SENP3-mediated de-conjugation of SUMO2/3 from promyelocytic leukemia is correlated with accelerated cell proliferation under mild oxidative stress

Abstract

Small ubiquitin-like modifier (SUMO) 2/3 is known to conjugate to substrates in response to a variety of cellular stresses. However, whether and how SUMO2/3-specific proteases are involved in de-conjugation under cell stress is unclear. Here, we show that low doses of hydrogen peroxide (H(2)O(2)) induce an increase of the SENP3 protein, which removes SUMO2/3 from promyelocytic leukemia (PML). Low dose H(2)O(2) causes SENP3 to co-localize with PML bodies and reduces the number of PML bodies in a SENP3-dependent manner. Furthermore, de-conjugation of SUMO2/3 from PML is responsible for the accelerated cell proliferation caused by low dose H(2)O(2). Knocking down PML promotes basal cell proliferation as expected. This can be reversed by reconstitution with wild-type PML but not its mutant lacking SUMOylation, indicating that only the SUMOylated PML can play an inhibitory role for cell proliferation. Thus, SENP3 appears to be a key mediator in mild oxidative stress-induced cell proliferation via regulation of the SUMOylation status of PML. Furthermore, SENP3 is over-accumulated in a variety of primary human cancers including colon adenocarcinoma in which PML is hypo-SUMOylated. These results reveal an important role of SENP3 and the SUMOylation status of PML in the regulation of cell proliferation under oxidative stress.

FIGURE 1.

Mild oxidative stress accelerates cell proliferation in a SENP3-dependent manner. A, three types of cells were treated with the indicated concentrations of H₂O₂ for 1 h. IB was performed as indicated. B, three types of cells were seeded in 24-well plates with 20% confluence and treated with H₂O₂ once a day from the second day. The concentrations of H₂O₂ were 10 μm for HUVEC and NIH3T3 cells and 50 μm for HeLa cells. Cell proliferation was assessed and is indicated by a -fold increase in cell number based on the results of MTT and cell counting. The values are the means ± S.D. of three independent experiments. SENP3 protein was analyzed by IB in a parallel setting every day after H₂O₂ addition for 1 h. C, control. For the fourth day, p < 0.01. C, HUVEC were cultured with 2% serum for 24 h and exposed to 10 μm H₂O₂ treatment for 6 h before labeling with 30 μm BrdUrd for 1 h. The BrdUrd incorporation rate was quantified by automatically counting the BrdUrd-positive cells among DAPI-stained cells within four random fields (×40) using Zeiss KS400 software (upper panel). IB was performed after H₂O₂ treatment for 6 h as indicated (bottom panel). D, NIH3T3 cells were transfected with the 100 nm oligonucleotides for NS siRNA or SENP3 siRNA for 24 h and then subcultured for an additional 4 days. The efficiency of the SENP3 siRNA was determined by IB (upper left panel). Cell proliferation was assessed (upper right panel). Cells with intact or knocked-down SENP3 were treated with 10 μm H₂O₂ for 6 h, and cell cycle analysis was assessed by propidium iodide staining (bottom panel).

FIGURE 2.

Mild oxidative stress decreases the PML nuclear bodies via SENP3 (Scale bar = 5 μm). A, immunofluorescence was performed with SENP3, PML, and fibrillarin antibodies in HUVEC. Cell nuclei were stained with DAPI. B, HUVEC were treated with 10 μm H₂O₂ for 1 h, and then immunofluorescence was performed with PML antibody. Cell nuclei were stained with DAPI (left panel). Image quantification shows the means ± S.D. of the number of PML bodies counted in 50 cells in each group (right panel). C, in HUVEC that were stably transfected with RH-SENP3 or pcDNA3 vector, immunofluorescence was performed with PML and RH antibodies. Cell nuclei were stained with DAPI (left panel). Quantification for the number of PML bodies was shown (right panel). D, after HUVEC were transfected with nonspecific shRNA or SENP3 shRNA viral constructs for 72 h, cells were treated with 10 μm H₂O₂ for 1 h. Immunofluorescence was performed with PML antibody. Cell nuclei were stained with DAPI (left panel). The efficiency of the SENP3 siRNA was determined by IB (right upper panel). Quantification for the number of PML bodies was shown (right bottom panel).

