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1.
Res Vet Sci ; 138: 167-177, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34153557

RESUMO

Adhesion molecules play an important role in urinary calculus formation. The expressions of adhesion molecules in renal tubular has been reported in some animals. However, the role of adhesion molecules in the process of sheep urinary calculus formation is still unclear. The magnesium ammonium phosphate (MAP) is the main component of sheep urinary calculus. In this paper, the sheep renal tubular epithelial cells (RTECs) were isolated and treated with MAP, the expressions of osteopontin (OPN), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and apoptosis-related indicators caspase-3, Bcl-2 and Bax in RTECs were observed, the viability of RTECs was detected by Cell Counting Kit-8 (CCK-8). The levels of superoxide dismutase (SOD) and malondialdehyde (MDA), and the expressions of inflammatory factors Interleukin-6 (IL-6), Interleukin-1 (IL-1), Interleukin-17 (IL-17) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent (ELISA). The histopathological observation of kidney in urolithiasis sheep was made. The results showed that MAP could reduce the viability and SOD activity, enhance the activity of MDA significantly and promote the expressions of IL-1, IL-6, IL-17 and TNF-α of RTECs. By western blot and qPCR methods, the expressions of ICAM-1, VCAM-1 and OPN increased in 48 h. In addition, the expression of caspase-3 increased significantly and the ratio of Bcl-2/Bax reduced with exposure to MAP. The renal tissue structure was seriously damaged, the RTECs in urolithiasis sheep were degenerative and necrotic.


Assuntos
Apoptose , Moléculas de Adesão Celular/metabolismo , Sobrevivência Celular , Citocinas/imunologia , Células Epiteliais/fisiologia , Estresse Oxidativo , Estruvita/metabolismo , Animais , Células Cultivadas , Molécula 1 de Adesão Intercelular/metabolismo , Rim/fisiologia , Osteopontina/metabolismo , Carneiro Doméstico/metabolismo , Carneiro Doméstico/urina , Cálculos Urinários/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo
2.
Nat Commun ; 12(1): 3709, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140509

RESUMO

Fibrotic skin disease represents a major global healthcare burden, characterized by fibroblast hyperproliferation and excessive accumulation of extracellular matrix. Fibroblasts are found to be heterogeneous in multiple fibrotic diseases, but fibroblast heterogeneity in fibrotic skin diseases is not well characterized. In this study, we explore fibroblast heterogeneity in keloid, a paradigm of fibrotic skin diseases, by using single-cell RNA-seq. Our results indicate that keloid fibroblasts can be divided into 4 subpopulations: secretory-papillary, secretory-reticular, mesenchymal and pro-inflammatory. Interestingly, the percentage of mesenchymal fibroblast subpopulation is significantly increased in keloid compared to normal scar. Functional studies indicate that mesenchymal fibroblasts are crucial for collagen overexpression in keloid. Increased mesenchymal fibroblast subpopulation is also found in another fibrotic skin disease, scleroderma, suggesting this is a broad mechanism for skin fibrosis. These findings will help us better understand skin fibrotic pathogenesis, and provide potential targets for fibrotic disease therapies.


Assuntos
Colágeno/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Queloide/metabolismo , Mesoderma/citologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Colágeno/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibroblastos/patologia , Regulação da Expressão Gênica/genética , Ontologia Genética , Humanos , Queloide/genética , Queloide/patologia , Ligantes , Mesoderma/metabolismo , Mesoderma/patologia , RNA-Seq , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Análise de Célula Única , Dermatopatias/genética , Dermatopatias/metabolismo , Dermatopatias/patologia
3.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069715

RESUMO

Intestinal metaplasia (IM) is an intermediate step in the progression from premalignant to malignant stages of gastric cancer (GC). The Popeye domain containing (POPDC) gene family encodes three transmembrane proteins, POPDC1, POPDC2, and POPDC3, initially described in muscles and later in epithelial and other cells, where they function in cell-cell interaction, and cell migration. POPDC1 and POPDC3 downregulation was described in several tumors, including colon and gastric cancers. We questioned whether IM-to-GC transition involves POPDC gene dysregulation. Gastric endoscopic biopsies of normal, IM, and GC patients were examined for expression levels of POPDC1-3 and several suggested IM biomarkers, using immunohistochemistry and qPCR. Immunostaining indicated lower POPDC1 and POPDC3 labeling in IM compared with normal tissues. Significantly lower POPDC1 and POPDC3 mRNA levels were measured in IM and GC biopsies and in GC-derived cell lines. The reduction in focal IM was smaller than in extensive IM that resembled GC tissues. POPDC1 and POPDC3 transcript levels were highly correlated with each other and inversely correlated with LGR5, OLFM4, CDX2, and several mucin transcripts. The association of POPDC1 and POPDC3 downregulation with IM-to-GC transition implicates a role in tumor suppression and highlights them as potential biomarkers for GC progression and prospective treatment targets.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas Musculares/metabolismo , Lesões Pré-Cancerosas/patologia , Idoso , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Sobrevivência Celular/genética , Feminino , Mucosa Gástrica/patologia , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Masculino , Proteínas de Membrana/metabolismo , Metaplasia/patologia , Pessoa de Meia-Idade , Proteínas Musculares/genética , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Estudos Prospectivos , RNA Mensageiro/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
4.
Int J Mol Sci ; 22(10)2021 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-34065633

