Age affects ERK1/2 and NRF2 signaling in the regulation of GCLC expression

Abstract

We previously reported that activator protein-1 (AP-1) DNA binding activity was increased in vascular smooth muscle cells (VSMC) from old rats when exposed to high glucose or tumor necrosis factor (TNF-a) (Li et al., 2003. J Cell Physiol 197:418-425). We have now examined the relationship between the age-dependent activation of the ERK1/2-AP-1 pathway and modulation of constitutive gene expression of the catalytic subunit of glutamate-cysteine ligase (GCLC) in response to high glucose and TNF-a. GCLC mRNA levels were higher in VSMC from old rats compared to young, a pattern consistent with its protein levels. To determine whether agerelated activation of ERK1/2-AP-1 signaling is responsible for the up-regulation of GCLC, the MEK inhibitors, PD98059 and U0126, were used to block ERK1/2 in VSMC from old rats. An increase in GCLC with inhibitors was observed, diminishing the likelihood of ERK1/2-AP-1 activation as the up-regulating signal for GCLC. However, the transcription factor Nrf2 was higher in nuclei and accompanied by increased Nrf2-ARE binding in VSMC from old rats. Furthermore, MEK inhibitors increased nuclear Nrf2 and Nrf2/ ARE binding. These data suggest opposing effects of Nrf2 and ERK1/2 signaling in the modulation of GCLC expression in old animals.

Age-Related Changes in Redox Signaling and VSMC Function

“…An increase in nuclear Nrf2 was found in VSMC from old compared to young F344 rats (60). There are a large number of genes regulated by Keap1-Nrf2-ARE, and some of the phase II enzymes were also found to be upregulated in VSMC with aging, including the enzymes involved in GSH metabolism and heme oxygenase1 (HO1) (81), as shown in Table 1.…”

Down-regulation of Manganese-Superoxide Dismutase through Phosphorylation of FOXO3a by Akt in Explanted Vascular Smooth Muscle Cells from Old Rats

“…The probes were labeled with [␥-32 P]ATP using T4 polynucleotide kinase, and EMSA and supershift EMSA were performed as described previously (30). For competition experiments, the competitive double-stranded oligonucleotides were added before loading of labeled probes at 100:1 molar ratio.…”

Identification and characterization of an Nrf2‐mediated ARE upstream of the rat glutamate cysteine ligase catalytic subunit gene (GCLC)

“…We have previously shown higher constitutive GCLC expression in VSMC explanted from old rats [Li et al, 2006a]. Changes in GCLC in these VSMC in response to 40, 80, and 160 mM of TBH-treatments for 16 h, or to transfection with the Nrf2 expression vector for 24 h were examined by Western blots using cytoplasmic proteins extracted from the cells (Fig.…”

“…The oligonucleotide probes were labeled with [g-32 P] ATP using T4 polynucleotide kinase. EMSA and supershift EMSA were performed as described previously [Li et al, 2006a]. Antibodies for the Nrf2, c-Fos (specifically recommended for supershifts), c-Jun, and Mafs proteins were purchased from Santa Cruz.…”

“…Explanted vascular smooth muscle cells (VSMC) were isolated from a group of F344 rats (24 months) and grown in Dulbecco's Modified Eagle Media (DMEM), supplemented with 10% (v/v) FBS and 100 unit/ml penicillin/streptomycin (PS) (Invitrogen, Carlsbad, CA), as previously described [Li et al, 2006a]. Cells were treated with TBH (Sigma-Aldrich, St. Louis, MO) at 40, 80, 120, and 160 mM for 4 or 16 h, respectively.…”