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In human colon cancer cells, cisplatin-induced apoptosis involves the Fas death receptor pathway independent of Fas ligand. The present study explores the role of ezrin and actin cytoskeleton in relation with Fas receptor in this cell death pathway. In response to cisplatin treatment, a rapid and transient actin reorganisation is observed at the cell membrane by fluorescence microscopy after Phalloidin-FITC staining. This event is dependent on the membrane fluidification studied by electron paramagnetic resonance and necessary for apoptosis induction. Moreover, early after the onset of cisplatin treatment, ezrin co-localised with Fas at the cell membrane was visualised by membrane microscopy and was redistributed with Fas, FADD and procaspase-8 into membrane lipid rafts as shown on Western blots. In fact, cisplatin exposure results in an early small GTPase RhoA activation demonstrated by RhoA-GTP pull down, Rho kinase (ROCK)-dependent ezrin phosphorylation and actin microfilaments remodelling. Pretreatment with latrunculin A, an inhibitor of actin polymerisation, or specific extinction of ezrin or ROCK by RNA interference prevents both cisplatin-induced actin reorganisation and apoptosis. Interestingly, specific extinction of Fas receptor by RNA interference abrogates cisplatin-induced ROCK-dependent ezrin phosphorylation, actin reorganisation and apoptosis suggesting that Fas is a key regulator of cisplatin-induced actin remodelling and is indispensable for apoptosis. Thus, these findings show for the first time that phosphorylation of ezrin by ROCK via Fas receptor is involved in the early steps of cisplatin-induced apoptosis. [ABSTRACT FROM AUTHOR]

Purpose: Uveal melanoma (UM) is a deadly cancer with limited therapeutic options. At advanced stages, UM cells metastasize almost exclusively into the liver, where targeting metastatic UM cells remain a clinical challenge. Transforming growth factor beta (TGF-β) exhibits a functional duality in cancer, with one arm limiting tumor growth at an early stage and the second arm promoting metastasis at an advanced stage, notably by inducing an epithelial-to-mesenchymal transition. Thus, we hypothesized that targeting the TGF-β pathway could be relevant in the treatment of metastatic UM., Methods: In this study, we first characterized the pseudoepithelial/mesenchymal phenotype of UM cell lines Mel270 and 92.1. We then treated the cell lines with TGF-β to evaluate their responsiveness to the cytokine and to characterize the functional impact of TGF-β on their cell viability., Results: The results demonstrated, first, that the UM cell lines exhibited a mesenchymal phenotype and responded to TGF-β treatment in vitro and, second, that TGF-β promoted a cytostatic effect on the UM cell lines., Conclusions: Our findings indicate that UM cells are sensitive to the two arms of TGF-β signaling, which suggests that targeting the TGF-β pathway could be challenging in UM and would require a precise selection of patients in which only the prometastatic arm of TGF-β is activated., (Copyright © 2024 Molecular Vision.)

Cholangiocarcinoma is a devastating liver cancer characterized by high aggressiveness and therapy resistance, resulting in poor prognosis. Long non-coding RNAs and signals imposed by oncogenic pathways, such as transforming growth factor β (TGFβ), frequently contribute to cholangiocarcinogenesis. Here, we explore novel effectors of TGFβ signalling in cholangiocarcinoma. LINC00313 is identified as a novel TGFβ target gene. Gene expression and genome-wide chromatin accessibility profiling reveal that nuclear LINC00313 transcriptionally regulates genes involved in Wnt signalling, such as the transcriptional activator TCF7. LINC00313 gain-of-function enhances TCF/LEF-dependent transcription, promotes colony formation in vitro and accelerates tumour growth in vivo. Genes affected by LINC00313 over-expression in CCA tumours are associated with KRAS and TP53 mutations and reduce overall patient survival. Mechanistically, ACTL6A and BRG1, subunits of the SWI/SNF chromatin remodelling complex, interact with LINC00313 and affect TCF7 and SULF2 transcription. We propose a model whereby TGFβ induces LINC00313 in order to regulate the expression of hallmark Wnt pathway genes, in co-operation with SWI/SNF. By modulating key genes of the Wnt pathway, LINC00313 fine-tunes Wnt/TCF/LEF-dependent transcriptional responses and promotes cholangiocarcinogenesis., (© 2024. The Author(s).)

Premature ovarian insufficiency (POI) affects 1 in 100 women and is a leading cause of female infertility. There are over 80 genes in which variants can cause POI, with these explaining only a minority of cases. Whole exome sequencing (WES) can be a useful tool for POI patient management, allowing clinical care to be personalized to underlying cause. We performed WES to investigate two French sisters, whose only clinical complaint was POI. Surprisingly, they shared one known and one novel likely pathogenic variant in the Perrault syndrome gene, LARS2. Using amino-acylation studies, we established that the novel missense variant significantly impairs LARS2 function. Perrault syndrome is characterized by sensorineural hearing loss in addition to POI. This molecular diagnosis alerted the sisters to the significance of their difficulty in following conversation. Subsequent audiology assessment revealed a mild bilateral hearing loss. We describe the first cases presenting with perceived isolated POI and causative variants in a Perrault syndrome gene. Our study expands the phenotypic spectrum associated with LARS2 variants and highlights the clinical benefit of having a genetic diagnosis, with prediction of potential co-morbidity and prompt and appropriate medical care, in this case by an audiologist for early detection of hearing loss., (© 2022. The Author(s), under exclusive licence to European Society of Human Genetics.)

Ganglioside-monosialic acid (GM1) gangliosidosis, a rare autosomal recessive disorder, is frequently caused by deleterious single nucleotide variants (SNVs) in GLB1 gene. These variants result in reduced β-galactosidase (β-gal) activity, leading to neurodegeneration associated with premature death. Currently, no effective therapy for GM1 gangliosidosis is available. Three ongoing clinical trials aim to deliver a functional copy of the GLB1 gene to stop disease progression. In this study, we show that 41% of GLB1 pathogenic SNVs can be replaced by adenine base editors (ABEs). Our results demonstrate that ABE efficiently corrects the pathogenic allele in patient-derived fibroblasts, restoring therapeutic levels of β-gal activity. Off-target DNA analysis did not detect off-target editing activity in treated patient's cells, except a bystander edit without consequences on β-gal activity based on 3D structure bioinformatics predictions. Altogether, our results suggest that gene editing might be an alternative strategy to cure GM1 gangliosidosis.

