Etude des effets de différentes formes de la protéine RBF de drosophile sur le devenir cellulaire

Le gène Rb est un gène suppresseur de tumeur dont les effets apoptotiques sont mal compris à ce jour. Rb peut être pro- ou anti-apoptotique, et des formes clivées de la protéine pRb sont générées par des caspases au cours de l apoptose. Seul un des sites de clivage par les caspases semble conservé entre les protéines pRb de mammifère et RBF1 de drosophile, le site TELD. J ai montré que RBF1 est pro-apoptotique dans les cellules en prolifération, possède un effet anti-apoptotique dans les cellules post-mitotiques, et que le clivage de RBF1 peut avoir lieu au cours du développement. Une forme de RBF1 mutée au niveau du site TELD, RBFD253A, est pro-apoptotique, et induit également une prolifération excessive de manière non-autonome cellulaire. Donc le clivage de RBF1 pourrait réguler ses fonctions in vivo. La forme p76 CRBF, correspondant à la forme résultant d un clivage au site TELD et présentant une délétion de son extrémité C-terminale, ne possède plus d'activité pro-apoptotique.The Rb gene is a tumor suppressor gene. Its effects on apoptosis are poorly known. Indeed, Rb can be pro-or anti-apoptotic, and cleaved forms of pRb are generated by caspases during apoptosis. Only one of the caspase cleavage sites seems to be conserved between mammalian pRb and Drosophila RBF1, the TELD site. I showed that RBF1 is pro-apoptotic in proliferating cells whereas it is anti-apoptotic in post-mitotic cells, and that RBF1 cleavage occurs during development. A mutated RBF1 form at the TELD site, RBFD253A, is pro-apoptotic but also induces excessive proliferation in a non-cell autonomous manner, which seems to indicate that RBF1 cleavage could regulate its activities in vivo. The p76 CRBF forms that corresponds to the form that would result from a cleavage at the TELD site and deleted of its C-terminus extremity is no longer pro-apoptotic and will certainly contribute to better understand RBF1 pro-apoptotic effects.VERSAILLES-BU Sciences et IUT (786462101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

The Mkx homeoprotein promotes tenogenesis in stem cells and improves tendon repair

International audienceTendons are essential structures that transmit mechanical forces generated by skeletal muscles to bones to cause body motion. Acute tendon injury and tendinopathy are common pathologies of the musculoskeletal system. However, a poor understanding of tendon biology has impaired the design of efficient treatments for tendon repair following injury and tendinopathy (1). A recent paper by Liu et al. , published in Stem Cells in 2014 (2) highlighted an essential role for the Mohawk (Mkx) homeodomain protein in promoting tenogenesis in mouse stem cells and improving tendon repair in a mouse model for tendon injury

TGFβ and FGF promote tendon progenitor fate and act downstream of muscle contraction to regulate tendon differentiation during chick limb development

International audienceThe molecular programme underlying tendon development has not been fully identified. Interactions with components of the musculoskeletal system are important for limb tendon formation. Limb tendons initiate their development independently of muscles; however, muscles are required for further tendon differentiation. We show that both FGF/ERK MAPK and TGFβ/SMAD2/3 signalling pathways are required and sufficient for SCX expression in chick undifferentiated limb cells, whereas the FGF/ERK MAPK pathway inhibits Scx expression in mouse undifferentiated limb mesodermal cells. During differentiation, muscle contraction is required to maintain SCX, TNMD and THBS2 expression in chick limbs. The activities of FGF/ERK MAPK and TGFβ/SMAD2/3 signalling pathways are decreased in tendons under immobilisation conditions. Application of FGF4 or TGFβ2 ligands prevents SCX downregulation in immobilised limbs. TGFβ2 but not FGF4 prevent TNMD and THBS2 downregulation under immobilisation conditions. We did not identify any intracellular crosstalk between both signalling pathways in their positive effect on SCX expression. Independently of each other, both FGF and TGFβ promote tendon commitment of limb mesodermal cells and act downstream of mechanical forces to regulate tendon differentiation during chick limb development

