Congenital cystic adenomatoid malformations of the lung: an epithelial transcriptomic approach.

BACKGROUND: The pathophysiology of congenital cystic adenomatoid malformations (CCAM) of the lung remains poorly understood. AIM: This study aimed to identify more precisely the molecular mechanisms limited to a compartment of lung tissue, through a transcriptomic analysis of the epithelium of macrocystic forms. METHODS: Tissue fragments displaying CCAM were obtained during planned surgical resections. Epithelial mRNA was obtained from cystic and normal areas after laser capture microdissection (LCM). Transcriptomic analyses were performed and the results were confirmed by RT-PCR and immunohistochemistry in independent samples. RESULTS: After controlling for RNA quality, we analysed the transcriptomes of six cystic areas and five control areas. In total, 393 transcripts were differentially expressed in the epithelium, between CCAM and control areas. The most highly redundant genes involved in biological functions and signalling pathways differentially expressed between CCAM and control epithelium included TGFB2, TGFBR1, and MAP 2 K1. These genes were considered particularly relevant as they have been implicated in branching morphogenesis. RT-qPCR analysis confirmed in independent samples that TGFBR1 was more strongly expressed in CCAM than in control tissues (p < 0.03). Immunohistochemistry analysis showed TGFBR1 (p = 0.0007) and TGFB2 (p < 0.02) levels to be significantly higher in the epithelium of CCAM than in that of control tissues. CONCLUSIONS: This compartmentalised transcriptomic analysis of the epithelium of macrocystic lung malformations identified a dysregulation of TGFB signalling at the mRNA and protein levels, suggesting a possible role of this pathway in CCAM pathogenesis. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01732185.

Polycomb antagonizes p300/CREB-binding protein-associated factor to silence FOXP3 in a Kruppel-like factor-dependent manner.

Inducible gene expression underlies the epigenetically inherited differentiation program of most immune cells. We report that the promoter of the FOXP3 gene possesses two distinct functional states: an "off state" mediated by the polycomb histone methyltransferase complex and a histone acetyltransferase-dependent "on state." Regulating these states is the presence of a Kruppel-like factor (KLF)-containing Polycomb response element. In the KLF10(-/-) mouse, the FOXP3 promoter is epigenetically silenced by EZH2 (Enhancer of Zeste 2)-mediated trimethylation of Histone 3 K27; thus, impaired FOXP3 induction and inappropriate adaptive T regulatory cell differentiation results in vitro and in vivo. The epigenetic transmittance of adaptive T regulatory cell deficiency is demonstrated throughout more than 40 generations of mice. These results provide insight into chromatin remodeling events key to phenotypic features of distinct T cell populations.

Transcriptomic analysis of peritoneal cells in a mouse model of sepsis: confirmatory and novel results in early and late sepsis.

BACKGROUND: The events leading to sepsis start with an invasive infection of a primary organ of the body followed by an overwhelming systemic response. Intra-abdominal infections are the second most common cause of sepsis. Peritoneal fluid is the primary site of infection in these cases. A microarray-based approach was used to study the temporal changes in cells from the peritoneal cavity of septic mice and to identify potential biomarkers and therapeutic targets for this subset of sepsis patients. RESULTS: We conducted microarray analysis of the peritoneal cells of mice infected with a non-pathogenic strain of Escherichia coli. Differentially expressed genes were identified at two early (1 h, 2 h) and one late time point (18 h). A multiplexed bead array analysis was used to confirm protein expression for several cytokines which showed differential expression at different time points based on the microarray data. Gene Ontology based hypothesis testing identified a positive bias of differentially expressed genes associated with cellular development and cell death at 2 h and 18 h respectively. Most differentially expressed genes common to all 3 time points had an immune response related function, consistent with the observation that a few bacteria are still present at 18 h. CONCLUSIONS: Transcriptional regulators like PLAGL2, EBF1, TCF7, KLF10 and SBNO2, previously not described in sepsis, are differentially expressed at early and late time points. Expression pattern for key biomarkers in this study is similar to that reported in human sepsis, indicating the suitability of this model for future studies of sepsis, and the observed differences in gene expression suggest species differences or differences in the response of blood leukocytes and peritoneal leukocytes.