Sexual development is a complex process relying on numerous genes. Disruptions in some of these genes are known to cause differences of sexual development (DSDs). Advances in genome sequencing allowed the discovery of new genes implicated in sexual development, such as PBX1. We present here a fetus with a new PBX1 NM_002585.3: c.320G>A,p.(Arg107Gln) variant, presenting with severe DSD along with renal and lung malformations. Using CRISPR-Cas9 gene editing on HEK293T cells, we generated a KD cell line for PBX1. The KD cell line showed reduced proliferation and adhesion properties compared with HEK293T cells. HEK293T and KD cells were then transfected plasmids coding either PBX1 WT or PBX1-320G>A (mutant). WT or mutant PBX1 overexpression rescued cell proliferation in both cell lines. RNA-seq analyses showed less than 30 differentially expressed genes, in ectopic mutant-PBX1-expressing cells compared with WT-PBX1. Among them, U2AF1 , encoding a splicing factor subunit, is an interesting candidate. Overall, mutant PBX1 seems to have modest effects compared with WT PBX1 in our model. However, the recurrence of PBX1 Arg107 substitution in patients with closely related phenotypes calls for its impact in human diseases. Further functional studies are needed to explore its effects on cellular metabolism.

Uveal melanoma (UM), the most common primary intraocular tumor in adults, has been extensively characterized by omics technologies during the last 5 yr. Despite the discovery of gene signatures, the molecular actors driving cancer aggressiveness are not fully understood, and UM is still associated with very poor overall survival (OS) at the metastatic stage. By defining the miR-16 interactome, we revealed that miR-16 mainly interacts via non-canonical base-pairing to a subset of RNAs, promoting their expression levels. Consequently, the canonical miR-16 activity, involved in the RNA decay of oncogenes, such as <i>cyclin D3</i>, is impaired. This non-canonical base-pairing can explain both the derepression of miR-16 targets and the promotion of oncogene expression observed in patients with poor OS in two cohorts. miR-16 activity, assessment using our RNA signature, discriminates the patient's OS as effectively as current methods. To the best of our knowledge, this is the first time that a predictive signature has been composed of genes belonging to the same mechanism (miR-16) in UM. Altogether, our results strongly suggest that UM is a miR-16 disease., (© 2022 Quéméner et al.)

Premature ovarian insufficiency (POI) is a leading form of female infertility, characterised by menstrual disturbance and elevated follicle-stimulating hormone before age 40. It is highly heterogeneous with variants in over 80 genes potentially causative, but the majority of cases having no known cause. One gene implicated in POI pathology is TP63. TP63 encodes multiple p63 isoforms, one of which has been shown to have a role in the surveillance of genetic quality in oocytes. TP63 C-terminal truncation variants and N-terminal duplication have been described in association with POI, however, functional validation has been lacking. Here we identify three novel TP63 missense variants in women with nonsyndromic POI, including one in the N-terminal activation domain, one in the C-terminal inhibition domain, and one affecting a unique and poorly understood p63 isoform, TA*p63. Via blue-native page and luciferase reporter assays we demonstrate that two of these variants disrupt p63 dimerization, leading to constitutively active p63 tetramer that significantly increases the transcription of downstream targets. This is the first evidence that TP63 missense variants can cause isolated POI and provides mechanistic insight that TP63 variants cause POI due to constitutive p63 activation and accelerated oocyte loss in the absence of DNA damage., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

PBX1 is a highly conserved atypical homeodomain transcription factor (TF) belonging to the TALE (three amino acid loop extension) family. Dimerized with other TALE proteins, it can interact with numerous partners and reach dozens of regulating sequences, suggesting its role as a pioneer factor. PBX1 is expressed throughout the embryonic stages (as early as the blastula stage) in vertebrates. In human, PBX1 germline variations are linked to syndromic renal anomalies (CAKUTHED). In this review, we summarized available data on PBX1 functions, PBX1-deficient animal models, and PBX1 germline variations in humans. Two types of genetic alterations were identified in PBX1 gene. PBX1 missense variations generate a severe phenotype including lung hypoplasia, cardiac malformations, and sexual development defects (DSDs). Conversely, truncating variants generate milder phenotypes (mainly cryptorchidism and deafness). We suggest that defects in PBX1 interactions with various partners, including proteins from the HOX (HOXA7, HOXA10, etc.), WNT (WNT9B, WNT3), and Polycomb (BMI1, EED) families are responsible for abnormal proliferation and differentiation of the embryonic mesenchyme. These alterations could explain most of the defects observed in humans. However, some phenotype variability (especially DSDs) remains poorly understood. Further studies are needed to explore the TALE family in greater depth., (© 2022 Wiley Periodicals LLC.)

Background: The essential amino acid, tryptophan, is predominantly metabolised through the kynurenine pathway (KP) to generate kynurenine, an aryl-hydrocarbon receptor (AhR) pro-ligand that exerts its effects in a ligand-dependent manner. Interaction between kynurenine and the AhR is an effector mechanism of immunosuppression. We previously found that the KP is involved in multiple sclerosis (MS) disease progression. We postulated that AhR activation by kynurenine might be neuroprotective by encouraging differentiation of Tregs. In this study, we assess both the prophylactic and therapeutic efficiency of kynurenine on disease severity and progression in mice with experimental autoimmune encephalomyelitis (EAE), an MS model., Methods: Myelin oligodendrocyte glycoprotein induced EAE mice (n = 6 per group) were treated with 200 mg/kg L-kynurenine once daily for 10 days beginning on either day 1 of EAE induction (prophylactic) or once they demonstrated motor weakness (therapeutic). Clinical disease severity measured by disease score, time on rotarod, and body weight., Results: The prophylactic kynurenine treatment significantly ( P < .0001) prevented the development of a more severe disease course with mice demonstrating diminished relapse rate and improved clinical and behavioural outcomes. However, therapeutic kynurenine did not significantly ( P = .4463) decrease the clinical signs until 36 days following induction of disease; after 36 days, it also significantly ( P = .0479) reduced disease relapse. Mean body weight measurements only correlated with time on rotarod ( r = -.6410; P = .0007) but not clinical scores ( r = .1925; P = .3674)., Conclusions: Kynurenine ameliorates EAE disease progression prophylactically and reduces relapses therapeutically. Further investigations are needed to elucidate the molecular mechanism explaining the therapeutic role of kynurenine for MS., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

