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1.
J Biol Chem ; 300(4): 107145, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38460941

RESUMO

Extracellular ATP activates P2 purinergic receptors. Whether purinergic signaling is functionally coupled to cellular senescence is largely unknown. We find that oxidative stress induced release of ATP and caused senescence in human lung fibroblasts. Inhibition of P2 receptors limited oxidative stress-induced senescence, while stimulation with exogenous ATP promoted premature senescence. Pharmacological inhibition of P2Y11 receptor (P2Y11R) inhibited premature senescence induced by either oxidative stress or ATP, while stimulation with a P2Y11R agonist was sufficient to induce cellular senescence. Our data show that both extracellular ATP and a P2Y11R agonist induced calcium (Ca++) release from the endoplasmic reticulum (ER) and that either inhibition of phospholipase C or intracellular Ca++ chelation impaired ATP-induced senescence. We also find that Ca++ that was released from the ER, following ATP-mediated activation of phospholipase C, entered mitochondria in a manner dependent on P2Y11R activation. Once in mitochondria, excessive Ca++ promoted the production of reactive oxygen species in a P2Y11R-dependent fashion, which drove development of premature senescence of lung fibroblasts. Finally, we show that conditioned medium derived from senescent lung fibroblasts, which were induced to senesce through the activation of ATP/P2Y11R-mediated signaling, promoted the proliferation of triple-negative breast cancer cells and their tumorigenic potential by secreting amphiregulin. Our study identifies the existence of a novel purinergic signaling pathway that links extracellular ATP to the development of a protumorigenic premature senescent phenotype in lung fibroblasts that is dependent on P2Y11R activation and ER-to-mitochondria calcium signaling.


Assuntos
Trifosfato de Adenosina , Cálcio , Senescência Celular , Fibroblastos , Receptores Purinérgicos P2 , Humanos , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Pulmão/metabolismo , Pulmão/citologia , Mitocôndrias/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Linhagem Celular , Proliferação de Células
2.
J Biol Chem ; 298(10): 102405, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35988650

RESUMO

Cellular senescence is a form of irreversible growth arrest that cancer cells evade. The cell division cycle protein 20 homolog (Cdc20) is a positive regulator of cell division, but how its dysregulation may relate to senescence is unclear. Here, we find that Cdc20 mRNA and protein expression are downregulated in stress-induced premature senescent lung fibroblasts in a p53-dependent manner. Either Cdc20 downregulation or inhibition of anaphase-promoting complex/cyclosome (APC/C) is sufficient to induce premature senescence in lung fibroblasts, while APC/C activation inhibits stress-induced premature senescence. Mechanistically, we show both Cdc20 downregulation and APC/C inhibition induce premature senescence through glycogen synthase kinase (GSK)-3ß-mediated phosphorylation and downregulation of securin expression. Interestingly, we determined Cdc20 expression is upregulated in human lung adenocarcinoma. We find that downregulation of Cdc20 in non-small cell lung cancer (NSCLC) cells is sufficient to inhibit cell proliferation and growth in soft agar and to promote apoptosis, but not senescence, in a manner dependent on downregulation of securin following GSK-3ß-mediated securin phosphorylation. Similarly, we demonstrate securin expression is downregulated and cell viability is inhibited in NSCLC cells following inhibition of APC/C. Furthermore, we show chemotherapeutic drugs downregulate both Cdc20 and securin protein expression in NSCLC cells. Either Cdc20 downregulation by siRNA or APC/C inhibition sensitize, while securin overexpression inhibits, chemotherapeutic drug-induced NSCLC cell death. Together, our findings provide evidence that Cdc20/APC/C/securin-dependent signaling is a key regulator of cell survival, and its disruption promotes premature senescence in normal lung cells and induces apoptosis in lung cancer cells that have bypassed the senescence barrier.


Assuntos
Apoptose , Carcinoma Pulmonar de Células não Pequenas , Senescência Celular , Neoplasias Pulmonares , Humanos , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Securina/genética , Securina/metabolismo
3.
J Biol Chem ; 296: 100242, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33380422

RESUMO

Oncogenic K-Ras (K-RasG12V) promotes senescence in normal cells but fuels transformation of cancer cells after the senescence barrier is bypassed. The mechanisms regulating this pleiotropic function of K-Ras remain to be fully established and bear high pathological significance. We find that K-RasG12V activates the angiotensinogen (AGT) gene promoter and promotes AGT protein expression in a Kruppel-like factor 6-dependent manner in normal cells. We show that AGT is then converted to angiotensin II (Ang II) in a cell-autonomous manner by cellular proteases. We show that blockade of the Ang II receptor type 1 (AT1-R) in normal cells inhibits oncogene-induced senescence. We provide evidence that the oncogenic K-Ras-induced synthesis of Ang II and AT1-R activation promote senescence through caveolin-1-dependent and nicotinamide adenine dinucleotide phosphate oxidase 2-mediated oxidative stress. Interestingly, we find that expression of AGT remains elevated in lung cancer cells but in a Kruppel-like factor 6-independent and high-mobility group AT-hook 1-dependent manner. We show that Ang II-mediated activation of the AT1-R promotes cell proliferation and anchorage-independent growth of lung cancer cells through a STAT3-dependent pathway. Finally, we find that expression of AGT is elevated in lung tumors of K-RasLA2-G12D mice, a mouse model of lung cancer, and human lung cancer. Treatment with the AT1-R antagonist losartan inhibits lung tumor formation in K-RasLA2-G12D mice. Together, our data provide evidence of the existence of a novel cell-autonomous and pleiotropic Ang II-dependent signaling pathway through which oncogenic K-Ras promotes oncogene-induced senescence in normal cells while fueling transformation in cancer cells.


