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1.
Am J Physiol Heart Circ Physiol ; 319(6): H1414-H1437, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33064567

RESUMO

Increased matrix metalloprotease 9 (MMP9) after myocardial infarction (MI) exacerbates ischemia-induced chronic heart failure (CHF). Autophagy is cardioprotective during CHF; however, whether increased MMP9 suppresses autophagic activity in CHF is unknown. This study aimed to determine whether increased MMP9 suppressed autophagic flux and MMP9 inhibition increased autophagic flux in the heart of rats with post-MI CHF. Sprague-Dawley rats underwent either sham surgery or coronary artery ligation 6-8 wk before being treated with MMP9 inhibitor for 7 days, followed by cardiac autophagic flux measurement with lysosomal inhibitor bafilomycin A1. Furthermore, autophagic flux was measured in vitro by treating H9c2 cardiomyocytes with two independent pharmacological MMP9 inhibitors, salvianolic acid B (SalB) and MMP9 inhibitor-I, and CRISPR/cas9-mediated MMP9 genetic ablation. CHF rats showed cardiac infarct, significantly increased left ventricular end-diastolic pressure (LVEDP), and increased MMP9 activity and fibrosis in the peri-infarct areas of left ventricular myocardium. Measurement of the autophagic markers LC3B-II and p62 with lysosomal inhibition showed decreased autophagic flux in the peri-infarct myocardium. Treatment with SalB for 7 days in CHF rats decreased MMP9 activity and cardiac fibrosis but increased autophagic flux in the peri-infarct myocardium. As an in vitro corollary study, measurement of autophagic flux in H9c2 cardiomyocytes and fibroblasts showed that pharmacological inhibition or genetic ablation of MMP9 upregulates autophagic flux. These data are consistent with our observations that MMP9 inhibition upregulates autophagic flux in the heart of rats with CHF. In conclusion, the results in this study suggest that the beneficial outcome of MMP9 inhibition in pathological cardiac remodeling is in part mediated by improved autophagic flux.NEW & NOTEWORTHY This study elucidates that the improved cardiac extracellular matrix (ECM) remodeling and cardioprotective effect of matrix metalloprotease 9 (MMP9) inhibition in chronic heart failure (CHF) are via increased autophagic flux. Autophagy is cardioprotective; however, the mechanism of autophagy suppression in CHF is unknown. We for the first time demonstrated here that increased MMP9 suppressed cardiac autophagy and ablation of MMP9 increased cardiac autophagic flux in CHF rats. Restoring the physiological level of autophagy in the failing heart is a challenge, and our study addressed this challenge. The novelty and highlights of this report are as follows: 1) MMP9 regulates cardiomyocyte and fibroblast autophagy, 2) MMP9 inhibition protects CHF after myocardial infarction (MI) via increased cardiac autophagic flux, 3) MMP9 inhibition increased cardiac autophagy via activation of AMP-activated protein kinase (AMPK)α, Beclin-1, Atg7 pathway and suppressed mechanistic target of rapamycin (mTOR) pathway.


Assuntos
Autofagia/efeitos dos fármacos , Benzofuranos/farmacologia , Fibroblastos/efeitos dos fármacos , Insuficiência Cardíaca/tratamento farmacológico , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Animais , Linhagem Celular , Modelos Animais de Doenças , Fibroblastos/enzimologia , Fibroblastos/patologia , Fibrose , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Masculino , Metaloproteinase 9 da Matriz/genética , Camundongos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Ratos Sprague-Dawley , Transdução de Sinais , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos
2.
Am J Physiol Heart Circ Physiol ; 319(4): H765-H774, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32822210

RESUMO

Ubiquitylation is a key event that regulates protein turnover, and induction of the ubiquitin ligase E3 WWP1 has been associated with age. Left ventricular hypertrophy (LVH) commonly occurs as a function of age and can cause heart failure (HF) with a preserved ejection fraction (EF; HFpEF). We hypothesized that overexpression (O/E) of WWP1 in the heart would cause LVH as well as functional and structural changes consistent with the aging HFpEF phenotype. Global WWP1 O/E was achieved in mice (n = 11) and echocardiography (40 MHz) performed to measure LV mass, EF, Doppler velocities (early E, late/atrial A), myocardial relaxation (E'), and isovolumetric relaxation time (IVRT) at 4, 6, and 8 wk. Age-matched wild-type animals (n = 15) were included as referent controls. LV EF was identical (60 ± 1 vs. 60 ± 1%, P > 0.90) with no difference in LV mass (67 ± 3 vs. 75 ± 5, P > 0.25) at 4 wk. However, at 8 wk of age, LV mass increased over twofold, E/A fell (impaired passive filling), and E/E' was lower and IVRT prolonged (impaired LV relaxation) - all P < 0.05. Collagen percent area increased by over twofold and fibrillar collagen expression (RT-PCR) over 1.5-fold (P < 0.05) with WWP1 O/E. WWP1 with an anti-WWP1 antibody could be identified in isolated cardiac fibroblasts, with WWP1 increased over twofold in O/E fibroblasts (P < 0.05). Inducing WWP1 expression caused LVH and preserved systolic function but impaired diastolic dysfunction, consistent with the HFpEF phenotype. Targeting the WWP1 pathway may be a novel therapeutic target for this intractable form of HF associated with aging.NEW & NOTEWORTHY Heart failure (HF) with a preserved ejection fraction (HFpEF) is a growing cause of HF and commonly afflicts the elderly. Milestones for HFpEF include diastolic dysfunction and an abnormal extracelluar matrix (ECM). The ubiquitin ligases, such as WWP1, change with aging and regulate critical protein turnover/stability processes, such as the ECM. The present study demonstrated that induction of WWP1 in mice induced LV hypertrophy, diastolic dysfunction, and ECM accumulation, consistent with the HFpEF phenotype, and thus may identify a new therapeutic pathway.


