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1.
Cell ; 186(14): 3013-3032.e22, 2023 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-37352855

RESUMO

Mitochondrial DNA (mtDNA) is a potent agonist of the innate immune system; however, the exact immunostimulatory features of mtDNA and the kinetics of detection by cytosolic nucleic acid sensors remain poorly defined. Here, we show that mitochondrial genome instability promotes Z-form DNA accumulation. Z-DNA binding protein 1 (ZBP1) stabilizes Z-form mtDNA and nucleates a cytosolic complex containing cGAS, RIPK1, and RIPK3 to sustain STAT1 phosphorylation and type I interferon (IFN-I) signaling. Elevated Z-form mtDNA, ZBP1 expression, and IFN-I signaling are observed in cardiomyocytes after exposure to Doxorubicin, a first-line chemotherapeutic agent that induces frequent cardiotoxicity in cancer patients. Strikingly, mice lacking ZBP1 or IFN-I signaling are protected from Doxorubicin-induced cardiotoxicity. Our findings reveal ZBP1 as a cooperative partner for cGAS that sustains IFN-I responses to mitochondrial genome instability and highlight ZBP1 as a potential target in heart failure and other disorders where mtDNA stress contributes to interferon-related pathology.


Assuntos
Cardiotoxicidade , DNA Mitocondrial , Animais , Camundongos , DNA Mitocondrial/metabolismo , Imunidade Inata , Interferons/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Fosforilação
2.
Hum Mol Genet ; 32(15): 2422-2440, 2023 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-37129502

RESUMO

The recognition that cytosolic mitochondrial DNA (mtDNA) activates cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) innate immune signaling has unlocked novel disease mechanisms. Here, an uncharacterized variant predicted to affect TOP1MT function, P193L, was discovered in a family with multiple early onset autoimmune diseases, including Systemic Lupus Erythematosus (SLE). Although there was no previous genetic association between TOP1MT and autoimmune disease, the role of TOP1MT as a regulator of mtDNA led us to investigate whether TOP1MT could mediate the release of mtDNA to the cytosol, where it could then activate the cGAS-STING innate immune pathway known to be activated in SLE and other autoimmune diseases. Through analysis of cells with reduced TOP1MT expression, we show that loss of TOP1MT results in release of mtDNA to the cytosol, which activates the cGAS-STING pathway. We also characterized the P193L variant for its ability to rescue several TOP1MT functions when expressed in TOP1MT knockout cells. We show that the P193L variant is not fully functional, as its re-expression at high levels was unable to rescue mitochondrial respiration deficits, and only showed partial rescue for other functions, including repletion of mtDNA replication following depletion, nucleoid size, steady state mtDNA transcripts levels and mitochondrial morphology. Additionally, expression of P193L at endogenous levels was unable to rescue mtDNA release-mediated cGAS-STING signaling. Overall, we report a link between TOP1MT and mtDNA release leading to cGAS-STING activation. Moreover, we show that the P193L variant has partial loss of function that may contribute to autoimmune disease susceptibility via cGAS-STING mediated activation of the innate immune system.


Assuntos
Doenças Autoimunes , Lúpus Eritematoso Sistêmico , Humanos , DNA Mitocondrial/genética , Imunidade Inata/genética , Interferons , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo
3.
J Biol Chem ; 298(10): 102420, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36030054

RESUMO

TOP1MT encodes a mitochondrial topoisomerase that is important for mtDNA regulation and is involved in mitochondrial replication, transcription, and translation. Two variants predicted to affect TOP1MT function (V1 - R198C and V2 - V338L) were identified by exome sequencing of a newborn with hypertrophic cardiomyopathy. As no pathogenic TOP1MT variants had been confirmed previously, we characterized these variants for their ability to rescue several TOP1MT functions in KO cells. Consistent with these TOP1MT variants contributing to the patient phenotype, our comprehensive characterization suggests that both variants had impaired activity. Critically, we determined neither variant was able to restore steady state levels of mitochondrial-encoded proteins nor to rescue oxidative phosphorylation when re-expressed in TOP1MT KO cells. However, we found the two variants behaved differently in some respects; while the V1 variant was more efficient in restoring transcript levels, the V2 variant showed better rescue of mtDNA copy number and replication. These findings suggest that the different TOP1MT variants affect distinct TOP1MT functions. Altogether, these findings begin to provide insight into the many roles that TOP1MT plays in the maintenance and expression of the mitochondrial genome and how impairments in this important protein may lead to human pathology.