Animal genomes are pervasively transcribed into multiple RNA molecules, of which many will not be translated into proteins. One major component of this transcribed non-coding genome is the long non-coding RNAs (lncRNAs), which are defined as transcripts longer than 200 nucleotides with low coding-potential capabilities. Domestic animals constitute a unique resource for studying the genetic and epigenetic basis of phenotypic variations involving protein-coding and non-coding RNAs, such as lncRNAs. This review presents the current knowledge regarding transcriptome-based catalogues of lncRNAs in major domesticated animals (pets and livestock species), covering a broad phylogenetic scale (from dogs to chicken), and in comparison with human and mouse lncRNA catalogues. Furthermore, we describe different methods to extract known or discover novel lncRNAs and explore comparative genomics approaches to strengthen the annotation of lncRNAs. We then detail different strategies contributing to a better understanding of lncRNA functions, from genetic studies such as GWAS to molecular biology experiments and give some case examples in domestic animals. Finally, we discuss the limitations of current lncRNA annotations and suggest research directions to improve them and their functional characterisation., (© 2021. The Author(s).)

Transforming growth factor beta (TGF-β) plays a key role in tumor progression, notably as a potent inducer of epithelial-mesenchymal transition (EMT). However, all of the molecular effectors driving TGFβ-induced EMT are not fully characterized. Here, we report that forkhead box S1 (FOXS1) is a SMAD (mothers against decapentaplegic)-dependent TGFβ-induced transcription factor, which regulates the expression of genes required for the initial steps of EMT (e.g., snail family transcription repressor 1) and to maintain a mesenchymal phenotype in hepatocellular carcinoma (HCC) cells. In human HCC, we report that FOXS1 is a biomarker of poorly differentiated and aggressive tumor subtypes. Importantly, FOXS1 expression level and activity are associated with a poor prognosis (e.g., reduced patient survival), not only in HCC but also in colon, stomach, and kidney cancers. Conclusion: FOXS1 constitutes a clinically relevant biomarker for tumors in which the pro-metastatic arm of TGF-β is active (i.e., patients who may benefit from targeted therapies using inhibitors of the TGF-β pathway)., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Lineage dedifferentiation toward a mesenchymal-like state displaying myofibroblast and fibrotic features is a common mechanism of adaptive and acquired resistance to targeted therapy in melanoma. Here, we show that the anti-fibrotic drug nintedanib is active to normalize the fibrous ECM network, enhance the efficacy of MAPK-targeted therapy, and delay tumor relapse in a preclinical model of melanoma. Acquisition of this resistant phenotype and its reversion by nintedanib pointed to miR-143/-145 pro-fibrotic cluster as a driver of this mesenchymal-like phenotype. Upregulation of the miR-143/-145 cluster under BRAFi/MAPKi therapy was observed in melanoma cells in vitro and in vivo and was associated with an invasive/undifferentiated profile. The 2 mature miRNAs generated from this cluster, miR-143-3p and miR-145-5p, collaborated to mediate transition toward a drug-resistant undifferentiated mesenchymal-like state by targeting Fascin actin-bundling protein 1 (FSCN1), modulating the dynamic crosstalk between the actin cytoskeleton and the ECM through the regulation of focal adhesion dynamics and mechanotransduction pathways. Our study brings insights into a novel miRNA-mediated regulatory network that contributes to non-genetic adaptive drug resistance and provides proof of principle that preventing MAPKi-induced pro-fibrotic stromal response is a viable therapeutic opportunity for patients on targeted therapy., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Mucosal melanoma (MM) is a rare, aggressive clinical cancer. Despite recent advances in genetics and treatment, the prognosis of MM remains poor. Canine MM offers a relevant spontaneous and immunocompetent model to decipher the genetic bases and explore treatments for MM. We performed an integrative genomic and transcriptomic analysis of 32 canine MM samples, which identified two molecular subgroups with a different microenvironment and structural variant (SV) content. The overexpression of genes related to the microenvironment and T-cell response was associated with tumors harboring a lower content of SVs, whereas the overexpression of pigmentation-related pathways and oncogenes, such as TERT , was associated with a high SV burden. Using whole-genome sequencing, we showed that focal amplifications characterized complex chromosomal rearrangements targeting oncogenes, such as MDM2 or CDK4 , and a recurrently amplified region on canine chromosome 30. We also demonstrated that the genes TRPM7 , GABPB1 , and SPPL2A , located in this CFA30 region, play a role in cell proliferation, and thus, may be considered as new candidate oncogenes for human MM. Our findings suggest the existence of two MM molecular subgroups that may benefit from dedicated therapies, such as immune checkpoint inhibitors or targeted therapies, for both human and veterinary medicine.

As the second and third leading cancer-related death in men and the world, respectively, primary liver cancer remains a major concern to human health. Despite advances in diagnostic technology, patients with primary liver cancer are often diagnosed at an advanced stage. Treatment options for patients with advanced hepatocarcinoma (HCC) are limited to systemic treatment with multikinase inhibitors and immunotherapy. Furthermore, the 5-year survival rate for these late-stage HCC patients is approximately 12% worldwide. There is an unmet need to identify novel treatment options and/or sensitive blood-based biomarker(s) to detect this cancer at an early stage. Given that the liver harbours the largest proportion of immune cells in the human body, understanding the tumour-immune microenvironment has gained increasing attention as a potential target to treat cancer. The kynurenine pathway (KP) has been proposed to be one of the key mechanisms used by the tumour cells to escape immune surveillance for proliferation and metastasis. In an inflammatory environment such as cancer, the KP is elevated, suppressing local immune cell populations and enhancing tumour growth. In this review, we collectively describe the roles of the KP in cancer and provide information on the latest research into the KP in primary liver cancer.

