Loss of MT 1‐ MMP causes cell senescence and nuclear defects which can be reversed by retinoic acid
2015; Springer Nature; Volume: 34; Issue: 14 Linguagem: Inglês
10.15252/embj.201490594
ISSN1460-2075
AutoresAna Gutiérrez‐Fernández, Clara Soria‐Valles, Fernando G. Osorio, Jesús Gutiérrez‐Abril, Cecilia Garabaya, Alina Aguirre, Antonio Fueyo, María Soledad Fernández‐García, Xosé S. Puente, Carlos López-Otı́n,
Tópico(s)Retinoids in leukemia and cellular processes
ResumoArticle19 May 2015free access Loss of MT1-MMP causes cell senescence and nuclear defects which can be reversed by retinoic acid Ana Gutiérrez-Fernández Ana Gutiérrez-Fernández Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Clara Soria-Valles Clara Soria-Valles Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Fernando G Osorio Fernando G Osorio Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Jesús Gutiérrez-Abril Jesús Gutiérrez-Abril Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Cecilia Garabaya Cecilia Garabaya Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Alina Aguirre Alina Aguirre Área de Fisiología, Departamento de Biología Funcional, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Antonio Fueyo Antonio Fueyo Área de Fisiología, Departamento de Biología Funcional, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author María Soledad Fernández-García María Soledad Fernández-García Servicio de Anatomía Patológica, Hospital Universitario Central de Asturias, Oviedo, Spain Search for more papers by this author Xose S Puente Xose S Puente Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Carlos López-Otín Corresponding Author Carlos López-Otín Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Ana Gutiérrez-Fernández Ana Gutiérrez-Fernández Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Clara Soria-Valles Clara Soria-Valles Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Fernando G Osorio Fernando G Osorio Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Jesús Gutiérrez-Abril Jesús Gutiérrez-Abril Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Cecilia Garabaya Cecilia Garabaya Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Alina Aguirre Alina Aguirre Área de Fisiología, Departamento de Biología Funcional, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Antonio Fueyo Antonio Fueyo Área de Fisiología, Departamento de Biología Funcional, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author María Soledad Fernández-García María Soledad Fernández-García Servicio de Anatomía Patológica, Hospital Universitario Central de Asturias, Oviedo, Spain Search for more papers by this author Xose S Puente Xose S Puente Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Carlos López-Otín Corresponding Author Carlos López-Otín Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Author Information Ana Gutiérrez-Fernández1, Clara Soria-Valles1, Fernando G Osorio1, Jesús Gutiérrez-Abril1, Cecilia Garabaya1, Alina Aguirre2, Antonio Fueyo2, María Soledad Fernández-García3, Xose S Puente1 and Carlos López-Otín 1 1Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain 2Área de Fisiología, Departamento de Biología Funcional, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain 3Servicio de Anatomía Patológica, Hospital Universitario Central de Asturias, Oviedo, Spain *Corresponding author. Tel: +34 985 104 201; Fax: +34 985 103 564; E-mail: [email protected] The EMBO Journal (2015)34:1875-1888https://doi.org/10.15252/embj.201490594 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract MT1-MMP (MMP14) is a collagenolytic enzyme located at the cell surface and implicated in extracellular matrix (ECM) remodeling. Mmp14−/− mice present dwarfism, bone abnormalities, and premature death. We demonstrate herein that the loss of MT1-MMP also causes cardiac defects and severe metabolic changes, and alters the cytoskeleton and the nuclear lamina structure. Moreover, the absence of MT1-MMP induces a senescent phenotype characterized by up-regulation of p16INK4a and p21CIP1/WAF1, increased activity of senescence-associated β-galactosidase, generation of a senescence-associated secretory phenotype, and somatotroph axis alterations. Consistent with the role of retinoic acid signaling in nuclear lamina stabilization, treatment of Mmp14−/− mice with all-trans retinoic acid reversed the nuclear lamina alterations, partially rescued the cell senescence phenotypes, ameliorated the pathological defects in bone, skin, and heart, and extended their life span. These results demonstrate that nuclear architecture and cell senescence