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1.
Int J Cancer ; 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31469414

RESUMO

Methylation of the promoter of the BRCA1 gene in DNA derived from peripheral blood cells is a possible risk factor for breast cancer. It is not clear if this association is restricted to certain types of breast cancer or is a general phenomenon. We evaluated BRCA1 methylation status in peripheral blood cells from 942 breast cancer patients and from 500 controls. We also assessed methylation status in 262 paraffin-embedded breast cancer tissues. Methylation status was assessed using methylation-sensitive high-resolution melting and was categorized as positive or negative. BRCA1 methylation in peripheral blood cells was strongly associated with the risk of triple-negative breast cancer (TNBC) (odds ratio [OR] 4.70; 95% confidence interval [CI]: 3.13-7.07; p < 0.001), but not of estrogen-receptor positive breast cancer (OR 0.80; 95% CI: 0.46-1.42; p = 0.46). Methylation was also overrepresented among patients with high-grade cancers (OR 4.53; 95% CI: 2.91-7.05; p < 0.001) and medullary cancers (OR 3.08; 95% CI: 1.38-6.88; p = 0.006). Moreover, we detected a significant concordance of BRCA1 promoter methylation in peripheral blood and paired tumor tissue (p < 0.001). We found that BRCA1 promoter methylation in peripheral blood cells is associated with approximately five times greater risk of TNBC. We propose that BRCA1 methylation in blood-derived DNA could be a novel biomarker of increased breast cancer susceptibility, in particular for triple-negative tumors.

2.
Br J Cancer ; 121(2): 180-192, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31213659

RESUMO

BACKGROUND: Height and body mass index (BMI) are associated with higher ovarian cancer risk in the general population, but whether such associations exist among BRCA1/2 mutation carriers is unknown. METHODS: We applied a Mendelian randomisation approach to examine height/BMI with ovarian cancer risk using the Consortium of Investigators for the Modifiers of BRCA1/2 (CIMBA) data set, comprising 14,676 BRCA1 and 7912 BRCA2 mutation carriers, with 2923 ovarian cancer cases. We created a height genetic score (height-GS) using 586 height-associated variants and a BMI genetic score (BMI-GS) using 93 BMI-associated variants. Associations were assessed using weighted Cox models. RESULTS: Observed height was not associated with ovarian cancer risk (hazard ratio [HR]: 1.07 per 10-cm increase in height, 95% confidence interval [CI]: 0.94-1.23). Height-GS showed similar results (HR = 1.02, 95% CI: 0.85-1.23). Higher BMI was significantly associated with increased risk in premenopausal women with HR = 1.25 (95% CI: 1.06-1.48) and HR = 1.59 (95% CI: 1.08-2.33) per 5-kg/m2 increase in observed and genetically determined BMI, respectively. No association was found for postmenopausal women. Interaction between menopausal status and BMI was significant (Pinteraction < 0.05). CONCLUSION: Our observation of a positive association between BMI and ovarian cancer risk in premenopausal BRCA1/2 mutation carriers is consistent with findings in the general population.

3.
Nat Commun ; 10(1): 1741, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988301

RESUMO

Genome-wide association studies (GWAS) have identified more than 170 breast cancer susceptibility loci. Here we hypothesize that some risk-associated variants might act in non-breast tissues, specifically adipose tissue and immune cells from blood and spleen. Using expression quantitative trait loci (eQTL) reported in these tissues, we identify 26 previously unreported, likely target genes of overall breast cancer risk variants, and 17 for estrogen receptor (ER)-negative breast cancer, several with a known immune function. We determine the directional effect of gene expression on disease risk measured based on single and multiple eQTL. In addition, using a gene-based test of association that considers eQTL from multiple tissues, we identify seven (and four) regions with variants associated with overall (and ER-negative) breast cancer risk, which were not reported in previous GWAS. Further investigation of the function of the implicated genes in breast and immune cells may provide insights into the etiology of breast cancer.


Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Locos de Características Quantitativas
4.
PLoS One ; 14(1): e0208610, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30640897

RESUMO

BACKGROUND: Lung cancer is the most common adult malignancy accounting for the largest proportion of cancer related deaths. Iron (Fe) is an essential trace element and is a component of several major metabolic pathways playing an important role in many physiological processes. In this study we evaluated the association between Fe concentration in serum, iron metabolism parameters and genetic variaton in 7 genes involved in iron metabolism and anti-oxidative processes with the incidence of lung cancer in Poland. MATERIALS AND METHODS: The study included 200 lung cancer patients and 200 matched healthy control subjects. We analyzed serum iron concentration and iron metabolism parameters (TIBC, UIBC, serum ferritin and transferrin saturation), and genotyped seven variants in seven genes: HFE, TFR1, HAMP, TF, SOD2, CAT and GPX1. RESULTS: Lung cancer patients compared to their matched controls had significantly higher mean serum iron level (p = 0.01), ferritin level (p = 0.007) and TIBC (p = 0.006). Analysis revealed that higher concentration of iron and ferritin (IVth quartile) compared to the lower concentration (Ist quartile) was associated with over 2-fold increased lung cancer incidence. We also found that higher transferrin saturation (p = 0.01) and lower TIBC (p<0.01) are associated with better survival of lung cancer patients. The analysis of polymorphisms in iron related genes did not reveal a significant difference between lung cancer patients and controls. However, rs10421768 in HAMP showed a borderline statistically significant correlation with lung cancer risk (OR = 2.83, p = 0.05). CONCLUSIONS: The results of this case control study indicate that higher body iron represented by higher Fe and ferritin levels may be associated with lung cancer incidence. Rs10421768 in HAMP may be associated with about 3-times higher lung cancer risk. Higher Fe body content may be associated with better survival of lung cancer patients.


Assuntos
Antioxidantes/metabolismo , Ferro/sangue , Ferro/metabolismo , Neoplasias Pulmonares/epidemiologia , Neoplasias Pulmonares/genética , Variação Genética , Humanos , Incidência , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Estadiamento de Neoplasias , Fatores de Risco , Análise de Sobrevida
5.
Nat Commun ; 9(1): 4465, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30367047

RESUMO

Dynamic communication between integrin-containing complexes (focal adhesions, FAs) and actin filaments is critical for regulating cell adhesion. Pseudokinase ILK plays a key role in this process but the underlying mechanism remains highly elusive. Here we show that by recruiting FA adaptors PINCH and Parvin into a heterotrimeric complex (IPP), ILK triggers F-actin filament bundling - a process known to generate force/mechanical signal to promote cytoskeleton reassembly and dynamic cell adhesion. Structural, biochemical, and functional analyses revealed that the F-actin bundling is orchestrated by two previously unrecognized WASP-Homology-2 actin binding motifs within IPP, one from PINCH and the other from Parvin. Strikingly, this process is also sensitized to Mg-ATP bound to the pseudoactive site of ILK and its dysregulation severely impairs stress fibers formation, cell spreading, and migration. These data identify a crucial mechanism for ILK, highlighting its uniqueness as a pseudokinase to transduce non-catalytic signal and regulate cell adhesion.

6.
PLoS One ; 13(7): e0201065, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30036379

RESUMO

INTRODUCTION: Prostate cancer is one of the most commonly diagnosed malignancies among men in Western populations. Evidence reported in the literature suggests that zinc may be related to prostate cancer. In this study we evaluated the association of serum zinc levels and polymorphisms in genes encoding zinc-dependent proteins with prostate cancer in Poland. METHODS: The study group consisted of 197 men affected with prostate cancer and 197 healthy men. Serum zinc levels were measured and 5 single nucleotide polymorphisms in MMP-1, MMP-2, MMP-7, MMP-13, MT2A genes were genotyped. RESULTS: The mean serum zinc level was higher in prostate cancer patients than in healthy controls (898.9±12.01 µg/l vs. 856.6±13.05 µg/l, p<0.01). When compared in quartiles a significant association of higher zinc concentration with the incidence of prostate cancer was observed. The highest OR (OR = 4.41, 95%CI 2.07-9.37, p<0.01) was observed in 3rd quartile (>853.0-973.9 µg/l). Among five analyzed genetic variants, rs11568818 in MMP-7 appeared to be correlated with 2-fold increased prostate cancer risk (OR = 2.39, 95% CI = 1.19-4.82, p = 0.015). CONCLUSION: Our results suggest a significant correlation of higher serum zinc levels with the diagnosis of prostate cancer. The polymorphism rs11568818 in MMP-7 gene was also associated with an increased prostate cancer risk in Poland.