Assuntos
Angiotensinogênio/genética , Fator 6 Semelhante a Kruppel/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptor Tipo 1 de Angiotensina/genética , Angiotensina II/genética , Angiotensina II/metabolismo , Angiotensinogênio/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/genética , Hipertensão/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/inervação , Losartan/farmacologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Estresse Oxidativo/genética , Sistema Renina-Angiotensina/genética , Fator de Transcrição STAT3/genética
4.
Cancer Metastasis Rev ; 39(2): 397-414, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32279119

RESUMO

Cellular senescence is a feature of most somatic cells. It is characterized by an irreversible cell cycle arrest and by the ability to secrete a plethora of mediators of inflammation and growth factors, which can alter the senescent cell's microenvironment. Senescent cells accumulate in tissues over time and contribute to both aging and the development of age-associated diseases. Senescent cells have antagonistic pleiotropic roles in cancer. Given the inability of senescent cells to proliferate, cellular senescence is a powerful tumor suppressor mechanism in young individuals. However, accumulation of senescent stromal cells during aging can fuel cancer cell growth in virtue of their capacity to release factors that stimulate cell proliferation. Caveolin-1 is a structural protein component of caveolae, invaginations of the plasma membrane involved in a variety of cellular processes, including signal transduction. Mounting evidence over the last 10-15 years has demonstrated a central role of caveolin-1 in the development of a senescent phenotype and the regulation of both the anti-tumorigenic and pro-tumorigenic properties of cellular senescence. In this review, we discuss the cellular mechanisms and functions of caveolin-1 in the context of cellular senescence and their relevance to the biology of cancer.


Assuntos
Caveolina 1/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Senescência Celular/fisiologia , Humanos , Transdução de Sinais
5.
FASEB J ; 33(4): 4866-4882, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30596512

RESUMO

Aurora kinase A (AURKA) is necessary for proper primary cilium disassembly before mitosis. We found that depletion of caveolin-1 expression promotes primary cilia formation through the proteasomal-dependent degradation of aurora kinase A and induces premature senescence in human fibroblasts. Down-regulation of intraflagellar transport-88, a protein essential for ciliogenesis, inhibits premature senescence induced by the depletion of caveolin-1. In support of these findings, we showed that alisertib, a pharmacological inhibitor of AURKA, causes primary cilia formation and cellular senescence by irreversibly arresting cell growth. Suppression of primary cilia formation limits cellular senescence induced by alisertib. The primary cilium must be disassembled to free its centriole to form the centrosome, a necessary structure for mitotic spindle assembly and cell division. We showed that the use of the centriole to form primary cilia blocks centrosome formation and mitotic spindle assembly and prevents the completion of mitosis in cells in which cellular senescence is caused by the inhibition of AURKA. We also found that AURKA is down-regulated and primary cilia formation is enhanced when cellular senescence is promoted by other senescence-inducing stimuli, such as oxidative stress and UV light. Thus, we propose that impaired AURKA function induces premature senescence by preventing reabsorption of the primary cilium, which inhibits centrosome and mitotic spindle formation and consequently prevents the completion of mitosis. Our study causally links the inability of the cell to disassemble the primary cilium, a microtubule-based cellular organelle, to the development of premature senescence, a functionally and pathologically relevant cellular state.-Jeffries, E. P., Di Filippo, M., Galbiati, F. Failure to reabsorb the primary cilium induces cellular senescence.


Assuntos
Caveolina 1/metabolismo , Senescência Celular/fisiologia , Cílios/metabolismo , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Azepinas/farmacologia , Western Blotting , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Células HeLa , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirimidinas/farmacologia
6.
J Biol Chem ; 293(5): 1794-1809, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29247004