Assuntos
Matriz Extracelular/enzimologia , Insuficiência Cardíaca/enzimologia , Hipertrofia Ventricular Esquerda/enzimologia , Miocárdio/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Disfunção Ventricular Esquerda/enzimologia , Função Ventricular Esquerda , Remodelação Ventricular , Fatores Etários , Animais , Células Cultivadas , Diástole , Modelos Animais de Doenças , Feminino , Fibroblastos/enzimologia , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/fisiopatologia , Hipertrofia Ventricular Esquerda/diagnóstico por imagem , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Proteólise , Volume Sistólico , Ubiquitina-Proteína Ligases/genética , Disfunção Ventricular Esquerda/diagnóstico por imagem , Disfunção Ventricular Esquerda/fisiopatologia
3.
Proc Natl Acad Sci U S A ; 117(28): 16500-16508, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601199

RESUMO

Despite the implementation of multiple HER2-targeted therapies, patients with advanced HER2+ breast cancer ultimately develop drug resistance. Stromal fibroblasts represent an abundant cell type in the tumor microenvironment and have been linked to poor outcomes and drug resistance. Here, we show that fibroblasts counteract the cytotoxic effects of HER2 kinase-targeted therapy in a subset of HER2+ breast cancer cell lines and allow cancer cells to proliferate in the presence of the HER2 kinase inhibitor lapatinib. Fibroblasts from primary breast tumors, normal breast tissue, and lung tissue have similar protective effects on tumor cells via paracrine factors. This fibroblast-mediated reduction in drug sensitivity involves increased expression of antiapoptotic proteins and sustained activation of the PI3K/AKT/MTOR pathway, despite inhibition of the HER2 and the RAS-ERK pathways in tumor cells. HER2 therapy sensitivity is restored in the fibroblast cocultures by combination treatment with inhibitors of MTOR or the antiapoptotic proteins BCL-XL and MCL-1. Expression of activated AKT in tumor cells recapitulates the effects of fibroblasts resulting in sustained MTOR signaling and poor lapatinib response. Lapatinib sensitivity was not altered by fibroblasts in tumor cells that exhibited sustained MTOR signaling due to a strong gain-of-function PI3KCA mutation. These findings indicate that in addition to tumor cell-intrinsic mechanisms that cause constitutive PI3K/AKT/MTOR pathway activation, secreted factors from fibroblasts can maintain this pathway in the context of HER2 inhibition. Our integrated proteomic-phenotypic approach presents a strategy for the discovery of protective mechanisms in fibroblast-rich tumors and the design of rational combination therapies to restore drug sensitivity.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Fibroblastos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Feminino , Fibroblastos/citologia , Fibroblastos/enzimologia , Humanos , Lapatinib/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética
4.
Toxicol Lett ; 331: 112-121, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32534005

RESUMO

Roxadustat is the first orally administered, small-molecule hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor that has been submitted for FDA regulatory approval to treat anemia secondary to chronic kidney diseases. Its usage has also been suggested for pulmonary fibrosis; however, the corresponding therapeutic effects remain to be investigated. The in vitro effects of roxadustat on cobalt chloride (CoCl2)-stimulated pulmonary fibrosis with L929 mouse fibroblasts as well as on an in vivo pulmonary fibrosismice model induced with bleomycin (BLM; intraperitoneal injection, 50 mg/kg twice a week for 4 continuous weeks) were investigated. It found that the proliferation of L929 cells was inhibited and the production of collagen I, collagen III, prolyl hydroxylase domain protein 2 (PHD2), HIF-1α, α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), transforming growth factor-ß1 (TGF-ß1) and p-Smad3 were reduced relative to that in the CoCl2 or BLM group after roxadustat treatment. Roxadustat ameliorated pulmonary fibrosis by reducing the pathology score and collagen deposition as well as decreasing the expression of collagen I, collagen III, PHD2, HIF-1α, α-SMA, CTGF, TGF-ß1 and p-Smad3/Smad3. Our cumulative results demonstrate that roxadustat administration can attenuate experimental pulmonary fibrosis via the inhibition of TGF-ß1/Smad activation.


