RESUMO
Objective: To investigate the relationship between plasma heat shock proteins 90α(Hsp90α) levels and the white matter hyperintensity(WMH) in patients with cerebral small vessel disease(SVD). Methods: Patients admitted to the Department of Neurology, the First Affiliated Hospital of Zhengzhou University from March to August 2021 and diagnosed with WMH by magnetic resonance examination (MRI) were selected as the case group, matched with physical examination patients who visited the Department of Medical Examination during the same period and showed no WMH on MRI and no history of neurological diseases as the control group, and the level of plasma Hsp90α was quantitatively detected by enzyme-linked immunosorbent assay. Mann-Whitney U test was used to compare whether there was a difference in plasma Hsp90α levels between the control group and the case group.Multivariate logistic regression analysis was used to explore the related factors of WMH in patients with SVD. Results: Of the 183 subjects, the control group (n=73) consisted of 28 males and 45 females, aged (54±10) years, while the case group (n=110) consisted of 71 males and 39 females, aged (64±10) years old. Plasma Hsp90α level was higher in the case group than that of the control group [53.33(35.33, 70.09) ng/ml vs 35.02(18.51, 54.95) ng/ml, P<0.001]. After adjusting for confounding factors by multivariate analysis, the results showed that plasma Hsp90α levels greater than 58.34 ng/ml was associated with WMH (P=0.002, OR=5.931, 95%CI:1.955-17.995). Conclusion: Higher level of plasma Hsp90α is associated with WMH in patients with SVD.
Assuntos
Doenças de Pequenos Vasos Cerebrais , Proteínas de Choque Térmico HSP90/metabolismo , Leucoaraiose , Substância Branca , Idoso , Feminino , Proteínas de Choque Térmico , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Substância Branca/patologiaRESUMO
BACKGROUND: Obesity is a risk factor for metabolic diseases, while preadipocyte differentiation or adipogenesis is closely related to obesity occurrence. Long noncoding RNAs (lncRNAs) are a unique class of transcripts in regulation of a variety of biological processes. Using cDNA microarray, we found lncRNA U90926 is negatively correlated with 3T3-L1 preadipocyte differentiation. OBJECTIVE: The aim of this study was to explore the role of lncRNA U90926 (lnc-U90926) in adipogenesis and the underlying mechanisms. METHODS: Quantitative real-time PCR (qPCR) was performed to determine lnc-U90926 expression in 3T3-L1 preadipocytes, differentiated adipocytes, and in adipose tissues form mice. RNA fluorescent in situ hybridization (FISH) was performed to determine the localization of lnc-U90926 in 3T3-L1 preadipocytes. The effects of lnc-U90926 on 3T3-L1 adipogenesis were analyzed with lentivirus-mediated gain- and loss-of-function experiments. Lipid accumulation was evaluated by oil red O staining; several adipogenesis makers were analyzed by qPCR and western blotting. Dual luciferase assay was applied to explore the transactivation of target genes modulated by lnc-U90926. All measurements were performed at least for three times. RESULTS: Lnc-U90926 expression decreased along the differentiation of 3T3-L1 preadipocytes. In mice, lnc-U90926 is predominantly expressed in adipose tissue. Obese mice have lower lnc-U90926 expression in subcutaneous and visceral adipose tissue than non-obese mice. FISH results showed that lnc-U90926 was mainly located in the cytoplasm. Overexpression lnc-U90926 attenuated 3T3-L1 adipocyte differentiation as evidenced by its ability to inhibit lipid accumulation, to decrease the mRNA levels of peroxisome proliferator-activated receptor gamma 2 (PPARγ2), fatty acid binding protein 4 (FABP4) and adiponectin (AdipoQ) as well as to reduce the protein levels of PPARγ and FABP4 (P<0.05). Knockdown of lnc-U90926 showed opposite effects, which increased mRNA expression of PPARγ2, FABP4, CCAAT/enhancer-binding proteinα (C/EBPα) and AdipoQ. CONCLUSION: Lnc-U90926 attenuates 3T3-L1 adipocyte differentiation via inhibiting the transactivation of PPARγ2 or PPARγ.
Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Adipogenia/genética , RNA Longo não Codificante/genética , Células 3T3-L1 , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Proliferação de Células , Modelos Animais de Doenças , Proteínas de Ligação a Ácido Graxo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Neuregulin receptor degradation protein-1 (Nrdp1) is a newly discovered E3 ligase that plays a role in the apoptosis process of multiple diseases. Previous studies has shown that Nrdp1 exerted a proapoptotic effect in cardiac diseases. The purpose of this study is to investigate the potential involvement of Nrdp1 in the pathological processes of inflammatory bowel disease (IBD). To create a mouse model of experimental colitis, trinitrobenzenesulfonic acid (TNBS) was administered and the severity of colitis was assessed based on changes in weight and histological scores. Using Western blot and immunohistochemistry, significant increase in Nrdp1 expression was observed in intestinal epithelial cells (IECs). This was accompanied with the up-regulation of cleaved PARP and active caspase-3 in IECs, indicating a potential function in IECs. To study this further, we built an in vitro model of tumor necrosis factor-alpha (TNF-α)-induced apoptosis using human IEC line HT-29 cells. When Nrdp1 was knocked down, a decrease in apoptosis was observed, suggesting that Nrdp1 may play a proapoptotic role in IEC apoptosis. The mechanism behind this phenomenon is associated with the suppression of downstream targets of Nrdp1, such as protein kinase B (AKT). Furthermore, immunohistochemistry analysis in patients with Crohn's disease (CD) and normal controls supported the same results as observed in experimental colitis. We conclude that Nrdp1 may be a promising new therapeutic target for ameliorating IBD in humans.
Assuntos
Colite , Doença de Crohn , Animais , Humanos , Camundongos , Apoptose , Colite/metabolismo , Doença de Crohn/tratamento farmacológico , Mucosa Intestinal , Intestinos/patologia , Neurregulinas/metabolismo , Neurregulinas/farmacologia , Neurregulinas/uso terapêuticoRESUMO
Seven novel MhcMamu-DQB1 alleles were observed in a population of rhesus macaques of Chinese origin.
Assuntos
Alelos , Antígenos de Histocompatibilidade Classe II/genética , Macaca mulatta/genética , Animais , China , Antígenos de Histocompatibilidade Classe II/imunologia , Macaca mulatta/imunologiaRESUMO
Interaction between a T cell receptor (TCR) and various ligands, i.e. , anti-TCR antibodies, superantigens, peptides, or altered peptide ligands in the context of major histocompatibility complex (MHC) molecules can trigger different T helper cell (Th) effector functions. Herein, we studied the T cell response induced by a soluble, dimeric peptide/MHC class II chimera, namely hemagglutinin (HA)110-120/I-E(d)alphabeta/Fcgamma2a (DEF). We have previously demonstrated that the soluble DEF molecule binds stably and specifically to HA110-120-specific TCRs expressed by a T cell hybridoma. Administration of DEF in vivo induced differentiation of resting and activated peptide-specific T cells toward a Th2 response, as indicated by the increase of interleukin (IL)-4, IL-10, and specific immunoglobulin (Ig)G1 antibodies and decrease of IL-2, specific IgG2a antibodies, and cytotoxic T lymphocyte activity. In contrast to HA110-120 peptide presented by the DEF molecule to T cells, the nominal synthetic peptide induced a predominant Th1 response, and the PR8 virus-derived HA110-120 peptides induced a mixed Th1/Th2 response. Independent of antigen processing, soluble DEF was almost 2 logs more potent in stimulating cognate T cells than the nominal peptide. Polarization of cognate T cells toward the Th2 response occurred upon interaction of soluble DEF with TCR and CD4 molecules followed by early activation of p56(lck) and ZAP-70 tyrosine kinases, and negative signaling of the signal transducer and activator of transcription (STAT)4 pathway of Th1 differentiation. DEF-like molecules may provide a new tool to study the mechanisms of signaling toward Th2 differentiation and may also provide a potential immunotherapeutic approach to modulate autoreactive T cells toward protective Th2 immune responses.
Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Oligopeptídeos/imunologia , Células Th2/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Proteínas de Ligação a DNA , Dimerização , Hemaglutininas , Antígenos de Histocompatibilidade Classe II/genética , Fragmentos Fc das Imunoglobulinas/genética , Interleucina-10/biossíntese , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Oligopeptídeos/genética , Fragmentos de Peptídeos , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão , Fator de Transcrição STAT4 , Transdução de Sinais , Solubilidade , Células Th2/citologia , TransativadoresRESUMO
The aim of this study was to investigate the protective effect of hydrogen in a rat model of osteoradionecrosis of the jaw (ORNJ). The rats and bone marrow-derived mesenchymal stem cells (BMSCs) were pre-treated with hydrogen before receiving irradiation (7Gy per fraction, five fractions in total once a day for rats, 4Gy for BMSCs). Reactive oxygen species (ROS) and cell differentiation were measured in the BMSCs. Also, the radioprotective effect of hydrogen for ORNJ in Sprague-Dawley rats was examined by gross clinical manifestations, micro-computed tomography, and histology. Hydrogen significantly reduced the production of ROS in BMSCs after irradiation. The cell viability was significantly decreased after irradiation (P= 0.001), but pre-treatment with hydrogen before irradiation increased the cell viability (P= 0.025). Hydrogen considerably increased the cellular differentiation potential of the irradiated cells. Comparing with the rats underwent irradiaton only, those rats treated by hydrogen-rich saline significantly appeared improved occlusion, salivation, alopecia, oral ulcer, and less bone necrosis. Myofibroblasts accumulated overwhelmingly in the fibrosis medulla and around the sequestrum after irradiation, and this was decreased in the group pre-treated with hydrogen. Hydrogen may represent a strategy for the prevention and treatment of ORNJ. Its high efficacy and low toxicity suggest possible therapeutic application.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Osteorradionecrose , Animais , Células da Medula Óssea , Hidrogênio , Osteorradionecrose/prevenção & controle , Ratos , Ratos Sprague-Dawley , Microtomografia por Raio-XRESUMO
The c-Mer receptor tyrosine kinase (RTK) is most closely related to chicken c-Eyk and belongs to the Axl RTK subfamily. Although not detected in normal lymphocytes, c-Mer is expressed in B- and T-cell leukemia cell lines, suggesting an association with lymphoid malignancies. To gain an understanding of the role of this receptor in lymphoid cells, we expressed in murine interleukin-3 (IL-3)-dependent Ba/F3 pro-B-lymphocyte cells a constitutively active receptor, CDMer, formed from the CD8 extracellular domain and the c-Mer intracellular domain. Cells transfected with a plasmid encoding the CDMer receptor became IL-3 independent. When tyrosine (Y)-to-phenylalanine (F) mutations were introduced into c-Mer, only the Y867 change significantly reduced the IL-3-independent cell proliferation. The Y867 residue in the CDMer receptor mediated the binding of Grb2, which recruited the p85 phosphatidylinositol 3-kinase (PI 3-kinase). Despite the difference in promotion of proliferation, both the CDMer and mutant F867 receptors activated Erk in transfected cells. On the other hand, we found that both transcriptional activation of NF-kappaB and activation of PI 3-kinase were significantly suppressed with the F867 mutant receptor, suggesting that the activation of antiapoptotic pathways is the major mechanism for the observed phenotypic difference. Consistent with this notion, apoptosis induced by IL-3 withdrawal was strongly prevented by CDMer but not by the F867 mutant receptor.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Sistema Hematopoético/citologia , Sistema Hematopoético/metabolismo , NF-kappa B/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases , Animais , Apoptose , Sítios de Ligação/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular , Linhagem Celular , Transformação Celular Neoplásica , Ativação Enzimática , Proteína Adaptadora GRB2 , Interleucina-3/farmacologia , Camundongos , Mutagênese Sítio-Dirigida , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Ativação Transcricional , Transfecção , c-Mer Tirosina QuinaseRESUMO
Two chimeric receptors, ER1 and ER2, were constructed. ER1 contains the extracellular and transmembrane (TM) domains derived from epidermal growth factor receptor and the cytoplasmic domain from c-Ros; ER2 is identical to ER1 except that its TM domain is derived from c-Ros. Both chimeras can be activated by epidermal growth factor and are capable of activating or phosphorylating an array of cellular signaling proteins. Both chimeras promote colony formation in soft agar with about equal efficiency. Surprisingly, ER1 inhibits while ER2 stimulates cell growth on monolayer culture. Cell cycle analysis revealed that all phases, in particular the S and G2/M phases, of the cell cycle in ER1 cells were elongated whereas G1 phase of ER2 cells was shortened threefold. Comparison of signaling pathways mediated by the two chimeras revealed several differences. Several early signaling proteins are activated or phosphorylated to a higher extent in ER1 than in ER2 cells in response to epidermal growth factor. ER1 is less efficiently internalized and remains tyrosine phosphorylated for a longer time than ER2. However, phosphorylation of the 66-kDa She protein, activation of mitogen activated protein kinase, and induction of c-fos and c-jun occur either to a lesser extent or for a shorter time in ER1 cells. Cellular protein phosphorylation patterns are also different in ER1 and ER2 cells. In particular, a 190-kDa Shc-associated protein is tyrosine phosphorylated in ER2 but not in ER1 cells. Our results indicate that the TM domains have a profound effect on the signal transduction and biological activity of those chimeric receptors. The results also imply that sustained stimulation of ER1 due to its retarded internalization apparently triggers an inhibitory response that dominantly counteracts the receptor-mediated mitogenic signals. These two chimeras, expressed at similar levels in the same cell type but having opposite effects on cell growth, provide an ideal system to study the mechanism by which a protein tyrosine kinase inhibits cell growth.
