Spt6 levels are modulated by PAAF1 and proteasome to regulate the HIV-1 LTR

<p>Abstract</p> <p>Background</p> <p>Tat-mediated activation of the HIV-1 promoter depends upon a proteasome-associated factor, PAAF1, which dissociates 26S proteasome to produce 19S RP that is essential for transcriptional elongation. The effect of PAAF1 on proteasome activity could also potentially shield certain factors from proteolysis, which may be implicated in the transcriptional co-activator activity of PAAF1 towards the LTR.</p> <p>Results</p> <p>Here, we show that Spt6 is targeted by proteasome in the absence of PAAF1. PAAF1 interacts with the N-terminus of Spt6, suggesting that PAAF1 protects Spt6 from proteolysis. Depletion of either PAAF1 or Spt6 reduced histone occupancy at the HIV-1 promoter, and induced the synthesis of aberrant transcripts. Ectopic Spt6 expression or treatment with proteasome inhibitor partially rescued the transcription defect associated with loss of PAAF1. Transcriptional profiling followed by ChIP identified a subset of cellular genes that are regulated in a similar fashion to HIV-1 by Spt6 and/or PAAF1, including many that are involved in cancer, such as BRCA1 and BARD1.</p> <p>Conclusion</p> <p>These results show that intracellular levels of Spt6 are fine-tuned by PAAF1 and proteasome, which is required for HIV-1 transcription and extends to cellular genes implicated in cancer.</p

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. © 2013 Lino Cardenas et al

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

La Fibrose Pulmonaire idiopathique (FPI) est une maladie fibroproliférative pour laquelle il n’existe aucun traitement efficace. Les mécanismes à l’origine de cette pathologie sont méconnus et impliquent plusieurs types cellulaires et facteurs de croissance, comme le TGF-β responsable de la différenciation de fibroblastes en myofibroblastes. Pour mieux comprendre ces mécanismes physiopathologiques, nous nous sommes intéressés à l’implication des miARN dans ce processus. Une analyse par puces à ADN de l’ensemble des miARN modulés dans des échantillons pulmonaires de souris, résistantes ou sensibles à la fibrose pulmonaire induite par la bléomycine, nous a permis d’identifier miR-199a-5p comme le meilleur candidat associé à la fibrose pulmonaire mais aussi fibrose rénale et hépatique. J’ai ensuite démontré que l’expression de miR-199a-5p était induite par le TGF-β in vitro, et que sa surexpression ectopique induisait la différenciation des fibroblastes. Une combinaison d’approche in silico et expérimentale, m’a permis d’identifier la Cavéoline-1 (CAV-1) comme cible de ce miARN. La CAV-1 est impliquée dans la dégradation du récepteur TGF-β. Ainsi, l’inhibition de CAV-1 par miR-199a-5p constitue une boucle de rétrocontrôle positif exacerbant la voie TGF-β. De manière intéressante, l’inhibition de miR-199a-5p in vitro régule la différenciation, la prolifération et la migration des fibroblastes pulmonaires par le TGF-β. Par ailleurs, nos résultats précliniques indiquent que l’inhibition de ce miARN diminue les marqueurs de fibrose, permettant d’envisager le développement de nouvelles approches thérapeutiques dans le traitement de la FPI et d’autres maladies fibroprolifératives.Idiopathic Pulmonary Fibrosis (IPF) is a fibroproliferative disease with poor prognosis and for which no effective treatment exists. The mechanisms of this disease remain poorly understood and involve numerous cell types and growth factors such as TGF-β, which leads to the activation of lung fibroblasts into myofibroblasts; the key cell type driving the fibrogenic process. In this context, we focused the involvement of miRNAs in fibrosis process. 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 lung fibrosis after bleomycin exposure. We identified miR- 199a-5p as the best candidate associated with lung fibrosis but also kidney and liver fibrosis. I observed that miR-199a-5p expression was induced upon TGF-β exposure, and that its ectopic expression was sufficient to promote the pathogenic activation of pulmonary fibroblasts. Using combination of targets miRNA prediction tools and a transcriptomic approach we identified the Caveolin-1 (CAV-1), a critical mediator of pulmonary fibrosis, as a specific target of miR-199a-5p. Thus, we shown that miR-199a-5p is a key effector of TGF-β signaling in lung fibroblasts by regulating CAV1. Interestingly, inhibition of miR-199a-5p in vitro prevents the differentiation, proliferation and migration of fibroblasts after TGF-β stimulation. Finally, our preclinical results indicate that inhibition of this miRNA decreases fibrosis markers. Thus, miR-199a-5p behaves as a major regulator of tissue fibrosis with therapeutic potency for the treatment of IPF and fibroproliferative diseases

MiR-199a-5p, un « fibromiR » amplificateur de la voie du TGF-beta dans la fibrose pulmonaire idiopathique