Assuntos
Fibroblastos/efeitos dos fármacos , Glicina/análogos & derivados , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Isoquinolinas/uso terapêutico , Pulmão/efeitos dos fármacos , Fibrose Pulmonar/prevenção & controle , Animais , Bleomicina/farmacologia , Técnicas de Cultura de Células , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Fibroblastos/enzimologia , Glicina/farmacologia , Glicina/uso terapêutico , Hidroxiprolina/metabolismo , Isoquinolinas/farmacologia , Pulmão/enzimologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/enzimologia
5.
Nat Commun ; 11(1): 2219, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32376827

RESUMO

Heterochromatin in the eukaryotic genome is rigorously controlled by the concerted action of protein factors and RNAs. Here, we investigate the RNA binding function of ATRX, a chromatin remodeler with roles in silencing of repetitive regions of the genome and in recruitment of the polycomb repressive complex 2 (PRC2). We identify ATRX RNA binding regions (RBRs) and discover that the major ATRX RBR lies within the N-terminal region of the protein, distinct from its PHD and helicase domains. Deletion of this ATRX RBR (ATRXΔRBR) compromises ATRX interactions with RNAs in vitro and in vivo and alters its chromatin binding properties. Genome-wide studies reveal that loss of RNA interactions results in a redistribution of ATRX on chromatin. Finally, our studies identify a role for ATRX-RNA interactions in regulating PRC2 localization to a subset of polycomb target genes.


Assuntos
Cromatina/metabolismo , Complexo Repressor Polycomb 2/metabolismo , RNA/metabolismo , Proteína Nuclear Ligada ao X/genética , Animais , Montagem e Desmontagem da Cromatina/genética , Feminino , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Heterocromatina/metabolismo , Histonas/química , Histonas/metabolismo , Metilação , Camundongos , Ligação Proteica , Domínios Proteicos/genética , Proteína Nuclear Ligada ao X/metabolismo
6.
Arterioscler Thromb Vasc Biol ; 40(7): 1680-1694, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32375544

RESUMO

OBJECTIVE: The recessive disease arterial calcification due to deficiency of CD73 (ACDC) presents with extensive nonatherosclerotic medial layer calcification in lower extremity arteries. Lack of CD73 induces a concomitant increase in TNAP (tissue nonspecific alkaline phosphatase; ALPL), a key enzyme in ectopic mineralization. Our aim was to investigate how loss of CD73 activity leads to increased ALPL expression and calcification in CD73-deficient patients and assess whether this mechanism may apply to peripheral artery disease calcification. Approach and Results: We previously developed a patient-specific disease model using ACDC primary dermal fibroblasts that recapitulates the calcification phenotype in vitro. We found that lack of CD73-mediated adenosine signaling reduced cAMP production and resulted in increased activation of AKT. The AKT/mTOR (mammalian target of rapamycin) axis blocks autophagy and inducing autophagy prevented calcification; however, we did not observe autophagy defects in ACDC cells. In silico analysis identified a putative FOXO1 (forkhead box O1 protein) binding site in the human ALPL promoter. Exogenous AMP induced FOXO1 nuclear localization in ACDC but not in control cells, and this was prevented with a cAMP analogue or activation of A2a/2b adenosine receptors. Inhibiting FOXO1 reduced ALPL expression and TNAP activity and prevented calcification. Mutating the FOXO1 binding site reduced ALPL promoter activation. Importantly, we provide evidence that non-ACDC calcified femoropopliteal arteries exhibit decreased CD73 and increased FOXO1 levels compared with control arteries. CONCLUSIONS: These data show that lack of CD73-mediated cAMP signaling promotes expression of the human ALPL gene via a FOXO1-dependent mechanism. Decreased CD73 and increased FOXO1 was also observed in more common peripheral artery disease calcification.


Assuntos
5'-Nucleotidase/deficiência , Fibroblastos/enzimologia , Proteína Forkhead Box O1/metabolismo , Doença Arterial Periférica/enzimologia , Artéria Poplítea/enzimologia , Calcificação Vascular/enzimologia , 5'-Nucleotidase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Autofagia , Estudos de Casos e Controles , Células Cultivadas , Feminino , Fibroblastos/patologia , Proteína Forkhead Box O1/genética , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Humanos , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/genética , Doença Arterial Periférica/patologia , Artéria Poplítea/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Calcificação Vascular/genética , Calcificação Vascular/patologia , Adulto Jovem
7.
J Cardiovasc Pharmacol ; 75(6): 535-544, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32168151

RESUMO

The pathogenesis of cardiorenal syndrome (CRS) is very complex, and currently there is no effective treatment for CRS. Higenamine (HI) has been shown to improve cardiac function in rats with heart failure. However, the role of higenamine in CRS remains unknown. Here, in vitro, higenamine treatment markedly reduced neonatal rat cardiac fibroblast collagen synthesis and inhibited neonatal rat cardiac myocyte hypertrophy. In our study, a rat model of type 2 CRS was induced by left anterior descending coronary artery ligation combined with 5/6 subtotal nephrectomy (STNx). Higenamine treatment decreased serum creatinine (Scr), blood urea nitrogen, and brain natriuretic peptide levels and was capable of improving left ventricular remodeling and systolic function in CRS rats, accompanied with decreased expression of transforming growth factor-ß1 (TGF-ß1), α-smooth muscle actin (α-SMA) and collagen I (Col1A1). Moreover, higenamine significantly inhibited the protein expression of phosphorylated apoptosis signal-regulated kinase 1 (p-ASK1) and downstream mitogen-activated protein kinases (MAPK) (ERK, P38)/NF-κB in cardiorenal tissues of CRS rats and neonatal rat cardiac fibroblast/neonatal rat cardiac myocyte cells. Our study demonstrated that higenamine improved cardiorenal function in CRS rats and attenuated heart and kidney fibrosis possibly via targeting ASK1/MAPK (ERK, P38)/NF-κB signaling pathway. This finding extends our knowledge on the role of higenamine in cardiorenal fibrosis, providing a potential target to prevent the progression of CRS.


