Christian Lacks Lino Cardenas

Identification of Keratinocyte Growth Factor as a Target of microRNA-155 in Lung Fibroblasts: Implication in Epithelial-Mesenchymal Interactions

Background Epithelial-mesenchymal interactions are critical in regulating many aspects of vertebrate embryo development, and for the maintenance of homeostatic equilibrium in adult tissues. The interactions between epithelium and mesenchyme are believed to be mediated by paracrine signals such as cytokines and extracellular matrix components secreted from fibroblasts that affect adjacent epithelia. In this study, we sought to identify the repertoire of microRNAs (miRNAs) in normal lung human fibroblasts and their potential regulation by the cytokines TNF-α, IL-1β and TGF-β. Methodology/Principal Findings MiR-155 was significantly induced by inflammatory cytokines TNF-α and IL-1β while it was down-regulated by TGF-β. Ectopic expression of miR-155 in human fibroblasts induced modulation of a large set of genes related to “cell to cell signalling”, “cell morphology” and “cellular movement”. This was consistent with an induction of caspase-3 activity and with an increase in cell migration in fibroblasts tranfected with miR-155. Using different miRNA bioinformatic target prediction tools, we found a specific enrichment for miR-155 predicted targets among the population of down-regulated transcripts. Among fibroblast-selective targets, one interesting hit was keratinocyte growth factor (KGF, FGF-7), a member of the fibroblast growth factor (FGF) family, which owns two potential binding sites for miR-155 in its 3′-UTR. Luciferase assays experimentally validated that miR-155 can efficiently target KGF 3′-UTR. Site-directed mutagenesis revealed that only one out of the 2 potential sites was truly functional. Functional in vitro assays experimentally validated that miR-155 can efficiently target KGF 3′-UTR. Furthermore, in vivo experiments using a mouse model of lung fibrosis showed that miR-155 expression level was correlated with the degree of lung fibrosis. Conclusions/Significance Our results strongly suggest a physiological function of miR-155 in lung fibroblasts. Altogether, this study implicates this miRNA in the regulation by mesenchymal cells of surrounding lung epithelium, making it a potential key player during tissue injury.

miR-199a-5p Is upregulated during fibrogenic response to tissue injury and mediates TGFbeta-induced lung fibroblast activation by targeting caveolin-1.

As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.

Reconstructing dynamic microRNA-regulated interaction networks

The regulation of gene expression in cells, including by microRNAs (miRNAs), is a dynamic process. Current methods for identifying miRNA targets by combining sequence and miRNA and mRNA expression data do not adequately use the temporal information and thus miss important miRNAs and their targets. We developed the MIRna Dynamic Regulatory Events Miner (mirDREM), a probabilistic modeling method that uses input–output hidden Markov models to reconstruct dynamic regulatory networks that explain how temporal gene expression is jointly regulated by miRNAs and transcription factors. We measured miRNA and mRNA expression for postnatal lung development in mice and used mirDREM to study the regulation of this process. The reconstructed dynamic network correctly identified known miRNAs and transcription factors. The method has also provided predictions about additional miRNAs regulating this process and the specific developmental phases they regulate, several of which were experimentally validated. Our analysis uncovered links between miRNAs involved in lung development and differentially expressed miRNAs in idiopathic pulmonary fibrosis patients, some of which we have experimentally validated using proliferation assays. These results indicate that some disease progression pathways in idiopathic pulmonary fibrosis may represent partial reversal of lung differentiation.

Hereditary Influence in Thoracic Aortic Aneurysm and Dissection

Thoracic aortic aneurysm is a potentially life-threatening condition in that it places patients at risk for aortic dissection or rupture. However, our modern understanding of the pathogenesis of thoracic aortic aneurysm is quite limited. A genetic predisposition to thoracic aortic aneurysm has been established, and gene discovery in affected families has identified several major categories of gene alterations. The first involves mutations in genes encoding various components of the transforming growth factor beta (TGF-β) signaling cascade (FBN1, TGFBR1, TGFBR2, TGFB2, TGFB3, SMAD2, SMAD3 and SKI), and these conditions are known collectively as the TGF-β vasculopathies. The second set of genes encode components of the smooth muscle contractile apparatus (ACTA2, MYH11, MYLK, and PRKG1), a group called the smooth muscle contraction vasculopathies. Mechanistic hypotheses based on these discoveries have shaped rational therapies, some of which are under clinical evaluation. This review discusses published data on genes involved in thoracic aortic aneurysm and attempts to explain divergent hypotheses of aneurysm origin.