RESUMO

Oncogene-induced senescence (OIS) is considered a powerful tumor suppressor mechanism. Caveolin-1 acts as a scaffolding protein to functionally regulate signaling molecules. We demonstrate that a lack of caveolin-1 expression inhibits oncogenic K-Ras (K-RasG12V)-induced premature senescence in mouse embryonic fibroblasts and normal human bronchial epithelial cells. Oncogenic K-Ras induces senescence by limiting the detoxification function of MTH1. We found that K-RasG12V promotes the interaction of caveolin-1 with MTH1, which results in inhibition of MTH1 activity. Lung cancer cells expressing oncogenic K-Ras have bypassed the senescence barrier. Interestingly, overexpression of caveolin-1 restores cellular senescence in both A549 and H460 lung cancer cells and inhibits their transformed phenotype. In support of these findings, our in vivo data demonstrate that overexpression of oncogenic K-Ras (K-RasG12D) induces cellular senescence in the lung of wildtype but not caveolin-1-null mice. A lack of K-RasG12D-induced premature senescence in caveolin-1-null mice results in the formation of more abundant lung tumors. Consistent with these data, caveolin-1-null mice overexpressing K-RasG12D display accelerated mortality. Finally, our animal data were supported by human sample analysis in which we show that caveolin-1 expression is dramatically down-regulated in lung adenocarcinomas from lung cancer patients, both at the mRNA and protein levels, and that low caveolin-1 expression is associated with poor survival. Together, our data suggest that lung cancer cells escape oncogene-induced premature senescence through down-regulation of caveolin-1 expression to progress from premalignant lesions to cancer.


Assuntos
Adenocarcinoma/metabolismo , Caveolina 1/biossíntese , Senescência Celular , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células A549 , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Substituição de Aminoácidos , Animais , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos Knockout , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética
7.
J Biol Chem ; 290(7): 4202-14, 2015 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-25512378

RESUMO

Oxidative stress can induce premature cellular senescence. Senescent cells secrete various growth factors and cytokines, such as IL-6, that can signal to the tumor microenvironment and promote cancer cell growth. Sirtuin 1 (Sirt1) is a class III histone deacetylase that regulates a variety of physiological processes, including senescence. We found that caveolin-1, a structural protein component of caveolar membranes, is a direct binding partner of Sirt1, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82-101) to the caveolin-binding domain of Sirt1 (amino acids 310-317). Our data show that oxidative stress promotes the sequestration of Sirt1 into caveolar membranes and the interaction of Sirt1 with caveolin-1, which lead to inhibition of Sirt1 activity. Reactive oxygen species stimulation promotes acetylation of p53 and premature senescence in wild-type but not caveolin-1 null mouse embryonic fibroblasts (MEFs). Either down-regulation of Sirt1 expression or re-expression of caveolin-1 in caveolin-1 null MEFs restores reactive oxygen species-induced acetylation of p53 and premature senescence. In addition, overexpression of caveolin-1 induces stress induced premature senescence in p53 wild-type but not p53 knockout MEFs. Phosphorylation of caveolin-1 on tyrosine 14 promotes the sequestration of Sirt1 into caveolar membranes and activates p53/senescence signaling. We also identified IL-6 as a caveolin-1-specific cytokine that is secreted by senescent fibroblasts following the caveolin-1-mediated inhibition of Sirt1. The caveolin-1-mediated secretion of IL-6 by senescent fibroblasts stimulates the growth of cancer cells. Therefore, by inhibiting Sirt1, caveolin-1 links free radicals to the activation of the p53/senescence pathway and the protumorigenic properties of IL-6.


Assuntos
Caveolina 1/metabolismo , Senescência Celular , Interleucina-6/metabolismo , Neoplasias/metabolismo , Estresse Oxidativo , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Animais , Western Blotting , Cavéolas , Caveolina 1/genética , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Imunoprecipitação , Camundongos , Camundongos Knockout , Células NIH 3T3 , Neoplasias/genética , Neoplasias/patologia , Fosforilação , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética
8.
Proc Natl Acad Sci U S A ; 108(40): 16657-62, 2011 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-21930911

RESUMO

Glucocorticoids (GCs) are used to treat pregnant women at risk for preterm delivery; however, prenatal exposure to GCs may trigger adverse neurological side effects due to reduced neural progenitor cell (NPC) proliferation. Whereas many established cell-cycle regulators impact NPC proliferation, other signaling molecules, such as the gap junction protein connexin-43 (Cx43), also influence proliferation. Gap junction intercellular communication (GJIC) is influenced by GCs in some cells, but such hormone effects have not been examined in coupled stem cells. We found that both continuous and transient exposure of embryonic day 14.5 mouse neurosphere cultures to dexamethasone (DEX) limits proliferation of coupled NPCs, which is manifested by both a reduction in S-phase progression and enhanced cell-cycle exit. A short (i.e., 1-h) DEX treatment also reduced GJIC as measured by live-cell fluorescence recovery after photobleaching, and altered the synchrony of spontaneous calcium transients in coupled NPCs. GC effects on GJIC in NPCs are transcription-independent and mediated through plasma membrane glucocorticoid receptors (GRs). This nongenomic pathway operates through lipid raft-associated GRs via a site-specific, MAPK-dependent phosphorylation of Cx43, which is linked to GR via caveolin-1 (Cav-1) and c-src. Cav-1 is essential for this nongenomic action of GR, as DEX effects on GJIC, Cx43 phosphorylation, and MAPK activation are not observed in Cav-1 knockout NPCs. As transient pharmacologic inhibition of GJIC triggers reduced S-phase progression but not enhanced cell-cycle exit, the nongenomic GR signaling pathway may operate via distinct downstream effectors to alter the proliferative capacity of NPCs.