RESUMO

The circadian clock coordinates biological and physiological functions to day/night cycles. The perturbation of the circadian clock increases cancer risk and affects cancer progression. Here, we studied how BMAL1 knockdown (BMAL1-KD) by shRNA affects the epithelial-mesenchymal transition (EMT), a critical early event in the invasion and metastasis of colorectal carcinoma (CRC). In corresponding to a gene set enrichment analysis, which showed a significant enrichment of EMT and invasive signatures in BMAL1_high CRC patients as compared to BMAL1_low CRC patients, our results revealed that BMAL1 is implicated in keeping the epithelial-mesenchymal equilibrium of CRC cells and influences their capacity of adhesion, migration, invasion, and chemoresistance. Firstly, BMAL1-KD increased the expression of epithelial markers (E-cadherin, CK-20, and EpCAM) but decreased the expression of Twist and mesenchymal markers (N-cadherin and vimentin) in CRC cell lines. Finally, the molecular alterations after BMAL1-KD promoted mesenchymal-to-epithelial transition-like changes mostly appeared in two primary CRC cell lines (i.e., HCT116 and SW480) compared to the metastatic cell line SW620. As a consequence, migration/invasion and drug resistance capacities decreased in HCT116 and SW480 BMAL1-KD cells. Together, BMAL1-KD alerts the delicate equilibrium between epithelial and mesenchymal properties of CRC cell lines, which revealed the crucial role of BMAL1 in EMT-related CRC metastasis and chemoresistance.


Assuntos
Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Neoplasias do Colo/genética , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/genética , Antígenos CD/metabolismo , Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Bases de Dados Genéticas , Molécula de Adesão da Célula Epitelial/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Técnicas de Silenciamento de Genes , Humanos , Queratina-20/metabolismo , Invasividade Neoplásica/genética , Oxaliplatina/farmacologia , Transporte Proteico , Vimentina/metabolismo , beta Catenina/metabolismo
5.
Life Sci ; 278: 119620, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34004251

RESUMO

AIMS: Accumulating evidence indicates that a number of microRNAs (miRNAs) serve as essential regulators during adipogenesis and adipolysis in humans and animals and play critical roles in the development of fat tissue. In this study, we aimed to determine the functional role and underlying regulatory mechanism of microRNA-489-3p (miR-489) in adipocytes. MATERIALS AND METHODS: The expression patterns of miR-489 in mice were measured by qRT-PCR. Overexpression and knockdown of miR-489 by mimic and inhibitor transfections in 3T3-L1 preadipocytes revealed the regulatory effect of miR-489 on cellular proliferation and differentiation and energy turnover. Furthermore, RNA-seq, bioinformatics prediction, and dual luciferase reporter assays were used to identify the direct target of miR-489. KEY FINDINGS: The results showed that miR-489 was highly expressed in the visceral fat tissue of adult mice, and obese mice exhibited higher levels of miR-489 than normal mice. Overexpression of miR-489 suppressed proliferation but promoted adipogenic differentiation and lipid accumulation in the cells. Mitochondrial oxidation also fluctuated in the cells due to the high expression of miR-489. Notably, knockdown of miR-489 did not have a strong opposing effect on the cells. Periostin (Postn) was identified as a direct target gene for miR-489, and silencing the Postn gene similarly stimulated adipogenesis and differentiation of adipocytes. SIGNIFICANCE: miR-489 provides a strong driving force for adipogenesis metabolism and adipocyte differentiation by targeting the Postn gene. This result may contribute to the treatment of obesity.


