Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 120
Filtrar
1.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070901

RESUMO

Glycosaminoglycans (GAGs) and proteoglycans (PGs) are major components of the glycocalyx. The secreted GAG and CD44 ligand hyaluronic acid (HA), and the cell surface PG syndecan-1 (Sdc-1) modulate the expression and activity of cytokines, chemokines, growth factors, and adhesion molecules, acting as critical regulators of tumor cell behavior. Here, we studied the effect of Sdc-1 siRNA depletion and HA treatment on hallmark processes of cancer in breast cancer cell lines of different levels of aggressiveness. We analyzed HA synthesis, and parameters relevant to tumor progression, including the stem cell phenotype, Wnt signaling constituents, cell cycle progression and apoptosis, and angiogenic markers in luminal MCF-7 and triple-negative MDA-MB-231 cells. Sdc-1 knockdown enhanced HAS-2 synthesis and HA binding in MCF-7, but not in MDA-MB-231 cells. Sdc-1-depleted MDA-MB-231 cells showed a reduced CD24-/CD44+ population. Furthermore, Sdc-1 depletion was associated with survival signals in both cell lines, affecting cell cycle progression and apoptosis evasion. These changes were linked to the altered expression of KLF4, MSI2, and miR-10b and differential changes in Erk, Akt, and PTEN signaling. We conclude that Sdc-1 knockdown differentially affects HA metabolism in luminal and triple-negative breast cancer model cell lines and impacts the stem phenotype, cell survival, and angiogenic factors.


Assuntos
Regulação Neoplásica da Expressão Gênica , Glicocálix/metabolismo , Ácido Hialurônico/metabolismo , Sindecana-1/genética , Neoplasias de Mama Triplo Negativas/genética , Via de Sinalização Wnt/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Antígeno CD24/genética , Antígeno CD24/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Bases de Dados Factuais , Feminino , Glicocálix/química , Glicocálix/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Hialuronan Sintases/genética , Hialuronan Sintases/metabolismo , Ácido Hialurônico/farmacologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Células MCF-7 , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sobrevida , Sindecana-1/antagonistas & inibidores , Sindecana-1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologia
2.
Viruses ; 13(6)2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064104

RESUMO

Patients with coronavirus disease 2019 (COVID-19) predominantly have a respiratory tract infection with various symptoms and high mortality is associated with respiratory failure second to severe disease. The risk factors leading to severe disease remain unclear. Here, we reanalyzed a published single-cell RNA-Seq (scRNA-Seq) dataset and found that bronchoalveolar lavage fluid (BALF) of patients with severe disease compared to those with mild disease contained decreased TH17-type cells, decreased IFNA1-expressing cells with lower expression of toll-like receptor 7 (TLR7) and TLR8, increased IgA-expressing B cells, and increased hyperactive epithelial cells (and/or macrophages) expressing matrix metalloproteinases (MMPs), hyaluronan synthase 2 (HAS2), and plasminogen activator inhibitor-1 (PAI-1), which may together contribute to the pulmonary pathology in severe COVID-19. We propose IFN-I (and TLR7/TLR8) and PAI-1 as potential biomarkers to predict the susceptibility to severe COVID-19.


Assuntos
COVID-19/patologia , Pulmão/patologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Biomarcadores/metabolismo , Líquido da Lavagem Broncoalveolar/imunologia , COVID-19/imunologia , COVID-19/metabolismo , Bases de Dados Genéticas , Humanos , Hialuronan Sintases/metabolismo , Imunoglobulina A/metabolismo , Interferon-alfa/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Metaloproteinases da Matriz/metabolismo , Mucina-1/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , RNA-Seq , SARS-CoV-2 , Células Th17/metabolismo , Células Th17/patologia
3.
J Histochem Cytochem ; 69(6): 415-428, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34080894

RESUMO

Although many studies have focused on a role for hyaluronan (HA) of interstitial extracellular matrix (presumably produced by non-vascular "stromal" cells) in regulating vascular growth, we herein examine the influence of "autocrine HA" produced by vascular endothelial cells themselves on tubulogenesis, using human umbilical vein endothelial cells (HUVECs) in angiogenic and vasculogenic three-dimensional collagen gel cultures. Relative to unstimulated controls, tubulogenic HUVECs upregulated HAS2 mRNA and increased the synthesis of cell-associated HA (but not HA secreted into media). Confocal microscopy/immunofluorescence on cultures fixed with neutral-buffered 10% formalin (NBF) revealed cytoplasmic HAS2 in HUVEC cords and tubes. Cultures fixed with NBF (with cetylpyridinium chloride added to retain HA), stained for HA using "affinity fluorescence" (biotinylated HA-binding protein with streptavidin-fluor), and viewed by confocal microscopy showed HA throughout tube lumens, but little/no HA on the abluminal sides of the tubes or in the surrounding collagen gel. Lumen formation in angiogenic and vasculogenic cultures was strongly suppressed by metabolic inhibitors of HA synthesis (mannose and 4-methylumbelliferone). Hyaluronidase strongly inhibited lumen formation in angiogenic cultures, but not in vasculogenic cultures (where developing lumens are not open to culture medium). Collectively, our results point to a role for autocrine, luminal HA in microvascular sprouting and lumen development. (J Histochem Cytochem 69: 415-428, 2021).


