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Impaired alveolar formation and maintenance are features of many pulmonary diseases that are associated with significant morbidity and mortality. In a forward genetic screen for modulators of mouse lung development, we identified the non-muscle myosin II heavy chain gene, Myh10. Myh10 mutant pups exhibit cyanosis and respiratory distress, and die shortly after birth from differentiation defects in alveolar epithelium and mesenchyme. From omics analyses and follow up studies, we find decreased Thrombospondin expression accompanied with increased matrix metalloproteinase activity in both mutant lungs and cultured mutant fibroblasts, as well as disrupted extracellular matrix (ECM) remodeling. Loss of Myh10 specifically in mesenchymal cells results in ECM deposition defects and alveolar simplification. Notably, MYH10 expression is downregulated in the lung of emphysema patients. Altogether, our findings reveal critical roles for Myh10 in alveologenesis at least in part via the regulation of ECM remodeling, which may contribute to the pathogenesis of emphysema.
Transcription factors can serve as links between tumor microenvironment signaling and oncogenesis. Interferon regulatory factor 9 (IRF9) is recruited and expressed upon interferon stimulation and is dependent on cofactors that exert in tumor-suppressing or oncogenic functions via the JAK-STAT pathway. IRF9 is frequently overexpressed in human lung cancer and is associated with decreased patient survival; however, the underlying mechanisms remain to be elucidated. Here, we used stably transduced lung adenocarcinoma cell lines (A549 and A427) to overexpress or knockdown IRF9. Overexpression led to increased oncogenic behavior in vitro, including enhanced proliferation and migration, whereas knockdown reduced these effects. These findings were confirmed in vivo using lung tumor xenografts in nude mice, and effects on both tumor growth and tumor mass were observed. Using RNA sequencing, we identified versican (VCAN) as a novel downstream target of IRF9. Indeed, IRF9 and VCAN expression levels were found to be correlated. We showed for the first time that IRF9 binds at a newly identified response element in the promoter region of VCAN to regulate its transcription. Using an siRNA approach, VCAN was found to enable the oncogenic properties (proliferation and migration) of IRF9 transduced cells, perhaps with CDKN1A involvement. The targeted inhibition of IRF9 in lung cancer could therefore be used as a new treatment option without multimodal interference in microenvironment JAK-STAT signaling.
The mouse bitter taste receptors Tas2r143, Tas2r135, and Tas2r126 are encoded by genes that cluster on chromosome 6 and have been suggested to be expressed under common regulatory elements. Previous studies indicated that the Tas2r143/Tas2r135/Tas2r126 cluster is expressed in the heart, but other organs had not been systematically analyzed. In order to investigate the expression of this bitter taste receptor gene cluster in non-gustatory tissues, we generated a BAC (bacterial artificial chromosome) based transgenic mouse line, expressing CreERT2 under the control of the Tas2r143 promoter. After crossing this line with a mouse line expressing EGFP after Cre-mediated recombination, we were able to validate the Tas2r143-CreERT2 transgenic mouse line and monitor the expression of Tas2r143. EGFP-positive cells, indicating expression of members of the cluster, were found in about 47% of taste buds, and could also be found in several other organs. A population of EGFP-positive cells was identified in thymic epithelial cells, in the lamina propria of the intestine and in vascular smooth muscle cells of cardiac blood vessels. EGFP-positive cells were also identified in the epithelium of organs readily exposed to pathogens including lower airways, the gastrointestinal tract, urethra, vagina, and cervix. With respect to the function of cells expressing this bitter taste receptor cluster, RNA-seq analysis in EGFP-positive cells isolated from the epithelium of trachea and stomach showed expression of genes related to innate immunity. These data further support the concept that bitter taste receptors serve functions outside the gustatory system.