Assuntos
Cardiomiopatia Hipertrófica , DNA Topoisomerases Tipo I , Genoma Mitocondrial , Mitocôndrias , Humanos , Recém-Nascido , Cardiomiopatia Hipertrófica/genética , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Mitocondrial/metabolismo , Variação Genética , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo
4.
Arterioscler Thromb Vasc Biol ; 41(6): e338-e353, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33792343
5.
FASEB J ; 34(11): 14287-14301, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32888229

RESUMO

Blood levels of heat shock protein (HSP27) and natural IgG auto-antibodies to HSP27 (AAbs) are higher in healthy controls compared to cardiovascular disease patients. Vaccination of mice with recombinant HSP25 (rHSP25, murine ortholog of human rHSP27) increased AAb levels, attenuated atherogenesis and reduced plaque inflammation and cholesterol content. We sought to determine if the HSP27 immune complex (IC) altered MΦ inflammation signaling (Toll Like Receptor 4; TLR4), and scavenger receptors involved in cholesterol uptake (SR-AI, CD-36). Combining a validated polyclonal IgG anti-HSP27 antibody (PAb) with rHSP27 enhanced binding to THP-1 MΦ cell membranes and activation of NF-κB signaling via TLR4, competing away LPS and effecting an anti-inflammatory cytokine profile. Similarly, adding the PAb with rHSP27 enhanced binding to SR-AI and CD-36, as well as lowered oxLDL binding in HEK293 cells separately transfected with SR-AI and CD-36, or THP-1 MΦ. Finally, the PAb enhanced the uptake and internalization of rHSP27 in THP-1 MΦ. Thus, the HSP27 IC potentiates HSP27 cell membrane signaling with receptors involved in modulating inflammation and cholesterol uptake, as well as HSP27 internalization. Going forward, we are focusing on the development of HSP27 Immune Complex Altered Signaling and Transport (ICAST) as a means of modulating inflammation.


Assuntos
Anti-Inflamatórios/farmacologia , Complexo Antígeno-Anticorpo/farmacologia , Aterosclerose/prevenção & controle , Autoanticorpos/imunologia , Proteínas de Choque Térmico HSP27/imunologia , Sistema Imunitário/imunologia , Inflamação/prevenção & controle , Animais , Aterosclerose/etiologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Fosforilação
6.
FASEB J ; 33(2): 1617-1630, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30188755

RESUMO

Previously, we reported that elevated serum levels of heat shock protein 27 (HSP27) are predictive of a lower risk of having a heart attack, stroke, or death from cardiovascular disease. Moreover, augmenting HSP27 (or the murine ortholog, HSP25) attenuated experimental atherogenesis, reduced inflammation, and lowered cholesterol levels. Recently, we noted that HSP27 activates NF-κB via TLR-4, resulting in attenuation of plaque inflammation; however, the precise anti-atherosclerosis mechanisms mediated by extracellular HSP27 are incompletely understood. Our purpose in this study was to investigate the existence of HSP27 in extracellular vesicles (EVs) and whether HSP27 elicited atheroprotective effects on target cells. Here, we provide evidence that HSP27 localizes to EVs derived from THP-1 cells using transmission electron microscopy (TEM) and immunogold labeling, Western blotting, ELISA, and fluorescence-activated cell sorting. TEM imaging indicated that HSP27 is found at the exosomal membrane. Multiple reactor monitor-mass spectrometric analysis of large vesicles, which included microparticles and exosomes, isolated from human plasma, also led to detection of HSP27 using the unique signature peptide, R.LFDQAFGLPR.L. Studies using THP-1 and human embryonic kidney cells show that HSP27-laden exosomes significantly stimulated NF-κB activation ( P < 0.001) and release of IL-10 ( P < 0.0001), suggesting that HSP27 may be important exosomal cargo with beneficial anti-inflammatory effects.-Shi, C., Ulke-Lemée, A., Deng, J., Batulan, Z., O'Brien, E. R. Characterization of heat shock protein 27 in extracellular vesicles: a potential anti-inflammatory therapy.