Epidermal-growth-factor-induced proliferation of astrocytes requires Egr transcription factors.

Stimulation of astrocytes with epidermal growth factor (EGF) induced proliferation and triggered the biosynthesis of the transcription factor Egr-1, involving the activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway. No differences in the proliferation rate of astrocytes prepared from wild-type or Egr-1-deficient mice were detected. However, expression of a dominant-negative mutant of Egr-1 that interfered with DNA-binding of all Egr proteins prevented EGF-induced proliferation of astrocytes. Site-directed mutagenesis of two crucial cysteine residues within the zinc finger DNA-binding domain revealed that DNA-binding of the Egr-1 mutant was essential to inhibit proliferation of EGF-stimulated astrocytes. Expression of NAB2 (a negative co-regulator of Egr-1, Egr-2 and Egr-3) or a dominant-negative mutant of Elk-1 (a key regulator of Egr-1 biosynthesis) abolished EGF-induced proliferation of astrocytes. Chromatin immunoprecipitation experiments showed that Egr-1, Egr-2 and Egr-3 bound to the gene expressing basic fibroblast growth factor (bFGF) in EGF-stimulated astrocytes. Egr-2 and Egr-3 also interacted with the bFGF gene in EGF-stimulated astrocytes prepared from Egr-1-deficient mice, indicating that loss of Egr-1 is compensated by other Egr proteins. Together, these data show that Egr transcription factors are essential for conversion of the mitogenic signal of EGF into a proliferative response.

Neurturin evokes MAPK-dependent upregulation of Egr4 and KCC2 in developing neurons.

The K-Cl cotransporter KCC2 plays a crucial role in the functional development of GABA(A)-mediated responses rendering GABA hyperpolarizing in adult neurons. We have previously shown that BDNF upregulates KCC2 in immature neurons through the transcription factor Egr4. The effect of BDNF on Egr4 and KCC2 was shown to be dependent on the activation of ERK1/2. Here we demonstrate that the trophic factor neurturin can also trigger Egr4 expression and upregulate KCC2 in an ERK1/2-dependent manner. These results show that Egr4 is an important component in the mechanism for trophic factor-mediated upregulation of KCC2 in immature neurons involving the activation of specific intracellular pathways common to BDNF and Neurturin.

KLF10 is upregulated in osteoarthritis and inhibits chondrocyte proliferation and migration by upregulating Acvr1 and suppressing inhbb expression.

BACKGROUD: Osteoarthritis (OA) is a common disease caused by chondrocyte dysfunction. KLF10 is a member of the Sp1-like transcription factor family that is involved in regulating osteoblasts, but its expression and regulatory mechanism(s) in chondrocytes are unclear. In the present study, we aimed to investigate the regulatory role of KLF10 on the pathological process of OA. METHODS: KLF10 expression in the cartilaginous tissue of patients with osteoarthritis (OA) was evaluated by immunohistochemistry (IHC). Next, we generated an OA mouse model, and the histological changes in cartilage tissue were verified using H&E staining, Safranin O-Fast Green staining, and IHC assays. KLF10 expression in the articular chondrocytes of OA mice was determined by qRT-PCR and Western blotting. To investigate the role of KLF10 in regulating cell proliferation and migration, a KLF10 overexpression plasmid was constructed and transfected into primary mouse chondrocytes. Subsequently, we used RNA sequencing (RNA-seq) to screen differentially expressed genes in chondrocytes with or without KLF10 overexpression. qRT-PCR was used for verification purposes. RESULTS: We found that KLF10 was upregulated in the cartilaginous tissue of patients with OA as well as in cartilaginous tissue of the OA mouse model. KLF10 overexpression inhibited the proliferation and migration of chondrocytes. Furthermore, RNA sequencing results identified increased expression of Acvr1 and decreased expression of Inhbb in cells overexpressing KLF10. Changes in mRNA expression of Acvr1 and Inhbb were confirmed by qRT-PCR. CONCLUSIONS: KLF10 inhibits chondrocyte proliferation and migration by regulating the expression of Acvr1 and Inhbb in both human and mouse OA. These data suggest that KLF10 may be a potential therapeutic target for the treatment of OA.