can be modulated by a membrane protease, in a process involving the ECM as a key regulator of nuclear stiffness under cell stress conditions. Synopsis Alterations in the extracellular matrix caused by loss of protease MT1-MMP lead to cellular senescence, nuclear lamina abnormalities, and features of premature ageing that can be rescued by treatment with retinoic acid. Loss of MT1-MMP metalloprotease activity induces a cellular senescence phenotype. Deficient remodeling of the extracellular matrix alters the cytoskeleton and nuclear structure. All-trans retinoic acid treatment ameliorates the cellular senescence process. Introduction The extracellular matrix (ECM) provides an attachment substrate for most cells, but its composition and stiffness are also able to modify many cellular characteristics (Eyckmans et al, 2011). Furthermore, certain ECM proteins can limit the access of cells to specific growth factors, by sequestering them within the ECM (Bergers et al, 2000). Therefore, the ECM constitutes an essential player during development and differentiation. Due to the dynamic nature of the ECM, a cellular response to different stimuli can result in a massive remodeling of this matrix. This response can be slow, by modifying its composition through secretion of alternative ECM proteins, or fast, by activating specific proteases which cleave and modify the ECM surrounding the cell. Matrix metalloproteinases (MMPs) constitute a group of extra- or pericellular proteases with the ability to process virtually all components of the ECM (Fanjul-Fernandez et al, 2010; Kessenbrock et al, 2010). Among them, collagenases represent a small subgroup of MMPs which precisely cleave fibrillar collagens and initiate collagen degradation and ECM remodeling. Collagenase-1, collagenase-2, and collagenase-3 (MMP-1, MMP-8, and MMP-13) were initially discovered by their ability to cleave collagen in different cell types (Overall & Lopez-Otin, 2002). However, the generation of mice deficient in specific MMPs has revealed that membrane-bound MT1-MMP (MMP14) is a powerful collagenase, necessary for cell invasion through collagen-rich ECMs (Sabeh et al, 2004). The importance of MT1-MMP in cell function has also been reinforced by the analysis of animals lacking different MMPs. Thus, mutant mice deficient in any of the three secreted collagenases (Mmp1a, Mmp8, and Mmp13) are viable and fertile (Balbin et al, 2003; Fanjul-Fernandez et al, 2013; Inada et al, 2004). In contrast, loss of mouse MT1-MMP (Mmp14) results in a complex phenotype that leads to premature death as soon as 3 weeks after birth (Holmbeck et al, 1999; Zhou et al, 2000). Mmp14-deficient mice show dwarfism, osteopenia, and severe connective tissue abnormalities, as well as defects in adipose tissue formation and alveolar development (Atkinson et al, 2005; Chun et al, 2006). Nevertheless, the systemic effects caused by the loss of MT1-MMP and the effect of a deficient ECM remodeling on cell structure and function are largely unknown. The ECM and the cytoskeleton constitute dynamic structures which are tightly interconnected at the plasma membrane by integrins (Hynes, 2002). These heterodimeric proteins are able to bind specific ECM components at the outer surface and, at the same time, recruit adaptor proteins to the cytoplasmic side, thus linking integrins with components of the cytoskeleton and intracellular signaling networks. The cytoskeleton is connected to the nucleus by a series of proteins that constitute the LINC complex (linker of the nucleoskeleton and the cytoskeleton) (Crisp et al, 2006; Mellad et al, 2011). This intricate network of proteins and filaments provides mechanosensor properties to cells, allowing them to adapt to different forms of mechanical stress (Martins et al, 2012). Genetic inactivation of the murine genes encoding lamin A or other components of the nuclear envelope causes progeroid syndromes characterized by short life span, lipodystrophy, and cardiac and skeletal abnormalities, as well as inability to produce a functional ECM (Hernandez et al, 2010; Osorio et al, 2011; Pendas et al, 2002). It is remarkable that some of these phenotypes have also been reported in Mmp14-deficient mice (Chun et al, 2006; Holmbeck et al, 1999; Zhou et al, 2000). Interestingly, depletion of the Sun-1 protein from the LINC complex in lamin A-deficient progeroid animals extends their longevity and ameliorates most pathological phenotypes (Chen et al, 2012), underscoring the importance of the nucleus–cytoskeleton connection in premature aging and cellular senescence. It is