Identification, Structural, and Functional Characterization of a New Early Gene (6A3-5, 7 kb): Implication in the Proliferation and Differentiation of Smooth Muscle Cells

Arterial smooth muscle cells (SMCs) play a major role in atherosclerosis and restenosis. Differential display was used to compare transcription profiles of synthetic SMCs to proliferating rat cultured SMC line. An isolated cDNA band (6A3-5) was shown by northern (7 kb) to be upregulated in the proliferating cell line. A rat tissue northern showed differential expression of this gene in different tissues. Using 5′ RACE and screening of a rat brain library, part of the cDNA was cloned and sequenced (5.4 kb). Sequence searches showed important similarities with a new family of transcription factors, bearing ARID motifs. A polyclonal antibody was raised and showed a protein band of 175 kd, which is localized intracellularly. We also showed that 6A3-5 is upregulated in dedifferentiated SMC (P9) in comparison to contractile SMC ex vivo (P0). This work describes cloning, structural, and functional characterization of a new early gene involved in SMC phenotype modulation

Caractérisation structurale et fonctionnelle d'un nouveau gène au niveau des cellules musculaires lisses vasculaires proliférantes

Les événements moléculaires contrôlant la réponse proliférative des cellules musculaires lisses vasculaires (CMLV), au cours du développement de pathologies vasculaires, restent mal compris. L'objectif de ce doctorat est de caractériser, sur un plan structural et fonctionnel, un nouveau gène surexprimé au niveau des CMLV proliférantes. Sur un plan structural, le clonage de l'ADNc du 6A3-5 ((1) criblage d'une banque d'expression de cerveau de rat par PCR complété par (2) une analyse in silico du génome) nous a permis d'isoler l'ADNc de ce nouveau gène chez le rat. L'analyse bioinformatique a révélé la présence d'un domaine ARID de liaison à l'ADN ("AT-rich interaction domain"), de deux motifs d'interactions protéiques ("Osa homology domain") et d'un signal de localisation nucléaire. La caractérisation des anticorps anti-6A3-5 a révélé une localisation périnucléaire et nucléaire de la protéine 6A3-5 (180kDa). Sur un plan fonctionnel, le sérum, le PDGF et l'Angiotensine II induisent, in vitro, une surexpression précoce et dose dépendante du 6A3-5. La transfection de CMLV par des oligonucléotides antisens, dirigés contre le 6A3-5, induit une déplétion du 6A3-5 associée à une réduction significative (dose et séquence dépendante) de la réponse proliférative des CMLV au PDGF. In vivo, la perfusion d'Angiotensine II, chez des rats traités par un inhibiteur de l'enzyme de conversion, induit une surexpression du 6A3-5 au niveau des CMLV de l'aorte de rats hypertendus. L'ischémie induite par clampage du pédicule rénal, initie une surexpression rapide du 6A3-5 au niveau des CMLV des artérioles de petits et moyens calibes, des cellules mésangiales et des myofibroblastes. Une expression analogue du 6A3-5 a été mise en évidence, par immunohistochimie, au niveau de biopsies de transplantation rénale. En conclusion, le 6A3-5 apparaît être un gène de réponse précoce potentiellement impliqué dans le contrôle moléculaire des CMLVLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Ischemia Induces Early Expression of a New Transcription Factor (6A3-5) in Kidney Vascular Smooth Muscle Cells: Studies in Rat and Human Renal Pathology

Acute renal failure, characterized by rapid decline in glomerular filtration rate, is a major cause of morbidity and mortality. During the evolution of renal diseases chronic ischemia develops. Indeed, acute or chronic renal failure may occur as a result of renal ischemia, which induces cells to dedifferentiate, proliferate, or become apoptotic. In this study, we have investigated the expression of a newly identified transcription factor, 6A3-5, under in vitro and in vivo conditions. Proliferating vascular smooth muscle were investigated in response to different mitogenic agents. The 6A3-5 expression was then studied in ischemic rat kidney, induced by renal pedicle clamping, followed, or not, by reperfusion. Subsequently human renal biopsies from early kidney grafts and chronic renal diseases were also investigated for 6A3-5 protein expression by immunohistochemistry. In vitro study shows an over-expression of 6A3-5 following 2 to 4 hours stimulation by serum or Angiotensin II, of rat proliferating aortic smooth muscle cell. Moreover, in vivo study shows that this new protein is over expressed in rat kidney submitted to 45 minutes ischemia. An anti-6A3-5 antibody shows the protein to be expressed in smooth muscle cells of the arterioles and intermediate size arteries, in mesangial cells and interstitial myofibroblasts. In human biopsies of early kidney grafts and renal disease, the same up-regulation of 6A3-5, as in acute ischemic situation, is observed. This 6A3-5 expression is intimately associated with α-smooth muscle cell actin expression in mesangial cells, arteriolar smooth muscle cells as well as interstitial myofibroblasts. Transcription factor 6A3-5 could potentially be a novel early vascular marker of acute and chronic renal ischemic stress implicated in tissue remodeling

Netrin-1 blockade inhibits tumour growth and EMT features in endometrial cancer

Netrin-1 is upregulated in cancers as a protumoural mechanism1. Here we describe netrin-1 upregulation in a majority of human endometrial carcinomas (ECs) and demonstrate that netrin-1 blockade, using an anti-netrin-1 antibody (NP137), is effective in reduction of tumour progression in an EC mouse model. We next examined the efficacy of NP137, as a first-in-class single agent, in a Phase I trial comprising 14 patients with advanced EC. As best response we observed 8 stable disease (8 out of 14, 57.1%) and 1 objective response as RECIST v.1.1 (partial response, 1 out of 14 (7.1%), 51.16% reduction in target lesions at 6 weeks and up to 54.65% reduction during the following 6 months). To evaluate the NP137 mechanism of action, mouse tumour gene profiling was performed, and we observed, in addition to cell death induction, that NP137 inhibited epithelial-to-mesenchymal transition (EMT). By performing bulk RNA sequencing (RNA-seq), spatial transcriptomics and single-cell RNA-seq on paired pre- and on-treatment biopsies from patients with EC from the NP137 trial, we noted a net reduction in tumour EMT. This was associated with changes in immune infiltrate and increased interactions between cancer cells and the tumour microenvironment. Given the importance of EMT in resistance to current standards of care2, we show in the EC mouse model that a combination of NP137 with carboplatin-paclitaxel outperformed carboplatin-paclitaxel alone. Our results identify netrin-1 blockade as a clinical strategy triggering both tumour debulking and EMT inhibition, thus potentially alleviating resistance to standard treatments