Assuntos
Divisão Celular/genética , Receptores ErbB/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/genética , Células 3T3 , Animais , Sequência de Bases , Membrana Celular/metabolismo , Ensaio de Unidades Formadoras de Colônias , Receptores ErbB/metabolismo , Cinética , Camundongos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Hemorrhagic cystitis is one of the complications of allogeneic hematopoietic stem cell transplantation. Treatment of hemorrhagic cystitis is difficult, especially in pediatric patients. A pediatric case of severe hemorrhagic cystitis after hematopoietic stem cell transplantation was treated in our hospital with arterial embolization combined with corticosteroid therapy because the conventional therapy was invalid for him. After the treatment, hemorrhagic cystitis was cured. During follow-up, the patient was in stable condition, with normal urine, blood cells returned to normal, bone marrow was in complete remission state, and disease-free survival for more than 8 months. Selective bladder arterial embolism followed by corticosteroid therapy successfully treated the patient.
Assuntos
Cistite/terapia , Embolização Terapêutica/métodos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Hemorragia/terapia , Adolescente , Cistite/etiologia , Hemorragia/etiologia , Humanos , Masculino , Transplante Homólogo/efeitos adversosRESUMO
Proto-oncogene c-ros codes for a receptor-type protein tyrosine kinase (PTK) sharing high homology with the Drosophila sevenless protein. Recent studies of c-ros expression in mouse by in situ hybridization showed that c-ros was expressed specifically and transiently in the epithelial cells of late embryonic kidney collecting duct and intestine villi. Those investigators suggested that c-ros may play a role in the development of those organelles. In the present study, we have examined the expression profile of c-ros in chicken by RNAase protection and in situ hybridization with riboprobes. Our results showed that in addition to kidney and intestine, low levels of c-ros mRNA could also be detected in lung, testis, thymus and bursa. Expression of c-ros commences during middle to late embryonic stages in those organs and persists into the adult life. In situ hybridization revealed that expression of c-ros was restricted to the epithelial cells of all the tissues examined including kidney, intestine, bursa, thymus and testis. In kidney c-ros was detected in all the epithelial cells of the collecting ducts, in intestine it was detected in the epithelial cells of villi and the underneath crypts. Our finding of c-ros expression in chicken differs from those in mouse in (1) instead of transient expression during the embryonic stage, expression of c-ros in chicken kidney and intestine persists into the adult life and (2) expression of c-ros in renal collecting ducts is not restricted to its growing tips, instead it is expressed in the entire epithelial layer of the ducts. Our results suggest that c-ros may play a role not only in the initial induction events in the organogenesis, but also in the mature function of those organs.
