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1.
Cell Rep ; 43(6): 114284, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38814785

RESUMO

Nuclear envelope (NE) ruptures are emerging observations in Lamin-related dilated cardiomyopathy, an adult-onset disease caused by loss-of-function mutations in Lamin A/C, a nuclear lamina component. Here, we test a prevailing hypothesis that NE ruptures trigger the pathological cGAS-STING cytosolic DNA-sensing pathway using a mouse model of Lamin cardiomyopathy. The reduction of Lamin A/C in cardio-myocyte of adult mice causes pervasive NE ruptures in cardiomyocytes, preceding inflammatory transcription, fibrosis, and fatal dilated cardiomyopathy. NE ruptures are followed by DNA damage accumulation without causing immediate cardiomyocyte death. However, cGAS-STING-dependent inflammatory signaling remains inactive. Deleting cGas or Sting does not rescue cardiomyopathy in the mouse model. The lack of cGAS-STING activation is likely due to the near absence of cGAS expression in adult cardiomyocytes at baseline. Instead, extracellular matrix (ECM) signaling is activated and predicted to initiate pro-inflammatory communication from Lamin-reduced cardiomyocytes to fibroblasts. Our work nominates ECM signaling, not cGAS-STING, as a potential inflammatory contributor in Lamin cardiomyopathy.


Assuntos
Matriz Extracelular , Proteínas de Membrana , Miócitos Cardíacos , Membrana Nuclear , Nucleotidiltransferases , Transdução de Sinais , Animais , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Membrana Nuclear/metabolismo , Matriz Extracelular/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/genética , Dano ao DNA
2.
FEBS J ; 291(10): 2155-2171, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38462947

RESUMO

Mammalian somatic cells undergo terminal proliferation arrest after a limited number of cell divisions, a phenomenon termed cellular senescence. However, cells acquire the ability to proliferate infinitely (cellular immortalization) through multiple genetic alterations. Inactivation of tumor suppressor genes such as p53, RB and p16 is important for cellular immortalization, although additional molecular alterations are required for cellular immortalization to occur. Here, we aimed to gain insights into these molecular alterations. Given that cellular immortalization is the escape of cells from cellular senescence, genes that regulate cellular senescence are likely to be involved in cellular immortalization. Because senescent cells show altered heterochromatin organization, we investigated the implications of lamin A/C, lamin B1 and lamin B receptor (LBR), which regulate heterochromatin organization, in cellular immortalization. We employed human immortalized cell lines, KMST-6 and SUSM-1, and found that expression of LBR was upregulated upon cellular immortalization and downregulated upon cellular senescence. In addition, knockdown of LBR induced cellular senescence with altered chromatin configuration. Additionally, enforced expression of LBR increased cell proliferation likely through suppression of genome instability in human primary fibroblasts that expressed the simian virus 40 large T antigen (TAg), which inactivates p53 and RB. Furthermore, expression of TAg or knockdown of p53 led to upregulated LBR expression. These observations suggested that expression of LBR might be upregulated to suppress genome instability in TAg-expressing cells, and, consequently, its upregulated expression assisted the proliferation of TAg-expressing cells (i.e. p53/RB-defective cells). Our findings suggest a crucial role for LBR in the process of cellular immortalization.


Assuntos
Proliferação de Células , Senescência Celular , Instabilidade Genômica , Receptor de Lamina B , Lamina Tipo B , Receptores Citoplasmáticos e Nucleares , Humanos , Proliferação de Células/genética , Senescência Celular/genética , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Regulação para Cima , Heterocromatina/genética , Heterocromatina/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fibroblastos/metabolismo , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo
3.
bioRxiv ; 2023 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-37693381

RESUMO

Mutations in the nuclear Lamin A/C gene (LMNA) cause diverse degenerative disorders, including malignant dilated cardiomyopathy in adults. A prevailing hypothesis postulates that LMNA mutations cause nuclear envelope ruptures that trigger pathogenic inflammatory signaling via the cGAS-STING cytosolic DNA-sensing pathway. Here, we provide evidence against this hypothesis, using a mouse model of LMNA-related cardiomyopathy that mimics Lamin A/C protein reduction observed in patient cardiomyocytes. We observed that pervasive nuclear envelope ruptures preceded the onset of cardiac transcriptional modulation and dilated cardiomyopathy. Nuclear ruptures activated DNA damage response without causing immediate cardiomyocyte death. However, cGAS-STING downstream cytokine genes remained inactive in the mutant cardiomyocytes. Deleting cGas or Sting did not alleviate cardiomyopathy. Instead, extracellular matrix signaling was predicted to emanate from Lamin A/C-reduced cardiomyocytes to communicate with fibroblasts in the heart. These findings suggest that cGAS-STING is not a major pathogenetic contributor to LMNA-related dilated cardiomyopathy in adult humans.