Assuntos
Adipócitos/patologia , Adipogenia , Moléculas de Adesão Celular/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , Obesidade/patologia , Adipócitos/metabolismo , Animais , Moléculas de Adesão Celular/genética , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Perfilação da Expressão Gênica , Camundongos , Camundongos Obesos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Obesidade/genética , Obesidade/metabolismo
6.
Methods Mol Biol ; 2256: 217-236, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34014525

RESUMO

Viruses have evolved to interact with their hosts. Some viruses such as human papilloma virus, dengue virus, SARS-CoV, or influenza virus encode proteins including a PBM that interact with cellular proteins containing PDZ domains. There are more than 400 cellular protein isoforms with these domains in the human genome, indicating that viral PBMs have a high potential to influence the behavior of the cell. In this review we analyze the most relevant cellular processes known to be affected by viral PBM-cellular PDZ interactions including the establishment of cell-cell interactions and cell polarity, the regulation of cell survival and apoptosis and the activation of the immune system. Special attention has been provided to coronavirus PBM conservation throughout evolution and to the role of the PBMs of human coronaviruses SARS-CoV and MERS-CoV in pathogenesis.


Assuntos
Moléculas de Adesão Celular/metabolismo , Interações Hospedeiro-Patógeno , Proteínas Virais/metabolismo , Viroses/metabolismo , Vírus/metabolismo , Apoptose/fisiologia , Proliferação de Células/fisiologia , Humanos , Domínios PDZ , Ligação Proteica , Estrutura Secundária de Proteína , Viroses/virologia , Vírus/isolamento & purificação
7.
PLoS Pathog ; 17(5): e1009576, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34015061

RESUMO

The efficient spread of SARS-CoV-2 resulted in a unique pandemic in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLRS) of antigen-presenting cells, widely present in respiratory mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2+ Vero E6 cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. These data have been then confirmed using authentic SARS-CoV-2 virus and human respiratory cell lines. Thus, we described a mechanism potentiating viral spreading of infection.


Assuntos
COVID-19/transmissão , Lectinas Tipo C/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Antígenos CD/metabolismo , COVID-19/prevenção & controle , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Chlorocebus aethiops , Humanos , Células Jurkat , Pulmão/metabolismo , Lectinas de Ligação a Manose/metabolismo , Manosídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Mucosa Respiratória/metabolismo , Células Vero
8.
Nat Commun ; 12(1): 2522, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947846

RESUMO

Haematopoietic stem cells (HSCs) tightly regulate their quiescence, proliferation, and differentiation to generate blood cells during the entire lifetime. The mechanisms by which these critical activities are balanced are still unclear. Here, we report that Macrophage-Erythroblast Attacher (MAEA, also known as EMP), a receptor thus far only identified in erythroblastic island, is a membrane-associated E3 ubiquitin ligase subunit essential for HSC maintenance and lymphoid potential. Maea is highly expressed in HSCs and its deletion in mice severely impairs HSC quiescence and leads to a lethal myeloproliferative syndrome. Mechanistically, we have found that the surface expression of several haematopoietic cytokine receptors (e.g. MPL, FLT3) is stabilised in the absence of Maea, thereby prolonging their intracellular signalling. This is associated with impaired autophagy flux in HSCs but not in mature haematopoietic cells. Administration of receptor kinase inhibitor or autophagy-inducing compounds rescues the functional defects of Maea-deficient HSCs. Our results suggest that MAEA provides E3 ubiquitin ligase activity, guarding HSC function by restricting cytokine receptor signalling via autophagy.


Assuntos
Autofagossomos/genética , Autofagia/genética , Moléculas de Adesão Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Moléculas de Adesão Celular/genética , Proteínas do Citoesqueleto/genética , Perfilação da Expressão Gênica , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Estabilidade Proteica , Receptores de Trombopoetina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Tirosina Quinase 3 Semelhante a fms/metabolismo
9.
Theranostics ; 11(12): 5634-5649, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33897872