Assuntos
Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Bolsa Sinovial/crescimento & desenvolvimento , Bolsa Sinovial/metabolismo , Embrião de Galinha , Galinhas , Expressão Gênica , Rim/crescimento & desenvolvimento , Rim/metabolismo , Masculino , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes/fisiologia , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/biossíntese , Receptores Proteína Tirosina Quinases/genética , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Timo/crescimento & desenvolvimento , Timo/metabolismoRESUMO
The proto-oncogene ErbB2 is known to be amplified and to play an important role in the development of about one-third of human breast cancers. Phosphatidylinositol 3-kinase (PI3K), which is often activated in ErbB2-overexpressing breast cancer cells, is known to regulate cell proliferation and cell survival. Selective inhibitors of the PI3K pathway were used to assess the relevance of PI3K signaling in the anchorage-independent growth of a series of human mammary carcinoma cell lines. Wortmannin, LY294002, and rapamycin at concentrations that did not affect MAPK phosphorylation but substantially inhibited PI3K, Akt, and p70(S6K) significantly suppressed the soft agar growth of tumor cell lines that overexpress ErbB2 but not the growth of tumor lines with low ErbB2 expression. A similar growth inhibition of ErbB2-overexpressing carcinoma lines was observed when a dominant negative p85(PI3K) mutant was introduced into these cells. Forced expression of ErbB2 in breast cancer lines originally expressing low ErbB2 levels augmented receptor expression and sensitized those lines to LY294002- and rapamycin-mediated inhibition of colony formation. Furthermore, treatment with LY294002 resulted in the selective increase of cyclin-dependent kinase inhibitors p21(Cip1) or p27(Kip1) and suppression of cyclin E-associated Cdk2 kinase activity in ErbB2-overexpressing lines, which may account for their hypersensitivity toward inhibitors of the PI3K pathway in anchorage-independent growth. Our results indicate that the PI3K/Akt/p70(S6K) pathway plays an enhanced role in the anchorage-independent growth of ErbB2-overexpressing breast cancer cells, therefore providing a molecular basis for the selective targeting of this signaling pathway in the treatment of ErbB2-related human breast malignancies.
Assuntos
Neoplasias da Mama/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptor ErbB-2/biossíntese , Androstadienos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Adesão Celular , Divisão Celular , Sobrevivência Celular , Cromonas/farmacologia , Ciclina E/metabolismo , Inibidores Enzimáticos/farmacologia , Genes Dominantes , Humanos , Immunoblotting , Sistema de Sinalização das MAP Quinases , Morfolinas/farmacologia , Fosforilação , Ligação Proteica , Proto-Oncogene Mas , Transdução de Sinais , Sirolimo/farmacologia , Transfecção , Células Tumorais Cultivadas , WortmaninaRESUMO
A 5,403 bp cDNA encoding chicken protein tyrosine phosphatase gamma (PTPgamma) was isolated and sequenced. The predicted open reading frame of 1,422 amino acids (aa) includes 742 aa of extracellular (EC) domain, 26 aa of transmembrane (TM) domain and 634 aa of intracellular domain. The chicken PTPgamma has a 86.7% aa identity to its human homolog and contains the carbonic anhydrase-like domain and fibronectin type III homologous regions in the EC domain, as well as the tandem linked catalytic sequences in the cytoplasmic domain. However, the chicken PTPgamma lacks 29 aa immediate downstream of the putative TM domain in comparison with its human counterpart. Northern analysis revealed the presence of two transcripts of 6.3 and 9.5 kb in various tissues. The cytoplasmic domain of the PTPgamma could be expressed as an enzymatically active form in SF9 insect cells. PTPgamma could also be expressed in normal and rsc-transformed NIH3T3 and Rat 1 cells as a gag-PTP fusion protein, but no detectable effects on growth and colony formation of these cells were observed. Copyright 1996 S. Karger AG, Basel
RESUMO
We studied the effect of vasoactive intestinal peptide (VIP) on angiogenesis in the ischemic boundary area after focal cerebral ischemia. Adult male Sprague-Dawley rats underwent middle cerebral artery occlusion for 2 h. A single dose of VIP was given via i.c.v. injection at the beginning of reperfusion. Immunohistochemistry and Western blotting were performed to assay angiogenesis and brain levels of vascular endothelial growth factor (VEGF) protein, respectively. In addition, the expression of VEGF and its receptors (flt-1 and flk-1), as well as endothelial proliferation, was measured using rat brain microvascular endothelial cells. Immunohistochemical analyses revealed significant (P<0.05) increases in the numbers of bromodeoxyuridine (BrdU) positive endothelial cells and microvessels at the boundary of the ischemic lesion in rats treated with VIP compared with rats treated with saline. Western blotting analysis showed that treatment with VIP significantly (P<0.05) raised VEGF levels in the ischemic hemisphere. In addition, treatment with VIP increased flt-1 and flk-1 immunoreactivity in endothelial cells. In vitro, incubation with VIP significantly (P<0.01) increased the proliferation of endothelial cells and induced the expression of VEGF, flt-1 and flk-1 in endothelial cells. The stimulatory effect of VIP on the proliferation of endothelial cells was significantly (P<0.01) inhibited by SU5416, a selective inhibitor of VEGF receptor tyrosine kinase. Our data suggest that treatment with VIP enhances angiogenesis in the ischemic brain, and this effect may be mediated by increases in levels of VEGF and its receptors.