Assuntos
Anti-Inflamatórios/farmacologia , Exossomos/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Sequência de Aminoácidos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Humanos , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Chaperonas Moleculares , NF-kappa B/metabolismo , Células THP-1
7.
Pharmacol Res ; 161: 105222, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33022407

RESUMO

AIMS: The estrogen-inducible protein Heat Shock Protein 27 (HSP27) as well as anti-HSP27 antibodies are elevated in healthy subjects compared to cardiovascular disease patients. Vaccination of ApoE-/- mice with recombinant HSP25 (rHSP25, the murine ortholog), boosts anti- HSP25 levels and attenuates atherogenesis. As estrogens promote HSP27 synthesis, cellular release and blood levels, we hypothesize that menopause will result in loss of HSP27 atheroprotection. Hence, the rationale for this study is to compare the efficacy of rHSP25 vaccination vs. estradiol (E2) therapy for the prevention of post-menopausal atherogenesis. METHODS AND RESULTS: ApoE-/- mice subjected to ovariectomy (OVX) showed a 65 % increase atherosclerotic burden compared to sham mice after 5 weeks of a high fat diet. Relative to vaccination with rC1, a truncated HSP27 control peptide, atherogenesis was reduced by 5-weekly rHSP25 vaccinations (-43 %), a subcutaneous E2 slow release pellet (-52 %) or a combination thereof (-82 %). Plasma cholesterol levels declined in parallel with the reductions in atherogenesis, but relative to rC1/OVX mice plasma PCSK9 levels were 52 % higher in E2/OVX and 41 % lower in rHSP25/OVX mice (p < 0.0001 for both). Hepatic LDLR mRNA levels did not change with E2 treatment but increased markedly with rHSP25 vaccination. Conversely, hepatic PCSK9 mRNA increased 148 % with E2 treatment vs. rC1/OVX but did not change with rHSP25 vaccination. In human HepG2 hepatocytes E2 increased PCSK9 promoter activity 303 %, while the combination of [rHSP27 + PAb] decreased PCSK9 promoter activity by 64 %. CONCLUSION: The reduction in post-OVX atherogenesis and cholesterol levels with rHSP25 vaccination is associated with increased LDLR but not PCSK9 expression. Surprisingly, E2 therapy attenuates atherogenesis and cholesterol levels post-OVX without altering LDLR but increases PCSK9 expression and promoter activity. This is the first documentation of increased PCSK9 expression with E2 therapy and raises questions about balancing physiological estrogenic / PCSK9 homeostasis and targeting PCSK9 in women - are there effects beyond cholesterol?


Assuntos
Aterosclerose/prevenção & controle , Colesterol/sangue , Estradiol/administração & dosagem , Terapia de Reposição de Estrogênios , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico/administração & dosagem , Fígado/efeitos dos fármacos , Chaperonas Moleculares/administração & dosagem , Pró-Proteína Convertase 9/metabolismo , Receptores de LDL/metabolismo , Vacinas/administração & dosagem , Animais , Aterosclerose/sangue , Aterosclerose/enzimologia , Aterosclerose/imunologia , Biomarcadores/sangue , Modelos Animais de Doenças , Regulação para Baixo , Implantes de Medicamento , Feminino , Proteínas de Choque Térmico/imunologia , Células Hep G2 , Humanos , Fígado/enzimologia , Menopausa , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Chaperonas Moleculares/imunologia , Ovariectomia , Pró-Proteína Convertase 9/genética , Receptores de LDL/genética , Vacinação
8.
Cell Stress Chaperones ; 28(6): 877-887, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37966617

RESUMO

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure in intensive care units that has increased dramatically as a result of the COVID-19 pandemic. In both COVID-19 and non-COVID ARDS, the pathogenesis of lung injury involves local (pulmonary) and systemic inflammation, leading to impaired gas exchange, requirement for mechanical ventilation, and a high risk of mortality. Heat shock protein 27 (HSP27) is a chaperone protein expressed in times of cell stress with roles in modulation of systemic inflammation via the NF-κB pathway. Given its important role as a modulator of inflammation, we sought to investigate the role of HSP27 and its associated auto-antibodies in ARDS caused by both SARS-CoV-2 and non-COVID etiologies. A total of 68 patients admitted to the intensive care unit with ARDS requiring mechanical ventilation were enrolled in a prospective, observational study that included 22 non-COVID-19 and 46 COVID-19 patients. Blood plasma levels of HSP27, anti-HSP27 auto-antibody (AAB), and cytokine profiles were measured on days 1 and 3 of ICU admission along with clinical outcome measures. Patients with COVID-19 ARDS displayed significantly higher levels of HSP27 in plasma, and a higher ratio of HSP27:AAB on both day 1 and day 3 of ICU admission. In patients with COVID-19, higher levels of circulating HSP27 and HSP27:AAB ratio were associated with a more severe systemic inflammatory response and adverse clinical outcomes including more severe hypoxemic respiratory failure. These findings implicate HSP27 as a marker of advanced pathogenesis of disease contributing to the dysregulated systemic inflammation and worse clinical outcomes in COVID-19 ARDS, and therefore may represent a potential therapeutic target.


