The Long Non-Coding RNA MALAT1 Modulates NR4A1 Expression through a Downstream Regulatory Element in Specific Cancer Cell Types.

Long non-coding RNAs (lncRNAs) have been shown to modulate gene expression and are involved in the initiation and progression of various cancer types. Despite the wealth of studies describing transcriptome changes upon lncRNA knockdown, there is limited information describing lncRNA-mediated effects on regulatory elements (REs) modulating gene expression. In this study, we investigated how the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) lncRNA regulates primary target genes using time-resolved MALAT1 knockdown followed by parallel RNA-seq and ATAC-seq assays. The results revealed that MALAT1 primarily regulates specific protein-coding genes and a substantial decrease in the accessibility downstream of the NR4A1 gene that was associated with a decreased NR4A1 expression. Moreover, the presence of an NR4A1-downstream RE was demonstrated by CRISPR-i assays to define a functional MALAT1/NR4A1 axis. By analyzing TCGA data, we identified a positive correlation between NR4A1 expression and NR4A1-downstream RE accessibility in breast cancer but not in pancreatic cancer. Accordingly, this regulatory mechanism was experimentally validated in breast cancer cells (MCF7) but not in pancreatic duct epithelial carcinoma (PANC1) cells. Therefore, our results demonstrated that MALAT1 is involved in a molecular mechanism that fine-tunes NR4A1 expression by modulating the accessibility of a downstream RE in a cell type-specific manner.

Recursos educativos digitales como herramientas de apoyo para la enseñanza de patología general en cursos de pregrado del área de la salud / Digital educational resources as support tools for teaching general pathology in undergraduate health courses

Los recursos educativos digitales se han transformado en un importante material de apoyo al proceso de enseñanza- aprendizaje, especialmente durante la pandemia por COVID-19. Estos corresponden a recursos de autoaprendizaje, generalmente en línea y de dominio público cuya disponibilidad inmediata a todo tipo de dispositivos electrónicos permite una rápida interacción del estudiante con materiales didácticos programados. El objetivo de este estudio fue evaluar el grado de satisfacción de cinco recursos educativos digitales, desarrollados como herramientas de apoyo para la enseñanza de la patología general, en estudiantes de carreras de pregrado del área de la salud de la Universidad Austral de Chile. Estudio descriptivo y exploratorio. Se desarrollaron cinco recursos educativos digitales donde se visualizan imágenes microscópicas correspondientes a procesos patológicos ocurridos en diferentes tejidos. Estos recursos fueron alojados en repositorios de la universidad y se encuentran actualmente disponibles en el canal de YouTube. Para conocer el grado de satisfacción, en sus aspectos pedagógicos y técnicos, se realizó una encuesta digital, anónima y voluntaria a estudiantes que cursaron asignaturas de patología, la que contempló cuatro dominios con sus respectivas preguntas: forma; control de usuario; contenido educativo y valoración global. El 94 % de los estudiantes calificaron el recurso de excelente o muy bueno y todos los dominios obtuvieron sobre el 80 % de satisfacción. Los contenidos representan lo que el recurso dice ofrecer, ayuda a resolver dudas y facilita la comprensión de la materia. El tamaño y color del texto es el adecuado y las imágenes presentan una excelente calidad y resolución. Los recursos cumplen con una alta calidad técnica y pedagógica, que asegura un gran potencial de uso para la enseñanza de la patología general, guiar el trabajo autónomo del estudiante y las actividades prácticas con el microscopio.

SUMMARY: Digital educational resources have become an important material to support the teaching-learning process, especially during the COVID-19 pandemic. These correspond to self-learning resources, generally online and the public domain, whose immediate availability to all types of electronic devices allows for rapid learner interaction with programmed didactic materials. The public domain and its immediate availability to all types of electronic devices allows a quick interaction of the student with self-explanatory didactic materials. The objective of this study was to evaluate the degree of satisfaction of five digital educational resources, developed as support tools for the teaching of general pathology, in undergraduate students of the health area of the Universidad Austral de Chile. Descriptive and exploratory study. Five digital educational resources have been developed where microscopic images corresponding to pathological processes occurring in different tissues are visualized these resources were hosted in university repositories and uploaded to the YouTube channel. To determine the degree of satisfaction, in their pedagogical and technical aspects, an anonymous and voluntary digital survey was carried out among students taking pathology courses, which included four domains with their respective questions: form; user control; educational content and overall assessment. The 94 % of the students evaluated the resource as excellent or very good and all domains obtained over 80 % satisfaction. The contents represent what the resource says it offers, helps to resolve doubts and facilitates the understanding of the subject. The size and color of the text is adequate, and the images present excellent quality and resolution. The resources developed offer a high technical and pedagogical quality, which guarantees a great potential for use in the teaching of general pathology, guiding the student's autonomous work and practical activities with the microscope.

