RESUMEN
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.
Asunto(s)
COVID-19 , Síndrome de Dificultad Respiratoria , Insuficiencia Respiratoria , Humanos , COVID-19/complicaciones , Proteínas de Choque Térmico HSP27 , Inflamación , Pandemias , Estudios Prospectivos , Síndrome de Dificultad Respiratoria/terapia , SARS-CoV-2RESUMEN
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.
Asunto(s)
Cardiotoxicidad , ADN Mitocondrial , Animales , Ratones , ADN Mitocondrial/metabolismo , Inmunidad Innata , Interferones/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , FosforilaciónRESUMEN
(+)-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.
Asunto(s)
Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Ratones , Humanos , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Proteínas Nucleares , Factores de Transcripción/metabolismo , Aorta/metabolismo , Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteínas de Ciclo CelularRESUMEN
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.
Asunto(s)
Enfermedades Autoinmunes , Lupus Eritematoso Sistémico , Humanos , ADN Mitocondrial/genética , Inmunidad Innata/genética , Interferones , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismoRESUMEN
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.
Asunto(s)
Cardiomiopatía Hipertrófica , ADN-Topoisomerasas de Tipo I , Genoma Mitocondrial , Mitocondrias , Humanos , Recién Nacido , Cardiomiopatía Hipertrófica/genética , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Mitocondrial/metabolismo , Variación Genética , Mitocondrias/enzimología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismoRESUMEN
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.
Asunto(s)
Enfermedad de la Arteria Coronaria , Proproteína Convertasa 9 , Estudios de Casos y Controles , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Humanos , Lipoproteínas LDL , Receptores de LDL , SubtilisinaRESUMEN
[Figure: see text].
Asunto(s)
Aorta/efectos de los fármacos , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Colesterol/metabolismo , Proteínas de Choque Térmico/administración & dosificación , Chaperonas Moleculares/administración & dosificación , Proproteína Convertasa 9/metabolismo , Receptores de LDL/metabolismo , Vacunas Sintéticas/administración & dosificación , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos/sangre , Aorta/enzimología , Aorta/inmunología , Aorta/patología , Enfermedades de la Aorta/enzimología , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/patología , Aterosclerosis/enzimología , Aterosclerosis/inmunología , Aterosclerosis/patología , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Femenino , Proteínas de Choque Térmico/inmunología , Proteínas de Choque Térmico/metabolismo , Células Hep G2 , Humanos , Inmunogenicidad Vacunal , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Persona de Mediana Edad , Chaperonas Moleculares/inmunología , Chaperonas Moleculares/metabolismo , Placa Aterosclerótica , Receptores de LDL/genética , Vacunación , Vacunas Sintéticas/inmunologíaRESUMEN
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?
Asunto(s)
Aterosclerosis/prevención & control , Colesterol/sangre , Estradiol/administración & dosificación , Terapia de Reemplazo de Estrógeno , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico/administración & dosificación , Hígado/efectos de los fármacos , Chaperonas Moleculares/administración & dosificación , Proproteína Convertasa 9/metabolismo , Receptores de LDL/metabolismo , Vacunas/administración & dosificación , Animales , Aterosclerosis/sangre , Aterosclerosis/enzimología , Aterosclerosis/inmunología , Biomarcadores/sangre , Modelos Animales de Enfermedad , Regulación hacia Abajo , Implantes de Medicamentos , Femenino , Proteínas de Choque Térmico/inmunología , Células Hep G2 , Humanos , Hígado/enzimología , Menopausia , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Chaperonas Moleculares/inmunología , Ovariectomía , Proproteína Convertasa 9/genética , Receptores de LDL/genética , VacunaciónRESUMEN
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.
Asunto(s)
Antiinflamatorios/farmacología , Complejo Antígeno-Anticuerpo/farmacología , Aterosclerosis/prevención & control , Autoanticuerpos/inmunología , Proteínas de Choque Térmico HSP27/inmunología , Sistema Inmunológico/inmunología , Inflamación/prevención & control , Animales , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Ratones , FosforilaciónRESUMEN
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.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Músculo Liso Vascular/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Miosinas/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Arterias/citología , Arterias/metabolismo , Células Cultivadas , Proteínas Quinasas Asociadas a Muerte Celular/antagonistas & inhibidores , Proteínas Quinasas Asociadas a Muerte Celular/genética , Humanos , Músculo Liso Vascular/citología , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/genética , Fosfatasa de Miosina de Cadena Ligera/genética , Fosforilación , ARN Interferente Pequeño/genética , Suero/metabolismo , Transducción de Señal , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genéticaRESUMEN
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.
