metaloproteinases da Matriz/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Interferência de RNA , Transdução de Sinais , Células Estromais , Transplante Heterólogo , Microambiente TumoralRESUMO
Although the female genital tract is the main portal of entry for sexually transmitted infections in women, we still have limited understanding of the generation, maintenance and characteristics of memory T cells in the local tissue. Here, we utilized a mouse model of intravaginal HSV-2 infection and tetramers against the immunodominant HSV glycoprotein B epitope recognized by CD8+ T cells to examine the generation, maintenance and characteristics of anti-HSV memory T cells in the genital tract following acute infection. Our results show that the highest percentage of HSVgB-specific CD8+ T cells was found in the genital tract compared to the spleen or iliac lymphnode. Indeed, although the actual number of CD8+ T cells contracted following viral clearance, approximately one quarter of the CD8+ population that remained in the genital tissue was HSVgB-specific. Memory gB-tetramer+CD8 T cells in the genital tract were positive for CD127 and KLRG1 and negative for CD62L and CCR7, thus confirming that HSV-specific CD8 cells were effector memory T cells that lack the capacity for homing to lymphoid tissues. Functionally, both memory CD8+ and CD4+ HSV-specific populations in the genital tract produced IFNγ when stimulated in vitro and CD4+ cells also produced TNFα. Genital HSVgB-specific memory T cells expressed tissue-homing integrins CD103 (αE integrin) and CD49a (VLA-1 or α1 integrin). Our findings suggest that HSV-specific memory T cells are retained in the genital tract, poised to act as an early line of defense against future virus encounter.
Assuntos
Genitália Feminina/imunologia , Herpes Genital/imunologia , Herpesvirus Humano 2/imunologia , Memória Imunológica , Linfócitos T/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Antígenos CD/biossíntese , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Feminino , Citometria de Fluxo , Genitália Feminina/virologia , Herpes Genital/virologia , Cadeias alfa de Integrinas/biossíntese , Integrina alfa1/biossíntese , Interferon gama/biossíntese , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores Imunológicos , Baço/imunologia , Linfócitos T/metabolismo , Transativadores/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Neuroblastoma is a childhood tumor thought to arise through improper differentiation of neural crest cells. MYCN amplification is a prognostic factor that indicates a highly malignant disease and poor patient prognosis. Integrins are important regulators of neuroblastoma attachment and migration and participate in many aspects of metastasis. However, the role of integrins in neuroblastoma metastasis, the leading cause of death from this disease, remains less well understood. Screening of neuroblastoma cell lines for integrin mRNA expression showed that integrin alpha1 expression was higher in lines such as SK-N-SH and NB69 that do not have MYCN amplification than in cell lines such as IMR32, NB1, NB9 and NB19 that have MYCN amplification. A knockdown of MYCN in NB1 and NB19 cells resulted in increased expression of integrin alpha1, which correlated with enhanced attachment to the extracellular matrix and reduced migratory activity. In contrast, the overexpression of MYCN in SK-N-SH and NB69 cells resulted in decreased expression of integrin alpha1, which correlated with reduced attachment to the extracellular matrix and enhanced migratory activity. These results show that MYCN may limit cell adhesion to the extracellular matrix and promote cell migration by downregulating integrin alpha1.
Assuntos
Neoplasias Encefálicas/metabolismo , Regulação para Baixo , Genes myc , Integrina alfa1/biossíntese , Neuroblastoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina alfa1/genética , Modelos Biológicos , Invasividade Neoplásica , Metástase Neoplásica , Neuroblastoma/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Irregular or low expression of integrins, which are cell adhesion molecules, may be associated with infertility. We conducted a prospective controlled study evaluating the effects of supraphysiological levels of estrogen and progesterone created by human menopausal gonadotropins (HMG) and progesterone support on alpha(1)-integrin immunolocalisation in the endometrium. Three groups were enrolled in the study. The first group of patients (group 1) had unexplained infertility and had been treated with HMG and progesterone (n=27). The second group of patients (group 2) was an untreated fertile group (n=24). The third group (group 3) consisted of patients who had unexplained infertility and had received no treatment (n=11). Endometrial biopsy specimens were taken from individuals from each group during the ovulation induction period. alpha(1)-integrin immunohistochemistry was performed. Serum estradiol and progesterone levels were also measured in parallel with histological dating of endometrial biopsies. Group 1 showed no statistical difference from group 2 in alpha(1)-integrin or histological dating. Group 3 showed less alpha(1)-integrin in the glandular epithelium in the secretory phase. We observed that alpha(1)-integrin was specific to the secretory phase. Its localization was denser in group 2 when compared with group 3, which supports the conclusion that alpha(1)-integrin may be a useful marker for luteal phase quality. Moreover, the supraphysiological estrogen and progesterone levels created by HMG and progesterone support may affect the alpha(1)-integrin in the endometrium in the secretory phase in the case of unexplained infertile patients.