Assuntos
COVID-19 , Síndrome do Desconforto Respiratório , Insuficiência Respiratória , Humanos , COVID-19/complicações , Proteínas de Choque Térmico HSP27 , Inflamação , Pandemias , Estudos Prospectivos , Síndrome do Desconforto Respiratório/terapia , SARS-CoV-2
9.
Cells ; 12(11)2023 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-37296583

RESUMO

(+)-JQ1, a specific chemical inhibitor of bromodomain and extraterminal (BET) family protein 4 (BRD4), has been reported to inhibit smooth muscle cell (SMC) proliferation and mouse neointima formation via BRD4 regulation and modulate endothelial nitric oxide synthase (eNOS) activity. This study aimed to investigate the effects of (+)-JQ1 on smooth muscle contractility and the underlying mechanisms. Using wire myography, we discovered that (+)-JQ1 inhibited contractile responses in mouse aortas with or without functional endothelium, reducing myosin light chain 20 (LC20) phosphorylation and relying on extracellular Ca2+. In mouse aortas lacking functional endothelium, BRD4 knockout did not alter the inhibition of contractile responses by (+)-JQ1. In primary cultured SMCs, (+)-JQ1 inhibited Ca2+ influx. In aortas with intact endothelium, (+)-JQ1 inhibition of contractile responses was reversed by NOS inhibition (L-NAME) or guanylyl cyclase inhibition (ODQ) and by blocking the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. In cultured human umbilical vein endothelial cells (HUVECs), (+)-JQ1 rapidly activated AKT and eNOS, which was reversed by PI3K or ATK inhibition. Intraperitoneal injection of (+)-JQ1 reduced mouse systolic blood pressure, an effect blocked by co-treatment with L-NAME. Interestingly, (+)-JQ1 inhibition of aortic contractility and its activation of eNOS and AKT were mimicked by the (-)-JQ1 enantiomer, which is structurally incapable of inhibiting BET bromodomains. In summary, our data suggest that (+)-JQ1 directly inhibits smooth muscle contractility and indirectly activates the PI3K/AKT/eNOS cascade in endothelial cells; however, these effects appear unrelated to BET inhibition. We conclude that (+)-JQ1 exhibits an off-target effect on vascular contractility.


Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Camundongos , Humanos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Aorta/metabolismo , Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas de Ciclo Celular
10.
Mol Med ; 18: 159-66, 2012 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-22064971

RESUMO

The fundamental mechanisms that underlie platelet activation in atherothrombosis are still obscure. Oxidative stress is involved in central features of atherosclerosis. Platelet-derived microvesicles (PMVs) could be important mediators between oxidative stress and platelet activation. CD36 could be a receptor of PMVs, thus generating a PMV-CD36 complex. We aimed to investigate the detailed pathway by which oxidative damage contributes to platelet activation by the PMV-CD36 complex. We found that oxidized low-density lipoprotein stimulated the generation of PMVs. PMVs enhanced normal platelet activation, as assessed by the expression of integrin α(IIb)ß3, secretion of soluble P-selectin and platelet aggregation, but CD36-deficient platelets were not activated by PMVs. The function of the PMV-CD36 complex was mediated by the MKK4/JNK2 signaling axis. Meanwhile, PMVs increased the level of 8-iso-prostaglandin-F2α, a marker of oxidative stress, in a CD36- and phosphatidylserine-dependent manner. We concluded that PMVs are important mediators between oxidative stress and platelet activation. PMVs and CD36 may be effective targets for preventing platelet activation in cardiovascular diseases.


