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1.
Exp Mol Med ; 56(5): 1206-1220, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38760513

RESUMO

The etiology of preeclampsia (PE), a severe complication of pregnancy with several clinical manifestations and a high incidence of maternal and fetal morbidity and mortality, remains unclear. This issue is a major hurdle for effective treatment strategies. We recently demonstrated that PE exhibits an Alzheimer-like etiology of impaired autophagy and proteinopathy in the placenta. Targeting of these pathological pathways may be a novel therapeutic strategy for PE. Stimulation of autophagy with the natural disaccharide trehalose and its lacto analog lactotrehalose in hypoxia-exposed primary human trophoblasts restored autophagy, inhibited the accumulation of toxic protein aggregates, and restored the ultrastructural features of autophagosomes and autolysosomes. Importantly, trehalose and lactotrehalose inhibited the onset of PE-like features in a humanized mouse model by normalizing autophagy and inhibiting protein aggregation in the placenta. These disaccharides restored the autophagy-lysosomal biogenesis machinery by increasing nuclear translocation of the master transcriptional regulator TFEB. RNA-seq analysis of the placentas of mice with PE indicated the normalization of the PE-associated transcriptome profile in response to trehalose and lactotrehalose. In summary, our results provide a novel molecular rationale for impaired autophagy and proteinopathy in patients with PE and identify treatment with trehalose and its lacto analog as promising therapeutic options for this severe pregnancy complication.


Assuntos
Autofagia , Lisossomos , Pré-Eclâmpsia , Trealose , Autofagia/efeitos dos fármacos , Pré-Eclâmpsia/tratamento farmacológico , Pré-Eclâmpsia/metabolismo , Feminino , Humanos , Gravidez , Animais , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Trealose/análogos & derivados , Trealose/farmacologia , Trealose/uso terapêutico , Camundongos , Trofoblastos/metabolismo , Trofoblastos/efeitos dos fármacos , Trofoblastos/patologia , Placenta/metabolismo , Placenta/efeitos dos fármacos , Modelos Animais de Doenças
2.
Biochim Biophys Acta Mol Basis Dis ; 1870(3): 167031, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38253214

RESUMO

Chronic psychological stress contributes to the occurrence of cancer and activates the renin-angiotensin system (RAS). However, the mechanisms by which RAS promotes the progression of breast cancer (BRCA) under chronic psychological stress are remain unknown. In this study, we observed elevated levels of Angiotensin II (Ang II) in both serum and BRCA tissue under chronic stress, leading to accelerated BRCA growth in a mouse model. An antihypertensive drug, candesartan (an AT1 inhibitor), effectively attenuated Ang II-induced cell proliferation and metastasis. Utilizing mass spectrometry and weighted gene co-expression network analysis (WGCNA), we identified fibronectin 1 (FN1) as the hub protein involved in chronic stress-Ang II/AT1 axis. Focal adhesion pathway was identified as a downstream signaling pathway activated during the progression of chronic stress. Depletion of FN1 significantly attenuated Ang II-induced proliferation and metastasis of BRCA cells. Poly (ADP-ribose) polymerase 1 (PARP1) was found to bind to the DNA promoter of FN1, leading to the transcription of FN1. Ang II upregulated PARP1 expression, resulting in increased FN1 levels. Recombinant FN1 partially restored the progress of BRCA malignancy induced by the Ang II/PARP1 pathway. In vivo, candesartan reversed the progressive effect of chronic psychological stress on BRCA. In clinical samples, Ang II levels in both serum and tumor tissues are higher in stressed patients compared to control patients. Serum Ang II levels were positively correlated with chronic stress indicators. In conclusion, our study demonstrated that chronic psychological stress accelerates the malignancy of BRCA, and the AT1 inhibitor candesartan counteracts these effects by suppressing the Ang II-AT1 axis and the downstream PARP1/FN1/focal adhesion pathway.


Assuntos
Angiotensina II , Benzimidazóis , Compostos de Bifenilo , Neoplasias da Mama , Tetrazóis , Camundongos , Animais , Humanos , Feminino , Angiotensina II/metabolismo , Anti-Hipertensivos , Fibronectinas , Neoplasias da Mama/tratamento farmacológico , Poli(ADP-Ribose) Polimerase-1/genética
3.
Biology (Basel) ; 12(8)2023 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-37626934

