Mitogenic effect of orphan receptor TR3 and its regulation by MEKK1 in lung cancer cells

Abstract

TR3, also known as NGFI-B or nur77, is an immediate-early response gene and an orphan member of the steroid/thyroid/retinoid receptor superfamily. We previously reported that TR3 expression was induced by apoptotic stimuli and was required for their apoptotic effect in lung cancer cells. Here, we present evidence that TR3 was also induced by epidermal growth factor (EGF) and serum and was required for their mitogenic effect in lung cancer cells. Ectopic expression of TR3 in both H460 and Calu-6 lung cancer cell lines promoted their cell cycle progression and BrdU incorporation, while inhibition of TR3 expression by the small interfering RNA approach suppressed the mitogenic effect of EGF and serum. Analysis of TR3 mutants showed that both TR3 DNA binding and transactivation were required for its mitogenic effect. In contrast, they were dispensable for its apoptotic activity. Furthermore, confocal microscopy analysis demonstrated that TR3 functioned in the nucleus to induce cell proliferation, whereas it acted on mitochondria to induce apoptosis. In examining the signaling that regulates the mitogenic function of TR3, we observed that coexpression of constitutive-active MEKK1 inhibited TR3 transcriptional activity and TR3-induced proliferation. The inhibitory effect of MEKK1 was mediated through activation of Jun N-terminal kinase, which efficiently phosphorylated TR3, resulting in loss of its DNA binding. Together, our results demonstrate that TR3 is capable of inducing both proliferation and apoptosis in the same cells depending on the stimuli and its cellular localization.

FIG. 1.

Induction of TR3 by serum and EGF. (A) H460 cells were serum starved for 16 h, replaced with medium containing 10% FBS or treated EGF (200 ng/ml) for 3 h, and then analyzed for TR3 expression by immunoblotting. α-Tubulin expression served as a control for similar loading of proteins in each lane. One of three similar experiments is shown. (B) Effect of EGF and serum on lung cancer cell proliferation. H460 cells were seeded (1,000 cells/well) in 96-well plates and maintained in serum-free medium for 24 h. The cells were then grown in serum-free medium or medium containing 10% FBS or treated with EGF (200 ng/ml) for 48 h. Viable cell numbers in quadruplicate were then determined by the MTS assay. Cells maintained in serum-free medium were set to 100%. The increase in cell numbers after EGF and FBS stimulation was normalized relative to cell numbers maintained in serum-free medium. The bars represent averages ± mean from two experiments.

FIG. 2.

TR3 expression promotes cell cycle progression in lung cancer cells. GFP-TR3 or GFP control expression vector (5 μg) was transfected into H460 (A) or Calu-6 (B) lung cancer cells which were seeded in 90-mm-diameter dishes and maintained in medium containing 0.5% FBS. The transfected and GFP-expressing subpopulation of cells was identified by the high level of green fluorescence compared to nontransfected cells using flow cytometry (24). The cell cycle distribution of the transfected cells was then determined by flow cytometry after 45 h of transfection. One of three similar experiments is shown.

FIG. 3.

TR3 expression stimulates cell proliferation. H460 or Calu-6 cells were seeded in 90-mm-diameter dishes, transfected with GFP-TR3 or GFP control expression vector (5 μg), and maintained in medium containing 0.5% FBS. Forty-five hours after transfection, cells were maintained in BrdU containing medium for 2 h. Transfected and nontransfected cells were identified by flow cytometry, and BrdU-positive cells in each population were identified by immunostaining, using anti-BrdU antibody conjugated to phycoerythrin (Pharmingen). (A) BrdU immunofluorescence of GFP-transfected cells is overlaid with that from GFP-TR3-transfected cells. (B) The bars represent increases in BrdU cells after GFP or GFP-TR3 transfection compared to the nontransfected cells from the same culture dish. The means ± standard deviations from three experiments are shown.

FIG. 4.