Assuntos
Plaquetas/metabolismo , Lipoproteínas LDL/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Antígenos CD36/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Ativação Plaquetária/efeitos dos fármacos
11.
Mol Cell Biochem ; 361(1-2): 305-14, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22012613

RESUMO

To characterize the roles of tribble 2 (TRB2) and its targeted microRNAs (miRNAs) in the pathogenesis of the early vascular injury involved in diabetic-2 rat. Goto-Kakizaki (GK) rat and Wistar rat were used as the animal models. Each eligible rat was killed and the rat aorta tissues were analyzed by immunohistochemistry, ELISA, reverse transcription-polymerase chain reaction (RT-PCR), and real-time PCR detection. GFP expression in RAOEC cells (rat vascular aortic endothelial cell)were detected by flow cytometry and fluorescent microscope. TRB2 gene expression was increased in endothelia cell and the adventitia of Goto-Kakizaki (GK) rat compared with Wistar rat. Next, studies using RAOEC cells showed that the TRB2 expression was inhibited by the treatment of miR-98. We further showed that the expression of miR-98 was significantly decreased in the adventitia and endomembrane at different degrees in GK rats compared with control. Finally, we validated the changes in TRB2 by studying one of the TRB2's substrates, Akt, in animal models. We expected a corresponding change in the levels of phosphorylated Akt. Indeed, our results showed that the phosphorylation of Akt at Thr 308 in the endothelial cells and phosphorylation of Akt at Ser 473 in adventitia was decreased in GK rats, compared with Wistar control. TRB2 plays important roles in the pathogenesis of diabetic-2 large artery complications at early stage, and these effects may be modulated by miR-98. Thus, targeting TRB2 and miR-98 could be considered as novel therapeutic strategies for the early large artery deficits in diabetic-2.


Assuntos
Doenças da Aorta/etiologia , Aterosclerose/etiologia , Diabetes Mellitus Tipo 2/complicações , Angiopatias Diabéticas/etiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , MicroRNAs/genética , Regulação para Cima , Regiões 3' não Traduzidas , Animais , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/metabolismo , Células Endoteliais/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar
12.
Int J Cardiol ; 350: 9-15, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35007650

RESUMO

BACKGROUND: Low-density lipoprotein receptor (LDLR) is the primary pathway for removal of cholesterol from the circulation, pro-protein convertase subtilisin-like kexin type 9 (PCSK9) is a secreted protease that binds to and promotes degradation of the LDLR protein. The goal of this case-control study was to investigate the role of soluble LDLR (sLDLR) and PCSK9 in coronary artery disease (CAD) and investigate the relationship between these two indices and CAD. METHODS: In a sample of 144 Chinese patients recruited between January 2018 and August 2018, 81 cases with mild and severe stenosis characterized by coronary angiograph (CAG) and 63 healthy controls were selected using the propensity score matching (PSM) based on demographics and medical history. sLDLR and PCSK9 concentrations were determined using enzyme-linked immunosorbent assay (ELISA), Immuno-precipitation (IP) and western blotting. Multivariable logistic models were used to assess the associations between the degree of coronary artery stenosis and the biomarkers of interest. RESULTS: Higher PCSK9 was found to be a significant predictor of coronary artery stenosis when comparing cases who had severe stenosis vs. controls (OR=1.016, 95%CI: 1.009, 1.024), and cases who had mild stenosis vs. controls (OR=1.009, 95%CI: 1.003, 1.015). sLDLR was positively corrected with PCSK9, which confounded the association between CAD and PCSK9. Compared to patients with mild-stenosis, patients with severe-stenosis also showed a higher level of PCSK9 (OR=1.007, 95%CI: 1.001, 1.013). CONCLUSIONS: These findings suggest that elevated PSCK9 may contribute to the odds of developing CAD, with a higher degree of coronary artery stenosis.


Assuntos
Doença da Artéria Coronariana , Pró-Proteína Convertase 9 , Estudos de Casos e Controles , Doença da Artéria Coronariana/diagnóstico por imagem , Humanos , Lipoproteínas LDL , Receptores de LDL , Subtilisina
13.
Clin Exp Pharmacol Physiol ; 37(1): 51-5, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19566842

