MRGPRX2 signaling involves the Lysyl-tRNA synthetase and MITF pathway.

MRGPRX2, a G-protein-coupled-seven transmembrane domain receptor, is mainly expressed in mast cells and neurons and is involved in skin immunity and pain. It is implicated in the pathophysiology of non-IgE-mediated immediate hypersensitivity and has been related to adverse drug reactions. Moreover, a role has been proposed in asthma, atopic dermatitis, contact dermatitis, and chronic spontaneous urticaria. Although it has a prominent role in disease, its signaling transduction is poorly understood. This study shows that MRGPRX2 activation with substance P increased Lysyl t-RNA synthetase (LysRS) translocation to the nucleus. LysRS is a moonlighting protein with a dual role in protein translation and IgE signaling in mast cells. Upon allergen- IgE-FcεRI crosslinking, LysRS is translocated to the nucleus and activates microphthalmia-associated transcription factor (MITF) activity. In this study, we found that MRGPRX2 triggering led to MITF phosphorylation and increased MITF activity. Therefore, overexpression of LysRS increased MITF activity after MRGPRX2 activation. MITF silencing reduced MRGPRX2-dependent calcium influx and mast cell degranulation. Furthermore, a MITF pathway inhibitor, ML329, impaired MITF expression, calcium influx, and mast cell degranulation. Moreover, drugs such as atracurium, vancomycin, and morphine, reported to induce MRGPRX2-dependent degranulation, increased MITF activity. Altogether, our data show that MRGPRX2 signaling enhances MITF activity, and its abrogation by silencing or inhibition resulted in defective MRGPRX2 degranulation. We conclude that MRGPRX2 signaling involves the LysRS and MITF pathway. Thus, MITF and MITF-dependent targets may be considered therapeutic approaches to treat pathologies where MRGPRX2 is implicated.

MITF Downregulation Induces Death in Human Mast Cell Leukemia Cells and Impairs IgE-Dependent Degranulation.

Activating mutations in KIT (CD117) have been associated with several diseases, including gastrointestinal stromal tumors and mastocytosis. Rapidly progressing pathologies or drug resistance highlight the need for alternative treatment strategies. Previously, we reported that the adaptor molecule SH3 binding protein 2 (SH3BP2 or 3BP2) regulates KIT expression at the transcriptional level and microphthalmia-associated transcription factor (MITF) expression at the post-transcriptional level in human mast cells and gastrointestinal stromal tumor (GIST) cell lines. Lately, we have found that the SH3BP2 pathway regulates MITF through miR-1246 and miR-5100 in GIST. In this study, miR-1246 and miR-5100 were validated by qPCR in the SH3BP2-silenced human mast cell leukemia cell line (HMC-1). MiRNA overexpression reduces MITF and MITF-dependent target expression in HMC-1. The same pattern was observed after MITF silencing. In addition, MITF inhibitor ML329 treatment reduces MITF expression and affects the viability and cell cycle progression in HMC-1. We also examine whether MITF downregulation affected IgE-dependent mast cell degranulation. MiRNA overexpression, MITF silencing, and ML329 treatment reduced IgE-dependent degranulation in LAD2- and CD34+-derived mast cells. These findings suggest MITF may be a potential therapeutic target for allergic reactions and deregulated KIT mast-cell-mediated disorders.

The microphthalmia-associated transcription factor is involved in gastrointestinal stromal tumor growth.

Gastrointestinal stromal tumors (GISTs) are the most common neoplasms of mesenchymal origin, and most of them emerge due to the oncogenic activation of KIT or PDGFRA receptors. Despite their relevance in GIST oncogenesis, critical intermediates mediating the KIT/PDGFRA transforming program remain mostly unknown. Previously, we found that the adaptor molecule SH3BP2 was involved in GIST cell survival, likely due to the co-regulation of the expression of KIT and Microphthalmia-associated transcription factor (MITF). Remarkably, MITF reconstitution restored KIT expression levels in SH3BP2 silenced cells and restored cell viability. This study aimed to analyze MITF as a novel driver of KIT transforming program in GIST. Firstly, MITF isoforms were characterized in GIST cell lines and GIST patients' samples. MITF silencing decreases cell viability and increases apoptosis in GIST cell lines irrespective of the type of KIT primary or secondary mutation. Additionally, MITF silencing leads to cell cycle arrest and impaired tumor growth in vivo. Interestingly, MITF silencing also affects ETV1 expression, a linage survival factor in GIST that promotes tumorigenesis and is directly regulated by KIT signaling. Altogether, these results point to MITF as a key target of KIT/PDGFRA oncogenic signaling for GIST survival and tumor growth.