Assuntos
Alcaloides/farmacologia , Síndrome Cardiorrenal/tratamento farmacológico , Colágeno/biossíntese , Fibroblastos/efeitos dos fármacos , Rim/efeitos dos fármacos , MAP Quinase Quinase Quinase 5/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Tetra-Hidroisoquinolinas/farmacologia , Animais , Síndrome Cardiorrenal/enzimologia , Síndrome Cardiorrenal/patologia , Células Cultivadas , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/enzimologia , Fibroblastos/patologia , Fibrose , Rim/enzimologia , Rim/patologia , Masculino , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , NF-kappa B/metabolismo , Fosforilação , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
Nat Cell Biol ; 22(4): 412-424, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203415

RESUMO

Although the transition metal copper (Cu) is an essential nutrient that is conventionally viewed as a static cofactor within enzyme active sites, a non-traditional role for Cu as a modulator of kinase signalling is emerging. Here, we found that Cu is required for the activity of the autophagic kinases ULK1 and ULK2 (ULK1/2) through a direct Cu-ULK1/2 interaction. Genetic loss of the Cu transporter Ctr1 or mutations in ULK1 that disrupt the binding of Cu reduced ULK1/2-dependent signalling and the formation of autophagosome complexes. Increased levels of intracellular Cu are associated with starvation-induced autophagy and are sufficient to enhance ULK1 kinase activity and, in turn, autophagic flux. The growth and survival of lung tumours driven by KRASG12D is diminished in the absence of Ctr1, is dependent on ULK1 Cu binding and is associated with reduced levels of autophagy and signalling. These findings suggest a molecular basis for exploiting Cu-chelation therapy to prevent autophagy signalling to limit proliferation and improve patient survival in cancer.


Assuntos
Adenocarcinoma de Pulmão/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Autofagia/genética , Cobre/metabolismo , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/genética , Proteínas Serina-Treonina Quinases/genética , Adenocarcinoma de Pulmão/enzimologia , Adenocarcinoma de Pulmão/patologia , Sequência de Aminoácidos , Animais , Autofagossomos/enzimologia , Proteína 5 Relacionada à Autofagia/deficiência , Proteína 5 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Transportador de Cobre 1/deficiência , Transportador de Cobre 1/genética , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/deficiência , Proteínas Proto-Oncogênicas p21(ras)/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Sci Rep ; 10(1): 3930, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32127618

RESUMO

A splice site mutation in the canine pyruvate dehydrogenase kinase 4 (PDK4) gene has been shown to be associated with the development of dilated cardiomyopathy (DCM) in Doberman Pinchers (DPs). Subsequent studies have successfully demonstrated the use of dermal fibroblasts isolated from DPs as models for PDK4 deficiency and have shown activation of the intrinsic (mitochondrial mediated) apoptosis pathway in these cells under starvation conditions. For this study, we sought to further explore the functional consequences of PDK4 deficiency in DP fibroblasts representing PDK4wt/wt, PDK4wt/del, and PDK4del/del genotypes. Our results show that starvation conditions cause increased perinuclear localization of mitochondria and decreased cell proliferation, altered expression levels of pyruvate dehydrogenase phosphatase (PDP) and pyruvate dehydrogenase (PDH), dramatically increased PDH activity, and an impaired response to mitochondrial stress in affected cells. In sum, these results show the broad impact of PDK4 deficiency and reveal mechanistic pathways used by these cells in an attempt to compensate for the condition. Our data help to elucidate the mechanisms at play in this extremely prevalent DP disorder and provide further support demonstrating the general importance of metabolic flexibility in cell health.


Assuntos
Fibroblastos/enzimologia , Proteínas Quinases/deficiência , Western Blotting , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Microscopia de Fluorescência , Consumo de Oxigênio/genética , Consumo de Oxigênio/fisiologia , Fosforilação/genética , Fosforilação/fisiologia , Proteínas Quinases/genética , Piruvato Desidrogenase (Lipoamida)-Fosfatase/genética , Piruvato Desidrogenase (Lipoamida)-Fosfatase/metabolismo , Complexo Piruvato Desidrogenase/genética , Complexo Piruvato Desidrogenase/metabolismo
10.
Int J Mol Sci ; 21(5)2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32120828

RESUMO

UVB irradiation can induce generation of reactive oxygen species (ROS) that cause skin aging or pigmentation. Lactobacillus acidophilus is a well-known probiotic strain that regulates skin health through antimicrobial peptides and organic products produced by metabolism and through immune responses. In this study, we investigated the antioxidative, antiwrinkle, and antimelanogenesis effects of tyndallized Lactobacillus acidophilus KCCM12625P (AL). To analyze the effects of AL on UV irradiation-induced skin wrinkle formation in vitro, human keratinocytes and human dermal fibroblasts were exposed to UVB. Subsequent treatment with AL induced antiwrinkle effects by regulating wrinkle-related genes such as matrix metalloproteinases (MMPs), SIRT-1, and type 1 procollagen (COL1AL). In addition, Western blotting assays confirmed that regulation of MMPs by AL in keratinocytes was due to regulation of the AP-1 signaling pathway. Furthermore, we confirmed the ability of AL to regulate melanogenesis in B16F10 murine melanoma cells treated with α-melanocyte-stimulating hormone (α-MSH). In particular, AL reduced the mRNA expression of melanogenesis-related genes such as tyrosinase, TYRP-1, and TYRP-2. Finally, we used Western blotting assays to confirm that the antimelanogenesis role of AL was due to its regulation of the cyclic adenosine monophosphate (cAMP) signaling pathway. Collectively, these results indicate that AL has an antiwrinkle activity in damaged skin and can inhibit melanogenesis. Thus, AL should be considered an important substance for potential use in anti-aging drugs or cosmetics.