TR3 is required for EGF- or serum-induced proliferation of lung cancer cells. (A) Suppression of endogenous TR3 expression by siRNA. H460 cells seeded in six-well plates were transfected with control scrambled or TR3 siRNA for 48 h. Cells were then treated with EGF or 10% FBS as described for Fig. 1A. Cell extracts were prepared and analyzed for TR3 expression by Western blotting. Two nonspecific bands around 105 kDa detected by anti-TR3 antibody and α-tubulin expression served as controls for similar loading of proteins in each lane. One out of four similar experiments is shown. (B) Suppression of proliferation by TR3 siRNA in H460 lung cancer cells. H460 cells were transfected with control scrambled or TR3 siRNA for 48 h in serum-free medium in 96-well plates and stimulated with EGF (200 ng/ml) or 10% FBS for 48 h or left untreated. Viable cell numbers in quadruplicate were then determined by the MTS assay. Absorbances (490 nM) representing the viable cells were plotted for nonstimulated cells maintained in serum-free medium (Control) and cells treated with EGF or FBS. The bars represent means ± standard deviation from four independent experiments. Similar results were also obtained by physically counting viable cells using trypan blue dye exclusion after transfection of H460 cells with control or TR3 siRNA in 24-well plates (n = 4).

FIG. 5.

DNA binding and transactivation of TR3 are required for its mitogenic effect. (A) Schematic representation of TR3 mutants. (B) Transcriptional activity of TR3 mutants. (NurRE)₂-tk-CAT (100 ng) and β-Gal gene expression vector (50 ng) were transiently transfected into CV-1 cells in 24-well plates with or without the expression vector for TR3 or its mutants (25 ng). CAT activity was determined and normalized relative to β-Gal activity. The bars represent averages ± mean from two experiments. (C) TR3 mutants with a deletion of the DNA-binding domain or the transactivation domain fail to promote lung cancer cell proliferation. H460 or Calu-6 cells were transfected with expression vectors for GFP-TR3 or its mutants. Transfected and nontransfected cells were identified by flow cytometry, and the number of BrdU-positive cells in each population was determined by immunostaining as described for Fig. 3. The bars represent the averages ± mean from two experiments. (D) TR3 with a deletion of its DNA-binding domain is capable of inducing apoptosis. The GFP-TR3/ΔDBD expression vector was transiently transfected into H460 cells. Nuclei were stained by DAPI 36 h after transfection. GFP-TR3/ΔDBD expression and nuclear morphology were visualized by fluorescence microscopy, and the two images were overlaid to show the effect of TR3/ΔDBD expression on nuclear condensation and fragmentation (shown by arrowheads). Apoptotic cells were scored by examining 300 transfected cells. The bars represent averages ± mean from two experiments.

FIG. 6.

Cellular localization of GFP-TR3 and GFP-TR3/ΔDBD. The GFP-TR3 (A) or GFP-TR3/ΔDBD (B) expression vector was transiently transfected into H460 cells seeded onto coverslips. After 20 h, cells were immunostained with anti-Hsp60 antibody followed by Cy3-conjugated secondary antibody to detect mitochondria. GFP-TR3 or GFP-TR3/ΔDBD and mitochondria (Hsp60) were visualized using confocal microscopy, and the two images were overlaid (Overlay). About 80% of the transfected cells showed the pattern presented.

FIG. 7.

