RESUMO
Serological assays detecting IgM antibodies in addition to IgG antibodies have a diagnostic advantage in finding early infections. Staphylococcal protein A (SpA), widely used as an antibody-detecting reagent in various immunoassays, is considered to have a high binding affinity mainly to IgG, although its interaction with other classes of immunoglobulins has also been documented. Using 28 samples from 22 HIV-1 seroconversion panels, the present study demonstrated detection of early IgM antibodies by SpA-based rapid point-of-care tests, including DPP HIV 1/2, DPP HIV-Syphilis, STAT-PAK HIV 1/2, and Sure Check HIV 1/2. Samples with predominant IgM antibodies were identified by in-house IgM assays and confirmed by pretreatment with 0.1 M 2-mercaptoethanol. Likewise, the detection of treponemal IgM antibodies was shown by DPP HIV-Syphilis assay in eight samples collected at early syphilis infection. Direct interaction between IgM and SpA immobilized in solid phase or in solution was demonstrated with purified human polyclonal IgM. A strong correlation was found between the antibody levels detected by SpA and anti-IgM reagent in the early seroconversion samples, thus supporting the evidence for IgM binding by SpA. These assays demonstrated the ability to detect IgM antibodies, which may increase test sensitivity in early infections due to a reduced serodiagnostic window. IMPORTANCE Sexually transmitted infections, including HIV and syphilis, remain a global public health concern. The main laboratory testing approach for HIV and syphilis relies on serological assays. Detection of the IgM class of antibodies may have a diagnostic advantage in finding early infections. The present study using well-characterized HIV-1 and syphilis samples has demonstrated that staphylococcal protein A employed for antibody detection in rapid point-of-care tests, including DPP HIV 1/2, DPP HIV-Syphilis, STAT-PAK HIV 1/2, and Sure Check HIV 1/2, can capture IgM antibodies in addition to IgG antibodies. The findings strongly suggest that the ability to detect IgM antibodies by these immunoassays may facilitate the identification of acute-stage HIV and syphilis infections.
Assuntos
Infecções por HIV , Imunoglobulina M , Sífilis , Humanos , Anticorpos Antibacterianos , Infecções por HIV/diagnóstico , HIV-1 , Imunoglobulina G , Testes Imediatos , Sensibilidade e Especificidade , Proteína Estafilocócica A , Sífilis/diagnóstico , Treponema pallidumRESUMO
IQ motif-containing GTPase-activating protein 2 (IQGAP2) is a multidomain scaffolding protein that plays a role in cytoskeleton regulation by juxtaposing Rho GTPase and Ca2+/calmodulin signals. While IQGAP2 suppresses tumorigenesis in liver, its role in pathophysiology of the gastrointestinal tract remains unexplored. Here we report that IQGAP2 is required for the inflammatory response in colon. Mice lacking Iqgap2 gene (Iqgap2-/- mice) were resistant to chemically-induced colitis. Unlike wild-type controls, Iqgap2-/- mice treated with 3% dextran sulfate sodium (DSS) in water for 13 days displayed no injury to colonic epithelium. Mechanistically, resistance to colitis was associated with suppression of colonic NF-κB signaling and IL-6 synthesis, along with diminished neutrophil and macrophage production and recruitment in Iqgap2-/- mice. Finally, alterations in IQGAP2 expression were found in colons of patients with inflammatory bowel disease (IBD). Our findings indicate that IQGAP2 promotes inflammatory response at two distinct levels; locally, in colonic epithelium through TLR4/NF-κB signaling pathway, and systemically, via control of maturation and recruitment of myeloid immune cells. This work identifies a novel mechanism of colonic inflammation mediated by signal transducing scaffolding protein IQGAP2. IQGAP2 domain-specific blocking agents may represent a conceptually novel strategy for therapy of IBD and other inflammation-associated disorders, including cancer.