Assuntos
Fibroblastos/efeitos da radiação , Queratinócitos/efeitos da radiação , Lactobacillus acidophilus , Probióticos , Raios Ultravioleta , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Humanos , Oxirredutases Intramoleculares/metabolismo , Queratinócitos/enzimologia , Queratinócitos/metabolismo , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Monofenol Mono-Oxigenase/metabolismo , Oxirredutases/metabolismo , Pró-Colágeno/genética , Pró-Colágeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Pele/enzimologia , Pele/efeitos da radiação , Fator de Transcrição AP-1/metabolismo , Raios Ultravioleta/efeitos adversos , alfa-MSH/farmacologia
11.
Clin Sci (Lond) ; 134(7): 889-905, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32219338

RESUMO

Senescence and mitochondrial stress are mutually reinforcing age-related processes that contribute to idiopathic pulmonary fibrosis (IPF); a lethal disease that manifests primarily in the elderly. Whilst evidence is accumulating that GMP-AMP synthase (cGAS) is crucial in perpetuating senescence by binding damaged DNA released into the cytosol, its role in IPF is not known. The present study examines the contributions of cGAS and self DNA to the senescence of lung fibroblasts from IPF patients (IPF-LFs) and age-matched controls (Ctrl-LFs). cGAS immunoreactivity was observed in regions of fibrosis associated with fibroblasts in lung tissue of IPF patients. Pharmacological inhibition of cGAS or its knockdown by silencing RNA (siRNA) diminished the escalation of IPF-LF senescence in culture over 7 days as measured by decreased p21 and p16 expression, histone 2AXγ phosphorylation and/or IL-6 production (P < 0.05, n = 5-8). The targeting of cGAS also attenuated etoposide-induced senescence in Ctrl-LFs (P < 0.05, n = 5-8). Levels of mitochondrial DNA (mDNA) detected by qPCR in the cytosol and medium of IPF-LFs or senescence-induced Ctrl-LFs were higher than Ctrl-LFs at baseline (P < 0.05, n = 5-7). The addition of DNAse I (100 U/ml) deaccelerated IPF-LF senescence (P < 0.05, n = 5), whereas ectopic mDNA or the induction of endogenous mDNA release augmented Ctrl-LF senescence in a cGAS-dependent manner (P < 0.05, n = 5). In conclusion, we provide evidence that cGAS reinforces lung fibroblast senescence involving damaged self DNA. The targeting of cGAS to supress senescent-like responses may have potential important therapeutic implications in the treatment of IPF.


Assuntos
Proliferação de Células , Senescência Celular , DNA Mitocondrial/metabolismo , Fibroblastos/enzimologia , Fibrose Pulmonar Idiopática/enzimologia , Pulmão/enzimologia , Nucleotidiltransferases/metabolismo , Estudos de Casos e Controles , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Histonas/metabolismo , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Interleucina-6/genética , Interleucina-6/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/genética , Comunicação Parácrina , Fosforilação , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
Toxicology ; 437: 152438, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32199159

RESUMO

Polychlorinated biphenyls (PCBs) are persistent organic pollutants with human carcinogenicity. Many lower chlorinated and non-dioxin-like PCBs have been observed to be mutagenic following activation by human CYP2E1, while activation of dioxin-like (DL-) PCBs by this enzyme has never been evidenced. In this study, each DL-PCB was analyzed by molecular docking to human CYP2E1 protein for predicting a substrate interaction. All compounds demonstrated high affinities with the active site of human CYP2E1, binding energy being -8.7 ∼ -9.7 kcal/mol. However, most compounds demonstrated ligand-heme distances as ≥ 6.8 Å, while the values for 2,3,3',4,4'- (PCB 105) and 2,3',4,4',5-pentachlorobiphenyl (PCB 118) were 5.3 and 5.4 Å, respectively (valid for electron transfer). Experimentally, both PCB 105 and 118 induced micronuclei in a V79-derived cell line engineered for expression of human CYP2E1 at low micromolar concentrations, while inactive or weakly positive in V79-Mz control cells; these effects were blocked or reduced by 1-aminobenzotriazole, a suicide CYP inhibitor. However, DL-PCBs 77, 81 and 126 were all negative in both cell lines. In a human hepatoma (C3A) cell line, PCB 105 and 118 induced micronuclei marginally, while with ethanol pretreatment (to stabilize CYP2E1) both compounds induced micronuclei efficiently, and co-exposure to trans-1,2-dichloroethylene (a selective CYP2E1 inhibitor) led to clearly negative results with both compounds. Finally, both PCB 105 and 118 induced PIG-A gene mutations in C3A cells, which was blocked by trans-1,2-dichloroethylene. In summary, in silico and experimental results consistently suggest that DL- PCBs 105 and 118 may be activated by human CYP2E1 for mutagenic activities.