RESUMO

Our prior studies have shown that protein misfolding and aggregation in the placenta are linked to the development of preeclampsia, a severe pregnancy complication. We identified transthyretin (TTR) as a key component of the aggregated protein complex. However, the regulation of native TTR in normal pregnancy remains unclear. In this study, we found that pregnant mice exhibited a remarkable and progressive decline in serum TTR levels through gestational day (gd) 12-14, followed by an increase in late pregnancy and postpartum. Meanwhile, serum albumin levels showed a modest but statistically significant increase throughout gestation. TTR protein and mRNA levels in the liver, a primary source of circulating TTR, mirrored the changes observed in serum TTR levels during gestation. Intriguingly, a similar pattern of TTR alteration was also observed in the serum of pregnant women and pregnant interleukin-10-knockout (IL-10-/-) mice with high inflammation background. In non-pregnant IL-10-/- mice, serum TTR levels were significantly lower than those in age-matched wild-type mice. Administration of IL-10 to non-pregnant IL-10-/- mice restored their serum TTR levels. Notably, dysregulation of TTR resulted in fewer implantation units, lower fetal weight, and smaller litter sizes in human TTR-overexpressing transgenic mice. Thus, TTR may play a pivotal role as a crucial regulator in normal pregnancy, and inflammation during pregnancy may contribute to the downregulation of serum TTR presence.

4.
J Pineal Res ; 75(1): e12870, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37002641

RESUMO

Arylacetamide deacetylase (AADAC) is a deacetylation enzyme present in the mammalian liver, gastrointestinal tract, and brain. During our search for mammalian enzymes capable of metabolizing N-acetylserotonin (NAS), AADAC was identified as having the ability to convert NAS to serotonin. Both human and rodent recombinant AADAC proteins can deacetylate NAS in vitro, although the human AADAC shows markedly higher activity compared with rodent enzyme. The AADAC-mediated deacetylation reaction can be potently inhibited by eserine in vitro. In addition to NAS, recombinant hAADAC can deacetylate melatonin (to form 5-methoxytryptamine) and N-acetyltryptamine (NAT) (to form tryptamine). In addition to the in vitro deacetylation of NAS by the recombinant AADAC proteins, liver (mouse and human) and brain (human) extracts were able to deacetylate NAS; these activities were sensitive to eserine. Taken together, these results demonstrate a new role for AADAC and suggest a novel pathway for the AADAC-mediated metabolism of pineal indoles in mammals.


Assuntos
Hidrolases de Éster Carboxílico , Melatonina , Animais , Humanos , Camundongos , Hidrolases de Éster Carboxílico/metabolismo , Mamíferos/metabolismo , Fisostigmina , Serotonina
5.
Int J Mol Sci ; 23(10)2022 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-35628454

RESUMO

We have previously described that placental activation of autophagy is a central feature of normal pregnancy, whereas autophagy is impaired in preeclampsia (PE). Here, we show that hypoxia-reoxygenation (H/R) treatment dysregulates key molecules that maintain autophagy-lysosomal flux in primary human trophoblasts (PHTs). Ultrastructural analysis using transmission electron microscopy reveals a significant reduction in autophagosomes and autolysosomes in H/R-exposed PHTs. H/R-induced accumulation of protein aggregates follows a similar pattern that occurs in PHTs treated with a lysosomal disruptor, chloroquine. Importantly, the placenta from early-onset PE deliveries exhibits the same features as seen in H/R-treated PHTs. Taken together, our results indicate that H/R disrupts autophagic machinery in PHTs and that impaired autophagy in the placenta from early-onset PE deliveries mimics the events in H/R-treated PHTs. Notably, assessment of key regulators at each stage of autophagic processes, especially lysosomal integrity, and verification of autophagic ultrastructure are essential for an accurate evaluation of autophagy activity in human trophoblasts and placental tissue from PE deliveries.


Assuntos
Pré-Eclâmpsia , Trofoblastos , Autofagia/fisiologia , Feminino , Humanos , Hipóxia/metabolismo , Lisossomos/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Gravidez , Trofoblastos/metabolismo
6.
Hypertension ; 79(8): 1738-1754, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35607996

RESUMO

BACKGROUND: We have demonstrated that protein aggregation plays a pivotal role in the pathophysiology of preeclampsia and identified several aggregated proteins in the circulation of preeclampsia patients, the most prominent of which is the serum protein TTR (transthyretin). However, the mechanisms that underlie protein aggregation remain poorly addressed. METHODS: We examined TTR aggregates in hypoxia/reoxygenation-exposed primary human trophoblasts (PHTs) and the preeclampsia placenta using complementary approaches, including a novel protein aggregate detection assay. Mechanistic analysis was performed in hypoxia/reoxygenation-exposed PHTs and Ttr transgenic mice overexpressing transgene-encoded wild-type human TTR or Ttr-/- mice. High-resolution ultrasound analysis was used to measure placental blood flow in pregnant mice. RESULTS: TTR aggregation was inducible in PHTs and the TCL-1 trophoblast cell line by endoplasmic reticulum stress inducers or autophagy-lysosomal disruptors. PHTs exposed to hypoxia/reoxygenation showed increased intracellular BiP (binding immunoglobulin protein), phosphorylated IRE1α (inositol-requiring enzyme-1α), PDI (protein disulfide isomerase), and Ero-1, all markers of the unfolded protein response, and the apoptosis mediator caspase-3. Blockade of IRE1α inhibited hypoxia/reoxygenation-induced upregulation of Ero-1 in PHTs. Excessive unfolded protein response activation was observed in the early-onset preeclampsia placenta. Importantly, pregnant human TTR mice displayed aggregated TTR in the junctional zone of the placenta and severe preeclampsia-like features. High-resolution ultrasound analysis revealed low blood flow in uterine and umbilical arteries in human TTR mice compared with control mice. However, Ttr-/- mice did not show any pregnancy-associated abnormalities. CONCLUSIONS: These observations in the preeclampsia placenta, cultured trophoblasts, and Ttr transgenic mice indicate that TTR aggregation is an important causal contributor to preeclampsia pathophysiology.