Assuntos
Colite/genética , Colo/metabolismo , Resistência a Medicamentos/genética , Proteínas Ativadoras de ras GTPase/genética , Animais , Western Blotting , Colite/induzido quimicamente , Colite/metabolismo , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Feminino , Expressão Gênica , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Humanos , Hiperplasia , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Contagem de Leucócitos , Camundongos da Linhagem 129 , Camundongos Knockout , Microscopia de Fluorescência , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
YopM is a leucine-rich repeat (LRR)-containing effector in several Yersinia species, including Yersinia pestis and Y. pseudotuberculosis. Different Yersinia strains encode distinct YopM isoforms with variable numbers of LRRs but conserved C-terminal tails. A 15-LRR isoform in Y. pseudotuberculosis YPIII was recently shown to bind and inhibit caspase-1 via a YLTD motif in LRR 10, and attenuation of YopM(-) YPIII was reversed in mice lacking caspase-1, indicating that caspase-1 inhibition is a major virulence function of YopM(YPIII). To determine if other YopM proteins inhibit caspase-1, we utilized Y. pseudotuberculosis strains natively expressing a 21-LRR isoform lacking the YLTD motif (YopM(32777)) or ectopically expressing a Y. pestis 15-LRR version with a functional (YopM(KIM)) or inactivated (YopM(KIM) D271A) YLTD motif. Results of mouse and macrophage infections with these strains showed that YopM(32777), YopM(KIM), and YopM(KIM) D271A inhibit caspase-1 activation, indicating that the YLTD motif is dispensable for this activity. Analysis of YopM(KIM) deletion variants revealed that LRRs 6 to 15 and the C-terminal tail are required to inhibit caspase-1 activation. YopM(32777), YopM(KIM), and YopM(KIM) deletion variants were purified, and binding partners in macrophage lysates were identified. Caspase-1 bound to YopM(KIM) but not YopM(32777). Additionally, YopM(KIM) bound IQGAP1 and the use of Iqgap1(-/-) macrophages revealed that this scaffolding protein is important for caspase-1 activation upon infection with YopM(-) Y. pseudotuberculosis. Thus, while multiple YopM isoforms inhibit caspase-1 activation, their variable LRR domains bind different host proteins to perform this function and the LRRs of YopM(KIM) target IQGAP1, a novel regulator of caspase-1, in macrophages. Importance: Activation of caspase-1, mediated by macromolecular complexes termed inflammasomes, is important for innate immune defense against pathogens. Pathogens can, in turn, subvert caspase-1-dependent responses through the action of effector proteins. For example, the Yersinia effector YopM inhibits caspase-1 activation by arresting inflammasome formation. This caspase-1 inhibitory activity has been studied in a specific YopM isoform, and in this case, the protein was shown to act as a pseudosubstrate to bind and inhibit caspase-1. Different Yersinia strains encode distinct YopM isoforms, many of which lack the pseudosubstrate motif. We studied additional isoforms and found that these YopM proteins inhibit caspase-1 activation independently of a pseudosubstrate motif. We also identified IQGAP1 as a novel binding partner of the Yersinia pestis YopM(KIM) isoform and demonstrated that IQGAP1 is important for caspase-1 activation in macrophages infected with Yersinia. Thus, this study reveals new insights into inflammasome regulation during Yersinia infection.
Assuntos
Proteínas de Bactérias/metabolismo , Caspase 1/metabolismo , Macrófagos/enzimologia , Yersinia pestis/metabolismo , Animais , Caspase 1/genética , Células Cultivadas , Feminino , Camundongos , Camundongos KnockoutRESUMO
Elevated bile acid levels increase hepatocellular carcinoma by unknown mechanisms. Here, we show that mice with a severe defect in bile acid homeostasis due to the loss of the nuclear receptors FXR and SHP have enlarged livers, progenitor cell proliferation, and Yes-associated protein (YAP) activation and develop spontaneous liver tumorigenesis. This phenotype mirrors mice with loss of hippo kinases or overexpression of their downstream target, YAP. Bile acids act as upstream regulators of YAP via a pathway dependent on the induction of the scaffold protein IQGAP1. Patients with diverse biliary dysfunctions exhibit enhanced IQGAP1 and nuclear YAP expression. Our findings reveal an unexpected mechanism for bile acid regulation of liver growth and tumorigenesis via the Hippo pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ácidos e Sais Biliares/metabolismo , Carcinogênese , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Fosfoproteínas/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Animais , Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular , Células Cultivadas , Criança , Pré-Escolar , Ativação Enzimática/genética , Fator de Crescimento de Hepatócito/genética , Via de Sinalização Hippo , Humanos , Lactente , Recém-Nascido , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Receptores Citoplasmáticos e Nucleares/genética , Serina-Treonina Quinase 3 , Fatores de Transcrição , Proteínas de Sinalização YAP , Proteínas Ativadoras de ras GTPase/biossíntese , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