7.
J Physiol ; 595(20): 6443-6462, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28799653

RESUMO

KEY POINTS: A reduction in Kindlin-2 levels in endothelial cells compromises vascular barrier function. Kindlin-2 is a previously unrecognized component of endothelial adherens junctions. By interacting directly and simultaneously with ß- or γ-catenin and cortical actin filaments, Kindlin-2 stabilizes adherens junctions. The Kindlin-2 binding sites for ß- and γ-catenin reside within its F1 and F3 subdomains. Although Kindlin-2 does not associate directly with tight junctions, its downregulation also destabilizes these junctions. Thus, impairment of both adherens and tight junctions may contribute to enhanced leakiness of vasculature in Kindlin-2+/- mice. ABSTRACT: Endothelial cells (EC) establish a physical barrier between the blood and surrounding tissue. Impairment of this barrier can occur during inflammation, ischaemia or sepsis and cause severe organ dysfunction. Kindlin-2, which is primarily recognized as a focal adhesion protein in EC, was not anticipated to have a role in vascular barrier. We tested the role of Kindlin-2 in regulating vascular integrity using several different approaches to decrease Kindlin-2 levels in EC. Reduced levels of Kindlin-2 in Kindlin-2+/- mice aortic endothelial cells (MAECs) from these mice, and human umbilical ECs (HUVEC) treated with Kindlin-2 siRNA showed enhanced basal and platelet-activating factor (PAF) or lipopolysaccharide-stimulated vascular leakage compared to wild-type (WT) counterparts. PAF preferentially disrupted the Kindlin-2+/- MAECs barrier to BSA and dextran and reduced transendothelial resistance compared to WT cells. Kindlin-2 co-localized and co-immunoprecipitated with vascular endothelial cadherin-based complexes, including ß- and γ-catenin and actin, components of adherens junctions (AJ). Direct interaction of Kindlin-2 with ß- and γ-catenin and actin was demonstrated in co-immunoprecipitation and surface plasmon resonance experiments. In thrombin-stimulated HUVECs, Kindlin-2 and cortical actin dissociated from stable AJs and redistributed to radial actin stress fibres of remodelling focal AJs. The ß- and γ-catenin binding site resides within the F1 and F3 subdomains of Kindlin-2 but not the integrin binding site in F3. These results establish a previously unrecognized and vital role of Kindlin-2 with respect to maintaining the vascular barrier by linking Vascuar endothelial cadherin-based complexes to cortical actin and thereby stabilizing AJ.


Assuntos
Junções Aderentes/fisiologia , Proteínas do Citoesqueleto/fisiologia , Células Endoteliais/fisiologia , Proteínas Musculares/fisiologia , Animais , Aorta/citologia , Sítios de Ligação , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células Endoteliais/metabolismo , Feminino , Células HEK293 , Humanos , Pulmão/irrigação sanguínea , Pulmão/fisiologia , Masculino , Camundongos Transgênicos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Domínios Proteicos , Pele/irrigação sanguínea , Fenômenos Fisiológicos da Pele , Traqueia/irrigação sanguínea , Traqueia/fisiologia , Veias Umbilicais/citologia , beta Catenina/metabolismo
8.
Cancer Res ; 77(18): 5129-5141, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28687620

RESUMO

Interplay between tumor cells and host cells in the tumor microenvironment dictates the development of all cancers. In breast cancer, malignant cells educate host macrophages to adopt a protumorigenic phenotype. In this study, we show how the integrin-regulatory protein kindlin-2 (FERMT2) promotes metastatic progression of breast cancer through the recruitment and subversion of host macrophages. Kindlin-2 expression was elevated in breast cancer biopsy tissues where its levels correlated with reduced patient survival. On the basis of these observations, we used CRISPR/Cas9 technology to ablate Kindlin-2 expression in human MDA-MB-231 and murine 4T1 breast cancer cells. Kindlin-2 deficiency inhibited invasive and migratory properties in vitro without affecting proliferation rates. However, in vivo tumor outgrowth was inhibited by >80% in a manner associated with reduced macrophage infiltration and secretion of the macrophage attractant and growth factor colony-stimulating factor-1 (CSF-1). The observed loss of CSF-1 appeared to be caused by a more proximal deficiency in TGFß-dependent signaling in Kindlin-2-deficient cells. Collectively, our results illuminate a Kindlin-2/TGFß/CSF-1 signaling axis employed by breast cancer cells to capture host macrophage functions that drive tumor progression. Cancer Res; 77(18); 5129-41. ©2017 AACR.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos Peritoneais/patologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Apoptose , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Humanos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Biol Chem ; 292(34): 14258-14269, 2017 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-28652408