Genetic polymorphism of CYP4A11 and CYP4A22 genes and in silico insights from comparative 3D modelling in a French population

The CYP4A subfamily is known to ω-hydroxylate the endogenous arachidonic acid into 20-hydroxyeicosatetranoic acid, which has renovascular and tubular functions. The aim of this work was to report a comprehensive investigation of the CYP4A11 and CYP4A22 genetic polymorphisms in a French population. Using PCR-SSCP and sequencing strategies, a total of 26 sequence variations were identified comprising 3 missense mutations for CYP4A11 (Ser404Phe, Phe434Ser and Arg505His) and 7 missense mutations for CYP4A22 (Arg126Trp, Gly130Ser, Asn152Tyr, Val185Phe, Cys231Arg, Leu428Pro and Leu509Phe). In comparison with SNPs reported in the database (dbSNP) of the National Center for Biotechnology information (NCBI), 6 and 3 novel polymorphisms were identified in CYP4A11 and CYP4A22, respectively. The potential impact of the amino acid substitutions on the structure and/or catalytic activity of the enzymes has been estimated by the construction and validation of the CYP4A 3D models. These results could be helpful for further investigations of the potential role of CYP4A variants in the genetic susceptibility to cardiovascular diseases in humans such as arterial hypertension.

Genetic polymorphisms of Glycine N-acyltransferase (GLYAT) in a French Caucasian population

In humans, the glycine N-acyltransferase enzyme (GLYAT) is thought to be important in the detoxification of endogenous and xenobiotic compounds which contain a carboxylic acid group, such as benzoic, isovaleric, or acetylsalicylic acids. The aim of this work was to report a comprehensive investigation of GLYAT genetic polymorphisms in DNA samples from 55 subjects of French Caucasian origin, using polymerase chain reaction–single-strand conformation polymorphism and sequencing strategies. Seven different polymorphisms of the GLYAT gene were identified, including two polymorphisms in the 5′ flanking region of the gene (g.−8457C>T and g.−8010A>G), two polymorphisms in intron 5 (g.13931A>G and g.13944C>T) and three missense mutations in exon 2 (g.49T>A; p.Ser17Thr), exon 5 (g.13886A>G; p.Asn156Ser) and exon 6 (g.14435C>T; p.Arg199Cys). In addition to the wild-type allele GLYAT*1 (2.7%), four novel alleles were identified: GLYAT*2A (75.5%), *2B (4.5%), *3 (16.4%) and *4 (0.9%), and five different genotypes. Localisation of the p.Ser17Thr and p.Arg199Cys missense mutations in predicted secondary structures suggest that these variants might have a potential role on the GLYAT protein activity. These results could be helpful in investigating the potential association of GLYAT variants with an incidence of reduced efficiency in xenobiotic carboxylic acids detoxification in humans.

Arachidonic acid ω-hydroxylase CYP4A11: inter-ethnic variations in the 8590T>C loss-of-function variant

The human Cytochrome P450 4A11 (CYP4A11) is a major ω-hydroxylase involved in the regulation of blood pressure in the kidney through the conversion of arachidonic acid into 20-hydroxyeicosatetraenoic acid (20-HETE). Previous studies have reported a significant association between the 8590T>C genetic variant of CYP4A11 and hypertension. Interestingly, several population-based studies have reported ethnic differences in the prevalence of hypertension, with the highest prevalence in African populations. The aim of this work was to determine the frequency and inter-ethnic comparison of the CYP4A11 (8590T>C) functional polymorphism, in five new ethnic groups: European (99 French Caucasians), African (36 Gabonese and 50 Senegalese), South American (60 Peruvians) and North African (53 Tunisians) populations, using polymerase chain reaction-single strand conformational polymorphism and sequencing strategies. We confirmed that the CYP4A11 (8590T>C) functional polymorphism exhibits inter-ethnic frequency differences. Noteworthy, the highest 8590C allele frequency was observed in the Tunisian (30.2%), followed by Senegalese (20%) populations. In addition, the CC genotype was only found in the Gabonese and Tunisian populations (5.6% and 8.4%, respectively). These populations may be of major interest to help to clarify the linkage between hypertension and CYP4A11 (8590T>C) genotype in African populations. These findings provide data for further studies that investigate the potential association of CYP4A11 (8590T>C) variant with an incidence of hypertension genesis in respect of ethnicity.