SH3BP2 Silencing Increases miRNAs Targeting ETV1 and Microphthalmia-Associated Transcription Factor, Decreasing the Proliferation of Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Gain of function in receptor tyrosine kinases type III, KIT, or PDGFRA drives the majority of GIST. Previously, our group reported that silencing of the adaptor molecule SH3 Binding Protein 2 (SH3BP2) downregulated KIT and PDGFRA and microphthalmia-associated transcription factor (MITF) levels and reduced tumor growth. This study shows that SH3BP2 silencing also decreases levels of ETV1, a required factor for GIST growth. To dissect the SH3BP2 pathway in GIST cells, we performed a miRNA array in SH3BP2-silenced GIST cell lines. Among the most up-regulated miRNAs, we found miR-1246 and miR-5100 to be predicted to target MITF and ETV1. Overexpression of these miRNAs led to a decrease in MITF and ETV1 levels. In this context, cell viability and cell cycle progression were affected, and a reduction in BCL2 and CDK2 was observed. Interestingly, overexpression of MITF enhanced cell proliferation and significantly rescued the viability of miRNA-transduced cells. Altogether, the KIT-SH3BP2-MITF/ETV1 pathway deserves to be considered in GIST cell survival and proliferation.

Anaphylaxis: Focus on Transcription Factor Activity.

Anaphylaxis is a severe allergic reaction, rapid in onset, and can lead to fatal consequences if not promptly treated. The incidence of anaphylaxis has risen at an alarming rate in past decades and continues to rise. Therefore, there is a general interest in understanding the molecular mechanism that leads to an exacerbated response. The main effector cells are mast cells, commonly triggered by stimuli that involve the IgE-dependent or IgE-independent pathway. These signaling pathways converge in the release of proinflammatory mediators, such as histamine, tryptases, prostaglandins, etc., in minutes. The action and cell targets of these proinflammatory mediators are linked to the pathophysiologic consequences observed in this severe allergic reaction. While many molecules are involved in cellular regulation, the expression and regulation of transcription factors involved in the synthesis of proinflammatory mediators and secretory granule homeostasis are of special interest, due to their ability to control gene expression and change phenotype, and they may be key in the severity of the entire reaction. In this review, we will describe our current understanding of the pathophysiology of human anaphylaxis, focusing on the transcription factors' contributions to this systemic hypersensitivity reaction. Host mutation in transcription factor expression, or deregulation of their activity in an anaphylaxis context, will be updated. So far, the risk of anaphylaxis is unpredictable thus, increasing our knowledge of the molecular mechanism that leads and regulates mast cell activity will enable us to improve our understanding of how anaphylaxis can be prevented or treated.

MYO1F Regulates IgE and MRGPRX2-Dependent Mast Cell Exocytosis.

The activation and degranulation of mast cells is critical in the pathogenesis of allergic inflammation and modulation of inflammation. Recently, we demonstrated that the unconventional long-tailed myosin, MYO1F, localizes with cortical F-actin and mediates adhesion and migration of mast cells. In this study, we show that knockdown of MYO1F by short hairpin RNA reduces human mast cell degranulation induced by both IgE crosslinking and by stimulation of the Mas-related G protein-coupled receptor X2 (MRGPRX2), which has been associated with allergic and pseudoallergic drug reactions, respectively. Defective degranulation was accompanied by a reduced reassembly of the cortical actin ring after activation but reversed by inhibition of actin polymerization. Our data show that MYO1F is required for full Cdc42 GTPase activation, a critical step in exocytosis. Furthermore, MYO1F knockdown resulted in less granule localization in the cell membrane and fewer fissioned mitochondria along with deficient mitochondria translocation to exocytic sites. Consistent with that, AKT and DRP1 phosphorylation are diminished in MYO1F knockdown cells. Altogether, our data point to MYO1F as an important regulator of mast cell degranulation by contributing to the dynamics of the cortical actin ring and the distribution of both the secretory granules and mitochondria.

