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1.
Zool Res ; 42(5): 650-659, 2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34472226

RESUMO

Phosphatidylserine (PS) is distributed asymmetrically in the plasma membrane of eukaryotic cells. Phosphatidylserine flippase (P4-ATPase) transports PS from the outer leaflet of the lipid bilayer to the inner leaflet of the membrane to maintain PS asymmetry. The ß subunit TMEM30A is indispensable for transport and proper function of P4-ATPase. Previous studies have shown that the ATP11A and TMEM30A complex is the molecular switch for myotube formation. However, the role of Tmem30a in skeletal muscle regeneration remains elusive. In the current study, Tmem30a was highly expressed in the tibialis anterior (TA) muscles of dystrophin-null ( mdx) mice and BaCl 2-induced muscle injury model mice. We generated a satellite cell (SC)-specific Tmem30a conditional knockout (cKO) mouse model to investigate the role of Tmem30a in skeletal muscle regeneration. The regenerative ability of cKO mice was evaluated by analyzing the number and diameter of regenerated SCs after the TA muscles were injured by BaCl 2-injection. Compared to the control mice, the cKO mice showed decreased Pax7 + and MYH3 + SCs, indicating diminished SC proliferation, and decreased expression of muscular regulatory factors (MYOD and MYOG), suggesting impaired myoblast proliferation in skeletal muscle regeneration. Taken together, these results demonstrate the essential role of Tmem30a in skeletal muscle regeneration.


Assuntos
Proteínas de Membrana/metabolismo , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/metabolismo , Animais , Proliferação de Células , Distrofina/genética , Distrofina/metabolismo , Antagonistas de Estrogênios/toxicidade , Regulação da Expressão Gênica/fisiologia , Genótipo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Músculo Esquelético/efeitos dos fármacos , Proteína MyoD/genética , Proteína MyoD/metabolismo , Miogenina/genética , Miogenina/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Regeneração/genética , Tamoxifeno/toxicidade
2.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445121

RESUMO

Cell signaling mediated by the αv integrin plays a pivotal role in macrophage activation in various inflammatory processes, but its involvement in the pathogenesis of dry eye disease (DED) remains unclear. In a murine model of DED, we found increased αv integrin expression in ocular surface macrophages. The αv integrins inhibitor c(RGDfK) ameliorated the corneal damage caused by DED, suggesting a pathogenic role for αv integrin. Because tear hyperosmolarity induces ocular inflammation in DED, a hyperosmolar culture of murine bone marrow-derived macrophages (BMDMs) is used to reproduce inflammation in vitro. However, the expression of proinflammatory cytokine mRNA was minimal, even though αv integrin was induced. In searching for components that are involved in αv integrin-mediated inflammation but that are missing from the culture model, we showed that the levels of vitronectin (VTN), a binding ligand of αv integrins, were increased in the tear fluid and conjunctival stroma of DED animals. The addition of VTN prominently enhanced hyperosmolarity-induced inflammation in BMDMs. Mechanistically, we showed that VTN/αv integrins mediated NF-κB activation to induce inflammatory gene expression in the BMDMs. Our findings indicate that interaction the of VTN with αv integrins is a crucial step in the inflammatory process in DED and suggests a novel therapeutic target.


Assuntos
Síndromes do Olho Seco/metabolismo , Inflamação/metabolismo , Integrina alfaV/metabolismo , Macrófagos/metabolismo , Vitronectina/metabolismo , Animais , Linhagem Celular , Citocinas/metabolismo , Olho/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Células THP-1 , Lágrimas/metabolismo
3.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445787