Effects of exercise on irisin in subjects with overweight or obesity. A systematic review of clinical studies.

Introduction: Irisin is an adipomyokine involved in white adipose tissue browning, therefore, could be a key protein in metabolic health. However, exercise effects on irisin in subjects with overweight and/or obesity are conflicting. Therefore, this systematic review aims to search and analyse the literature available on this topic. From three databases: PubMed, ScienceDirect, and Medline, clinical studies published between 2010 and 2021 were considered. From 134 found, 14 studies were included. Only six reported plasma increases after exercise (~1.2 to 3-fold from pre-exercise levels). In addition, only 1 reported significant increases in skeletal muscle irisin mRNA levels (~2-fold). Also, irisin was measured from subcutaneous adipose tissue and saliva, where a ~2-fold increase in its protein levels was found in the latter. Exercise seems to increase the circulatory concentrations of irisin in subjects with overweight or obesity. However, this response is highly variable, therefore, a more integrative approach is urgently needed.

Introducción: La irisina es una adipomioquina relacionada a la transformación del tejido adiposo blanco a marrón, por tanto, podría ser una proteína clave para la salud metabólica. Sin embargo, los efectos del ejercicio sobre la irisina en personas con sobrepeso u obesidad son poco claros. Por lo anterior, esta revisión sistemática apunta a buscar y analizar la literatura disponible en este tema. Desde tres bases de datos: PubMed, ScienceDirect y Medline se buscaron estudios clínicos publicados entre el 2010 y 2021. De 134 estudios encontrados, 14 fueron incluidos. Solo 6 reportaron incrementos plasmáticos de irisina después del ejercicio (~1.2 a 3-veces respecto a niveles preejercicio). Además, solo 1 estudio describió incrementos significativos en el ARNm de irisina en el músculo esquelético (~2 veces sobre niveles preejercicio). La irisina también se medió desde tejido adiposo subcutáneo y saliva, encontrándose una elevación de (~2 veces sobre niveles preejercicio) en esta última. El ejercicio físico incrementaría las concentraciones circulatorias de irisina en personas con sobrepeso u obesidad. Sin embargo, esta respuesta es muy variable, por lo que se requiere una mirada más integrativa a la hora de estudiar este fenómeno.

Effects of exercise on irisin in subjects with overweight or obesity. A systematic review of clinical studies / Efectos del ejercicio sobre la irisina en personas con sobrepeso u obesidad. Una revisión sistemática de estudios clínicos

Irisin is an adipomyokine involved in white adipose tissue browning, therefore, could be a key protein in metabolic health. However, exercise effects on irisin in subjects with overweight and/or obesity are conflicting. Therefore, this systematic review aims to search and analyse the literature available on this topic. From three databases: PubMed, ScienceDirect, and Medline, clinical studies published between 2010 and 2021 were considered. From 134 found, 14 studies were included. Only six reported plasma increases after exercise (~1.2 to 3-fold from pre-exercise levels). In addition, only 1 reported significant increases in skeletal muscle irisin mRNA levels (~2-fold). Also, irisin was measured from subcutaneous adipose tissue and saliva, where a ~2-fold increase in its protein levels was found in the latter. Exercise seems to increase the circulatory concentrations of irisin in subjects with overweight or obesity. However, this response is highly variable, therefore, a more integrative approach is urgently needed. (AU)