Idiopathic Pulmonary Fibrosis (IPF) is a fibroproliferative disease with poor prognosis and for which no effective treatment exists. The mechanisms of this disease remain poorly understood and involve numerous cell types and growth factors such as TGF-β, which leads to the activation of lung fibroblasts into myofibroblasts; the key cell type driving the fibrogenic process. In this context, we focused the involvement of miRNAs in fibrosis process. 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 lung fibrosis after bleomycin exposure. We identified miR- 199a-5p as the best candidate associated with lung fibrosis but also kidney and liver fibrosis. I observed that miR-199a-5p expression was induced upon TGF-β exposure, and that its ectopic expression was sufficient to promote the pathogenic activation of pulmonary fibroblasts. Using combination of targets miRNA prediction tools and a transcriptomic approach we identified the Caveolin-1 (CAV-1), a critical mediator of pulmonary fibrosis, as a specific target of miR-199a-5p. Thus, we shown that miR-199a-5p is a key effector of TGF-β signaling in lung fibroblasts by regulating CAV1. Interestingly, inhibition of miR-199a-5p in vitro prevents the differentiation, proliferation and migration of fibroblasts after TGF-β stimulation. Finally, our preclinical results indicate that inhibition of this miRNA decreases fibrosis markers. Thus, miR-199a-5p behaves as a major regulator of tissue fibrosis with therapeutic potency for the treatment of IPF and fibroproliferative diseases.La Fibrose Pulmonaire idiopathique (FPI) est une maladie fibroproliférative pour laquelle il n’existe aucun traitement efficace. Les mécanismes à l’origine de cette pathologie sont méconnus et impliquent plusieurs types cellulaires et facteurs de croissance, comme le TGF-β responsable de la différenciation de fibroblastes en myofibroblastes. Pour mieux comprendre ces mécanismes physiopathologiques, nous nous sommes intéressés à l’implication des miARN dans ce processus. Une analyse par puces à ADN de l’ensemble des miARN modulés dans des échantillons pulmonaires de souris, résistantes ou sensibles à la fibrose pulmonaire induite par la bléomycine, nous a permis d’identifier miR-199a-5p comme le meilleur candidat associé à la fibrose pulmonaire mais aussi fibrose rénale et hépatique. J’ai ensuite démontré que l’expression de miR-199a-5p était induite par le TGF-β in vitro, et que sa surexpression ectopique induisait la différenciation des fibroblastes. Une combinaison d’approche in silico et expérimentale, m’a permis d’identifier la Cavéoline-1 (CAV-1) comme cible de ce miARN. La CAV-1 est impliquée dans la dégradation du récepteur TGF-β. Ainsi, l’inhibition de CAV-1 par miR-199a-5p constitue une boucle de rétrocontrôle positif exacerbant la voie TGF-β. De manière intéressante, l’inhibition de miR-199a-5p in vitro régule la différenciation, la prolifération et la migration des fibroblastes pulmonaires par le TGF-β. Par ailleurs, nos résultats précliniques indiquent que l’inhibition de ce miARN diminue les marqueurs de fibrose, permettant d’envisager le développement de nouvelles approches thérapeutiques dans le traitement de la FPI et d’autres maladies fibroprolifératives

The Long Noncoding RNA DNM3OS Is a Reservoir of FibromiRs with Major Functions in Lung Fibroblast Response to TGF-β and Pulmonary Fibrosis

International audienc

CAV1 is a direct target of miR-199a-5p.

<p>(A) Position of miR-199a-5p target site in CAV1 3′ UTR and sequence alignment of miR-199a-5p and the CAV1 3′ UTR from various species are shown. The “seed” region with a conserved WC match to the eighth nucleotide of the miRNA is highlighted. Bases that have been mutated in the psiCHECK-2 construct are underlined. (B) Co-transfection of pre-miR-199a-5p or pre-miR-Neg and human CAV1 3′UTR-derived psiCHECK-2 construct (wild type or mutated in the putative miR-199a-5p seed region) in HEK293 cells. Cells were harvested two days after transfection and luciferase activities were analyzed. All renilla luciferase activities were normalized with firefly luciferase activity. * p<0.05. (C) and (D) Normalized expression values of CAV1 in hFL1 lung fibroblasts after transfection with pre-miR-199a-5p, pre-miR-21 or si-CAV1 from microarrays (C, n = 2) or real time PCR (D, n = 3) experiments. Data are expressed as mean ± SEM (n = 2). **p<0.01 (E) Western blot analysis showing the down regulated expression of CAV1 protein after transfection of hFL1 lung fibroblasts with pre-miR-199a-5p, pre-miR-21 or si-CAV1. One representative experiment out of two is shown.</p

miR-199a-5p and its target CAV1 are dysregulated in IPF.

<p>(A,B) Box plots showing the normalized expression of log₂-transformed CAV1 and miR-199a-5p in both IPF (n = 94) and control (n = 83) lungs. The box represents the 25–75% quartiles, the line in the box represents the median and whiskers represent the range. (**p<0.001). (C) Immunohistochemical analysis of CAV1 expression in lung tissue sections from IPF patients (i, ii and iii) and normal lung (iv). Three representative sections are shown (IPF n = 8 and control n = 8). (D) <i>In situ</i> hybridization (ISH) was performed to determine the localization of miR-199a-5p in lung tissue of IPF patients (i, ii and iii). ISH with scrambled probes (iv). Three representative sections are shown (n = 6).</p

Altered expression of miR-199a-5p and CAV1 in the unilateral ureteral obstruction (UUO) mouse model of kidney fibrosis.

<p>(A) miR-199a-5p expression in kidney from C57BL/6 mice after UUO at the indicated time points. n = 5 to 7 mice in each group. Data are expressed as mean ± SEM. * p<0.05 and ** p<0.01. (B) Paraffin sections were prepared from kidneys of C57BL/6 mice harvested 28 days after UUO. <i>In situ</i> hybridization assay was performed to determine the localization of miR-199a-5p in the kidney. Results represent one out of three independent experiments. (C) Immunohistochemical analysis of CAV1 expression in kidney tissue sections from C57BL/6 mice 28 days after UUO. One representative section out of two is shown.</p