RESUMO

Mechanical stress is an important factor affecting bone tissue homeostasis. We focused on the interactions among mechanical stress, glucose uptake via glucose transporter 1 (Glut1), and the cellular energy sensor sirtuin 1 (SIRT1) in osteoblast energy metabolism, since it has been recognized that SIRT1, an NAD+-dependent deacetylase, may function as a master regulator of the mechanical stress response as well as of cellular energy metabolism (glucose metabolism). In addition, it has already been demonstrated that SIRT1 regulates the activity of the osteogenic transcription factor runt-related transcription factor 2 (Runx2). The effects of mechanical loading on cellular activities and the expressions of Glut1, SIRT1, and Runx2 were evaluated in osteoblasts and chondrocytes in a 3D cell-collagen sponge construct. Compressive mechanical loading increased osteoblast activity. Mechanical loading also significantly increased the expression of Glut1, significantly decreased the expression of SIRT1, and significantly increased the expression of Runx2 in osteoblasts in comparison with non-loaded osteoblasts. Incubation with a Glut1 inhibitor blocked mechanical stress-induced changes in SIRT1 and Runx2 in osteoblasts. In contrast with osteoblasts, the expressions of Glut1, SIRT1, and Runx2 in chondrocytes were not affected by loading. Our present study indicated that mechanical stress induced the upregulation of Glut1 following the downregulation of SIRT1 and the upregulation of Runx2 in osteoblasts but not in chondrocytes. Since SIRT1 is known to negatively regulate Runx2 activity, a mechanical stress-induced downregulation of SIRT1 may lead to the upregulation of Runx2, resulting in osteoblast differentiation. Incubation with a Glut1 inhibitor the blocked mechanical stress-induced downregulation of SIRT1 following the upregulation of Runx2, suggesting that Glut1 is necessary to mediate the responses of SIRT1 and Runx2 to mechanical loading in osteoblasts.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Osteoblastos/metabolismo , Osteogênese/fisiologia , Transdução de Sinais/fisiologia , Sirtuína 1/metabolismo , Idoso , Diferenciação Celular/fisiologia , Células Cultivadas , Condrócitos/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Homeostase/fisiologia , Humanos , NAD/metabolismo , Estresse Mecânico
4.
FASEB J ; 35(9): e21831, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383982

RESUMO

The nuclear factor of activated T-cells 5 (NFAT5) is a transcriptional regulator of macrophage activation and T-cell development, which controls stabilizing responses of cells to hypertonic and biomechanical stress. In this study, we detected NFAT5 in the media layer of arteries adjacent to human arteriosclerotic plaques and analyzed its role in vascular smooth muscle cells (VSMCs) known to contribute to arteriosclerosis through the uptake of lipids and transformation into foam cells. Exposure of both human and mouse VSMCs to cholesterol stimulated the nuclear translocation of NFAT5 and increased the expression of the ATP-binding cassette transporter Abca1, required to regulate cholesterol efflux from cells. Loss of Nfat5 promoted cholesterol accumulation in these cells and inhibited the expression of genes involved in the management of oxidative stress or lipid handling, such as Sod1, Plin2, Fabp3, and Ppard. The functional relevance of these observations was subsequently investigated in mice fed a high-fat diet upon induction of a smooth muscle cell-specific genetic ablation of Nfat5 (Nfat5(SMC)-/- ). Under these conditions, Nfat5(SMC)-/- but not Nfat5fl/fl mice developed small, focal lipid-rich lesions in the aorta after 14 and 25 weeks, which were formed by intracellular lipid droplets deposited in the sub-intimal VSMCs layer. While known for being activated by external stimuli, NFAT5 was found to mediate the expression of VSMC genes associated with the handling of lipids in response to a cholesterol-rich environment. Failure of this protective function may promote the formation of lipid-laden arterial VSMCs and pro-atherogenic vascular responses.


Assuntos
Aorta/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fatores de Transcrição/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Idoso , Animais , Aterosclerose/metabolismo , Células Cultivadas , Colesterol/metabolismo , Feminino , Células Espumosas/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Hipercolesterolemia/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Estresse Oxidativo/fisiologia , Túnica Íntima/metabolismo
5.
Zoolog Sci ; 38(4): 317-325, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34342952

RESUMO

Many insects in temperate regions avoid environmental adversity for reproduction, and thus enter reproductive diapause according to photoperiod. This reproductive diapause is induced by inhibition of juvenile hormone biosynthesis in the corpus allatum. Some neuropeptides that have an effect on juvenile hormone biosynthesis have been detected in insect brains. Thus, the reproductive diapause may be photoperiodically regulated by these juvenile hormones-controlling neuropeptides. However, there is limited understanding of how the neurons expressing these neuropeptides respond to the photoperiod and control the peptide release accordingly. Here, we performed electrophysiological analyses in the pars intercerebralis (PI) of Plautia stali, where juvenile hormone inhibitory neuropeptides, Plautia stali myoinhibitory peptides (Plast-MIPs) are expressed. We found that the large neurons in the PI showed very high firing activity under diapause-inducing short day conditions. Neurotracer staining revealed that all recorded neurons projected to the nervus corporis cardiaci 1, which is known to be connected to the corpus cardiacum-corpus allatum complex. Finally, we determined how many of the large PI cells expressed Plast-MIP by single cell reverse transcription PCR. About half of large PI neurons coexpressed Plast-Mip and other neuropeptides, Diuretic hormone 44 and insulin-like peptide 1. The remaining cells only expressed Diuretic hormone 44 and insulin-like peptide 1. The present results suggested that large PI neurons, including Plast-MIP neurons, have enhanced activity under short day conditions, which may increase Plast-MIP release to the corpus cardiacum-corpus allatum complex and thus contribute to reproductive diapause.


