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1.
Oncol Rep ; 49(2)2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36633146

RESUMO

Structural alterations of collagen impact signaling that helps control tumor progression and the responses to therapeutic intervention. Integrins represent a class of receptors that include members that mediate collagen signaling. However, a strategy of directly targeting integrins to control tumor growth has demonstrated limited activity in the clinical setting. New molecular understanding of integrins have revealed that these receptors can regulate both pro­ and anti­tumorigenic functions in a cell type­dependent manner. Therefore, designing strategies that block pro­tumorigenic signaling, without impeding anti­tumorigenic functions, may lead to development of more effective therapies. In the present study, evidence was provided for a novel signaling cascade in which ß3­integrin­mediated binding to a secreted RGDKGE­containing collagen fragment stimulates an autocrine­like signaling pathway that differentially governs the activity of both YAP and (protein kinase­A) PKA, ultimately leading to alterations in the levels of immune checkpoint molecule PD­L1 by a proteasome dependent mechanism. Selectively targeting this collagen fragment, reduced nuclear YAP levels, and enhanced PKA and proteasome activity, while also exhibiting significant antitumor activity in vivo. The present findings not only provided new mechanistic insight into a previously unknown autocrine­like signaling pathway that may provide tumor cells with the ability to regulate PD­L1, but our findings may also help in the development of more effective strategies to control pro­tumorigenic ß3­integrin signaling without disrupting its tumor suppressive functions in other cellular compartments.


Assuntos
Antígeno B7-H1 , Colágeno , Integrinas , Neoplasias , Fragmentos de Peptídeos , Complexo de Endopeptidases do Proteassoma , Humanos , Antígeno B7-H1/metabolismo , Colágeno/química , Colágeno/metabolismo , Integrinas/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas de Sinalização YAP/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo
2.
Am J Pathol ; 191(3): 527-544, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33307038

RESUMO

The growth and spread of malignant tumors, such as ovarian carcinomas, are governed in part by complex interconnected signaling cascades occurring between stromal and tumor cells. These reciprocal cross-talk signaling networks operating within the local tissue microenvironment may enhance malignant tumor progression. Understanding how novel bioactive molecules generated within the tumor microenvironment regulate signaling pathways in distinct cellular compartments is critical for the development of more effective treatment paradigms. Herein, we provide evidence that blocking cellular interactions with an RGDKGE-containing collagen peptide that selectively binds integrin ß3 on ovarian tumor cells enhances the phosphorylation of the hippo effector kinase large tumor suppressor kinase-1 and reduces nuclear accumulation of yes-associated protein and its target gene c-Myc. Selectively targeting this RGDKGE-containing collagen fragment inhibited ovarian tumor growth and the development of ascites fluid in vivo. These findings suggest that this bioactive collagen fragment may represent a previously unknown regulator of the hippo effector kinase large tumor suppressor kinase-1 and regulate ovarian tumor growth by a yes-associated protein-dependent mechanism. Taken together, these data not only provide new mechanistic insight into how a unique collagen fragment may regulate ovarian cancer, but in addition may help provide a useful new alternative strategy to control ovarian tumor progression based on selectively disrupting a previously unappreciated signaling cascade.


Assuntos
Biomarcadores Tumorais/metabolismo , Colágeno/metabolismo , Neoplasias Ovarianas/patologia , Fragmentos de Peptídeos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-yes/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL , Camundongos Nus , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-yes/genética , Transdução de Sinais , Células Tumorais Cultivadas , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Acc Chem Res ; 54(1): 120-131, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33291882