Assuntos
Comunicação Celular/fisiologia , Junções Comunicantes/fisiologia , Glucocorticoides/farmacologia , Células-Tronco Neurais/fisiologia , Receptores de Glucocorticoides/metabolismo , Animais , Western Blotting , Caveolina 1/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Conexina 43/metabolismo , Dexametasona/farmacologia , Recuperação de Fluorescência Após Fotodegradação , Camundongos , Fosforilação
9.
J Biol Chem ; 286(33): 28657-28661, 2011 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-21705337

RESUMO

According to the "free radical theory" of aging, premature senescence induced by oxidative stress contributes to organismal aging. Polymerase I and transcript release factor (PTRF)/cavin-1 is a structural protein component of caveolae, invaginations of the plasma membrane involved in signal transduction. We show that oxidative stress up-regulates PTRF/cavin-1 protein expression and promotes the interaction between PTRF/cavin-1 and caveolin-1, another structural protein component of caveolae. Consistent with these data, the number of caveolae is dramatically increased in cells subjected to oxidative stress. We demonstrate that down-regulation of PTRF/cavin-1 by shRNA significantly inhibits oxidative stress-induced premature senescence. Mechanistically, we found that PTRF/cavin-1 expression is necessary for the oxidant-induced sequestration of Mdm2, a negative regulator of p53, into caveolar membranes, away from p53, and activation of the p53/p21(Waf1/Cip1) pathway. Expression of a mutant form of PTRF/cavin-1, which fails to localize to caveolar membranes after oxidative stress, inhibits oxidative stress-induced activation of p53 and induction of premature senescence. Thus, PTRF/cavin-1 is a novel regulator of oxidative stress-induced premature senescence by acting as a link between free radicals and activation of the p53/p21(Waf1/Cip1) pathway.


Assuntos
Cavéolas/metabolismo , Senescência Celular/fisiologia , Estresse Oxidativo/fisiologia , Proteínas de Ligação a RNA/biossíntese , Regulação para Cima/fisiologia , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Mutação , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas de Ligação a RNA/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
10.
EMBO Rep ; 10(12): 1334-40, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19820694

RESUMO

Thioredoxin reductase 1 (TrxR1) is an important antioxidant enzyme that controls cellular redox homeostasis. By using a proteomic-based approach, here we identify TrxR1 as a caveolar membrane-resident protein. We show that caveolin 1, the structural protein component of caveolae, is a TrxR1-binding protein by demonstrating that the scaffolding domain of caveolin 1 (amino acids 82-101) binds directly to the caveolin-binding motif (CBM) of TrxR1 (amino acids 454-463). We also show that overexpression of caveolin 1 inhibits TrxR activity, whereas a lack of caveolin 1 activates TrxR, both in vitro and in vivo. Expression of a peptide corresponding to the caveolin 1 scaffolding domain is sufficient to inhibit TrxR activity. A TrxR1 mutant lacking the CBM, which fails to localize to caveolae and bind to caveolin 1, is constitutively active and inhibits oxidative-stress-mediated activation of the p53/p21(Waf1/Cip1) pathway and induction of premature senescence. Finally, we show that caveolin 1 expression inhibits TrxR1-mediated cell transformation. Thus, caveolin 1 links free radicals to activation of the p53/p21(Waf1/Cip1) pathway and induction of cellular senescence by acting as an endogenous inhibitor of TrxR1.


Assuntos
Caveolina 1/fisiologia , Senescência Celular , Estresse Oxidativo/fisiologia , Tiorredoxina Redutase 1/antagonistas & inibidores , Animais , Caveolina 1/antagonistas & inibidores , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Nus , Modelos Biológicos , Células NIH 3T3 , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Tiorredoxina Redutase 1/genética , Tiorredoxina Redutase 1/metabolismo , Fatores de Tempo , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cancer Res ; 66(22): 10805-14, 2006 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-17108117

RESUMO

Cellular senescence is believed to represent a natural tumor suppressor mechanism. We have previously shown that up-regulation of caveolin-1 was required for oxidative stress-induced premature senescence in fibroblasts. However, the molecular mechanisms underlying caveolin-1 up-regulation in senescent cells remain unknown. Here, we show that subcytotoxic oxidative stress generated by hydrogen peroxide application promotes premature senescence and stimulates the activity of a (-1,296) caveolin-1 promoter reporter gene construct in fibroblasts. Functional deletion analysis mapped the oxidative stress response elements of the mouse caveolin-1 promoter to the sequences -244/-222 and -124/-101. The hydrogen peroxide-mediated activation of both Cav-1 (-244/-222) and Cav-1 (-124/-101) was prevented by the antioxidant quercetin. Combination of electrophoretic mobility shift studies, chromatin immunoprecipitation analysis, Sp1 overexpression experiments, as well as promoter mutagenesis identifies enhanced Sp1 binding to two GC-boxes at -238/-231 and -118/-106 as the core mechanism of oxidative stress-triggered caveolin-1 transactivation. In addition, signaling studies show p38 mitogen-activated protein kinase (MAPK) as the upstream regulator of Sp1-mediated activation of the caveolin-1 promoter following oxidative stress. Inhibition of p38 MAPK prevents the oxidant-induced Sp1-mediated up-regulation of caveolin-1 protein expression and development of premature senescence. Finally, we show that oxidative stress induces p38-mediated up-regulation of caveolin-1 and premature senescence in normal human mammary epithelial cells but not in MCF-7 breast cancer cells, which do not express caveolin-1 and undergo apoptosis. This study delineates for the first time the molecular mechanisms that modulate caveolin-1 gene transcription upon oxidative stress and brings new insights into the redox control of cellular senescence in both normal and cancer cells.