La irisina es una adipomioquina relacionada a la transformación del tejido adiposo blanco a marrón, por tanto, podría ser una proteína clave para la salud metabólica. Sin embargo, los efectos del ejercicio sobre la irisina en personas con sobrepeso u obesidad son poco claros. Por lo anterior, esta revisión sistemática apunta a buscar y analizar la literatura disponible en este tema. Desde tres bases de datos: PubMed, ScienceDirect y Medline se buscaron estudios clínicos publicados entre el 2010 y 2021. De 134 estudios encontrados, 14 fueron incluidos. Solo 6 reportaron incrementos plasmáticos de irisina después del ejercicio (~1.2 a 3-veces respecto a niveles preejercicio). Además, solo 1 estudio describió incrementos significativos en el ARNm de irisina en el músculo esquelético (~2 veces sobre niveles preejercicio). La irisina también se medió desde tejido adiposo subcutáneo y saliva, encontrándose una elevación de (~2 veces sobre niveles preejercicio) en esta última. El ejercicio físico incrementaría las concentraciones circulatorias de irisina en personas con sobrepeso u obesidad. Sin embargo, esta respuesta es muy variable, por lo que se requiere una mirada más integrativa a la hora de estudiar este fenómeno. (AU)

Effects of aerobic exercise on fibroblast growth factor 21 in overweight and obesity. A systematic review.

Fibroblast growth factor 21 (FGF21) has been suggested to improve metabolism during aerobic exercise in obesity. However, the variability of exercise interventions gives rise to discrepancies in the field. Therefore, we aimed to systematically review the available literature regarding the effects of aerobic exercise on FGF21 in the context of overweight and obesity. Our search included original articles published between 2009 and November 2021 found in PubMed, Science Direct, and Medline. Clinical and preclinical studies were included. Studies, where subjects or animals presented with other conditions (e.g., cancer, stroke), were excluded. From an initial 43 studies, 19 (clinical studies = 9; preclinical studies = 10) were eligible for inclusion in this review. The main findings were that acute exercise tended to increase circulatory levels of FGF21. In contrast, chronic exercise programs (≥4 weeks) had the opposite effect along with inducing mRNA and protein increases of FGF receptors and ß-klotho in adipose tissue, liver, and skeletal muscle. In conclusion, both clinical and preclinical studies showed that aerobic exercise exerts changes in circulatory and tissue FGF21, along with its receptors and co-receptor. Future research is needed to elucidate the mechanisms, along with the physiological and clinical implications of these changes.

Combining Materials Obtained by 3D-Printing and Electrospinning from Commercial Polylactide Filament to Produce Biocompatible Composites.

The design of scaffolds to reach similar three-dimensional structures mimicking the natural and fibrous environment of some cells is a challenge for tissue engineering, and 3D-printing and electrospinning highlights from other techniques in the production of scaffolds. The former is a well-known additive manufacturing technique devoted to the production of custom-made structures with mechanical properties similar to tissues and bones found in the human body, but lacks the resolution to produce small and interconnected structures. The latter is a well-studied technique to produce materials possessing a fibrillar structure, having the advantage of producing materials with tuned composition compared with a 3D-print. Taking the advantage that commercial 3D-printers work with polylactide (PLA) based filaments, a biocompatible and biodegradable polymer, in this work we produce PLA-based composites by blending materials obtained by 3D-printing and electrospinning. Porous PLA fibers have been obtained by the electrospinning of recovered PLA from 3D-printer filaments, tuning the mechanical properties by blending PLA with small amounts of polyethylene glycol and hydroxyapatite. A composite has been obtained by blending two layers of 3D-printed pieces with a central mat of PLA fibers. The composite presented a reduced storage modulus as compared with a single 3D-print piece and possessing similar mechanical properties to bone tissues. Furthermore, the biocompatibility of the composites is assessed by a simulated body fluid assay and by culturing composites with 3T3 fibroblasts. We observed that all these composites induce the growing and attaching of fibroblast over the surface of a 3D-printed layer and in the fibrous layer, showing the potential of commercial 3D-printers and filaments to produce scaffolds to be used in bone tissue engineering.

[Relationship between fibroblast growth factor-21, muscle mass, and function outcomes in overweight and obese older adults living in the community. An exploratory study]. / Relación del factor de crecimiento de fibroblastos 21 con indicadores de masa y función muscular en personas mayores con sobrepeso u obesidad. Estudio exploratorio.