RESUMO

Pore forming toxins (PFTs) are the largest class of bacterial toxins playing a central role in bacterial pathogenesis. They are proteins specifically designed to form nanochannels in the membranes of target cells, ultimately resulting in cell death and establishing infection. PFTs are broadly classified as α- and ß-PFTs, depending on secondary structures that form the transmembrane channel. A unique feature about this class of proteins is the drastic conformational changes and complex oligomerization pathways that occur upon exposure to the plasma membrane. A molecular understanding of pore formation has implications in designing novel intervention strategies to combat rising antimicrobial resistance, targeted-cancer therapy, as well as designing nanopores for specialized technologies. Central to unraveling the pore formation pathway is the availability of high resolution crystal structures. In this regard, ß-toxins are better understood, when compared with α-toxins whose pore forming mechanisms are complicated by an incomplete knowledge of the driving forces for amphiphatic membrane-inserted helices to organize into functional pores. With the publication of the first crystal structure for an α-toxin, cytolysin A (ClyA), in 2009 we embarked on an extensive multiscale study to unravel its pore forming mechanism. This Account represents the collective mechanistic knowledge gained in our laboratories using a variety of experimental and theoretical techniques which include large scale molecular dynamics (MD) simulations, kinetic modeling studies, single-molecule fluorescence imaging, and super-resolution spectroscopy. We reported MD simulations of the ClyA protomer, oligomeric intermediates, and full pore complex in a lipid bilayer and mapped the conformational transitions that accompany membrane binding. Using single-molecule fluorescence imaging, the conformational transition was experimentally verified by analysis of various diffusion states of membrane bound ClyA. Importantly, we have uncovered a hitherto unknown putative cholesterol binding motif in the membrane-inserted helix of ClyA. Distinct binding pockets for cholesterol formed by adjacent membrane-inserted helices are revealed in MD simulations. Cholesterol appears to play a dual role by stabilizing both the membrane-inserted protomer as well as oligomeric intermediates. Molecular dynamics simulations and kinetic modeling studies suggest that the membrane-inserted arcs oligomerize reversibly to form the predominant transmembrane oligomeric intermediates during pore formation. We posit that this mechanistic understanding of the complex action of α-PFTs has implications in unraveling pore assembly across the wider family of bacterial toxins. With emerging antimicrobial resistance, alternate therapies may rely on disrupting pore functionality or oligomerization of these pathogenic determinants utilized by bacteria, and our study includes assessing the potential for dendrimers as pore blockers.


Assuntos
Bicamadas Lipídicas/metabolismo , Perforina/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Colesterol/química , Colesterol/metabolismo , Escherichia coli/metabolismo , Cinética , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Perforina/química , Estrutura Terciária de Proteína
4.
Oncogene ; 39(3): 722, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31530933

RESUMO

The original version of this Article did not acknowledge Pradeep Sathyanarayana as an author. His affiliation is Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine, USA.

5.
Proc Natl Acad Sci U S A ; 115(31): E7323-E7330, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30012608

RESUMO

Pore-forming toxins (PFTs) form nanoscale pores across target membranes causing cell death. Cytolysin A (ClyA) from Escherichia coli is a prototypical α-helical toxin that contributes to cytolytic phenotype of several pathogenic strains. It is produced as a monomer and, upon membrane exposure, undergoes conformational changes and finally oligomerizes to form a dodecameric pore, thereby causing ion imbalance and finally cell death. However, our current understanding of this assembly process is limited to studies in detergents, which do not capture the physicochemical properties of biological membranes. Here, using single-molecule imaging and molecular dynamics simulations, we study the ClyA assembly pathway on phospholipid bilayers. We report that cholesterol stimulates pore formation, not by enhancing initial ClyA binding to the membrane but by selectively stabilizing a protomer-like conformation. This was mediated by specific interactions by cholesterol-interacting residues in the N-terminal helix. Additionally, cholesterol stabilized the oligomeric structure using bridging interactions in the protomer-protomer interfaces, thereby resulting in enhanced ClyA oligomerization. This dual stabilization of distinct intermediates by cholesterol suggests a possible molecular mechanism by which ClyA achieves selective membrane rupture of eukaryotic cell membranes. Topological similarity to eukaryotic membrane proteins suggests evolution of a bacterial α-toxin to adopt eukaryotic motifs for its activation. Broad mechanistic correspondence between pore-forming toxins hints at a wider prevalence of similar protein membrane insertion mechanisms.