Mutating RBF Can Enhance Its Pro-Apoptotic Activity and Uncovers a New Role in Tissue Homeostasis

International audienceThe tumor suppressor retinoblastoma protein (pRb) is inactivated in a wide variety of cancers. While its role during cell cycle is well characterized, little is known about its properties on apoptosis regulation and apoptosis-induced cell responses. pRb shorter forms that can modulate pRB apoptotic properties, resulting from cleavages at caspase specific sites are observed in several cellular contexts. A bioinformatics analysis showed that a putative caspase cleavage site (TELD) is found in the Drosophila homologue of pRb (RBF) at a position similar to the site generating the p76Rb form in mammals. Thus, we generated a punctual mutant form of RBF in which the aspartate of the TELD site is replaced by an alanine. This mutant form, RBF D253A , conserved the JNK-dependent pro-apoptotic properties of RBF but gained the ability of inducing overgrowth phenotypes in adult wings. We show that this overgrowth is a consequence of an abnormal proliferation in wing imaginal discs, which depends on the JNK pathway activation but not on wingless (wg) ectopic expression. These results show for the first time that the TELD site of RBF could be important to control the function of RBF in tissue homeostasis in vivo

Screening of suppressors of bax-induced cell death identifies glycerophosphate oxidase-1 as a mediator of debcl-induced apoptosis in Drosophila

International audienceMembers of the Bcl-2 family are key elements of the apoptotic machinery. In mammals, this multigenic family contains about twenty members, which either promote or inhibit apoptosis. We have previously shown that the mammalian pro-apoptotic Bcl-2 family member Bax is very efficient in inducing apoptosis in Drosophila, allowing the study of bax-induced cell death in a genetic animal model. We report here the results of the screening of a P[UAS]-element insertion library performed to identify gene products that modify the phenotypes induced by the expression of bax in Drosophila melanogaster. We isolated 17 putative modifiers involved in various function or process: the ubiquitin/proteasome pathway; cell growth, proliferation and death; pathfinding and cell adhesion; secretion and extracellular signaling; metabolism and oxidative stress. Most of these suppressors also inhibit debcl-induced phenotypes, suggesting that the activities of both proteins can be modulated in part by common signaling or metabolic pathways. Among these suppressors, Glycerophosphate oxidase-1 is found to participate in debcl-induced apoptosis by increasing mitochondrial reactive oxygen species accumulation

The World’s First Acne Dysbiosis-like Model of Human 3D Ex Vivo Sebaceous Gland Colonized with <i>Cutibacterium acnes</i> and <i>Staphylococcus epidermidis</i>

Acne-prone skin is associated with dysbiosis involving Cutibacterium acnes (C. acnes) and Staphylococcus epidermidis (S. epidermidis) causing increased seborrhea in sebaceous glands (SG) and inflammation. Human primary sebocytes were cultivated using 1.106 UFC/mL C. acnes Type IA (facial acne, ATCC6919) and/or 1.105 UFC/mL S. epidermidis (unknown origin, ATCC12228) for 48 h in our SEB4GLN-optimized media without antibiotics. Bacteria and sebocytes were enumerated and assessed to determine their viability. Lipid production was imaged and quantified via Nile Red staining. SG with hair follicles were microdissected from healthy skin and cultured using 1.105 UFC/mL C. acnes Type 1A and/or 1.104 UFC/mL S. epidermidis (wild-type facial skin strain) through prior fixation and immunostaining for MC5R, C. acnes and nuclei (DAPI) via Z-stack confocal microscopy bioimaging (Leica SP5X & FIJI software, Version 2.9.0). C. acnes growth was not impacted when co-cultivated with sebocytes (2D) or SG (3D) models. Phylotype IA stimulated sebocyte lipid production, which had no impact on viability. The S. epidermidis reference strain overproliferated, inducing sebocyte mortality. For 3D SG model, culture conditions were optimized using a wild-type facial skin strain at a lower concentration, 1:10 ratio to C. acnes, reduced contact time, sequential inoculation and rinsing step. Bioimaging revealed strong C. acnes labeling in the active areas of the pilosebaceous unit. S. epidermidis formed biofilm, which was distributed across the SG via non-specific fluorescence imaging. We developed an innovative model of a sebaceous gland that mimics acne-prone skin with lipid overproduction and virulent phylotype IA C. acnes inoculation

RBF^D253A is pro-apoptotic in the ZNC and induces more apoptosis than RBF in third instar larvae wing imaginal discs.