RESUMO

Although a small number of cardiomyocytes may reenter the cell cycle after injury, the adult mammalian heart is incapable of a robust cardiomyocyte proliferation. Periostin, a secreted extracellular matrix protein, has been implicated as a regulator of cardiomyocyte proliferation; however, this role remains controversial. Alternative splicing of the human periostin gene results in 6 isoforms lacking sequences between exons 17 and 21, in addition to full-length periostin. We previously showed that exosomes (Exo) secreted by human cardiac explant-derived progenitor cells (CPC) carried periostin. Here, we aimed to investigate their cell cycle activity. Methods: CPC were derived as the cellular outgrowth of ex vivo cultured cardiac atrial explants. Exo were purified from CPC conditioned medium using size exclusion chromatography. Exosomal periostin was analyzed by Western blotting using a pair of antibodies (one raised against aa 537-836, and one raised against amino acids mapping at exon 17 of human periostin), by ELISA, and by cryo-EM with immune-gold labeling. Cell cycle activity was assessed in neonatal rat cardiomyocytes, in human induced pluripotent stem cell (iPS)-derived cardiomyocytes, and in adult rat cardiomyocytes after myocardial infarction. The role of periostin in cell cycle activity was investigated by transfecting donor CPC with a siRNA against this protein. Results: Periostin expression in CPC-secreted exosomes was detected using the antibody raised against aa 537-836 of the human protein, but not with the exon 17-specific antibody, consistent with an isoform lacking exon 17. Periostin was visualized on vesicle surfaces by cryo-EM and immune-gold labeling. CPC-derived exosomes induced cell proliferation in neonatal rat cardiomyocytes both in vitro and in vivo, in human iPS-derived cardiomyocytes, and in adult rat cardiomyocytes after myocardial infarction. Exo promoted phosphorylation of focal adhesion kinase (FAK), actin polymerization, and nuclear translocation of Yes-associated protein (YAP) in cardiomyocytes. Knocking down of periostin or YAP, or blocking FAK phosphorylation with PF-573228 nullified Exo-induced proliferation. A truncated human periostin peptide (aa 22-669), but not recombinant human full-length periostin, mimicked the pro-proliferative activity of exosomes. Conclusions: Our results show, for the first time, that CPC-secreted exosomes promote cardiomyocyte cell cycle-reentry via a short periostin isoform expressed on their surfaces, whereas recombinant full-length periostin does not. These findings highlight isoform-specific roles of periostin in cardiomyocyte proliferation.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proliferação de Células/fisiologia , Exossomos/metabolismo , Miócitos Cardíacos/metabolismo , Isoformas de Proteínas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Ciclo Celular/fisiologia , Células Cultivadas , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/metabolismo , Ratos , Ratos Wistar
10.
Am J Physiol Heart Circ Physiol ; 320(6): H2222-H2239, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33834866

RESUMO

Extracellular matrix (ECM) exerts a series of biological functions and contributes to almost 30% of the osteogenic process. Periostin is a secreted protein that can alter ECM remodeling in response to vascular injury. However, the role of periostin in vascular calcification has yet to be fully investigated. As found in this study, recombinant periostin accelerated the thoracic aortas calcification, increased the expression of glycolysis key enzymes, and disturbed the normal oxidative phosphorylation (OXPHOS) ex vivo, which could be alleviated by the peroxisome proliferation-activated receptor γ (PPARγ) agonist pioglitazone. In vascular smooth muscle cells (VSMCs), periostin promoted VSMC-osteoblastic phenotype transition and calcium deposition and suppressed PPARγ expression. Mechanistically, periostin caused overactivation of glycolysis and mitochondrial dysfunction in VSMCs as assessed by extracellular acidification rate, oxygen consumption rate, and mitochondrial respiratory chain complex activities. Targeted glycolysis inhibitors reduced mitochondrial calcium overload, apoptosis, and periostin-induced VSMCs calcification. PPARγ agonists preserved glycolysis and OXPHOS in the stimulated microenvironment and reversed periostin-promoted VSMC calcification. Furthermore, plasma periostin, lactate, and matrix Gla protein levels were measured in 274 patients undergoing computed tomography to determine coronary artery calcium score (Agatston score). Plasma periostin and lactate levels were both linked to an Agatston score in patients with coronary artery calcification (CAC). There was also a positive correlation between plasma periostin and lactate levels. This study suggests that downregulation of PPARγ is involved in the mechanism by which periostin accelerates arterial calcification partly through excessive glycolysis activation and unbalanced mitochondrial homeostasis.NEW & NOTEWORTHY Periostin caused arterial calcification, overactivated glycolysis, and damaged OXPHOS. PPARγ agonists alleviated periostin-promoted arterial calcification and corrected abnormal glycolysis and unbalanced mitochondrial homeostasis. There exists a relationship between periostin and lactate in patients with CAC.