Assuntos
Colesterol/química , Proteínas de Escherichia coli/toxicidade , Proteínas Hemolisinas/toxicidade , Membrana Celular/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas Hemolisinas/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Multimerização Proteica
6.
Exp Hematol ; 50: 77-83.e6, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28408238

RESUMO

Podocalyxin (Podxl) is a CD34 orthologue and cell surface sialomucin reported to have roles in renal podocyte diaphragm slit development; vascular cell integrity; and the progression of blood, breast, and prostate cancers. Roles for Podxl during nonmalignant hematopoiesis, however, are largely undefined. We have developed a Vav-Cre Podxl knockout (KO) mouse model, and report on novel roles for Podxl in governing stress myelopoiesis. At steady state, Podxl expression among hematopoietic progenitor cells was low level but was induced by granulocyte colony-stimulating factor (G-CSF) in myeloid progenitors and by thrombopoietin in human stem cells. In keeping with low-level Podxl expression at steady state, Vav-Cre deletion of Podxl did not markedly alter peripheral blood cell levels. A G-CSF challenge in Podxl-KO mice, in contrast, hyperelevated peripheral blood neutrophil and monocyte levels. Podxl-KO also substantially heightened neutrophil levels after 5-fluorouracil myeloablation. These loss-of-function phenotypes were selective, and Podxl-KO did not alter lymphocyte, basophil, or eosinophil levels. Within bone marrow (and after G-CSF challenge), Podxl deletion moderately decreased colony forming units-granulocytes, eyrthrocytes, monocyte/macrophages, megakaryocytes and CD16/32posCD11bpos progenitors but did not affect Gr-1pos cell populations. Notably, Podxl-KO did significantly heighten peripheral blood neutrophil migration capacities. To interrogate Podxl's action mechanisms, a co-immunoprecipitation plus liquid chromatography-mass spectrometry approach was applied using hematopoietic progenitors from G-CSF-challenged mice. Rap1a, a Ras-related small GTPase, was a predominant co-retrieved Podxl partner. In bone marrow human progenitor cells, Podxl-KO led to heightened G-CSF activation of Rap1aGTP, and Rap1aGTP inhibition attenuated Podxl-KO neutrophil migration. Studies have revealed novel roles for Podxl as an important modulator of neutrophil and monocyte formation and of Rap1a activation during stress hematopoiesis.


Assuntos
Mielopoese , Neutrófilos/fisiologia , Sialoglicoproteínas/genética , Estresse Fisiológico , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Ordem dos Genes , Loci Gênicos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mielopoese/genética , Sialoglicoproteínas/metabolismo , Estresse Fisiológico/genética , Proteínas rap1 de Ligação ao GTP
7.
Biochemistry ; 55(42): 5952-5961, 2016 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-27682503

RESUMO

Pore-forming toxins (PFTs) bind to cell membranes and form nanoscale pores that allow leakage of cellular components, resulting in cell death. The water-soluble, monomeric form of these toxins shows a dramatic conformational change during pore formation, as exemplified by crystal structures of the monomer and functional pore of cytolysin A (ClyA). The solvent-exposed, C-terminal residues of the protein are essential for activity, but the mechanism by which this region regulates pore formation remains unknown. We show here that deletion of the C-terminus of ClyA did not alter its ability to bind to the membrane or oligomerize in detergent. However, the truncated toxin lysed erythrocytes poorly, was more susceptible to proteolysis and thermal unfolding, and showed low calcein leakage from small unilamellar vesicles. Using fully atomistic molecular dynamics (MD) simulations, we find that deletion of C-terminal residues from the ClyA monomer significantly altered stability and unfolding trajectories in the transmembrane N-terminal helix, a region that is pivotal in maintaining the structural integrity of the helical bundle. MD simulations of pores with or without the C-terminus showed minor differences, implying that if oligomerization could be induced prior to the addition to vesicles, then an active pore could be generated. Via generation of oligomers in a detergent prior to the addition to vesicles, the truncated toxin could induce calcein leakage from vesicles, albeit to a lower extent. Therefore, regions of pore-forming toxins, not directly involved in the pore structure, are not passive players but have important roles in undergoing the transition through intermediary steps leading to successful pore formation in a membrane environment.