Assuntos
Heterópteros/fisiologia , Fotoperíodo , Animais , Encéfalo/citologia , Diapausa , Feminino , Regulação da Expressão Gênica/fisiologia , Regulação da Expressão Gênica/efeitos da radiação , Neurônios/fisiologia , Neuropeptídeos/genética , Neuropeptídeos/metabolismo
6.
J Immunol ; 207(4): 1078-1086, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34341172

RESUMO

Emergency granulopoiesis, also known as demand-adapted granulopoiesis, is defined as the response of an organism to systemic bacterial infections, and it results in neutrophil mobilization from reservoir pools and increased myelopoiesis in the bone marrow. Indirect and direct initiating mechanisms of emergency granulopoiesis have been hypothesized. However, the detailed mechanism of hyperactive myelopoiesis in the bone marrow, which leads to granulocyte left shift, remains unknown. In this study, we report that TLR4 is expressed on granulo-monocytic progenitors, as well as mobilized human peripheral blood CD34+ cells, which account for 0.2% of monocytes in peripheral blood, and ∼ 10% in bone marrow. LPS, a component of Gram-negative bacteria that results in a systemic bacterial infection, induces the differentiation of peripheral blood CD34+ cells into myelocytes and monocytes in vitro via the TLR4 signaling pathway. Moreover, CD34+ cells directly responded to LPS stimulation by activating the MAPK and NF-κB signaling pathways, and they produced IL-6 that promotes emergency granulopoiesis by phosphorylating C/EBPα and C/EBPß, and this effect was suppressed by the action of an IL-6 receptor inhibitor. This work supports the finding that TLR is expressed on human hematopoietic stem and progenitor cells, and it provides evidence that human hematopoietic stem and progenitor cells can directly sense pathogens and produce cytokines exerting autocrine and/or paracrine effects, thereby promoting differentiation.


Assuntos
Granulócitos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Interleucina-6/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Receptor 4 Toll-Like/metabolismo , Adaptação Fisiológica/fisiologia , Antígenos CD34/metabolismo , Medula Óssea/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular/fisiologia , Citocinas/metabolismo , Regulação da Expressão Gênica/fisiologia , Células Precursoras de Granulócitos/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Monócitos/metabolismo , Mielopoese/fisiologia
7.
Zool Res ; 42(5): 562-573, 2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34355875

RESUMO

Inositol requiring mutant 80 (INO80) is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells. However, the roles and mechanisms of INO80 in porcine pre-implantation embryonic development remain largely unknown. Here, we show that INO80 modulates trophectoderm epithelium permeability to promote porcine blastocyst development. The INO80 protein is highly expressed in the nuclei during morula-to-blastocyst transition. Functional studies revealed that RNA interference (RNAi)-mediated knockdown of INO80 severely blocks blastocyst formation and disrupts lineage allocation between the inner cell mass and trophectoderm. Mechanistically, single-embryo RNA sequencing revealed that INO80 regulates multiple genes, which are important for lineage specification, tight junction assembly, and fluid accumulation. Consistent with the altered expression of key genes required for tight junction assembly, a permeability assay showed that paracellular sealing is defective in the trophectoderm epithelium of INO80 knockdown blastocysts. Importantly, aggregation of 8-cell embryos from the control and INO80 knockdown groups restores blastocyst development and lineage allocation via direct complementation of the defective trophectoderm epithelium. Taken together, these results demonstrate that INO80 promotes blastocyst development by regulating the expression of key genes required for lineage specification, tight junction assembly, and fluid accumulation.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Blastocisto/fisiologia , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mórula/fisiologia , Suínos , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Proteínas de Ligação a DNA/genética , Técnicas de Cultura Embrionária/veterinária , Fertilização In Vitro , Regulação da Expressão Gênica/fisiologia , Oócitos/fisiologia , Permeabilidade
8.
FASEB J ; 35(8): e21821, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34325487

