Regulation of the Activity of the Dual Leucine Zipper Kinase by Distinct Mechanisms.

The dual leucine zipper kinase (DLK) alias mitogen-activated protein 3 kinase 12 (MAP3K12) has gained much attention in recent years. DLK belongs to the mixed lineage kinases, characterized by homology to serine/threonine and tyrosine kinase, but exerts serine/threonine kinase activity. DLK has been implicated in many diseases, including several neurodegenerative diseases, glaucoma, and diabetes mellitus. As a MAP3K, it is generally assumed that DLK becomes phosphorylated and activated by upstream signals and phosphorylates and activates itself, the downstream serine/threonine MAP2K, and, ultimately, MAPK. In addition, other mechanisms such as protein-protein interactions, proteasomal degradation, dephosphorylation by various phosphatases, palmitoylation, and subcellular localization have been shown to be involved in the regulation of DLK activity or its fine-tuning. In the present review, the diverse mechanisms regulating DLK activity will be summarized to provide better insights into DLK action and, possibly, new targets to modulate DLK function.

Increase of c-FOS promoter transcriptional activity by the dual leucine zipper kinase.

The dual leucine zipper kinase (DLK) and the ubiquitously expressed transcription factor c-FOS have important roles in beta-cell proliferation and function. Some studies in neuronal cells suggest that DLK can influence c-FOS expression. Given that c-FOS is mainly regulated at the transcriptional level, the effect of DLK on c-FOS promoter activity was investigated in the beta-cell line HIT. The methods used in this study are the following: Luciferase reporter gene assays, immunoblot analysis, CRISPR-Cas9-mediated genome editing, and real-time quantitative PCR. In the beta-cell line HIT, overexpressed DLK increased c-FOS promoter activity twofold. Using 5'-,3'-promoter deletions, the promoter regions from - 348 to - 339 base pairs (bp) and from a - 284 to - 53 bp conferred basal activity, whereas the promoter region from - 711 to - 348 bp and from - 53 to + 48 bp mediated DLK responsiveness. Mutation of the cAMP response element within the promoter prevented the stimulatory effect of DLK. Treatment of HIT cells with KCl and the adenylate cyclase activator forskolin increased c-FOS promoter transcriptional activity ninefold. Since the transcriptional activity of those promoter fragments activated by KCl and forskolin was decreased by DLK, DLK might interfere with KCl/forskolin-induced signaling. In a newly generated, genome-edited HIT cell line lacking catalytically active DLK, c-Fos mRNA levels were reduced by 80% compared to the wild-type cell line. DLK increased c-FOS promoter activity but decreased stimulated transcriptional activity, suggesting that DLK fine-tunes c-FOS promoter-dependent gene transcription. Moreover, at least in HIT cells, DLK is required for FOS mRNA expression.

Arginine:Glycine Amidinotransferase Is Essential for Creatine Supply in Mice During Chronic Hypoxia.

Objective: Chronic hypoxia induces pulmonary and cardiovascular pathologies, including pulmonary hypertension (PH). L-arginine:glycine amidinotransferase (AGAT) is essential for homoarginine (hArg) and guanidinoacetate synthesis, the latter being converted to creatine by guanidinoacetate methyltransferase. Low hArg concentrations are associated with cardiovascular morbidity and predict mortality in patients with PH. We therefore aimed to investigate the survival and cardiac outcome of AGAT knockout (Agat -/-) mice under hypoxia and a possible rescue of the phenotype. Methods: Agat -/- mice and wild-type (WT) littermates were subjected to normoxia or normobaric hypoxia (10% oxygen) for 4 weeks. A subgroup of Agat -/- mice was supplemented with 1% creatine from weaning. Survival, hematocrit, blood lactate and glucose, heart weight-to-tibia length (HW/TL) ratio, hArg plasma concentration, and Agat and Gamt expression in lung, liver, and kidneys were evaluated. Results: After 6 h of hypoxia, blood lactate was lower in Agat -/--mice as compared to normoxia (p < 0.001). Agat -/- mice died within 2 days of hypoxia, whereas Agat -/- mice supplemented with creatine and WT mice survived until the end of the study. In WT mice, hematocrit (74 ± 4 vs. 55 ± 2%, mean ± SD, p < 0.001) and HW/TL (9.9 ± 1.3 vs. 7.3 ± 0.7 mg/mm, p < 0.01) were higher in hypoxia, while hArg plasma concentration (0.25 ± 0.06 vs. 0.38 ± 0.12 µmol/L, p < 0.01) was lower. Agat and Gamt expressions were differentially downregulated by hypoxia in lung, liver, and kidneys. Conclusion: Agat and Gamt are downregulated in hypoxia. Agat-/- mice are nonviable in hypoxia. Creatine rescues the lethal phenotype, but it does not reduce right ventricular hypertrophy of Agat-/- mice in hypoxia.