Assuntos
Caveolina 1/biossíntese , Fator de Transcrição Sp1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Antioxidantes/farmacologia , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Senescência Celular/efeitos dos fármacos , Senescência Celular/fisiologia , Ativação Enzimática , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Células NIH 3T3 , Oxidantes/farmacologia , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Regiões Promotoras Genéticas , Quercetina/farmacologia , Elementos de Resposta , Fator de Transcrição Sp1/biossíntese , Fator de Transcrição Sp1/genética , Transcrição Gênica , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
12.
FEBS Lett ; 581(26): 5099-104, 2007 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-17935719

RESUMO

Caveolin-3 (Cav-3) is the main scaffolding protein present in myofiber caveolae. We transfected C2C12 myoblasts with dominant negative forms of Cav-3, P104L or DeltaTFT, respectively, which cause the limb-girdle muscular dystrophy 1-C. Both these forms triggered Cav-3 loss during C2C12 cell differentiation. The P104L mutation reduced myofiber formation by impaired AKT signalling, accompanied by dramatic expression of the E3 ubiquitin ligase Atrogin. On the other hand, the DeltaTFT mutation triggered hypertrophic myotubes sustained by prolonged AKT activation, but independent of increased levels of follistatin and interleukin 4 expression. These data suggest that separated mutations within the same dystrophy-related gene may cause muscle degeneration through different mechanisms.


Assuntos
Caveolina 3/metabolismo , Diferenciação Celular , Mioblastos/citologia , Animais , Caveolina 3/genética , Humanos , Camundongos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutação , Mioblastos/metabolismo , Fenótipo , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
13.
FASEB J ; 20(6): 705-7, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16455755

RESUMO

Skeletal muscle tissue is one of the main sites where glucose uptake occurs in response to insulin. The glucose transporter type-4 (GLUT4) is primarily responsible for the insulin-stimulated increase in glucose uptake. Upon insulin stimulation, GLUT4 is recruited from intracellular reserves to the plasma membrane. The molecular mechanisms that regulate the translocation of GLUT4 to the sarcolemma remain to be fully identified. Here, we demonstrate that GLUT4 is localized to perinuclear stores that contain flotillin-1, a marker of lipid rafts, in skeletal muscle cells. Stimulation with insulin for 10 min results in the translocation of flotillin-1/GLUT4-containing domains to the plasma membrane in a PI3K- and PKCzeta-dependent manner. We also demonstrate that caveolin-3, a marker of caveolae, is required for the insulin receptor-mediated activation of the PI3K-dependent pathway, which occurs 2 min after insulin stimulation. In fact, we demonstrate that lack of caveolin-3 significantly reduces insulin-stimulated glucose uptake in caveolin-3 null myotubes by inhibiting both PI3K and Akt, as well as the movement of GLUT4 to the plasma membrane. Interestingly, caveolin-3 moves away from the plasma membrane toward the cytoplasm 5 min after insulin stimulation and temporarily interacts with flotillin-1/GLUT4-containing domains before they reach the sarcolemma, with the consequent movement of the insulin receptor from caveolin-3-containing domains to flotillin-1-containing domains. Such translocation temporally matches the insulin-stimulated movement of Cbl and CrkII in flotillin-1/GLUT4-containing domains, as well as the activation of the GDP-GTP exchange factor C3G. Disruption of flotillin-1-based domains prevents the activation of C3G, movement of GLUT4 to the sarcolemma, and glucose uptake in response to insulin. Thus, the activation of the Cbl/C3G/TC10-dependent pathway, which occurs before flotillin-1/GLUT4-containing domains reach the plasma membrane, is flotillin-1 mediated and follows the activation of the PI3K-mediated signaling. Taken together, these results indicate that flotillin-1 and caveolin-3 may regulate muscle energy metabolism through the spatial and temporal segregation of key components of the insulin signaling.