Infección por VPH y su correlación clínica en población indígena de la región altoandina del Ecuador / Infección por VPH y su correlación clínica en población indígena de la región altoandina del Ecuador VPH infection and its clinical correlation in indigenous population of the high andean region of Ecuador

El cáncer cervico-uterino (CaCu) constituye un problema de salud pública. Estudios epidemiológicos han identificado una estrecha relación entre el CaCu y la infección por el virus del papiloma humano (VPH). El objetivo del estudio fue realizar la detección molecular de VPH, identificación de infecciones mixtas y el tamizaje de CaCu a través de un estudio transversal a 189 mujeres entre 21 a 65 años pertenecientes a la comunidad de Pilahuin- Provincia del Tungurahua a las cuales se les realizó detección molecular de VPH y citología convencional. Los resultados fueron analizados con los programas EPI INFO y SPSS. Resultados: La edad promedio de las participantes fue de 35,2 años, en relación estable, multíparas, con un nivel medio, bajo de instrucción, sin trabajo remunerado. El 100% de las participantes presentó una actitud favorable frente a la citología cervico-uterina sin mostrar relación con la edad, nivel de instrucción o estado civil. El 10,5% de las participantes presentaron infección por VPH y un 70% de las pacientes presentaron inflamación severa con una asociación positiva a infección bacteriana mixta.

Cervical-uterine cancer (CaCu) is a public health problem. Epidemiological studies have identified a close relationship between CaCu and human papillomavirus (HPV) infection. The purpose of this study was to perform the molecular detection of HPV, identification of mixed infections and the screening of CaCu through conventional cytology in an indigenous population of the high Andean highlands of Ecuador in order to correlate the data obtained with the presence of lesions. Methodology: A cross-sectional study was carried out on 189 women between the ages of 21 and 65; who belonged to the community of Pilahuin in the province of Tungurahua, who assisted to gynecological check-ups. Data were analyzed with the EPI-INFO and SPSS statistical programs. Results: the average age of the participants was 35.2 years, most of the participants identified themselves as indigenous, in a stable relationship, multiparous, with a medium to low level of education and no paid work. The participants showed a favorable attitude towards the cervico-uterine cytology in 100% of the cases without showing a relation with age, level of education or civil status. HPV infection was positive in 10.5% of the participants, 70% of the patients had severe inflammation with a positive association with mixed bacterial infection.

Medio de cultivo convencional enriquecido con extractos de quinoa, amaranto y chía promueve el crecimiento de Staphylococcus aureus / Conventional culture media enriched with extracts of quinoa, amaranth and chia promotes the Staphylococcus aureus growth

La identificación bacteriana durante las infecciones microbiológicas es un aspecto crítico a la hora de escoger un tratamiento específico para evitar complicaciones del paciente o en algunos casos su muerte. Por ello, incrementar el crecimiento celular durante el diagnóstico clínico por cultivo bacteriano puede reducir el tiempo para determinar el patógeno que causa la enfermedad. En este trabajo, se utilizó el cultivo bacteriano como metodología y se evaluó el crecimiento de Staphylococcus aureus en un medio de cultivo convencional enriquecido con extractos de Chenopodium quinoa, Amaranthus caudatus y Salvia hispanica. Los resultados obtenidos muestran que estos extractos, a bajas concentraciones, tienen un efecto protector contra la citotoxicidad que se podría generar por el estrés oxidativo producto del metabolismo celular de las bacterias cultivadas in vitro e incrementan significativamente el crecimiento bacteriano. La adicción de estos extractos a los medios convencionales podría mejorar el crecimiento bacteriano durante un diagnóstico bacteriológico y reducir el tiempo de identificación del patógeno.

Increasing the bacterial growth rate reduces the time getting the bacteria identification. This is helpful to choose an accurate and quick therapeutic strategy during microbiological infections, avoiding illness complications or in some cases the death. Here, we used bacterial growth method and we evaluated the growth of Staphylococcus aureus including an extract of Chenopodium quinoa, Amaranthus caudatus and Salvia hispanica in the routine culture media. Results show that adding these extracts, at low concentrations, have a protective effect against the cytotoxicity that could be generated by the oxidative stress product of the cellular metabolism of the bacteria growing in vitro and significantly increase the bacterial growth. The addition of these extracts to conventional culture media could improve bacterial growth during a bacteriological diagnosis and to reduce the time of pathogen identification.