Recent advances in genome editing tools, especially novel developments in the clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases (CRISPR/Cas9)-derived editing machinery, have revolutionized not only basic science but, importantly, also the gene therapy field. Their flexibility and ability to introduce precise modifications in the genome to disrupt or correct genes or insert expression cassettes in safe harbors in the genome underline their potential applications as a medicine of the future to cure many genetic diseases. In this review, we give an overview of the recent progress made by French researchers in the field of therapeutic genome editing, while putting their work in the general context of advances made in the field. We focus on recent hematopoietic stem cell gene editing strategies for blood diseases affecting the red blood cells or blood coagulation as well as lysosomal storage diseases. We report on a genome editing-based therapy for muscular dystrophy and the potency of T cell gene editing to increase anticancer activity of chimeric antigen receptor T cells to combat cancer. We will also discuss technical obstacles and side effects such as unwanted editing activity that need to be surmounted on the way toward a clinical implementation of genome editing. We propose here improvements developed today, including by French researchers to overcome the editing-related genotoxicity and improve editing precision by the use of novel recombinant nuclease-based systems such as nickases, base editors, and prime editors. Finally, a solution is proposed to resolve the cellular toxicity induced by the systems employed for gene editing machinery delivery.

In the animal kingdom, skin pigmentation is highly variable between species, and it contributes to phenotypes. In humans, skin pigmentation plays a part in sun protection. Skin pigmentation depends on the ratio of the two pigments pheomelanin and eumelanin, both synthesized by a specialized cell population, the melanocytes. In this review, we explore one important factor in pigmentation: the tyrosinase-related protein 1 (TYRP1) gene which is involved in eumelanin synthesis via the TYRP1 protein. Counterintuitively, high TYRP1 mRNA expression is associated with a poor clinical outcome for patients with metastatic melanomas. Recently, we were able to explain this unexpected TYRP1 function by demonstrating that TYRP1 mRNA sequesters microRNA-16, a tumor suppressor miRNA. Here, we focus on actors influencing TYRP1 mRNA abundance, particularly transcription factors, single nucleotide polymorphisms (SNPs), and miRNAs, as they all dictate the indirect oncogenic activity of TYRP1., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Sustained transcriptional activation of the aryl hydrocarbon receptor (AhR) promotes tumour growth and impairs the immune defence, at least for cutaneous melanoma and glioma. AhR ligands are produced by the tumour microenvironment (TME) and by the tumour itself (intracrine). The recent identification of interleukin-4-induced-1 (IL4I1), a parallel pathway to indoleamine 2 3-dioxygenase 1 (IDO1)/ tryptophan 2,3-dioxygenase (TDO), and its ability to generate AhR ligands, confirms that a complete inhibition of AhR ligand production might be difficult to reach. Here, we have focused on recent discoveries explaining the large varieties of AhR ligands and the functional consequences in terms of cancer cell plasticity and consecutive therapy resistance. We also examined therapeutic strategies targeting the AhR signalling pathway and their possible adverse effects. Since the end of 2019, two phase I clinical trials have investigated the ability of the AhR antagonist to 'reset' the immune system and re-sensitize the cancer cells to therapies by preventing their dedifferentiation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Most genetic alterations that drive melanoma development and resistance to targeted therapy have been uncovered. In contrast, and despite their increasingly recognized contribution, little is known about the non-genetic mechanisms that drive these processes. Here, we performed in vivo gain-of-function CRISPR screens and identified SMAD3, BIRC3, and SLC9A5 as key actors of BRAFi resistance. We show that their expression levels increase during acquisition of BRAFi resistance and remain high in persister cells and during relapse. The upregulation of the SMAD3 transcriptional activity (SMAD3-signature) promotes a mesenchymal-like phenotype and BRAFi resistance by acting as an upstream transcriptional regulator of potent BRAFi-resistance genes such as EGFR and AXL. This SMAD3-signature predicts resistance to both current melanoma therapies in different cohorts. Critically, chemical inhibition of SMAD3 may constitute amenable target for melanoma since it efficiently abrogates persister cells survival. Interestingly, decrease of SMAD3 activity can also be reached by inhibiting the Aryl hydrocarbon Receptor (AhR), another druggable transcription factor governing SMAD3 expression level. Our work highlights novel drug vulnerabilities that can be exploited to develop long-lasting antimelanoma therapies., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

In humans, histiocytic sarcoma (HS) is an aggressive cancer involving histiocytes. Its rarity and heterogeneity explain that treatment remains a challenge. Sharing high clinical and histopathological similarities with human HS, the canine HS is conversely frequent in specific breeds and thus constitutes a unique spontaneous model for human HS to decipher the genetic bases and to explore therapeutic options. We identified sequence alterations in the MAPK pathway in at least 63.9% (71/111) of HS cases with mutually exclusive BRAF (0.9%; 1/111), KRAS (7.2%; 8/111) and PTPN11 (56.75%; 63/111) mutations concentrated at hotspots common to human cancers. Recurrent PTPN11 mutations are associated to visceral disseminated HS subtype in dogs, the most aggressive clinical presentation. We then identified PTPN11 mutations in 3/19 (15.7%) human HS patients. Thus, we propose PTPN11 mutations as key events for a specific subset of human and canine HS: the visceral disseminated form. Finally, by testing drugs targeting the MAPK pathway in eight canine HS cell lines, we identified a better anti-proliferation activity of MEK inhibitors than PTPN11 inhibitors in canine HS neoplastic cells. In combination, these results illustrate the relevance of naturally affected dogs in deciphering genetic mechanisms and selecting efficient targeted therapies for such rare and aggressive cancers in humans., (© 2020 UICC.)

Antisense oligonucleotides (ASOs) represent a new and highly promising class of drugs for personalized medicine. In the last decade, major chemical developments and improvements of the backbone structure of ASOs have transformed them into true approved and commercialized drugs. ASOs target both DNA and RNA, including pre-mRNA, mRNA, and ncRDA, based on sequence complementary. They are designed to be specific for each identified molecular and genetic alteration to restore a normal, physiological situation. Thus, the characterization of the underpinning mechanisms and alterations that sustain pathology is critical for accurate ASO-design. ASOs can be used to cure both rare and common diseases, such as orphan genetic alterations and cancer. Through pioneering examples, this review shows the versatility of the mechanisms of action that provide ASOs with the potential capacity to achieve custom treatment, revolutionizing personalized medicine. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions., (© 2020 The Authors. WIREs RNA published by Wiley Periodicals, Inc.)