Assuntos
Ataque Isquêmico Transitório/fisiopatologia , Neovascularização Fisiológica , Peptídeo Intestinal Vasoativo/fisiologia , Animais , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Encéfalo/patologia , Proliferação de Células , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Masculino , Microvasos/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
There is a 3-aa insertion in the transmembrane (TM) domain of the p68gag-ros protein-tyrosine kinase encoded by avian sarcoma virus UR2 v-ros as compared with that of the protooncogene c-ros. The effect of this insertion on biological function and biochemical properties of v-Ros protein was investigated by deleting these 3 aa to generate the mutant TM1. This mutant has greatly reduced transforming, mitogenic, and tumorigenic activities despite the fact that the protein-tyrosine kinase activity and cell-surface localization of TM1 protein are unaffected. However, unlike UR2 protein, mutant TM1 protein becomes glycosylated, is differentially phosphorylated, and fails to induce tyrosine phosphorylation of a 88-kDa protein and a major substrate of insulin receptor, insulin receptor substrate 1. The TM1 protein is unable to associate with phosphatidylinositol 3-kinase and fails to promote association of insulin receptor substrate 1 with phosphatidylinositol 3-kinase. By contrast, tyrosine phosphorylation of Shc protein and phospholipase C gamma as well as interaction of Grb2 protein with Shc and SOS protein signaling components are unaltered in the TM1 infected cells. Our results show that the TM-domain sequence of p68gag-ros profoundly affects its function and substrate interaction. The mutant defines a signaling pathway including phosphatidylinositol 3-kinase, insulin receptor substrate 1, and possibly an 88-kDa protein that does not overlap the Ras pathway and is important for full transforming and mitogenic potency of v-ros protein-tyrosine kinase.
Assuntos
Proteínas Oncogênicas Virais/química , Proteínas Tirosina Quinases/química , Proteínas Proto-Oncogênicas/química , Receptores Proteína Tirosina Quinases/química , Animais , Transformação Celular Neoplásica , Células Cultivadas , Embrião de Galinha , Técnicas In Vitro , Glicoproteínas de Membrana/química , Mitógenos , Mutagênese Sítio-Dirigida , Mapeamento de Peptídeos , Fosfotirosina , Relação Estrutura-Atividade , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Mitogen-activated protein (MAP) kinase mediates cell proliferation, cell differentiation, and cell survival by regulating signaling pathways activated by receptor protein tyrosine kinases (RPTKs), including the insulin-like growth factor 1 receptor (IGF-IR). We analyzed the upstream signaling components of the MAP kinase pathway, including RPTKs, in human breast cancer cell lines and found that some of those components were overexpressed. Importantly, signaling molecules such as IGF-IR, insulin receptor, and insulin receptor substrate 1, leading to the MAP kinase pathway, were found to be concomitantly overexpressed within certain tumor lines, i.e., MCF-7 and T-47D. When compared with the nonmalignant and other breast tumor lines examined, MCF-7 and T-47D cells displayed a more rapid, robust, and sustained MAP kinase activation in response to insulin-like growth factor I (IGF-I) stimulation. By contrast, IGF-I treatment led to a sustained down-regulation of MAP kinase in those lines overexpressing ErbB2-related RPTKs. Interestingly, blocking the MAP kinase pathway with PD098059 had the greatest antiproliferative effect on MCF-7 and T-47D among the normal and tumor lines tested. Furthermore, addition of an IGF-IR blocking antibody to growth medium attenuated the ability of PD098059 to suppress the growth of MCF-7 and T-47D cells. Thus, our study suggests that concomitant overexpression of multiple signaling components of the IGF-IR pathway leads to the amplification of IGF-I-mediated MAP kinase signaling and resultant sensitization to PD098059. The enhanced sensitivity to PD098059 implies an increased requirement for the MAP kinase pathway in those breast cancer cells, making this pathway a potential target in the treatment of selected breast malignancies.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Fator de Crescimento Insulin-Like I/metabolismo , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Neoplasias da Mama/metabolismo , Diferenciação Celular , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Feminino , Flavonoides/farmacologia , Humanos , Immunoblotting , Fosforilação , Testes de Precipitina , Transdução de Sinais , Fatores de Tempo , Células Tumorais Cultivadas , Tirosina/metabolismo , Regulação para CimaRESUMO