It is broadly accepted that genetically engineered animal models do not always recapitulate human pathobiology. Therefore identifying best-fit mouse models of human cancers that truly reflect the corresponding human disease is of vital importance in elucidating molecular mechanisms of tumorigenesis and developing preventive and therapeutic approaches. A new hepatocellular carcinoma (HCC) mouse model lacking a novel putative tumor suppressor IQGAP2 has been generated by our laboratory. The aim of this study was to obtain the molecular signature of Iqgap2(-/-) HCC tumors and establish the relevance of this model to human disease. Here we report a comprehensive transcriptome analysis of Iqgap2(-/-) livers and a cross-species comparison of human and Iqgap2(-/-) HCC tumors using Significance Analysis of Microarray (SAM) and unsupervised hierarchical clustering analysis. We identified the Wnt/ß-catenin signaling pathway as the top canonical pathway dysregulated in Iqgap2(-/-) livers. We also demonstrated that Iqgap2(-/-) hepatic tumors shared genetic signatures with HCC tumors from patients with advanced disease as evidenced by a 78% mouse-to-human microarray data set concordance rate with 117 out of 151 identified ortholog genes having similar expression profiles across the two species. Collectively, these results indicate that the Iqgap2 knockout mouse model closely recapitulates human HCC at the molecular level and supports its further application for the study of this disease.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Perfilação da Expressão Gênica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas Ativadoras de ras GTPase/deficiência , Animais , Carcinoma Hepatocelular/metabolismo , Transformação Celular Neoplásica/genética , Análise por Conglomerados , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Estadiamento de Neoplasias , Reprodutibilidade dos Testes , Transdução de Sinais , Transcriptoma , Via de Sinalização Wnt , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
IQ motif-containing GTPase-activating proteins IQGAP1 and IQGAP2 are highly homologous multidomain scaffolding proteins. Their major function consists of integration of Rho GTPase and Ca(2+)/calmodulin signals with cell adhesive and cytoskeletal reorganizational events. Recent studies showed that they play an important role in carcinogenesis. There is growing evidence that IQGAP2 is a novel tumor suppressor counteracting the effects of IQGAP1, an oncogene, in several cancers, especially in hepatocellular carcinoma (HCC). While HCC is highly prevalent and one of the deadliest cancers worldwide, the signaling pathways involved are not fully understood and treatment of advanced disease still represents an area of high unmet medical need. This paper compiles various findings from studies in mouse models, cell lines, and patient samples that support future development of IQGAPs into new therapeutic targets. It also discusses distinct features of IQGAP2 in an attempt to provide insight into the mechanism of the seemingly paradoxical opposing roles of the two very similar IQGAP proteins in carcinogenesis.
RESUMO
Loss of IQGAP2 contributes to the tumorigenesis of hepatocellular carcinoma and gastric cancer. However, whether IQGAP2 also suppresses prostate tumorigenesis remains unclear. We report here that IQGAP2 is a candidate tumour suppressor of prostate cancer (PC). Elevated IQGAP2 was detected in prostatic intraepithelial neoplasia (PIN), early stages of PCs (Gleason score ≤3), and androgen-dependent LNCaP PC cells. However, IQGAP2 was expressed at substantially reduced levels not only in prostate glands and non-tumorigenic BPH-1 prostate epithelial cells but also in advanced (Gleason score 4 or 5) and androgen-independent PCs. Furthermore, xenograft tumours that were derived from stem-like DU145 cells displayed advanced features and lower levels of IQGAP2 in comparison to xenograft tumours that were produced from non stem-like DU145 cells. Collectively, these results suggest that IQGAP2 functions in the surveillance of prostate tumorigenesis. Consistent with this concept, ectopic IQGAP2 reduced the proliferation of DU145, PC3, and 293T cells as well as the invasion ability of DU145 cells. While ectopic IQGAP2 up-regulated E-cadherin in DU145 and PC3 cells, knockdown of IQGAP2 reduced E-cadherin expression. In primary PC and DU145 cells-derived xenograft tumours, the majority of tumours with high levels of IQGAP2 were strongly-positive for E-cadherin. Therefore, IQGAP2 may suppress PC tumorigenesis, at least in part, by up-regulation of E-cadherin. Mechanistically, overexpression of IQGAP2 significantly reduced AKT activation in DU145 cells and inhibition of AKT activation upregulated E-cadherin, suggesting that IQGAP2 increases E-cadherin expression by inhibiting AKT activation. Taken together, we demonstrate here that IQGAP2 is a candidate tumour suppressor of PC.
