RESUMEN

Interleukin-1 receptor-associated kinase (IRAK) 4 mediates host defense against infections. As an active kinase, IRAK4 elicits full spectra of myeloid differentiation primary response protein (MyD) 88-dependent responses, while kinase-inactive IRAK4 induces a subset of cytokines and negative regulators whose expression is not regulated by mRNA stability. IRAK4 kinase activity is critical for resistance against Streptococcus pneumoniae, but its involvement in autoimmunity is incompletely understood. In this study, we determined the role of IRAK4 kinase activity in murine lupus. Lupus development in BXSB mice expressing the Y chromosome autoimmunity accelerator (Yaa) increased basal and Toll-like receptor (TLR) 4/7-induced phosphorylation of mitogen-activated protein kinases, p65 nuclear factor-κB (NF-κB), enhanced tumor necrosis factor (TNF)-α and C-C motif chemokine ligand (CCL) 5 gene expression in splenic macrophages, but decreased levels of Toll-interacting protein and IRAK-M, without affecting IRAK4 or IRAK1 expression. Mice harboring kinase-inactive IRAK4 on the lupus-prone Yaa background manifested blunted TLR signaling in macrophages and reduced glomerulonephritis, splenomegaly, serum anti-nuclear antibodies, numbers of splenic macrophages, total and TNF-α+ dendritic cells, activated T- and B-lymphocytes, and lower TNF-α expression in macrophages compared with lupus-prone mice with functional IRAK4. Thus, IRAK4 kinase activity contributes to murine lupus and could represent a new therapeutic target.

Asunto(s)

Autoinmunidad , Citocinas/inmunología , Síndromes de Inmunodeficiencia , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Nefritis Lúpica/inmunología , Animales , Quimiocina CCL5/genética , Quinasas Asociadas a Receptores de Interleucina-1/deficiencia , Macrófagos/inmunología , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fosforilación , Enfermedades de Inmunodeficiencia Primaria , Transducción de Señal , Bazo/citología , Bazo/inmunología , Receptores Toll-Like/inmunología , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Endotoxin tolerance protects the host by limiting excessive 'cytokine storm' during sepsis, but compromises the ability to counteract infections in septic shock survivors. It reprograms Toll-like receptor (TLR) 4 responses by attenuating the expression of proinflammatory cytokines without suppressing anti-inflammatory and antimicrobial mediators, but the mechanisms of reprogramming remain unclear. In this study, we demonstrate that the induction of endotoxin tolerance in human monocytes, THP-1 and MonoMac-6 cells inhibited lipopolysaccharide (LPS)-mediated phosphorylation of Lyn, c-Src and their recruitment to TLR4, but increased total protein phosphatase (PP) activity and the expression of protein tyrosine phosphatase (PTP) 1B, PP2A, PTP nonreceptor type (PTPN) 22 and mitogen-activated protein kinase phosphatase (MKP)-1. Chemical PP inhibitors, okadaic acid, dephostatin and cantharidic acid markedly decreased or completely abolished LPS tolerance, indicating the importance of phosphatases in endotoxin tolerization. Overexpression of PTPN22 decreased LPS-mediated nuclear factor (NF)-x03BA;B activation, p38 phosphorylation and CXCL8 gene expression, while PTPN22 ablation upregulated LPS-induced p65 NF-x03BA;B and p38 phosphorylation and the expression of TNF-α and pro-IL-1ß mRNA, indicating PTPN22 as an inhibitor of TLR4 signaling. Thus, LPS tolerance interferes with TLR4 signaling by inhibiting Lyn and c-Src phosphorylation and their recruitment to TLR4, while increasing the phosphatase activity and expression of PP2A, PTPN22, PTP1B and MKP1.

Asunto(s)

Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Tolerancia Inmunológica/efectos de los fármacos , Lipopolisacáridos/farmacología , Monocitos/inmunología , Fosfoproteínas Fosfatasas/inmunología , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/inmunología , Familia-src Quinasas/inmunología , Proteína Tirosina Quinasa CSK , Línea Celular Tumoral , Regulación Enzimológica de la Expresión Génica/inmunología , Humanos , Tolerancia Inmunológica/genética , Lipopolisacáridos/inmunología , Fosfoproteínas Fosfatasas/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 4/genética , Familia-src Quinasas/genética