Assuntos
Citocromo P-450 CYP2E1/metabolismo , Proteínas de Membrana/genética , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Simulação de Acoplamento Molecular , Mutação , Bifenilos Policlorados/toxicidade , Ativação Metabólica , Animais , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Domínio Catalítico , Cricetulus , Citocromo P-450 CYP2E1/química , Citocromo P-450 CYP2E1/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/patologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Pulmão/patologia , Bifenilos Policlorados/química , Bifenilos Policlorados/metabolismo , Ligação Proteica , Conformação Proteica
13.
J Am Heart Assoc ; 9(3): e013518, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32000579

RESUMO

Background Pressure overload of the heart occurs in patients with hypertension or valvular stenosis and induces cardiac fibrosis because of excessive production of extracellular matrix by activated cardiac fibroblasts. This initially provides essential mechanical support to the heart, but eventually compromises function. Osteopontin is associated with fibrosis; however, the underlying signaling mechanisms are not well understood. Herein, we examine the effect of thrombin-cleaved osteopontin on fibrosis in the heart and explore the role of syndecan-4 in regulating cleavage of osteopontin. Methods and Results Osteopontin was upregulated and cleaved by thrombin in the pressure-overloaded heart of mice subjected to aortic banding. Cleaved osteopontin was higher in plasma from patients with aortic stenosis receiving crystalloid compared with blood cardioplegia, likely because of less heparin-induced inhibition of thrombin. Cleaved osteopontin and the specific osteopontin peptide sequence RGDSLAYGLR that is exposed after thrombin cleavage both induced collagen production in cardiac fibroblasts. Like osteopontin, the heparan sulfate proteoglycan syndecan-4 was upregulated after aortic banding. Consistent with a heparan sulfate binding domain in the osteopontin cleavage site, syndecan-4 was found to bind to osteopontin in left ventricles and cardiac fibroblasts and protected osteopontin from cleavage by thrombin. Shedding of the extracellular part of syndecan-4 was more prominent at later remodeling phases, at which time levels of cleaved osteopontin were increased. Conclusions Thrombin-cleaved osteopontin induces collagen production by cardiac fibroblasts. Syndecan-4 protects osteopontin from cleavage by thrombin, but this protection is lost when syndecan-4 is shed in later phases of remodeling, contributing to progression of cardiac fibrosis.


Assuntos
Cardiomiopatias/enzimologia , Colágeno Tipo I/metabolismo , Fibroblastos/enzimologia , Miocárdio/enzimologia , Osteopontina/metabolismo , Sindecana-4/metabolismo , Função Ventricular Esquerda , Remodelação Ventricular , Animais , Estenose da Valva Aórtica/sangue , Estenose da Valva Aórtica/complicações , Cardiomiopatias/genética , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Linhagem Celular Tumoral , Colágeno Tipo I/genética , Modelos Animais de Doenças , Fibroblastos/patologia , Fibrose , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/patologia , Osteopontina/sangue , Ligação Proteica , Sindecana-4/genética , Trombina/metabolismo
14.
Protein Expr Purif ; 170: 105589, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32027983

RESUMO

The cation-independent mannose-6-phosphate receptor (CI-M6PR, aka insulin-like growth factor II receptor or IGFIIR) is a membrane protein that plays a central role in the trafficking of lysosomal acid hydrolases into lysosomes via mannose-6-phosphate (M6P) binding domains. In order to maintain cellular metabolic/catabolic homeostasis, newly synthesized lysosomal acid hydrolases are required to bind to M6PR for transit. Acid hydrolases secreted by cells can also be internalized via M6PR residing on the cell membrane and are transported to the lysosomes, a feature that enables enzyme replacement therapy for the treatment of several lysosomal storage disorders. Therefore, a thorough characterization of this receptor is critical to the development of lysosomal enzyme-based therapeutics that utilize M6PR for drug delivery to the lysosome. However, the extracellular domain (ECD) of M6PR is highly complex, containing 15-mannose receptor homology (MRH) domains. In addition, homodimerization of the receptor can occur at the membrane, making its characterization challenging. In this study, a novel human M6PR (hM6PR)-overexpressing cell line originally established for hM6PR cellular uptake assay was utilized for production of hM6PR-ECD, and a novel small molecule biomimetic (aminophenyl-M6P) affinity resin was developed for the purification of M6PR-ECD. The affinity-purified hM6PR-ECD was monomeric, contained 14 intact MRH domains (1-14) and a partial MRH domain 15, and was successfully employed in ELISA-based and surface plasmon resonance-based binding assays to demonstrate its ligand-binding functionality, making it suitable for the evaluation of biotherapeutics that utilize M6PR for cellular internalization.