RESUMO

Skeletal muscle atrophy is a debilitating complication of many chronic disease states and disuse conditions including denervation. However, molecular and signaling mechanisms of muscle wasting remain less understood. Here, we demonstrate that the levels of several toll-like receptors (TLRs) and their downstream signaling adaptor, myeloid differentiation primary response 88 (MyD88), are induced in skeletal muscle of mice in response to sciatic nerve denervation. Muscle-specific ablation of MyD88 mitigates denervation-induced skeletal muscle atrophy in mice. Targeted ablation of MyD88 suppresses the components of ubiquitin-proteasome system, autophagy, and FOXO transcription factors in skeletal muscle during denervation. We also found that specific inhibition of MyD88 reduces the activation of canonical nuclear factor-kappa (NF-κB) pathway and expression of receptors for inflammatory cytokines in denervated muscle. In contrast, inhibition of MyD88 stimulates the activation of non-canonical NF-κB signaling in denervated skeletal muscle. Ablation of MyD88 also inhibits the denervation-induced increase in phosphorylation of AMPK without having any effect on the phosphorylation of mTOR. Moreover, targeted ablation of MyD88 inhibits the activation of a few components of the unfolded protein response (UPR) pathways, especially X-box protein 1 (XBP1). Importantly, myofiber-specific ablation of XBP1 mitigates denervation-induced skeletal muscle atrophy in mice. Collectively, our experiments suggest that TLR-MyD88 signaling mediates skeletal muscle wasting during denervation potentially through the activation of canonical NF-κB signaling, AMPK and UPR pathways.


Assuntos
Músculo Esquelético/inervação , Atrofia Muscular/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Animais , Biomarcadores/sangue , Estresse do Retículo Endoplasmático/fisiologia , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , NF-kappa B/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Resposta a Proteínas não Dobradas
9.
FASEB J ; 35(8): e21826, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34320244

RESUMO

In pancreatic cancer, autocrine insulin-like growth factor-1 (IGF-1) and paracrine insulin stimulate both IGF-1 receptor (IGF1R) and insulin receptor (IR) to increase tumor growth and glycolysis. In pancreatic cancer patients, cancer-induced glycolysis increases hepatic gluconeogenesis, skeletal muscle proteolysis, and fat lipolysis and, thereby, causes cancer cachexia. As a protein coexisting with IGF1R and IR, caveolin-1 (cav-1) may be involved in pancreatic cancer-induced cachexia. We undertook the present study to test this hypothesis. Out of wild-type MiaPaCa2 and AsPC1 human pancreatic cancer cell lines, we created their stable sub-lines whose cav-1 expression was diminished with RNA interference or increased with transgene expression. When these cells were studied in vitro, we found that cav-1 regulated IGF1R/IR expression and activation and also regulated cellular glycolysis. We transplanted the different types of MiaPaCa2 cells in growing athymic mice for 8 weeks, using intact athymic mice as tumor-free controls. We found that cav-1 levels in tumor grafts were correlated with expression levels of the enzymes that regulated hepatic gluconeogenesis, skeletal muscle proteolysis, and fat lipolysis in the respective tissues. When the tumors had original or increased cav-1, their carriers' body weight gain was less than the tumor-free reference. When cav-1 was diminished in tumors, the tumor carriers' body weight gain was not changed significantly, compared to the tumor-free reference. In conclusion, cav-1 in pancreatic cancer cells stimulated IGF1R/IR and glycolysis in the cancer cells and triggered cachectic states in the tumor carrier.


Assuntos
Caquexia/etiologia , Caveolina 1/metabolismo , Glicólise/fisiologia , Neoplasias Pancreáticas/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Animais , Caveolina 1/genética , Linhagem Celular Tumoral , Deleção de Genes , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/metabolismo , Receptor IGF Tipo 1/genética , Receptor de Insulina/genética
10.
FASEB J ; 35(8): e21796, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34324238

RESUMO

Polycystin-1 (PC1) is a transmembrane protein found in different cell types, including cardiomyocytes. Alterations in PC1 expression have been linked to mitochondrial damage in renal tubule cells and in patients with autosomal dominant polycystic kidney disease. However, to date, the regulatory role of PC1 in cardiomyocyte mitochondria is not well understood. The analysis of mitochondrial morphology from cardiomyocytes of heterozygous PC1 mice (PDK1+/- ) using transmission electron microscopy showed that cardiomyocyte mitochondria were smaller with increased mitochondria density and circularity. These parameters were consistent with mitochondrial fission. We knocked-down PC1 in cultured rat cardiomyocytes and human-induced pluripotent stem cells (iPSC)-derived cardiomyocytes to evaluate mitochondrial function and morphology. The results showed that downregulation of PC1 expression results in reduced protein levels of sub-units of the OXPHOS complexes and less functional mitochondria (reduction of mitochondrial membrane potential, mitochondrial respiration, and ATP production). This mitochondrial dysfunction activates the elimination of defective mitochondria by mitophagy, assessed by an increase of autophagosome adapter protein LC3B and the recruitment of the Parkin protein to the mitochondria. siRNA-mediated PC1 knockdown leads to a loss of the connectivity of the mitochondrial network and a greater number of mitochondria per cell, but of smaller sizes, which characterizes mitochondrial fission. PC1 silencing also deregulates the AKT-FoxO1 signaling pathway, which is involved in the regulation of mitochondrial metabolism, mitochondrial morphology, and processes that are part of cell quality control, such as mitophagy. Together, these data provide new insights about the controls that PC1 exerts on mitochondrial morphology and function in cultured cardiomyocytes dependent on the AKT-FoxO1 signaling pathway.