We have shown previously that the extracellular sequences of the human insulin receptor (IR) and the insulin-like growth factor I receptor (IGFR) have an inhibitory effect on protein tyrosine kinase (PTK) activity and on the biological functions of their respective Gag-receptor fusion proteins. To study the role of IGFR carboxyl sequence in modulation of the Gag-IGFR PTK and biological activities, five mutants, CM1, CM2, CM3, CM4, and CM5, containing carboxyl deletions of 17, 27, 47, 67, and 88 amino acids (aa), respectively, were constructed from the parental virus UIGFR encoding the Gag-IGFR. Deletion of up to 27 aa had little effect on the cell-transforming and PTK activities of UIGFR. Deletions of 47 aa in CM3 abolished PTK and transforming activities. Surprisingly, a further deletion of 20 aa in CM4 beyond that in CM3 reactivated the kinase and transforming activities. CM5, containing a deletion of 20 aa beyond that in CM4, had only marginal transforming and PTK activities. We conclude that deletion of the carboxyl region of the Gag-IGFR inactivates, instead of activating as in the case with Gag-IR, its transforming activity and the amino acid sequence 1250 to 1310 is essential for PTK and transforming activities. Analysis of the ability of the full-length IGFR and its mutant receptors described above to associate with phosphatidylinositol 3 kinase indicated that the association required PTK activity and tyrosine phosphorylation of the receptors and correlated well with their transforming activities. The carboxyl 88 aa are not essential for the association.
Assuntos
Receptores Proteína Tirosina Quinases/química , Receptor IGF Tipo 1/química , Sequência de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , Humanos , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotirosina , Deleção de Sequência , Transdução de Sinais , Relação Estrutura-Atividade , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
At least eight herpes simplex virus type 1 (HSV-1) and five HSV-2 proteins were tyrosine phosphorylated in infected cells. The first viral tyrosine phosphoprotein identified was the HSV-1 regulatory protein ICP22. Also, two novel phosphotyrosine proteins were bound by anti-ICP22 antibodies. H(R22) is a cellular protein, while the F(R10) protein is observed only in HSV-1-infected cells.
Assuntos
Epitopos/imunologia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/imunologia , Proteínas Imediatamente Precoces/imunologia , Fosfoproteínas/imunologia , Tirosina/metabolismo , Proteínas Virais , Células 3T3 , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Células HeLa , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 2/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Fosforilação , Fosfotirosina/imunologia , Coelhos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Células Vero , Proteínas Virais Reguladoras e AcessóriasRESUMO
The role of signal transducers and activators of transcription (STATs) in receptor protein-tyrosine kinase (PTK)-induced cell growth and transformation was investigated using an inducible epidermal growth factor receptor-Ros chimeric receptor called ER2 and a constitutively activated insulin-like growth factor I receptor called NM1, both of which are able to induce anchorage-independent growth of NIH 3T3 cells. ER2 and NM1 receptor PTKs are able to cause Stat3 activation. Co-expressing the dominant negative Stat3 mutant with ER2 or NM1 in transiently or stable transfected cells resulted in a dramatic inhibition of colonies induced by these receptor PTKs and a moderate inhibition of their mitogenicity in monolayer. Therefore, Stat3 is not only important for initiation of transformation, as demonstrated by inhibition of the epidermal growth factor-inducible colony formation of the ER2 cells by the mutant, but it is also required for the maintenance of transformation, as evidenced by reversion of the NM1 transformed cells. The DNA binding and transcriptional activities of the endogenous Stat3 were greatly inhibited in the ER2 and NM1 cells co-expressing the Stat3 mutants. We conclude that activated function of Stat3 is required for the establishment and maintenance of Ros and insulin-like growth factor I receptor PTK-induced cell transformation.