Assuntos
Pré-Eclâmpsia , Trofoblastos , Animais , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Feminino , Humanos , Hipóxia/metabolismo , Camundongos , Camundongos Transgênicos , Placenta/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Pré-Albumina/análise , Pré-Albumina/genética , Pré-Albumina/metabolismo , Gravidez , Agregados Proteicos , Proteínas Serina-Treonina Quinases , Trofoblastos/metabolismo
7.
Front Cell Infect Microbiol ; 11: 694298, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34485175

RESUMO

Understanding of sterile inflammation and its associated biological triggers and diseases is still at the elementary stage. This becomes more warranted in cases where infections are not associated with the pathology. Detrimental effects of bacterial and viral infections on the immune responses at the maternal-fetal interface as well as pregnancy outcomes have been well documented. However, an infection-induced etiology is not thought to be a major contributing component to severe pregnancy complications such as preeclampsia (PE) and gestational diabetes. How is then an inflammatory signal thought to be associated with these pregnancy complications? It is not clear what type of inflammation is involved in the onset of PE-like features. We opine that sterile inflammation regulated by the inflammasome-gasdermins-caspase-1 axis is a contributory factor to the onset of PE. We hypothesize that increased production and release of damage-associated molecular patterns (DAMPs) or Alarmins such as high-mobility group box1 (HMGB1), cell-free fetal DNA, uric acid, the NOD-like receptor pyrin-containing receptor 3 (NLRP3) inflammasome, IL-1ß and IL-18 occur in the PE placenta. Some of these molecules have already been observed in the placenta from women with PE. Mechanistically, emerging evidence has demonstrated that excessive placental endoplasmic reticulum (ER) stress, impaired autophagy and gasdermine D (GSDMD)-mediated intrinsic pyroptosis are key events that contribute to systemic sterile inflammation in patients with PE, especially early-onset PE (e-PE). In this review, we highlight the advances on the roles of sterile inflammation and inflammatory signaling cascades involving ER stress, autophagy deficiency and pyroptosis in PE pathophysiology. Deciphering the mechanisms underlying these inflammatory pathways may provide potential diagnostic biomarkers and facilitate the development of therapeutic strategies to treat this devastating disease.


Assuntos
Pré-Eclâmpsia , Feminino , Humanos , Inflamassomos , Inflamação , Proteína 3 que Contém Domínio de Pirina da Família NLR , Placenta , Pré-Eclâmpsia/etiologia , Gravidez
8.
Sci Rep ; 11(1): 15934, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354200

RESUMO

A non-invasive and sensitive blood test has long been a goal for early stage disease diagnosis and treatment for Alzheimer's disease (AD) and other proteinopathy diseases. We previously reported that preeclampsia (PE), a severe pregnancy complication, is another proteinopathy disorder with impaired autophagy. We hypothesized that induced autophagy deficiency would promote accumulation of pathologic protein aggregates. Here, we describe a novel, sensitive assay that detects serum protein aggregates from patients with PE (n = 33 early onset and 33 late onset) and gestational age-matched controls (n = 77) as well as AD in both dementia and prodromal mild cognitive impairment (MCI, n = 24) stages with age-matched controls (n = 19). The assay employs exposure of genetically engineered, autophagy-deficient human trophoblasts (ADTs) to serum from patients. The aggregated protein complexes and their individual components, including transthyretin, amyloid ß-42, α-synuclein, and phosphorylated tau231, can be detected and quantified by co-staining with ProteoStat, a rotor dye with affinity to aggregated proteins, and respective antibodies. Detection of protein aggregates in ADTs was not dependent on transcriptional upregulation of these biomarkers. The ROC curve analysis validated the robustness of the assay for its specificity and sensitivity (PE; AUC: 1, CI: 0.949-1.00; AD; AUC: 0.986, CI: 0.832-1.00). In conclusion, we have developed a novel, noninvasive diagnostic and predictive assay for AD, MCI and PE.