BACKGROUND: Age-related decreases in muscle mass and function are associated with the development of metabolic impairments, particularly in the context of obesity. Fibroblast growth factor21 (FGF-21) has been suggested as a common mediator of both processes. No known studies have examined the association between FGF-21 and muscle mass and function in overweight or obese older adults. With this in mind, this study aimed to investigate the association between plasma levels of FGF-21 and muscle mass and function outcomes in overweight or obese older adults. MATERIALS AND METHODS: Exploratory study, which included 39 adults of 60-70years old with body mass indexes >25kg/m2. As study outcomes, measurements were made of appendicular muscle mass (AMM), grip strength, 5 times sit-to-stand test (5xSTT), as well as plasma levels of FGF-21, fasting glucose, and insulin. The homeostatic model assessment index (HOMA-IR) was also calculated to determine the presence of insulin resistance. RESULTS: Significant relationships were found between plasma levels of FGF-21 vs 5xSTT (rho=0.49; P<.05). Moreover, FGF-21 levels were significantly higher in those with insulin resistance (P<.05), as well as with having lower levels of AMM (P<.05). CONCLUSION: There is a relationship between the plasma levels of FGF-21 and muscle function outcomes in overweight or obese older adults. Future studies should investigate the potential causalities between these relationships.

Biodentine Induce Proteínas Apoptóticas Caspasa 3 y PARP-1 en Células de la Pulpa Dental Humana / Biodentine Induces Apoptotic Proteins Caspase 3 and PARP - 1 on Human Dental Pulp Cells

RESUMEN: Las patologías pulpares han sido un verdadero reto para la odontología principalmente por su tratamiento. Actualmente, existen numerosos biomateriales en el mercado que reportan tener propiedades inherentes en los tejidos dentarios. Sin embargo, diferentes estudios sobre múltiples líneas celulares expuestas a estos biomateriales demuestran resultados controversiales como biocompatiblidad y citotoxicidad celular. Biodentine, es un cemento endodóntico en base a silicatos cálcico de múltiples aplicaciones, que prestaría propiedades de biocompatibilidad como bioactividad celular, características que le permitirían incluso ser utilizado en contacto directo con la pulpa dental. El objetivo de este estudio es la evaluación in-vitro de Biodentine, sobre cultivos de células de la pulpa dental humana (CCPDH). Se prepararon discos de cemento de Biodentine™ de 2 x 6 mm, los que se expusieron a cultivos de células aisladas de la pulpa dental humana. Luego de 24, 48 y 72 horas de exposición, se realizaron ensayos de viabilidad celular utilizando el método colorimétrico MTT. También se realizaron ensayos de expresión proteica de dos proteínas involucradas en la vía de señalización de la apoptosis celular: Caspasa - 3 clivada y Poli (ADP-Ribosa) Polimerasa, PARP - 1. Existen diferencias estadísticamente significativas (p<0,05) en los ensayos de viabilidad celular entre las células expuestas a Biodentine y el grupo control, como también a medida que aumenta el tiempo de exposición (p<0,05). Por otra parte, también existen diferencias significativas (p<0,05) en la expresión de PARP- 1 en los grupos sometidos a Biodentine. Los resultados obtenidos en este estudio demuestran que Biodentine genera citotoxicidad celular en cultivos celulares de pulpa dental humana, por disminución de la viabilidad celular como por la expresión de proteínas apoptóticas. Es por esto que la utilización de este biomaterial debería ser estudiado y considerarse en cada caso clínico, especialmente como recubridor pulpar directo.

ABSTRACT: Oral pathologies have been a real challenge for dentistry, mainly due to its treatment. Currently, there are numerous biomaterials on the market that may present inherent properties in dental tissues. However, studies on multiple cell lines are based on biocompatible results such as biocompatibility and cellular cytotoxicity. Biodentine is endodontic cement based on calcium silicates of multiple applications, which would provide biocompatibility properties as cellular bioactivity, characteristics that will allow it to be used in direct contact with the dental pulp. The objective of this study is the in vitro evaluation of Biodentine, on cultures of cells of the human dental pulp (HDPC). Biodentine cement disks of 2 x 6 mm were prepared, and HDPC culture plates were introduced. After 24, 48 and 72 hours of exposure, cell viability tests were performed using the MTT colorimetric method. On the other hand, protein expression assays of two proteins involved in the signaling pathway of cell apoptosis Caspase-3 cleaved (cas-3 clv) and PARP-1 are carried out. There are statistically significant differences (p <0,05) in the cell viability tests between Biodentine and control group, as well as the exposure time increases (p <0,05). Otherwise, there are also significant differences (p <0,05) in the expression of PARP-1 in the groups, sometimes a Biodentine. The results in this study that Biodentine generates a cellular cytotoxicity in HDPC cultures, therefore, cell viability as the expression of apoptotic proteins. This is why the use of this biomaterial should be studied for each particular clinical case, especially as a direct pulp capping agent.