Assuntos
Proteína Forkhead Box O1/metabolismo , Mitofagia/fisiologia , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Canais de Cátion TRPP/metabolismo , Animais , Animais Recém-Nascidos , Proteína Forkhead Box O1/genética , Regulação da Expressão Gênica/fisiologia , Inativação Gênica , Mitocôndrias/metabolismo , Mitofagia/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPP/genética
11.
Nat Struct Mol Biol ; 28(6): 501-511, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34117481

RESUMO

The mammalian SWI/SNF complex, or BAF complex, has a conserved and direct role in antagonizing Polycomb-mediated repression. Yet, BAF also promotes repression by Polycomb in stem cells and cancer. How BAF both antagonizes and promotes Polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF-Polycomb axis in mouse embryonic stem cells on short timescales. We report that rapid BAF depletion redistributes Polycomb repressive complexes PRC1 and PRC2 from highly occupied domains, like Hox clusters, to weakly occupied sites normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 and PRC2, we identify a conventional role for BAF in Polycomb-mediated repression, in addition to global Polycomb redistribution. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb-Trithorax axis.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Repressão Epigenética/fisiologia , Regulação da Expressão Gênica/fisiologia , Complexos Multiproteicos/fisiologia , Proteínas do Grupo Polycomb/fisiologia , Animais , Sistemas CRISPR-Cas , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/fisiologia , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Repressão Epigenética/genética , Edição de Genes , Regulação da Expressão Gênica/genética , Genes Homeobox , Genoma , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Mutação com Perda de Função , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteólise , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia
12.
Zool Res ; 42(4): 450-460, 2021 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-34156172

RESUMO

Over the last several hundred years, donkeys have adapted to high-altitude conditions on the Tibetan Plateau. Interestingly, the kiang, a closely related equid species, also inhabits this region. Previous reports have demonstrated the importance of specific genes and adaptive introgression in divergent lineages for adaptation to hypoxic conditions on the Tibetan Plateau. Here, we assessed whether donkeys and kiangs adapted to the Tibetan Plateau via the same or different biological pathways and whether adaptive introgression has occurred. We assembled a de novo genome from a kiang individual and analyzed the genomes of five kiangs and 93 donkeys (including 24 from the Tibetan Plateau). Our analyses suggested the existence of a strong hard selective sweep at the EPAS1 locus in kiangs. In Tibetan donkeys, however, another gene, i.e., EGLN1, was likely involved in their adaptation to high altitude. In addition, admixture analysis found no evidence for interspecific gene flow between kiangs and Tibetan donkeys. Our findings indicate that despite the short evolutionary time scale since the arrival of donkeys on the Tibetan Plateau, as well as the existence of a closely related species already adapted to hypoxia, Tibetan donkeys did not acquire adaptation via admixture but instead evolved adaptations via a different biological pathway.


Assuntos
Adaptação Fisiológica/genética , Altitude , Equidae/genética , Equidae/fisiologia , Genoma , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Evolução Biológica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Especificidade da Espécie
13.
Mol Cell ; 81(14): 2944-2959.e10, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34166609

RESUMO

A number of regulatory factors are recruited to chromatin by specialized RNAs. Whether RNA has a more general role in regulating the interaction of proteins with chromatin has not been determined. We used proteomics methods to measure the global impact of nascent RNA on chromatin in embryonic stem cells. Surprisingly, we found that nascent RNA primarily antagonized the interaction of chromatin modifiers and transcriptional regulators with chromatin. Transcriptional inhibition and RNA degradation induced recruitment of a set of transcriptional regulators, chromatin modifiers, nucleosome remodelers, and regulators of higher-order structure. RNA directly bound to factors, including BAF, NuRD, EHMT1, and INO80 and inhibited their interaction with nucleosomes. The transcriptional elongation factor P-TEFb directly bound pre-mRNA, and its recruitment to chromatin upon Pol II inhibition was regulated by the 7SK ribonucleoprotein complex. We postulate that by antagonizing the interaction of regulatory proteins with chromatin, nascent RNA links transcriptional output with chromatin composition.