<p>(A, E) <i>C96-Gal4</i> and <i>vg-Gal4</i> expression patterns are visualized by <i>UAS-mtGFP</i> expression in third instar larvae wing imaginal discs. (B-D, F-H) Apoptotic cells are labeled by TUNEL in wing imaginal discs; specific staining of apoptotic cells corresponds to bright white patches. (B, F) <i>C96-Gal4/+</i> and <i>vg-Gal4/+</i> control discs have few apoptotic cells. (C) <i>C96-Gal4/UAS-RBF</i> wing discs are similar to control. (D) Some apoptotic cells are observed within the <i>C96-Gal4</i> expression domain in <i>UAS-RBF^D253A/X</i>; <i>C96-Gal4/+</i> discs (white arrow). (G, H) Apoptotic cells are observed within the <i>vg-Gal4</i> expression domain in <i>vg-Gal4/+</i>; <i>UAS-RBF/+</i> and <i>UAS-RBF^D253A/X</i>; <i>vg-Gal4/+</i> wing discs (white arrows). (G) The white arrowhead indicates a zone at the center of the pouch where cells are not TUNEL-labeled in <i>vg-Gal4/+</i>; <i>UAS-RBF/+</i> wing discs. All discs are shown with posterior to the top.</p

RBF- and RBF^D253A-induced apoptosis as well as RBF^D253A-induced overgrowth depend on the JNK pathway activity.

<p>(A–C) Anti-◯P-JNK staining in young third instar larvae wing imaginal discs. (A) No staining is observed in <i>ptc-Gal4/+</i> control. (B, C) <i>UAS-RBF/ptc-Gal4</i> and <i>UAS-RBF^D253A/X; ptc-Gal4/+</i> discs present anti-◯P-JNK staining at the dorso-ventral boundary. All discs are shown with posterior to the top. (D) Notch phenotypes in adult wings of <i>vg-Gal4/+</i>; <i>UAS-RBF</i> and <i>vg-Gal4/+</i>; <i>UAS-RBF/UAS-bsk-RNAi</i> flies are grouped into four categories (wild type, weak, intermediate and strong) according to the number and size of notches observed on the wing margin (asterisks). <i>Bsk-RNAi</i> partially suppresses <i>RBF</i>-induced notch phenotypes (Wilcoxon test, α<10⁻¹⁵, n = 540). (E) The frequency of RBF^D253A-induced ectopic tissue growth is strongly decreased in <i>UAS-bsk-RNAi</i> co-expressing flies (Chi² test, α = 8.8 10⁻¹⁵). (F) TUNEL-labeling of apoptotic cells in wing imaginal discs; specific staining of apoptotic cells corresponds to bright patches in wing discs <i>of vg-Gal4/+</i>; <i>UAS-bsk-RNAi/+</i> (left panel), <i>vg-Gal4/+</i>; <i>UAS-RBF^D253A/+</i> (center panel), <i>vg-Gal4/+</i>; <i>UAS-RBF^D253A/UAS-bsk-RNAi</i> (right panel) <i>larvae</i>. RNAi-mediated knockdown of <i>bsk</i> strongly decreased RBF^D253A-induced apoptosis. All discs are shown with posterior to the top.</p

RBF^D253A-induced overgrowth does not depend on Wg.

<p>(A, B) Anti-Wg staining in wing imaginal discs of control (A) or <i>en-Gal4, UAS-wg-RNAi</i> third instar larvae (B). No staining is observed in the posterior compartment of discs that express <i>wg-RNAi</i>. (C) The frequency of RBF^D253A-induced ectopic tissue growth is not affected in <i>UAS-wg-RNAi</i> co-expressing flies (Chi² test, α = 0.15). All discs are shown with posterior to the top.</p