Dataset Information

Hif-1? Knockdown Reduces Glycolytic Metabolism and Induces Cell Death of Human Synovial Fibroblasts Under Normoxic Conditions.

ABSTRACT: Increased glycolysis and HIF-1? activity are characteristics of cells under hypoxic or inflammatory conditions. Besides, in normal O2 environments, elevated rates of glycolysis support critical cellular mechanisms such as cell survival. The purpose of this study was to analyze the contribution of HIF-1? to the energy metabolism and survival of human synovial fibroblasts (SF) under normoxic conditions. HIF-1? was silenced using lentiviral vectors or small-interfering RNA (siRNA) duplexes. Expression analysis by qRT-PCR and western blot of known HIF-1? target genes in hypoxia demonstrated the presence of functional HIF-1? in normoxic SF and confirmed the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a HIF-1? target even in normoxia. HIF-1? silencing induced apoptotic cell death in cultured SF and, similarly, treatment with glycolytic, but not with OXPHOS inhibitors, induced SF death. Finally, in vivo HIF-1? targeting by siRNA showed a significant reduction in the viability of human SF engrafted into a murine air pouch. Our results demonstrate that SF are highly dependent on glycolytic metabolism and that HIF-1? plays a regulatory role in glycolysis even under aerobic conditions. Local targeting of HIF-1? provides a feasible strategy to reduce SF hyperplasia in chronic arthritic diseases.

SUBMITTER: Del Rey MJ

PROVIDER: S-EPMC5473902 | biostudies-other | 2017 Jun

REPOSITORIES: biostudies-other

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Hif-1α Knockdown Reduces Glycolytic Metabolism and Induces Cell Death of Human Synovial Fibroblasts Under Normoxic Conditions.

Del Rey Manuel J MJ Valín Álvaro Á Usategui Alicia A García-Herrero Carmen M CM Sánchez-Aragó María M Cuezva José M JM Galindo María M Bravo Beatriz B Cañete Juan D JD Blanco Francisco J FJ Criado Gabriel G Pablos José L JL

Scientific reports 20170616 1

Increased glycolysis and HIF-1α activity are characteristics of cells under hypoxic or inflammatory conditions. Besides, in normal O<sub>2</sub> environments, elevated rates of glycolysis support critical cellular mechanisms such as cell survival. The purpose of this study was to analyze the contribution of HIF-1α to the energy metabolism and survival of human synovial fibroblasts (SF) under normoxic conditions. HIF-1α was silenced using lentiviral vectors or small-interfering RNA (siRNA) duplex ...[more]

PMID: 28623342

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Project description:BackgroundMacrophages adapt to microenvironments, and change metabolic status and functions to regulate inflammation and/or maintain homeostasis. In joint cavities, synovial macrophages (SM) and synovial fibroblasts (SF) maintain homeostasis. However, under inflammatory conditions such as rheumatoid arthritis (RA), crosstalk between SM and SF remains largely unclear.MethodsImmunofluorescent staining was performed to identify localization of SM and SF in synovium of collagen antibody induced arthritis (CAIA) model mice and normal mice. Murine arthritis tissue-derived SM (ADSM), arthritis tissue-derived SF (ADSF) and normal tissue-derived SF (NDSF) were isolated and the purity of isolated cells was examined by RT-qPCR and flow cytometry analysis. RNA-seq was conducted to reveal gene expression profile in ADSM, NDSF and ADSF. Cellular metabolic status and expression levels of metabolic genes and inflammatory genes were analyzed in ADSM treated with ADSM-conditioned medium (ADSM-CM), NDSF-CM and ADSF-CM.ResultsSM and SF were dispersed in murine hyperplastic synovium. Isolations of ADSM, NDSF and ADSF to analyze the crosstalk were successful with high purity. From gene expression profiles by RNA-seq, we focused on secretory factors in ADSF-CM, which can affect metabolism and inflammatory activity of ADSM. ADSM exposed to ADSF-CM showed significantly upregulated glycolysis and mitochondrial respiration as well as glucose and glutamine uptake relative to ADSM exposed to ADSM-CM and NDSF-CM. Furthermore, mRNA expression levels of metabolic genes, such as Slc2a1, Slc1a5, CD36, Pfkfb1, Pfkfb3 and Irg1, were significantly upregulated in ADSM treated with ADSF-CM. Inflammation marker genes, including Nos2, Tnf, Il-1b and CD86, and the anti-inflammatory marker gene, Il-10, were also substantially upregulated by ADSF-CM. On the other hand, NDSF-CM did not affect metabolism and gene expression in ADSM.ConclusionsThese findings suggest that crosstalk between SM and SF under inflammatory conditions can induce metabolic reprogramming and extend SM viability that together can contribute to chronic inflammation in RA. Video Abstract.

Project description:In cancer tumors, lactate accumulation was initially attributed to high glucose consumption associated with the Warburg Effect. Now it is evident that lactate can also serve as an energy source in cancer cell metabolism. Additionally, lactate has been shown to promote metastasis, generate gene expression patterns in cancer cells consistent with "cancer stem cell" phenotypes, and result in treatment resistant tumors. Therefore, the goal of this work was to quantify the impact of lactate on metabolism in three breast cell lines (one normal and two breast cancer cell lines-MCF 10A, MCF7, and MDA-MB-231), in order to better understand the role lactate may have in different disease cell types. Parallel labeling metabolic flux analysis (13C-MFA) was used to quantify the intracellular fluxes under normal and high extracellular lactate culture conditions. Additionally, high extracellular lactate cultures were labelled in parallel with [U-13C] lactate, which provided qualitative information regarding the lactate uptake and metabolism. The 13C-MFA model, which incorporated the measured extracellular fluxes and the parallel labeling mass isotopomer distributions (MIDs) for five glycolysis, four tricarboxylic acid cycle (TCA), and three intracellular amino acid metabolites, predicted lower glycolysis fluxes in the high lactate cultures. All three cell lines experienced reductive carboxylation of glutamine to citrate in the TCA cycle as a result of high extracellular lactate. Reductive carboxylation previously has been observed under hypoxia and other mitochondrial stresses, whereas these cultures were grown aerobically. In addition, this is the first study to investigate the intracellular metabolic responses of different stages of breast cancer progression to high lactate exposure. These results provide insight into the role lactate accumulation has on metabolic reaction distributions in the different disease cell types while the cells are still proliferating in lactate concentrations that do not significantly decrease exponential growth rates.

Dataset Information

Hif-1? Knockdown Reduces Glycolytic Metabolism and Induces Cell Death of Human Synovial Fibroblasts Under Normoxic Conditions.

Publications

Hif-1α Knockdown Reduces Glycolytic Metabolism and Induces Cell Death of Human Synovial Fibroblasts Under Normoxic Conditions.

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