8.
Exp Hematol Oncol ; 5: 4, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26848406

RESUMO

BACKGROUND: Dysregulation of miRNAs that can act as tumor suppressors or oncogenes can result in tumorigenesis. Previously we demonstrated that miR-199b was significantly downregulated in acute myeloid leukemia (AML) and targets podocalyxin and discoidin domain receptor 1. Herein we investigated the functional role of miR-199b in AML and its prognostic implications. METHODS: Major approaches include transduction of hematopoietic stem cells and bone marrow transplantation, analyses of blood lineages, histone deacetylases (HDAC) inhibitors, and molecular and clinical data analyses of AML patients using The Cancer Genome Atlas (TCGA). RESULTS: We first examined the relative miR-199b expression in steady state hematopoiesis and showed CD33(+) myeloid progenitors had the highest miR-199b expression. Further, silencing of miR-199b in CD34(+) cells resulted in significant increases in CFU-GM colonies. Via TCGA we analyzed the molecular and clinical characteristics of 166 AML cases to investigate a prognostic role for miR-199b. The Kaplan-Meier curves for high and low expression values of miR-199b and the observed distribution of miRNA expression revealed the highly expressed group had significantly better survival outcomes (p < 0.016, log rank test). Additionally, there was significant difference between miR-199b expression across the AML subtypes with particularly low expression found in the FAB-M5 subtype. Furthermore, FAB-M5 subtype showed a poor prognosis with a 1-year survival rate of only 25 %, compared with 51 % survival in the overall sample (p < 0.024). Furthermore, significant inverse correlation of HoxA7 and HoxB6 expression with miR-199b was observed in FAB-M5 AML patients. Molecular mutations were analyzed among miR-199b high and low AML cases. Significant correlations in terms of association and survival outcomes were observed for NPMc and IDH1 mutations. Treatment of THP-1 cells (represents M5-subtype) with HDAC inhibitors AR-42, Panobinostat, or Decitabine showed miR-199b expression was significantly elevated upon AR-42 and Panobinostat treatment. To further understand the hematopathological consequences of decreased miR-199b, we employed a bone-marrow transduce/transplant (BMT) mouse model. Interestingly, in vivo miR-199b silencing per-se in HSCs did not result in profound perturbations. CONCLUSIONS: Loss of miR-199b can lead to myeloproliferation while HDAC inhibitors restore miR-199b expression and promote apoptosis. Low miR-199b in AML patients correlates with worse overall survival and has prognostic significance for FAB-M5 subtype.

9.
Front Genet ; 5: 23, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24600468

RESUMO

The human genome encodes for over 1800 microRNAs (miRNAs), which are short non-coding RNA molecules that function to regulate gene expression post-transcriptionally. Due to the potential for one miRNA to target multiple gene transcripts, miRNAs are recognized as a major mechanism to regulate gene expression and mRNA translation. Computational prediction of miRNA targets is a critical initial step in identifying miRNA:mRNA target interactions for experimental validation. The available tools for miRNA target prediction encompass a range of different computational approaches, from the modeling of physical interactions to the incorporation of machine learning. This review provides an overview of the major computational approaches to miRNA target prediction. Our discussion highlights three tools for their ease of use, reliance on relatively updated versions of miRBase, and range of capabilities, and these are DIANA-microT-CDS, miRanda-mirSVR, and TargetScan. In comparison across all miRNA target prediction tools, four main aspects of the miRNA:mRNA target interaction emerge as common features on which most target prediction is based: seed match, conservation, free energy, and site accessibility. This review explains these features and identifies how they are incorporated into currently available target prediction tools. MiRNA target prediction is a dynamic field with increasing attention on development of new analysis tools. This review attempts to provide a comprehensive assessment of these tools in a manner that is accessible across disciplines. Understanding the basis of these prediction methodologies will aid in user selection of the appropriate tools and interpretation of the tool output.

10.
Cancer Med ; 3(2): 265-72, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24519883

RESUMO

Previously, we showed that discoidin domain receptor 1 (DDR1), a class of collagen-activated receptor tyrosine kinase (RTK) was highly upregulated on bone marrow (BM)-derived CD33+ leukemic blasts of acute myeloid leukemia (AML) patients. Herein as DDR1 is a class of collagen-activated RTK, we attempt to understand the role of native and remodeled collagen IV in BM microenvironment and its functional significance in leukemic cells. Exposure to denatured collagen IV significantly increased the migration and adhesion of K562 cells, which also resulted in increased activation of DDR1 and AKT. Further, levels of MMP9 were increased in conditioned media (CM) of denatured collagen IV exposed cells. Mass spectrometric liquid chromatography/tandem mass spectrometry QSTAR proteomic analysis revealed exclusive presence of Secretogranin 3 and InaD-like protein in the denatured collagen IV CM. Importantly, BM samples of AML patients exhibited increased levels of remodeled collagen IV compared to native as analyzed via anti-HUIV26 antibody. Taken together, for the first time, we demonstrate that remodeled collagen IV is a potent activator of DDR1 and AKT that also modulates both migration and adhesion of myeloid leukemia cells. Additionally, high levels of the HUIV26 cryptic collagen IV epitope are expressed in BM of AML patients. Further understanding of this phenomenon may lead to the development of therapeutic agents that directly modulate the BM microenvironment and attenuate leukemogenesis.