Assuntos
Células Endoteliais/metabolismo , Ácido Hialurônico/metabolismo , Neovascularização Fisiológica , Técnicas de Cultura de Células , Colágeno/metabolismo , Células Endoteliais/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Hialuronan Sintases/genética , Hialuronan Sintases/metabolismo , Regulação para Cima
4.
Mol Cell Biochem ; 476(9): 3383-3391, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33954907

RESUMO

Although luminal breast cancer cells are typically highly cohesive epithelial cells and have low invasive ability, many eventually develop metastasis. Until now, the underlying mechanisms remain obscure. In this work, we showed that the level of hyaluronic acid synthase 2 (HAS2) was positively correlated with the malignant phenotype of breast cancer cells. Notably, the increased expression of HAS2 promoted the invasive and migratory abilities of luminal breast cancer cells in vitro, followed by a reduced expression of E-cadherin, ß-catenin, and ZO-1, and an elevated expression of N-cadherin and vimentin. Furthermore, overexpression of HAS2 promoted while knockdown of HAS2 impeded invadopodia formation, which subsequently increased or decreased the activation of cortactin, Tks5, and metalloproteinases (MMPs). Activation of these invadopodia-related proteins was prevented by inhibition of HAS2 or disruption of HA, which in turn attenuated the increased motility and invasiveness. Further, in vivo study showed that, HAS2 increased tumor growth and the rate of lung metastasis via driving transition to an invasive cell phenotype in SCID mice that were orthotopically transplanted with luminal breast cancer cells. Collectively, our results showed that HAS2 promoted cell invasion by inducing transition to an invasive phenotype and by enhancing invadopodia formation in luminal breast cancer cells, which may provide new mechanistic insights into its role in tumor metastasis.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Hialuronan Sintases/metabolismo , Podossomos/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Humanos , Hialuronan Sintases/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Podossomos/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
5.
BMC Cancer ; 21(1): 456, 2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33892667

RESUMO

BACKGROUND: Endometrial cancer (UCEC) is one of the most common gynecological malignancies. We previously found that overexpression of G protein α subunit 14 (GNA14) promoted UCEC growth. Krüppel-like factor 7 (KLF7) acts as an oncogene in various cancer types, whereas the connection between GNA14 and KLF7 in UCEC is unclear. We herein explored the involvement of GNA14/KLF7 in UCEC development. METHODS: Clinical relevance of GNA14, KLF7 and HAS2 in UCEC was analyzed from TCGA and by immunohistochemical staining. Knockdown and overexpression of indicated genes were conducted by transfecting the cells with siRNAs and lentivirus, respectively. mRNA and protein expression was detected by qRT-PCR and Western blot. CCK8, colony formation, cell cycle, apoptosis, transwell and wound healing were performed to check cell biology function in vitro. Tumor growth in nude mice was conducted to check in vivo function. RNA sequencing was used to determine dys-regulated genes. RESULTS: We demonstrated that GNA14 stimulated the expression of KLF7 in UCEC cells. There was a positive correlation between GNA14 and KLF7 in normal and UCEC tissues. In vitro, KLF7 promoted cell proliferation, colony formation, cell cycle progression, and migration of UCEC cells. Apoptosis was inhibited by KLF7. Xenografted tumorigenesis of UCEC cells was suppressed by KLF7 knockdown. Furthermore, RNA sequencing results showed that KLF7 regulated the expression of a large amount of genes, among which hyaluronan synthase 2 (HAS2) was downregulated in KLF7 knockdown cells. Based on TCGA database and immunoblotting assays, KLF7 positively regulated HAS2 in UCEC cells and tissues. Lastly, knockdown of HAS2 reversed the oncogenic role of KLF7 on UCEC cell proliferation, migration, and xenografted tumor development. CONCLUSION: Taken together, we reveal that GNA14/KLF7/HAS2 signaling cascade exerts tumor promoting function during UCEC development.


