Modulation of nuclear factor E2-related factor-2 (Nrf2) activation by the stress response gene immediate early response-3 (IER3) in colonic epithelial cells: a novel mechanism of cellular adaption to inflammatory stress

Abstract

Although nuclear factor E2-related factor-2 (Nrf2) protects from carcinogen-induced tumorigenesis, underlying the rationale for using Nrf2 inducers in chemoprevention, this antioxidative transcription factor may also act as a proto-oncogene. Thus, an enhanced Nrf2 activity promotes formation and chemoresistance of colon cancer. One mechanism causing persistent Nrf2 activation is the adaptation of epithelial cells to oxidative stress during chronic inflammation, e.g. colonocytes in inflammatory bowel diseases, and the multifunctional stress response gene immediate early response-3 (IER3) has a crucial role under these conditions. We now demonstrate that colonic tissue from Ier3(-/-) mice subject of dextran sodium sulfate colitis exhibit greater Nrf2 activity than Ier3(+/+) mice, manifesting as increased nuclear Nrf2 protein level and Nrf2 target gene expression. Likewise, human NCM460 colonocytes subjected to shRNA-mediated IER3 knockdown exhibit greater Nrf2 activity compared with control cells, whereas IER3 overexpression attenuated Nrf2 activation. IER3-deficient NCM460 cells exhibited reduced reactive oxygen species levels, indicating increased antioxidative protection, as well as lower sensitivity to TRAIL or anticancer drug-induced apoptosis and greater clonogenicity. Knockdown of Nrf2 expression reversed these IER3-dependent effects. Further, the enhancing effect of IER3 deficiency on Nrf2 activity relates to the control of the inhibitory tyrosine kinase Fyn by the PI3K/Akt pathway. Thus, the PI3K inhibitor LY294002 or knockdown of Akt or Fyn expression abrogated the impact of IER3 deficiency on Nrf2 activity. In conclusion, the interference of IER3 with the PI3K/Akt-Fyn pathway represents a novel mechanism of Nrf2 regulation that may get lost in tumors and by which IER3 exerts its stress-adaptive and tumor-suppressive activity.

Keywords: Colitis; Colon Cancer; Nrf2; Oxidative Stress; Transcription Factors.

FIGURE 1.

Greater Nrf2 activity in the inflamed colon tissue of DSS-treated mice lacking Ier3 expression. a and b, cryostat sections of colon tissues from DSS-treated Ier3^−/− or Ier3^+/+ mice were submitted to immunofluorescent staining of the macrophage marker F4/80 to detect inflamed areas enriched for tissue macrophages (a) or to TUNEL staining for detection of apoptotic cells (b). Tissue stainings from three different animals (I–III) each genotype are shown; scale bars, 20 μm. c, cryostat sections were analyzed for Nrf2 activation by immunostaining with an antibody detecting activated (P-Ser⁴⁰)-Nrf2. Tissue stainings from three different animals each genotype are shown; scale bars, 20 μm. d, two series of colon tissues from Ier3^−/− or Ier3^+/+ mice (three animals either (I–III)) subjects of chronic DSS colitis were analyzed by Western blotting for Nrf2 protein level in nuclear extracts (n.e., top panel), or for the expression of the Nrf2 target genes Nqo1 and Gclc in total cell lysates (t.l., middle panel). Lamin A/C and Hsp90 were used as loading controls. Protein band intensities were determined by densitometry analysis (bottom panel) normalizing to the corresponding loading controls (mean ± S.D. (error bars), n = 6).

FIGURE 2.

Ier3 deficiency enhances Nrf2 activation in murine colonic tissue. Colon organ cultures from untreated Ier3^−/− or Ier3^+/+ mice (data from two independent experiments are shown) were subjected to treatment with 50 μm tBHQ for 16 h or not. a, nuclear extracts (n.e., upper panel) were analyzed by Western blotting for Nrf2 protein level using lamin A/C as loading control, or total cell lysates (t.l., lower panel) were analyzed for Nqo1 and Gclc (Hsp90 as loading control). n-fold protein band intensities were determined by densitometry analysis normalizing to the corresponding loading controls. b, total RNA samples were submitted to RT-qPCR analyses of Gclc, Nqo1, and Ier3 mRNA levels normalized to β-actin mRNA (molecular mass ± S.D. (error bars), n = 4). * indicates statistical significance between the two genotypes. c and d, colon organ cultures were analyzed for viability by MTS staining for 2 h, and A_{490 nm} values were normalized for protein content (mean ± S.D., n = 4) or by Western blotting (results from two independent experiments) for PARP1 and caspase-3 protein expression using tubulin as loading control.