Assuntos
Colágeno Tipo IV/metabolismo , Leucemia Mieloide/patologia , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Colágeno Tipo IV/genética , Humanos , Células K562 , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Transfecção , Células Tumorais Cultivadas
11.
Leuk Res ; 38(3): 402-10, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24484870

RESUMO

microRNA profiling of acute myeloid leukemia patient samples identified miR-125a as being decreased. Current literature has investigated miR-125a's role in normal hematopoiesis but not within acute myeloid leukemia. Analysis of the upstream region of miR-125a identified several CpG islands. Both precursor and mature miR-125a increased in response to a de-methylating agent, Decitabine. Profiling revealed the ErbB pathway as significantly decreased with ectopic miR-125a. Either ectopic expression of miR-125a or inhibition of ErbB via Mubritinib resulted in inhibition of cell cycle proliferation and progression with enhanced apoptosis revealing ErbB inhibitors as potential novel therapeutic agents for treating miR-125a-low AML.


Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , MicroRNAs/genética , Proteínas Oncogênicas v-erbB/antagonistas & inibidores , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Ilhas de CpG/genética , Decitabina , Hematopoese/genética , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , MicroRNAs/metabolismo , Proteínas Oncogênicas v-erbB/genética , Proteínas Oncogênicas v-erbB/metabolismo , Transdução de Sinais
12.
PLoS One ; 7(7): e38530, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22808010

RESUMO

Certain concepts concerning EPO/EPOR action modes have been challenged by in vivo studies: Bcl-x levels are elevated in maturing erythroblasts, but not in their progenitors; truncated EPOR alleles that lack a major p85/PI3K recruitment site nonetheless promote polycythemia; and Erk1 disruption unexpectedly bolsters erythropoiesis. To discover novel EPO/EPOR action routes, global transcriptome analyses presently are applied to interrogate EPO/EPOR effects on primary bone marrow-derived CFUe-like progenitors. Overall, 160 EPO/EPOR target transcripts were significantly modulated 2-to 21.8-fold. A unique set of EPO-regulated survival factors included Lyl1, Gas5, Pim3, Pim1, Bim, Trib3 and Serpina 3g. EPO/EPOR-modulated cell cycle mediators included Cdc25a, Btg3, Cyclin-d2, p27-kip1, Cyclin-g2 and CyclinB1-IP-1. EPO regulation of signal transduction factors was also interestingly complex. For example, not only Socs3 plus Socs2 but also Spred2, Spred1 and Eaf1 were EPO-induced as negative-feedback components. Socs2, plus five additional targets, further proved to comprise new EPOR/Jak2/Stat5 response genes (which are important for erythropoiesis during anemia). Among receptors, an atypical TNF-receptor Tnfr-sf13c was up-modulated >5-fold by EPO. Functionally, Tnfr-sf13c ligation proved to both promote proerythroblast survival, and substantially enhance erythroblast formation. The EPOR therefore engages a sophisticated set of transcriptome response circuits, with Tnfr-sf13c deployed as one novel positive regulator of proerythroblast formation.


