Dystrophy-associated caveolin-3 mutations reveal that caveolae couple IL6/STAT3 signaling with mechanosensing in human muscle cells

Caveolin-3 is the major structural protein of caveolae in muscle. Mutations in the CAV3 gene cause different types of myopathies with altered membrane integrity and repair, expression of muscle proteins, and regulation of signaling pathways. We show here that myotubes from patients bearing the CAV3P28L and R26Q mutations present a dramatic decrease of caveolae at the plasma membrane, resulting in abnormal response to mechanical stress. Mutant myotubes are unable to buffer the increase in membrane tension induced by mechanical stress. This results in impaired regulation of the IL6/STAT3 signaling pathway leading to its constitutive hyperactivation and increased expression of muscle genes. These defects are fully reversed by reassembling functional caveolae through expression ofcaveolin-3. Our study reveals that under mechanical stress the regulation of mechan-oprotection by caveolae is directly coupled with the regulation of IL6/STAT3 signaling inmuscle cells and that this regulation is absent in Cav3-associated dystrophic patients

Rôle de la cavéoline-3 et de la mécanique des cavéoles dans la physiopathologie du muscle

Caveolae are plasma membrane invaginations that require caveolin proteins for their biogenesis. Recently, our laboratory reported a new role for caveolae in the cell response to mechanical stress (Sinha et al, Cell, 2011). Mutations in the CAV3 gene (muscle isoform), which lead to Cav3 retention in the Golgi apparatus, are associated with muscular dystrophies (MD). My project consists in identifying the functional link between Cav3 mutations and MDs, which exhibit defects in membrane integrity and repair, and in muscle homeostasis.In Cav3-P28L and Cav3-R26Q mutated human myotubes, I showed a lack of caveolae structures at the plasma membrane. This results in a failed buffering of membrane tension increase upon mechanical stress, which leads to membrane integrity defects. I also showed that the interleukin-6 (IL6) pathway, important for muscle homeostasis, is overactivated in mutant myotubes, showing evidence of a negative regulation of the pathway by Cav3. Furthermore, the IL6 pathway is no longer negatively regulated upon mechanical stress, as it is the case in wild-type (WT) myotubes. Interestingly, mutated myotubes phenocopy Cav3 depletion, and the phenotype is reversible with caveolae reformation upon expression of the WT form of Cav3. This confirms the direct link between Cav3 mutations and the absence of caveolae with failed mechano-protection and IL6/STAT3 mechano-signaling.Les cavéoles sont des invaginations de la membrane plasmique qui nécessitent les cavéolines pour leur biogénèse. Récemment, mon laboratoire d’accueil a décrit un nouveau rôle pour les cavéoles dans la réponse au stress mécanique (Sinha et al, Cell, 2011). Des mutations de la Cavéoline-3 (Cav3), isoforme spécifique du muscle, qui mènent à la rétention de la protéine dans l’appareil de Golgi, ont été décrites dans certaines dystrophies musculaires (DM). Mon projet consiste en l’identification du lien fonctionnel entre les mutations de la Cavéoline-3 et les dystrophies musculaires, qui ont comme phénotype principal un défaut d’intégrité et de réparation membranaire et des dérégulations dans l’homéostasie du muscle.Dans des myotubes humains provenant d’un patient portant la mutation Cav3-P28L ou Cav3-R26Q, j’ai pu montré une diminution de la quantité de cavéoles à la membrane plasmique. En conséquence, les myotubes mutants ne sont plus capables de tamponner l’augmentation de la tension membranaire provoquée par un stress mécanique, ce qui conduit à un défaut d’intégrité membranaire. J’ai aussi montré que la voie de l’interleukin-6 (IL6), importante pour l’homéostasie du muscle, est hyperactivée dans les myotubes mutants, révélant un rôle de régulateur négatif de la voie IL6 par Cav3. De plus, cette voie n’est plus régulée négativement quand un stress mécanique est appliqué comme c’est le cas dans les myotubes sauvages (WT). De manière intéressante, les myotubes mutés phénocopient une déplétion de Cav3 et ce phénotype est réversible lorsque l’on reforme des cavéoles à la membrane plasmiques des myotubes mutés en exprimant la forme WT de Cav3. Ceci confirme un lien direct entre les mutations de Cav3 induisant l’absence de cavéoles et le défaut de mécano-protection et mécano-signalisation de la voie IL6