Assuntos
Transformação Celular Neoplásica , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Caderinas/biossíntese , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Próstata/metabolismo , Próstata/patologia , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasia Prostática Intraepitelial/patologia , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Transplante Heterólogo , Proteínas Supressoras de Tumor/genéticaRESUMO
UNLABELLED: Long-chain fatty acids (LCFA) serve as structural components for membrane biogenesis and as primary energy sources during mitochondrial ß-oxidation reactions. Hepatic LCFA uptake is complex, with characteristics suggestive of a dual-kinetic model manifested by rapid (carrier-assisted/facilitated) and delayed (passive diffusional) phases. Our previous work using mice deficient of the Iqgap2 gene established a highly novel link between IQGAP2, a putative GTPase-activating protein, and hepatocarcinogenesis. Now we report that Iqgap2 deficiency also results in selective loss of the facilitated phase of hepatocyte LCFA uptake with preservation of the diffusional component. This molecular defect was seen in Iqgap2(-/-) hepatocytes of all ages studied (1-, 4-, 8-months). The loss of facilitated LCFA uptake protected against development of hepatic triglyceride accumulation in Iqgap2-deficient mice fed high-fat diet, consistent with a fundamental role in physiological fat partitioning. These phenotypic changes could not be explained by genetic loss of fatty acid processing proteins known to regulate lipid uptake or metabolic processing pathways. Iqgap2-deficient livers also displayed enhanced insulin sensitivity. CONCLUSION: These observations identify a novel property of the putative GTPase-activating protein IQGAP2 in LCFA uptake in vitro and in vivo, and implicate IQGAP2 in an intracellular signaling pathway necessary for functional fatty acid uptake, lipid processing, and, possibly, glucose homeostasis.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos não Esterificados/metabolismo , Fígado Gorduroso/etiologia , Proteínas Ativadoras de ras GTPase/genética , Animais , Células Cultivadas , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Perfilação da Expressão Gênica , Teste de Tolerância a Glucose , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , PPAR alfa/genética , PPAR alfa/metabolismo , Transcrição Gênica , Triglicerídeos/sangue , Triglicerídeos/metabolismo , Aumento de Peso , Proteínas Ativadoras de ras GTPase/deficiênciaRESUMO
BACKGROUND: IQGAP1 and IQGAP2 are homologous members of the IQGAP family of scaffold proteins. Accumulating evidence implicates IQGAPs in tumorigenesis. We recently reported that IQGAP2 deficiency leads to the development of hepatocellular carcinoma (HCC) in mice. In the current study we extend these findings, and investigate IQGAP1 and IQGAP2 expression in human HCC. METHODS: IQGAP1 and IQGAP2 protein expression was assessed by Western blotting and immunohistochemistry. IQGAP mRNA was measured by quantitative RT-PCR. The methylation status of the Iqgap2 promoter was determined by pyrosequencing of bisulfite-treated genomic DNA. RESULTS: IQGAP1 and IQGAP2 expression was reciprocally altered in 6/6 liver cancer cell lines. Similarly, immunohistochemical staining of 82 HCC samples showed that IQGAP2 protein expression was reduced in 64/82 (78.0%), while IQGAP1 was present in 69/82 (84.1%). No IQGAP1 staining was detected in 23/28 (82.1%) normal livers, 4/4 (100.0%) hepatic adenomas and 23/23 (100.0%) cirrhosis cases, while IQGAP2 was increased in 22/28 (78.6%), 4/4 (100.0%) and 23/23 (100.0%), respectively. Although the Iqgap2 promoter was not hypermethylated in HCC at any of the 25 CpG sites studied (N = 17), IQGAP2 mRNA levels were significantly lower in HCC specimens (N = 23) than normal livers (N = 6). CONCLUSIONS: We conclude that increased IQGAP1 and/or decreased IQGAP2 contribute to the pathogenesis of human HCC. Furthermore, downregulation of IQGAP2 in HCC occurs independently of hypermethylation of the Iqgap2 promoter. Immunostaining of IQGAP1 and IQGAP2 may aid in the diagnosis of HCC, and their pharmacologic modulation may represent a novel therapeutic strategy for the treatment of liver cancer.