RESUMO

1. C/EBP homologueueueous protein (CHOP), an endoplasmic reticulum (ER) stress-inducible protein, has a critical role in regulation of the cell cycle and apoptosis by forming heterodimers with other C/EBP proteins. However, how CHOP function is regulated remains to be determined. The human homologue of Drosophila tribbles (TRIB3) is associated with CHOP and is upregulated by oxidized low-density lipoprotein (ox-LDL). The aim of the present study was to investigate the role of CHOP in ox-LDL-induced TRIB3 expression in macrophages. 2. Human monocyte-derived macrophages were treated with various concentrations of ox-LDL (0, 2.5, 5, 10, 25 and 50 microg/mL) or 2 microg/mL tunicamycin for 0, 4, 8, 16, 24 and 48 h or were transfected with CHOP or TRIB3 expression plasmid and TRIB3 targeting short interference RNA (siRNA). The expression of CHOP and activating transcription factor 4 (ATF4) mRNA in treated cells was detected by quantitative real-time polymerase chain reaction (PCR). 3. The expression of CHOP and ATF4 mRNA increased with increasing concentrations of ox-LDL and duration of time. The ox-LDL-induced expression of TRIB3 mRNA was upregulated later than the expression of CHOP and ATF4 mRNA. Overexpression of CHOP increased the mRNA expression of TRIB3, which was further increased in CHOP-overexpressing macrophages treated with ox-LDL. Overexpression of TRIB3 suppressed the expression of CHOP, whereas TRIB3 silencing increased CHOP expression following ox-LDL stimulation by a negative feedback mechanism. 4. In conclusion, the expression of ATF4 and CHOP is upregulated by ox-LDL in a dose- and time-dependent manner in naturally differentiated human macrophages. Oxidized LDL induces TRIB3 expression via an ATF4/CHOP-dependent ER stress pathway.


Assuntos
Fator 4 Ativador da Transcrição/fisiologia , Proteínas de Ciclo Celular/metabolismo , Lipoproteínas LDL/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Fator de Transcrição CHOP/fisiologia , Regulação para Cima/efeitos dos fármacos , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/farmacologia , Proteínas Repressoras/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Tunicamicina/farmacologia
14.
Clin Sci (Lond) ; 116(3): 241-8, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18643775

RESUMO

Mammalian orthologues of the Drosophila tribbles protein (Trb1, Trb2 and Trb3) are a recently described family of signalling molecules that regulate gene expression by modulation of protein kinase signalling pathways. In the present study, a screen for mRNA species specifically regulated in vulnerable regions of human atherosclerotic plaque demonstrated the up-regulation of both Trb1 and Trb2, the latter by more than 8-fold. In vitro experiments in primary human monocyte-derived macrophages showed that Trb2 expression was up-regulated by treatment with oxidized LDL (low-density lipoprotein), and that expression of recombinant Trb2 specifically reduced macrophage levels of IL-10 (interleukin-10) mRNA. Our results thus identify Trb2 as a highly regulated gene in vulnerable atherosclerotic lesions, and demonstrate inhibition of macrophage IL-10 biosynthesis as a potential pro-inflammatory consequence of high Trb2 expression, which may contribute to plaque instability.


Assuntos
Doenças das Artérias Carótidas/metabolismo , Interleucina-10/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Doenças das Artérias Carótidas/cirurgia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Endarterectomia das Carótidas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-10/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Lipoproteínas LDL/farmacologia , Macrófagos/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
15.
PLoS One ; 14(12): e0226406, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31834925

RESUMO

Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30-120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Quinases Associadas com Morte Celular/metabolismo , Músculo Liso Vascular/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Miosinas/metabolismo , Quinases Associadas a rho/metabolismo , Proteínas Reguladoras de Apoptose/genética , Artérias/citologia , Artérias/metabolismo , Células Cultivadas , Proteínas Quinases Associadas com Morte Celular/antagonistas & inibidores , Proteínas Quinases Associadas com Morte Celular/genética , Humanos , Músculo Liso Vascular/citologia , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/genética , Fosfatase de Miosina-de-Cadeia-Leve/genética , Fosforilação , RNA Interferente Pequeno/genética , Soro/metabolismo , Transdução de Sinais , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética
16.
Methods Mol Biol ; 365: 209-23, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17200564

RESUMO

CPI-17 is a cytosolic protein of 17 kDa that becomes a potent inhibitor of certain type 1 protein serine/threonine phosphatases, including smooth muscle myosin light-chain phosphatase (MLCP), when phosphorylated at Thr38. Several protein kinases are capable of phosphorylating CPI-17 at this site in vitro; however, in intact tissue, compelling evidence only exists for phosphorylation by protein kinase C (PKC). Agonist-induced activation of heterotrimeric G proteins of the Gq/11 family via seven-transmembrane domain-containing, G protein-coupled receptors results in phospholipase Cbeta-mediated hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 triggers Ca2+ release from the sarcoplasmic reticulum. DAG and Ca2+ together activate classical isoforms of PKC, and DAG activates novel PKC isoforms without a requirement for Ca2+. Activated PKC phosphorylates CPI-17 at Thr38, enhancing its potency of inhibition of MLCP approx 1000-fold. The myosin light-chain kinase (MLCK):MLCP activity ratio is thereby increased at the prevailing cytosolic free-Ca2+ concentration ([Ca2+]i), resulting in an increase in phosphorylation of the 20-kDa light chains of myosin II (LC20) catalyzed by Ca2+- and calmodulin-dependent MLCK and contraction of the smooth muscle. Physiologically, this mechanism can account for some instances of Ca2+ sensitization of smooth muscle contraction (i.e., an increase in force in response to agonist stimulation without a change in [Ca2+]i).