Assuntos
Cromatina/metabolismo , RNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos , Nucleossomos/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Ligação Proteica/fisiologia , Proteômica/métodos , RNA Polimerase II/metabolismo , Transcrição Genética/fisiologia , Fatores de Elongação da Transcrição/metabolismo
14.
FASEB J ; 35(7): e21725, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34110636

RESUMO

Renal fibrosis leads to chronic kidney disease, which affects over 15% of the U.S. population. PAI-1 is highly upregulated in the tubulointerstitial compartment in several common nephropathies and PAI-1 global ablation affords protection from fibrogenesis in mice. The precise contribution of renal tubular PAI-1 induction to disease progression, however, is unknown and surprisingly, appears to be independent of uPA inhibition. Human renal epithelial (HK-2) cells engineered to stably overexpress PAI-1 underwent dedifferentiation (E-cadherin loss, gain of vimentin), G2/M growth arrest (increased p-Histone3, p21), and robust induction of fibronectin, collagen-1, and CCN2. These cells are also susceptible to apoptosis (elevated cleaved caspase-3, annexin-V positivity) compared to vector controls, demonstrating a previously unknown role for PAI-1 in tubular dysfunction. Persistent PAI-1 expression results in a loss of klotho expression, p53 upregulation, and increases in TGF-ßRI/II levels and SMAD3 phosphorylation. Ectopic restoration of klotho in PAI-1-transductants attenuated fibrogenesis and reversed the proliferative defects, implicating PAI-1 in klotho loss in renal disease. Genetic suppression of p53 reversed the PA1-1-driven maladaptive repair, moreover, confirming a pathogenic role for p53 upregulation in this context and uncovering a novel role for PAI-1 in promoting renal p53 signaling. TGF-ßRI inhibition also attenuated PAI-1-initiated epithelial dysfunction, independent of TGF-ß1 ligand synthesis. Thus, PAI-1 promotes tubular dysfunction via klotho reduction, p53 upregulation, and activation of the TGF-ßRI-SMAD3 axis. Since klotho is an upstream regulator of both PAI-1-mediated p53 induction and SMAD3 signaling, targeting tubular PAI-1 expression may provide a novel, multi-level approach to the therapy of CKD.


Assuntos
Células Epiteliais/metabolismo , Glucuronidase/metabolismo , Rim/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Insuficiência Renal Crônica/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular , Fibroblastos/metabolismo , Fibrose/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Fosforilação/fisiologia , Transdução de Sinais , Proteína Smad3/metabolismo , Regulação para Cima/fisiologia
15.
Exp Eye Res ; 207: 108610, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33940009

RESUMO

Our earlier decorin (Dcn) gene overexpression studies found that the targeted Dcn gene transfer into the cornea inhibited corneal angiogenesis in vivo using a rabbit model. In this study, we tested the hypothesis that anti-angiogenic effects of decorin in the cornea are mediated by alterations in a normal physiologic balance of pro- and anti-angiogenic factors using decorin deficient (Dcn-/-) and wild type (Dcn+/+) mice. Corneal neovascularization (CNV) in Dcn-/- and Dcn+/+ mice was produced with a standard chemical injury technique. The clinical progression of CNV in mice was monitored with stereo- and slit-lamp microscopes, and histopathological hematoxylin and eosin (H&E) staining. Protein and mRNA expression of pro- and anti-angiogenic factors in the cornea were evaluated using immunofluorescence and quantitative real-time PCR, respectively. Slit-lamp clinical eye examinations revealed significantly more CNV in Dcn-/- mice than the Dcn+/+ mice post-injury (p < 0.05) and AAV5-Dcn gene therapy significantly reduced CNV in Dcn-/- mice compered to no AAV5-Dcn gene therapy controls (p < 0.001). H&E-stained corneal sections exhibited morphology with several neovessels in injured corneas of the Dcn-/- mice than the Dcn+/+ mice. Immunofluorescence of corneal sections displayed significantly higher expression of α-smooth muscle actin (α-SMA) and endoglin proteins in Dcn-/- mice than Dcn+/+ mice (p < 0.05). Quantitative real-time PCR found significantly increased mRNA levels of pro-angiogenic factors endoglin (2.53-fold; p < 0.05), Vegf (2.47-fold; p < 0.05), and Pecam (2.14-fold; p < 0.05) and anti-angiogenic factor Vegfr2 (1.56-fold; p < 0.05) in the normal cornea of the Dcn-/- mice than the Dcn+/+ mice. Furthermore, neovascularized Dcn-/- mice corneas showed greater increase in mRNA expression of pro-angiogenic factors endoglin (4.58-fold; p < 0.0001), Vegf (4.16-fold; p < 0.0001), and Pdgf (2.15-fold; p < 0.0001) and reduced expression of anti-angiogenic factors Ang2 (0.12-fold; p < 0.05), Timp1 (0.22-fold; p < 0.05), and Vegfr2 (0.67-fold; p > 0.05) compared to neovascularized Dcn+/+ mice corneas. These gene deficience studies carried with transgenic Dcn-/- mice revealed decorin's role in influencing a physiologic balance between pro-and anti-angiogenic factors in the normal and injured cornea. We infer that the functional deletion of Dcn promotes irregular corneal repair and aggravates CNV.


