Jean-Marc Lo-Guidice

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.

Increased Circulating miR-21 Levels Are Associated with Kidney Fibrosis

MicroRNAs (miRNAs) are a class of noncoding RNA acting at a post-transcriptional level to control the expression of large sets of target mRNAs. While there is evidence that miRNAs deregulation plays a causative role in various complex disorders, their role in fibrotic kidney diseases is largely unexplored. Here, we found a strong up-regulation of miR-21 in the kidneys of mice with unilateral ureteral obstruction and also in the kidneys of patients with severe kidney fibrosis. In addition, mouse primary fibroblasts derived from fibrotic kidneys exhibited higher miR-21 expression level compared to those derived from normal kidneys. Expression of miR-21 in normal primary kidney fibroblasts was induced upon TGFβ exposure, a key growth factor involved in fibrogenesis. Finally, ectopic expression of miR-21 in primary kidney fibroblasts was sufficient to promote myofibroblast differentiation. As circulating miRNAs have been suggested as promising non-invasive biomarkers, we further assess whether circulating miR-21 levels are associated with renal fibrosis using sera from 42 renal transplant recipients, categorized according to their renal fibrosis severity, evaluated on allograft biopsies (Interstitial Fibrosis/Tubular Atrophy (IF/TA). Circulating miR-21 levels are significantly increased in patients with severe IF/TA grade (IF/TA grade 3: 3.0±1.0 vs lower grade of fibrosis: 1.5±1.2; p = 0.001). By contrast, circulating miR-21 levels were not correlated with other renal histological lesions. In a multivariate linear regression model including IF/TA grade and estimated GFR, independent associations were found between circulating miR-21 levels and IF/TA score (ß = 0.307, p = 0.03), and between miR-21 levels and aMDRD (ß = −0.398, p = 0.006). Altogether, these data suggest miR-21 has a key pathogenic role in kidney fibrosis and may represent a novel, predictive and reliable blood marker of kidney fibrosis.

A novel CYP2D6 allele with an abolished splice recognition site associated with the poor metabolizer phenotype.

A novel loss-of function allele of the CYP2D6 gene was characterized in a PM individual using exon-by-exon PCR-SSCP analysis. This allele, we termed CYP2D6(F), harbours four mutations including a new mutation (D6-F) which abolishes the splice acceptor site of the 1st intron and results in a premature stop codon. DNA samples from a large population of healthy unrelated volunteers were tested for D6-F using a PCR-assay we developed for the specific identification of the mutation in genomic DNA. The prevalence of D6-F was very low. However, its identification combined with that of the previously reported gene inactivating mutations would further increase the phenotype prediction rate by genotyping.

Profiling gene expression of whole cytochrome P450 superfamily in human bronchial and peripheral lung tissues: Differential expression in non-small cell lung cancers.

Susceptibility to lung diseases, such as lung cancer and chronic obstructive pulmonary disease, is largely influenced by the metabolic capacity of lung tissues. This capacity is partly determined by the expression profile of the cytochromes P450 (CYPs), a superfamily of enzymes that have relevant catalytic properties toward exogenous and endogenous compounds. Using quantitative real-time RT-PCR, we conducted a comprehensive analysis of the expression profile of the 57 human CYP genes in non-tumoral (bronchial mucosa and pulmonary parenchyma) and tumoral lung tissues of 18 patients with non-small cell lung cancer. This study highlights (i) inter-individual variations in lung expression for some CYPs, (ii) different CYP expression patterns between bronchial mucosa and pulmonary parenchyma, that indicate distinctive susceptibility of these tissues toward the deleterious effects of inhaled chemical toxicants and carcinogens, (iii) high intertumoral variability, that could have major implications on lung tumor response to anti-cancer drugs.

Xenobiotic Metabolism and Disposition in Human Lung Cell Models: Comparison with In Vivo Expression Profiles

Numerous lung cell lines are currently used as in vitro models for pharmacological and toxicological studies. However, no exhaustive report about the metabolic capacities of these models in comparison with those of lung tissues is available. In the present study, we used a high-throughput quantitative real-time reverse transcription-polymerase chain reaction strategy to characterize the expression profiles of 380 genes encoding proteins involved in the metabolism and disposition of xenobiotics in 10 commonly used lung cell lines (A549, H292, H358, H460, H727, Calu-1, 16HBE, 1 HAEO, BEAS-2B, and L-132) and four primary cultures of human bronchial epithelial cells. Expression results were then compared with those previously obtained in human nontumoral and tumoral lung tissues. Our results revealed disparities in gene expression between lung cell lines or when comparing lung cell lines with primary cells or lung tissues. Primary cell cultures displayed the highest similarities with bronchial mucosa in terms of transcript profiling and therefore seem to be the most relevant in vitro model for investigating the metabolism and bioactivation of toxicants and drugs in bronchial epithelium. H292 and BEAS-2B cell lines, which exhibited the highest homology in gene expression pattern with primary cells and the lowest number of dysregulated genes compared with nontumoral lung tissues, could be used as surrogates for toxicological and pharmacological studies. Overall, our study should provide references for researchers to choose the most appropriate in vitro model for analyzing the cellular effects of drugs or airborne toxicants on the airway.