Role of caveolin-3 and caveolae mechanics in muscle pathophysiology

Les cavéoles sont des invaginations de la membrane plasmique qui nécessitent les cavéolines pour leur biogénèse. Récemment, mon laboratoire d’accueil a décrit un nouveau rôle pour les cavéoles dans la réponse au stress mécanique (Sinha et al, Cell, 2011). Des mutations de la Cavéoline-3 (Cav3), isoforme spécifique du muscle, qui mènent à la rétention de la protéine dans l’appareil de Golgi, ont été décrites dans certaines dystrophies musculaires (DM). Mon projet consiste en l’identification du lien fonctionnel entre les mutations de la Cavéoline-3 et les dystrophies musculaires, qui ont comme phénotype principal un défaut d’intégrité et de réparation membranaire et des dérégulations dans l’homéostasie du muscle.Dans des myotubes humains provenant d’un patient portant la mutation Cav3-P28L ou Cav3-R26Q, j’ai pu montré une diminution de la quantité de cavéoles à la membrane plasmique. En conséquence, les myotubes mutants ne sont plus capables de tamponner l’augmentation de la tension membranaire provoquée par un stress mécanique, ce qui conduit à un défaut d’intégrité membranaire. J’ai aussi montré que la voie de l’interleukin-6 (IL6), importante pour l’homéostasie du muscle, est hyperactivée dans les myotubes mutants, révélant un rôle de régulateur négatif de la voie IL6 par Cav3. De plus, cette voie n’est plus régulée négativement quand un stress mécanique est appliqué comme c’est le cas dans les myotubes sauvages (WT). De manière intéressante, les myotubes mutés phénocopient une déplétion de Cav3 et ce phénotype est réversible lorsque l’on reforme des cavéoles à la membrane plasmiques des myotubes mutés en exprimant la forme WT de Cav3. Ceci confirme un lien direct entre les mutations de Cav3 induisant l’absence de cavéoles et le défaut de mécano-protection et mécano-signalisation de la voie IL6.Caveolae are plasma membrane invaginations that require caveolin proteins for their biogenesis. Recently, our laboratory reported a new role for caveolae in the cell response to mechanical stress (Sinha et al, Cell, 2011). Mutations in the CAV3 gene (muscle isoform), which lead to Cav3 retention in the Golgi apparatus, are associated with muscular dystrophies (MD). My project consists in identifying the functional link between Cav3 mutations and MDs, which exhibit defects in membrane integrity and repair, and in muscle homeostasis.In Cav3-P28L and Cav3-R26Q mutated human myotubes, I showed a lack of caveolae structures at the plasma membrane. This results in a failed buffering of membrane tension increase upon mechanical stress, which leads to membrane integrity defects. I also showed that the interleukin-6 (IL6) pathway, important for muscle homeostasis, is overactivated in mutant myotubes, showing evidence of a negative regulation of the pathway by Cav3. Furthermore, the IL6 pathway is no longer negatively regulated upon mechanical stress, as it is the case in wild-type (WT) myotubes. Interestingly, mutated myotubes phenocopy Cav3 depletion, and the phenotype is reversible with caveolae reformation upon expression of the WT form of Cav3. This confirms the direct link between Cav3 mutations and the absence of caveolae with failed mechano-protection and IL6/STAT3 mechano-signaling

The caveolae dress code: structure and signaling

International audienceOver the past decade, interest in caveolae biology has peaked. These small bulb-shaped plasma membrane invaginations of 50-80nm diameter present in most cell types have been upgraded from simple membrane structures to a more complex bona fide organelle. However, although caveolae are involved in several essential cellular functions and pathologies, the underlying molecular mechanisms remain poorly defined. Following the identification of caveolins and cavins as the main caveolae constituents, recent studies have brought new insight into their structural organization as a coat. In this review, we discuss how these new data on caveolae can be integrated in the context of their role in signaling and pathophysiology