Assuntos
Doença de Alzheimer/sangue , Análise Química do Sangue/métodos , Pré-Eclâmpsia/sangue , Adulto , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides , Biomarcadores/sangue , Proteínas Sanguíneas/análise , Disfunção Cognitiva/diagnóstico , Feminino , Testes Hematológicos/métodos , Humanos , Imuno-Histoquímica , Fragmentos de Peptídeos , Pré-Eclâmpsia/diagnóstico , Gravidez , Agregados Proteicos/fisiologia , Curva ROC , Trofoblastos/efeitos dos fármacos , alfa-Sinucleína , Proteínas tau
9.
Autophagy ; 16(10): 1771-1785, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-31856641

RESUMO

The etiology of preeclampsia (PE), a serious pregnancy complication, remains an enigma. We have demonstrated that proteinopathy, a pathologic feature of neurodegenerative diseases, is a key observation in the placenta and serum from PE patients. We hypothesize that the macroautophagy/autophagy machinery that mediates degradation of aggregated proteins and damaged organelles is impaired in PE. Here, we show that TFEB (transcription factor EB), a master transcriptional regulator of lysosomal biogenesis, and its regulated proteins, LAMP1, LAMP2, and CTSD (cathepsin D), were dysregulated in the placenta from early and late onset PE deliveries. Primary human trophoblasts and immortalized extravillous trophoblasts (EVTs) showed reduced TFEB expression and nuclear translocation as well as lysosomal protein content in response to hypoxia. Hypoxia-exposed trophoblasts also showed decreased PPP3/calcineurin phosphatase activity and increased XPO1/CRM1 (exportin 1), events that inhibit TFEB nuclear translocation. These proteins were also dysregulated in the PE placenta. These results are supported by observed lysosomal ultrastructural defects with decreased number of autolysosomes in hypoxia-treated primary human trophoblasts. Autophagy-deficient human EVTs exhibited poor TFEB nuclear translocation, reduced lysosomal protein expression and function, and increased MTORC1 activity. Sera from PE patients induced these features and protein aggregation in EVTs. Importantly, trophoblast-specific conditional atg7 knockout mice exhibited reduced TFEB expression with increased deposition of protein aggregates in the placenta. These results provide compelling evidence for a regulatory link between accumulation of protein aggregates and TFEB-mediated impaired lysosomal biogenesis and autophagy in the placenta of PE patients. Abbreviation:atg7: autophagy related 7; CTSD: cathepsin D; ER: endoplasmic reticulum; EVTs: extravillous trophoblasts; KRT7: keratin 7; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; mSt: mStrawberry; MTORC1: mechanistic target of rapamycin complex 1; NP: normal pregnancy; NPS: normal pregnancy serum; PE: preeclampsia; PES: preeclampsia serum; p-RPS6KB: phosphorylated ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; XPO1/CRM1: exportin 1.


Assuntos
Autofagia , Hipóxia , Lisossomos/metabolismo , Pré-Eclâmpsia/metabolismo , Proteoma/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Catepsina D/biossíntese , Linhagem Celular , Citoplasma/metabolismo , Feminino , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/biossíntese , Proteínas de Membrana Lisossomal/biossíntese , Camundongos , Camundongos Knockout , Placenta/metabolismo , Gravidez , Prenhez , Proteína Sequestossoma-1/biossíntese , Trofoblastos/metabolismo
10.
Cell Death Dis ; 10(12): 927, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804457

RESUMO

Systemic manifestation of preeclampsia (PE) is associated with circulating factors, including inflammatory cytokines and damage-associated molecular patterns (DAMPs), or alarmins. However, it is unclear whether the placenta directly contributes to the increased levels of these inflammatory triggers. Here, we demonstrate that pyroptosis, a unique inflammatory cell death pathway, occurs in the placenta predominantly from early onset PE, as evidenced by elevated levels of active caspase-1 and its substrate or cleaved products, gasdermin D (GSDMD), IL-1ß, and IL-18. Using cellular models mimicking pathophysiological conditions (e.g., autophagy deficiency, hypoxia, and endoplasmic reticulum (ER) stress), we observed that pyroptosis could be induced in autophagy-deficient human trophoblasts treated with sera from PE patients as well as in primary human trophoblasts exposed to hypoxia. Exposure to hypoxia elicits excessive unfolded protein response (UPR) and ER stress and activation of the NOD-like receptor pyrin-containing 3 (NLRP3) inflammasome in primary human trophoblasts. Thioredoxin-interacting protein (TXNIP), a marker for hyperactivated UPR and a crucial signaling molecule linked to NLRP3 inflammasome activation, is significantly increased in hypoxia-treated trophoblasts. No evidence was observed for necroptosis-associated events. Importantly, these molecular events in hypoxia-treated human trophoblasts are significantly observed in placental tissue from women with early onset PE. Taken together, we propose that placental pyroptosis is a key event that induces the release of factors into maternal circulation that possibly contribute to severe sterile inflammation and early onset PE pathology.