3-Cl-AHPC induces H460 cell apoptosis and TR3 mitochondrial targeting. (A) Induction of TR3 expression by AHPN analog 3-Cl-AHPC. H460 cells were treated with 3-Cl-AHPC (10⁻⁶ M) or not treated for 3 h and analyzed for TR3 expression by immunoblotting. (B) Regulation of transactivation activity of TR3 by 3-Cl-AHPC. (NurRE)₂-tk-CAT (400 ng) and the β-Gal gene expression vector (100 ng) were transiently transfected into H460 cells in six-well plates by Lipofectamine Plus reagent (Invitrogen) together with or without the GFP-TR3 expression vector (50 ng). Cells were then treated with 3-Cl-AHPC (10⁻⁶ M) for 24 h. Reporter gene (CAT) activity was determined and normalized relative to the cotransfected β-Gal gene activity. The bars represent the averages ± mean from two experiments. (C) 3-Cl-AHPC disrupts mitochondrial membrane potential in H460 cells. H460 cells were treated with 10⁻⁶ M 3-Cl-AHPC or not treated for 18 h. Cells were then incubated with Rh123 for 30 min and analyzed by flow cytometry. Cells fluorescing within the range of Rh123 were considered depolarized. (D) 3-Cl-AHPC inhibits growth of H460 cells. H460 cells were treated with 10⁻⁶ M 3-Cl-AHPC for 48 h. Viable cell numbers in quadruplicate were determined by the 3-(4,5-dimethythiazol-2-yl)-2-5-diphenyl-tetrazolium assay. (E) 3-Cl-AHPC induces apoptosis in H460 cells. H460 cells were treated with 10⁻⁶ M 3-Cl-AHPC or not treated for 30 h. Nuclei were stained by DAPI. Apoptotic cells displaying nuclear fragmentation and/or condensation were scored by examining 600 cells. The bars represent the averages ± mean from two experiments. (F) 3-Cl-AHPC induces TR3 mitochondrial localization. H460 cells were treated with 10⁻⁶ M 3-Cl-AHPC or not treated (control) for 3 h, fixed, and then immunostained with mouse monoclonal anti-TR3 antibody followed by Cy3-conjugated secondary antibody or with anti-Hsp60 antibody followed by fluorescein isothiocyanate-conjugated secondary antibody. TR3 and Hsp60 were visualized using confocal microscopy, and images were overlaid (Overlay) as indicated. About 50% of the cells showed mitochondrial localization of endogenous TR3 after 3-Cl-AHPC treatment (n = 2).

FIG. 8.

Effect of MEKK1 on transcriptional activity of TR3. (A) Inhibition of TR3 transcriptional activity by MEKK1. TR3 expression vector (25 ng), β-Gal expression vector (50 ng), and the indicated reporter gene (200 ng) were cotransfected into CV-1 cells in 24-well plates with or without the indicated amount (expressed in nanograms) of MEKK1-DA expression vector (5). Reporter gene activity was determined and normalized relative to cotransfected β-Gal gene activity. One of six (left panel) or three (right panel) similar experiments is shown. (B) TR3 transcriptional activity is unaffected by kinase-deficient MEKK1. TR3 expression vector (25 ng), (NurRE)₂-tk-CAT (100 ng), β-Gal expression vector (50 ng) with or without the indicated amount (in nanograms) of MEKK1-DN expression vector (5) was transfected, and reporter gene activity was measured as described for panel A. The bars are averages ± mean from two experiments. (C) TR3 expression is not altered by MEKK1.GFP-TR3 expression vector (5 μg) was transfected into H460 cells with or without MEKK1-DA (5 μg) expression vector. Forty-five hours after transfection, amounts of GFP-TR3-expressing cells (percent green cells) were determined by flow cytometry. Similar results were observed in Calu-6 cells. The bars represent the averages ± mean from two experiments. (D) MEKK1 expression does not alter TR3 nuclear localization. GFP-TR3 alone (1.0 μg) or together with MEKK1-DA (1.0 μg) was transiently expressed in H460 cells. Fourteen hours later, cells were immunostained with anti-Hsp60 antibody followed by Cy3-conjugated secondary antibody to detect mitochondria. GFP-TR3 was visualized using confocal microscopy, and the two images were overlaid (Overlay). (E) Effect of MEKK1 expression on transactivation of other nuclear receptors and AP-1. The expression vector for ER or TR (25 ng) and the corresponding reporter gene (ERE-tk-CAT or TREpal-tk-CAT, respectively) were cotransfected into CV-1 cells with or without MEKK1-DA expression vector (30 ng). Twenty hours after transfection, cells were treated with estradiol (10⁻⁸ M) or T₃ (10⁻⁷ M), and CAT activity was determined and normalized relative to cotransfected β-Gal gene activity. For measuring the effect of MEKK1 on AP-1 activity, the −73Col-CAT (250 ng) reporter (37) was transfected with or without MEKK1-DA expression vector (30 ng). Reporter gene activity was measured as described earlier. One of three similar experiments is shown. (F) MEKK1 does not affect the transrepression activity of TR3. TR3 expression vector (20 ng) with or without MEKK1-DA expression vector (30 ng) and/or GR expression vector (20 ng) was cotransfected into CV-1 cells with the GRE-tk-CAT reporter vector (50). Twenty hours after transfection, cells were treated with Dex (10⁻⁷ M), and CAT activity was determined and normalized relative to cotransfected β-Gal gene activity. One of two similar experiments is shown.