Assuntos
Endométrio/metabolismo , Infertilidade/metabolismo , Integrina alfa1/biossíntese , Progesterona/metabolismo , Adulto , Biomarcadores , Biópsia , Índice de Massa Corporal , Estrogênios/biossíntese , Feminino , Gonadotropinas/metabolismo , Humanos , Imuno-Histoquímica/métodos , Infertilidade/etiologia , Progesterona/biossíntese , Estudos Prospectivos , Fatores de TempoRESUMO
Primary viral infections of the lung induce potent effector CD8 T cell responses. To function in the influenza-infected airways, CD8 T cells must be able to resist cell death. The majority of the CD8 T cells in the airways and lung parenchyma expressed CD49a, the alpha-chain of the type IV collagen receptor VLA-1, and these cells were highly activated, producing both IFN-gamma and TNF-alpha. In the airways, where type IV collagen is abundant, but not the spleen, the CD49a(+) CD8 cells had reduced proportions of annexin V and caspase 8, and >80% expressed the TNF-alpha receptor II, while Fas, TNFR-I, and CD27 expression were similar to CD49a(-) cells. Furthermore, the CD49a(+), but not CD49a(-), CD8 T cells from the airways were resistant to active induction of apoptosis in the presence of type IV collagen and TNF-alpha in vitro. We propose that TNFR-II and the VLA-1 synergize to protect effector CD8 T cells in the infected airways from apoptosis during the acute infection.
Assuntos
Apoptose/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Integrina alfa1beta1/fisiologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/fisiologia , Mucosa Respiratória/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Separação Celular , Feminino , Imunofenotipagem , Vírus da Influenza A Subtipo H3N2/imunologia , Integrina alfa1/biossíntese , Integrina alfa1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/patologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologiaRESUMO
The alpha1beta1 integrin, very late antigen (VLA)-1, characterizes collagen adherent interferon (IFN) gamma producing memory T cells in inflamed synovium. We now report that the mean percentage of VLA-1+ T cells is significantly lower among peripheral blood mononuclear cells of rheumatoid patients responsive to antitumor necrosis factor (TNF) alpha therapy than of those with active disease not receiving therapy. Neutralization of TNFalpha during in vitro polyclonal activation of VLA-1- T cells reduced differentiation to expression of VLA-1 and inhibited secretion of IFNgamma, but did not affect integrin expression on in vivo differentiated VLA-1+ T cells. Moreover, synovial fluids of patients relapsing during and after therapy were enriched in VLA-1+ T cells and lines derived from VLA-1+ T cells in peripheral blood of treated patients retained collagen binding and secreted IFN gamma. Thus, whereas therapy decreases VLA-1+ T cells in rheumatoid arthritis patients, a subset is resistant and contributes to residual and recurring inflammation.
Assuntos
Artrite Reumatoide/imunologia , Integrina alfa1/biossíntese , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto , Idoso , Anticorpos Bloqueadores/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Artrite Reumatoide/terapia , Linhagem Celular , Regulação para Baixo/imunologia , Feminino , Humanos , Infliximab , Integrina alfa1/fisiologia , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Recidiva , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The integrin collagen receptor locus on human chromosome 5q11.2 includes the integrin genes ITGA1 and ITGA2, and the cell cycle regulation gene PELO, embedded within ITGA1 intron 1. ITGA1 contains a CArG box that is bound by serum response factor (SRF), while PELO contains two Sp1 binding elements. A comparison of mRNA levels in megakaryocytic (MK) and non-megakaryocytic (non-MK) cell lines and an analysis of the transcriptional activity of promoter-LUC reporter gene constructs in transfected cells revealed that ITGA1 is selectively suppressed in the MK lineage. Sodium bisulfite genomic sequencing established that a CpG-rich ITGA1 promoter region (-209/+115) is fully methylated at 19 CpG sites in MK cells that do not express alpha1beta1, but completely demethylated in expressing cells. In vitro methylation of ITGA1 suppresses transcription, while treatment of megakaryocytic cells with 5-aza-2'-deoxycytidine, but not Trichostatin A, resulted in de novo expression of ITGA1. During thrombopoietin-induced in vitro differentiation of primary human cord blood mononuclear cells into megakaryocytes, we observed rapid, progressive CpG methylation of ITGA1, but not PELO or ITGA2. Thus, selective CpG methylation of the ITGA1 promoter is a specific feature of alpha1beta1 regulation that coincides with the initiation of megakaryocyte differentiation.