Gene Expression Profiling of Systems Involved in the Metabolism and the Disposition of Xenobiotics: Comparison between Human Intestinal Biopsy Samples and Colon Cell Lines

Intestinal cell lines are used as in vitro models for pharmacological and toxicological studies. However, a general report of the gene expression spectrum of proteins that are involved in the metabolism and the disposition of xenobiotics in these in vitro systems is not currently available. To fill this information gap, we systematically characterized the expression profile of 377 genes encoding xenobiotic-metabolizing enzymes, transporters, and nuclear receptors and transcription factors in intestinal mucosa (ileum, ascending colon, transverse colon, descending colon, and rectum) from five healthy subjects and in five commonly used intestinal cell lines (Caco-2, C2BBe1, HT29, T84, and FHC). For this, we performed a quantitative real-time reverse transcription-polymerase chain reaction analysis using TaqMan low-density arrays and analyzed the results by different statistical approaches: Spearman correlation coefficients, hierarchical clustering, and principal component analysis (PCA). A large variation in gene expression spectra was observed between intestinal cell lines and intestinal tissues. Both hierarchical clustering and PCA showed that two distinct clusters are visible, of which one corresponds to all cultured cell lines and the other to all intestinal biopsies. The best agreement between human tissue and the representative cell line was observed for human colonic tissues and HT29 and T84 cell lines. Altogether, these data demonstrated that gene expression profiling represents a new valuable tool for investigating in vitro and in vivo expression level correlation. This study has pointed out interesting expression profiles for various colon cell lines, which will be useful for choosing the appropriate in vitro model for pharmacological and toxicological studies.

NAT2 genotyping and efficacy of sulfasalazine in patients with chronic discoid lupus erythematosus

Sulfasalazine is an effective agent for chronic discoid lupus erythematosus (CDLE) but the response to treatment is considerably variable between patients and is also unpredictable. The reason for this might relate to differences in metabolism of the drug which is extensively acetylated by the polymorphic enzyme N-acetyltransferase 2 (NAT2). To test this possibility, the N-acetylation phenotype of eleven patients with CDLE and treated by standard doses of sulfasalazine was retrospectively determined by genotyping. A clear-cut difference in the outcome of treatment was observed according to whether the patients were slow acetylators (SA) or rapid acetylators (RA). Eight out of 11 patients responded to treatment with a complete or marked remission of the disease. Seven of them were RA. The three other patients who did not respond at all to the drug were SA. In addition, SA seem to be more prone to toxic events. These findings strongly suggest that the genetic polymorphism of NAT2 is responsible for differences in the response to sulfasalazine in patients with CDLE. Therefore, candidates for sulfasalazine therapy should be genotyped to identify those patients who might benefit from the drug.

An additional allelic variant of the CYP2D6 gene causing impaired metabolism of sparteine.

The identification of a novelCYP2D6 allele from a healthy Caucasian poor metabolizer was achieved by using a previously described polymerase chain reaction/single-strand conformation polymorphism strategy. Among the four point mutations that this allele carries, a missense mutation in exon 1 (212 G → A or D6-H) seems to be responsible for the loss of CYP2D6 function. Although the mutation D6-H has a low prevalence in a randomly selected population of healthy Caucasians, its identification should further increase the phenotype prediction rate by genotyping.

Xenobiotic metabolism and disposition in human lung: transcript profiling in non-tumoral and tumoral tissues.

The lung is directly exposed to a wide variety of inhaled toxicants and carcinogens. In order to improve our knowledge of the cellular processing of these compounds in the respiratory tract, we investigated the mRNA expression level of 380 genes encoding xenobiotic-metabolizing enzymes (XME), transporters, nuclear receptors and transcription factors, in pulmonary parenchyma (PP), bronchial mucosa (BM) and tumoral lung tissues from 12 patients with non-small cell lung cancer (NSCLC). Using a high throughput quantitative real-time RT-PCR method, we found that ADH1B, CYP4B1, CES1 and GSTP1 are the major XME genes expressed both in BM and PP. Our results also documented the predominant role played by the xenosensor AhR in human lung. The gene expression profiles were different for BM and PP, with a tendency toward increased mRNA levels of phase I and phase II XME genes in BM, suggesting major differences in the initial stages of xenobiotic metabolism. Some of the significantly overexpressed genes in BM (i.e. CYP2F1, CYP2A13, CYP2W1, NQO1…) encode proteins involved in the bioactivation of procarcinogens, pointing out distinct susceptibility to xenobiotics and their toxic effects between these two tissue types. Additionally, interindividual differences in transcript levels observed for some genes may be of genetic origin and may contribute to the variability in response to environmental exposure and, consequently, in the risk of developing lung diseases. A global decrease in gene expression was observed in tumoral specimens. Some of the proteins are involved in the metabolism or transport of anti-cancer drugs and their influence in the response of tumors to chemotherapy should be considered. In conclusion, the present study provides an overview of the cellular response to toxicants and drugs in healthy and cancerous human lung tissues, and thus improves our understanding of the mechanisms of chemical carcinogenesis as well as cellular resistance to chemotherapy.