also remarkable the recent finding that ECM stiffness increases the relative abundance of lamin A through a process modulated by the retinoid receptor signaling pathway (Swift et al, 2013), reflecting the critical role of lamin A in mechanosensing (Buxboim et al, 2014). Herein, we show that the abnormal proteolytic processing of ECM components in Mmp14-deficient mice triggers signaling events that alter the nuclear lamina and cytoskeleton structure. Moreover, loss of Mmp14 elicits a cellular senescence response characterized by major metabolic changes. We also show that the Mmp14-mediated proteolytic activity prevents the senescent phenotype of Mmp14-deficient cells, providing evidence of a cellular senescence response caused by abnormal ECM remodeling. Finally, we report that treatment of Mmp14-null mice with retinoic acid rescues some of their senescent features and increases their life span. Results Generation and characterization of a new strain of Mmp14-deficient mice We generated mice deficient in Mmp14 by deleting exons 4 and 5, encoding the MT1-MMP catalytic domain (Supplementary Fig S1A). MT1-MMP protein could not be detected in Mmp14−/− tissues by Western blot analysis, and a reduction in the activation of pro-MMP2 (a substrate for MT1-MMP) was observed by gelatin zymography (Supplementary Fig S1B). In agreement with previous studies (Holmbeck et al, 1999; Zhou et al, 2000), Mmp14−/− mice were smaller than their wild-type littermates and showed a reduced growth which was evident as soon as 4 days after birth (Supplementary Fig S1C and D). These mutant mice failed to grow and their mean survival was 14 days (Supplementary Fig S1E) (Zhou et al, 2000). Mmp14 deficiency also resulted in a severe skeletal phenotype which has been thoroughly investigated (Holmbeck et al, 1999; Zhou et al, 2000). We also found prominent cranial sutures in Mmp14-null mice (Supplementary Fig S1F) and a marked impairment of the collagenolytic activity in Mmp14-null fibroblasts (Supplementary Fig S1G). A more detailed analysis of these mutant mice allowed us to identify other alterations, including an enlargement of the distal airways and alveoli, the accumulation of extravasated blood cells in the lung matrix of Mmp14−/− mice, and the presence of inflammatory cells around biliar ducts, which were not detected in wild-type mice (Supplementary Fig S1H). We also observed that the skeletal muscle structure was altered in Mmp14-deficient mice as assessed by the detection of changes in the activity of several mitochondrial enzymes such as succinate dehydrogenase and by the accumulation of collagen fibers in muscle from Mmp14−/− mice (Supplementary Fig S1I). Furthermore, muscle fiber length was 30% shorter in Mmp14−/− animals than that in control mice (29 versus 20 μm, P < 0.05) (Supplementary Fig S1J). Together, and despite the clear skeletal abnormalities observed in Mmp14−/− mice, these data show additional alterations in non-skeletal tissues which might contribute to the early death of these animals. Loss of MT1-MMP induces cellular senescence Notably, we also observed that Mmp14-null mice exhibit cardiovascular defects which have not been previously reported in other strains of Mmp14-null mice. We found a thickened muscular wall of the right ventricle and ventricular septum hypertrophy in 16-day-old Mmp14−/− hearts (Fig 1A; Supplementary Fig S2A), suggesting abnormal cardiac function in these animals. These cardiac abnormalities were accompanied by a strong accumulation of type I collagen in hearts from Mmp14−/− mice as determined by picrosirius red staining to visualize collagen. Quantitative PCR analysis of different factors involved in cardiomyocyte function revealed that hearts from Mmp14−/− mice accumulate β-myosin heavy chain (β-MHC/Myh7), gap junction connexin 40 (Cx40) and NKx2.5 (Supplementary Fig S1K–M). These findings reflect either an improper maturation of cardiomyocytes during heart development or the presence of cardiac stress in these animals (Lowes et al, 1997). Interestingly, Mmp14−/− mice also exhibited an accumulation of sudan black staining in heart, which detects the complex lysosomal aggregate known as lipofucsin, that usually accumulates in aged tissues, suggesting the occurrence of a senescence process in their cardiac tissue (Georgakopoulou et al, 2013) (Fig 1B; Supplementary Fig S2B). The finding of these cardiac defects and cell senescence features in mice lacking MT1-MMP led us to evaluate the putative presence of senescence in other tissues from these mutant animals. Analysis of the senescence-associated