FIGURE 3.

IER3 interferes with Nrf2 activation in human NCM460 colonocytes. NCM460 cells were transfected with an IER3 expression vector or the empty vector (mock) and then analyzed for Nrf2 activation. a, nuclear and cytoplasmic extracts from untreated or tBHQ- (50 μm, 16 h) or SFN- (10 μm, 16 h) treated cells were analyzed by Nrf2 Western blotting (lamin A/C and tubulin, respectively, served as loading control). Three of six biological replicate experiments are shown from which n-fold band intensities of Nrf2 (100 kDa) were determined by densitometry analysis normalizing to the corresponding loading controls (mean ± S.D. (error bars), n = 6). b, ARE-Luc assays were conducted in IER3-transfected or untransfected cells subject of treatment (16 h) with 50 μm tBHQ or 10 μm SFN or not. Data are expressed as ARE-specific relative luciferase units (RLU) and represent the mean ± S.D., n = 4. c, NQO1 and GCLC mRNA levels were analyzed by qPCR (using TATA-binding protein (TBP) mRNA for normalization) in IER3-transfected or untransfected cells subjected to treatment (16 h) with 50 μm tBHQ or 10 μm SFN or not. Data represent the mean ± S.D., n = 4. d, total cell lysates from untreated or tBHQ- (50 μm, 24 h) or SFN- (10 μm, 24 h) treated cells were analyzed by Western blotting for NQO1 and GCLC as well as IER3 expression (Hsp90 served as loading control). Three of four biological replicate experiments are shown from which n-fold band intensities were determined by densitometry analysis normalizing to Hsp90 (mean ± S.D., n = 4). * indicates statistical significance between IER3 and mock transfectants.

FIGURE 4.

Increased Nrf2 activation in human NCM460 colonocytes with suppressed IER3 expression. NCM460 stably transfected with an IER3-shRNA or control-shRNA vector were analyzed for Nrf2 activation. a, nuclear and cytoplasmic extracts from untreated or tBHQ- (50 μm, 16 h) or SFN- (10 μm, 16 h) treated cells were analyzed by Nrf2 Western blotting (lamin A/C and tubulin, respectively, served as loading control). Three of six biological replicate experiments are shown from which n-fold band intensities of Nrf2 (100 kDa) were determined by densitometry analysis normalizing to the corresponding loading controls (mean ± S.D. (error bars), n = 6). b, ARE-luciferase assays were conducted in IER3 shRNA- or control shRNA-transfected cells subjected to treatment (16 h) with 50 μm tBHQ or 10 μm SFN or not. Data are expressed as ARE-specific relative luciferase units (RLU) and represent the mean ± S.D., n = 4. c, NQO1 and GCLC as well as IER3 mRNA levels were analyzed by qPCR (using TATA-binding protein (TBP) mRNA for normalization) in IER3 shRNA- or control shRNA-transfected cells subjected to treatment (16 h) with 50 μm tBHQ or 10 μm SFN or not. Data represent the mean ± S.D., n = 6. d, total cell lysates from untreated or tBHQ- (50 μm, 24 h) or SFN- (10 μm, 24 h) treated cells were analyzed by Western blot for NQO1 and GCLC expression (Hsp90 served as loading control). Three of six biological replicate experiments are shown from which n-fold band intensities were determined by densitometry analysis normalizing to Hsp90 (mean ± S.D., n = 6).* indicates statistical significance between IER3 shRNA- and control shRNA-expressing cells.

FIGURE 5.

Decreased ROS level in human NCM460 colonocytes with suppressed IER3 expression depending on Nrf2. a–c, NCM460 cells overexpressing IER3 or not (mock) (a) or NCM460 cells stably transfected with control or IER3 shRNA (b and c) were subjected to tBHQ treatment (24 h), or not, and then stained with cH2DCFdA to detect intracellular ROS (a and b) or with MitoSOX Red to detect mitochondrial ROS (c). Fluorescence was determined 4 h later; data represent the mean ± S.D. (error bars), n = 4. d, NCM460 stably transfected with control or IER3 shRNA were treated with control or Nrf2 siRNA for 40 h. Then, tBHQ was added, or not, for 24 h followed by cH2DCFdA staining and fluorescence measurement 4 h later; data represent the mean ± S.D., n = 6. * indicates statistical significance between the IER3 shRNA- and control shRNA-expressing cells.

FIGURE 6.