Assuntos
Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/metabolismo , Proteína Quinase C/metabolismo , Animais , Cálcio/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosforilação , Suínos
17.
Cell Mol Biol Lett ; 12(4): 482-92, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17468839

RESUMO

Human PCAN1 (prostate cancer gene 1) is a prostate-specific gene that is highly expressed in prostate epithelial tissue, and frequently mutated in prostate tumors. To better understand the regulation of the PCAN1 gene, a 2.6-kb fragment of its 5' flanking region was obtained by PCR. Its promoter activity was examined via the dual-luciferase reporter assay after it had been cloned into a pGL(3)-basic vector generating pGL(3)-p2.6 kb and transfected into LNCaP cells. pGL(3)-basic and pGL(3)-control were respectively used as the negative and positive controls. Sequence analysis with the MatInspector database showed that some possible binding sites for the transcriptional factors, NKX3.1, P53, SP1, cEBP and the PPAR/RXR heterodimers may locate on a 2.6-kb region upstream of the PCAN1 gene. To examine the relevant regulation of PCAN1, pGL(3)-p2.6 kb was transfected into the prostate cancer cell line LNCaP, which was treated with R1881 (10(-7) approximately 10(-9) mol/l), 17beta-estradiol (17beta-E(2), 10(-7) approximately 10(-9) mol/l), all-trans-retinoic acid (all-trans-RA, 10(-5) approximately 10(-7) mol/l) or 9-cis-retinoic acid (9-cis-RA, 10(-5) approximately 10(-7) mol/l), and eukaryotic expression plasmids of NKX3.1, p53, Sp1, Pten, PPARgamma or cEBPalpha were cotransfected with pGL(3)-p2.6 kb into LNCaP cells. pRL-TK, a Renilla luciferase reporter vector, was cotransfected into all the transfection lines as an internal control. The activities of pGL(3)-p2.6 kb (PCAN1 promoter) were analyzed via the dual-luciferase reporter assay 48 h after transfection. The results showed that 9-cis-RA enhanced the PCAN1 promoter activity in a dose-dependent manner, while R1881, 17beta-E(2) and all-trans-RA had no significant effect on PCAN1 promoter activities. Cotransfection with pGL(3)-p2.6kb and the expression plasmids of NKX3.1, p53, Sp1 or Pten respectively resulted in 1.66-, 2.48-, 2.00-and 1.72-fold 2.6 kb PCAN1 promoter activity increases relative to the controls, which were cotransfected with pcDNA3.1(+), while cotransfection of PPARgamma and cEBPalpha yielded no significant effect on PCAN1 promoter activities. These results could be applied for further study of the function and transcription regulation of the PCAN1 gene in prostate development and carcinogenesis.


Assuntos
Clonagem Molecular , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , Alitretinoína , Sequência de Bases , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Luciferases/genética , Luciferases/metabolismo , Masculino , Dados de Sequência Molecular , Próstata , Neoplasias da Próstata/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Transfecção , Tretinoína/metabolismo
18.
J Diabetes Res ; 2017: 3918681, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28182086

RESUMO

This study was designed to investigate the changes of urinary microvesicle-bound uromodulin and total urinary uromodulin levels in human urine and the correlations with the severity of diabetic kidney disease (DKD). 31 healthy subjects without diabetes and 100 patients with type 2 diabetes mellitus (T2DM) were included in this study. The patients with T2DM were divided into three groups based on the urinary albumin/creatinine ratio (UACR): normoalbuminuria group (DM, n = 46); microalbuminuria group (DN1, n = 32); and macroalbuminuria group (DN2, n = 22). We use a specific monoclonal antibody AD-1 to capture the urinary microvesicles. Urinary microvesicle-bound uromodulin and total urinary uromodulin levels were determined by enzyme-linked immunosorbent assay (ELISA). Our results showed that the levels of urinary microvesicle-bound uromodulin in DN1 and DN2 groups were significantly higher than those in control group and DM group (P < 0.01). Multiple stepwise linear regression analysis showed that UACR was independent determinant for urinary microvesicle-bound uromodulin (P < 0.05) but not for total urinary uromodulin. These findings suggest that the levels of urinary microvesicle-bound uromodulin are associated with the severity of DKD. The uromodulin in urinary microvesicles may be a specific marker of DKD and potentially may be used to predict the onset and/or monitor the progression of DKD.