Coupling of melanocyte signaling and mechanics by caveolae is required for human skin pigmentation

International audienceTissue homeostasis requires regulation of cell-cell communication, which relies on signaling molecules and cell contacts. In skin epidermis, keratinocytes secrete factors transduced by melanocytes into signaling cues promoting their pigmentation and dendrite outgrowth, while melanocytes transfer melanin pigments to keratinocytes to convey skin photoprotection. How epidermal cells integrate these functions remains poorly characterized. Here, we show that caveolae are asymmetrically distributed in melanocytes and particularly abundant at the melanocyte-keratinocyte interface in epidermis. Caveolae in melanocytes are modulated by ultraviolet radiations and keratinocytes-released factors, like miRNAs. Preventing caveolae formation in melanocytes increases melanin pigment synthesis through upregulation of cAMP signaling and decreases cell protrusions, cell-cell contacts, pigment transfer and epidermis pigmentation. Altogether, we identify that caveolae serve as molecular hubs that couple signaling outputs from keratinocytes to mechanical plasticity of pigment cells. The coordination of intercellular communication and contacts by caveolae is thus crucial to skin pigmentation and tissue homeostasis

New Blocking Antibodies Impede Adhesion, Migration and Survival of Ovarian Cancer Cells, Highlighting MFGE8 as a Potential Therapeutic Target of Human Ovarian Carcinoma

<div><p>Milk Fat Globule – EGF – factor VIII (MFGE8), also called lactadherin, is a secreted protein, which binds extracellularly to phosphatidylserine and to αvβ3 and αvβ5 integrins. On human and mouse cells expressing these integrins, such as endothelial cells, phagocytes and some tumors, MFGE8/lactadherin has been shown to promote survival, epithelial to mesenchymal transition and phagocytosis. A protumoral function of MFGE8 has consequently been documented for a few types of human cancers, including melanoma, a subtype of breast cancers, and bladder carcinoma. Inhibiting the functions of MFGE8 could thus represent a new type of therapy for human cancers. Here, we show by immunohistochemistry on a collection of human ovarian cancers that MFGE8 is overexpressed in 45% of these tumors, and we confirm that it is specifically overexpressed in the triple-negative subtype of human breast cancers. We have established new <i>in vitro</i> assays to measure the effect of MFGE8 on survival, adhesion and migration of human ovarian and triple-negative breast cancer cell lines. Using these assays, we could identify new MFGE8-specific monoclonal antibodies, which efficiently blocked these three tumor-promoting effects of MFGE8. Our results suggest future use of MFGE8-blocking antibodies as new anti-cancer therapeutics in subgroups of ovarian carcinoma, and triple-negative breast carcinoma patients.</p> </div

Expression of MFGE8 in biopsies of human ovarian carcinoma.

<p>(<b>a</b>) Examples of Human MFGE8 immunohistochemistry on ovarian carcinoma sections showing: no staining of tumor cells (score 0), but MFGE8-positive endothelial cells (black arrow) in the left panel. Areas of weak expression (w) and of medium expression (m) of MFGE8 in the same section, thus ranked as score 2 in the middle panel. Strong MFGE8 expression (s) throughout the tumor, ranked as score 3 (right panel). (<b>b</b>) Scoring of MFGE8 expression (y-axis) in 48 ovarian tumor biopsies from Institut Curie’s patients, as a function of tumor grade (x-axis). No statistically significant difference was observed in distribution of tumors overexpressing MFGE8 (score 2-3) or not (score 0-1) in grade 2 versus grade 3 tumors.</p

Expression of MFGE8 in human breast carcinoma and effect of MFGE8 on MDA-MB-231 cells.