Assuntos
Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Hialuronan Sintases/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação para Baixo , Neoplasias do Endométrio/genética , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Xenoenxertos , Humanos , Hialuronan Sintases/genética , Fatores de Transcrição Kruppel-Like/genética , Lentivirus , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Transfecção/métodos , Regulação para Cima
6.
Int J Mol Sci ; 22(6)2021 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-33803805

RESUMO

During biomineralization, the cells generating the biominerals must be able to sense the external physical stimuli exerted by the growing mineralized tissue and change their intracellular protein composition according to these stimuli. In molluscan shell, the myosin-chitin synthases have been suggested to be the link for this communication between cells and the biomaterial. Hyaluronan synthases (HAS) belong to the same enzyme family as chitin synthases. Their product hyaluronan (HA) occurs in the bone and is supposed to have a regulatory function during bone regeneration. We hypothesize that HASes' expression and activity are controlled by fluid-induced mechanotransduction as it is known for molluscan chitin synthases. In this study, bone marrow-derived human mesenchymal stem cells (hMSCs) were exposed to fluid shear stress of 10 Pa. The RNA transcriptome was analyzed by RNA sequencing (RNAseq). HA concentrations in the supernatants were measured by ELISA. The cellular structure of hMSCs and HAS2-overexpressing hMSCs was investigated after treatment with shear stress using confocal microscopy. Fluid shear stress upregulated the expression of genes that encode proteins belonging to the HA biosynthesis and bone mineralization pathways. The HAS activity appeared to be induced. Knowledge about the regulation mechanism governing HAS expression, trafficking, enzymatic activation and quality of the HA product in hMSCs is essential to understand the biological role of HA in the bone microenvironment.


Assuntos
Hialuronan Sintases/metabolismo , Células-Tronco Mesenquimais/enzimologia , Reologia , Estresse Mecânico , Idoso , Idoso de 80 Anos ou mais , Forma Celular , Células Cultivadas , Humanos , Ácido Hialurônico/biossíntese , Masculino , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Transcrição Genética , Regulação para Cima/genética
7.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669634

RESUMO

Little is known about the effects on hyaluronan (HA) metabolism of UVA radiation. This study demonstrates that the secretion of HA by human dermal fibroblasts (HDFs) is downregulated by UVA, accompanied by the down- and upregulation of mRNA and protein levels of the HA-synthesizing enzyme (HAS2) and the HA-degrading protein, HYaluronan Binding protein Involved in HA Depolymerization(HYBID), respectively. Signaling analysis revealed that the exposure distinctly elicits activation of the p38/MSK1/CREB/c-Fos/AP-1 axis, the JNK/c-Jun axis, and the p38/ATF-2 axis, but downregulates the phosphorylation of NF-kB and JAK/STAT3. A signal inhibition study demonstrated that the inhibition of p38 significantly abrogates the UVA-accentuated mRNA level of HYBID. Furthermore, the inhibition of STAT3 significantly downregulates the level of HAS2 mRNA in non-UVA exposed HDFs. Analysis using siRNAs demonstrated that transfection of ATF-2 siRNA but not c-Fos siRNA abrogates the increased protein level of HYBID in UVA-exposed HDFs. An inhibitor of protein tyrosine phosphatase but not of protein serine/threonine phosphatase restored the diminished phosphorylation level of STAT3 at Tyr 705, accompanied by a significant abolishing effect on the decreased mRNA expression level of HAS2. Silencing with a protein tyrosine phosphatase PTP-Meg2 siRNA revealed that it abrogates the decreased phosphorylation of STAT3 at Tyr 705 in UVA-exposed HDFs. These findings suggest that the UVA-induced decrease in HA secretion by HDFs is attributable to the down- and upregulation of HAS2 and HYBID expression, respectively, changes that are mainly ascribed to the inactivated signaling of the STAT3 axis due to the activated tyrosine protein phosphatase PTP-Meg2 and the activated signaling of the p38/ATF2 axis, respectively.