Assuntos
Albuminúria/diagnóstico , Diabetes Mellitus Tipo 2/urina , Nefropatias Diabéticas/diagnóstico , Uromodulina/urina , Adulto , Idoso , Albuminúria/urina , Biomarcadores/urina , Nefropatias Diabéticas/urina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
19.
Sci Rep ; 7(1): 956, 2017 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-28424476

RESUMO

The fidelity of synaptic transmission depends on the integrity of the protein machinery at the synapse. Unfolded synaptic proteins undergo refolding or degradation in order to maintain synaptic proteostasis and preserve synaptic function, and buildup of unfolded/toxic proteins leads to neuronal dysfunction. Many molecular chaperones contribute to proteostasis, but one in particular, cysteine string protein (CSPα), is critical for proteostasis at the synapse. In this study we report that exported vesicles from neurons contain CSPα. Extracellular vesicles (EV's) have been implicated in a wide range of functions. However, the functional significance of neural EV's remains to be established. Here we demonstrate that co-expression of CSPα with the disease-associated proteins, polyglutamine expanded protein 72Q huntingtinex°n1 or superoxide dismutase-1 (SOD-1G93A) leads to the cellular export of both 72Q huntingtinex°n1 and SOD-1G93A via EV's. In contrast, the inactive CSPαHPD-AAA mutant does not facilitate elimination of misfolded proteins. Furthermore, CSPα-mediated export of 72Q huntingtinex°n1 is reduced by the polyphenol, resveratrol. Our results indicate that by assisting local lysosome/proteasome processes, CSPα-mediated removal of toxic proteins via EVs plays a central role in synaptic proteostasis and CSPα thus represents a potential therapeutic target for neurodegenerative diseases.


Assuntos
Vesículas Extracelulares/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/citologia , Proteostase , Animais , Células Cultivadas , Humanos , Camundongos , Neurônios/metabolismo , Dobramento de Proteína , Sinapses/química , Sinapses/metabolismo
20.
Sci Rep ; 6: 32118, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27573465

RESUMO

The selectivity of (4Z)-2-(4-chloro-3-nitrophenyl)-4-(pyridin-3-ylmethylidene)-1,3-oxazol-5-one (DI) for zipper-interacting protein kinase (ZIPK) was previously described by in silico computational modeling, screening a large panel of kinases, and determining the inhibition efficacy. Our assessment of DI revealed another target, the Rho-associated coiled-coil-containing protein kinase 2 (ROCKII). In vitro studies showed DI to be a competitive inhibitor of ROCKII (Ki, 132 nM with respect to ATP). This finding was supported by in silico molecular surface docking of DI with the ROCKII ATP-binding pocket. Time course analysis of myosin regulatory light chain (LC20) phosphorylation catalyzed by ROCKII in vitro revealed a significant decrease upon treatment with DI. ROCKII signaling was investigated in situ in human coronary artery vascular smooth muscle cells (CASMCs). ROCKII down-regulation using siRNA revealed several potential substrates involved in smooth muscle contraction (e.g., LC20, Par-4, MYPT1) and actin cytoskeletal dynamics (cofilin). The application of DI to CASMCs attenuated LC20, Par-4, LIMK, and cofilin phosphorylations. Notably, cofilin phosphorylation was not significantly decreased with a novel ZIPK selective inhibitor (HS-38). In addition, CASMCs treated with DI underwent cytoskeletal changes that were associated with diminution of cofilin phosphorylation. We conclude that DI is not selective for ZIPK and is a potent inhibitor of ROCKII.


Assuntos
Vasos Coronários/enzimologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Oxazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Vasos Coronários/citologia , Humanos , Simulação de Acoplamento Molecular , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Oxazóis/química , Inibidores de Proteínas Quinases/química , Quinases Associadas a rho/química , Quinases Associadas a rho/metabolismo
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