Assuntos
Aorta Torácica/metabolismo , Moléculas de Adesão Celular/metabolismo , Doença da Artéria Coronariana/metabolismo , Glucose/metabolismo , Mitocôndrias/metabolismo , Miócitos de Músculo Liso/metabolismo , PPAR gama/metabolismo , Calcificação Vascular/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/patologia , Apoptose/efeitos dos fármacos , Moléculas de Adesão Celular/farmacologia , Angiografia por Tomografia Computadorizada , Regulação para Baixo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Ácido Láctico/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio , PPAR gama/agonistas , Pioglitazona/farmacologia , Ratos
11.
Mol Cell ; 81(11): 2445-2459.e13, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33905682

RESUMO

How are E3 ubiquitin ligases configured to match substrate quaternary structures? Here, by studying the yeast GID complex (mutation of which causes deficiency in glucose-induced degradation of gluconeogenic enzymes), we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryoelectron microscopy (cryo-EM) structures show that, to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two minimally functional GID E3s assemble into the 20-protein Chelator-GIDSR4, which resembles an organometallic supramolecular chelate. The Chelator-GIDSR4 assembly avidly binds multiple Fbp1 degrons so that multiple Fbp1 protomers are simultaneously ubiquitylated at lysines near the allosteric and substrate binding sites. Importantly, key structural and biochemical features, including capacity for supramolecular assembly, are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical, and cell biological data, we propose that higher-order E3 ligase assembly generally enables multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Moléculas de Adesão Celular/química , Frutose-Bifosfatase/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Complexos Multienzimáticos/química , Proteínas de Saccharomyces cerevisiae/química , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sítios de Ligação , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Microscopia Crioeletrônica , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/metabolismo , Expressão Gênica , Gluconeogênese/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células K562 , Cinética , Modelos Moleculares , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Células Sf9 , Spodoptera , Homologia Estrutural de Proteína , Especificidade por Substrato , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação
12.
Biomed Pharmacother ; 139: 111624, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33915503

RESUMO

Acute kidney injury (AKI) is a sudden insult of the kidney that happens within a short period of time, which is associated with poor prognosis in diabetic patients with myocardial infarction (MI). Subclinical AKI is a condition in which tubular damage biomarkers [Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1(KIM-1)] are positive even in the absence of elevated serum creatinine. Recent studies reported that SGLT-2 inhibitors could protect against subclinical AKI in diabetic patients by elevating the level of ß-Hydroxybutyric acid (ßOHB). This study aims to examine the reno-protective potential of empagliflozin (EMPA) against MI associated AKI in diabetic rats. Eighty Albino Wistar rats were divided into: (1) nondiabetic sham group (CS), (2) nondiabetic + myocardial infarction group (CM), (3) diabetic + myocardial infarction group (DM) and (4) diabetic + myocardial infarction + empagliflozin group (DME). At the end of the experiment, blood samples and kidneys were collected for biochemical analysis, histopathological, and immunohistochemical studies. After induction of myocardial infarction, there was a significant decrease in serum creatinine and NGAL levels in DME. After EMPA administration, mesangial matrix index and glomerular area were lowered in DME if compared to DM group. As a marker for tubular injury, we used anti-NGAL and anti-KIM-1 immunohistochemistry. Strong positive reaction was noticed in DM group if compared to DME group which showed weak positive reaction. Levels of renal mRNAs [NGAL; KIM-1; Nox-2,4; TLR-2,4; MyD88; TNF- α and IL-1 ß, 18] in DME group were reduced significantly compared to DM group. In conclusion, empagliflozin can protect against subclinical acute kidney injury in diabetic albino Wistar rats after myocardial infarction induction, which could improve the clinical outcome of SGLT-2 inhibitors in diabetic patients.


Assuntos
Compostos Benzidrílicos/uso terapêutico , Diabetes Mellitus Experimental/tratamento farmacológico , Glucosídeos/uso terapêutico , Rim/efeitos dos fármacos , Rim/metabolismo , Infarto do Miocárdio/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Injúria Renal Aguda/patologia , Injúria Renal Aguda/prevenção & controle , Animais , Moléculas de Adesão Celular/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/prevenção & controle , Eletrocardiografia , Rim/patologia , Lipocalina-2/metabolismo , Masculino , Infarto do Miocárdio/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar
13.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33809984

RESUMO

The Perlecan-Semaphorin 3A-Plexin A1-Neuropilin-1 (PSPN) Complex at the cell surface of prostate cancer (PCa) cells influences cell-cell cohesion and dyscohesion. We investigated matrix metalloproteinase-7/matrilysin (MMP-7)'s ability to digest components of the PSPN Complex in bone metastatic PCa cells using in silico analyses and in vitro experiments. Results demonstrated that in addition to the heparan sulfate proteoglycan, perlecan, all components of the PSPN Complex were degraded by MMP-7. To investigate the functional consequences of PSPN Complex cleavage, we developed a preformed microtumor model to examine initiation of cell dispersion after MMP-7 digestion. We found that while perlecan fully decorated with glycosaminoglycan limited dispersion of PCa microtumors, MMP-7 initiated rapid dyscohesion and migration even with perlecan present. Additionally, we found that a bioactive peptide (PLN4) found in perlecan domain IV in a region subject to digestion by MMP-7 further enhanced cell dispersion along with MMP-7. We found that digestion of the PSPN Complex with MMP-7 destabilized cell-cell junctions in microtumors evidenced by loss of co-registration of E-cadherin and F-actin. We conclude that MMP-7 plays a key functional role in PCa cell transition from a cohesive, indolent phenotype to a dyscohesive, migratory phenotype favoring production of circulating tumor cells and metastasis to bone.