Staphylococcus aureus, a versatile Gram-positive bacterium, is the main cause of bone and joint infections (BJI), which are prone to recurrence. The inflammasome is an immune signaling platform that assembles after pathogen recognition. It activates proteases, most notably caspase-1 that proteolytically matures and promotes the secretion of mature IL-1β and IL-18. The role of inflammasomes and caspase-1 in the secretion of mature IL-1β and in the defence of S. aureus-infected osteoblasts has not yet been fully investigated. We show here that S. aureus-infected osteoblast-like MG-63 but not caspase-1 knock-out CASP1 -/- MG-63 cells, which were generated using CRISPR-Cas9 technology, activate the inflammasome as monitored by the release of mature IL-1β. The effect was strain-dependent. The use of S. aureus deletion and complemented phenole soluble modulins (PSMs) mutants demonstrated a key role of PSMs in inflammasomes-related IL-1β production. Furthermore, we found that the lack of caspase-1 in CASP1 -/- MG-63 cells impairs their defense functions, as bacterial clearance was drastically decreased in CASP1 -/- MG-63 compared to wild-type cells. Our results demonstrate that osteoblast-like MG-63 cells play an important role in the immune response against S. aureus infection through inflammasomes activation and establish a crucial role of caspase-1 in bacterial clearance., (© 2020 John Wiley & Sons Ltd.)

Trans-encoded bacterial regulatory RNAs (sRNAs) are functional analogues of eukaryotic microRNAs (miRNAs). These RNA classes act by base-pairing complementarity with their RNA targets to modulate gene expression (transcription, half-life and/or translation). Based on base-pairing, algorithms predict binding and the impact of small RNAs on targeted-RNAs expression and fate. However, other actors are involved such as RNA binding proteins and epigenetic modifications of the targeted and small RNAs. Post-transcriptional base modifications are widespread in all living organisms where they lower undesired RNA folds through conformation adjustments and influence RNA pairing and stability, especially if remodeling their ends. In bacteria, sRNAs possess RNA modifications either internally (methylation, pseudouridinylation) or at their ends. Nicotinamide adenine dinucleotide were detected at 5'-ends, and polyadenylation can occur at 3'-ends. Eukaryotic miRNAs possess N ⁶-methyladenosine (m⁶A), A editing into I, and non-templated addition of uridines at their 3'-ends. Biological functions and enzymes involved in those sRNA and micro RNA epigenetic modifications, when known, are presented and challenged.

BRAF inhibitors target the BRAF-V600E/K mutated kinase, the driver mutation found in 50% of cutaneous melanoma. They give unprecedented anti-tumor responses but acquisition of resistance ultimately limits their clinical benefit. The master regulators driving the expression of resistance-genes remain poorly understood. Here, we demonstrate that the Aryl hydrocarbon Receptor (AhR) transcription factor is constitutively activated in a subset of melanoma cells, promoting the dedifferentiation of melanoma cells and the expression of BRAFi-resistance genes. Typically, under BRAFi pressure, death of BRAFi-sensitive cells leads to an enrichment of a small subpopulation of AhR-activated and BRAFi-persister cells, responsible for relapse. Also, differentiated and BRAFi-sensitive cells can be redirected towards an AhR-dependent resistant program using AhR agonists. We thus identify Resveratrol, a clinically compatible AhR-antagonist that abrogates deleterious AhR sustained-activation. Combined with BRAFi, Resveratrol reduces the number of BRAFi-resistant cells and delays tumor growth. We thus propose AhR-impairment as a strategy to overcome melanoma resistance.

microRNA (miRNA) are critical post-transcriptional regulators and key players in diseases development. We demonstrated that non-canonical microRNA Responsive Elements (here MRE-16) could sequester miR-16, dampening miR-16 tumor suppressor function. We developed small oligonucleotides, masking specifically these unusual miR-16 binding sites, that restored miR-16 function. This constitutes a promising targeted approach.

Canine cancers represent a tremendous natural resource due to their incidence and striking similarities to human cancers, sharing similar clinical and pathologic features as well as oncogenic events, including identical somatic mutations. Considering the importance of gene fusions as driver alterations, we explored their relevance in canine cancers. We focused on three distinct human-comparable canine cancers representing different tissues and embryonic origins. Through RNA-Seq, we discovered similar gene fusions as those found in their human counterparts: IGK - CCND3 in B-cell lymphoma, MPB - BRAF in glioma, and COL3A1 - PDGFB in dermatofibrosarcoma protuberans-like. We showed not only similar partner genes but also identical breakpoints leading to oncogene overexpression. This study demonstrates similar gene fusion partners and mechanisms in human-dog corresponding tumors and allows for selection of targeted therapies in preclinical and clinical trials with pet dogs prior to human trials, within the framework of personalized medicine. Cancer Res; 77(21); 5721-7. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Competition among RNAs to bind miRNA is proposed to influence biological systems. However, the role of this competition in disease onset is unclear. Here, we report that TYRP1 mRNA, in addition to encoding tyrosinase-related protein 1 (TYRP1), indirectly promotes cell proliferation by sequestering miR-16 on non-canonical miRNA response elements. Consequently, the sequestered miR-16 is no longer able to repress its mRNA targets, such as RAB17, which is involved in melanoma cell proliferation and tumour growth. Restoration of miR-16 tumour-suppressor function can be achieved in vitro by silencing TYRP1 or increasing miR-16 expression. Importantly, TYRP1-dependent miR-16 sequestration can also be overcome in vivo by using small oligonucleotides that mask miR-16-binding sites on TYRP1 mRNA. Together, our findings assign a pathogenic non-coding function to TYRP1 mRNA and highlight miRNA displacement as a promising targeted therapeutic approach for melanoma.