RESUMO

Kindlin-2 (K2), a 4.1R-ezrin-radixin-moesin (FERM) domain adaptor protein, mediates numerous cellular responses, including integrin activation. The C-terminal 15-amino acid sequence of K2 is remarkably conserved across species but is absent in canonical FERM proteins, including talin. In CHO cells expressing integrin αIIbß3, co-expression of K2 with talin head domain resulted in robust integrin activation, but this co-activation was lost after deletion of as few as seven amino acids from the K2 C terminus. This dependence on the C terminus was also observed in activation of endogenous αIIbß3 in human erythroleukemia (HEL) cells and ß1 integrin activation in macrophage-like RAW264.1 cells. Kindlin-1 (K1) exhibited a similar dependence on its C terminus for integrin activation. Expression of the K2 C terminus as an extension of membrane-anchored P-selectin glycoprotein ligand-1 (PSGL-1) inhibited integrin-dependent cell spreading. Deletion of the K2 C terminus did not affect its binding to the integrin ß3 cytoplasmic tail, but combined biochemical and NMR analyses indicated that it can insert into the F2 subdomain. We suggest that this insertion determines the topology of the K2 FERM domain, and its deletion may affect the positioning of the membrane-binding functions of the F2 subdomain and the integrin-binding properties of its F3 subdomain. Free C-terminal peptide can still bind to K2 and displace the endogenous K2 C terminus but may not restore the conformation needed for integrin co-activation. Our findings indicate that the extreme C terminus of K2 is essential for integrin co-activation and highlight the importance of an atypical architecture of the K2 FERM domain in regulating integrin activation.


Assuntos
Integrina alfa2/metabolismo , Integrina beta3/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Substituição de Aminoácidos , Animais , Células CHO , Linhagem Celular Tumoral , Cricetulus , Deleção de Genes , Humanos , Integrina alfa2/química , Integrina alfa2/genética , Integrina beta3/química , Integrina beta3/genética , Leucemia Eritroblástica Aguda/patologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Macrófagos/citologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Mutação , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Células RAW 264.7 , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Talina/química , Talina/genética , Talina/metabolismo
10.
Discoveries (Craiova) ; 4(2)2016 Apr-Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27500205

RESUMO

Kindlins are 4.1-ezrin-ridixin-moesin (FERM) domain containing proteins. There are three kindlins in mammals, which share high sequence identity. Kindlin-1 is expressed primarily in epithelial cells, kindlin-2 is widely distributed and is particularly abundant in adherent cells, and kindlin-3 is expressed primarily in hematopoietic cells. These distributions are not exclusive; some cells express multiple kindlins, and transformed cells often exhibit aberrant expression, both in the isoforms and the levels of kindlins. Great interest in the kindlins has emerged from the recognition that they play major roles in controlling integrin function. In vitro studies, in vivo studies of mice deficient in kindlins, and studies of patients with genetic deficiencies of kindlins have clearly established that they regulate the capacity of integrins to mediate their functions. Kindlins are adaptor proteins; their function emanate from their interaction with binding partners, including the cytoplasmic tails of integrins and components of the actin cytoskeleton. The purpose of this review is to provide a brief overview of kindlin structure and function, a consideration of their binding partners, and then to focus on the relationship of each kindlin family member with cancer. In view of many correlations of kindlin expression levels and neoplasia and the known association of integrins with tumor progression and metastasis, we consider whether regulation of kindlins or their function would be attractive targets for treatment of cancer.

11.
J Cell Biol ; 213(1): 97-108, 2016 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-27044892

RESUMO

Reduced levels of kindlin-2 (K2) in endothelial cells derived from K2(+/-)mice or C2C12 myoblastoid cells treated with K2 siRNA showed disorganization of their actin cytoskeleton and decreased spreading. These marked changes led us to examine direct binding between K2 and actin. Purified K2 interacts with F-actin in cosedimentation and surface plasmon resonance analyses and induces actin aggregation. We further find that the F0 domain of K2 binds actin. A mutation, LK(47)/AA, within a predicted actin binding site (ABS) of F0 diminishes its interaction with actin by approximately fivefold. Wild-type K2 and K2 bearing the LK(47)/AA mutation were equivalent in their ability to coactivate integrin αIIbß3 in a CHO cell system when coexpressed with talin. However, K2-LK(47)/AA exhibited a diminished ability to support cell spreading and actin organization compared with wild-type K2. The presence of an ABS in F0 of K2 that influences outside-in signaling across integrins establishes a new foundation for considering how kindlins might regulate cellular responses.