Assuntos
Eritroblastos/metabolismo , Eritropoese/genética , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Receptores da Eritropoetina/genética , Receptores do Fator de Necrose Tumoral/genética , Transcriptoma , Animais , Medula Óssea/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Eritroblastos/citologia , Eritropoetina/metabolismo , Eritropoetina/farmacologia , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Camundongos , Camundongos Transgênicos , Isoformas de Proteínas/metabolismo , Receptores da Eritropoetina/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais
13.
Blood ; 119(23): 5522-31, 2012 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-22508938

RESUMO

Sprouty proteins are established modifiers of receptor tyrosine kinase (RTK) signaling and play important roles in vasculogenesis, bone morphogenesis, and renal uteric branching. Little is understood, however, concerning possible roles for these molecular adaptors during hematopoiesis. Within erythroid lineage, Spry1 was observed to be selectively and highly expressed at CFU-e to erythroblast stages. In analyses of possible functional roles, an Mx1-Cre approach was applied to conditionally delete Spry1. At steady state, Spry1 deletion selectively perturbed erythroid development and led to reticulocytosis plus heightened splenic erythropoiesis. When challenged by hemolysis, Spry1-null mice exhibited worsened anemia and delayed recovery. During short-term marrow transplantation, Spry1-null donor marrow also failed to efficiently rescue the erythron. In each anemia model, however, hyperexpansion of erythroid progenitors was observed. Spry function depends on phosphorylation of a conserved N-terminal PY motif. Through an LC-MS/MS approach, Spry1 was discovered to be regulated via the erythropoietin receptor (EPOR), with marked EPO-induced Spry1-PY53 phosphorylation observed. When EPOR signaling pathways were analyzed within Spry1-deficient erythroid progenitors, hyperactivation of not only Erk1,2 but also Jak2 was observed. Studies implicate Spry1 as a novel regulator of erythropoiesis during anemia, transducer of EPOR signals, and candidate suppressor of Jak2 activity.


Assuntos
Eritropoese , Eritropoetina/metabolismo , Janus Quinase 2/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Receptores da Eritropoetina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Anemia/genética , Anemia/metabolismo , Animais , Transplante de Medula Óssea , Células Cultivadas , Ativação Enzimática , Eritroblastos/citologia , Eritroblastos/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfoproteínas/genética , Reticulócitos/citologia , Reticulócitos/metabolismo
15.
Leuk Res ; 36(3): 334-41, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22015065

RESUMO

Aberrations in IL-3, GM-CSF and G-CSF induced signaling are frequently reported in acute myeloid leukemia (AML). Herein, we utilized a unique human myeloid leukemic cell line, AML-193, which responds to all three cytokines to analyze the regulation at microRNA level. Using real-time PCR-based miRNA expression profiling, we investigated miRNA signatures regulated by IL-3, GM-CSF and G-CSF for n=704 miRNAs. We discovered that in addition to regulating specific miRNAs, these cytokines also regulate common set of miRNAs, which includes miR-590-5p, miR-219-5p, miR-15b and miR-628-5p. Taken together, we have identified novel candidate miRNAs that may be instructive during leukemic and normal hematopoiesis.


Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interleucina-3/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , MicroRNAs/genética , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Células Tumorais Cultivadas
16.
Blood ; 116(24): 5334-46, 2010 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-20810925

RESUMO

Investigations of bone marrow (BM) erythroblast development are important for clinical concerns but are hindered by progenitor cell and tissue availability. We therefore sought to more specifically define dynamics, and key regulators, of the formation of developing BM erythroid cell cohorts. A unique Kit(-)CD71(high)Ter119(-) "stage E2" proerythroblast pool first is described, which (unlike its Kit(+) "stage E1" progenitors, or maturing Ter119(+) "stage E3" progeny) proved to selectively expand ∼ 7-fold on erythropoietin challenge. During short-term BM transplantation, stage E2 proerythroblasts additionally proved to be a predominantly expanded progenitor pool within spleen. This E1→E2→E3 erythroid series reproducibly formed ex vivo, enabling further characterizations. Expansion, in part, involved E1 cell hyperproliferation together with rapid E2 conversion plus E2 stage restricted BCL2 expression. Possible erythropoietin/erythropoietin receptor proerythroblast stage specific events were further investigated in mice expressing minimal erythropoietin receptor alleles. For a hypomorphic erythropoietin receptor-HM allele, major defects in erythroblast development occurred selectively at stage E2. In addition, stage E2 cells proved to interact productively with primary BM stromal cells in ways that enhanced both survival and late-stage development. Overall, findings reveal a novel transitional proerythroblast compartment that deploys unique expansion devices.