Assuntos
Aminofenóis/química , Materiais Biomiméticos/química , Membrana Celular/enzimologia , Manosefosfatos/química , Receptor IGF Tipo 2/isolamento & purificação , Sequência de Aminoácidos , Aminofenóis/metabolismo , Materiais Biomiméticos/metabolismo , Linhagem Celular Tumoral , Membrana Celular/química , Cromatografia de Afinidade , Ensaios Enzimáticos , Ensaio de Imunoadsorção Enzimática , Fibroblastos/química , Fibroblastos/enzimologia , Expressão Gênica , Humanos , Cinética , Manosefosfatos/metabolismo , Domínios Proteicos , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Ressonância de Plasmônio de Superfície
15.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32040547

RESUMO

Cell proliferation exerts a high demand on protein synthesis, yet the mechanisms coupling the two processes are not fully understood. A kinase and phosphatase screen for activators of translation, based on the formation of stress granules in human cells, revealed cell cycle-associated kinases as major candidates. CDK1 was identified as a positive regulator of global translation, and cell synchronization experiments showed that this is an extramitotic function of CDK1. Different pathways including eIF2α, 4EBP, and S6K1 signaling contribute to controlling global translation downstream of CDK1. Moreover, Ribo-Seq analysis uncovered that CDK1 exerts a particularly strong effect on the translation of 5'TOP mRNAs, which includes mRNAs encoding ribosomal proteins and several translation factors. This effect requires the 5'TOP mRNA-binding protein LARP1, concurrent to our finding that LARP1 phosphorylation is strongly dependent on CDK1. Thus, CDK1 provides a direct means to couple cell proliferation with biosynthesis of the translation machinery and the rate of protein synthesis.


Assuntos
Proteína Quinase CDC2/metabolismo , Proliferação de Células , Neoplasias do Colo do Útero/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Feminino , Fibroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Cinética , Camundongos Endogâmicos C57BL , Fosforilação , Biossíntese de Proteínas , Sequência de Oligopirimidina na Região 5' Terminal do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
16.
Proc Natl Acad Sci U S A ; 117(10): 5463-5471, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32079726

RESUMO

Chronic pain is a major clinical problem of which the mechanisms are incompletely understood. Here, we describe the concept that PI16, a protein of unknown function mainly produced by fibroblasts, controls neuropathic pain. The spared nerve injury (SNI) model of neuropathic pain increases PI16 protein levels in fibroblasts in dorsal root ganglia (DRG) meninges and in the epi/perineurium of the sciatic nerve. We did not detect PI16 expression in neurons or glia in spinal cord, DRG, and nerve. Mice deficient in PI16 are protected against neuropathic pain. In vitro, PI16 promotes transendothelial leukocyte migration. In vivo, Pi16 -/- mice show reduced endothelial barrier permeability, lower leukocyte infiltration and reduced activation of the endothelial barrier regulator MLCK, and reduced phosphorylation of its substrate MLC2 in response to SNI. In summary, our findings support a model in which PI16 promotes neuropathic pain by mediating a cross-talk between fibroblasts and the endothelial barrier leading to barrier opening, cellular influx, and increased pain. Its key role in neuropathic pain and its limited cellular and tissue distribution makes PI16 an attractive target for pain management.


Assuntos
Fibroblastos/enzimologia , Neuralgia/genética , Proteínas Secretadas Inibidoras de Proteinases/genética , Animais , Movimento Celular , Dor Crônica , Modelos Animais de Doenças , Células Endoteliais/fisiologia , Gânglios Espinais , Leucócitos/fisiologia , Meninges/citologia , Camundongos Knockout , Traumatismos dos Nervos Periféricos/fisiopatologia , Nervo Isquiático/enzimologia
17.
PLoS One ; 15(1): e0220348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935221

RESUMO

In a process linked to DNA replication, duplicated chromosomes are entrapped in large, circular cohesin complexes and functional sister chromatid cohesion (SCC) is established by acetylation of the SMC3 cohesin subunit. Roberts Syndrome (RBS) and Warsaw Breakage Syndrome (WABS) are rare human developmental syndromes that are characterized by defective SCC. RBS is caused by mutations in the SMC3 acetyltransferase ESCO2, whereas mutations in the DNA helicase DDX11 lead to WABS. We found that WABS-derived cells predominantly rely on ESCO2, not ESCO1, for residual SCC, growth and survival. Reciprocally, RBS-derived cells depend on DDX11 to maintain low levels of SCC. Synthetic lethality between DDX11 and ESCO2 correlated with a prolonged delay in mitosis, and was rescued by knockdown of the cohesin remover WAPL. Rescue experiments using human or mouse cDNAs revealed that DDX11, ESCO1 and ESCO2 act on different but related aspects of SCC establishment. Furthermore, a DNA binding DDX11 mutant failed to correct SCC in WABS cells and DDX11 deficiency reduced replication fork speed. We propose that DDX11, ESCO1 and ESCO2 control different fractions of cohesin that are spatially and mechanistically separated.


Assuntos
Acetiltransferases/genética , Proteínas de Ciclo Celular/genética , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/genética , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Células Epiteliais/enzimologia , Fibroblastos/enzimologia , Acetiltransferases/metabolismo , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Proliferação de Células , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Quebra Cromossômica , Segregação de Cromossomos , Anormalidades Craniofaciais/enzimologia , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/patologia , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Ectromelia/enzimologia , Ectromelia/genética , Ectromelia/patologia , Células Epiteliais/patologia , Fibroblastos/patologia , Expressão Gênica , Humanos , Hipertelorismo/enzimologia , Hipertelorismo/genética , Hipertelorismo/patologia , Camundongos , Mitose , Modelos Biológicos , Mutação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
18.
Scand J Gastroenterol ; 55(1): 82-89, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31917931