RESUMEN

Development of endotoxin tolerance in macrophages during sepsis reprograms Toll-like receptor 4 signaling to inhibit proinflammatory cytokines without suppressing anti-inflammatory and antimicrobial mediators and protects the host from excessive inflammation and tissue damage. However, endotoxin tolerance renders septic patients immunocompromised and unable to control secondary infections. Although previous studies have revealed the importance of several negative regulators of Toll-like receptor signaling in endotoxin tolerance, the role of Pellino proteins has not been addressed. The present report shows that the induction of endotoxin tolerance in vivo in mice and in vitro in human monocytes and THP-1 and MonoMac-6 macrophages increases the expression of Pellino-3. Overexpression of Pellino-3 in human embryonic kidney 293/Toll-like receptor 2 or 293/Toll-like receptor 4/myeloid differentiation factor-2 cells inhibited Toll-like receptor 2/4-mediated activation of nuclear factor-κB and induction of CXCL-8 mRNA, and Pellino-3 ablation increased these responses. Pellino-3-deficient THP-1 cells had elevated Toll-like receptor 2/4-driven tumor necrosis factor-α, interleukin-6 mRNA, and Toll-like receptor 4-driven CCL5 gene expression in response to Toll-like receptor agonists and heat-killed Escherichia coli and Staphylococcus aureus, cytokines controlled by the MyD88 and Toll-interleukin-1R domain-containing protein inducing interferon-ß-mediated pathways, respectively. In addition, deficiency in Pellino-3 slightly increased phagocytosis of heat-killed bacteria. Transfected Pellino-3 inhibited nuclear factor-κB activation driven by overexpression of MyD88, TIR domain-containing adapter inducing interferon-ß, interleukin-1R-associated kinase-1, and tumor necrosis factor receptor activator of nuclear factor-κB-binding kinase-1, TGF-ß-activated kinase 1, and tumor necrosis factor receptor-associated factor-6, and inhibited interleukin-1R-associated kinase 1 modifications and tumor necrosis factor receptor activator of nuclear factor-κB-binding kinase 1 phosphorylation. Finally, Pellino-3 ablation in THP-1 decreased the extent of endotoxin tolerization. Thus, Pellino-3 is involved in endotoxin tolerance and functions as a negative regulator of Toll-like receptor 2/4 signaling.

Asunto(s)

Tolerancia Inmunológica/efectos de los fármacos , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/inmunología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Animales , Línea Celular Tumoral , Citocinas/genética , Citocinas/inmunología , Escherichia coli/inmunología , Humanos , Tolerancia Inmunológica/genética , Ratones , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , FN-kappa B/genética , FN-kappa B/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Staphylococcus aureus/inmunología , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genética , Ubiquitina-Proteína Ligasas/genética

RESUMEN

INTRODUCTION: The role of miRNAs in acquired endocrine-resistant breast cancer is not fully understood. One hallmark of tumor progression is epithelial-to-mesenchymal transition (EMT), characterized by a loss of cell adhesion resulting from reduced E-cadherin and increased cell mobility. miR-200 family members regulate EMT by suppressing expression of transcriptional repressors ZEB1/2. Previously we reported that the expression of miR-200a, miR-200b, and miR-200c was lower in LY2 endocrine-resistant, mesenchymal breast cancer cells compared to parental, endocrine sensitive, epithelial MCF-7 breast cancer cells. Here we investigated the regulation of miR-200 family members and their role in endocrine-sensitivity in breast cancer cells. RESULTS: miR-200 family expression was progressively reduced in a breast cancer cell line model of advancing endocrine/tamoxifen (TAM) resistance. Concomitant with miR-200 decrease, there was an increase in ZEB1 mRNA expression. Overexpression of miR-200b or miR-200c in LY2 cells altered cell morphology to a more epithelial appearance and inhibited cell migration. Further, miR-200b and miR-200c overexpression sensitized LY2 cells to growth inhibition by estrogen receptor (ER) antagonists TAM and fulvestrant. Knockdown of ZEB1 in LY2 cells recapitulated the effect of miR-200b and miR-200c overexpression resulting in inhibition of LY2 cell proliferation by TAM and fulvestrant, but not the aromatase inhibitor exemestane. Demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) in combination with histone deacetylase inhibitor trichostatin A (TSA) increased miR-200b and miR-200c in LY2 cells. Concomitant with the increase in miR-200b and miR-200c, ZEB1 expression was decreased and cells appeared more epithelial in morphology and were sensitized to TAM and fulvestrant inhibition. Likewise, knockdown of ZEB1 increased antiestrogen sensitivity of LY2 cells resulting in inhibition of cell proliferation. CONCLUSIONS: Our data indicate that reduced miRNA-200b and miR-200c expression contributes to endocrine resistance in breast cancer cells and that the reduced expression of these miR-200 family members in endocrine-resistant cells can be reversed by 5-aza-dC+TSA.