Assuntos
Metaloproteinase 7 da Matriz/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neoplasias da Próstata/metabolismo , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Imunofluorescência , Humanos , Masculino , Modelos Biológicos , Neuropilina-1/metabolismo , Neoplasias da Próstata/etiologia , Ligação Proteica , Proteólise
14.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810326

RESUMO

Musashi-1 (MSI1) is an RNA-binding protein that regulates progenitor cells in adult and developing organisms to maintain self-renewal capacities. The role of musashi-1 in the bone healing environment and its relation with other osteogenic factors is unknown. In the current study, we analyze the expression of MSI1 in an experimental model of rat femoral bone fractures. We also analyze the relation between MSI1 expression and the expression of two osteogenic markers: periostin (POSTN) and runt-related transcription factor 2 (RUNX2). We use histological, immunohistochemical, and qPCR techniques to evaluate bone healing and the expression of MSI1, POSTN, and RUNX2 over time (4, 7, and 14 days). We compare our findings with non-fractured controls. We find that in bone calluses, the number of cells expressing MSI1 and RUNX2 increase over time and the intensity of POSTN expression decreases over time. Within bone calluses, we find the presence of MSI1 expression in mesenchymal stromal cells, osteoblasts, and osteocytes but not in hypertrophic chondrocytes. After 14 days, the expression of MSI1, POSTN, and RUNX2 was significantly correlated. Thus, we conclude that musashi-1 potentially serves in the osteogenic differentiation of mesenchymal stromal cells and bone healing. Therefore, further studies are needed to determine the possibility of musashi-1's role as a clinical biomarker of bone healing and therapeutic agent for bone regeneration.


Assuntos
Consolidação da Fratura , Proteínas do Tecido Nervoso/metabolismo , Osteogênese , Proteínas de Ligação a RNA/metabolismo , Animais , Calo Ósseo/citologia , Calo Ósseo/metabolismo , Calo Ósseo/fisiologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Condrócitos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Proteínas do Tecido Nervoso/genética , Osteoblastos/metabolismo , Osteócitos/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Ratos Wistar
15.
BMC Cancer ; 21(1): 444, 2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33882870

RESUMO

BACKGROUND: DNA methylation is frequently observed in the development and progression of many human tumors as well as renal cell cancer (RCC). Tumor Associated Calcium Signal Transducer 2 (TACSTD2) participates in cell cycle progression through MAPK signalling pathway activation. Moreover, tumor-specific hypermethylation and association with aggressive cancer characteristics has been found for lung adenocarcinoma, hepatocellular carcinoma and cholangiocarcinoma. Whether TACSTD2 is tumor specifically hypermethylated in RCC or shows association of methylation with adverse clinicopathological parameters and survival of patients has not been investigated at yet. METHODS: Quantitative methylation-specific PCR (qMSP) analysis of a locus in the intron 1 region of TACSTD2 gene was carried out in a cross-sectional study of 127 paired RCC and normal samples. In silico analysis of TACSTD2 methylation in the TCGA Kidney Renal Clear Cell Carcinoma (KIRC) dataset of 280 patients served as validation cohort. Statistical analyses were carried out using the two-sided paired t-test for matched tumor and normal sample comparisons, logistic regression for subgroup comparisons, Cox regression for analysis of recurrence free survival (RFS) and Pearson correlation analysis for correlation of TACSTD2 methylation and TACSTD2 mRNA in KIRC data. RESULTS: Higher methylation levels in RCC were significantly associated with advanced disease (p < 0.001), high tumor stage (p = 0.003), tumor differentiation (p = 0.033) and presence of lymph node (p = 0.021) or distant metastases (p = 0.008). TACSTD2 hypermethylation was associated with a shorter RFS of patients and demonstrate statistical independency from clinical parameters as state of metastasis, tumor stage, grade and state of advanced disease. In silico validation using TCGA KIRC data also demonstrated association of TACSTD2 loci with adverse clinicopathology and shortened RFS of patients. In addition, in silico analyses of TCGA KIRC data showed an inverse correlation between DNA methylation levels of TACSTD2 and mRNA expression. CONCLUSIONS: Our results suggest an association between TACSTD2 methylation and disease progression and clinical course of RCC.


Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Carcinoma de Células Renais/etiologia , Carcinoma de Células Renais/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Metilação de DNA , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Ilhas de CpG , Progressão da Doença , Suscetibilidade a Doenças , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico
16.
Int J Mol Sci ; 22(7)2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807264

RESUMO

Skeletal muscle regeneration is a well-organized process that requires remodeling of the extracellular matrix (ECM). In this study, we revealed the protective role of periostin, a matricellular protein that binds to several ECM proteins during muscle regeneration. In intact muscle, periostin was localized at the neuromuscular junction, muscle spindle, and myotendinous junction, which are connection sites between muscle fibers and nerves or tendons. During muscle regeneration, periostin exhibited robustly increased expression and localization at the interstitial space. Periostin-null mice showed decreased muscle weight due to the loss of muscle fibers during repeated muscle regeneration. Cultured muscle progenitor cells from periostin-null mice showed no deficiencies in their proliferation, differentiation, and the expression of Pax7, MyoD, and myogenin, suggesting that the loss of muscle fibers in periostin-null mice was not due to the impaired function of muscle stem/progenitor cells. Periostin-null mice displayed a decreased number of CD31-positive blood vessels during muscle regeneration, suggesting that the decreased nutritional supply from blood vessels was the cause of muscle fiber loss in periostin-null mice. These results highlight the novel role of periostin in maintaining muscle mass during muscle regeneration.


Assuntos
Moléculas de Adesão Celular/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Regeneração/fisiologia , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/fisiologia , Diferenciação Celular , Junções Célula-Matriz/metabolismo , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/metabolismo , Doenças Musculares/metabolismo , Tendões/metabolismo , Cicatrização/fisiologia
17.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799588

RESUMO

We have been studying mesenchymal stem cells (MSCs) in synovial fluid and the intra-articular injection of synovial MSCs in osteoarthritis (OA) knees. Here, mainly based on our own findings, we overview the characteristics of endogenous MSCs in the synovial fluid of OA knees and their mode of action when injected exogenously into OA knees. Many MSCs similar to synovial MSCs were detected in the synovial fluid of human OA knees, and their number correlated with the radiological OA grade. Our suspended synovium culture model demonstrated the release of MSCs from the synovium through a medium into a non-contacting culture dish. In OA knees, endogenous MSCs possibly mobilize in a similar manner from the synovium through the synovial fluid and act protectively. However, the number of mobilized MSCs is limited; therefore, OA progresses in its natural course. Synovial MSC injections inhibited the progression of cartilage degeneration in a rat OA model. Injected synovial MSCs migrated into the synovium, maintained their MSC properties, and increased the gene expressions of TSG-6, PRG-4, and BMP-2. Exogenous synovial MSCs can promote anti-inflammation, lubrication, and cartilage matrix synthesis in OA knees. Based on our findings, we have initiated a human clinical study of synovial MSC injections in OA knees.


Assuntos
Condrogênese/genética , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Osteoartrite do Joelho/terapia , Líquido Sinovial/fisiologia , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Injeções Intra-Articulares , Células-Tronco Mesenquimais/citologia , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Proteoglicanas/genética , Proteoglicanas/metabolismo , Ratos , Líquido Sinovial/citologia , Transplante Heterólogo , Resultado do Tratamento
18.
Int J Mol Sci ; 22(7)2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33805974

RESUMO

The semicarbazide-sensitive amine oxidase (SSAO), also known as vascular adhesion protein-1 (VAP-1) or primary amine oxidase (PrAO), is a deaminating enzyme highly expressed in vessels that generates harmful products as a result of its enzymatic activity. As a multifunctional enzyme, it is also involved in inflammation through its ability to bind and promote the transmigration of circulating leukocytes into inflamed tissues. Inflammation is present in different systemic and cerebral diseases, including stroke and Alzheimer's disease (AD). These pathologies show important affectations on cerebral vessels, together with increased SSAO levels. This review summarizes the main roles of SSAO/VAP-1 in human physiology and pathophysiology and discusses the mechanisms by which it can affect the onset and progression of both stroke and AD. As there is an evident interrelationship between stroke and AD, basically through the vascular system dysfunction, the possibility that SSAO/VAP-1 could be involved in the transition between these two pathologies is suggested. Hence, its inhibition is proposed to be an interesting therapeutical approach to the brain damage induced in these both cerebral pathologies.