Targeting RNAs appears as an important opportunity to modulate biological processes. Here, we overviewed critical parameters implied in RNAs competition to bind small RNAs. These competitions influence small RNA availability and thereby gene expression and cell fate. We focused on the ability of RNAs to sequester small RNA, mainly the microRNAs (miRNAs) and proposed experimental workflows to demonstrate the existence and activity of RNA-sponge. From this basic science, we detailed tailored oligonucleotides, developed to challenge the binding of small RNA. In vitro and in vivo, these tailored oligonucleotides efficiently restore small RNA activity by preventing their sequestration on RNA-sponges., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The stress-activated p38α MAP Kinase is an integral and critical component of the UV-induced inflammatory response. Despite the advances in recent years in the development of p38 kinase inhibitors, validation of these compounds in the diseased models remains limited. Based on the pharmacological profile of p38α inhibitor lead compound, SB203580, we synthesized a series of pyrrole-derivatives. Using UV-irradiated human skin punch-biopsies and cell cultures, we identified and validated the inhibitory activity of the derivatives by quantitatively measuring their effect on the expression of p38α target genes using real-time PCR. This approach not only identified pyrrole-2 as a unique derivative of this series that specifically inhibited the UV-activated p38α kinase, but also documented the skin permeation, bioavailability and reversible properties of this derivative in a 3D structure. The successful skin permeation of pyrrole-2 and its impact on AREG, COX-2 and MMP-9 gene expression demonstrates its potential use in modulating inflammatory processes in the skin. This study underscored the importance of using adapted biological models to identify accurate bioactive compounds.

Background: The aryl hydrocarbon receptor (AhR) has gradually emerged as a regulator of inflammation in the lung and other tissues. AhR may interact with the p65-subunit of the nuclear factor (NF)-κB transcription factors, but reported outcomes of AhR/NF-κB-interactions are conflicting. Some studies suggest that AhR possess pro-inflammatory activities while others suggest that AhR may be anti-inflammatory. The present study explored the impact of AhR and its binding partner AhR nuclear translocator (Arnt) on p65-activation and two differentially regulated chemokines, CXCL8 (IL-8) and CCL5 (RANTES), in human bronchial epithelial cells (BEAS-2B)., Results: Cells were exposed to CXCL8- and CCL5-inducing chemicals, 1-nitropyrene (1-NP) and 1-aminopyrene (1-AP) respectively, or the synthetic double-stranded RNA analogue, polyinosinic-polycytidylic acid (Poly I:C) which induced both chemokines. Only CXCL8, and not CCL5, appeared to be p65-dependent. Yet, constitutively active unligated AhR suppressed both CXCL8 and CCL5, as shown by siRNA knock-down and the AhR antagonist α-naphthoflavone. Moreover, AhR suppressed activation of p65 by TNF-α and Poly I:C as assessed by luciferase-assay and p65-phosphorylation at serine 536, without affecting basal p65-activity. In contrast, Arnt suppressed only CXCL8, but did not prevent the p65-activation directly. However, Arnt suppressed expression of the NF-κB-subunit RelB which is under transcriptional regulation by p65. Furthermore, AhR-ligands alone at high concentrations induced a moderate CXCL8-response, without affecting CCL5, but suppressed both CXCL8 and CCL5-responses by Poly I:C., Conclusion: AhR and Arnt may differentially and independently regulate chemokine-responses induced by both inhaled pollutants and pulmonary infections. Constitutively active, unligated AhR suppressed the activation of p65, while Arnt may possibly interfere with the action of activated p65. Moreover, ligand-activated AhR suppressed CXCL8 and CCL5 responses by other agents, but AhR ligands alone induced CXCL8 responses when given at sufficiently high concentrations, thus underscoring the duality of AhR in regulation of inflammation. We propose that AhR-signaling may be a weak activator of p65-signaling that suppresses p65-activity induced by strong activators of NF-κB, but that its anti-inflammatory properties also are due to interference with additional pathways.

Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness.

Genomic instability is a major hallmark of cancer. To maintain genomic integrity, cells are equipped with dedicated sensors to monitor DNA repair or to force damaged cells into death programs. The tumor suppressor p53 is central in this process. Here, we report that the ubiquitous transcription factor Upstream Stimulatory factor 1 (USF1) coordinates p53 function in making proper cell fate decisions. USF1 stabilizes the p53 protein and promotes a transient cell cycle arrest, in the presence of DNA damage. Thus, cell proliferation is maintained inappropriately in Usf1 KO mice and in USF1-deficient melanoma cells challenged by genotoxic stress. We further demonstrate that the loss of USF1 compromises p53 stability by enhancing p53-MDM2 complex formation and MDM2-mediated degradation of p53. In USF1-deficient cells, the level of p53 can be restored by the re-expression of full-length USF1 protein similarly to what is observed using Nutlin-3, a specific inhibitor that prevents p53-MDM2 interaction. Consistent with a new function for USF1, a USF1 truncated protein lacking its DNA-binding and transactivation domains can also restore the induction and activity of p53. These findings establish that p53 function requires the ubiquitous stress sensor USF1 for appropriate cell fate decisions in response to DNA-damage. They underscore the new role of USF1 and give new clues of how p53 loss of function can occur in any cell type. Finally, these findings are of clinical relevance because they provide new therapeutic prospects in stabilizing and reactivating the p53 pathway.

The metabolically competent hepatocyte-like human HepaRG cells represent a suitable alternative in vitro cell model to human primary hepatocytes. Here, we describe the culture procedure required to expand progenitor HepaRG cells and to differentiate them into hepatocyte-like cells. Transient transfection of gene and siRNA into cultured cells, using nonviral strategies, is an invaluable technique to decipher gene functions. In this chapter, we detail transfection protocols for efficient transfer of plasmid DNA or siRNAs into proliferating progenitor or quiescent differentiated HepaRG cells as well as into primary hepatocytes.

Background: The aryl hydrocarbon receptor (AhR) is a transcription factor activated by several environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and involved in carcinogenesis and various physiological processes, including immune response and endocrine functions. Characterization of kinases-related AhR transduction pathway remains an important purpose., Results: We performed a kinome-wide siRNA screen in human mammary MCF-7 cells to identify non redundant protein kinases implicated in the up-regulation of cytochrome P-450 (CYP) 1A1 activity, an AhR referent target, in response to TCDD exposure. To this aim, we monitored CYP1A1-related ethoxyresorufin-O-deethylase (EROD) activity and quantified cell density. This normalization was crucial since it allowed us to focus only on siRNA affecting EROD activity and discard siRNA affecting cell density. Analyses of the cell density data allowed us to identify several hits already well-characterized as effectors of the cell cycle and original hits. Collectively, these data fully validated the protocol and the siRNA library. Next, 22 novel candidates were identified as kinases potentially implicated in the up-regulation of CYP1A1 in response to TCDD, without alteration of cell survival or cell proliferation. The siRNA library screen gave a limited number of hits (approximately 3%). Interestingly, four of them are able to bind calmodulin among which the IP3 kinase A (ITPKA) and pregnancy up-regulated non-ubiquitously expressed CaM kinase (PNCK, also named CaMKIβ). Remarkably, for both proteins, their kinase activity depends on the calmodulin binding. Involvement of ITPKA and PNCK in TCDD-mediated CYP1A1 up-regulation was further validated by screening-independent expression knock-down. PNCK was finally shown to regulate activation of CaMKIα, a CaMKI isoform previously reported to interplay with the AhR pathway., Conclusions: These data fully support a role for both IP3-related kinase and CaMK isoforms in the AhR signaling cascade. More generally, this study also highlights the interest of large scale loss-of-function screens for characterizing the molecular mechanism of action of environmental contaminants.