Assuntos
Carcinoma Hepatocelular/genética , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Proteínas Ativadoras de ras GTPase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Feminino , Predisposição Genética para Doença , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Células Tumorais Cultivadas , Proteínas Ativadoras de ras GTPase/biossínteseRESUMO
IQGAPs are multidomain scaffolding proteins that integrate Rho GTPase and Ca2+/calmodulin signals with cell adhesive and cytoskeletal reorganizational events. Targeted disruption of the murine Iqgap2 gene resulted in the age-dependent development of apoptosis and hepatocellular carcinoma (HCC), characterized by the overexpression of IQGAP1, the loss of membrane E-cadherin expression, the cytoplasmic translocation (and activation) of beta-catenin, and the overexpression of a nuclear target of beta-catenin, cyclin D1. In normal hepatocytes, IQGAP2 was found to exist as one component of a multifunctional scaffolding complex comprising IQGAP1, beta-catenin, and E-cadherin, with no evidence for direct IQGAP1-IQGAP2 interactions. Interbreeding of Iqgap2(-/-) mice into the Iqgap1(-/-) background resulted in the phenotypic correction of the preexisting hepatopathy, decreases in the incidence and sizes of HCC tumors, and the normalization of overall survival rates compared to those of Iqgap2(-/-) mice, suggesting that maximal penetrance of the Iqgap2(-/-) HCC phenotype requires the coordinate expression of IQGAP1. These results identify Iqgap2 as a novel tumor suppressor gene specifically linked to the development of HCC and the activation of the Wnt/beta-catenin signaling pathway, while also suggesting that IQGAP1 and IQGAP2 retain functionally divergent roles in hepatocellular carcinogenesis.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteínas Supressoras de Tumor/deficiência , Proteínas Ativadoras de ras GTPase/metabolismo , Envelhecimento/fisiologia , Fosfatase Alcalina/sangue , Alelos , Animais , Apoptose , Bilirrubina/sangue , Carcinoma Hepatocelular/genética , Cruzamentos Genéticos , Eletroporação , Células-Tronco Embrionárias/citologia , Fígado/ultraestrutura , Neoplasias Hepáticas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/ultraestrutura , Músculo Esquelético/ultraestrutura , Penetrância , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
OBJECTIVE: Two major protease-activated receptors (PARs), PAR1 and PAR4, are involved in the activation of human platelets by thrombin. A third, PAR3, is preferentially expressed by tissues of hematopoietic origin and megakaryocytes. Although PAR3 is also a thrombin substrate, its low-level expression on human platelets suggests a function distinct from that of PAR1, the major receptor involved in thrombin-mediated platelet activation. We studied the expression of PARs during megakaryocyte differentiation of human erythroleukemia (HEL) cells in order to determine the role of PAR3 in megakaryocytopoiesis. METHODS: HEL cells exposed to phorbol 12-myristate 13-acetate (PMA) to induce megakaryocyte differentiation were examined by light microscopy and flow cytometry (DNA ploidy, surface expression of PAR1, PAR3, GPIIb-IIIa). Northern blot, RT-PCR, and quantitative RT-PCR were used to evaluate the expression of PARs 1, 3, and 4 mRNA. HEL cells were also exposed to thrombin and thrombopoietin (TPO). RESULTS: In baseline studies, unstimulated HEL cells were found to express comparable levels of PAR1 and PAR3 by Northern blot. Minimal expression of PAR4 was detected by RT-PCR, but not by Northern analysis. Exposure to PMA, but not thrombin or TPO, resulted in megakaryocytic differentiation as evident by increased cell size and nuclear complexity, increased ploidy, and enhanced expression of GPIIb-IIIa, a specific marker of megakaryocytes/platelets. PMA-stimulated HEL cells showed enhanced PAR3 cell-surface expression (approximately threefold increase by day 2) by flow cytometry. In contrast, there was no change in cell-surface PAR1 expression. Northern blot analysis (approximately 10-fold) and quantitative RT-PCR (approximately threefold) confirmed the upregulation of PAR3 mRNA expression (by 24 hours) in cells exposed to PMA. This did not occur with exposure to TPO. CONCLUSION: These data demonstrate increased expression of PAR3 mRNA and protein in HEL cells undergoing megakaryocytic maturation following PMA exposure, suggesting a developmental role for PAR3. Furthermore, regulation of PAR3 expression appears to be specifically coupled to the protein kinase C system, but independent of the Ras/Raf/MAP kinase pathway.