Assuntos
Proteínas de Transporte/genética , Inflamação/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Pré-Eclâmpsia/genética , Adulto , Hipóxia Celular/genética , Estresse do Retículo Endoplasmático/genética , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Ligação a Fosfato/genética , Placenta/metabolismo , Placenta/patologia , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Piroptose/genética , Transdução de Sinais , Trofoblastos/metabolismo , Trofoblastos/patologia , Resposta a Proteínas não Dobradas/genética
11.
Endocrinology ; 158(9): 2837-2847, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28645193

RESUMO

Aberrant proinflammatory and suppressed anti-inflammatory (alternative; M2) macrophage activation underlies the chronic inflammation associated with obesity and other metabolic disorders. This study demonstrates a critical role for interferon regulatory factor 6 (IRF6) in regulating macrophage M2 activation by suppressing peroxisome proliferator-activated receptor-γ (PPARγ) expression, a critical regulator of alternative macrophage polarization. The data demonstrate suppression of IRF6 in both M2 macrophages and obese adipose tissue macrophages. Using gain- and loss-of-function strategies, we confirmed that IRF6 knockdown enhanced M2 activation, whereas IRF6 overexpression dramatically attenuated M2 activation. Computational target prediction analysis coupled with chromatin immunoprecipitation indicated that IRF6 suppresses PPARγ through binding IRF recognition sites located upstream of the PPARγ coding region. Taken together, our results suggest that an IRF6/PPARγ regulatory axis suppresses anti-inflammatory responses in bone marrow-derived macrophages and provides references for future study addressing dysregulated metabolic and immunologic homeostasis of obese adipose tissue.


Assuntos
Fatores Reguladores de Interferon/fisiologia , Ativação de Macrófagos/genética , Macrófagos/metabolismo , PPAR gama/genética , Animais , Células Cultivadas , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Inflamação/genética , Inflamação/metabolismo , Fatores Reguladores de Interferon/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/metabolismo , Transdução de Sinais/genética
12.
Sci Rep ; 7: 39885, 2017 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-28071720

RESUMO

The intrauterine environment is particularly vulnerable to environmental exposures. We previously established a mouse model that provided evidence for pregnancy complications and placental anti-angiogenesis in response to Aroclor 1254 (A-1254), a mixture of polychlorinated biphenyls (PCBs). Importantly, these effects were observed in IL-10-/-, but not wild type, mice, suggesting that IL-10 deficiency predisposes to pregnancy disruptive effects of environmental toxicants. However, the mechanisms by which PCBs cause anti-angiogenic effects are unclear. Here, we evaluated PCB-mediated anti-angiogenic effects by diverse but complementary approaches, including HUVEC-mediated trophoblast invasion in nude mice, in vitro three-dimensional capillary tube formation involving HUVEC and/or HTR8 trophoblasts, and aortic ring endothelial cell outgrowth/sprouting. Taken together, our data suggest that PCBs act as potent anti-angiogenic agents. Importantly, we show that treatment of pregnant IL-10-/- mice with A-1254 resulted in placental activation of the Notch/Delta-like ligand (Dll) pathway, a master regulator of cell-cell interaction and vascular patterning. Similar results were obtained with HUVEC and HTR8 trophoblasts. Rescue of A-1254-induced disruption of HUVEC-based tube formation by γ-secretase inhibitor L1790 confirmed the critical role of the Notch/Dll pathway. Our data suggest that PCBs impart pregnancy disruptive functions by activating the Notch/Dll pathway and by inducing anti-angiogenic effects at the maternal-fetal interface.


Assuntos
Inibidores da Angiogênese/toxicidade , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Bifenilos Policlorados/toxicidade , Complicações na Gravidez/metabolismo , Receptor Notch4/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação ao Cálcio , Modelos Animais de Doenças , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Interleucina-10/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Complicações na Gravidez/induzido quimicamente , Transdução de Sinais
13.
J Biol Chem ; 291(33): 17178-96, 2016 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-27339900