Salvia miltiorrhiza bunge exerts anti-oxidative effects through inhibiting KLF10 expression in vascular smooth muscle cells exposed to high glucose.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicinal herb Salvia miltiorrhiza Bunge(Danshen) and its components have been widely used to treat cardiovascular diseases for hundreds of years in China, including hypertension, diabetes, atherosclerosis, and chronic heart failure. Salvia miltiorrhiza injection (SMI), an aqueous extracts of Salvia miltiorrhiza Bunge, is one of most widely used traditional Chinese medicine injections. SMI is widely used in the treatment of diabetic vascular complications, However, the mechanisms remain to be defined. AIM OF THE STUDY: To investigate protective mechanism of Salvia miltiorrhiza Bunge against ROS generation in VSMCs of diabetic mice and patients. MATERIALS AND METHODS: Salvia miltiorrhiza injection (hereinafter referred to as SMI, 1.5 g mL-1), which was approved by the State Food and Drug Administration (approval number: Z32020161), was obtained from Shenlong Pharmaceutical Co., Ltd. (batch number: 11040314). SMI or vehicle were intraperitoneally administrated to the HFD-fed db/db mice, artery was harvested after 24weeks later. qRT-PCR and Western blot analysis were used to detect the expression of KLF6, KLF5, KLF4, KLF10, KLF12, and HO-1. DCFH-DA staining detected intracellular ROS production. Loss- and gain-of-function experiments of KLF10 were used to investigate the effect of KLF10 on the expression of HO-1. Dual-luciferase reporter assay evaluated the effect of KLF10 on the activity of the HO-1 promoter. RESULTS: KLF10 expression and ROS generation are significantly increased in the arteries of HFD-fed db/db mice, VSMCs of diabetic patients, as well as in high glucose-treated VSMCs. KLF10 overexpression suppresses, while its knockdown facilitates the expression of heme oxygenase (HO-1) mRNA and protein. Further, Salvia miltiorrhiza injection (SMI) abrogates KLF10 upregulation and reduces ROS generation induced by high glucose in VSMCs. Mechanistically, KLF10 negatively regulates the HO-1 gene transcription via directly binding to its promoter. Accordingly, SMI treatment of VSMCs reduces ROS generation through inhibiting KLF10 expression and thus relieving KLF10 repression of the expression of HO-1 gene, subsequently contributing to upregulation of HO-1. CONCLUSION: SMI exerts anti-oxidative effects on VSMCs exposed to high glucose through inhibiting KLF10 expression and thus upregulating HO-1.

Upregulation of the neuron-specific K+/Cl- cotransporter expression by transcription factor early growth response 4.

The expression of the neuron-specific K+/Cl- cotransporter (KCC2) is restricted to the CNS and is strongly upregulated during neuronal maturation, yielding a low intracellular chloride concentration that is required for fast synaptic inhibition in adult neurons. To elucidate the mechanisms of KCC2 gene regulation, we analyzed the KCC2 (alias Slc12a5) promoter and proximal intron-1 regions and revealed 10 candidate transcription factor binding sites that are highly conserved in mammalian KCC2 genes. Here we focus on one of these factors, early growth response 4 (Egr4), which shows a similar developmental upregulation in CNS neurons as KCC2. KCC2 luciferase reporter constructs containing the Egr4 site (Egr4(KCC2)) were strongly induced by Egr4 overexpression in neuro-2a neuroblastoma cells and in cultured neurons. Egr4-mediated induction was decreased significantly by point-mutating the Egr4(KCC2). Insertion of Egr4(KCC2) into the KCC2 basal promoter in the endogenous reverse, but not in the opposite, orientation reestablished Egr4-mediated induction. Electrophoretic mobility shift assay confirmed specific Egr4 binding to Egr4(KCC2). Interference RNA-mediated knock-down of Egr4 and a dominant-negative isoform of Egr4 significantly inhibited KCC2 reporter induction and endogenous KCC2 expression in cultured neurons. Together, the results indicate an important role for Egr4 in the developmental upregulation of KCC2 gene expression.