Inter-ethnic variability of three functional polymorphisms affecting the IMPDH2 gene

Human type II inosine monophosphate dehydrogenase (IMPDH2) is a key enzyme in the purine nucleotide biosynthetic pathway and constitutes a pivotal biological target for immunosuppressant and antiviral drugs. Several Single Nucleotide Polymorphisms (SNP) affecting the IMPDH2 gene sequence have been reported with potential functional relevance and could impact drugs response. We aimed to determine the frequency of three of these polymorphisms, namely g.3375C>T (Leu263Phe), c.-95T>G and IVS7+10T>C, in Caucasians, Tunisians, Peruvians and Black Africans (Gabonese and Senegalese). The g.3375C>T and c.-95T>G polymorphisms are rare with a Minor Allele Frequency ≤1.0% in our populations, whereas the third variant, IVS7+10T>C, is more frequent and displays large interethnic variations, with an allelic frequency ranging from 14.6% in the French Caucasian population studied to less than 2% in Black African and Peruvian populations. This ethnic-related data might contribute to a better understanding of the variability in clinical outcome and/or dose adjustments of drugs that are IMPDH inhibitors such as mycophenolic acid.

High Concentrations of Rosiglitazone Reduce mRNA and Protein Levels of LRP1 in HepG2 Cells

Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic receptor involved in the uptake of a variety of molecules, such as apoE, α2-macroglobulin, and the amyloid β peptide (Aβ), for either transcellular transport, protein trafficking or lysosomal degradation. The LRP1 gene can be transcribed upon activation of peroxisome proliferator receptor activated-γ (PPARγ) by the potent PPARγ agonist, rosiglitazone (RGZ). In previous studies, RGZ was shown to upregulate LRP1 levels in concentrations between 0.1 and 5 μM in HepG2 cells. In this study, we sought to replicate previous studies and to investigate the molecular mechanism by which high concentrations of RGZ reduce LRP1 levels in HepG2 cells. Our data confirmed that transcriptional activation of LRP1 occurred in response to RGZ at 3 and 10 μM, in agreement with the study reported by Moon et al. (2012a). On the other hand, we found that high concentrations of RGZ decreased both mRNA and protein levels of LRP1. Mechanistically, transcriptional dysregulation of LRP1 was affected by the downregulation of PPARγ in a time- and concentration-dependent manner. However, downregulation of PPARγ was responsible for only 40% of the LRP1 reduction and thereby the remaining loss of LRP1 (60%) was found to be through degradation in the lysosomal system. In conclusion, our findings demonstrate the mechanisms by which high concentrations of RGZ caused LRP1 levels to be reduced in HepG2 cells. Taken together, this data will be helpful to better explain the pharmacological modulation of this pivotal membrane receptor by PPARγ agonists.

An HDAC9-MALAT1-BRG1 complex mediates smooth muscle dysfunction in thoracic aortic aneurysm

Thoracic aortic aneurysm (TAA) has been associated with mutations affecting members of the TGF-β signaling pathway, or components and regulators of the vascular smooth muscle cell (VSMC) actomyosin cytoskeleton. Although both clinical groups present similar phenotypes, the existence of potential common mechanisms of pathogenesis remain obscure. Here we show that mutations affecting TGF-β signaling and VSMC cytoskeleton both lead to the formation of a ternary complex comprising the histone deacetylase HDAC9, the chromatin-remodeling enzyme BRG1, and the long noncoding RNA MALAT1. The HDAC9–MALAT1–BRG1 complex binds chromatin and represses contractile protein gene expression in association with gain of histone H3-lysine 27 trimethylation modifications. Disruption of Malat1 or Hdac9 restores contractile protein expression, improves aortic mural architecture, and inhibits experimental aneurysm growth. Thus, we highlight a shared epigenetic pathway responsible for VSMC dysfunction in both forms of TAA, with potential therapeutic implication for other known HDAC9-associated vascular diseases.Vascular smooth muscle cell (VSMC) dysfunction is a common feature of thoracic aortic aneurysms (TAAs). Here, Lino Cardenas and colleagues show that the formation of a HDAC9-MALAT1-BRG1 complex promotes VSMC dysfunction in TAA by epigenetically altering the expression of key components of the cytoskeleton in VSMCs.