Assuntos
Actinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação/fisiologia , Células CHO , Adesão Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Cricetulus , Proteínas do Citoesqueleto/metabolismo , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Talina/metabolismo
12.
J Lipid Res ; 56(3): 515-25, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25593327

RESUMO

Cells produce two cholesteryl ester transfer protein (CETP) isoforms, full-length and a shorter variant produced by alternative splicing. Blocking synthesis of both isoforms disrupts lipid metabolism and storage. To further define the role of CETP in cellular lipid metabolism, we stably overexpressed full-length CETP in SW872 cells. These CETP(+) cells had several-fold higher intracellular CETP and accumulated 50% less TG due to a 26% decrease in TG synthesis and 2.5-fold higher TG turnover rate. Reduced TG synthesis was due to decreased fatty acid uptake and impaired conversion of diglyceride to TG even though diacylglycerol acyltransferase activity was normal. Sterol-regulatory element binding protein 1 mRNA levels were normal, and although PPARγ expression was reduced, the expression of several of its target genes including adipocyte triglyceride lipase, FASN, and APOE was normal. CETP(+) cells contained smaller lipid droplets, consistent with their higher levels of perilipin protein family (PLIN) 3 compared with PLIN1 and PLIN2. Intracellular CETP was mostly associated with the endoplasmic reticulum, although CETP near lipid droplets poorly colocalized with this membrane. A small pool of CETP resided in the cytoplasm, and a subfraction coisolated with lipid droplets. These data show that overexpression of full-length CETP disrupts lipid homeostasis resulting in the formation of smaller, more metabolically active lipid droplets.


Assuntos
Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Citoplasma/metabolismo , Metabolismo dos Lipídeos/fisiologia , Triglicerídeos/metabolismo , Apolipoproteínas E/biossíntese , Apolipoproteínas E/genética , Linhagem Celular Tumoral , Proteínas de Transferência de Ésteres de Colesterol/genética , Citoplasma/genética , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , PPAR gama/biossíntese , PPAR gama/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/genética
13.
J Biol Chem ; 290(10): 6226-42, 2015 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-25609252

RESUMO

The contributions of integrins to cellular responses depend upon their activation, which is regulated by binding of proteins to their cytoplasmic tails. Kindlins are integrin cytoplasmic tail binding partners and are essential for optimal integrin activation, and kindlin-3 fulfills this role in hematopoietic cells. Here, we used human platelets and human erythroleukemia (HEL) cells, which express integrin αIIbß3, to investigate whether phosphorylation of kindlin-3 regulates integrin activation. When HEL cells were stimulated with thrombopoietin or phorbol 12-myristate 13-acetate (PMA), αIIbß3 became activated as evidenced by binding of an activation-specific monoclonal antibody and soluble fibrinogen, adherence and spreading on fibrinogen, colocalization of ß3 integrin and kindlin-3 in focal adhesions, and enhanced ß3 integrin-kindlin-3 association in immunoprecipitates. Kindlin-3 knockdown impaired adhesion and spreading on fibrinogen. Stimulation of HEL cells with agonists significantly increased kindlin-3 phosphorylation as detected by mass spectrometric sequencing. Thr(482) or Ser(484) was identified as a phosphorylation site, which resides in a sequence not conserved in kindlin-1 or kindlin-2. These same residues were phosphorylated in kindlin-3 when platelets were stimulated with thrombin. When expressed in HEL cells, T482A/S484A kindlin-3 decreased soluble ligand binding and cell spreading on fibrinogen compared with wild-type kindlin-3. A membrane-permeable peptide containing residues 476-485 of kindlin-3 was introduced into HEL cells and platelets; adhesion and spreading of both cell types were blunted compared with a scrambled control peptide. These data identify a role of kindlin-3 phosphorylation in integrin ß3 activation and provide a basis for functional differences between kindlin-3 and the two other kindlin paralogs.