Asunto(s)
Antiinflamatorios/farmacología , Exosomas/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Secuencia de Aminoácidos , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Técnicas de Silenciamiento del Gen , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Humanos , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Chaperonas Moleculares , FN-kappa B/metabolismo , Células THP-1RESUMEN
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.
Asunto(s)
Vesículas Extracelulares/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/citología , Proteostasis , Animales , Células Cultivadas , Humanos , Ratones , Neuronas/metabolismo , Pliegue de Proteína , Sinapsis/química , Sinapsis/metabolismoRESUMEN
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.
Asunto(s)
Albuminuria/diagnóstico , Diabetes Mellitus Tipo 2/orina , Nefropatías Diabéticas/diagnóstico , Uromodulina/orina , Adulto , Anciano , Albuminuria/orina , Biomarcadores/orina , Nefropatías Diabéticas/orina , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
Vasos Coronarios/enzimología , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Oxazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidores , Vasos Coronarios/citología , Humanos , Simulación del Acoplamiento Molecular , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Oxazoles/química , Inhibidores de Proteínas Quinasas/química , Quinasas Asociadas a rho/química , Quinasas Asociadas a rho/metabolismoRESUMEN
Inhibition of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) represents a therapeutic target for treating hyperglycemia in type 2 diabetes. Here, we investigate the effects of 11ß-HSD1 on the innate immune response of adipocytes to produce proinflammatory cytokines. The 11ß-HSD1 inhibitor emodin, or 11ß-HSD1-targeted small interfering RNA, dose dependently suppressed IL-6, IL-1ß, and TNF-α expression in lipopolysaccharide-treated 3T3-L1 adipocytes. Inhibiting 11ß-HSD1 also reduced phosphatase and tensin homologue (PTEN) expression, a negative regulator of phosphatidylinositol 3-kinase effects, whereas 1pM cortisone or dexamethasone induced IL-6 and PTEN levels. PTEN-targeted small interfering RNA decreased IL-6, IL-1ß, and TNF-α without affecting 11ß-HSD1 levels. Correspondingly, emodin increased phosphorylated protein kinase B (p-PKB) (Ser473) to PKB ratio but not p-PKB (Thr308) to PKB ratio. Emodin did not increase the p-PKB (Ser473) to PKB ratio when the rapamycin-insensitive companion of mTOR was depleted, further supporting the involvement of mammalian target of rapamycin complex 2 in PKB phosphorylation. Moreover, emodin suppressed phosphorylated inhibitor of κB α (p-IκBα) to IκBα ratio and reduced nuclear factor κ B subunit p50 in the nuclear fraction. In contrast, 1pM cortisone or dexamethasone decreased p-PKB (Ser473) to PKB ratio, increased p-IκBα to IκBα ratio, and increased nuclear NF-κB subunit p50. Additionally, wortmannin had similar effects on IL-6, p-PKB (Ser473) to PKB ratio, and p-IκBα to IκBα ratio as 1pM cortisone or dexamethasone. Finally, emodin treatment of streptozotocin diabetic rats on a high-fat diet reduced levels of IL-6, PTEN, Cluster of Differentiation 68, and the ratio of p-IκBα to IκBα in visceral fat, indicating that our findings in vitro may also apply to visceral fat in vivo. Together, these results suggest that inhibiting 11ß-HSD1 reduces lipopolysaccharide-induced proinflammatory innate immune responses in adipocytes by down-regulating PTEN expression, leading to activation of the PI3K/PKB pathway.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Adipocitos/metabolismo , Citocinas/metabolismo , Inmunidad Innata/fisiología , Lipopolisacáridos/farmacología , Fosfohidrolasa PTEN/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Animales , Diabetes Mellitus Experimental/metabolismo , Dieta Alta en Grasa , Emodina/farmacología , Inmunidad Innata/efectos de los fármacos , Masculino , Ratones , Fosfohidrolasa PTEN/genética , ARN Interferente Pequeño , RatasRESUMEN
Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC). Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1ß mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude that ROCK1 and ZIPK have diverse, but predominantly distinct regulatory functions in vascular SMC and that ROCK1-mediated activation of ZIPK is not involved in most of these functions.