Assuntos
Regulação para Baixo/efeitos da radiação , Fibroblastos/efeitos da radiação , Hialuronan Sintases/metabolismo , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/metabolismo , Transdução de Sinais/efeitos da radiação , Raios Ultravioleta , Regulação para Cima/efeitos da radiação , Fator 2 Ativador da Transcrição/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Derme/citologia , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Janus Quinase 2/metabolismo , Masculino , Modelos Biológicos , Peso Molecular , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
BMB Rep ; 54(2): 136-141, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33407998

RESUMO

Thyroid eye disease (TED) is a complex autoimmune disease with a spectrum of signs. we previously reported that trisialoganglioside (GT)1b is significantly overexpressed in the orbital tissue of TED patients, and that exogenous GT1b strongly induced HA synthesis in orbital fibroblasts. However, the signaling pathway in GT1b-induced hyaluronic acid synthase (HAS) expression in orbital fibroblasts from TED patients have rarely been investigated. Here, we demonstrated that GT1b induced phosphorylation of Akt/mTOR in a dose-dependent manner in orbital fibroblasts from TED patients. Both co-treatment with a specific inhibitor for PI3K and siRNA knockdown of TLR2 attenuated GT1b-induced Akt phosphorylation. GT1b significantly induced HAS2 expression at both the transcriptional and translational level, which was suppressed by specific inhibitors of PI3K or Akt/mTOR, and by siRNA knockdown of TLR2. In conclusion, GT1b induced HAS2 in orbital fibroblasts from TED patients via activation of the PI3Krelated signaling pathway, dependent on TLR2. [BMB Reports 2021; 54(2): 136-141].


Assuntos
Fibroblastos/efeitos dos fármacos , Gangliosídeos/farmacologia , Oftalmopatia de Graves/tratamento farmacológico , Hialuronan Sintases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptor 2 Toll-Like/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Oftalmopatia de Graves/metabolismo , Oftalmopatia de Graves/patologia , Humanos , Hialuronan Sintases/genética , Ácido Hialurônico/biossíntese
9.
Biochem Biophys Res Commun ; 539: 77-82, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33422943

RESUMO

In the skin, the metabolism of hyaluronan (HA) is highly regulated. Aging leads to chronic low-grade inflammation, which is characterized by elevated levels of pro-inflammatory cytokines; however, the relationship between inflammation and HA metabolism is not clear. Herein, we investigated the effects of a mixture of pro-inflammatory cytokines containing TNF-α, IL-1ß, and IL-6 on HA metabolism in human skin fibroblasts. Treatment with the cytokine mixture for 24 h suppressed HA depolymerization via downregulation of HYBID (HA-binding protein involved in HA depolymerization/KIAA1199/CEMIP) and promoted HA synthesis via upregulation of HAS2 in human skin fibroblasts. Moreover, HAS2-dependent HA synthesis was driven mainly by IL-1ß with partial contribution from TNF-α. Transmembrane protein 2 (TMEM2/CEMIP2), which was previously reported as a candidate hyaluronidase, was upregulated by the cytokine mixture, suggesting that TMEM2 might not function as a hyaluronidase in human skin fibroblasts. Furthermore, the effects of the cytokine mixture on HA metabolism were observed in fibroblasts after 8 days of treatment with cytokines during three passages. Thus, we have shown that HYBID-mediated HA metabolism is negatively regulated by the pro-inflammatory cytokine mixture, providing novel insights into the relationship between inflammation and HA metabolism in the skin.


Assuntos
Receptores de Hialuronatos/metabolismo , Hialuronan Sintases/metabolismo , Ácido Hialurônico/química , Hialuronoglucosaminidase/antagonistas & inibidores , Interleucina-1beta/farmacologia , Pele/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Hialuronan Sintases/genética , Ácido Hialurônico/metabolismo , Pele/metabolismo , Pele/patologia , Envelhecimento da Pele/efeitos dos fármacos , Envelhecimento da Pele/genética , Envelhecimento da Pele/patologia
10.
Biomed Res Int ; 2021: 8861766, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33506044

RESUMO

Objective: SPHK1 and HAS2 have been reported to play important roles in tumorigenesis and development. However, their expression and prognostic value in pancreatic cancer (PC) remain unclear. This study is aimed at investigating the expression of SPHK1 and HAS2 on the prognosis of pancreatic cancer. Materials and Methods: The expression of SPHK1 and HAS2 in pancreatic cancer tissues was analyzed through TCGA and GTEx databases and validated by qRT-PCR and Western blot in pancreatic cancer cell lines. χ 2 test was used to explore the correlation of the SPHK1 and HAS2 expressions with clinical characteristics. Kaplan-Meier survival analysis and ROC curve were used to evaluate the prognostic and diagnostic roles of SPHK1 and HAS2 in pancreatic cancer. Additionally, Spearman correlation analysis was applied to assess the correlation between the SPHK1 and HAS2 in pancreatic cancer. GO analysis and KEGG analysis were applied to explore the possible signaling pathway that SPHK1 and HAS2 coregulated genes mediated. Results: The expression of SPHK1 and HAS2 was markedly upregulated in pancreatic cancer tissue and cell lines, respectively. Furthermore, there was a significant positive correlation between SPHK1 and HAS2 expressions. ROC curves showed that SPHK1 combine with HAS2 has good diagnostic value in pancreatic cancer patients with 85% sensitivity and 99.4% specificity. Kaplan-Meier analysis showed that increased expression of SPHK1 and HAS2 was significantly associated with short overall survival (OS) of pancreatic cancer patients. GO and KEGG results revealed that SPHK1 and HAS2 mainly involved cell proliferation and invasion mediated by extracellular matrix- (ECM-) receptor interaction, focal adhesion, and PI3K-AKT signaling pathways. Conclusions: Overexpression of SPHK1 and HAS2 could be important markers for the prognosis of pancreatic cancer.