Ionic Nanocomplexes of Hyaluronic Acid and Polyarginine to Form Solid Materials: A Green Methodology to Obtain Sponges with Biomedical Potential.

We report on the design, development, characterization, and a preliminary cellular evaluation of a novel solid material. This material is composed of low-molecular-weight hyaluronic acid (LMWHA) and polyarginine (PArg), which generate aqueous ionic nanocomplexes (INC) that are then freeze-dried to create the final product. Different ratios of LMWHA/PArg were selected to elaborate INC, the size and zeta potential of which ranged from 100 to 200 nm and +25 to -43 mV, respectively. Turbidimetry and nanoparticle concentration analyses demonstrated the high capacity of the INC to interact with increasing concentrations of LMWHA, improving the yield of production of the nanostructures. Interestingly, once the selected formulations of INC were freeze-dried, only those comprising a larger excess of LMWHA could form reproducible sponge formulations, as seen with the naked eye. This optical behavior was consistent with the scanning transmission electron microscopy (STEM) images, which showed a tendency of the particles to agglomerate when an excess of LMWHA was present. Mechanical characterization evidenced low stiffness in the materials, attributed to the low density and high porosity. A preliminary cellular evaluation in a fibroblast cell line (RMF-EG) evidenced the concentration range where swollen formulations did not affect cell proliferation (93-464 µM) at 24, 48, or 72 h. Considering that the reproducible sponge formulations were elaborated following inexpensive and non-contaminant methods and comprised bioactive components, we postulate them with potential for biomedical purposes. Additionally, this systematic study provides important information to design reproducible porous solid materials using ionic nanocomplexes.

Aerogels made of chitosan and chondroitin sulfate at high degree of neutralization: Biological properties toward wound healing.

In this study, highly neutralized, highly porous, and ultralight polymeric aerogels prepared from aqueous colloidal suspensions of chitosan (CS) and chondroitin sulfate (ChS) nanocomplexes, formulated as quasi-equimolar amounts of both, are described. These aerogels were designed as healing agents under the inspiration of minimizing the amount of matter applied to wounds, reducing the electrostatic potential of the material and avoiding covalent cross-linkers in order to decrease metabolic stress over wounds. Aerogels synthesized under these criteria are biocompatible and provide specific properties for the induction of wound healing. They do not affect neither the metabolic activity of cultured 3T3 fibroblasts nor the biochemical parameters of experimental animals, open wounds close significantly faster and, unlike control wounds, complete reepithelialization and scarring can be attained 14 days after surgery. Because of its hydration abilities, rapid adaptation to the wound bed and the early accelerator effect of wound closure, the CS/ChS aerogels appear to be functional inducers of the healing. Previous information show that CS/ChS aerogels improve wound bed quality, increase granulation tissue and have pain suppressive effect. CS/ChS aerogels are useful as safe, inexpensive and easy to handle materials for topical applications, such as skin chronic wounds. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2464-2471, 2018.

Relevance of charge balance and hyaluronic acid on alginate-chitosan sponge microstructure and its influence on fibroblast growth.

The study of biomaterials by electrical charge scaling to explore the role of net charge on biocompatibility and suitability for tissue regeneration has been limited as has the search for products that could improve this first-rate variable. In the present study, we prepared sponges composed of chitosan/alginate (CS/ALG) with or without hyaluronic acid (HA) by mixing polymer stock solutions of different net electric charge ratios (n(+/) n(-) ), and then lyophilizing them to obtain porous materials. The electric charge ratios n(+/) n(-) studied were 0.3, 0.8, 1.0, and 2.5 for CS/ALG and 0.3, 1.0, 1.9, and 3.7 for CS/ALG/HA sponges. Under these conditions a role for net electric charge balance over sponge microstructure rearrangement, protection to dissolution, cellular proliferation, and cell-cell interactions was apparent, effects that were enhanced by copolymer modification with HA. Mass balance, electric charge, and specific products that influence both such as HA, have a potential in biomaterials for wound healing. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2537-2543, 2016.

Activation of the human keratinocyte B1 bradykinin receptor induces expression and secretion of metalloproteases 2 and 9 by transactivation of epidermal growth factor receptor.