RESUMO

Serotonin N-acetyltransferase (AANAT) converts serotonin to N-acetylserotonin (NAS), a distinct biological regulator and the immediate precursor of melatonin, a circulating hormone that influences circadian processes, including sleep. N-terminal sequences of AANAT enzymes vary among vertebrates. Mechanisms that regulate the levels of AANAT are incompletely understood. Previous findings were consistent with the possibility that AANAT may be controlled through its degradation by the N-end rule pathway. By expressing the rat and human AANATs and their mutants not only in mammalian cells but also in the yeast Saccharomyces cerevisiae, and by taking advantage of yeast genetics, we show here that two "complementary" forms of rat AANAT are targeted for degradation by two "complementary" branches of the N-end rule pathway. Specifically, the N(α)-terminally acetylated (Nt-acetylated) Ac-AANAT is destroyed through the recognition of its Nt-acetylated N-terminal Met residue by the Ac/N-end rule pathway, whereas the non-Nt-acetylated AANAT is targeted by the Arg/N-end rule pathway, which recognizes the unacetylated N-terminal Met-Leu sequence of rat AANAT. We also show, by constructing lysine-to-arginine mutants of rat AANAT, that its degradation is mediated by polyubiquitylation of its Lys residue(s). Human AANAT, whose N-terminal sequence differs from that of rodent AANATs, is longer-lived than its rat counterpart and appears to be refractory to degradation by the N-end rule pathway. Together, these and related results indicate both a major involvement of the N-end rule pathway in the control of rodent AANATs and substantial differences in the regulation of rodent and human AANATs that stem from differences in their N-terminal sequences.


Assuntos
Arilalquilamina N-Acetiltransferase/metabolismo , Mutação , Proteólise , Ubiquitinação/fisiologia , Acetilação , Animais , Arilalquilamina N-Acetiltransferase/genética , Células HEK293 , Humanos , Ratos , Saccharomyces cerevisiae
14.
Sci Rep ; 6: 19455, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26781643

RESUMO

Cisplatin resistance is a major obstacle in the treatment of NSCLC, and its mechanism has not been fully elucidated. The objectives of the study were to determine the role of miR-378 in the sensitivity of lung adenocarcinoma cells to cisplatin (cDDP) and its working mechanism. With TargetScan and luciferase assay, miR-378 was found to directly target sCLU. miR-378 and sCLU were regulated in A549/cDDP and Anip973/cDDP cells to investigate the effect of miR-378 on the sensitivity and apoptotic effects of cDDP. The effect of miR-378 upregulation on tumor growth was analyzed in a nude mouse xenograft model. The correlation between miR-378 and chemoresistance was tested in patient samples. We found that upregulation of miR-378 in A549/cDDP and Anip973/cDDP cells significantly down-regulated sCLU expression, and sensitized these cells to cDDP. miR-378 overexpression inhibited tumor growth and sCLU expression in a xenograft animal model. Analysis of human lung adenocarcinoma tissues revealed that the cDDP sensitive group expressed higher levels of miR-378 and lower levels of sCLU. miR-378 and sCLU were negatively correlated. To conclude, we identified sCLU as a novel miR-378 target, and we showed that targeting sCLU via miR-378 may help disable the chemoresistance against cisplatin in lung adenocarcinoma cells.


Assuntos
Adenocarcinoma/genética , Cisplatino/farmacologia , Clusterina/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , MicroRNAs/genética , Interferência de RNA , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Clusterina/química , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos Nus , MicroRNAs/química , Ensaios Antitumorais Modelo de Xenoenxerto
15.
J Clin Psychopharmacol ; 34(4): 495-8, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24943388

RESUMO

OBJECTIVE: This study aims to investigate the effects of low-dose olanzapine on preventing occurrence and controlling the severity of nausea and vomiting related to duloxetine in treating major depressive disorder. METHODS: Two hundred sixty-eight subjects with major depressive disorder were divided into control and trial groups. The control group had 165 subjects and was treated with duloxetine only, whereas the trial group had 103 subjects and received duloxetine combined with low-dose olanzapine. After the treatment for 2 weeks, both groups were evaluated for occurrence and severity of adverse effects on digestive tract using the Treatment Emergent Symptom Scale. RESULTS: The trial group, scored at 13, showed significantly less occurrence and severity of nausea and vomiting than the control group, scored at 38 (P < 0.05). The occurrence of nausea and vomiting in the trial group is significantly lower compared with that in the control group, and the occurrence decreases as the dose of olanzapine increases (P < 0.05). The antiemetic effect of olanzapine is valid to all subjects who received it. CONCLUSIONS: Duloxetine combined with low-dose olanzapine can effectively reduce the occurrence and severity of duloxetine-related nausea and vomiting. With the increase of olanzapine dose, the antiemetic effect becomes stronger. Olanzapine is a safe and efficient drug for the prevention of duloxetine-related nausea and vomiting.