Assuntos
Hialuronan Sintases/biossíntese , Hialuronan Sintases/genética , Neoplasias Pancreáticas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Biologia Computacional/métodos , Bases de Dados Genéticas , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hialuronan Sintases/metabolismo , Masculino , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Prognóstico , Curva ROC , Transdução de Sinais , Taxa de Sobrevida
11.
J Histochem Cytochem ; 69(1): 35-47, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32623953

RESUMO

Hyaluronan (HA) is a linear glycosaminoglycan (GAG) of extracellular matrix (ECM) synthesized by three hyaluronan synthases (HASes) at the plasma membrane using uridine diphosphate (UDP)-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc) as substrates. The production of HA is mainly regulated by hyaluronan synthase 2 (HAS2), that can be controlled at different levels, from epigenetics to transcriptional and post-translational modifications. HA biosynthesis is an energy-consuming process and, along with HA catabolism, is strongly connected to the maintenance of metabolic homeostasis. The cytoplasmic pool of UDP-sugars is critical for HA synthesis. UDP-GlcNAc is an important nutrient sensor and serves as donor substrate for the O-GlcNAcylation of many cytosolic proteins, including HAS2. This post-translational modification stabilizes HAS2 in the membrane and increases HA production. Conversely, HAS2 can be phosphorylated by AMP activated protein kinase (AMPK), a master metabolic regulator activated by low ATP/AMP ratios, which inhibits HA secretion. Similarly, HAS2 expression and the deposition of HA within the pericellular coat are inhibited by sirtuin 1 (SIRT1), another important energetic sensor, confirming the tight connection between nutrients availability and HA metabolism.


Assuntos
Vias Biossintéticas , Metabolismo Energético , Hialuronan Sintases/metabolismo , Ácido Hialurônico/metabolismo , Animais , Humanos , Uridina Difosfato Ácido Glucurônico/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismo
12.
J Histochem Cytochem ; 69(1): 9-11, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33180636

RESUMO

In times where many people have suffered loss and others of us are dealing with stress, disruption, and fear, there is a lot of comfort to be taken in reading. If we are not able to meet up and discuss our work in person, exploring published studies provides some succor, even without the cheese, wine, and other traditions of our usual get-togethers. Fortunately, recent months have seen many high-quality papers around the topic of glycosaminoglycans. I can only pick up on a very few here, those that I have particularly enjoyed, but the following collection of reviews will also be a treat and hopefully tide us over until our research community can regroup.


Assuntos
Glicosaminoglicanos/metabolismo , Animais , Vias Biossintéticas , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Glicocálix/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Hialuronan Sintases/metabolismo , Ácido Hialurônico/metabolismo , Proteoglicanas/metabolismo , Uridina Difosfato Glucose Desidrogenase/metabolismo
13.
Arterioscler Thromb Vasc Biol ; 41(2): 796-807, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33380173

RESUMO

OBJECTIVE: The aim of this study was to unravel mechanisms whereby deficiency of the transcription factor Id3 (inhibitor of differentiation 3) leads to metabolic dysfunction in visceral obesity. We investigated the impact of loss of Id3 on hyaluronic acid (HA) production by the 3 HAS isoenzymes (HA synthases; -1, -2, and -3) and on obesity-induced adipose tissue (AT) accumulation of proinflammatory B cells. Approach and Results: Male Id3-/- mice and respective wild-type littermate controls were fed a 60% high-fat diet for 4 weeks. An increase in inflammatory B2 cells was detected in Id3-/- epididymal AT. HA accumulated in epididymal AT of high-fat diet-fed Id3-/- mice and circulating levels of HA were elevated. Has2 mRNA expression was increased in epididymal AT of Id3-/- mice. Luciferase promoter assays showed that Id3 suppressed Has2 promoter activity, while loss of Id3 stimulated Has2 promoter activity. Functionally, HA strongly promoted B2 cell adhesion in the AT and on cultured vascular smooth muscle cells of Id3-/- mice, an effect sensitive to hyaluronidase. CONCLUSIONS: Our data demonstrate that loss of Id3 increases Has2 expression in the epididymal AT, thereby promoting HA accumulation. In turn, elevated HA content promotes HA-dependent binding of B2 cells and an increase in the B2 cells in the AT, which contributes to AT inflammation.