4.
FEBS J ; 290(3): 692-711, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-35882390

RESUMO

5-Bromodeoxyuridine (BrdU), a thymidine analogue, is an interesting reagent that modulates various biological phenomena. BrdU, upon incorporation into DNA, causes destabilized nucleosome positioning which leads to changes in heterochromatin organization and gene expression in cells. We have previously shown that BrdU effectively induces cellular senescence, a phenomenon of irreversible growth arrest in mammalian cells. Identification of the mechanism of action of BrdU would provide a novel insight into the molecular mechanisms of cellular senescence. Here, we showed that a basic domain in the histone H2B N-terminal tail, termed the HBR (histone H2B repression) domain, is involved in the action of BrdU. Notably, deletion of the HBR domain causes destabilized nucleosome positioning and derepression of gene expression, as does BrdU. We also showed that the genes up-regulated by BrdU significantly overlapped with those by deletion of the HBR domain, the result of which suggested that BrdU and deletion of the HBR domain act in a similar way. Furthermore, we showed that decreased HBR domain function induced cellular senescence or facilitated the induction of cellular senescence. These findings indicated that the HBR domain is crucially involved in the action of BrdU, and also suggested that disordered nucleosome organization may be involved in the induction of cellular senescence.


Assuntos
Histonas , Nucleossomos , Animais , Histonas/genética , Histonas/metabolismo , Bromodesoxiuridina/farmacologia , DNA/metabolismo , Senescência Celular/genética , Mamíferos/metabolismo
5.
Biochem Biophys Res Commun ; 634: 175-181, 2022 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-36244116

RESUMO

Oxygen is essential for aerobic organisms, but generates reactive oxygen species (ROS), which can cause cellular dysfunction by damaging cellular molecules. Many genes are involved in the regulation of ROS; however, much attention has not focused on them. To identify these genes, we screened for mutants with an altered sensitivity to oxidative stress in the nematode Caenorhabditis elegans. We isolated a novel mutant, oxy-7(qa5004) which showed an increased sensitivity to ROS in C. elegans. oxy-7 showed increased production of ROS and decreased longevity due to its increased oxidative stress. Genetic analysis revealed that oxy-7 has a causative mutation in Y71H2AM.9, a homologue of SLC30A9 which encodes a zinc transporter in mitochondria. We further showed that knockdown of human SLC30A9 caused increased ROS production in human cells as well. These results suggested an important role of mitochondrial zinc homeostasis in the regulation of ROS.


Assuntos
Proteínas de Caenorhabditis elegans , Proteínas de Transporte de Cátions , Nematoides , Animais , Humanos , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Oxidativo/genética , Longevidade/genética , Nematoides/metabolismo , Mutação , Fatores de Transcrição/genética , Proteínas de Ciclo Celular/genética , Proteínas de Transporte de Cátions/genética
6.
Exp Cell Res ; 390(1): 111927, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32126237

RESUMO

Cellular senescence is a phenomenon of irreversible growth arrest of mammalian somatic cells. Senescent cells increase the production of secretory proteins such as inflammatory cytokines, a phenomenon termed senescence-associated secretory phenotype (SASP). SASP is known to have profound effects on organismal health and aging; however, the molecular mechanisms of SASP are not precisely understood. In our previous studies, we have shown that senescent cells show decreased function of lamin B receptor (LBR), a nuclear membrane protein that regulates heterochromatin organization. Here we examined the implication of LBR in the regulation of SASP because senescent cells show altered heterochromatin organization, which would affect gene expression. We found that knock-down of LBR up-regulated the expression of the SASP factors such as IL-6, IL-8, and MMP1 in HeLa cells, even though cellular senescence was not induced by LBR knock-down. Conversely, enforced expression of LBR suppressed their up-regulated expression in senescent cells induced by excess thymidine. Further, our gene expression profile analysis also showed that many secretory proteins were up-regulated by LBR knock-down. We then analyzed the regulatory mechanisms of the expression of SASP factors by LBR, and found that the promoters of these SASP factors associated with LBR in normally growing cells, but dissociated from it in senescent cells. Additionally, we found that enforced expression of LBR decreased the generation of cytoplasmic DNA, which could be involved in SASP, in senescent cells. These findings suggested that LBR would play crucial roles in the regulation of SASP.