FIG. 9.

Effect of MEKK1 on TR3 mitogenic activity. (A) GFP-TR3 or GFP control expression vector (5 μg) and/or MEKK1-DA expression vector (5 μg) were transfected into H460 or Calu-6 cells, which were seeded in 9-mm-diameter dishes and then maintained in medium containing 0.5% FBS. The transfected and GFP-expressing subpopulations of cells were identified by their high level of green fluorescence compared to nontransfected cells using flow cytometry (24). The cell cycle distribution of the transfected cells was then determined as described for Fig. 2. The increase in S-G₂ phase population was calculated by comparing the S-G₂-phase cells from transfected cells to those from nontransfected cells in the same culture dish. (B) BrdU-positive cells were identified by immunostaining as described for Fig. 3, except that H460 cells were pulsed with BrdU for 6 h. The bars in panels A and B represent the averages ± mean from two experiments.

FIG. 10.

Activation of JNK, but not Erk or p38, mediates the effect of MEKK1 on TR3 transcriptional activity. (A) Effect of JNK dominant-negative mutants on MEKK-1-induced inhibition of TR3 transactivation. The NurRE-tk-CAT reporter vector was cotransfected with TR3 (25 ng) and/or MEKK1-DA (10 ng) expression vector(s) into CV-1 cells together with or without the indicated JNK dominant-negative (DN) mutant expression vectors (50 ng). CAT activity was determined and normalized to cotransfected β-Gal gene activity. The bars represent means ± standard deviations from three experiments. (B) Effect of a MEK1 or p38 inhibitor on MEKK1-induced inhibition of TR3 transactivation. The NurRE-tk-CAT reporter vector(s) was cotransfected with TR3 (25 ng) and/or MEKK1-DA (10 ng) expression vector into CV-1 cells. Cells were treated with or without either PD98059 (20 μM) or SB202190 (20 μM). CAT activity was determined and normalized relative to cotransfected β-Gal gene activity. One of two similar experiments is shown.

FIG. 11.

Phosphorylation of TR3 by JNK and its effect on TR3 DNA binding. (A) Phosphorylation of TR3 by JNK. Equal amounts of bacterially expressed and purified GST-TR3 (33), GST-TR3 mutant (1-151), or c-Jun were incubated with JNK in an in vitro kinase assay. One of four similar experiments is shown. (B) Phosphorylation of the TR3 N terminus. Expression vectors for GFP-TR3, GFP-TR3-1-151, and GFP-TR3/Δ122 (a TR3 mutant with a deletion of the first 122 amino acid residues) (2.5 μg) were transfected into HEK293T cells in six-well plates. Twenty-four hours after transfection, cells were treated for 2 h with 10 μM anisomycin (Sigma) with or without a 30-min pretreatment with 20 μM JNK inhibitor II (Cat-10-420119; CalBiochem). Cell lysates were prepared and subjected to Western blotting using anti-GFP antibody. * indicates the modification of TR3 induced by anisomycin. One of two similar experiments is shown. (C) Effect of phosphorylation on TR3 DNA binding. Bacterially expressed TR3 or TR3 that had been phosphorylated by JNK in vitro were incubated with ³²P-labeled NurRE and analyzed by gel retardation assay as described previously (36). One of two similar experiments is shown.