Assuntos
Caveolina 3/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Proteínas de Membrana/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Animais , Carcinoma de Células Renais , Membrana Celular , Células Cultivadas , Regulação da Expressão Gênica , Insulina , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
Mol Biol Cell ; 14(10): 4075-88, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14517320

RESUMO

Caveolae are vesicular invaginations of the plasma membrane. Caveolin-3 is the principal structural component of caveolae in skeletal muscle cells in vivo. We have recently generated caveolin-3 transgenic mice and demonstrated that overexpression of wild-type caveolin-3 in skeletal muscle fibers is sufficient to induce a Duchenne-like muscular dystrophy phenotype. In addition, we have shown that caveolin-3 null mice display mild muscle fiber degeneration and T-tubule system abnormalities. These data are consistent with the mild phenotype observed in Limb-girdle muscular dystrophy-1C (LGMD-1C) in humans, characterized by a approximately 95% reduction of caveolin-3 expression. Thus, caveolin-3 transgenic and null mice represent valid mouse models to study Duchenne muscular dystrophy (DMD) and LGMD-1C, respectively, in humans. Here, we derived conditionally immortalized precursor skeletal muscle cells from caveolin-3 transgenic and null mice. We show that overexpression of caveolin-3 inhibits myoblast fusion to multinucleated myotubes and lack of caveolin-3 enhances the fusion process. M-cadherin and microtubules have been proposed to mediate the fusion of myoblasts to myotubes. Interestingly, we show that M-cadherin is downregulated in caveolin-3 transgenic cells and upregulated in caveolin-3 null cells. For the first time, variations of M-cadherin expression have been linked to a muscular dystrophy phenotype. In addition, we demonstrate that microtubules are disorganized in caveolin-3 null myotubes, indicating the importance of the cytoskeleton network in mediating the phenotype observed in these cells. Taken together, these results propose caveolin-3 as a key player in myoblast fusion and suggest that defects of the fusion process may represent additional molecular mechanisms underlying the pathogenesis of DMD and LGMD-1C in humans.


Assuntos
Caderinas/metabolismo , Caveolinas/metabolismo , Distrofias Musculares/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Mioblastos Esqueléticos/metabolismo , Animais , Cavéolas/fisiologia , Caveolina 3 , Caveolinas/fisiologia , Células Cultivadas , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Fibras Musculares Esqueléticas/patologia , Mioblastos Esqueléticos/patologia
15.
Mol Biol Cell ; 13(7): 2502-17, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12134086

RESUMO

Caveolae are vesicular invaginations of the plasma membrane. Caveolin-1 is the principal structural component of caveolae in vivo. Several lines of evidence are consistent with the idea that caveolin-1 functions as a "transformation suppressor" protein. In fact, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). We have previously demonstrated that overexpression of caveolin-1 arrests mouse embryonic fibroblasts in the G(0)/G(1) phase of the cell cycle through activation of a p53/p21-dependent pathway, indicating a role of caveolin-1 in mediating growth arrest. However, it remains unknown whether overexpression of caveolin-1 promotes cellular senescence in vivo. Here, we demonstrate that mouse embryonic fibroblasts transgenically overexpressing caveolin-1 show: 1) a reduced proliferative lifespan; 2) senescence-like cell morphology; and 3) a senescence-associated increase in beta-galactosidase activity. These results indicate for the first time that the expression of caveolin-1 in vivo is sufficient to promote and maintain the senescent phenotype. Subcytotoxic oxidative stress is known to induce premature senescence in diploid fibroblasts. Interestingly, we show that subcytotoxic level of hydrogen peroxide induces premature senescence in NIH 3T3 cells and increases endogenous caveolin-1 expression. Importantly, quercetin and vitamin E, two antioxidant agents, successfully prevent the premature senescent phenotype and the up-regulation of caveolin-1 induced by hydrogen peroxide. Also, we demonstrate that hydrogen peroxide alone, but not in combination with quercetin, stimulates the caveolin-1 promoter activity. Interestingly, premature senescence induced by hydrogen peroxide is greatly reduced in NIH 3T3 cells harboring antisense caveolin-1. Importantly, induction of premature senescence is recovered when caveolin-1 levels are restored. Taken together, these results clearly indicate a central role for caveolin-1 in promoting cellular senescence and they suggest the hypothesis that premature senescence may represent a tumor suppressor function mediated by caveolin-1 in vivo.


Assuntos
Cavéolas/metabolismo , Caveolinas/metabolismo , Senescência Celular/fisiologia , Fibroblastos/fisiologia , Células 3T3/efeitos dos fármacos , Células 3T3/efeitos da radiação , Animais , Antioxidantes/farmacologia , Apoptose/fisiologia , Caveolina 1 , Caveolinas/genética , Divisão Celular/fisiologia , Tamanho Celular , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Fibroblastos/citologia , Genes Reporter , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Oxidantes/farmacologia , Estresse Oxidativo , Regiões Promotoras Genéticas , Quercetina/farmacologia , Fatores de Tempo , Transcrição Gênica , Transgenes , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta , Regulação para Cima/fisiologia , Vitamina E/farmacologia , beta-Galactosidase/metabolismo
16.
FASEB J ; 19(2): 237-9, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15545301