Asunto(s)
Arterias , Proteínas Quinasas Asociadas a Muerte Celular/genética , Regulación de la Expresión Génica , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Quinasas Asociadas a rho/genética , Proliferación Celular , Supervivencia Celular/genética , Células Cultivadas , Análisis por Conglomerados , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Miosinas/metabolismo , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Quinasas Asociadas a rho/metabolismoRESUMEN
As pro-inflammatory cytokines and chemokines contribute to the malignancy of many types of human cancer, we examined the anti-inflammatory effect of bisbibenzyls, a diverse bioactive group of naturally occurring compounds. Marchantin M (Mar M) was identified through a screening process of these compounds as a potent anti-inflammatory agent based on its capacity to inhibit LPS-induced IL6, IL1ß and CCL2 expression in HUVECs and PBMCs without affecting cell proliferation. Since Mar M has been found to exhibit anticancer activity, we observed that Mar M treatment also resulted in decreases in the expressions of IL6, IL1ß and TNFα in metastatic prostate cancer (PCa) cells. This effect was further confirmed in other cancer cell lines that express high level of pro-inflammatory cytokines. Furthermore, inactivation of NF-κB, a critical transcription factor controlling many pro-inflammatory cytokine expressions, was observed in Mar M-treated PCa cells as evidenced by decreased phosphor-p65 and subsequently phosphor-STAT3. Mar M also suppressed phosphorylation of IKBα, an inhibitor of NF-κB in the cytosol. However, reduced phosphor-p65 by Mar M was slightly increased when knockdown of IKBα, suggesting that Mar M may target upstream molecules of IKBα/NF-κB signaling. Finally, treatment with Mar M resulted in more enhanced-sensitivity of PCa cells to docetaxel-induced apoptosis than that of the IL6 blocking. Our study demonstrates the potential of the anti-inflammatory agent Mar M as an adjuvant to improve the efficacy of traditional anticancer agents such as docetaxel.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Próstata/patología , Antiinflamatorios/farmacología , Bibencilos/farmacología , Supervivencia Celular/efectos de los fármacos , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Docetaxel , Relación Dosis-Respuesta a Droga , Células HT29 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Mediadores de Inflamación/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Células K562 , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Masculino , Inhibidor NF-kappaB alfa , Éteres Fenílicos/farmacología , Fosforilación , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Interferencia de ARN , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Taxoides/farmacología , Factores de Tiempo , Factor de Transcripción ReIA/metabolismo , Transfección , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Changes in circulatory aminopeptidases [dipeptidyl-peptidase-IV (DPP-IV), Prolyl-oligopeptidase (POP) and Leucine aminopeptidase (LAP)] activities have been found to be associated with psychiatric illnesses and inflammatory diseases. METHODS: The discriminatory indices of aminopeptidases activities were assessed by enzymatic assays in plasma samples from 240 unipolar depression (UD) patients and 264 matched controls. In addition the relationship between soluble and cellular DPP-IV activity was determined in plasma and blood cells from healthy subjects. RESULTS: Greater than 95% of the plasma DPP-IV activity could be blocked by inhibitors, demonstrating the specificity of the assay. Also, DPP-IV protein and activity levels were strongly correlated. In contrast, only 50% of the membrane-bound activity in blood cells was inhibited, which suggested that other similar peptidases may be present in these cells. UD patients had decreased plasma levels of DPP-IV and POP activities compared to healthy controls with a concomitant increase in LAP activity. Finally, testing of the LAP/DPP-IV ratio resulted in good discrimination of UD patients from controls with an area under the curve-receiver operating characteristic of 0.70. LIMITATIONS: Further biological validation studies using different cohorts are warranted. CONCLUSIONS: The finding that plasma DPP-IV activity was decreased and LAP activity was increased in UD patients suggests the potential value for testing the levels of these enzymes for improved classification of patients. In addition, the changes in these enzymes, suggests that the proteolytic maturation of their proneuropeptide and prohormone subtrates may also be affected in UD, resulting in altered production of the associated bioactive peptides.