G-protein-coupled receptor (GPCR) expression is extensively studied in bulk cDNA, but heterogeneity and functional patterning of GPCR expression in individual vascular cells is poorly understood. Here, we perform a microfluidic-based single-cell GPCR expression analysis in primary smooth muscle cells (SMC) and endothelial cells (EC). GPCR expression is highly heterogeneous in all cell types, which is confirmed in reporter mice, on the protein level and in human cells. Inflammatory activation in murine models of sepsis or atherosclerosis results in characteristic changes in the GPCR repertoire, and we identify functionally relevant subgroups of cells that are characterized by specific GPCR patterns. We further show that dedifferentiating SMC upregulate GPCRs such as Gpr39, Gprc5b, Gprc5c or Gpr124, and that selective targeting of Gprc5b modulates their differentiation state. Taken together, single-cell profiling identifies receptors expressed on pathologically relevant subpopulations and provides a basis for the development of new therapeutic strategies in vascular diseases.
Tumor-associated macrophages (TAMs) influence lung tumor development by inducing immunosuppression. Transcriptome analysis of TAMs isolated from human lung tumor tissues revealed an up-regulation of the Wnt/β-catenin pathway. These findings were reproduced in a newly developed in vitro “trained” TAM model. Pharmacological and macrophage-specific genetic ablation of β-catenin reprogrammed M2-like TAMs to M1-like TAMs both in vitro and in various in vivo models, which was linked with the suppression of primary and metastatic lung tumor growth. An in-depth analysis of the underlying signaling events revealed that β-catenin–mediated transcriptional activation of FOS-like antigen 2 (FOSL2) and repression of the AT-rich interaction domain 5A (ARID5A) drive gene regulatory switch from M1-like TAMs to M2-like TAMs. Moreover, we found that high expressions of β-catenin and FOSL2 correlated with poor prognosis in patients with lung cancer. In conclusion, β-catenin drives a transcriptional switch in the lung tumor microenvironment, thereby promoting tumor progression and metastasis.
Tubulogenesis is essential for the formation and function of internal organs. One such organ is the trachea, which allows gas exchange between the external environment and the lungs. However, the cellular and molecular mechanisms underlying tracheal tube development remain poorly understood. Here, we show that the potassium channel KCNJ13 is a critical modulator of tracheal tubulogenesis. We identify Kcnj13 in an ethylnitrosourea forward genetic screen for regulators of mouse respiratory organ development. Kcnj13 mutants exhibit a shorter trachea as well as defective smooth muscle (SM) cell alignment and polarity. KCNJ13 is essential to maintain ion homeostasis in tracheal SM cells, which is required for actin polymerization. This process appears to be mediated, at least in part, through activation of the actin regulator AKT, as pharmacological increase of AKT phosphorylation ameliorates the Kcnj13-mutant trachea phenotypes. These results provide insight into the role of ion homeostasis in cytoskeletal organization during tubulogenesis.
Background: The Catechol-O-methyltransferase (COMT) represents the key enzyme in catecholamine degradation. Recent studies suggest that the COMT rs4680 polymorphism is associated with the response to endogenous and exogenous catecholamines. There are, however, conflicting data regarding the COMT Met/Met phenotype being associated with an increased risk of acute kidney injury (AKI) after cardiac surgery. The aim of the current study is to prospectively investigate the impact of the COMT rs4680 polymorphism on the incidence of AKI in patients undergoing cardiac surgery.
Methods: In this prospective single center cohort study consecutive patients hospitalized for elective cardiac surgery including cardiopulmonary-bypass (CPB) were screened for participation. Demographic clinical data, blood, urine and tissue samples were collected at predefined time points throughout the clinical stay. AKI was defined according to recent recommendations of the Kidney Disease Improving Global Outcome (KDIGO) group. Genetic analysis was performed after patient enrolment was completed.
Results: Between April and December 2014, 150 patients were recruited. The COMT genotypes were distributed as follows: Val/Met 48.7%, Met/Met 29.3%, Val/Val 21.3%. No significant differences were found for demography, comorbidities, or operative strategy according to the underlying COMT genotype. AKI occurred in 35 patients (23.5%) of the total cohort, and no differences were evident between the COMT genotypes (20.5% Met/Met, 24.7% Val/Met, 25.0% Val/Val, p = 0.66). There were also no differences in the post-operative period, including ICU or in-hospital stay.