Assuntos
Plaquetas/metabolismo , Hematopoese/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Animais , Células CHO , Adesão Celular/genética , Cricetinae , Cricetulus , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Fosforilação/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo
14.
Arterioscler Thromb Vasc Biol ; 34(9): 1961-7, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24969775

RESUMO

OBJECTIVE: Kindlin-3 is a critical supporter of integrin function in platelets. Lack of expression of kindlin-3 protein in patients impairs integrin αIIbß3-mediated platelet aggregation. Although kindlin-3 has been categorized as an integrin-binding partner, the functional significance of the direct interaction of kindlin-3 with integrin αIIbß3 in platelets has not been established. Here, we evaluated the significance of the binding of kindlin-3 to integrin αIIbß3 in platelets in supporting integrin αIIbß3-mediated platelet functions. APPROACH AND RESULTS: We generated a strain of kindlin-3 knockin (K3KI) mice that express a kindlin-3 mutant that carries an integrin-interaction defective substitution. K3KI mice could survive normally and express integrin αIIbß3 on platelets similar to their wild-type counterparts. Functional analysis revealed that K3KI mice exhibited defective platelet function, including impaired integrin αIIbß3 activation, suppressed platelet spreading and platelet aggregation, prolonged tail bleeding time, and absence of platelet-mediated clot retraction. In addition, whole blood drawn from K3KI mice showed resistance to in vitro thrombus formation and, as a consequence, K3KI mice were protected from in vivo arterial thrombosis. CONCLUSIONS: These observations demonstrate that the direct binding of kindlin-3 to integrin αIIbß3 is involved in supporting integrin αIIbß3 activation and integrin αIIbß3-dependent responses of platelets and consequently contributes significantly to arterial thrombus formation.


Assuntos
Plaquetas/fisiologia , Trombose das Artérias Carótidas/fisiopatologia , Proteínas do Citoesqueleto/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Substituição de Aminoácidos , Animais , Tempo de Sangramento , Plaquetas/ultraestrutura , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/induzido quimicamente , Forma Celular , Cloretos/toxicidade , Retração do Coágulo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Modelos Animais de Doenças , Feminino , Compostos Férricos/toxicidade , Técnicas de Introdução de Genes , Genes Reporter , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Ativação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão/metabolismo
15.
FASEB J ; 28(5): 2260-71, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24469992

RESUMO

The FERM domain containing protein Kindlin-3 has been recognized as a major regulator of integrin function in hematopoietic cells, but its role in neoplasia is totally unknown. We have examined the relationship between Kindlin-3 and breast cancer in mouse models and human tissues. Human breast tumors showed a ∼7-fold elevation in Kindlin-3 mRNA compared with nonneoplastic tissue by quantitative polymerase chain reaction. Kindlin-3 overexpression in a breast cancer cell line increased primary tumor growth and lung metastasis by 2.5- and 3-fold, respectively, when implanted into mice compared with cells expressing vector alone. Mechanistically, the Kindlin-3-overexpressing cells displayed a 2.2-fold increase in vascular endothelial growth factor (VEGF) secretion and enhanced ß1 integrin activation. Increased VEGF secretion resulted from enhanced production of Twist, a transcription factor that promotes tumor angiogenesis. Knockdown of Twist diminished VEGF production, and knockdown of ß1 integrins diminished Twist and VEGF production by Kindlin-3-overexpressing cells, while nontargeting small interfering RNA had no effect on expression of these gene products. Thus, Kindlin-3 influences breast cancer progression by influencing the crosstalk between ß1 integrins and Twist to increase VEGF production. This signaling cascade enhances breast cancer cell invasion and tumor angiogenesis and metastasis.


Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Animais , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Integrina beta1/metabolismo , Camundongos , Camundongos SCID , Metástase Neoplásica , Estrutura Terciária de Proteína , Receptor ErbB-2/metabolismo , Receptores Estrogênicos/metabolismo , Receptores de Progesterona/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
16.
Blood ; 122(14): 2491-9, 2013 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-23896409