Assuntos
Cromossomos Humanos Par 5/metabolismo , Metilação de DNA , Epigênese Genética/fisiologia , Integrina alfa1/biossíntese , Integrina alfa1beta1/biossíntese , Integrina alfa2/biossíntese , Megacariócitos/metabolismo , Proteínas Nucleares/biossíntese , Locos de Características Quantitativas/fisiologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Cromossomos Humanos Par 5/genética , Metilação de DNA/efeitos dos fármacos , Decitabina , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Integrina alfa1/genética , Integrina alfa1beta1/genética , Integrina alfa2/genética , Íntrons/fisiologia , Células K562 , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Megacariócitos/citologia , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/fisiologia , Trombopoetina/farmacologia , Transcrição GênicaRESUMO
Expression of collagen receptor integrins alpha1beta1 and alpha2beta1 has been associated with progression and metastatic potential of malignant melanoma. Integrin alpha2beta1 was originally characterized as a melanoma progression antigen. We have used real-time quantitative PCR to study the mRNA expression levels of three collagen receptor integrin chains, that is alpha1, alpha2 and alpha11 in metastases from 26 patients with melanoma. Interestingly, we find that survival after initiation of chemoimmunotherapy was significantly decreased in all patients whose tumours expressed high mRNA levels of alpha1 integrin, alpha2 integrin or alpha11 integrin when compared with lower tumour expression levels (P<0.05, log rank test). Moreover, those patients with high mRNA levels of all studied integrins had a significantly shorter survival from the appearance of the first metastasis than the patients with low levels of integrins (P<0.05). Furthermore, a high mRNA expression level of integrin alpha2 was found to be associated with poorer overall survival. High alpha2 mRNA levels (n=6) were associated with median survival of 35 months and low alpha2 mRNA levels (n=20), with median survival of 53 months (P=0.033). We conclude that collagen receptor integrins are important in the progression and prognosis of metastatic melanoma, and their measurements might be used as predictive markers when assessing disease progression.
Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Melanoma/mortalidade , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/mortalidade , Adulto , Idoso , Intervalo Livre de Doença , Feminino , Humanos , Integrina alfa1/biossíntese , Integrina alfa2/biossíntese , Masculino , Melanoma/tratamento farmacológico , Pessoa de Meia-Idade , Modelos Biológicos , Receptores de Colágeno/metabolismo , Neoplasias Cutâneas/tratamento farmacológicoRESUMO
STUDY DESIGN: In vitro investigation of vertebral endplate chondrocyte apoptosis. OBJECTIVES: To determine whether Fas antibody caused apoptosis in endplate chondrocytes, and whether insulin-like growth factor-1 (IGF-1) inhibited this effect. Integrin-alpha1 and focal adhesion kinase (FAK) expression in conjunction with apoptosis was also investigated. SUMMARY OF BACKGROUND DATA: Binding of Fas antibody to Fas mimics Fas-FasL ligation, which causes apoptosis. IGF-1 has been shown to have anti-apoptotic effects. MATERIALS AND METHODS: Rat cervical endplate chondrocytes were cultured and treated with Fas antibody, with or without IGF-1. Cellular morphology was examined by microscopy. Apoptotic changes were evaluated by transmission electron microscopy, TUNEL staining, and immunostaining. Apoptosis-induced changes in the expression of integrin-alpha1 chain and FAK were also investigated. RESULTS: Endplate chondrocytes were able to be cultured; a chondrocytic phenotype was maintained. Fas antibody induced apoptosis in endplate chondrocytes; this was confirmed by TUNEL staining. Bcl-2 expression was decreased by Fas antibody, while Bax expression increased. Integrin-alpha1 and FAK expression was decreased by Fas antibody. IGF-1 treatment inhibited these Fas antibody-induced changes. CONCLUSIONS: Fas antibody induces apoptosis and decreases Integrin-alpha1 and FAK expression in cultured endplate chondrocytes; IGF-1 is protective against these changes.