Assuntos
Tecido Adiposo/metabolismo , Linfócitos B/metabolismo , Hialuronan Sintases/metabolismo , Ácido Hialurônico/biossíntese , Proteínas Inibidoras de Diferenciação/metabolismo , Paniculite/metabolismo , Tecido Adiposo/imunologia , Animais , Linfócitos B/imunologia , Adesão Celular , Células Cultivadas , Técnicas de Cocultura , Dieta Hiperlipídica , Modelos Animais de Doenças , Hialuronan Sintases/genética , Proteínas Inibidoras de Diferenciação/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/imunologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/metabolismo , Paniculite/genética , Paniculite/imunologia , Fenótipo , Transdução de Sinais , Regulação para Cima
14.
J Neurochem ; 157(3): 781-801, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33345310

RESUMO

Perineuronal nets (PNNs) are presumed to limit plasticity in adult animals. Ischaemic stroke results in the massive breakdown of PNNs resulting in rejuvenating states of neuronal plasticity, but the mechanisms of this phenomenon are largely unknown. As hyaluronic acid (HA) is the structural backbone of PNNs, we hypothesized that these changes are a consequence of the altered expression of HA metabolism enzymes. Additionally, we investigated whether early hyaluronidase inhibition interferes with post-stroke PNN reduction and behavioural recovery. We investigated the mRNA/protein expression of these enzymes in the perilesional, remote and contralateral cortical regions in mice at different time points after photothrombosis, using quantitative real-time polymerase chain reaction and immunofluorescence. A skilled reaching test was employed to test hyaluronidase inhibitor L-ascorbic acid 6-hexadecanoate influence on post-stroke recovery. We found the simultaneous up-regulation of mRNA of HA synthesizing and degrading enzymes in the perilesional area early after stroke, suggesting an acceleration of HA turnover in ischaemic animals. Immunostaining revealed differential cellular localization of enzymes, with hyaluronidase 1 in astrocytes and hyaluronan synthase 2 in astrocytes and neurons, and post-stroke up-regulation of both of them in astrocytes. ß-glucuronidase was observed in neurons but post-stroke up-regulation occurred in microglia. Inhibition of hyaluronidase activity early after stroke resulted in improved performance in skilled reaching test, without affecting the numbers of PNNs. These results suggest that after stroke, a substantial reorganization of polysaccharide content occurs, and interfering with this process at early time has a beneficial effect on recovery.


Assuntos
Encéfalo/patologia , Inibidores Enzimáticos/uso terapêutico , Hialuronoglucosaminidase/antagonistas & inibidores , AVC Isquêmico/patologia , AVC Isquêmico/terapia , Animais , Astrócitos/metabolismo , Feminino , Glucuronidase/metabolismo , Hialuronan Sintases/metabolismo , Ácido Hialurônico/biossíntese , Ácido Hialurônico/metabolismo , AVC Isquêmico/psicologia , Camundongos , Camundongos Endogâmicos C57BL , Destreza Motora , Neurônios/metabolismo , Cultura Primária de Células , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Recuperação de Função Fisiológica , Células Satélites Perineuronais/metabolismo , Trombose
15.
Biomed Res Int ; 2020: 2905634, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33134374

RESUMO

Osteoarthritis (OA) is a joint disease characterized by cartilage degeneration. Osteopontin (OPN) is involved in the initiation, repair, and maintenance of metabolic homeostasis in normal articular cartilage. This study investigated the role of OPN and its interaction with the integrin ανß3 receptor in the expression of hyaluronic acid (HA) in OA chondrocytes. Overexpression of OPN significantly increased the expression of integrin ανß3 and hyaluronic acid synthases (HAS) and synthesis of HA. Depleting OPN in OA chondrocytes showed the opposite trend for integrin alpha;νß3, HAS, and HA. Nonspecifically and specifically blocking integrin receptor using GRGDSP and integrin ανß3 antibody downregulated HAS and HA; both were inhibited to similar extents. The expression of HAS and HA was predominantly regulated by the interaction between OPN and integrin ανß3. Taken together, we have delineated the importance of the OPN/integrin ανß3/HAS/HA axis in OA and identified OPN as a promising candidate for molecular therapy for use in patients with OA.