RESUMO

We have previously shown that caveolin-1, the principal structural protein component of caveolar membrane domains, inhibits cellular proliferation and induces cell cycle arrest. We demonstrate here for the first time that caveolin-1 is expressed in satellite cells but not in mature muscle fibers. Satellite cells are quiescent myogenic precursors that, after muscle injury, become mitotically active, proliferate, and fuse together or, to existing myofibers, to form new muscle fibers. We show that down-regulation of caveolin-1 expression occurs in satellite cells/myogenic precursor cells (MPCs) during muscle regeneration and that hepatocyte growth factor, which is produced after muscle injury, down-regulates caveolin-1. We also demonstrate that down-regulation of endogenous caveolin-1 expression activates ERK and that activation of the p42/44 MAP kinase pathway is necessary to promote muscle regeneration. Finally, we show that overexpression of caveolin-1 inhibits muscle repair mechanisms both in vitro and in vivo. Taken together, these results propose caveolin-1 as a novel regulator of satellite cell functions and suggest that the following signaling pathway modulates satellite cell activation during muscle repair: injured fibers release HGF --> HGF down-regulates caveolin-1 protein expression --> down-regulation of caveolin-1 activates ERK --> activation of ERK promotes muscle repair by stimulating the proliferation and migration of MPCs toward the wounded area.


Assuntos
Caveolinas/biossíntese , Caveolinas/fisiologia , Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/fisiologia , Animais , Caveolina 1 , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Linhagem Celular Transformada , Regulação para Baixo/genética , Regulação para Baixo/fisiologia , Ativação Enzimática/genética , Ativação Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/química , Músculo Esquelético/patologia , Mioblastos/química , Mioblastos/citologia , Mioblastos/metabolismo , Regeneração/genética , Regeneração/fisiologia , Cicatrização/genética
17.
Aging (Albany NY) ; 8(10): 2355-2369, 2016 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-27705926

RESUMO

Mitochondrial proteases ensure mitochondrial integrity and function after oxidative stress by providing mitochondrial protein quality control. However, the molecular mechanisms that regulate this basic biological function in eukaryotic cells remain largely unknown. Caveolin-1 is a scaffolding protein involved in signal transduction. We find that AFG3L2, a m-AAA type of mitochondrial protease, is a novel caveolin-1-interacting protein in vitro. We show that oxidative stress promotes the translocation of both caveolin-1 and AFG3L2 to mitochondria, enhances the interaction of caveolin-1 with AFG3L2 in mitochondria and stimulates mitochondrial protease activity in wild-type fibroblasts. Localization of AFG3L2 to mitochondria after oxidative stress is inhibited in fibroblasts lacking caveolin-1, which results in impaired mitochondrial protein quality control, an oxidative phosphorylation to aerobic glycolysis switch and reduced ATP production. Mechanistically, we demonstrate that a lack of caveolin-1 does not alter either mitochondrial number or morphology but leads to the cytoplasmic and proteasome-dependent degradation of complexes I, III, IV and V upon oxidant stimulation. Restoration of mitochondrial respiratory chain complexes in caveolin-1 null fibroblasts reverts the enhanced glycolysis observed in these cells. Expression of a mutant form of AFG3L2, which has reduced affinity for caveolin-1, fails to localize to mitochondria and promotes degradation of complex IV after oxidative stress. Thus, caveolin-1 maintains mitochondrial integrity and function when cells are challenged with free radicals by promoting the mitochondrial localization of m-AAA protease and its quality control functions.


Assuntos
Caveolina 1/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Fibroblastos/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Animais , Caveolina 1/genética , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos
18.
FASEB J ; 18(10): 1080-9, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15226268

RESUMO

The heme oxygenase (HO) and nitric oxide synthase (NOS) enzymes generate the gaseous signaling molecules carbon monoxide (CO) and nitric oxide, respectively. Constitutive NOSs localize to caveolae, and their activities are modulated by caveolin-1. Nothing is known of the localization of the inducible heme oxygenase-1 (HO-1) in plasma membrane caveolae. Thus, we examined the distribution and subcellular localization of HO-1, biliverdin reductase (BVR), and NADPH:cytochrome P450 reductase (NPR) in pulmonary artery endothelial cells. Each of these proteins localized in part to plasma membrane caveolae in endothelial cells. Inducers of HO-1 or overexpression of HO-1 increased the content of this protein in a detergent-resistant fraction containing caveolin-1. Inducible HO activity appeared in plasma membrane, cytosol, and isolated caveolae. In addition, caveolae contained endogenous BVR activity, supporting the same compartmentalization of both enzymes. Caveolin-1 physically interacted with HO-1, as shown by coimmunoprecipitation studies. HO activity dramatically increased in cells expressing caveolin-1 antisense transcripts, suggesting a negative regulatory role for caveolin-1. Conversely, caveolin-1 expression attenuated LPS-inducible HO activity. Since their initial characterization in 1969, HO enzymes have been described as endoplasmic reticulum-associated proteins. We demonstrate for the first time the localization of heme degradation enzymes to plasma membrane caveolae, and present novel evidence that caveolin-1 interacts with and modulates HO activity.