Aurora kinases belong to a conserved family of serine/threonine kinases key regulators of cell cycle progression. Aurora-A and Aurora-B are expressed in somatic cells and involved mainly in mitosis while Aurora-C is expressed during spermatogenesis and oogenesis and is involved in meiosis. Aurora-C is hardly detectable in normal somatic cells. However all three kinases are overexpressed in many cancer lines. Aurora-A possesses an oncogenic activity while Aurora-B does not. Here we investigated whether Aurora-C possesses such an oncogenic activity. We report that overexpression of Aurora-C induces abnormal cell division resulting in centrosome amplification and multinucleation in both transiently transfected cells and in stable cell lines. Only stable NIH3T3 cell clones overexpressing active Aurora-C formed foci of colonies when grown on soft agar, indicating that a gain of Aurora-C activity is sufficient to transform cells. Furthermore, we reported that NIH-3T3 stable cell lines overexpressing Aurora-C induced tumour formation when injected into nude mice, demonstrating the oncogenic activity of enzymatically active Aurora kinase C. Interestingly enough tumor aggressiveness was positively correlated with the quantity of active kinase, making Aurora-C a potential anti-cancer therapeutic target.

Deregulation of the phosphatidylinositol 3 (PI3) kinase/Akt pathway, resulting in enhanced Akt activity, is one of the most frequent changes in human cancer. Akt has therefore attracted significant attention as an anticancer target in recent years and many Akt inhibitors have been identified, especially Akti-1/2, a non-ATP competitive inhibitor of Akt isoforms 1 and 2. In this study, our results suggest that caution may be required when using Akti-1/2 as a specific inhibitor of Akt since it perfectly inhibits Ca(2+)/CaM-dependent protein kinase (CaMK) Iα activity. Akti-1/2 was thus able to inhibit recombinant CaMKIα activity as efficiently as the CaMK inhibitor KN-93. Moreover, Akti-1/2 prevented the nuclear translocation of aryl hydrocarbon receptor (AhR) in MCF-7 cells in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure, which has been demonstrated to require CaMKI activity. In addition, our results, obtained with a large panel of structurally-unrelated PI3K inhibitors, make unlikely any contribution of PI3K/Akt activity to the AhR pathway. To the best of our knowledge, this is the first report showing that Akti-1/2 has off-target effects at concentration equipotent with Akt inhibition. This may impact on the therapeutic application of Akti-1/2 and structurally-related compounds., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Unlabelled: We investigated the specific role of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1 (ERK1)/ERK2 pathway in the regulation of multiple cell cycles and long-term survival of normal hepatocytes. An early and sustained epidermal growth factor (EGF)-dependent MAPK activation greatly improved the potential of cell proliferation. In this condition, almost 100% of the hepatocytes proliferated, and targeting ERK1 or ERK2 via RNA interference revealed the specific involvement of ERK2 in this regulation. However, once their first cell cycle was performed, hepatocytes failed to undergo a second round of replication and stayed blocked in G1 phase. We demonstrated that sustained EGF-dependent activation of the MAPK/ERK kinase (MEK)/ERK pathway was involved in this blockage as specific transient inhibition of the cascade repotentiated hepatocytes to perform a new wave of replication and multiple cell cycles. We identified this mechanism by showing that this blockage was in part supported by ERK2-dependent p21 expression. Moreover, continuous MEK inhibition was associated with a lower apoptotic engagement, leading to an improvement of survival up to 3 weeks. Using RNA interference and ERK1 knockout mice, we extended these results by showing that this improved survival was due to the specific inhibition of ERK1 expression/phosphorylation and did not involve ERK2., Conclusion: Our results emphasize that transient MAPK inhibition allows multiple cell cycles in primary cultures of hepatocytes and that ERK2 has a key role in the regulation of S phase entry. Moreover, we revealed a major and distinct role of ERK1 in the regulation of hepatocyte survival. Taken together, our results represent an important advance in understanding long-term survival and cell cycle regulation of hepatocytes.

This study was designed to analyze the effects of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid) toward the aryl hydrocarbon receptor (AhR) pathway because Ca2+/calmodulin-dependent protein kinase (CaMK) Ialpha, known as a downstream CaMKK effector, has been recently shown to contribute to the AhR cascade. STO-609 failed to alter up-regulation of the AhR target CYP1A1 in response to the potent AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells. STO-609, used at a 25 muM concentration known to fully inhibit CaMKK activity, was surprisingly found to markedly induce CYP1A1 expression and activity by itself in MCF-7 cells; it similarly up-regulated various other AhR target genes in human macrophages. STO-609-related CYP1A1 induction was prevented by chemical inhibition or small interfering RNA-mediated knockdown expression of AhR. Moreover, STO-609 was demonstrated to physically interact with the ligand-binding domain of AhR, as assessed by TCDD binding competition assay, and to induce AhR translocation to the nucleus. As already reported for AhR agonists, STO-609 triggered the increase of [Ca2+](i) and activation of CaMKIalpha, whose inhibition through the use of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester or the CaMK inhibitor KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), respectively, prevented STO-609-mediated CYP1A1 activity induction. Taken together, these results demonstrate that the CaMKK inhibitor STO-609 can act as an AhR ligand and, in this way, fully activates the Ca2+/CaMKIalpha/AhR cascade. Such data, therefore, make unlikely any contribution of CaMKK activity to the AhR pathway and, moreover, suggest that caution may be required when using STO-609 as a specific inhibitor of CaMKKs.