Assuntos
Antieméticos/administração & dosagem , Benzodiazepinas/administração & dosagem , Transtorno Depressivo Maior/tratamento farmacológico , Náusea/tratamento farmacológico , Tiofenos/administração & dosagem , Vômito/tratamento farmacológico , Adolescente , Adulto , Antidepressivos/administração & dosagem , Antidepressivos/efeitos adversos , Transtorno Depressivo Maior/diagnóstico , Transtorno Depressivo Maior/psicologia , Esquema de Medicação , Quimioterapia Combinada , Cloridrato de Duloxetina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Náusea/induzido quimicamente , Náusea/diagnóstico , Olanzapina , Tiofenos/efeitos adversos , Resultado do Tratamento , Vômito/induzido quimicamente , Vômito/diagnóstico , Adulto Jovem
16.
Cancer Lett ; 350(1-2): 15-24, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24769073

RESUMO

The transcriptional factor liver receptor homolog 1 (LRH1) regulates pancreatic development, and may participate in pancreatic oncogenesis through activation of growth factor signaling transduction cascades. We measured transcriptional activity of ß-catenin in response to LRH1 stimulation by a Topflash reporter assay. The pancreatic cancer (PC) phenotype was then characterized by cell migration, wound healing, invasion, and sphere formation in vitro, as well as tumor formation and distant metastatic spread in vivo. We compared results between vector control and LRH1-overexpressing stable PC cell lines. In addition, tumor burden, angiogenesis, histologic characteristics, and hepatic spread were assessed in orthotopic and experimental liver metastatic murine models. Expression of downstream LRH1 related genes was evaluated by Western blot and immunohistochemistry in PC cell lines and human tumor specimens. Specific inhibition of LRH1 expression and function was accomplished by shRNAs "knockdown" experiments. It was found that LRH1 enhanced transcriptional activity of ß-catenin and the expression of downstream target genes (c-Myc, MMP2/9), as well as promoted migration, wound healing, invasion, and sphere formation of PC cell lines. Specific inhibition of LRH1 by shRNAs reduced cell migration, invasion, sphere formation and expression of c-Myc and MMP2/9 target genes. Mice injected with LRH1 overexpressing stable PC cells developed tumors with increased size and exhibited striking hepatic metastatic spread. More important, LRH1 was overexpressed in PC tumors compared to adjacent normal pancreas. Our findings demonstrate that LRH1 overexpression is associated with increased PC growth and metastatic spread, indicating that LRH1-targeted therapy could inhibit tumor progression.


Assuntos
Neoplasias Pancreáticas/patologia , Receptores Citoplasmáticos e Nucleares/biossíntese , beta Catenina/biossíntese , Animais , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/genética , Metástase Neoplásica , Proteínas Proto-Oncogênicas c-myc/biossíntese , Interferência de RNA , RNA Interferente Pequeno , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais , Esferoides Celulares/citologia , Transcrição Gênica , Células Tumorais Cultivadas , Cicatrização/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética
17.
Proc Natl Acad Sci U S A ; 111(13): E1230-9, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24639524

RESUMO

Golgi fragmentation occurs in neurons of patients with Alzheimer's disease (AD), but the underlying molecular mechanism causing the defects and the subsequent effects on disease development remain unknown. In this study, we examined the Golgi structure in APPswe/PS1E9 transgenic mouse and tissue culture models. Our results show that accumulation of amyloid beta peptides (Aß) leads to Golgi fragmentation. Further biochemistry and cell biology studies revealed that Golgi fragmentation in AD is caused by phosphorylation of Golgi structural proteins, such as GRASP65, which is induced by Aß-triggered cyclin-dependent kinase-5 activation. Significantly, both inhibition of cyclin-dependent kinase-5 and expression of nonphosphorylatable GRASP65 mutants rescued the Golgi structure and reduced Aß secretion by elevating α-cleavage of the amyloid precursor protein. Our study demonstrates a molecular mechanism for Golgi fragmentation and its effects on amyloid precursor protein trafficking and processing in AD, suggesting Golgi as a potential drug target for AD treatment.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/farmacologia , Complexo de Golgi/metabolismo , Doença de Alzheimer/patologia , Animais , Células CHO , Proteínas de Transporte/metabolismo , Células Cultivadas , Cricetinae , Cricetulus , Quinase 5 Dependente de Ciclina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Hipocampo/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mutantes/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura , Fosforilação/efeitos dos fármacos , Presenilina-1/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos
18.
Cancer Lett ; 345(1): 85-90, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24333731

RESUMO

Liver receptor homolog 1 (LRH1), directs the development and differentiation of embryonic pancreas, and is overexpressed in pancreatic cancer (PC). We hypothesized that LRH1 promotes PC growth. Cell proliferation and tumorigenicity in nude mice were compared between empty vector-transfected (control) and stable LRH1-overexpressed PC cell lines. The subsequent tumor burden, vasculature development, and histologic features were evaluated. LRH1 overexpression enhanced the expression of downstream target genes (cyclin D1/E1) and stimulated cell proliferation in PC cell lines. LRH1 upregulated cyclin E1 truncated T1/T2 isoforms expression which may occur through ERα-calpain1 signaling. Compared with the control, LRH1 overexpressing stable cells generated tumors with increased weight, proliferation index and enhanced angiogenesis. Cyclin D1/E1 and calpain1 were overexpressed in human PC tumors compared to adjacent normal pancreas. These observations demonstrate that LRH1 promotes PC growth and angiogenesis, suggesting that LRH1 is a driving factor in tumorigenesis and may serve as a potential therapeutic target.