Assuntos
Anticorpos Monoclonais/fisiologia , Apoptose/fisiologia , Condrócitos/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Receptor fas/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Murinos , Afinidade de Anticorpos , Apoptose/efeitos dos fármacos , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Proteína Ligante Fas , Integrina alfa1/biossíntese , Glicoproteínas de Membrana/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Necrose Tumoral/metabolismo , Receptor fas/metabolismoRESUMO
Stem cells responsible for maintenance and repair of tissues are found in a number of organs. The liver's remarkable capacity to regenerate after hepatectomy or chemical-induced injury does not involve proliferation of stem cells. However, recent studies suggest that liver stem cells exist in both embryonic and adult livers. Using fluorescence-activated cell sorting and a culture system in which primitive hepatic progenitor cells form colonies, a novel class of cells with the marker profile c-Met(+)CD49f(+/low)c-Kit(-)CD45(-)TER119(-) was found in the developing liver. This class apparently represents the population of cells that form colonies containing distinct hepatocytes and cholangiocytes. When cells in this class are transplanted into the spleen or liver of mice subjected to liver injury, the cells migrate and differentiate into liver parenchymal cells and cholangiocytes that are morphologically and functionally indistinguishable from their native counterparts. During mid-gestation, hematopoietic cells migrate into the liver from a region bounded by aorta, gonad, and mesonephros and produce oncostatin M (OSM). In combination with glucocorticoid hormones, OSM induces maturation of liver stem and progenitor cells, including those of the c-Met(+)CD49f(+/low)c-Kit(-)CD45(-)TER119(-) class. The ability to manipulate the proliferation and differentiation of liver stem cells in vitro will greatly aid in analyzing mechanisms of liver development and offers promise in stem cell therapy of liver diseases.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fígado/citologia , Fígado/embriologia , Células-Tronco/patologia , Animais , Proliferação de Células , Separação Celular , Transplante de Células , Citocinas/metabolismo , Matriz Extracelular/metabolismo , Citometria de Fluxo , Glicoforinas/biossíntese , Células-Tronco Hematopoéticas/metabolismo , Fatores Nucleares de Hepatócito/metabolismo , Hepatócitos/metabolismo , Humanos , Integrina alfa1/biossíntese , Antígenos Comuns de Leucócito/biossíntese , Fígado/metabolismo , Fígado/patologia , Camundongos , Modelos Biológicos , Oncostatina M , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transplante de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
To investigate the interaction between anticancer drug resistance and radioresistance in cervical cancer cells, 3 single cell-derived cyclophosphamide-resistant subclones were established from the drug- and radiosensitive human cervical squamous cell carcinoma cell line ME180 by chronic exposure cultures with 4-hydroxy-cyclophosphamide followed by limiting dilution. The established cyclophosphamide-resistant subclones were also radio- and multidrug-resistant to 7 other anticancer drugs. Flow cytometric analysis revealed significantly increased levels of CD40 expression on the 3 resistant subclones, whereas no CD40 expression was found on the parent ME180 cells. However, there were no changes in the expression levels of CD29, CD49a-CD49f or CD59 between the parent cells and resistant subclones. A recombinant human soluble CD40 ligand had no effect on the proliferation of the resistant subclones. Irradiation had no effect on the 4-hydroxy-cyclophosphamide sensitivity of the parent cells. These results indicate that the established cyclophosphamide-resistant subclones have impaired cell death signals, which are common to both drug- and radiation-induced apoptosis, and cyclophosphamide may not be an adequate drug for use in concurrent chemoradiotherapy. Furthermore, CD40 activation signals may be associated with the multidrug- and radioresistance in these cyclophosphamide-resistant subclones.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Antígenos CD40/biossíntese , Carcinoma de Células Escamosas/tratamento farmacológico , Ciclofosfamida/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Neoplasias do Colo do Útero/tratamento farmacológico , Antineoplásicos/farmacologia , Bleomicina/farmacologia , Ligante de CD40/metabolismo , Antígenos CD59/biossíntese , Linhagem Celular Tumoral , Proliferação de Células , Ciclofosfamida/análogos & derivados , Ciclofosfamida/metabolismo , Ciclofosfamida/farmacologia , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Resistência a Múltiplos Medicamentos , Feminino , Citometria de Fluxo , Raios gama , Humanos , Integrina alfa1/biossíntese , Integrina alfa2/biossíntese , Integrina alfa3/biossíntese , Integrina alfa4/biossíntese , Integrina alfa5/biossíntese , Integrina alfa6/biossíntese , Integrina beta1/biossíntese , Tolerância a RadiaçãoRESUMO