Assuntos
Hialuronan Sintases/genética , Ácido Hialurônico/metabolismo , Integrina beta3/genética , Integrinas/genética , Osteoartrite do Joelho/genética , Osteopontina/genética , Anticorpos Neutralizantes/farmacologia , Artroplastia do Joelho/métodos , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Cartilagem Articular/cirurgia , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Regulação da Expressão Gênica , Humanos , Hialuronan Sintases/metabolismo , Integrina beta3/metabolismo , Integrinas/antagonistas & inibidores , Integrinas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Oligopeptídeos/farmacologia , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Osteoartrite do Joelho/cirurgia , Osteopontina/metabolismo , Cultura Primária de Células , Ligação Proteica , Transdução de Sinais
16.
J Biol Chem ; 295(49): 16797-16812, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33020183

RESUMO

The extracellular matrix encompasses a reservoir of bioactive macromolecules that modulates a cornucopia of biological functions. A prominent body of work posits matrix constituents as master regulators of autophagy and angiogenesis and provides molecular insight into how these two processes are coordinated. Here, we review current understanding of the molecular mechanisms underlying hyaluronan and HAS2 regulation and the role of soluble proteoglycan in affecting autophagy and angiogenesis. Specifically, we assess the role of proteoglycan-evoked autophagy in regulating angiogenesis via the HAS2-hyaluronan axis and ATG9A, a novel HAS2 binding partner. We discuss extracellular hyaluronan biology and the post-transcriptional and post-translational modifications that regulate its main synthesizer, HAS2. We highlight the emerging group of proteoglycans that utilize outside-in signaling to modulate autophagy and angiogenesis in cancer microenvironments and thoroughly review the most up-to-date understanding of endorepellin signaling in vascular endothelia, providing insight into the temporal complexities involved.


Assuntos
Autofagia , Endotélio Vascular/metabolismo , Ácido Hialurônico/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Endotélio Vascular/citologia , Humanos , Hialuronan Sintases/metabolismo , Neovascularização Patológica , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
17.
Biochem Biophys Res Commun ; 533(4): 732-738, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-32993960

RESUMO

Hyaluronan synthase 2 (HAS2) is an integral membrane protein composed of multi-membrane-spanning regions and a large intracellular loop (HAS2-loop). We previously examined the effect of phorbol 12-myristate 13-acetate (PMA) and/or 4-methylumbelliferone (4-MU) on the synthesis of hyaluronan (HA) in human skin fibroblasts and found that TPA and 4-MU have opposing effects on HA synthesis by phosphorylation and O-linked ß-N-acetylglucosaminylation of HAS2, respectively. In this study, we constructed an expression vector for the HAS2-loop and analyzed its post-translational modification by PMA and 4-MU using mass spectrometry. We identified a phosphorylation site at the position corresponding to the Thr328 position of full-length HAS2, which was detected in the cells regardless of the presence of PMA or 4-MU. We next prepared T328A site-directed mutagenesis construct-transfected cells and investigated HA synthesis. The amount of HA was increased in cells expressing full-length HAS2 compared to in mock cells, whereas the amount of HA synthesized by cells transfected with the T328A site-directed mutagenesis construct was the same as that in mock cells. This phosphorylation site corresponded with the casein kinase 1 substrate motif. These results suggest that Thr328 phosphorylation is an essential factor for HA synthesis by HAS2 and the role of HAS2-loop may be useful in analyzing the regulation of HAS2 synthesis in physiological and pathological conditions.


Assuntos
Hialuronan Sintases/metabolismo , Ácido Hialurônico/biossíntese , Himecromona/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Animais , Células COS , Caseína Quinase I/genética , Chlorocebus aethiops , Células HEK293 , Humanos , Hialuronan Sintases/genética , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Fosforilação , Mutação Puntual , Processamento de Proteína Pós-Traducional , Regulação para Cima
18.
Int J Mol Sci ; 21(17)2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32825309

RESUMO

Hyaluronic acid (HA) plays a vital role in the extracellular matrix of neural tissues. Originally thought to hydrate tissues and provide mechanical support, it is now clear that HA is also a complex signaling molecule that can regulate cell processes in the developing and adult nervous systems. Signaling properties are determined by molecular weight, bound proteins, and signal transduction through specific receptors. HA signaling regulates processes such as proliferation, differentiation, migration, and process extension in a variety of cell types including neural stem cells, neurons, astrocytes, microglia, and oligodendrocyte progenitors. The synthesis and catabolism of HA and the expression of HA receptors are altered in disease and influence neuroinflammation and disease pathogenesis. This review discusses the roles of HA, its synthesis and breakdown, as well as receptor expression in neurodevelopment, nervous system function and disease.