Conclusions: We did not find statistically significant variations in the risk for postoperative AKI, length of ICU or in-hospital stay according to the underlying COMT genotype.
The transcription factor vitamin D receptor (VDR) is the high affinity nuclear target of the biologically active form of vitamin D3 (1,25(OH)2D3). In order to identify pure genomic transcriptional effects of 1,25(OH)2D3, we used VDR cistrome, transcriptome and open chromatin data, obtained from the human monocytic cell line THP-1, for a novel hierarchical analysis applying three bioinformatics approaches. We predicted 75.6% of all early 1,25(OH)2D3-responding (2.5 or 4 h) and 57.4% of the late differentially expressed genes (24 h) to be primary VDR target genes. VDR knockout led to a complete loss of 1,25(OH)2D3–induced genome-wide gene regulation. Thus, there was no indication of any VDR-independent non-genomic actions of 1,25(OH)2D3 modulating its transcriptional response. Among the predicted primary VDR target genes, 47 were coding for transcription factors and thus may mediate secondary 1,25(OH)2D3 responses. CEBPA and ETS1 ChIP-seq data and RNA-seq following CEBPA knockdown were used to validate the predicted regulation of secondary vitamin D target genes by both transcription factors. In conclusion, a directional network containing 47 partly novel primary VDR target transcription factors describes secondary responses in a highly complex vitamin D signaling cascade. The central transcription factor VDR is indispensable for all transcriptome-wide effects of the nuclear hormone.
Background: Previously, we used inhibitors blocking BET bromodomain binding proteins (BRDs) in Ewing sarcoma (EwS) and observed that long term treatment resulted in the development of resistance. Here, we analyze the possible interaction of BRD4 with cyclin-dependent kinase (CDK) 9. Methods: Co-immunoprecipitation experiments (CoIP) to characterize BRD4 interaction and functional consequences of inhibiting transcriptional elongation were assessed using drugs targeting of BRD4 or CDK9, either alone or in combination. Results: CoIP revealed an interaction of BRD4 with EWS-FLI1 and CDK9 in EwS. Treatment of EwS cells with CDKI-73, a specific CDK9 inhibitor (CDK9i), induced a rapid downregulation of EWS-FLI1 expression and block of contact-dependent growth. CDKI-73 induced apoptosis in EwS, as depicted by cleavage of Caspase 7 (CASP7), PARP and increased CASP3 activity, similar to JQ1. Microarray analysis following CDKI-73 treatment uncovered a transcriptional program that was only partially comparable to BRD inhibition. Strikingly, combined treatment of EwS with BRD- and CDK9-inhibitors re-sensitized cells, and was overall more effective than individual drugs not only in vitro but also in a preclinical mouse model in vivo. Conclusion: Treatment with BRD inhibitors in combination with CDK9i offers a new treatment option that significantly blocks the pathognomonic EWS-ETS transcriptional program and malignant phenotype of EwS.
With increasing distribution of endovascular stroke therapies, transient middle cerebral artery occlusion (tMCAO) in mice now more than ever depicts a relevant patient population with recanalized M1 occlusion. In this case, the desired therapeutic effect of blood flow restauration is accompanied by breakdown of the blood-brain barrier (BBB) and secondary reperfusion injury. The aim of this study was to elucidate short and intermediate-term transcriptional patterns and the involved pathways covering the different cellular players at the neurovascular unit after transient large vessel occlusion. To achieve this, male C57Bl/6J mice were treated according to an intensive post-stroke care protocol after 60 min occlusion of the middle cerebral artery or sham surgery to allow a high survival rate. After 24 h or 7 days, RNA from microvessel fragments from the ipsilateral and the contralateral hemispheres was isolated and used for mRNA sequencing. Bioinformatic analyses allowed us to depict gene expression changes at two timepoints of neurovascular post-stroke injury and regeneration. We validated our dataset by quantitative real time PCR of BBB-associated targets with well-characterized post-stroke dynamics. Hence, this study provides a well-controlled transcriptome dataset of a translationally relevant mouse model 24 h and 7 days after stroke which might help to discover future therapeutic targets in cerebral ischemia/reperfusion injury.