Assuntos
Regulação Neoplásica da Expressão Gênica , Leucemia Eritroblástica Aguda/patologia , Megacariócitos/citologia , Receptores Ativados por Proteinase/genética , Receptores de Trombina/genética , Diferenciação Celular , Linhagem Celular Tumoral , Linhagem da Célula , DNA/análise , Humanos , Megacariócitos/imunologia , RNA Mensageiro/análise , Receptores de Superfície Celular/análise , Transdução de Sinais , Acetato de Tetradecanoilforbol , Regulação para CimaRESUMO
Thrombin has a critical role in many adult and embryologic cellular processes, exerting its effects through two high-affinity thrombin receptor systems: protease-activated receptor 1 (PAR1) and the PAR3/PAR4 system. Both hPAR1 and hPAR3 are coclustered in the human genome, with hPAR3 encompassed within hIQGAP2, a putative GTPase activating protein with actin polymerizing functions linked to cytoskeletal reorganization. Since hPARs colocalize with hIQGAP2 in the human genome and function coordinately with this protein in platelet thrombin signaling pathways, we have further characterized these genes in developing embryonic and adult tissues. We confirmed the presence of a mIQGAP2/ mPAR gene cluster on murine Chromosome 13 and showed it to be organized similarly to that in humans, except that murine PAR3 is translated off the forward (sense) strand. Northern analysis demonstrated limited mPAR3 expression in adult tissues, although its expression during embryogenesis was evident at E15 in cartilage, brain, and keratinocytes. mIQGAPs 1 and 2 had congruent expression patterns in 11 of 15 adult tissues studied. In contrast, whole embryos demonstrated predominant mIQGAP1 expression starting at E7 and evident to E17. In situ hybridization of whole embryos (E9-E16) demonstrated distinct patterns of tissue-dependent mIQGAP1/ mIQGAP2 expression. Concordant expression (absence or presence) of mPAR1 with either mIQGAP1 or mIQGAP2 was seen in the majority (12 of 15) of adult tissues studied. Similarly, there was no evidence for mPAR3 expression during embryogenesis in the absence of either mIQGAP1 or mIQGAP2. These data provide a panoramic survey of PAR/ IQGAP expression as an initial approach to dissect thrombin signaling pathways linked to cytoskeletal reorganization.
Assuntos
Citoesqueleto/metabolismo , Camundongos/embriologia , Trombina/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Animais , Expressão Gênica/fisiologia , Perfilação da Expressão Gênica , Humanos , Hibridização In Situ , Família Multigênica , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Sintenia , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
Human blood platelets are anucleate cells whose response to extracellular stimuli results in actin cytoskeleton rearrangements, thereby providing the critical initial step in the regulation of hemostasis. The serine protease alpha-thrombin, known to activate platelets by cleavage of a family of protease-activated receptors (PARs), is the most potent physiologic activator of human platelets, though downstream effector proteins uniquely linked to platelet cytoskeletal actin polymerization remain largely uncharacterized. The gene encoding the putative rac1/cdc42 effector protein IQGAP2 was identified within the PAR gene cluster at 5q13, flanked telomeric by PAR1 and encompassing PAR3. Immunofluorescence microscopy demonstrated IQGAP2 expression in filopodial extensions of activated platelets and colocalized with F-actin in lamellipodia and filopodia of IQGAP2-transfected COS1 cells. Platelet activation by alpha-thrombin, but not saturating concentrations of fibrillar collagen or adenosine 5'-diphosphate, uniquely assemble an IQGAP2/arp2/3-actin cytoplasmic complex, an association regulated by guanosine triphosphate rac1 ([GTP]rac1) but not by [GTP]cdc42. Likewise, only thrombin-activated platelets resulted in rapid translocation of IQGAP2 to the platelet cytoskeleton. These observations identify a physiologic scaffolding function for IQGAP2 and establish the presence of a functional genomic unit in humans uniquely evolved to regulate thrombin-induced platelet cytoskeletal actin reorganization.