Greater Nrf2 activity in IER3-deficient human NCM460 colonocytes confers apoptosis protection. a, NCM460 cells stably transfected with control or IER3 shRNA and subjected to tBHQ treatment (24 h), or not, were left untreated or treated with either 10 ng/ml TRAIL (8 h) or 20 μg/ml etoposide (24 h). Caspase assays were conducted, and apoptosis was expressed as n-fold of untreated, mean ± S.D. (error bars); n = 4. b, IER3 shRNA or control shRNA NCM460 cells were treated with control or Nrf2 siRNA. After 24 h, tBHQ was added, or not, followed by TRAIL or etoposide treatment 24 h later. Caspase assays were conducted and apoptosis was expressed as n-fold of untreated, mean ± S.D.; n = 4. * indicates statistical significance between the IER3 shRNA- and control shRNA-expressing cells.

FIGURE 7.

Increased clonal growth of IER3-deficient NCM460 cells. NCM460 cells stably transfected with control or IER3 shRNA were seeded at a density of 200 or 500 cells/well on a 6-well plate and cultured for 1–2 weeks in the absence or presence of 50 μm tBHQ. Then, cells were fixed and stained with crystal violet. Visualized colonies with a diameter of >0.25 mm were counted, and the plating efficiency was calculated. Representative results (left panel) of four independent experiments performed in duplicates are shown, and the evaluation was carried out using the mean values ± S.D. (error bars, right panel) from these duplicate experiments. * indicates statistical significance between the IER3 shRNA- and control shRNA-expressing cells.

FIGURE 8.

Elevated Akt phosphorylation in IER3-deficient murine or human colonocytes and Akt dependence of the IER3 effect on Nrf2 activation. a, tissue sections from DSS-treated Ier3^−/− or Ier3^+/+mice were submitted to P-Akt immunofluorescence staining and DAPI counterstaining. b, colon organ cultures from untreated Ier3^−/− or Ier3^+/+ mice (data from two independent experiments are shown) were treated with 50 μm tBHQ, and cell lysates were analyzed by Western blotting detecting phospho-Akt, Akt, and tubulin. n-fold protein band intensities of Akt and P-Akt were determined by densitometry analysis normalizing to tubulin. c, NCM460 cells stably transfected with control or IER3 shRNA were incubated with 50 μm tBHQ or 10 μm SFN for 4 h or not. Cell lysates were analyzed by Western blotting for P-Akt (Hsp90 served as loading control) and three replicate of four experiments are shown. n-fold protein band intensities of P-Akt (lower panel) were determined by densitometry analysis normalizing to the Hsp90 (mean ± S.D. (error bars); n = 4). d and e, ARE-Luc assays were conducted with NCM460 cells stably transfected with control or IER3 shRNA and treated with 50 μm tBHQ or 10 μm SFN for 16 h, or not, either preincubated with 25 μm LY294002 for 1 h or not (d) or following pretreatment with Akt or control (co) siRNA for 48 h (e). The knockdown was verified by Western blotting and protein band densitometry (lower panel). Data are expressed as n-fold ARE-specific relative luciferase units (RLU) and represent the mean ± S.D., n = 4. * indicates statistical significance between the IER3 shRNA- and control shRNA-expressing cells.

FIGURE 9.

IER3 deficiency affects the nuclear accumulation of Fyn and its inhibitory effect on Nrf2 activation. a, colon organ cultures from untreated Ier3^−/− or Ier3^+/+ mice (data from two independent experiments are shown) were left untreated or were treated with 50 μm tBHQ for 8 h. Nuclear extracts were analyzed by Western blotting for Fyn (lamin A/C served as loading control). n-fold protein band intensities were determined by densitometry analysis normalizing to the corresponding loading control. b, nuclear and cytoplasmic extracts from untreated or tBHQ- (50 μm, 8 h) or SFN- (10 μm, 8 h) treated cells were analyzed by Fyn Western blotting (lamin A/C and tubulin, respectively, served as loading control). Three of six biological replicate experiments are shown from which n-fold band intensities of Fyn were determined by densitometry analysis normalizing to the corresponding loading controls (mean ± S.D. (error bars), n = 6). c, after pretreatment with Fyn or control (co) siRNA for 48 h, ARE-Luc assays were conducted with NCM460 cells stably transfected with control or IER3 shRNA and incubated with 50 μm tBHQ or 10 μm SFN for 16 h, or not. The knockdown was verified by Western blotting and protein band densitometry (lower panel). Data are expressed as n-fold ARE-specific relative luciferase units (RLU) and represent the mean ± S.D., n = 4. * indicates statistical significance between the IER3 shRNA- and control shRNA-expressing cells.