Glucagon-like peptide-1 receptor signaling modifies the extent of diabetic kidney disease through dampening the receptor for advanced glycation end products-induced inflammation.

Glucagon like peptide-1 (GLP-1) is a hormone produced and released by cells of the gastrointestinal tract following meal ingestion. GLP-1 receptor agonists (GLP-1RA) exhibit kidney-protective actions through poorly understood mechanisms. Here we interrogated whether the receptor for advanced glycation end products (RAGE) plays a role in mediating the actions of GLP-1 on inflammation and diabetic kidney disease. Mice with deletion of the GLP-1 receptor displayed an abnormal kidney phenotype that was accelerated by diabetes and improved with co-deletion of RAGE in vivo. Activation of the GLP-1 receptor pathway with liraglutide, an anti-diabetic treatment, downregulated kidney RAGE, reduced the expansion of bone marrow myeloid progenitors, promoted M2-like macrophage polarization and lessened markers of kidney damage in diabetic mice. Single cell transcriptomics revealed that liraglutide induced distinct transcriptional changes in kidney endothelial, proximal tubular, podocyte and macrophage cells, which were dominated by pathways involved in nutrient transport and utilization, redox sensing and the resolution of inflammation. The kidney-protective action of liraglutide was corroborated in a non-diabetic model of chronic kidney disease, the subtotal nephrectomised rat. Thus, our findings identify a novel glucose-independent kidney-protective action of GLP-1-based therapies in diabetic kidney disease and provide a valuable resource for exploring the cell-specific kidney transcriptional response ensuing from pharmacological GLP-1R agonism.

Characterization of K562 cells: uncovering novel chromosomes, assessing transferrin receptor expression, and probing pharmacological therapies.

Human erythroleukemic K562 cells represent the prototypical cell culture model of chronic myeloid leukemia (CML). The cells are pseudo-triploid and positive for the Philadelphia chromosome. Therefore, K562 cells have been widely used for investigating the BCR/ABL1 oncogene and the tyrosine kinase inhibitor, imatinib-mesylate. Further, K562 cells overexpress transferrin receptors (TfR) and have been used as a model for targeting cytotoxic therapies, via receptor-mediated endocytosis. Here, we have characterized K562 cells focusing on the karyotype of cells in prolonged culture, regulation of expression of TfR in wildtype (WT) and doxorubicin-resistant cells, and responses to histone deacetylase inhibition (HDACi). Karyotype analysis indicates novel chromosomes and gene expression analysis suggests a shift of cultured K562 cells away from patient-derived leukemic cells. We confirm the high expression of TfR on K562 cells using immunofluorescence and cell-surface receptor binding radioassays. Importantly, high TfR expression is observed in patient-derived cells, and we highlight the persistent expression of TfR following doxorubicin acquired resistance. Epigenetic analysis indicates that permissive histone acetylation and methylation at the promoter region regulates the transcription of TfR in K562 cells. Finally, we show relatively high expression of HDAC enzymes in K562 cells and demonstrate the chemotoxic effects of HDACi, using the FDA-approved hydroxamic acid, vorinostat. Together with a description of morphology, infrared spectral analysis, and examination of metabolic properties, we provide a comprehensive characterization of K562 cells. Overall, K562 cell culture systems remain widely used for the investigation of novel therapeutics for CML, which is particularly important in cases of imatinib-mesylate resistance.

Intestinal microbiota regulates diabetes and cancer progression by IL-1ß and NOX4 dependent signaling cascades.

