Perturbations of Glutathione and Sphingosine Metabolites in Port Wine Birthmark Patient-Derived Induced Pluripotent Stem Cells
Vi NguyenJacob KravitzChao GaoMarcelo HochmanDehao MengDong‐bao ChenYunguan WangAnil G. JeggaJ. Stuart NelsonWenbin Tan
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Abstract:
Port Wine Birthmarks (PWBs) are a congenital vascular malformation on the skin, occurring in 1–3 per 1000 live births. We have recently generated PWB-derived induced pluripotent stem cells (iPSCs) as clinically relevant disease models. The metabolites associated with the pathological phenotypes of PWB-derived iPSCs are unknown, and so we aim to explore them in this study. Metabolites were separated by ultra-performance liquid chromatography and screened with electrospray ionization mass spectrometry. Orthogonal partial least-squares discriminant, multivariate, and univariate analyses were used to identify differential metabolites (DMs). KEGG analysis was used to determine the enrichment of metabolic pathways. A total of 339 metabolites was identified. There were 22 DMs, among which nine were downregulated—including sphingosine—and 13 were upregulated, including glutathione in PWB iPSCs, as compared to controls. Pathway enrichment analysis confirmed the upregulation of glutathione and the downregulation of sphingolipid metabolism in PWB-derived iPSCs as compared to normal ones. We next examined the expression patterns of the key molecules associated with glutathione metabolism in PWB lesions. We found that hypoxia-inducible factor 1α (HIF1α), glutathione S-transferase Pi 1 (GSTP1), γ-glutamyl transferase 7 (GGT7), and glutamate cysteine ligase modulatory subunit (GCLM) were upregulated in PWB vasculatures as compared to blood vessels in normal skin. Other significantly affected metabolic pathways in PWB iPSCs included pentose and glucuronate interconversions; amino sugar and nucleotide sugars; alanine, aspartate, and glutamate; arginine, purine, D-glutamine, and D-glutamate; arachidonic acid, glyoxylate, and dicarboxylate; nitrogen, aminoacyl-tRNA biosynthesis, pyrimidine, galactose, ascorbate, and aldarate; and starch and sucrose. Our data demonstrated that there were perturbations in sphingolipid and cellular redox homeostasis in PWB vasculatures, which could facilitate cell survival and pathological progression. Our data implied that the upregulation of glutathione could contribute to laser-resistant phenotypes in some PWB vasculatures.Keywords:
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Glucuronates
Glucuronic acid (GH) was found to undergo coloring by hydroxamic acid and the degradation of ethyl glucuronate (GE) was re-examined by this method. The reaction scheme proposed in a previous (in the preceding paper), [chemical formula], was confirmed, where GL is glucuronic acid lactone. Further, in a weakly acid solution, hydrolysis of ethyl glucuronate proceeded according to the reaction schem [chemical formula]where G- is a glucuronate ion. Effect of pH and buffer concentration on the rate constants was examined and the following equations were obtained : [numerical formula]
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Several genes in cysteine (Cys) metabolism are transcriptionally regulated in response to either Cys deprivation or oxidative stress. These include GCLC and GCLM , which encode glutamate:Cys ligase subunits that catalyze the first step of glutathione synthesis, and SLC7A11 , which encodes the cystine‐glutamate exchanger responsible for cystine uptake. In Cys‐deprived HepG2/C3A cells, GCLC , GCLM and SLC7A11 were upregulated as assessed by microarray analysis and qRT‐PCR. Expression of many genes with antioxidant response elements was not affected by Cys deprivation while genes with amino acid response elements (AAREs) were consistently upregulated, suggesting that Cys deprivation can induce the eIF2α kinase 4 mediated amino acid deprivation pathway without inducing oxidative stress. Induction of GCLC, GCLM and SLC7A11 was also observed in rats fed 10% (w/w) protein diets, and activation of the AARE‐mediated pathway was evidenced by phosphorylation of eIF2α and upregulation of mRNA and protein levels for genes with AAREs. The pattern of gene expression in growing rats fed diets marginally deficient in sulfur amino acids suggests that the amino acid deprivation response can be activated in response to mild, as well as severe, amino acid deficiencies. SLC7A11 contains an AARE and is likely a direct target, but the details of the regulation of GCLC and GCLM expression in response to Cys deficiency need further study. Funded by NIH Grant PHS DK0664303.
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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) generates both hepatocellular injury and steatosis, processes that involve oxidative stress. Herein, we evaluated the role of the antioxidant glutathione (GSH) in TCDD-induced hepatotoxicity. Glutamate-cysteine ligase (GCL), comprising catalytic (GCLC) and modifier (GCLM) subunits, is rate limiting in de novo GSH biosynthesis; GCLM maintains GSH homeostasis by optimizing the catalytic efficiency of GCL holoenzyme. Gclm(-/-) transgenic mice exhibit 10-20% of normal tissue GSH levels. Gclm(-/-) and Gclm(+/+) wild-type (WT) female mice received TCDD for 3 consecutive days and were then examined 21 days later. As compared with WT littermates, Gclm(-/-) mice were more sensitive to TCDD-induced hepatocellular toxicity, exhibiting lower reduction potentials for GSH, lower ATP levels, and elevated levels of plasma glutamic oxaloacetic transaminase (GOT) and γ-glutamyl transferase (GGT). However, the histopathology showed that TCDD-mediated steatosis, which occurs in WT mice, was absent in Gclm(-/-) mice. This finding was consistent with cDNA microarray expression analysis, revealing striking deficiencies in lipid biosynthesis pathways in Gclm(-/-) mice; qrt-PCR analysis confirmed that Gclm(-/-) mice are deficient in expression of several lipid metabolism genes including Srebp2, Elovl6, Fasn, Scd1/2, Ppargc1a, and Ppara. We suggest that whereas GSH protects against TCDD-mediated hepatocellular damage, GSH deficiency confers resistance to TCDD-induced steatosis due to impaired lipid metabolism.
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Oxidative stress has been linked to the progression of mutations and cancer. Increased glutathione (GSH) contents have been observed in a number of different human cancer tissues. GSH is synthesized de novo in a two-step process catalyzed by glutamate cysteine ligase (GCL). The present study aimed to investigate whether GCL was associated with renal cell carcinoma (RCC). The protein expression levels of the GCL subunits (catalytic subunit, GCLc; and modulatory subunit, GCLm) and GCL activity were examined in renal cancer tissue. A total of 46 patients fulfilling the RCC criteria of the World Health Organization, revised in 2004, were enrolled. The tumor and adjacent tissues were sampled from all the subjects by surgery. The study demonstrated that GCLc and GCLm protein expression and the GCL activity were significantly increased in the tumor tissue from RCC patients. These results indicate that increased expression and enzymatic activity of GCL is closely associated with RCC and thus, this suggests an important role for GSH in the pathogenesis of RCC.
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