CRIP1 expression in monocytes related to hypertension.

Hypertension is a complex and multifactorial disorder caused by lifestyle and environmental factors, inflammation and disease-related genetic factors and is a risk factor for stroke, ischemic heart disease and renal failure. Although circulating monocytes and tissue macrophages contribute to the pathogenesis of hypertension, the underlying mechanisms are poorly understood. Cysteine rich protein 1 (CRIP1) is highly expressed in immune cells, and CRIP1 mRNA expression in monocytes associates with blood pressure (BP) and is up-regulated by proinflammatory modulation suggesting a link between CRIP1 and BP regulation through the immune system. To address this functional link, we studied CRIP1 expression in immune cells in relation to BP using a human cohort study and hypertensive mouse models. CRIP1 expression in splenic monocytes/macrophages and in circulating monocytes was significantly affected by angiotensin II (Ang II) in a BP-elevating dose (2 mg/kg/day). In the human cohort study, monocytic CRIP1 expression levels were associated with elevated BP, whereas upon differentiation of monocytes to macrophages this association along with the CRIP1 expression level was diminished. In conclusion, CRIP1-positive circulating and splenic monocytes seem to play an important role in hypertension related inflammatory processes through endogenous hormones such as Ang II. These findings suggest that CRIP1 may affect the interaction between the immune system, in particular monocytes, and the pathogenesis of hypertension.

Bicuspid aortopathy - molecular involvement of microRNAs and MMP-TIMP.

OBJECTIVES: We aimed to elucidate the correlation between expression patterns of aortic tissue microRNAs and the aortopathy formation in bicuspid aortic valve (BAV) disease. METHODS: All 65 patients who underwent elective aortic valve repair/replacement +/- proximal aortic replacement due to BAV disease with or without concomitant aortic aneurysm were identified from our BAV registry. Aortic tissue was collected intraoperatively from the ascending aorta at the greater and lesser curvature. Aortic tissue microRNAs analysis included 11 microRNAs (miR-1, miR-17, miR-18a, miR-19a, miR-20a, miR-21, miR-29b, miR-106a, miR-133a, miR-143 and miR-145). Furthermore, analysis of MMP2, TIMP1/2 mRNA and the protein expression was subsequently performed. The primary study endpoint was the correlation between microRNAs and MMP2, TIMP1/2 mRNA/protein expression. RESULTS: We found a significant association between miR-133a and TIMP1 mRNA (r = 0.870, p < 0.001), an inverse correlation between miR-143a and MMP2 protein expression (r= -0.614, p = 0.044) and a positive correlation between miR-133a and TIMP-2 protein expression (r = 0.583, p = 0.036) at the greater curvature. CONCLUSION: Our findings indicate that aortic tissue microRNAs may reflect remodelling processes of the proximal aorta in BAV aortopathy. Specific aortic tissue microRNAs may exert their regulatory effects on the aortopathy through their impact on MMPs/TIMPs homeostasis at the level of the greater curvature.

TNFα-induced DLK activation contributes to apoptosis in the beta-cell line HIT.

Reduction in beta-cell mass and function contributes to the pathogenesis of diabetes mellitus type 2. The proinflammatory cytokines tumor necrosis factor (TNF)α and interleukin (IL)-1ß have been implicated in the pathogenesis of this disease. Overexpression of the dual leucine zipper kinase (DLK) inhibits beta-cell function and induces apoptosis in the beta-cell line HIT. In the present study, it was investigated whether TNFα or IL-1ß stimulates DLK enzymatic activity. Immunoblot analysis, transient transfection with luciferase reporter gene assays, and immunofluorescence were used. In contrast to IL-1ß, TNFα stimulated DLK kinase activity, which was dependent on the c-Jun N-terminal kinase (JNK). Furthermore, DLK contributed to TNFα-induced JNK phosphorylation. The phosphorylation of DLK on Ser-302 within the activation loop was required for DLK to stimulate JNK and to inhibit CREB-dependent gene transcription. TNFα induced apoptosis in a time- and concentration-dependent manner and inhibited CREB-directed gene transcription in HIT cells. The reduction of endogenous DLK by small interfering or small hairpin RNA attenuated TNFα's effects on apoptosis and CREB-dependent transcription. These data suggest that TNFα induces beta-cell apoptosis through activation of DLK thereby inhibiting the beta-cell protective transcription factor CREB. Furthermore, activation of DLK by a well-known diabetic risk factor supports the role of DLK in the pathogenesis of diabetes mellitus. Thus, the inhibition of DLK might prevent or retard the pathogenesis of diabetes mellitus type 2.