RESUMO

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice. In the present study, we tested whether hemostasis might be perturbed in kindlin-2(+/-) mice. Bleeding time and carotid artery occlusion time were significantly prolonged in kindlin-2(+/-) mice. Whereas plasma concentrations/activities of key coagulation/fibrinolytic proteins and platelet counts and aggregation were similar in wild-type and kindlin-2(+/-) mice, kindlin-2(+/-) endothelial cells (ECs) showed enhanced inhibition of platelet aggregation induced by adenosine 5'-diphosphate (ADP) or low concentrations of other agonists. Cell-surface expression of 2 enzymes involved in ADP/adenosine 5'-monophosphate (AMP) degradation, adenosine triphosphate (ATP) diphosphohydrolase (CD39) and ecto-5'-nucleotidase (CD73) were increased twofold to threefold on kindlin-2(+/-) ECs, leading to enhanced ATP/ADP catabolism and production of adenosine, an inhibitor of platelet aggregation. Trafficking of CD39 and CD73 at the EC surface was altered in kindlin-2(+/-) mice. Mechanistically, this was attributed to direct interaction of kindlin-2 with clathrin heavy chain, thereby controlling endocytosis and recycling of CD39 and CD73. The interaction of kindlin-2 with clathrin was independent of its integrin binding site but still dependent on a site within its F3 subdomain. Thus, kindlin-2 regulates trafficking of EC surface enzymes that control platelet responses and hemostasis.


Assuntos
Plaquetas/metabolismo , Clatrina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células Endoteliais/metabolismo , Hemostasia/fisiologia , Proteínas Musculares/metabolismo , 5'-Nucleotidase/biossíntese , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Antígenos CD/biossíntese , Apirase/biossíntese , Membrana Celular/metabolismo , Feminino , Citometria de Fluxo , Imunoprecipitação , Masculino , Camundongos , Camundongos Knockout , Agregação Plaquetária/fisiologia , Transporte Proteico/fisiologia , Ressonância de Plasmônio de Superfície
17.
J Biol Chem ; 287(29): 24585-94, 2012 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-22648415

RESUMO

Both talin head domain and kindlin-2 interact with integrin ß cytoplasmic tails, and they function in concert to induce integrin activation. Binding of talin head domain to ß cytoplasmic tails has been characterized extensively, but information on the interaction of kindin-2 with this integrin segment is limited. In this study, we systematically examine the interactions of kindlin-2 with integrin ß tails. Kindlin-2 interacted well with ß(1) and ß(3) tails but poorly with the ß(2) cytoplasmic tail. This binding selectivity was determined by the non-conserved residues, primarily the three amino acids at the extreme C terminus of the ß(3) tail, and the sequence in ß(2) was non-permissive. The region at the C termini of integrin ß(1) and ß(3) tails recognized by kindlin-2 was a binding core of 12 amino acids. Kindlin-2 and talin head do not interact with one another but can bind simultaneously to the integrin ß(3) tail without enhancing or inhibiting the interaction of the other binding partner. Kindlin-2 itself failed to directly unclasp integrin α/ß tail complex, indicating that kindlin-2 must cooperate with talin to support the integrin activation mechanism.


Assuntos
Citoplasma/metabolismo , Cadeias beta de Integrinas/química , Cadeias beta de Integrinas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Talina/química , Talina/metabolismo , Animais , Western Blotting , Células CHO , Calorimetria , Cromatografia Líquida de Alta Pressão , Cricetinae , Espectroscopia de Ressonância Magnética , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ressonância de Plasmônio de Superfície
18.
Mol Cancer Res ; 9(11): 1500-8, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21875932

RESUMO

Integrins are adhesion receptors involved in bidirectional signaling that are crucial for various cellular responses during normal homeostasis and pathologic conditions such as cancer progression and metastasis. Aberrant expression of noncoding microRNAs (miRNA) has been implicated in the deregulation of integrin expression and activity, leading to the development and progression of cancer tumors, including their acquisition of the metastatic phenotype. miR-31 is a key regulator of several critical genes involved in the invasion-metastasis cascade in cancer. Using diverse cell-based, genetic, biochemical, flow cytometry, and functional analyses, we report that miR-31 is a master regulator of integrins as it targets multiple α subunit partners (α2, α5, and αV) of ß1 integrins and also ß3 integrins. We found that expression of miR-31 in cancer cells resulted in a significant repression of these integrin subunits both at the mRNA and protein levels. Loss of expression of α2, α5, αV, and ß3 was a direct consequence of miR-31 targeting conserved seed sequences in the 3' untranslated region of these integrin subunits leading to their posttranscriptional repression, which was reflected in their diminished surface expression in live cells. The biological consequence of decreased the cell surface of these integrins was a significant inhibition of cell spreading in a ligand-dependent manner. Although different reports have shown that a single integrin can be regulated by several miRNAs, here we show that a single miRNA, miR-31, is able to specifically target several integrin subunits to regulate key aspects of cancer cell invasion and metastasis.