Assuntos
Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/fisiopatologia , Decorina/fisiologia , Actinas/metabolismo , Animais , Neovascularização da Córnea/genética , Endoglina/genética , Endoglina/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
16.
J Nutr Biochem ; 95: 108761, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33965533

RESUMO

Inflammation in insulin-sensitive tissues (e.g., liver, visceral adipose tissue [VAT]) plays a major role in obesity and insulin resistance. Recruitment of innate immune cells drives the dysregulation of glucose and lipid metabolism. We aimed to seek the role of Toll like receptor 3 (TLR3), a pattern recognition receptor involved in innate immunity, obesity and the metabolic disorder. TLR3 expression in liver and VAT from diet induced obese mice and in VAT from overweight women was examined. Body weight, glucose homeostasis and insulin sensitivity were evaluated in TLR3 wild-type and knockout (KO) mice on a chow diet (CD) or high-fat diet for 15 weeks. At euthanasia, blood was collected, and plasma biochemical parameters and adipokines were determined with commercial kits. Flow cytometry was used to measure macrophage infiltration and activation in VAT. Standard western blot, immunohistochemistry and quantative PCR were used to assess molecules in pathways about lipid and glucose metabolism, insulin and inflammation in tissues of liver and VAT. Utilizing human and animal samples, we found that expression of TLR3 was upregulated in the liver and VAT in obese mice as well as VAT in overweight women. TLR3-deficiency protected against high-fat diet induced obesity, glucose intolerance, insulin resistance and lipid accumulation. Lipolysis was enhanced in VAT and hepatic lipogenesis was inhibited in TLR3 KO animals. Macrophages infiltration into adipose tissue was attenuated in TLR3 KO mice, accompanied with inhibition of NF-κB-dependent AMPK/Akt signaling pathway. These findings demonstrated that TLR3 ablation prevented obesity and metabolic disorders, thereby providing new mechanistic links between inflammation and obesity and associated metabolic abnormalities in lipid/glucose metabolism.


Assuntos
Gordura Intra-Abdominal/metabolismo , Receptor 3 Toll-Like/metabolismo , Adulto , Animais , Dieta Hiperlipídica , Feminino , Regulação da Expressão Gênica/fisiologia , Intolerância à Glucose , Transtornos do Metabolismo de Glucose , Humanos , Metabolismo dos Lipídeos , Fígado/metabolismo , Macrófagos/fisiologia , Masculino , Síndrome Metabólica , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica , Obesidade , Receptor 3 Toll-Like/genética
17.
Clin Nutr ; 40(5): 2683-2696, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33933734

RESUMO

BACKGROUND: Zinc deficiency is a worldwide public health problem. Currently, there are no established biomarkers available for the accurate diagnosis of zinc-deficiency in individuals. Additionally, a comprehensive view of the adverse effects of zinc deficiency is lacking. Our aim was to identify superior biomarkers of zinc deficiency and uncover the adverse effects of zinc deficiency. METHODS: We performed multi-omics analysis using serum proteomics-metabolomics and liver proteomics on zinc-deficient rats to identify candidate biomarkers and reveal the associated adverse effects of zinc deficiency. Secondly, the candidate biomarkers were validated in two zinc-deficient populations and an RCT zinc supplementation trial on a zinc-deficient population. RESULTS: Our integrated multi-omics approach revealed numerous biomarkers (>2000) and glutathione metabolism as the most important changed pathway in zinc deficiency. Three candidate biomarkers from glutathione metabolism were validated in repeated zinc-deficient rats by quantitative analysis. Only glutathione sulfotransferase omega-1 (GSTO1) (among 3 candidate biomarkers) was validated in the two zinc-deficient populations and zinc-supplemented population. Compared with serum zinc, serum GSTO1 yielded a better response to zinc supplementation and a higher correlation coefficient with zinc intake and the AUC value and has the potential for diagnosing zinc deficiency. By integrated multi-omics, we identified both established and novel adverse effects of zinc deficiency. CONCLUSIONS: Our integrated multi-omics analysis revealed more complete information about zinc deficiency; GSTO1 was found to be a reliable potential biomarker for diagnosis of zinc deficiency. This trial is registered at http://www.chictr.org.cn/registry.aspx as ChiCTR1900028162.