Multiple sclerosis (MS), an inflammatory, demyelinating disease of the central nervous system (CNS), is thought to be caused by a T cell-mediated attack on CNS myelin and axons. Recombinant interferon (IFN)-beta is an established treatment of multiple sclerosis, and is known to reduce the number of disease relapses and the development of irreversible symptoms and signs of disease. The mechanism of action of IFN-beta treatment is, however, not completely understood. Previous studies have suggested major effects on mononuclear cell cytokine production and T cell migration, but results have been inconsistent. We found decreases in CD4 and CD8 T cell expression of the CD49d/VLA-4 molecule, increases in plasma concentrations of soluble vascular cell adhesion molecule (sVCAM-1), and increases in plasma concentrations of tumor necrosis factor and interleukin (IL)-12 p40 chain in patients with MS who were initiated on de novo treatment with IFN-beta1b. We found only minor associations between the different changes induced by IFN-beta1b-treatment. Our findings are consistent with changes in T cell expression of CD49d/VLA-4 and induction of sVCAM-1 as important effects of treatment with IFN-beta1b in multiple sclerosis, whereas the role of changes in TNF and IL-12 p40 chain concentrations is more difficult to interpret.
Assuntos
Citocinas/metabolismo , Regulação da Expressão Gênica , Interferon beta/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Adesão Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Integrina alfa1/biossíntese , Molécula 1 de Adesão Intercelular/sangue , Interferon beta-1b , Interleucina-12/metabolismo , Subunidade p40 da Interleucina-12 , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/sangue , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely alpha1beta1, alpha2beta1, alpha10beta1, and alpha11beta1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express alpha1beta1, alpha2beta1, or alpha10beta1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant alphaI domains in Escherichia coli. For each of the four alphaI domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both alpha1I- and alpha2I-collagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by alpha2I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the alpha2I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix.
Assuntos
Cartilagem/metabolismo , Colágeno Tipo IX/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cartilagem/química , Adesão Celular , Linhagem Celular , Linhagem Celular Tumoral , Condrócitos/metabolismo , Condrossarcoma/metabolismo , Colágeno/química , Colágeno/metabolismo , Colágeno Tipo IX/química , Colágeno Tipo IX/metabolismo , Cricetinae , Escherichia coli/metabolismo , Humanos , Imunoprecipitação , Cadeias alfa de Integrinas/biossíntese , Integrina alfa1/biossíntese , Integrina alfa2/biossíntese , Ligantes , Camundongos , Microscopia Eletrônica , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Peptídeos/química , Reação em Cadeia da Polimerase , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
Vascular endothelial growth factor-A (VEGF-A) is strongly up-regulated in wounded cutaneous tissue and promotes repair-associated angiogenesis. However, little is known about its role in lymphatic regeneration of the healing skin. We studied wound healing in transgenic mice that overexpress VEGF-A specifically in the epidermis and in wild-type mice in the absence or presence of inhibitors of VEGF-A signaling. Surprisingly, transgenic overexpression of VEGF-A in the skin promoted lymphangiogenesis at the wound healing site, whereas systemic blockade of VEGFR-2 prevented lymphatic vessel formation. Studies in cultured lymphatic endothelial cells revealed that VEGF-A induced expression of the alpha1 and alpha2 integrins, which promoted their in vitro tube formation and their haptotactic migration toward type I collagen. VEGF-A-induced lymphatic endothelial cord formation and haptotactic migration were suppressed by anti-alpha1 and anti-alpha2 integrin blocking antibodies, and systemic blockade of the alpha1 and alpha2 integrins inhibited VEGF-A-driven lymphangiogenesis in vivo. We propose that VEGF-A promotes lymphatic vasculature formation via activation of VEGFR-2 and that lineage-specific differences of integrin receptor expression contribute to the distinct dynamics of wound-associated angiogenesis and lymphangiogenesis.