Benzo(a)pyrene (BP) is an environmental contaminant known to favor airway inflammation likely through up-regulation of pro-inflammatory cytokines. The present study was designed to characterize its effects toward interleukin-8 (IL-8), a well-established pulmonary inflammatory cytokine. In primary human macrophages, BP was shown to induce IL-8 expression at both mRNA and secretion levels in a dose-dependent manner. Such an up-regulation was likely linked to aryl hydrocarbon receptor (AhR)-activation since BP-mediated IL-8 induction was reduced after AhR expression knock-down through RNA interference. Moreover, electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation experiments showed BP-triggered binding of AhR to a consensus xenobiotic responsive element (XRE) found in the human IL-8 promoter. Finally, BP administration to mice led to over-expression of keratinocyte chemoattractant (KC), the murine functional homologue of IL-8, in lung. It also triggered the recruitment of neutrophils in bronchoalveolar lavage (BAL) fluids, which was however fully abolished in the presence of a chemical antagonist of the KC/IL-8 receptors CXCR1/CXCR2, thus supporting the functional and crucial involvement of KC in BP-induced lung inflammation. Overall, these data highlight an AhR-dependent regulation of IL-8 in response to BP that likely contributes to the airway inflammatory effects of this environmental chemical.

Regulation of genes targeted by the ligand-activated aryl hydrocarbon receptor (AhR) has been shown to be controlled by calcium (Ca(2+)) changes induced by AhR agonists such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The present study was designed to characterize this link between Ca(2+) and the AhR pathway. We report that fast elevation of intracellular Ca(2+) in TCDD-exposed mammary MCF-7 cells was associated with transient enhanced activity of the Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) pathway. Chemical inhibition of this pathway using the CaM antagonist W7 or the CaMK inhibitor KN-93 strongly reduced TCDD-mediated induction of the AhR target gene CYP1A1. Small interfering RNA (siRNA)-mediated knockdown expression of CaMKIalpha, one of the CaMK isoforms, similarly prevented CYP1A1 up-regulation. Both KN-93 and siRNA targeting CaMKIalpha were found to abolish TCDD-mediated activation of CYP1A1 promoter and TCDD-triggered nuclear import of AhR, a crucial step of the AhR signaling pathway. TCDD-mediated inductions of various AhR targets, such as the drug metabolizing CYP1B1, the cytokine interleukin-1beta, the chemokines interleukin-8 and CCL1, the adhesion molecule beta7 integrin, and the AhR repressor, were also prevented by KN-93 in human macrophages. Taken together, these data identified the Ca(2+)/CaM/CaMKIalpha pathway as an important contributing factor to AhR-mediated genomic response.

In order to identify molecular targets of environmental polycyclic aromatic hydrocarbons (PAHs), we have analysed regulation of integrin (ITG) expression in PAH-exposed human macrophages. Among ITG subunits, beta7 ITG was found to be markedly up-regulated at both mRNA and protein levels in response to the prototypical PAH benzo(a)pyrene (BP). Knock-down of the transcription factor c-maf, known to control beta7 ITG expression, markedly impaired BP-mediated beta7 ITG induction. Moreover, chromatin immunoprecipitation and electrophoretic mobility shift assays showed BP-triggered binding of c-maf to a specific maf-responsive element found in beta7 ITG promoter. Such a binding, and also beta7 ITG induction, were however abolished in response to chemical inhibition of the aryl hydrocarbon receptor (AhR), to which PAHs bind. Taken together, these data establish beta7 ITG as a new molecular target of PAHs, whose up-regulation by these environmental contaminants most likely requires activation of co-operative pathways involving both AhR and c-maf.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed immunotoxic environmental contaminants well known to regulate expression of pro-inflammatory cytokines such as interleukine-1beta and tumor necrosis factor-alpha. In the present study, we demonstrated that the chemokine CCL1, notably involved in cardiovascular diseases and inflammatory or allergic processes, constitutes a new molecular target for PAHs. Indeed, exposure to PAHs such as benzo[a]pyrene (BP) markedly increased mRNA expression and secretion of CCL1 in primary human macrophage cultures. Moreover, intranasal administration of BP to mice enhanced mRNA levels of TCA3, the mouse orthologue of CCL1, in lung. CCL1 induction in cultured human macrophages was fully prevented by targeting the aryl hydrocarbon receptor (AhR) through chemical inhibition or small interfering RNA-mediated down-modulation of its expression. In addition, BP and the potent AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin were found to enhance activity of a CCL1 promoter sequence containing a consensus xenobiotic-responsive element known to specifically interact with AhR. Moreover, 2,3,7,8-tetrachlorodibenzo-p-dioxin triggered AhR binding to this CCL1 promoter element as revealed by chromatin immunoprecipitation experiments and electrophoretic mobility shift assays. In an attempt to further characterize the mechanism of CCL1 induction, we demonstrated that BP was able to induce an early and transient increase of intracellular calcium concentration in human macrophages. Inhibition of this calcium increase, using the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester or the calcium store-operated channel inhibitor 2-aminoethoxydiphenyl borate, fully blocked CCL1 up-regulation. Taken together, these results bring the first demonstration that PAHs induce expression of the chemokine CCL1 in an AhR- and calcium-dependent manner.

Cigarette smoking, a well-known cardiovascular risk factor, has been recently demonstrated to decrease circulating endothelial progenitor cell (EPC) number. Owing to the fact that polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) constitute major components of tobacco smoke, the present study was designed to analyze the effects of these chemicals on the development of human EPC cultures from peripheral blood mononuclear cells. Treatment by BP markedly impaired EPC number and EPC colonies in a dose-dependent manner. Such deleterious effects were abrogated using 3'-methoxy-4'-nitroflavone, a pure antagonist of the aryl hydrocarbon receptor, highlighting the involvement of this receptor in PAH toxicity towards EPCs. Additional events such as cytochrome P-450-dependent PAH metabolism and formation of PAH-related adducts to cellular macromolecules were also required. Overall, these data established EPCs as new cellular targets of PAHs, which may contribute to the deleterious cardiovascular effects of environmental substances containing these chemicals, especially tobacco smoke.