GCLM
Glutamate--cysteine ligase regulatory subunit (GCS light chain) (Gamma-ECS regulatory subunit) (Gamma-glutamylcysteine synthetase regulatory subunit) (Glutamate--cysteine ligase modifier subunit) [GLCLR]
PublicationsПравить
Nrf2 is the master transcription factor regulating the basal and inducible expression of antioxidant genes. With aging, the basal Nrf2 activity is increased but oxidant/electrophile-enhanced activation of Nrf2 signaling is diminished, and these changes are accompanied by an increased expression of Bach1, a repressor of Nrf2 signaling. In this limited follow-up study, we explored how Bach1 may be involved in aging-related alteration in Nrf2 signaling in primary human bronchial epithelial (HBE) cells. Silencing Bach1 with siRNA increased the basal mRNA expression of Nrf2 regulated genes including glutamate cysteine ligase catalytic (GCLC) and modifier subunit (GCLM), NAD(P)H oxidoreductase 1(NQO-1) and heme oxygenase 1(HO-1), in HBE cells from both young (aged 21-29 years) and older (aged 61-69 years) donors. On the other hand, Bach1 silencing affected the induction of Nrf2-regulated genes differentially in young and older HBE cells. Bach1 silencing significantly enhanced sulforaphane-induced expression of HO-1 but had no effect on that of GCLC, GCLM, and NQO1 in young HBE cells. In contrast, Bach1 silencing enhanced sulforaphane-induced expression of GCLC, GCLM and HO-1 but had no effect on that of NQO-1 in older HBE cells. In conclusion, these results suggest that increased Bach1 contributes to aging-related loss of electrophile-enhanced Nrf2 signaling.
MeSH Terms
- Adult
- Aged
- Aging
- Basic-Leucine Zipper Transcription Factors
- Bronchi
- Epithelial Cells
- Gene Expression
- Gene Silencing
- Glutamate-Cysteine Ligase
- Heme Oxygenase-1
- Humans
- Isothiocyanates
- Middle Aged
- NAD(P)H Dehydrogenase (Quinone)
- NF-E2-Related Factor 2
- RNA, Messenger
- RNA, Small Interfering
- Signal Transduction
- Young Adult
Keywords
- Aging
- Bach1
- Glutamate cysteine ligase
- Heme oxygenase
- Nrf2
- Sulforaphane
The Nrf2/Keap1 antioxidant system plays important roles in protecting against oxidative stress and toxic stimuli, which may vary in infants, elderly, and females. The constitutive expression of the Nrf2 genes during development and aging in both sexes would help our understanding of the Nrf2/Keap1 pathway in toxicological studies. Sprague Dawley rat livers were collected at 11 age points from prenatal (-2 d), neonatal (1, 7, 14 and 21 d), at puberty (28 and 35 d), at adulthood (60 and 180 d), to aging (540 and 800 d) from both sexes. Total RNA and proteins were extracted for real-time RT-PCR and Western-blot analysis. The abundant mRNA expression was in the order of Nrf2, Gclm, Nqo1, Gclc, Ho-1, and Keap1. The expression of these genes except Gclc was high in fetal livers, decreased at birth, reached the first peak at 7 days of age, and gradually decreased to adult levels till 180 days of age. All these genes remained high at 540 days of age, but declined at 800 days of age, with more increases with Nqo1 and Ho-1. Females had lower fetal, neonatal, and aged levels than males. Protein expressions of Nrf2, Nqo1, Ho-1, GCLC and GCLM agree with mRNA analysis. This study characterized the age- and sex-related changes of Nrf2-related gene/proteins in livers of rats, and higher expressions in newborns and aged rats could cope with increased oxidative stress in infants and elderly.