Assuntos
Doenças do Sistema Nervoso Central/metabolismo , Receptores de Hialuronatos/metabolismo , Hialuronan Sintases/metabolismo , Ácido Hialurônico/fisiologia , Sistema Nervoso/crescimento & desenvolvimento , Animais , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Humanos , Ácido Hialurônico/química , Hialuronoglucosaminidase/metabolismo , Fenômenos Fisiológicos do Sistema Nervoso , Células-Tronco Neurais/metabolismo
19.
Biomed Res ; 41(4): 187-197, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32801268

RESUMO

As chondrocytes fail to retain their chondrogenic potential in two-dimensional monolayer cultures, several three-dimensional culture systems have been employed for investigating the physiology and pathophysiology in articular cartilage tissues. In this study, we introduced a polyethylene glycol-coated microfabricated chip that enables spheroid formation from ATDC5 cell line, commonly used as a model for in vitro chondrocyte research. ATDC5 cells cultured in our devices aggregated immediately and generated a single spheroid per well within 24 h. Most cells in spheroids cultured in differentiation medium were viable and the circular shape and smooth surface of the spheroid were maintained up to 14 d in culture. We also detected potent hypoxia conditions, a key factor in chondrogenesis, in whole lesions of ATDC5 spheroids. Expression of chondrogenesis-related genes and type X collagen protein was significantly increased in ATDC5 spheroids grown in differentiation medium, compared with monolayer-cultured ATDC5 cells. We also demonstrated that the differentiation medium-induced Akt protein phosphorylation was upregulated in ATDC5 cells cultured in our spheroid device, suggesting that enhancement of chondrogenic potential in ATDC5 spheroids results from PI3/Akt signaling activation. These results indicated that our spheroid culture system could constitute a high-throughput strategy approach towards elucidating the molecular mechanisms that regulate chondrogenesis.


Assuntos
Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/farmacologia , Polietilenoglicóis/farmacologia , Esferoides Celulares/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular , Condrócitos/citologia , Condrócitos/metabolismo , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Materiais Revestidos Biocompatíveis/química , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Expressão Gênica , Hialuronan Sintases/genética , Hialuronan Sintases/metabolismo , Dispositivos Lab-On-A-Chip , Camundongos , Platina/química , Polimetil Metacrilato/química , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo
20.
Bone ; 141: 115584, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32795674

RESUMO

OBJECTIVE: Although associations between dysregulated glucose metabolism and human rheumatoid arthritis have been reported, the disturbance and influence of glycolytic metabolism on temporomandibular joint osteoarthritis remains unclear. This study aimed to investigate the expression level and metabolite profile of the critical glycolytic enzyme, lactate dehydrogenase A (LDHA) in synovial fibroblasts (SFs) of TMJOA, assess the effect of glycolytic inhibition on synthesis of hyaluronan synthase 2 (HAS2) and inflammation progression in these cells. METHODS: Immunohistochemistry and western blotting were performed to detect the expression of LDHA in the lining and sub-lining layers of synovial tissue and SFs. MTT and EdU assays were used to measure the cell proliferation. The cell apoptosis were demonstrated by TUNEL staining and Annexin V/PI double staining. A potent and specific inhibitor of LDHA, GSK2837808A, was administrated to suppress the activity of LDHA and detect the potential efficacy on HAS2. RESULTS: LDHA expression was dramatically higher in the synovial tissue and SFs from TMJOA patients compared to control groups. LDHA inhibition impaired active LDHA performance, suppressed the glucose uptake and decreased lactate concentration. Furthermore, GSK2837808A reversed the occurrence of low ratio of ATP/AMP, high level of Adenosine Monophosphate-activated Protein Kinase (AMPK) activation, disturbed HAS2 synthesis and hyaluronic acid (HA) production by inhibiting LDHA. The cellular viability and cell cycle were not affected by GSK2837808A at the working concentration. CONCLUSIONS: Targeting LDHA using its specific suppressant GSK2837808A impeded lactate secretion and contributed to HAS2 and HA synthesis in TMJOA SFs, providing the vital role of LDHA associated with TMJOA pathogenesis and a novel therapeutic approach for TMJOA.


Assuntos
Glicólise , Osteoartrite , Linhagem Celular Tumoral , Fibroblastos , Humanos , Hialuronan Sintases/metabolismo , Lactato Desidrogenase 5 , Osteoartrite/metabolismo , Articulação Temporomandibular
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...