Asunto(s)
Trastorno Depresivo/enzimología , Dipeptidil Peptidasa 4/metabolismo , Leucil Aminopeptidasa/metabolismo , Serina Endopeptidasas/metabolismo , Adulto , Biomarcadores/sangre , Biomarcadores/metabolismo , Estudios de Cohortes , Trastorno Depresivo/sangre , Dipeptidil Peptidasa 4/sangre , Femenino , Humanos , Leucil Aminopeptidasa/sangre , Masculino , Persona de Mediana Edad , Prolil Oligopeptidasas , Serina Endopeptidasas/sangreRESUMEN
microRNAs (miRNAs) are small noncoding RNAs that regulate genes and contribute to many kinds of human diseases, including cancer. Two miRNAs, miR-511 and miR-1297, were investigated for a possible role in adenocarcinoma based on predicted binding sites for the TRIB2 oncogene by microRNA analysis software, and the pcDNA-GFP-TRIB2-3'UTR vector was constructed to investigate the interaction between TRIB2 and miR-511/1297 in the adenocarcinoma cell line A549. Green fluorescent protein (GFP) expression was estimated by fluorescence microscopy and flow cytometry after A549 cells were co-transfected with miR-511 (or miR-1297) and pcDNA-GFP-TRIB2-3'UTR vector. The expression of GFP in the miR-511- and miR-1297-treated cells was significantly downregulated in contrast with the negative-control (NC) miRNA-treated cells. The decreased expression of TRIB2 was further detected after miR-511 (or miR-1297) treatment by western blotting. The MTT test showed inhibition of A549 cell proliferation and Annexin V-FITC/PI dual staining showed increased apoptosis in the miR-511- and miR-1297-treated cells compared to the NC cultures. A transcription factor downstream of TRIB2, the CCAAT/enhancer-binding protein alpha (C/EBPα), was expression at higher levels after miR-511 (or miR-1297) decreasing TRIB2 expression. Our results illustrate that miR-511 and miR-1297 act as tumor suppressor genes, which could suppress A549 cell proliferation in vitro and in vivo by suppressing TRIB2 and further increasing C/EBPα expression.
Asunto(s)
Adenocarcinoma/patología , Proliferación Celular , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Pulmonares/patología , MicroARNs/fisiología , Oncogenes , Regiones no Traducidas 3' , Adenocarcinoma/genética , Animales , Secuencia de Bases , Western Blotting , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Línea Celular Tumoral , Cartilla de ADN , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Fluorescente , Reacción en Cadena de la PolimerasaRESUMEN
The present study was designed to identify the changes in microvesicle-dipeptidyl peptidase-IV (DPP IV) levels in human urine and serum, and to determine whether there were correlations with the severity of diabetic kidney disease (DKD). A total of 127 patients with type 2 diabetes mellitus (T2DM) were divided into three groups according to the urinary albumin/ creatinine ratio (UACR): microalbuminuria group (n = 50); macroalbuminuria group (n = 34) and normoalbuminuria group (n = 43), and 34 age- and sex-matched non-diabetic healthy subjects were selected as controls. Microvesicle-bound DPP IV and free urinary DPP IV were separated by a filtra-centrifugation method. The total microvesicles were captured by a specific monoclonal antibody, AD-1. DPP IV activity was determined by measuring the cleavage of chromogenic free 4-nitroaniline from Gly-Pro-p-nitroanilide at 405 nm with an ELISA plate reader. DPP IV protein levels were determined by ELISA and Western blot. Our results showed that the microvesicle-bound type was the major form of DPP IV in urine; the urinary microvesicle-DPP IV excretion of each T2DM group was significantly higher compared with controls. The urinary microvesicle-DPP IV level was positively correlated with UACR in patients with T2DM. These findings suggest that the urinary level of microvesicle-bound DPP IV is associated with the severity of DKD.