MeSH Terms
- Aging
- Animals
- Female
- Liver
- Male
- NF-E2-Related Factor 2
- Oxidative Stress
- RNA, Messenger
- Rats
- Rats, Sprague-Dawley
- Signal Transduction
Keywords
- Aging
- Development
- Ho-1
- Nqo1
- Nrf2/Keap1
- Rat liver
- Sex-difference
The mechanisms underlying life span extension by sulfur amino acid restriction (SAAR) are unclear. Cysteine and methionine are essential for the biosynthesis of proteins and glutathione (GSH), a major redox buffer in the endoplasmic reticulum (ER). We hypothesized that SAAR alters protein synthesis by modulating the redox milieu. Male F344-rats were fed control (CD: 0.86% methionine without cysteine) and SAAR diets (0.17% methionine without cysteine) for 12 weeks. Growth rates, food intake, cysteine and GSH levels, proteins associated with redox status and translation, and fractional protein synthesis rates (FSRs) were determined in liver. Despite a 40% higher food intake, growth rates for SAAR rats were 27% of those fed CD. Hepatic free cysteine in SAAR rats was 55% compared with CD rats. SAAR altered tissue distribution of GSH, as hepatic and erythrocytic levels were 56% and 196% of those in CD rats. Lower GSH levels did not induce ER stress (i.e., unchanged expression of Xbp1 , Chop, and Grp78), but activated PERK and its substrates eIF2-α and NRF2. SAAR-induced changes in translation-initiation machinery (higher p-eIF2-α and 4E-BP1, and lower eIF4G-1) resulted in slower protein synthesis rates (53% of CD). Proteins involved in the antioxidant response (NRF2, KEAP1, GCLM, and NQO1) and protein folding (PDI and ERO1-α) were increased in SAAR. Lower FSR and efficient protein folding might be improving proteostasis in SAAR.
MeSH Terms
- Amino Acids, Sulfur
- Animals
- Biomarkers
- Cysteine
- Diet
- Endoplasmic Reticulum Stress
- Erythrocytes
- Feeding Behavior
- Glutathione
- Growth
- Liver
- Longevity
- Male
- Organ Size
- Oxidation-Reduction
- Protein Biosynthesis
- Proteins
- Rats
- Rats, Inbred F344
Keywords
- NRF2
- cysteine
- endoplasmic reticulum
- life span
- protein turnover
Evidence from animal studies suggests that stress-induced increases in Nrf2-regulated antioxidant gene expression, a critical mechanism of cellular protection, declines with aging. This study examined whether this also occurs in humans. We measured the basal and inducible levels of Nrf2-regulated antioxidant genes in human bronchial epithelial (HBE) cells from subjects of young adult (21-29 years) and older (60-69 years) non-smokers, and explored factors affecting expresion. The basal expression of three representative Nrf2-regulated genes, the catalytic and modulator subunits of glutamate cysteine ligase (GCLC and GCLM, respectively), and NAD(P)H quinone oxidoreductase 1 (NQO1), was higher in cells from the older donors compared with cells from the young adult donors. Upon exposure to the Nrf2 activator, sulforaphane (SF), the expression of these antioxidant genes was increased in cells from both the young adults and the older donors; however, the induction by SF in older donor cells was significantly less than that seen in young adult cells. In addition, the activation of an EpRE-driven reporter by SF was lower in cells from older donors compared to cells from young adults. The basal expression of Nrf2 protein was also lower in cells from older donors than cells from young adults. Furthermore, we found that the basal expression of both Bach1 and c-Myc, two Nrf2 suppressors, was higher in cells from older adults than from young adult donors. In summary, our data suggest that, as in other species, basal expression of Nrf2-regulated genes increases with aging, while inducibility declines with aging. The increased expression of Nrf2 suppressors such as Bach1 and c-Myc may contribute to the impaired inducibility of the Nrf2-regulated antioxidant genes with aging in human bronchial epithelial cells.