Distinct functions of the dual leucine zipper kinase depending on its subcellular localization.

The dual leucine zipper kinase DLK induces ß-cell apoptosis by inhibiting the transcriptional activity conferred by the ß-cell protective transcription factor cAMP response element binding protein CREB. This action might contribute to ß-cell loss and ultimately diabetes. Within its kinase domain DLK shares high homology with the mixed lineage kinase (MLK) 3, which is activated by tumor necrosis factor (TNF) α and interleukin (IL)-1ß, known prediabetic signals. In the present study, the regulation of DLK in ß-cells by these cytokines was investigated. Both, TNFα and IL-1ß induced the nuclear translocation of DLK. Mutations within a putative nuclear localization signal (NLS) prevented basal and cytokine-induced nuclear localization of DLK and binding to the importin receptor importin α, thereby demonstrating a functional NLS within DLK. DLK NLS mutants were catalytically active as they phosphorylated their down-stream kinase c-Jun N-terminal kinase to the same extent as DLK wild-type but did neither inhibit CREB-dependent gene transcription nor transcription conferred by the promoter of the anti-apoptotic protein BCL-xL. In addition, the ß-cell apoptosis-inducing effect of DLK was severely diminished by mutation of its NLS. In a murine model of prediabetes, enhanced nuclear DLK was found. These data demonstrate that DLK exerts distinct functions, depending on its subcellular localization and thus provide a novel level of regulating DLK action. Furthermore, the prevention of the nuclear localization of DLK as induced by prediabetic signals with consecutive suppression of ß-cell apoptosis might constitute a novel target in the therapy of diabetes mellitus.

La bioética y el autocuidado de la salud: imperativos para la formación integral en la universidad / A bioética e o autocuidado em saúde: imperativos para a formação integral na universidade / Bioethics and auto health care: imperatives for integral training in the university

El autocuidado es responsabilidad del ser humano. La formación integral (FI), como proceso de desarrollo humano, ubica al docente universitario en la socialización terciaria de los estudiantes, reconociéndolos como seres dignos y sujetos de derechos y deberes. La FI fomenta un diálogo multidisciplinario y pluralista sobre cuestiones éticas entre todas las partes interesadas, incluyendo la sociedad en su conjunto. Objetivos: identificar significados que los docentes atribuyen al autocuidado en la FI, como deber bioético, y desentrañar motivos de los docentes para incluir el autocuidado en la FI de los estudiantes. Métodos: investigación cualitativa, de corte comprensivo con grupo focal; unidad de trabajo: docentes; unidad de análisis: expresiones de los participantes. Conclusiones: para asumir el compromiso bioético, es necesario el autocuidado en la FI, mediante estrategias concienciadoras en la cotidianidad, capaces de transformar el actuar de los universitarios.

Self care is the responsibility of human beings. Integral training (IT) as a process of human development places university professors as tertiary socialization for students, recognizing them as beings with dignity, subjects of rights and duties. IT promotes multidisciplinary and pluralist dialogue about bioethics topics among all interested parties, including society as a whole. Objectives: To identify meanings of auto health care attributed by professors in IT as bioethical duty and to unfold professors motives to include auto health care in IT for students. Methods: qualitative research of comprehensive nature by focus group discussion; unit of study: professors; unit of analysis: statements of participants. Conclusions: in order to compromise with bioethics, auto health care is needed in IT by taking conscientious strategies in the daily life, able to transform university students acting.

O autocuidado é responsabilidade do ser humano. A formação integral (FI) como processo de Desenvolvimento Humano situa o docente universitario na socialização terciária dos estudantes, reconhecendo-os como seres dignos, sujeitos de direitos e deveres, para fomentar um diálogo multidisciplinar e pluralista sobre as questões de bioética para todas as partes interessadas, incluindo a sociedade em seu conjunto. Objetivos: Identificar significados que atribuem docentes ao autocuidado na FI, como dever bioético e compreender os motivos dos docentes para incluir o autocuidado na FI dos estudantes. Métodos: investigação qualitativa, de corte compreensivo com grupo focal; unidade de trabalho: docentes; unidade de análise: expressões dos participantes. Conclusões: Para assumir o compromisso bioético é necessário o autocuidado na FI, mediante estratégias conscientizadoras na cotidianidade, capazes de transformar a atuação dos universitários.