β-galactosidase (SA-β-Gal) activity, a biomarker for senescence and aging cells, demonstrated that it was clearly augmented in kidney from Mmp14−/− mice (Fig 1C; Supplementary Fig S2C), reflecting an increased number of senescent cells in vivo. Similarly, higher SA-β-Gal activity was observed in adipose tissue and fibroblasts derived from Mmp14-deficient mice (Fig 1C; Supplementary Fig S2C). To further confirm the senescence phenotype, we performed a BrdU incorporation assay to corroborate the lack of proliferation in these fibroblasts (Fig 1D), and an immunofluorescence assay of HP1γ, a senescence-associated heterochromatin protein that is accumulated in the foci of senescence cells (Fig 1E). We next analyzed different molecular mediators implicated in the senescence process. The tumor suppressor protein p16INK4a, a marker of cellular senescence (Collado & Serrano, 2010; Krishnamurthy et al, 2004), showed a strong accumulation in fibroblasts derived from Mmp14−/− mice (Fig 1F). Gene expression analysis also revealed that the cyclin-dependent kinase inhibitor 1A (p21CIP1/WAF1), a direct target of p53 involved in senescence processes (Baker et al, 2013), was overexpressed in Mmp14−/− muscles (Fig 1G). Thus, quantitative RT–PCR analysis showed that p21CIP1/WAF1 expression was 11-fold higher in muscles from Mmp14−/− mice when compared with Mmp14+/+ animals (P < 0.01) (Fig 1G). Likewise, Mmp14 deficiency also caused a 6-fold (P < 0.01) and a 2-fold (P < 0.05) increase in p21CIP/WAF1 expression in kidney and liver, respectively (Supplementary Fig S3A and B), suggesting that cellular senescence is induced in different tissues from these mutant mice. Figure 1. Lack of MT1-MMP activates a cellular senescence signaling process and causes alterations in the somatotroph axis A. Cardiac defects and accumulation of collagen fibers in Mmp14-deficient mice. H&E and picrosirius red staining of hearts from 15-day-old mice. B. Sudan black staining counterstained with nuclear fast red to visualize senescence activity in hearts from Mmp14-deficient mice (40× magnification, and right panel shows a detailed view of the area indicated on the left panel). C. SA-β-Gal activity was assayed in kidney, adipose tissue (dashed square and H&E staining to confirm the adipose tissue), and fibroblasts from control and Mmp14−/− mice (n = 3) for each condition. D. Percentage of cells with positive staining for BrdU. E. Senescence heterochromatin foci were visualized by HP-1γ immunostaining. The number of foci per cell is represented. A representative image is shown. F. Western blot (left panel) and RT–qPCR analysis (right panel) of p16INK4a in control and Mmp14−/− fibroblasts. G. Gene expression analysis of p21CIP1/WAF1 by RT–qPCR in muscle from 15-day-old control and Mmp14−/− mice. H. Absence of MT1-MMP reduces cell proliferation. Control and Mmp14−/− cells (1 million cells), passage 1, were seeded onto plates and every 3 days cell populations were counted. The graphic shows the log2 of the number of million cells for each population. The experiment was done in triplicate using fibroblasts from three wild-type and three mutant mice. I. Plasma samples from 15-day-old Mmp14−/− and Mmp14+/+ mice were collected and pooled, and levels of IL-6 were measured by ELISA (n = 12). Each sample was measured in triplicate. J. Blood glucose concentrations in Mmp14+/+ (n = 12) and Mmp14−/− (n = 12) of 15-day-old mice. K, L. Plasma concentration of IGF-1 and GH was measured in 15-day-old Mmp14−/− mice (n = 4) and their wild-type littermates (n = 4). Concentrations were normalized to the mean control. Data information: Mean values are represented and error bars indicate SD (*P < 0.05, **P < 0.01; two-tailed Student's t-test). Download figure Download PowerPoint Further analysis of putative senescent features revealed that Mmp14−/− fibroblasts exhibited a significant proliferative decrease, when compared with wild-type fibroblasts (Fig 1H). Additionally, and consistent with the fact that MT1-MMP triggers anti-inflammatory responses (Shimizu-Hirota et al, 2012), we observed signs of a chronic inflammatory response in Mmp14−/− mice, characterized by a significant increase in plasma levels of interleukin-6 (Fig 1I) and the chemokine CXCL-1 (Supplementary Fig S3C). These results support the occurrence of a senescence-associated secretory phenotype in Mmp14−/− animals, as previously described in other mouse models of cellular senescence (Osorio et al, 2012; Rodier et al, 2009; Tchkonia et al, 2013). Furthermore, and in agreement with recent data showing that the TGF-β1 pathway