MeSH Terms
- Adult
- Aged
- Aging
- Antioxidants
- Basic-Leucine Zipper Transcription Factors
- Bronchi
- Cells, Cultured
- Epithelial Cells
- Glutamate-Cysteine Ligase
- Humans
- Isothiocyanates
- Middle Aged
- NAD(P)H Dehydrogenase (Quinone)
- NF-E2-Related Factor 2
- Proto-Oncogene Proteins c-myc
- Up-Regulation
- Young Adult
Age-related macular degeneration (AMD) represents the leading cause of blindness in the elderly, yet no definitive therapy exists for early, dry disease. Several lines of evidence have implicated oxidative stress-induced damage to the retinal pigment epithelium (RPE) in the pathogenesis of AMD, suggesting that the aging RPE may exhibit increased susceptibility to cell damage induced by exogenous stressors. The transcription factor Nrf2 serves as the master regulator of a highly coordinated antioxidant response in virtually all cell types. We compared Nrf2 signaling in the RPE of young (2 months) and old (15 months) mice under unstressed and stressed (sodium iodate) conditions. The aging RPE expressed higher levels of the Nrf2 target genes NQO1, GCLM, and HO1 compared with the RPE of younger mice under unstressed conditions, suggesting an age-related increase in basal oxidative stress. Moreover, the RPE of older mice demonstrated impaired induction of the protective Nrf2 pathway following oxidative stress induced with sodium iodate. The RPE of old mice exposed to sodium iodate also exhibited higher levels of superoxide anion and malondialdehyde than young mice, suggesting inadequate protection against oxidative damage. Induction of Nrf2 signaling in response to sodium iodate was partially restored in the RPE of aging mice with genetic rescue, using conditional knockdown of the Nrf2 negative regulator Keap1 (Tam-Cre; Keap1loxP) compared to Keap1loxP mice. These data indicate that the aging RPE is vulnerable to oxidative damage due to impaired Nrf2 signaling, and that Nrf2 signaling is a promising target for novel pharmacologic or genetic therapeutic strategies.
MeSH Terms
- Aging
- Animals
- Humans
- Macular Degeneration
- NF-E2-Related Factor 2
- Oxidative Stress
- Retinal Pigment Epithelium
- Signal Transduction
Keywords
- Nrf2
- age-related macular degeneration
- aging
- oxidative stress
- retinal pigment epithelium
Although the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2) regulated expression of multiple antioxidant and cytoprotective genes through the electrophile responsive element (EpRE) is well established, interaction of Nrf2/EpRE with Nrf1, a closely-related transcription factor, is less well understood. Due to either proteolysis or alternative translation, Nrf1 has been found as proteins of varying size, p120, p95, and p65, which have been described as either activators of EpRE or competitive inhibitors of Nrf2. We investigated the effect of Nrf1 on EpRE-regulated gene expression using the catalytic and modifier subunits of glutamate cysteine ligase (GCLC and GCLM) as models and explored the potential role of Nrf1 in altering their expression in aging and upon chronic exposure to airborne nano-sized particulate matter (nPM). Nrf1 knockout resulted in the increased expression of GCLC and GCLM in human bronchial epithelial (HBE1) cells. Overexpression Nrf2 in combination with either p120 or p65 diminished or failed to further increase the GCLC- and GLCM-EpRE luciferase activity. All known forms of Nrf1 protein, remained unchanged in the lungs of mice with age or in response to nPM. Our study shows that Nrf1 could inhibit EpRE activity in vitro, whereas the precise role of Nrf1 in vivo requires further investigations. We conclude that Nrf1 may not be directly responsible for the loss of Nrf2-dependent inducibility of antioxidant and cytoprotective genes observed in aged animals.
MeSH Terms
- Aging
- Animals
- Antioxidants
- Bronchi
- Cell Line
- Epithelial Cells
- Gene Expression Regulation
- Gene Knockout Techniques
- Glutamate-Cysteine Ligase
- HEK293 Cells
- Humans
- Lung
- Male
- Mice
- Mice, Inbred C57BL
- NF-E2 Transcription Factor, p45 Subunit
- Nuclear Respiratory Factor 1
- Particulate Matter
- Protein Isoforms
- Response Elements
Keywords
- Air pollution
- ER, endoplasmic reticulum
- Electrophile response element
- EpRE, electrophile response element
- GCL, glutamate cysteine ligase
- GCLC, catalytic subunit of GCL
- GCLM, modifier subunit of GCL
- Glutamate cysteine ligase
- HBE1, human bronchial epithelial cells
- HO-1, heme oxygenase
- Nrf1
- Nrf1, nuclear factor-erythroid 2 p45 subunit-related factor 1
- Nrf2
- Nrf2, nuclear factor-erythroid 2 p45 subunit-related factor 2
- Phase II genes
- nPM, nanoparticulate air pollution