Diabetes changes the host microbiota, a condition known as dysbiosis. Dysbiosis is an important factor for the pathogenesis of diabetes and colorectal cancer (CRC). We aimed at identifying the microbial signature associated with diabetes and CRC; and identifying the signaling mechanism altered by dysbiosis and leading to CRC progression in diabetes. MKR mice that can spontaneously develop type 2 diabetes were used. For CRC induction, another subset of mice was treated with azoxymethane and dextran sulfate sodium. To identify the role of microbiota, microbiota-depleted mice were inoculated with fecal microbial transplant from diabetic and CRC mice. Further, a mouse group was treated with probiotics. At the end of the treatment, 16S rRNA sequencing was performed to identify microbiota in the fecal samples. Blood was collected, and colons were harvested for molecular, anatomical, and histological analysis. Our results show that diabetes is associated with a microbial signature characterized by reduction of butyrate-forming bacteria. This dysbiosis is associated with gastrointestinal complications reflected by a reduction in colon lengths. These changes are reversed upon treatment with probiotics, which rectified the observed dysbiosis. Inoculation of control mice with diabetic or cancer microbiota resulted in the development of increased number of polyps. Our data also show that inflammatory cytokines (mainly interleukin (IL)-1ß) and NADPH oxidase (NOX)4 are over-expressed in the colon tissues of diabetic mice. Collectively our data suggest that diabetes is associated with dysbiosis characterized by lower abundance of butyrate-forming bacteria leading to over-expression of IL-1ß and NOX4 leading to gastrointestinal complications and CRC.

Chromatin context and ncRNA highlight targets of MeCP2 in brain.

The discovery that Rett syndrome (RTT) is caused by mutation of the methyl-CpG-binding-protein MeCP2 provided a major breakthrough in understanding the neurodevelopmental disorder and accelerated MeCP2 research. However, gene regulation by MeCP2 is complicated. The current consensus for MeCP2 remains as a classical repressor complex, with major emphasis on its role in methylation-dependent binding and repression. However, recent evidence indicates additional regulatory roles, suggesting non-classical mechanisms in gene activation. This has opened the field of MeCP2 research and suggests that the gene targets may not be the usual suspects, that is, dependent only on DNA methylation. Here we examine how chromatin binding and sequence preference may confer MeCP2 functionality, and connect relevant pathways in an active genome. Finding both genomic and proteomic evidence to indicate MeCP2 spliceosome interaction, we consequently discovered broad MeCP2 enrichment of the transcriptome while our focus toward long non-coding RNA (lncRNA) revealed MeCP2 association with RNCR3. Our data may indicate an as-yet-unappreciated role between lncRNA and MeCP2. We hypothesize that ncRNA may mediate chromatin-remodeling events by interacting with MeCP2, thereby conferring changes in gene expression. We consider that these results may suggest new mechanisms of gene regulation conferred by MeCP2 and its interactions upon chromatin structure and gene function.

Sex-Based Mhrt Methylation Chromatinizes MeCP2 in the Heart.

In the heart, primary microRNA-208b (pri-miR-208b) and Myheart (Mhrt) are long non-coding RNAs (lncRNAs) encoded by the cardiac myosin heavy chain genes. Although preclinical studies have shown that lncRNAs regulate gene expression and are protective for pathological hypertrophy, the mechanism underlying sex-based differences remains poorly understood. In this study, we examined DNA- and RNA-methylation-dependent regulation of pri-miR-208b and Mhrt. Expression of pri-miR-208b is elevated in the left ventricle of the female heart. Despite indistinguishable DNA methylation between sexes, the interaction of MeCP2 on chromatin is subject to RNase digestion, highlighting that affinity of the methyl-CG reader is broader than previously thought. A specialized procedure to isolate RNA from soluble cardiac chromatin emphasizes sex-based affinity of an MeCP2 co-repressor complex with Rest and Hdac2. Sex-specific Mhrt methylation chromatinizes MeCP2 at the pri-miR-208b promoter and extends the functional relevance of default transcriptional suppression in the heart.

NET silencing by let-7i in postural tachycardia syndrome.

While strongly implicated in postural tachycardia syndrome (POTS), considerable controversy exists regarding norepinephrine transporter (NET) loss of function. POTS is characterized by the clinical symptoms of orthostatic intolerance, lightheadedness, tachycardia, and syncope or near syncope with upright posture. Abnormal sympathetic nervous system activity is typical, of a type which suggests dysfunction of the NET, with evidence that the gene responsible is under tight epigenetic control. Using RNA of isolated chromatin combined with massive parallel sequencing (RICh-seq) we show that let-7i miRNA suppresses NET by methyl-CpG-binding protein 2 (MeCP2). Vorinostat restores epigenetic control and NET expression in leukocytes derived from POTS participants.