RESUMO

Objectives: The current survival of patients with distal cholangiocarcinoma (dCCA) is poor. There is a need to develop new prognostic and predictive biomarkers to improve the survival of patients. Fibroblast activation protein (FAP) expression has been associated with survival in several solid malignancies. The goal of this study was to evaluate the expression pattern and prognostic significance of FAP in dCCA.Materials and methods: FAP expression was examined in 57 resected dCCA specimens and 28 paired lymph node metastasis specimens, as well as 10 benign bile ducts using immunohistochemistry. FAP expression was scored in the epithelial and stromal component of the dCCA specimens. The association between FAP expression and prognosis was evaluated using univariable and multivariable statistical modeling.Results: FAP expression was absent in the benign controls. FAP expression was evident in the epithelial 43 (75%) and stromal compartment 34 (60%) of dCCA. There was no association between epithelial or stromal FAP expression and clinicopathological factors. Epithelial FAP expression (HR 0.4 95% CI 0.20-0.78; p=.007) but not stromal FAP expression was significantly associated with better survival in univariable and multivariable analysis.Conclusions: FAP overexpression is evident in dCCA. There was a positive association between epithelial FAP expression and better survival which merits further evaluation.


Assuntos
Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Gelatinases/metabolismo , Proteínas de Membrana/metabolismo , Serina Endopeptidases/metabolismo , Idoso , Idoso de 80 Anos ou mais , Neoplasias dos Ductos Biliares/mortalidade , Biomarcadores Tumorais/metabolismo , Colangiocarcinoma/mortalidade , Feminino , Fibroblastos/enzimologia , Humanos , Imuno-Histoquímica , Linfonodos/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Prognóstico , Análise de Sobrevida , Suécia
19.
Tohoku J Exp Med ; 250(1): 5-11, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31941852

RESUMO

Acid sphingomyelinase (ASM) is a lysosomal hydrolase that degrades sphingomyelin into ceramide and phosphocholine. Recent crystallographic studies revealed the functional role of the N-terminal ASM saposin domain. ASM deficiency due to mutations in the ASM-encoding sphingomyelin phosphodiesterase 1 (SMPD1) gene causes an autosomal recessive sphingolipid-storage disorder, known as Niemann-Pick disease Type A (NPA) or Type B (NPB). NPA is an early-onset neuronopathic disorder, while NPB is a late-onset non-neuronopathic disorder. A homozygous one-base substitution (c.398G>A) of the SMPD1 gene was identified in an infant with NPA, diagnosed with complete loss of ASM activity in the patient's fibroblasts. This mutation is predicted to substitute tyrosine for cysteine at amino acid residue 133, abbreviated as p.C133Y. The patient showed developmental delay, hepatosplenomegaly and rapid neurological deterioration leading to death at the age of 3 years. To characterize p.C133Y, which may disrupt one of the three disulfide bonds of the N-terminal ASM saposin domain, we performed immunoblotting analysis to explore the expression of a mutant ASM protein in the patient's fibroblasts, showing that the protein was detected as a 70-kDa protein, similar to the wild-type ASM protein. Furthermore, transient expression of p.C133Y ASM protein in COS-7 cells indicated complete loss of ASM enzyme activity, despite that the p.C133Y ASM protein was properly localized to the lysosomes. These results suggest that the proper three-dimensional structure of saposin domain may be essential for ASM catalytic activity. Thus, p.C133Y is associated with complete loss of ASM activity even with stable protein expression and proper subcellular localization.


Assuntos
Mutação/genética , Doença de Niemann-Pick Tipo A/enzimologia , Doença de Niemann-Pick Tipo A/genética , Saposinas/química , Esfingomielina Fosfodiesterase/química , Esfingomielina Fosfodiesterase/genética , Idade de Início , Sequência de Aminoácidos , Sequência de Bases , Pré-Escolar , Análise Mutacional de DNA , DNA Complementar/genética , Evolução Fatal , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Lactente , Domínios Proteicos
20.
Genomics ; 112(1): 484-493, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30946891

RESUMO

Exposing the skin to solar UV radiation induces cascades of signaling pathways and biological alterations such as redox imbalance, suppression of antioxidant genes and programmed cell death. Therefore, the aim of this study was to use RNA-Seq to unravel the effects of UV radiation on Normal Human Adult Fibroblast cells (NHDF). Cells were exposed to UV (20 mJ/cm2 for 3 mins) and incubated for 24 h. Total mRNA from the cells generated libraries of 72,080,648 and 40,750,939 raw reads from UV-treated and control cells respectively. Of the differentially expressed genes (DEGs) produced 2,007 were up-regulated and 2,791 were down-regulated (fold change ≥2, p < 0.05). The expression of 4 genes was validated with RT-qPCR. Chemokine signaling pathways in cancer were significantly activated and antioxidant genes were down-regulated. This study applied Next Generation Sequencing technology to reveal the genes and pathways involved in UV-induced human dermal fibroblast cells necrosis.


Assuntos
Derme/citologia , Fibroblastos/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Transcriptoma/efeitos da radiação , Raios Ultravioleta , Antioxidantes/metabolismo , Células Cultivadas , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Ontologia Genética , Humanos , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Redes e Vias Metabólicas/genética , Necrose , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Envelhecimento da Pele , Fator de Necrose Tumoral alfa/metabolismo
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