Assuntos
Senescência Celular , Receptores Citoplasmáticos e Nucleares/genética , Via Secretória , Células Cultivadas , Células HeLa , Heterocromatina/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Fenótipo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptor de Lamina B
7.
FEBS Open Bio ; 10(2): 237-250, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31825172

RESUMO

Cellular senescence is a terminal growth arrest phenomenon in mammalian cells. Coordinated regulation of protein synthesis and degradation is required to maintain protein homeostasis in cells; however, senescent cells exhibit decreased activity of the proteasome, a major cellular proteolytic machinery, with an accumulation of proteins. Indeed, we showed that MG132, a proteasome inhibitor, induced cellular senescence through an accumulation of proteins in human cells. We then investigated the mechanisms of cellular senescence induced by protein accumulation by treating cells with MG132. We found that lamin B receptor (LBR), a nuclear membrane protein that regulates heterochromatin organization, was mislocalized and down-regulated in cells on treatment with MG132. Importantly, enforced expression of LBR suppressed cellular senescence induced by MG132. We also showed that LBR was involved in the regulation of chromatin organization in senescent cells, and that endoplasmic reticulum stress and autophagy were likely to be involved in the mislocalization and down-regulation of LBR. These findings indicate that decreased LBR function was responsible for the induction of cellular senescence by MG132, and thus suggest that protein accumulation caused by inhibition of the proteasome induced cellular senescence probably through chromatin dysregulation in human cells.


Assuntos
Senescência Celular/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Linhagem Celular Tumoral , Senescência Celular/fisiologia , Cromatina , Citoplasma , Células HeLa , Humanos , Lamina Tipo B/metabolismo , Leupeptinas/farmacologia , Proteínas de Membrana , Complexo de Endopeptidases do Proteassoma/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptor de Lamina B
8.
Mech Ageing Dev ; 178: 25-32, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30615890

RESUMO

Cellular senescence is a phenomenon of irreversible growth arrest in mammalian somatic cells in culture. Various stresses induce cellular senescence and indeed, we have found that excess thymidine effectively induces cellular senescence in human cells. Further, many reports indicate the implication of chromatin proteins in cellular senescence. Here we analysed the role of lamin B receptor (LBR), a nuclear envelope protein that regulates heterochromatin organization, in cellular senescence induced by excess thymidine. We then found that the LBR protein was down-regulated and showed aberrant localization in cells upon induction of cellular senescence by excess thymidine. Additionally, we also found that knock-down of LBR facilitated the induction of cellular senescence by excess thymidine in cancerous HeLa cells, and importantly, it induced cellular senescence in normal human diploid fibroblast TIG-7 cells. These results suggested that decreased LBR function is involved in the induction of cellular senescence in human cells.


Assuntos
Senescência Celular/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Bromodesoxiuridina/farmacologia , Senescência Celular/genética , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HeLa , Heterocromatina/metabolismo , Humanos , Lamina Tipo B/metabolismo , Membrana Nuclear/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Timidina/farmacologia , Receptor de Lamina B
9.
Biochem Cell Biol ; 95(4): 517-523, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28380310

RESUMO

Enforced expression of GNG11, G-protein subunit γ 11, induces cellular senescence in normal human diploid fibroblasts. We here examined the effect of the expression of GNG11 on the growth of immortalized human cell lines, and found that it suppressed the growth of SUSM-1 cells, but not of HeLa cells. We then compared these two cell lines to understand the molecular basis for the action of GNG11. We found that expression of GNG11 induced the generation of reactive oxygen species (ROS) and abnormal nuclear morphology in SUSM-1 cells but not in HeLa cells. Increased ROS generation by GNG11 would likely be caused by the down-regulation of the antioxidant enzymes in SUSM-1 cells. We also found that SUSM-1 cells, even under normal culture conditions, showed higher levels of ROS and higher incidence of abnormal nuclear morphology than HeLa cells, and that abnormal nuclear morphology was relevant to the increased ROS generation in SUSM-1 cells. Thus, SUSM-1 and HeLa cells showed differences in the regulation of ROS and nuclear morphology, which might account for their different responses to the expression of GNG11. Thus, SUSM-1 cells may provide a unique system to study the regulatory relationship between ROS generation, nuclear morphology, and G-protein signaling.