Assuntos
Cavéolas/enzimologia , Caveolinas/fisiologia , Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Heme Oxigenase (Desciclizante)/metabolismo , Animais , Cavéolas/metabolismo , Caveolina 1 , Caveolinas/deficiência , Compartimento Celular , Hipóxia Celular , Membrana Celular/enzimologia , Células Cultivadas/enzimologia , Retículo Endoplasmático/enzimologia , Células Endoteliais/ultraestrutura , Endotélio Vascular/citologia , Heme Oxigenase (Desciclizante)/antagonistas & inibidores , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1 , Hemina/farmacologia , Hepatócitos/enzimologia , Humanos , Lipopolissacarídeos/farmacologia , Proteínas de Membrana , Camundongos , Células NIH 3T3/enzimologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Mapeamento de Interação de Proteínas , Artéria Pulmonar , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
19.
Endocrinology ; 145(6): 2815-23, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15016722

RESUMO

G protein-coupled receptors (GPCRs) mediate the action of many hormones, cytokines, and sensory and chemical signals. It is generally thought that receptor desensitization and internalization require occupancy and activation of the GPCR. PTH and PTHrP receptor (PTH1R) belongs to GPCR class B and is the major regulator of extracellular calcium homeostasis. Using kidney distal convoluted tubule cells transfected with a human PTH1R/enhanced green fluorescent protein fusion protein, quantitative, real-time fluorescence microscopy was used to analyze receptor internalization. In these cells, which are the target of the calcium-sparing action of PTH, PTH(1-34) activated adenylyl cyclase (AC) and phospholipase C (PLC) and PTH1R endocytosis. PTH(1-31), however, stimulated AC and PLC but not PTH1R endocytosis. Conversely, PTH(7-34) rapidly stimulated PTH1R internalization without activating AC or PLC. PTH(2-34) and (3-34) caused PTH1R internalization intermediate between PTH(1-34) and (7-34). PTH1R sequestration occurred in a dynamin- and clathrin-dependent manner. Directly activating AC inhibited PTH1R internalization in response to PTH(7-34). PTH1R endocytosis was sensitive to protein kinase C inhibition. PTH(1-34), (7-34), and (1-31) evoked PTH1R phosphorylation. Removal of most of the C terminus of the PTH1R eliminated receptor phosphorylation and the cAMP/protein kinase C sensitivity of internalization. PTH(1-34) and (7-34) internalized the truncated PTH1R with identical kinetics, and the response was unaffected by forskolin. Thus, the PTH1R C terminus contains regulatory sequences that are involved in, but not required for, PTH1R internalization. The results demonstrate that receptor activation and internalization can be selectively dissociated.


Assuntos
Endocitose , Receptor Tipo 1 de Hormônio Paratireóideo/fisiologia , Animais , Cavéolas/fisiologia , Células Cultivadas , Clatrina/fisiologia , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Humanos , Membranas Intracelulares/metabolismo , Túbulos Renais Distais/citologia , Túbulos Renais Distais/metabolismo , Ligantes , Camundongos , Hormônio Paratireóideo/química , Fragmentos de Peptídeos/farmacologia , Fosforilação , Fosfotransferases/fisiologia , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptores de Hormônios Paratireóideos/metabolismo , Receptores de Hormônios Paratireóideos/fisiologia , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais
20.
Mol Cell Biol ; 34(14): 2611-23, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24777604

RESUMO

While glucocorticoids (GCs) are used clinically to treat many conditions, their neonatal and prenatal usage is increasingly controversial due to reports of delayed adverse outcomes, especially their effects on brain development. Such alterations may reflect the impact of GCs on neural progenitor/stem cell (NPSC) function. We previously demonstrated that the lipid raft protein caveolin-1 (Cav-1) was required for rapid GC signaling in embryonic mouse NPSCs operating through plasma membrane-bound glucocorticoid receptors (GRs). We show here that genomic GR signaling in NPSCs requires Cav-1. Loss of Cav-1 impacts the transcriptional response of many GR target genes (e.g., the serum- and glucocorticoid-regulated kinase 1 gene) that are likely to mediate the antiproliferative effects of GCs. Microarray analysis of wild-type C57 or Cav-1-deficient NPSCs identified approximately 100 genes that are differentially regulated by GC treatment. These changes in hormone responsiveness in Cav-1 knockout NPSCs are associated with the loss of GC-regulated phosphorylation of GR at serine 211 but not at serine 226. Chromatin recruitment of total GR to regulatory regions of target genes such as Fkbp-5, RhoJ, and Sgk-1, as well as p211-GR recruitment to Sgk-1, are compromised in Cav-1 knockout NPSCs. Cav-1 is therefore a multifunctional regulator of GR in NPSCs influencing both rapid and genomic action of the receptor to impact cell proliferation.


Assuntos
Caveolina 1/metabolismo , Dexametasona/efeitos adversos , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/efeitos adversos , Células-Tronco Neurais/metabolismo , Receptores de Glucocorticoides/metabolismo , Elementos Reguladores de Transcrição , Animais , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , Cromatina/metabolismo , Embrião de Mamíferos , Técnicas de Inativação de Genes , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Receptores de Glucocorticoides/genética , Serina/metabolismo
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