Assuntos
Transformação Celular Neoplásica , Proteínas de Ligação a DNA/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptor IGF Tipo 1/metabolismo , Transativadores/metabolismo , Células 3T3 , Animais , Proteínas de Ligação a DNA/genética , Fator de Crescimento Epidérmico/farmacologia , Camundongos , Mutação , Fosforilação , Ligação Proteica , Fator de Transcrição STAT3 , Transdução de Sinais , Transativadores/genética , Transcrição GênicaRESUMO
The signaling functions of the oncogenic protein-tyrosine kinase v-Ros were studied by systematically mutating the tyrosine residues in its cytoplasmic domain. The carboxyl mutation of Tyr-564 produces the most pronounced inhibitory effect on v-Ros autophosphorylation and interaction with phospholipase Cgamma. A cluster of 3 tyrosine residues, Tyr-414, Tyr-418, and Tyr-419, within the PTK domain of v-Ros plays an important role in modulating its kinase activity. The mutant F419 and the mutant DI, deleting 6-amino acids near the catalytic loop, retain wild type protein tyrosine kinase and mitogenic activities, but have dramatically reduced oncogenicity. Both mutant proteins are able to phosphorylate or activate components in the Ras/microtubule-associated protein kinase signaling pathway. However, F419 mutant protein is unable to phosphorylate insulin receptor substrate 1 (IRS-1) or promote association of IRS-1 with phosphatidylinositol 3-kinase. This tyrosine residue in the context of the NDYY motif may define a novel recognition site for IRS-1. Both F419 and DI mutants display impaired ability to induce tyrosine phosphorylation of a series of cytoskeletal and cell-cell interacting proteins. Thus the F419 and DI mutations define v-Ros sequences important for cytoskeleton signaling, the impairment of which correlates with the reduced cell transforming ability.
Assuntos
Proteínas Oncogênicas Virais/metabolismo , Receptores Proteína Tirosina Quinases , Transdução de Sinais , Animais , Adesão Celular , Divisão Celular/genética , Linhagem Celular Transformada , Embrião de Galinha , Proteínas do Citoesqueleto/metabolismo , Ativação Enzimática , Proteínas Substratos do Receptor de Insulina , Mutagênese Sítio-Dirigida , Proteínas Oncogênicas Virais/genética , Fosfatidilinositol 3-Quinases , Fosfoproteínas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Especificidade por SubstratoRESUMO
To determine the sequences of the oncogenes src (encoded by Rous sarcoma virus [RSV]) and ros (encoded by UR2) that are responsible for causing different transformation phenotypes and to correlate those sequences with differences in substrate recognition, we constructed recombinants of the two transforming protein tyrosine kinases (PTKs) and studied their biological and biochemical properties. A recombinant with a 5' end from src and a 3' end from ros, called SRC x ROS, transformed chicken embryo fibroblasts (CEF) to a spindle shape morphology, mimicking that of UR2. Neither of the two reverse constructs, ROS x SRC I and ROS x SRC II, could transform CEF. However, a transforming variant of ROS x SRC II appeared during passages of the transfected cells and was called ROS x SRC (R). ROS x SRC (R) contains a 16-amino-acid deletion that includes the 3' half of the transmembrane domain of ros. Unlike RSV, ROS x SRC (R) also transformed CEF to an elongated shape similar to that of UR2. We conclude that distinct phenotypic changes of RSV- and UR2-infected cells do not depend solely on the kinase domains of their oncogenes. We next examined cellular proteins phosphorylated by the tyrosine kinases of UR2, RSV, and their recombinants as well as a number of other avian sarcoma viruses including Fujinami sarcoma virus Y73, and some ros-derived variants. Our results indicate that the UR2-encoded receptorlike PTK P68gag-ros and its derivatives have a very restricted substrate specificity in comparison with the nonreceptor PTKs encoded by the rest of the avian sarcoma viruses. Data from ros and src recombinants indicate that sequences both inside and outside the catalytic domains of ros and src exert a significant effect on the substrate specificity of the two recombinant proteins. Phosphorylation of most of the proteins in the 100- to 200-kDa range correlated with the presence of the 5' src domain, including the SH2 region, but not with the kinase domain in the recombinants. This corroborates the conclusion given above that the kinase domain of src or ros per se is not sufficient to dictate the transforming morphology of these two oncogenes. High-level tyrosyl phosphorylation of most of the prominent substrates of src is not sufficient to cause a round-shape transformation morphology.