mediates paracrine senescence and influences senescence in vivo (Acosta et al, 2013), we observed that Mmp14-deficient mice show a significant increase in TGF-β1 levels (Supplementary Fig S3D). Finally, we examined telomere length in cells from Mmp14-null mice, as telomere shortening is believed to promote cell senescence and it has been proposed to be a hallmark of biological aging (Lopez-Otin et al, 2013). By using a quantitative PCR method, we observed a significant reduction in telomeric TRF (terminal restriction fragment) in muscles from Mmp14-null mice (Supplementary Fig S3E). Collectively, these findings indicate that the loss of MT1-MMP induces a cell senescence process, which may contribute to explain the phenotypic alterations observed in Mmp14-null mice. Loss of MT1-MMP causes profound metabolic changes We next explored whether non-cell-autonomous alterations detected in mouse models with cellular senescence phenotypes could also be present in Mmp14-null mice. We found that Mmp14−/− mice are hypoglycemic, with blood glucose concentration three times lower in these mutant mice when compared to Mmp14+/+ littermates (71 versus 227 mg/dl, P < 0.01) (Fig 1J). Due to the short age of these mice, glucose levels were determined before weaning. Nevertheless, these alterations in blood glucose could not be attributed to differences in access to food, as animals from both genotypes had visible milk in their stomachs. Next, we observed that lack of MT1-MMP causes a profound alteration of the somatotroph axis, a major regulator of longevity from nematodes to man (Lopez-Otin et al, 2013; Niedernhofer et al, 2006; Russell & Kahn, 2007). Circulating levels of insulin-like growth factor 1 (IGF-1) were drastically reduced in plasma from Mmp14−/− animals in comparison with wild-type mice (73 versus 608 pg/ml, P < 0.01) (Fig 1K). As IGF-1 synthesis is mainly regulated by circulating growth hormone (GH), we also measured plasma GH concentration in these animals with the finding of very high levels of circulating GH in Mmp14−/− mice when compared to wild-type littermates (13 versus 2.3 pg/ml, P < 0.05) (Fig 1L). Furthermore, expression of miR-1, which targets Igf1 and is altered during premature aging (Mariño et al, 2010), was dramatically increased up to 159-fold in the liver of Mmp14−/− animals when compared with wild-type mice (P < 0.01) (Supplementary Fig S3F). Taken together, these results demonstrate that, in addition to cell intrinsic abnormalities, the loss of MT1-MMP causes profound systemic alterations which closely resemble those previously described in different models of cell senescence and premature aging. Mmp14 deficiency alters nuclear envelope structure and cytoskeleton organization The fact that mesenchymal tissues appeared to be more affected than other tissues by the lack of MT1-MMP likely reflects the sensitivity of these tissues to mechanical tensions created by their interactions with the ECM (Buxboim et al, 2010). Microscopic analysis of Mmp14−/− fibroblasts revealed profound aberrations in the nuclear envelope. Confocal microscopy with antibodies against the nuclear lamina component lamin A/C showed an abnormal morphology of the nucleus, including the presence of blebs and herniations of the nuclear lamina in Mmp14−/− fibroblasts, which were not observed in Mmp14+/+ cells (Fig 2A). Figure 2. Structural and morphological alterations of the nuclear envelope and cytoskeleton in Mmp14-deficient mice A. Immunofluorescence analysis of the nuclear envelope architecture in Mmp14−/− mice stained with an anti-lamin A/C antibody and counterstained with DAPI. The plot represents the percentage of nuclei with alterations, blebbing, or irregular shape in Mmp14+/+ and Mmp14−/− fibroblasts. B. Western blot analysis of proteins of the nuclear envelope from Mmp14+/+ and Mmp14−/− fibroblasts using specific antibodies against lamin A/C, nesprin-3, Sun-1, and Sun-2. β-actin was used as a loading control. C–E. Transcriptional analyses by RT–qPCR of nesprin-3, Sun1, and Sun2 were performed in skeletal muscle from control and Mmp14−/− mice. A GAPDH probe was used to normalize the expression level. F. Immunofluorescence staining of γH2AX foci in fibroblasts from Mmp14−/− and control mice (left panel). The percentage of nuclei that were positive for γH2AX is represented in the graphic (right panel). Three independent experiments were carried out for each genotype. G. Alteration of cytoskeleton structure around the nucleus in Mmp14−/− fibroblasts compared to wild-type cells. Confocal microscopy of cytoskeleton filaments, tubulin, F-actin (phalloidin), and vimentin stained in red; the nuclear lamin was stained with an anti-lamin A/C antibody (green) and counterstained with DAPI (blue). All the images in the z-stack were used to generate a maximum intensity projection (63× magnification). Data information: Mean values are represented and error bars indicate SD (*P < 0.05, **P < 0.01; two-tailed Student's t-test). Download figure Download PowerPoint Nuclear envelope abnormalities are a common feature of several progeroid syndromes caused by mutations which lead to an abnormal maturation of prelamin A (Agarwal et al, 2003; De Sandre-Giovannoli et al, 2003; Dechat et al, 2008; Eriksson et al, 2003; Gordon et al, 2014; Varela et al, 2008). Mmp14−/− fibroblasts showed marked differences in lamin A levels when compared with cells from wild-type mice, as revealed by Western blot analysis (Fig 2B). The connection between nucleus and cytoskeleton involves a series of LINC complex proteins, including nesprins and Sun proteins located in the nuclear membrane (Crisp et al, 2006). Nesprin-3 is an important member of this complex that links the nuclear envelope to intermediate filaments (Ketema & Sonnenberg, 2011). Analysis of nesprin-3 levels revealed a significant increase in this LINC component in Mmp14−/− cells when compared to wild-type cells, both at the protein and at the transcriptional level (Fig 2B and C). In addition, the expression of two other components of the LINC complex, Sun-1 and Sun-2, was also slightly altered in muscles from Mmp14-null animals (Fig 2B, D and E). These results suggest a deficient connectivity between nuclear envelope and cytoskeleton in mice lacking MT1-MMP, which likely contributes to the nuclear abnormalities observed in these animals. Previous studies have shown that the nuclear stress caused by alterations in the nuclear envelope is frequently accompanied by DNA damage, which triggers a DNA damage response (Saha et al, 2013). Accordingly, immunofluorescence staining for the histone γH2AX, an early marker of cell response to DNA damage, revealed that Mmp14−/− fibroblasts displayed an increased number of DNA damage foci when compared to control fibroblasts (Fig 2F). These nuclear structure abnormalities found in Mmp14−/− cells were also accompanied by marked alterations in the cytoskeletal organization around the nucleus (Fig 2G). Immunofluorescence analysis of cytoskeleton filaments showed that Mmp14+/+ cells presented a well-organized structure of the cytoskeleton around the nucleus. However, we observed a marked reduction in the number of actin fibers in the perinuclear and nuclear region of Mmp14−/− fibroblasts (Fig 2G). Furthermore, vimentin was also altered in Mmp14−/− cells, displaying an irregular distribution characterized by its marked accumulation at one side of the nucleus and its complete absence in other areas (Fig 2G). We also observed a diminished number of tubulin filaments in the nuclear region (Fig 2G). These alterations were similar to those reported in cells deficient in nesprin-3 (Postel et al, 2011). Then, and because the connection between ECM and cytoskeleton is mainly mediated by cell adhesion molecules, we evaluated the putative occurrence of changes in cell adhesion proteins in tissues from Mmp14-null mice. By using RT–qPCR, we found that the expression of integrin β6 was drastically reduced in Mmp14−/− muscles when compared to wild-type animals (0.059 versus 1, P < 0.01) (Supplementary Fig S3G). By contrast, other molecules implicated in cell adhesion, such as osteopontin, showed a marked increase in Mmp14−/− muscle (8.6 versus 1, P < 0.01) (Supplementary Fig S3H). These alterations were accompanied by changes in the activation of downstream effectors, such as the focal adhesion kinase (FAK). Western blot analysis of muscle tissue from Mmp14−/− mice and wild-type littermates showed that phosphorylation of Tyr397 in FAK was increased in Mmp14−/− muscles compared to Mmp14+/+ controls, while total levels of FAK were similar in both genotypes (Supplementary Fig S3I). Taken together, these results indicate that the loss of MT1-MMP causes marked alterations in both nuclear envelope structure and cytoskeletal organization. MT1-MMP proteolytic activity is required for maintaining cell integrity To further address the role of MT1-MMP in regulating the cytoskeletal and nuclear structure, we transduced Mmp14-deficient fibroblasts, seeded onto collagen plates, with expression vectors for either wild-type MT1-MMP or a catalytically inactive mutant (E
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