<p>(<b>a</b>–<b>b</b>) Scoring of MFGE8 expression, analyzed by immunohistochemistry as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072708#pone-0072708-g001" target="_blank">Figure 1</a>, in 59 breast tumor biopsies from Institut Curie’s patients, as a function of tumor grade (a) or phenotype in terms of Hormon Receptor (HR) or HER2 expression (<b>b</b>). ***: P = 0.0001 for distribution of tumors overexpressing (score 2-3) or not (score 0-1) MFGE8 between grade 2 versus grade 3 tumors (a), or between HR+/HER2- versus triple-negative tumors (b). (<b>c</b>) Analysis of mRNA expression levels (Log₂(Affymetrix U133plus2.0 signal)) of <i>MFGE8</i> in public results of Affymetrix microarray data of the Broad-Novartis Cancer Cell Line Encyclopedia as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072708#pone-0072708-g002" target="_blank">Figure 2</a>. Black arrow indicates the MDA-MB-231 cell line (HR-/HER2- triple negative phenotype) selected for subsequent experiments. (<b>d</b>) Quantification of MFGE8 by ELISA in MDA-MB-231 conditioned medium (24 hr). Secreted MFGE8 is expressed in ng/mL per 10⁶ cultured cells. Mean of two experiments + SD. (<b>e</b>) FACS analysis of αvβ3 (left panel) and αvβ5 (right panel) integrin expression at the surface of MDA-MB-231 cells. Specific antibody (blue line). Isotype control (red line). (<b>f</b>) MDA-MB-231 adhesion assay on 5µg/mL MFGE8, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072708#pone-0072708-g003" target="_blank">Figure 3-5</a>, and inhibition by 10µg/mL hMC3, represented as slope values between 0 and 1 hour. (<b>g</b>) MDA-MB-231 migration assay to 5µg/mL MFGE8 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072708#pone-0072708-g003" target="_blank">Figure 3-5</a>, and inhibition by 10µg/mL 215A9 antibody. Data represented as slope values calculated between 0 and 18 hours. (<b>h</b>) MDA-MB-231 survival assay in the presence of 10 µg/mL MFGE8, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072708#pone-0072708-g003" target="_blank">Figure 3-5</a>, and inhibition by 10 µg/mL 399A12. 100% survival corresponds to 5µg/mL MFGE8 without antibodies. Box-and-whiskers graphs as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072708#pone-0072708-g003" target="_blank">Figure 3</a>. N=4 (except in (g): PBS N=6, MFGE8 + 215A9 N=5, and in (h): ctrl N=6, MFGE8: N=5). ***: P<0.001, **: P<0.01, compared to Ctrl = MFGE8.</p

Effect of MFGE8 and anti-MFGE8 antibodies on IGROV-1 and SHIN-3 ovarian cancer cells.

<p>(<b>a</b>–<b>b</b>) Adhesion of IGROV-1 (a) and SHIN-3 (b) to 5 µg/mL MFGE8, as compared to PBS, quantified by slope value between 0 and 2h. The effect of three selected antibodies delivered at two doses (2,5 and 10 µg/mL) on MFGE8-coated wells is also shown. (<b>c</b>–<b>d</b>) IGROV-1 (c) and SHIN-3 (d) migration assay to 5µg/mL MFGE8 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072708#pone-0072708-g003" target="_blank">Figure 3-4</a>, and inhibition by antibodies as in panels a and b for adhesion. Slopes values calculated between 0 and 18 hours. (<b>d</b>–<b>e</b>) IGROV-1 (d) and SHIN-3 (e) survival assay in the presence of 10µg/mL MFGE8, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072708#pone-0072708-g003" target="_blank">Figure 3-4</a>, and inhibition by 2,5 or 10µg/mL antibodies. 100% survival corresponds to 10 µg/mL MFGE8 without antibodies. Box-and-whiskers graphs as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072708#pone-0072708-g003" target="_blank">Figure 3</a>. N=4 (except in (b): 399A12 -2.5 µg/mL, 346B6 -10 µg/mL, 311A7 -10 µg/mL: N = 3; in (e, f) PBS and MFGE8 : N=5; in (f) MFGE8 + 311A7: N=3). *** = P<0.001; ** = P<0.01; * = P<0.05, compared to Ctrl = MFGE8.</p