Assuntos
Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Células Hep G2 , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/genética , Receptores Citoplasmáticos e Nucleares/genética , Transfecção
19.
J Biol Chem ; 288(35): 25646-25657, 2013 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-23888051

RESUMO

Mechanical forces are critical for normal fetal lung development. However, the mechanisms regulating this process are not well-characterized. We hypothesized that strain-induced release of HB-EGF and TGF-α is mediated via integrin-ADAM17/TACE interactions. Employing an in vitro system to simulate mechanical forces in fetal lung development, we showed that mechanical strain of fetal epithelial cells actives TACE, releases HB-EGF and TGF-α, and promotes differentiation. In contrast, in samples incubated with the TACE inhibitor IC-3 or in cells isolated from TACE knock-out mice, mechanical strain did not release ligands or promote cell differentiation, which were both rescued after transfection of ADAM17. Cell adhesion assay and co-immunoprecipitation experiments in wild-type and TACE knock-out cells using several TACE constructs demonstrated not only that integrins α6 and ß1 bind to TACE via the disintegrin domain but also that mechanical strain enhances these interactions. Furthermore, force applied to these integrin receptors by magnetic beads activated TACE and shed HB-EGF and TGF-α. The contribution of integrins α6 and ß1 to differentiation of fetal epithelial cells by strain was demonstrated by blocking their binding site with specific antibodies and by culturing the cells on membranes coated with anti-integrin α6 and ß1 antibodies. In conclusion, mechanical strain releases HB-EGF and TGF-α and promotes fetal type II cell differentiation via α6ß1 integrin-ADAM17/TACE signaling pathway. These investigations provide novel mechanistic information on how mechanical forces promote fetal lung development and specifically differentiation of epithelial cells. This information could be also relevant to other tissues exposed to mechanical forces.


Assuntos
Proteínas ADAM/metabolismo , Diferenciação Celular/fisiologia , Células Epiteliais/metabolismo , Integrina alfa6beta1/metabolismo , Pulmão/embriologia , Mucosa Respiratória/embriologia , Transdução de Sinais/fisiologia , Proteínas ADAM/genética , Proteína ADAM17 , Animais , Células Epiteliais/citologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Integrina alfa6beta1/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Pulmão/citologia , Camundongos , Camundongos Knockout , Ligação Proteica , Mucosa Respiratória/citologia , Estresse Fisiológico/fisiologia , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismo
20.
PLoS One ; 8(3): e59598, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23527226

RESUMO

An imbalance between pro-inflammatory and anti-inflammatory cytokines is a key factor in the lung injury of premature infants exposed to mechanical ventilation. Previous studies have shown that lung cells exposed to stretch produces reduced amounts of the anti-inflammatory cytokine IL-10. The objective of these studies was to analyze the signaling mechanisms responsible for the decreased IL-10 production in fetal type II cells exposed to mechanical stretch. Fetal mouse type II epithelial cells isolated at embryonic day 18 were exposed to 20% stretch to simulate lung injury. We show that IL-10 receptor gene expression increased with gestational age. Mechanical stretch decreased not only IL-10 receptor gene expression but also IL-10 secretion. In contrast, mechanical stretch increased release of IL-6. We then investigated IL-10 signaling pathway-associated proteins and found that in wild-type cells, mechanical stretch decreased activation of JAK1 and TYK2 and increased STAT3 and SOCS3 activation. However, opposite effects were found in cells isolated from IL-10 knockout mice. Reduction in IL-6 secretion by stretch was observed in cells isolated from IL-10 null mice. To support the idea that stretch-induced SOCS3 expression via IL-6 leads to reduced IL-10 expression, siRNA-mediated inhibition of SOCS3 restored IL-10 secretion in cells exposed to stretch and decreased IL-6 secretion. Taken together, these studies suggest that the inhibitory effect of mechanical stretch on IL-10 secretion is mediated via activation of IL-6-STAT3-SOCS3 signaling pathway. SOCS3 could be a therapeutic target to increase IL-10 production in lung cells exposed to mechanical injury.


Assuntos
Células Epiteliais/metabolismo , Feto/citologia , Interleucina-10/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiologia , Análise de Variância , Animais , Western Blotting , Eletroporação , Feto/metabolismo , Idade Gestacional , Interleucina-10/genética , Interleucina-6/metabolismo , Modelos Lineares , Camundongos , Camundongos Knockout , Estimulação Física , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Interleucina-10/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismo
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