Assuntos
Núcleo Celular/metabolismo , Núcleo Celular/patologia , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proliferação de Células , Células Cultivadas , Células HeLa , Humanos
10.
Exp Cell Res ; 346(2): 216-23, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27443255

RESUMO

DNA damage response is crucially involved in cellular senescence. We have previously shown that excess thymidine, which stalls DNA replication forks, induces cellular senescence in human cells, and ERK1/2 play a key role in the induction of it. In this study, we found that Chk1 and ERK1/2 were activated to promote cell survival upon addition of excess thymidine. Knockdown of ERK1/2 activated Chk1, and conversely, knockdown of Chk1 activated ERK1/2, which observations suggested a mechanism for compensatory activation of Chk1 and ERK1/2 in the absence of ERK1/2 and Chk1, respectively. We also found that Chk1 functioned mainly at the onset of cellular senescence, and on the other hand, ERK1/2 functioned for a more extended period to induce cellular senescence. Our findings suggested that Chk1 and ERK1/2 were activated to promote cell survival upon addition of excess thymidine, but prolonged activation of ERK1/2 led to cellular senescence. This implies a pleiotropic effect of ERK1/2 in cellular senescence induced by excess thymidine.


Assuntos
Senescência Celular/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Timidina/farmacologia , Western Blotting , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo
11.
Exp Cell Res ; 345(2): 239-46, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27339653

RESUMO

Mammalian cells, when treated with sub-lethal doses of genotoxic stresses, slow down DNA synthesis but continue protein synthesis. Thus, these cells show an accumulation of proteins and undergo unbalanced growth. In the previous studies, we have shown that HeLa cells treated with excess thymidine or camptothecin undergo unbalanced growth, and prolonged unbalanced growth causes induction of cellular senescence, which is suppressed by restriction of protein synthesis or inhibition of ERK-signaling. In this study, we found that restriction of protein synthesis, inhibition of ERK-signaling, and elimination of reactive oxygen species showed a combinatorial effect on suppression of cellular senescence induced by excess thymidine or camptothecin. Of these, restriction of protein synthesis most effectively suppressed cellular senescence. Importantly, a similar combinatorial effect was observed in replicative senescence in normal human diploid fibroblasts. Our findings suggested that various stresses were cumulatively involved in cellular senescence, and suppression of cellular senescence was improved by combining the treatments that reduce the stresses.


Assuntos
Senescência Celular , Sistema de Sinalização das MAP Quinases , Biossíntese de Proteínas , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/farmacologia , Butadienos/farmacologia , Camptotecina/farmacologia , Tamanho Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Dano ao DNA , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nitrilas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Timidina/farmacologia
12.
Biochem Biophys Res Commun ; 473(4): 1078-1083, 2016 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-27059139

RESUMO

5-Bromodeoxyuridine (BrdU), a thymidine analogue, induces cellular senescence in mammalian cells. BrdU induces cellular senescence probably through the regulation of chromatin because BrdU destabilizes or disrupts nucleosome positioning and decondenses heterochromatin. Since heterochromatin is tethered to the nuclear periphery through the interaction with the nuclear envelope proteins, we examined the localization of the several nuclear envelope proteins such as lamins, lamin-interacting proteins, nuclear pore complex proteins, and nuclear transport proteins in senescent cells. We have shown here that lamin B receptor (LBR) showed a change in localization in both BrdU-induced and replicative senescent cells.


Assuntos
Senescência Celular/fisiologia , Laminas/metabolismo , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Receptores Citoplasmáticos e Nucleares/metabolismo , Bromodesoxiuridina/farmacologia , Células HeLa , Humanos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Distribuição Tecidual/efeitos dos fármacos , Receptor de Lamina B
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