Asunto(s)

Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Expresión Génica , MicroARNs/genética , Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Epigénesis Genética/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Estradiol/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/genética , Humanos , Hidroxitestosteronas/farmacología , Células MCF-7 , Factores de Transcripción/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc

RESUMEN

Fenhexamid and fludioxonil are antifungal agents used in agricultural applications, which are present at measurable amounts in fruits and vegetables. Fenhexamid and fludioxonil showed endocrine disruptor activity as antiandrogens in an androgen receptor reporter assay in engineered human breast cancer cells. Little is known about how environmental chemicals regulate microRNA (miRNA) expression. This study examined the effect of fenhexamid and fludioxonil on the expression of the oncomiR miR-21 in MCF-7, T47D, and MDA-MB-231 human breast cancer cells and downstream targets of miR-21 in MCF-7 cells. Fenhexamid and fludioxonil stimulated miR-21 expression in a concentration-dependent manner and reduced the expression of miR-21 target Pdcd4 protein. Antisense to miR-21 blocked the increase in Pdcd4 protein by fenhexamid and fludioxonil. Fenhexamid and fludioxonil reduced miR-125b and miR-181a, demonstrating specificity of miRNA regulation. Induction of miR-21 was inhibited by the estrogen receptor antagonist fulvestrant, by androgen receptor antagonist bicalutamide, by actinomycin D and cycloheximide, and by inhibitors of the mitogen-activated protein kinases and phosphoinositide 3-kinase pathways. Fenhexamid activation was inhibited by the arylhydrocarbon receptor antagonist α-napthoflavone. Fenhexamid and fludioxonil did not affect dihydrotestosterone-induced miR-21 expression. Fludioxonil, but not fenhexamid, inhibited MCF-7 cell viability, and both inhibited estradiol-induced cell proliferation and reduced cell motility. Together these data indicate that fenhexamid and fludioxonil use similar and distinct mechanisms to increase miR-21 expression with downstream antiestrogenic activity.

Asunto(s)

Amidas/toxicidad , Dioxoles/toxicidad , Disruptores Endocrinos/toxicidad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , MicroARNs/genética , Pirroles/toxicidad , Western Blotting , Femenino , Genes Reporteros , Humanos , Luciferasas/genética , Células MCF-7 , MicroARNs/antagonistas & inhibidores , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección

RESUMEN

Microarrays identified miRNAs differentially expressed and 4-hydroxytamoxifen (4-OHT) regulated in MCF-7 endocrine-sensitive versus resistant LY2 human breast cancer cells. 97 miRNAs were differentially expressed in MCF-7 versus LY2 cells. Opposite expression of miRs-10a, 21, 22, 29a, 93, 125b, 181, 200a, 200b, 200c, 205, and 222 was confirmed. Bioinformatic analyses to impute the biological significance of these miRNAs identified 36 predicted gene targets from those regulated by 4-OHT in MCF-7 cells. Agreement in the direction of anticipated regulation was detected for 12 putative targets. These miRNAs with opposite expression between the two cell lines may be involved in endocrine resistance.

Asunto(s)

Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Análisis por Conglomerados , Resistencia a Antineoplásicos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc

RESUMEN

Select changes in microRNA (miRNA) expression correlate with estrogen receptor alpha (ER alpha) expression in breast tumors. miR-21 is higher in ER alpha positive than negative tumors, but no one has examined how estradiol (E(2)) regulates miR-21 in breast cancer cells. Here we report that E(2) inhibits miR-21 expression in MCF-7 human breast cancer cells. The E(2)-induced reduction in miR-21 was inhibited by 4-hydroxytamoxifen (4-OHT), ICI 182 780 (Faslodex), and siRNA ER alpha indicating that the suppression is ER alpha-mediated. ER alpha and ER beta agonists PPT and DPN inhibited and 4-OHT increased miR-21 expression. E(2) increased luciferase activity from reporters containing the miR-21 recognition elements from the 3'-UTRs of miR-21 target genes, corroborating that E(2) represses miR-21 expression resulting in a loss of target gene suppression. The E(2)-mediated decrease in miR-21 correlated with increased protein expression of endogenous miR-21-targets Pdcd4, PTEN and Bcl-2. siRNA knockdown of ER alpha blocked the E(2)-induced increase in Pdcd4, PTEN and Bcl-2. Transfection of MCF-7 cells with antisense (AS) to miR-21 mimicked the E(2)-induced increase in Pdcd4, PTEN and Bcl-2. These results are the first to demonstrate that E(2) represses the expression of an oncogenic miRNA, miR-21, by activating estrogen receptor in MCF-7 cells.

Asunto(s)

Neoplasias de la Mama/genética , Estradiol/farmacología , Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Regiones no Traducidas 3'/química , Proteínas Reguladoras de la Apoptosis/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Cicloheximida/farmacología , Dactinomicina/farmacología , Regulación hacia Abajo , Estradiol/análogos & derivados , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/agonistas , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/genética , Receptor beta de Estrógeno/agonistas , Receptor beta de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/genética , Femenino , Fulvestrant , Genes Reporteros , Humanos , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Regiones Promotoras Genéticas , Inhibidores de la Síntesis de la Proteína/farmacología , ARN sin Sentido/metabolismo , Proteínas de Unión al ARN/genética , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Proteínas Activadoras de ras GTPasa/genética