Assuntos
Metabolômica/métodos , Proteômica/métodos , Zinco/deficiência , Adulto , Animais , Biomarcadores/sangue , Criança , Pré-Escolar , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas/genética , Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Zinco/metabolismo
18.
Nat Commun ; 12(1): 2617, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976169

RESUMO

Disruption of the transcription factor FoxP2, which is enriched in the basal ganglia, impairs vocal development in humans and songbirds. The basal ganglia are important for the selection and sequencing of motor actions, but the circuit mechanisms governing accurate sequencing of learned vocalizations are unknown. Here, we show that expression of FoxP2 in the basal ganglia is vital for the fluent initiation and termination of birdsong, as well as the maintenance of song syllable sequencing in adulthood. Knockdown of FoxP2 imbalances dopamine receptor expression across striatal direct-like and indirect-like pathways, suggesting a role of dopaminergic signaling in regulating vocal motor sequencing. Confirming this prediction, we show that phasic dopamine activation, and not inhibition, during singing drives repetition of song syllables, thus also impairing fluent initiation and termination of birdsong. These findings demonstrate discrete circuit origins for the dysfluent repetition of vocal elements in songbirds, with implications for speech disorders.


Assuntos
Corpo Estriado/metabolismo , Tentilhões/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/fisiologia , Vocalização Animal/fisiologia , Adulto , Animais , Animais Geneticamente Modificados , Dopamina/metabolismo , Técnicas de Silenciamento de Genes , Centro Vocal Superior , Humanos , Masculino , Modelos Animais , Vias Neurais/fisiologia , Optogenética , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/metabolismo , Fala/fisiologia , Técnicas Estereotáxicas
19.
Gene ; 791: 145724, 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34010703

RESUMO

Intravitreal anti-vascular endothelial growth factor agents are the gold standard treatment of ocular neovascular diseases. However, their short-term efficacy implies frequent intravitreal injections. Gene therapy has the ability to provide longer duration of the therapeutic effect. We have previously described the effectiveness of the self-replicating episomal vector, pEPito, in long-term gene expression in mouse retina. In this study, we evaluated different constructs to overexpress pigment epithelium-derived factor (PEDF), an angiogenesis inhibitor, and simultaneously, to silence placental growth factor (PlGF), a key player in neovascularization. We employed the human cytomegalovirus promoter to drive the expression of PEDF and PlGF shRNA, in conjunction with cis-acting ribozymes, using pEPito as expressing vector. Our results demonstrated that the non-viral systems were able to efficiently promote a sustained increase of the PEDF: PlGF ratio in the mice retina, decreased in pathological conditions. This innovative approach could open avenues for the development of new therapeutic strategies.


Assuntos
Terapia Genética/métodos , Retina/metabolismo , Neovascularização Retiniana/metabolismo , Inibidores da Angiogênese/metabolismo , Animais , Linhagem Celular , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Vetores Genéticos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/fisiopatologia , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Fator de Crescimento Placentário/genética , Fator de Crescimento Placentário/metabolismo , Retina/fisiologia , Neovascularização Retiniana/genética , Serpinas/genética , Serpinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética
20.
Am J Physiol Regul Integr Comp Physiol ; 321(1): R41-R48, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34009045

RESUMO

Preeclampsia (PE) is a life-threatening human gestational syndrome with incompletely understood etiopathogenesis. The disorder has a spectrum of clinical features, likely due to a complex interaction between maternal predisposing factors and abnormalities at the maternal-fetal interface. Poor trophoblast cell invasion, inadequate uterine vascular remodeling, and placental hypoperfusion are considered as key placental events leading to PE. Kisspeptins, a family of small peptides derived from the KISS1 gene, have been implicated in the development of this syndrome. Most studies of kisspeptin expression in PE have reported an upregulation of kisspeptins and/or their cognate receptor in preeclamptic placentas. Conversely, maternal peripheral blood concentration of kisspeptins is reportedly lower in PE than in uncomplicated pregnancies. This apparent paradox remains to be further elucidated. Although kisspeptins were initially known for inhibiting cellular migration and invasion, other biological activities attributed to these peptides include neuroendocrine regulation of reproduction, metabolism regulation, inhibition of angiogenesis, and induction of apoptosis. This review summarizes the current knowledge on expression and biological activity of kisspeptins at the maternal-fetal interface in the context of PE.


Assuntos
Apoptose/fisiologia , Regulação da Expressão Gênica/fisiologia , Kisspeptinas/metabolismo , Pré-Eclâmpsia/metabolismo , Trofoblastos/fisiologia , Feminino , Humanos , Gravidez
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