El autocuidado: un compromiso de la formación integral en educación superior / Self care: a comprenhensive education compromise in higher education / O autocuidado: um compromisso da formação integral em educação superior

Objetivo: descubrir en las expresiones de los docentes la relación del autocuidado con la formación integral en la Universidad de Caldas, Manizales, entre el 2008 y el 2010. Método: estudio cualitativo interpretativo, se indagó sobre los significados y sentidos que tienen para los docentes la formación integral y su relación con el autocuidado. Unidad de trabajo: 34 docentes; unidad de análisis: las expresiones aportadas por los docentes en grupos focales. Se solicitó el consentimiento informado a los participantes. Categorías de análisis: ser, saber, concienciar, actuar e interactuar. Hallazgos y discusión: “Yo parto del presupuesto de que es la capacidad negociadora del estudiante lo que le permite autorregularse; que implica una dimensión humana, porque esta se requiere al igual que la capacidad cognitiva, que deben integrarse como condición particular de la vida para trabajar todas las funciones cognitivas y ejecutivas complejas, la capacidad de autogobernarse permite manejar mi propia vida y situarme teniendo todas las posibilidades”. Parece ser que el problema del autocuidado hace referencia a la conciencia sobre lo que es la vida, el cuerpo, la salud y el bienestar, incluyendo las esferas que constituyen al ser humano. Conclusión: la formación integral se logra por medio de un proceso educativo, indica aquellas acciones dirigidas hacia adelante, que educan e involucran al docente, es decir, lo incluye como aquella persona que enseña. Permite enriquecer a los sujetos comprometidos en el sentido que orientan hacia el perfeccionamiento de sus cualidades y virtudes. Los profesores resaltan la importancia de su actuar académico como émulo para los estudiantes.

Objective: To discover the professors’ expressions related to self care in comprehensive education at Universidad de Caldas, Manizales, between 2008 and 2010. Method: An interpretive-qualitative study in which meaning and sense that comprehensive education has for the professors and their relation with self care, are investigated. Work unit: 34 professors; analysis unit: expressions provided by professors in focal groups. Informed consent from the participants was requested. Analysis categories: Being, knowledge, being aware of, acting and interacting. Findings and discussion: “I start from the supposition that it is the students’ negotiating ability which allows them to self-regulate. This capacity implies a human dimension because it is a requirement, as well as it is the cognitive ability, which must be integrated as a particular life condition to work in the complexity of cognitive and executive functions. The capacity to self-govern allows me to manage my own life and place myself having all possibilities at hand.” It seems to be that the problem of self care refers to the conscience of what life, body, health, and welfare are all about, including all spheres that constitute the human beings. Conclusion: comprehensive education is achieved by means of an educational process; it indicates those actions directed to the future which educate and involve professors. This is to say what includes the way in which a person teaches. It allows the enrichment of committed people in the sense that they guide students towards the perfectionism of their qualities and virtues. Professors highlight the importance of their academic actions as an emulator for the students.

Objetivo: descobrir nas expressões dos docentes a relação do autocuidado com a formação integral na Universidade de Caldas, Manizales, entre 2008 e 2010. Método: pesquisa qualitativa interpretativa, indagou se sobre os significados e sentidos que tem para os docentes a formação integral e sua relação com o autocuidado. Unidade de trabalho: 34 docentes, unidade de analise: expressões aportadas pelos docentes em grupos focais. Solicitou se o consentimento informado aos participantes. Categorias de analise: ser, saber, conscientização, atuar e interatuar. Descobrimentos e discussão: “Eu parto do pressuposto de que é a capacidade negociadora do estudante o que permite auto-regulasse; que implica uma dimensão humana, porque esta requeira se ao igual que a capacidade cognitiva, que devem integrar se como condição particular da vida para trabalhar todas as funções cognitivas e executivas complexas, a capacidade de auto-regularse permite manejar minha própria vida e situar me tendo todas as possibilidades”. Parece ser que o problema do autocuidado faz referência à consciência sobre o que é a vida, o corpo, a saúde e o bem- estar incluindo as esferas que constituem ao ser humano. Conclusão: a formação integral logra se por meio dum processo educativo, indica aquelas ações dirigidas para adiante, que educam e envolvem ao docente, é dizer, o inclui como aquela pessoa que ensina. Permite enriquecer aos sujeitos comprometidos no sentido que orientam até o aperfeiçoamento de suas qualidades e virtudes. Os professores ressaltam a importancia de seu atuar acadêmico como emulo para os estudantes.