Assuntos
Anemia , Proliferação de Células , Eritroblastos/citologia , Células Precursoras Eritroides/citologia , Eritropoetina/farmacologia , Alelos , Animais , Transplante de Medula Óssea , Comunicação Celular/fisiologia , Eritropoese , Camundongos , Camundongos Endogâmicos C57BL , Receptores da Eritropoetina/genética , Baço/citologia , Células Estromais
17.
Curr Opin Hematol ; 17(3): 169-76, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20173635

RESUMO

PURPOSE OF REVIEW: In 1985-1989, erythropoietin (EPO), its receptor (EPOR), and janus kinase 2 were cloned; established to be essential for definitive erythropoiesis; and initially intensely studied. Recently, new impetus, tools, and model systems have emerged to re-examine EPO/EPOR actions, and are addressed in this review. Impetus includes indications that EPO affects significantly more than standard erythroblast survival pathways, the development of novel erythropoiesis-stimulating agents, increasing evidence for EPO/EPOR cytoprotection of ischemically injured tissues, and potential EPO-mediated worsening of tumorigenesis. RECENT FINDINGS: New findings are reviewed in four functional contexts: (pro)erythroblast survival mechanisms, new candidate EPO/EPOR effects on erythroid cell development and new EPOR responses, EPOR downmodulation and trafficking, and novel erythropoiesis-stimulating agents. SUMMARY: As Current Opinion, this monograph seeks to summarize, and provoke, new EPO/EPOR action concepts. Specific problems addressed include: beyond (and before) BCL-XL, what key survival factors are deployed in early-stage proerythroblasts? Are distinct EPO/EPOR signals transduced in stage-selective fashions? Is erythroblast proliferation also modulated by EPO/EPOR signals? What functions are subserved by new noncanonical EPO/EPOR response factors (e.g. podocalyxin like-1, tribbles 3, reactive oxygen species, and nuclear factor kappa B)? What key regulators mediate EPOR inhibition and trafficking? And for emerging erythropoiesis-stimulating agents, to what extent do activities parallel EPOs (or differ in advantageous, potentially complicating ways, or both)?


Assuntos
Eritroblastos/fisiologia , Eritropoetina/metabolismo , Receptores da Eritropoetina/metabolismo , Transdução de Sinais , Animais , Humanos
18.
Cell Res ; 20(1): 89-98, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19918265

RESUMO

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-alpha (TNF-alpha) and specifically activates c-Jun N-terminal kinase (JNK) on TNF-alpha stimulation. The mechanism by which TNF-alpha activates MLK3 is still not known. TNF receptor-associated factors (TRAFs) are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half (amino acids 511-847). Endogenous TRAF2 and MLK3 associate with each other in response to TNF-alpha treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose-dependent manner, on TNF-alpha treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-alpha in a TRAF2-dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-alpha-induced activation of MLK3 and its downstream target, JNK.


Assuntos
MAP Quinase Quinase Quinases/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Domínio Catalítico/fisiologia , Relação Dose-Resposta a Droga , Fibroblastos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células Jurkat , Camundongos , Camundongos Knockout , Mutação/fisiologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Estrutura Terciária de Proteína/fisiologia , Fator 2 Associado a Receptor de TNF/química , Fator 2 Associado a Receptor de TNF/genética , Fator 5 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Ativação Transcricional , Fator de Necrose Tumoral alfa/farmacologia , MAP Quinase Quinase Quinase 11 Ativada por Mitógeno
19.
Blood ; 113(20): 4955-62, 2009 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-19264917

RESUMO

Anemia as associated with numerous clinical conditions can be debilitating, but frequently can be treated via administration of epoetin-alfa, darbepoietin-alfa, or methoxy-PEG epoetin-beta. Despite the complexity of EPO-EPO receptor interactions, the development of interesting EPO mimetic peptides (EMPs) also has been possible. CNTO 530 is one such novel MIMETIBODY Fc-domain dimeric EMP fusion protein. In a mouse model, single-dose CNTO 530 (unlike epoetin-alfa or darbepoietin-alfa) bolstered red cell production for up to 1 month. In 5-fluorouracil and carboplatin-paclitaxel models, CNTO 530 also protected against anemia with unique efficiency. These actions were not fully accounted for by half-life estimates, and CNTO 530 signaling events therefore were studied. Within primary bone marrow erythroblasts, kinetics of STAT5, ERK, and AKT activation were similar for CNTO 530 and epoetin-alfa. p70S6K activation by CNTO 530, however, was selectively sustained. In vivo, CNTO 530 uniquely stimulated the enhanced formation of PODXL(high)CD71(high) (pro)erythroblasts at frequencies multifold above epoetin-alfa or darbepoietin-alfa. CNTO 530 moreover supported the sustained expansion of a bone marrow-resident Kit(neg)CD71(high)Ter119(neg) progenitor pool. Based on these distinct erythropoietic and EPOR signaling properties, CNTO 530 holds excellent promise as a new EPO mimetic.