Assuntos
Neoplasias da Mama/metabolismo , Integrina beta1/biossíntese , MicroRNAs/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , MicroRNAs/genética , Transdução de Sinais , Transfecção
19.
Blood ; 117(18): 4978-87, 2011 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-21378273

RESUMO

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice and causes severe developmental defects in zebrafish. Knockdown of kindlin-2 levels in endothelial cells resulted in defective adhesive and migratory responses, suggesting that angiogenesis might be aberrant even with partial reduction of kindlin-2. This hypothesis has now been tested in the kindlin-2(+/-) mice. RM1 prostate tumors grown in kindlin-2(+/-) mice had fewer blood vessels, which were thinner and shorter and supported less tumor growth compared with wild-type littermates. The vessels that did form in the kindlin-2(+/-) mice lacked smooth muscle cells and pericytes and had thinner basement membranes, indicative of immature vessels. VEGF-induced angiogenesis in matrigel implants was also abnormal in the kindlin-2(+/-) mice. Vessels in the kindlin-2(+/-) mice were leaky, and BM transplantation from kindlin-2(+/-) to WT mice did not correct this defect. Endothelial cells derived from kindlin-2(+/-) mice had integrin expression levels similar to WT mice but reduced αVß3-dependent signaling, migration, adhesion, spreading, and tube formation. Developmental angiogenesis was markedly impaired by kindlin-2 morpholinos in zebrafish. Taken together, kindlin-2 plays an important role in pathologic and developmental angiogenesis, which arises from defective activation of integrin αVß3.


Assuntos
Proteínas do Citoesqueleto/fisiologia , Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Feminino , Técnicas de Silenciamento de Genes , Integrina alfaVbeta3/fisiologia , Masculino , Camundongos , Camundongos Knockout , Neovascularização Patológica/patologia , Oligodesoxirribonucleotídeos Antissenso/genética , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/fisiopatologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética
20.
J Cell Physiol ; 226(11): 2965-78, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21302295

RESUMO

14-3-3 is an adaptor protein that localizes to the leading edge of spreading cells, returning to the cytoplasm as spreading ceases. Previously, we showed that integrin-induced Rac1 activation and spreading were inhibited by sequestration of 14-3-3ζ and restored by its overexpression. Here, we determined whether 14-3-3 mediates integrin signaling by localizing a guanine nucleotide exchange factor (GEF) to Rac1-activating integrin complexes. We showed that GST-14-3-3ζ recruited the Rac1-GEF, Tiam1, from cell lysates through Tiam1 residues 1-182 (N(1-182) Tiam1). The physiological relevance of this interaction was examined in serum-starved Hela cells plated on fibronectin. Both Tiam1 and N(1-182) Tiam1 were recruited to 14-3-3-containing ß1-integrin complexes, as shown by co-localization and co-immunoprecipitation. Integrin-induced Rac1 activation was inhibited when Tiam1 was depleted with siRNA or by overexpression of catalytically inactive N(1-182) Tiam1, which was incorporated into 14-3-3/ß1-integrin complexes and inhibited spreading in a manner that was overcome by constitutively active Rac1. Integrin-induced Rac1 activation, spreading, and migration were also inhibited by overexpression of 14-3-3ζ S58D, which was unable to recruit Tiam1 from lysates, co-immunoprecipitate with Tiam1, or mediate its incorporation into ß1-integrin complexes. Taken together, these findings suggest a previously unrecognized mechanism of integrin-induced Rac1 activation in which 14-3-3 dimers localize Tiam1 to integrin complexes, where it mediates integrin-dependent Rac1 activation, thus initiating motility-inducing pathways. Moreover, since Tiam1 is recruited to other sites of localized Rac1 activation through its PH-CC-EX domain, the present findings show that a mechanism involving its N-terminal 182 residues is utilized to recruit Tiam1 to motility-inducing integrin complexes.


Assuntos
Proteínas 14-3-3/metabolismo , Plaquetas/metabolismo , Movimento Celular , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Integrina beta1/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T
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