RNA-seq Based Transcription Characterization of Fusion Breakpoints as a Potential Estimator for Its Oncogenic Potential.

Based on high-throughput sequencing technology, the detection of gene fusions is no longer a big challenge but estimating the oncogenic potential of fusion genes remains challenging. Recent studies successfully applied machine learning methods and gene structural and functional features of fusion mutation to predict their oncogenic potentials. However, the transcription characterizations features of fusion genes have not yet been studied. In this study, based on the clonal evolution theory, we hypothesized that a fusion gene is more likely to be an oncogenic genomic alteration, if the neoplastic cells harboring this fusion mutation have larger clonal size than other neoplastic cells in a tumor. We proposed a novel method, called iFCR (internal Fusion Clone Ratio), given an estimation of oncogenic potential for fusion mutations. We have evaluated the iFCR method in three public cancer transcriptome sequencing datasets; the results demonstrated that the fusion mutations occurring in tumor samples have higher internal fusion clone ratio than normal samples. And the most frequent prostate cancer fusion mutation, TMPRSS2-ERG, appears to have a remarkably higher iFCR value in all three independent patients. The preliminary results suggest that the internal fusion clone ratio might potentially advantage current fusion mutation oncogenic potential prediction methods.

RBFOX3/NeuN is Required for Hippocampal Circuit Balance and Function.

RBFOX3 mutations are linked to epilepsy and cognitive impairments, but the underlying pathophysiology of these disorders is poorly understood. Here we report replication of human symptoms in a mouse model with disrupted Rbfox3. Rbfox3 knockout mice displayed increased seizure susceptibility and decreased anxiety-related behaviors. Focusing on hippocampal phenotypes, we found Rbfox3 knockout mice showed increased expression of plasticity genes Egr4 and Arc, and the synaptic transmission and plasticity were defective in the mutant perforant pathway. The mutant dentate granules cells exhibited an increased frequency, but normal amplitude, of excitatory synaptic events, and this change was associated with an increase in the neurotransmitter release probability and dendritic spine density. Together, our results demonstrate anatomical and functional abnormality in Rbfox3 knockout mice, and may provide mechanistic insights for RBFOX3-related human brain disorders.

Early growth response proteins EGR-4 and EGR-3 interact with immune inflammatory mediators NF-kappaB p50 and p65.

Here, we characterize the basis for the T-cell-specific activity of the human zinc-finger protein early growth response factor 4 (EGR-4). A yeast two-hybrid screen showed interaction of EGR-4 with NF-kappaB p50. Using recombinant proteins, stable physical complex formation was confirmed for EGR-4 and EGR-3 with p50 and with p65 using glutathione-S-transferase pull-down assays and surface-plasmon-resonance and peptide-spot analyses. In vivo interaction of EGR-4 and EGR-3 with NF-kappaB p65 was demonstrated by immunoprecipitation experiments and fluorescence-resonance-energy transfer (FRET) analysis showing interaction in the nucleus of transfected Jurkat T cells. In transfection assays, EGR-p50 complexes were transcriptionally inactive and EGR-p65 complexes strongly activated transcription of the promoters of the human genes encoding the cytokines interleukin 2, tissue necrosis factor alpha and ICAM-1. The EGR-p65 complexes increased reporter-gene activity about 100-fold and thus exceeded the transcriptional activities of the p65 homodimer and the p65/p50 heterodimers. The major interaction domain for p65 was localized within the third zinc finger of EGR-4 using deletion mutants for pull-down assays and peptide-spot assays. By computer modeling, this interaction domain was localized to an alpha-helical region and shown to have the central amino acids surface exposed and thus accessible for interaction. In summary, in T cells, the two zinc-finger proteins EGR-4 and EGR-3 interact with the specific nuclear mediator NF-kappaB and control transcription of genes encoding inflammatory cytokines.