Assuntos
Células da Medula Óssea/efeitos dos fármacos , Eritroblastos/efeitos dos fármacos , Eritropoetina/análogos & derivados , Hematínicos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Anemia/patologia , Animais , Células da Medula Óssea/fisiologia , Contagem de Células , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Eritroblastos/fisiologia , Eritropoese/efeitos dos fármacos , Eritropoetina/química , Feminino , Hematínicos/química , Camundongos , Camundongos Endogâmicos C57BL , Mimetismo Molecular , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/química , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo
20.
Exp Hematol ; 37(2): 159-71, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19100679

RESUMO

OBJECTIVE: KITL/KIT can elicit diverse sets of signals within lymphoid, myeloid, mast, and erythroid lineages, and exert distinct effects on growth, survival, migration, adhesion, and secretory responses. Presently, we have applied a PY-mutant allele knockin approach to specifically assess possible roles for KIT-PY567 and KIT-PY719 sites, and coupled pathways, during erythropoiesis. MATERIALS AND METHODS: Mouse models used to investigate this problem include those harboring knocked-in KIT(Y567F/Y567F), KIT(Y569F/Y569F), KIT(Y719F,Y719F), and KIT(Y567F/Y567F:Y569F/Y569F) alleles. The erythron was stressed by myelosuppression using 5-fluorouracil, and by phenylhydrazine-induced hemolysis. In addition, optimized systems for ex vivo analyses of bone marrow and splenic erythropoiesis were employed to more directly analyze possible stage-specific effects on erythroid cell growth, survival, development and KIT signaling events. RESULTS: In Kit(Y567F/Y567F) mice, steady-state erythropoiesis was unperturbed while recovery from anemia due to 5-fluorouracil or phenylhydrazine was markedly impaired. Deficiencies in erythroid progenitor expansion occurred both in the bone marrow and the spleen. Responses to chronic erythropoietin dosing were also compromised. Ex vivo, Kit(Y567F/Y567F) (pro)erythroblast development was skewed from a Kit(pos)CD71(high) stage toward a subsequent Kit(neg)CD71(high) compartment. Proliferation and, to an extent, survival capacities were also compromised. Similar stage-specific defects existed for erythroid progenitors from Kit(Y567F/Y567F:Y569F/Y569F) but not KIT(Y719F/Y719F) mice. Kit(Y567F/Y567F) erythroblasts were used further to analyze KIT-PY567-dependent signals. MEK-1,2/ERK-1,2 signaling was unaffected while AKT, p70S6K, and especially JNK2/p54 pathways were selectively attenuated. CONCLUSIONS: Nonredundant KIT-PY567-directed erythroblast-intrinsic signals are selectively critical for stress erythropoiesis. Investigations also add to an understanding of how KIT directs distinct outcomes among diverse progenitors and lineages.


Assuntos
Anemia/metabolismo , Células Precursoras Eritroides/metabolismo , Eritropoese , Sistema de Sinalização das MAP Quinases , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Substituição de Aminoácidos , Anemia/induzido quimicamente , Anemia/genética , Animais , Antimetabólitos Antineoplásicos/efeitos adversos , Antimetabólitos Antineoplásicos/farmacologia , Eritropoese/genética , Fluoruracila/efeitos adversos , Fluoruracila/farmacologia , Hemólise/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Oxidantes/toxicidade , Fenil-Hidrazinas/toxicidade , Proteínas Quinases/genética , Proteínas Proto-Oncogênicas c-kit/genética , Fator de Células-Tronco/genética , Fator de Células-Tronco/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética
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