Assuntos
Doença de Alzheimer/terapia , Amina Oxidase (contendo Cobre)/metabolismo , Moléculas de Adesão Celular/metabolismo , Transtornos Cerebrovasculares/terapia , Acidente Vascular Cerebral/terapia , Doença de Alzheimer/metabolismo , Aminas/metabolismo , Animais , Adesão Celular , Angiopatia Amiloide Cerebral/metabolismo , Transtornos Cerebrovasculares/metabolismo , Progressão da Doença , Células Endoteliais/metabolismo , Glucose/metabolismo , Humanos , Inflamação/terapia , Leucócitos/citologia , Camundongos , Ratos , Acidente Vascular Cerebral/metabolismo
19.
Eur J Pharmacol ; 900: 174038, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-33737008

RESUMO

Subarachnoid hemorrhage (SAH) due to rupture of an intracranial aneurysm leads to vasospasm resulting in delayed cerebral ischemia. Therapeutic options are currently limited to hemodynamic optimization and nimodipine, which have marginal clinical efficacy. Nitric oxide (NO) modulates cerebral blood flow through activation of the cGMP-Protein Kinase G (PKG) pathway. Our hypothesis is that SAH results in downregulation of signaling components in the NO-PKG pathway which could explain why treatments for vasospasm targeting this pathway lack efficacy and that treatment with a cell permeant phosphopeptide mimetic of downstream effector prevents delayed vasospasm after SAH. Using a rat endovascular perforation model, reduced levels of NO-PKG pathway molecules were confirmed. Additionally, it was determined that expression and phosphorylation of a PKG substrate: Vasodilator-stimulated phosphoprotein (VASP) was downregulated. A family of cell permeant phosphomimetic of VASP (VP) was wasdesigned and shown to have vasorelaxing property that is synergistic with nimodipine in intact vascular tissuesex vivo. Hence, treatment targeting the downstream effector of the NO signaling pathway, VASP, may bypass receptors and signaling elements leading to vasorelaxation and that treatment with VP can be explored as a therapeutic strategy for SAH induced vasospasm and ameliorate neurological deficits.


Assuntos
Fosfopeptídeos/uso terapêutico , Hemorragia Subaracnóidea/tratamento farmacológico , Vasodilatadores/uso terapêutico , Vasoespasmo Intracraniano/tratamento farmacológico , Animais , Moléculas de Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/efeitos dos fármacos , Regulação para Baixo , Desenho de Fármacos , Sinergismo Farmacológico , Proteínas dos Microfilamentos/efeitos dos fármacos , Proteínas dos Microfilamentos/metabolismo , Mimetismo Molecular , Nimodipina/farmacologia , Óxido Nítrico/metabolismo , Fosfopeptídeos/farmacocinética , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Hemorragia Subaracnóidea/metabolismo , Suínos , Vasodilatadores/farmacocinética
20.
Aging (Albany NY) ; 13(5): 7416-7429, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33686968

RESUMO

Fc fragment of IgG-binding protein (FCGBP) is differentially expressed in various tumors. However, the correlation between FCGBP and immune cell infiltration in ovarian cancer remains unclear. FCGBP expression was analyzed using The Cancer Genome Atlas (TCGA) pan-cancer data, and the ovarian cancer expression profile was analyzed using the Gene Expression Omnibus database. The clinical prognostic value of FCGBP was evaluated using clinical survival data from TCGA. Enrichment analysis of FCGBP was performed using the R package clusterProfiler. Based on known immune cell infiltration scores for samples found in TCGA, we analyzed the association between immune cell infiltration level and FCGBP expression. FCGBP was highly expressed and associated with poorer overall survival (p = 0.00051) and disease-specific survival (p = 0.0012) in ovarian cancer and other tumors. Additionally, high FCGBP expression correlated significantly with immune-related gene sets, including those involved in chemokine signaling pathways and innate and adaptive immunity. Further analysis showed that M2 macrophage infiltration increased and M1 macrophage infiltration decreased in tissues with high FCGBP expression. Our study suggests that FCGBP contributes to M2 macrophage polarization by acting as an oncogene in ovarian cancer. FCGBP may represent a clinically helpful biomarker for predicting overall survival of ovarian cancer patients.


Assuntos
Moléculas de Adesão Celular/metabolismo , Neoplasias Ovarianas/metabolismo , Biologia Computacional , Feminino , Humanos , Linfócitos do Interstício Tumoral/patologia , Macrófagos/patologia , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Ovário/metabolismo , Ovário/patologia , Prognóstico , Análise de Sobrevida , Transcriptoma
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