RESUMEN
The mechanical environment generated through the adhesive interaction of endothelial cells (ECs) with the matrix controls nuclear tension, preventing aberrant gene synthesis and the transition from restrictive to leaky endothelium, a hallmark of acute lung injury (ALI). However, the mechanisms controlling tension transmission to the nucleus and EC-restrictive fate remain elusive. Here, we demonstrate that, in a kinase-independent manner, focal adhesion kinase (FAK) safeguards tension transmission to the nucleus to maintain EC-restrictive fate. In FAK-depleted ECs, robust activation of the RhoA-Rho-kinase pathway increased EC tension and phosphorylation of the nuclear envelope protein, emerin, activating DNMT3a. Activated DNMT3a methylates the KLF2 promoter, impairing the synthesis of KLF2 and its target S1PR1 to induce the leaky EC transcriptome. Repleting FAK (wild type or kinase dead) or inhibiting RhoA-emerin-DNMT3a activities in damaged lung ECs restored KLF2 transcription of the restrictive EC transcriptome. Thus, FAK sensing and control of tension transmission to the nucleus govern restrictive endothelium to maintain lung homeostasis.
Asunto(s)
Núcleo Celular , Células Endoteliales , Factores de Transcripción de Tipo Kruppel , Transcriptoma , Proteína de Unión al GTP rhoA , Animales , Humanos , Ratones , Núcleo Celular/metabolismo , ADN Metiltransferasa 3A , Células Endoteliales/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Fosforilación , Regiones Promotoras Genéticas/genética , Quinasas Asociadas a rho/metabolismo , Quinasas Asociadas a rho/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/genética , Transcriptoma/genética , Masculino , FemeninoRESUMEN
The transcription factor Kruppel-like factor 4 (KLF4) regulates the expression of immunosuppressive and anti-thrombotic proteins. Despite its importance in maintaining homeostasis, the signals that control its expression and the mechanism of its transactivation remain unclarified. CD55 [aka decay accelerating factor (DAF)], now known to be a regulator of T and B cell responses, biases between pro- and anti-inflammatory processes by controlling autocrine C3a and C5a receptor (C3ar1/C5ar1) signaling in cells. The similarity in CD55's and KLF4's regulatory effects prompted analyses of their functional relationship. In vascular endothelial cells (ECs), CD55 upregulation accompanied KLF4 expression via a p-CREB and CREB Binding Protein (CBP) mechanism. In both ECs and macrophages, CD55 expression was essential for KLF4's downregulation of pro-inflammatory/pro-coagulant proteins and upregulation of homeostatic proteins. Mechanistic studies showed that upregulation of KLF4 upregulated CD55. The upregulated CD55 in turn enabled the recruitment of p-CREB and CBP to KLF4 needed for its transcription. Activation of adenylyl cyclase resulting from repression of autocrine C3ar1/C5ar1 signaling by upregulated CD55 concurrently led to p-CREB and CBP recruitment to KLF4-regulated genes, thereby conferring KLF4's transactivation. Accordingly, silencing CD55 in statin-treated HUVEC disabled CBP transfer from the E-selectin to the eNOS promoter. Importantly, silencing CD55 downregulated KLF4's expression. It did the same in untreated HUVEC transitioning from KLF4low growth to KLF4hi contact inhibition. KLF4's and CD55's function in ECs and macrophages thus are linked via a novel mechanism of gene transactivation. Because the two proteins are co-expressed in many cell types, CD55's activity may be broadly tied to KLF4's immunosuppressive and antithrombotic activities.
Asunto(s)
Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Células Endoteliales/metabolismo , Regulación hacia Arriba , Regiones Promotoras GenéticasRESUMEN
Circadian time of intake determines the cardioprotective outcome of glucocorticoids in normal and infarcted hearts. The cardiomyocyte-specific glucocorticoid receptor (GR) is genetically required to preserve normal heart function in the long-term. The GR co-factor KLF15 is a pleiotropic regulator of cardiac metabolism. However, the cardiomyocyte-autonomous metabolic targets of the GR-KLF15 concerted epigenetic action remain undefined. Here we report that circadian time of intake determines the activation of a transcriptional and functional glucose oxidation program in heart by the glucocorticoid prednisone with comparable magnitude between sexes. We overlayed transcriptomics, epigenomics and cardiomyocyte-specific inducible ablation of either GR or KLF15. Downstream of a light-phase prednisone stimulation in mice, we found that both factors are non-redundantly required in heart to transactivate the adiponectin receptor expression (Adipor1) and promote insulin-stimulated glucose uptake, as well as transactivate the mitochondrial pyruvate complex expression (Mpc1/2) and promote pyruvate oxidation. We then challenged this time-specific drug effect in obese diabetic db/db mice, where the heart shows insulin resistance and defective glucose oxidation. Opposite to dark-phase dosing, light-phase prednisone rescued glucose oxidation in db/db cardiomyocytes and diastolic function in db/db hearts towards control-like levels with sex-independent magnitude of effect. In summary, our study identifies novel cardiomyocyte-autonomous metabolic targets of the GR-KLF15 concerted program mediating the time-specific cardioprotective effects of glucocorticoids on cardiomyocyte glucose utilization.