The B1 bradykinin receptor (BDKRB1) is a component of the kinin cascade localized in the human skin. Some of the effects produced by stimulation of BDKRB1 depend on transactivation of epidermal growth factor receptor (EGFR), but the mechanisms involved in this process have not been clarified yet. The primary purpose of this study was to determine the effect of a BDKRB1 agonist on wound healing in a mouse model and the migration and secretion of metalloproteases 2 and 9 from human HaCaT keratinocytes and delineate the signalling pathways that triggered their secretion. Although stimulation of BDKRB1 induces weak chemotactic migration of keratinocytes and wound closure in an in vitro scratch-wound assay, the BDKRB1 agonist improved wound closure in a mouse model. BDKRB1 stimulation triggers synthesis and secretion of both metalloproteases, effects that depend on the activity of EGFR and subsequent phosphorylation of ERK1/2 and p38 mitogen-activated protein kinases and PI3K/Akt. In the mouse model, immunoreactivity for both gelatinases was concentrated around wound borders. EGFR transactivation by BDKRB1 agonist involves Src kinases family and ADAM17. In addition to extracellular matrix degradation, metalloproteases 2 and 9 regulate cell migration and differentiation, cell functions that are associated with the role of BDKRB1 in keratinocyte differentiation. Considering that BDKRB1 is up-regulated by inflammation and/or by cytokines that are abundant in the inflammatory milieu, more stable BDKRB1 agonists may be of therapeutic value to modulate wound healing.

Intracellular signaling pathways involved in the release of IL-4 and VEGF from human keratinocytes by activation of kinin B1 receptor: functional relevance to angiogenesis.

The injured skin produces a number of mediators that directly or indirectly modulate cell chemotaxis, migration, proliferation, and angiogenesis. Components of the kinin pathway including the kinin B1 receptor (B1R) have been found to occur in the human skin, but information about its role on keratinocyte biology is still scarce. Our aim was to determine whether stimulation of B1R causes the secretion of IL-4 and/or VEGF from human keratinocytes and to evaluate the role of the B1R agonist Lys-des[Arg(9)]bradykinin and IL-4 on various stages of angiogenesis, such as cell migration, proliferation, and release of metalloproteases. By using ELISA and Western blotting, we showed that HaCaT keratinocytes stimulated with the B1R agonist release IL-4 and VEGF. Stimulation of B1R also caused transient c-JunN-terminal kinase phosphorylation and JunB nuclear translocation, transcription factor that regulates IL-4 expression. The 3D-angiogenesis assay, performed on spheroids of EA.hy923 endothelial cells embedded in a collagen matrix, showed that their cumulative sprout area increased significantly following stimulation with either IL-4 or B1R agonist. Furthermore, these ligands produced significant endothelial cell migration and release of metalloproteases 2 and 9, but did not increase endothelial cell proliferation as measured by 5-bromo-2'-deoxyuridine incorporation. Our results provide experimental evidence that establishes IL-4 and B1R agonist as important angiogenic factors of relevance for skin repair.

Kinin B1 receptor regulates interactions between neutrophils and endothelial cells by modulating the levels of Mac-1, LFA-1 and intercellular adhesion molecule-1.

Kinins are pro-inflammatory peptides that mimic the cardinal features of inflammation. We examined the concept that expression levels of endothelial intercellular adhesion molecule-1 (ICAM-1) and neutrophil integrins Mac-1 and LFA-1 are modulated by the kinin B1 receptor (B1R) agonist, Lys-des[Arg(9)]bradykinin (LDBK). Stimulation of endothelial cells with LDBK increased the levels of ICAM-1 mRNA transcripts/protein, and also of E-selectin and platelet endothelial adhesion molecule-1. ICAM-1 levels increased in a magnitude comparable with that produced by TNF-α. This stimulatory effect was reduced when endothelial cells, which had been previously transfected with a B1R small interfering RNA, were stimulated with LDBK, under comparable conditions. Similarly, LDBK produced a significant increase in protein levels of LFA-1 and Mac-1 integrins in human neutrophils, an effect that was reversed by pretreatment of cells with 10 µg/ml cycloheximide or a B1R antagonist. Functional experiments performed with post-confluent monolayers of endothelial cells stimulated with LDBK and neutrophils primed with TNF-α, and vice versa, resulted in enhanced adhesiveness between both cells. Neutralizing Abs to ICAM-1 and Mac-1 reduced the adhesion between them. Our results indicate that kinin B1R is a novel modulator that promotes adhesion of leukocytes to endothelial cells, critically enhancing the movement of neutrophils from the circulation to sites of inflammation.

Kinin B1 receptor activation turns on exocytosis of matrix metalloprotease-9 and myeloperoxidase in human neutrophils: involvement of mitogen-activated protein kinase family.

During neutrophil activation and degranulation, MMP-9 and MPO are released into the extracellular space to propagate inflammatory disorders. As kinin peptides are major participants in acute inflammatory responses, and the G-protein-coupled B(1)R mediates the chemotaxis of human neutrophils, we examined the release of the neutrophil enzymes MMP-9 and MPO by the B(1)R agonist LDBK and determined the signaling pathways that may regulate this cellular effect. Cytochalasin-treated and -untreated neutrophils were suspended in HBSS and stimulated with a range concentration of LDBK for 5 min. Zymography and Western blotting revealed that LDBK induced the release of MMP-9 and MPO. The use of specific signaling transduction inhibitors showed that release of MMP-9 depended on ERK1/2 and p38 MAPKs, whereas release of MPO involved only the p38 cascade. Inhibition of the key steps in these pathways showed that the release of both enzymes depended on PKC and PI3K. Stimulation of neutrophils with LDBK produced phosphorylation of ERK1/2 and p38 MAPK, which was inhibited by B(1)R antagonists. The phosphorylated ERK1/2 MAPK translocated to the neutrophil nucleus, suggesting that transcription of new genes may follow activation of B(1)R. Our results demonstrate that in human neutrophils, activation of kinin B(1)R by LDBK initiates separate signaling cascades that trigger the release of MMP-9 and MPO from tertiary and primary granules, respectively, suggesting that the B(1)R plays a pivotal role in inflammatory disorders.

Stimulation of the bradykinin B(1) receptor induces the proliferation of estrogen-sensitive breast cancer cells and activates the ERK1/2 signaling pathway.

Kinin peptides exert multiple biological effects by binding to two types of G protein-coupled receptors known as B(1) (B(1)R) and B(2) receptors. Expression of the B(1)R in human breast cancer was recently reported, but up to now the consequences of its stimulation are unknown. Our aims were (1) to investigate the capacity of B(1)R to trigger cell proliferation in breast cancer cells, (2) to explore some of the downstream events occurring after B(1)R stimulation that may be linked to cell proliferation, and (3) to determine whether human breast tumors express potentially active B(1)R assessed by the binding of a radiolabeled agonist. Breast cancer cells expressed both the mRNA and the immunoreactive protein of B(1)R that once stimulated triggered cell proliferation at nanomolar concentrations of the ligand. Inhibitor studies suggested that the proliferative effects depend on the activity of epidermal growth factor receptor and subsequent ERK1/2 mitogen-activated protein kinases phosphorylation. B(1)R binding sites, were detected in 3/4 fibroadenomas, in 4/4 ductal carcinomas in situ and in 11/13 invasive ductal carcinomas. The B(1)R-epidermal growth factor receptor crosstalk may be a key interaction that maintains tumor growth, and antagonism of B(1)R may be a valuable alternative for the treatment of breast cancer.

Kinin B2 receptor-coupled signal transduction in human cultured keratinocytes.

Kinins are key pro-inflammatory peptides that exhibit mitogenic effects in tissue-specific cellular systems. Since the life span of the keratinocyte is regulated by receptors that control proliferation and differentiation, and since both processes are affected during wound healing, we have examined the consequence of kinin B2 receptors (B2R) activation in cultured human keratinocytes. Stimulation of keratinocytes by Lys-bradykinin (LBK) induced a rapid and sustained phosphorylation of 42/44 mitogen-activated protein kinase (MAPK) that translocated to the nucleus, and decreased only after 120 min of stimulation. Kinin B1 and B2 receptor (B1R and B2R) antagonists showed that phosphorylation was mainly because of B2R activation. The GF109203X inhibitor almost completely abolished the effect of LBK, suggesting the involvement of protein kinase C in the signal cascade. MAPK phosphorylation was partially dependent on epidermal growth factor receptor transactivation as assessed by the selective inhibitor, AG1478. LBK stimulation did not result in cell proliferation, but produced a rapid c-Fos expression, nuclear translocation of nuclear factor-kappaB, and a moderated (pro)filaggrin synthesis, indicating that it may modulate cell differentiation. Our results support the view that kinins may affect the life span of human keratinocytes and highlight the importance that kinin peptides may have in the pathogenesis and/or progression of skin diseases.