FIGURE 3.

SENP3 de-conjugates SUMO2/3 from PML under mild oxidative stress. A, NIH3T3 cells were transfected with RH-PML. After transfection for 48 h and then the addition of 10 μm H₂O₂ for 1 h as indicated, cells were lysed, and pulldown assay was performed with Talon beads. IB was performed as indicated. B, HUVEC were treated with 10 μm H₂O₂ for 1 h as indicated. Immunofluorescence was performed with PML and SENP3 antibodies. Cell nuclei were stained with DAPI. Scale bar, 5 μm (left panel). Quantitative data show the percentage of PML bodies that co-localized with SENP3 versus all PML bodies counted in 30 cells in each group (right panel). C, NIH3T3 cells were transfected with RH-PML alone or plus Myc-SENP3 or Myc-SENP3 mutant as indicated for 48 h. Nickel bead pulldown assays and IB were performed as indicated. D, after transfection of SENP3 siRNA or NS siRNA for 72 h and then the addition of 10 μm H₂O₂ for 1 h as indicated, IP was performed with PML antibody, and IB was performed as indicated in NIH3T3 cells.

FIGURE 4.

SENP3-mediated de-conjugation of SUMO2/3 from PML is responsible for the accelerated cell proliferation and decreased PML bodies under mild oxidative stress. A, HUVEC that were stably transfected with RH-SENP3/m or pcDNA3 vector were cultured. Cell proliferation was assessed. B, after NIH3T3 cells were transfected with nonspecific siRNA or PML siRNA for 24 h, cells were subcultured and treated with H₂O₂ once a day as indicated. Cell proliferation was assessed (left panel). The efficiency of PML siRNA was determined by quantitative PCR (right panel). The differences between the pared values for proliferation at day 4 were statistically assessed (see “Results”). C, after NIH3T3 cells were transfected with PML siRNA for 24 h, cells were subcultured and reintroduced with the viral constructs for WT-PML and SUMOylation mutant (SM)-PML and the empty vector (V) containing HA tags. The viral constructs for SENP3 were simultaneously transfected as indicated. Cell proliferation was assessed 48 h post-viral transfections (bottom panel). IP and IB were performed as indicated (upper panel). D, HUVEC were transfected with HA-PML WT or HA-PML SM for 48 h and then were treated with 10 μm H₂O₂ for additional 1 h as indicated. Immunofluorescence was performed with HA antibody. Cell nuclei were stained with DAPI. Scale bar = 5 μm.

FIGURE 5.

SENP3-mediated de-conjugation of SUMOylated PML may be involved in tumor growth. A, HeLa cells stably transfected with pcDNA3, and SENP3 plasmids were injected subcutaneously into nude mice to form the xenografts. After 3 weeks, the mice were sacrificed. The tumor xenografts were dissected (left panel) and weighted (middle panel). The tumor tissues of the xenografts were homogenized, and IP was performed with PML antibody. IB was performed as indicated (right panel). B, immunohistochemistry for SENP3 was performed in the tissue chips. Image quantification shows the means ± S.D. of the percentage of SENP3-positive areas. n = the number of samples. *, p < 0.05. C, immunohistochemistry for SENP3 was performed in two colon adenocarcinomas and adjacent normal colon tissues. The brown staining represents a positive signal (left panel). Scale bar, 10 μm. Two colon adenocarcinomas and adjacent normal colon tissues were homogenized. IP was performed with PML antibody, and IB was performed as indicated (right panel). D, two colon adenocarcinomas and adjacent normal tissues were homogenized, and the GSH level was assessed. Quantification showed the means ± S.D. of the relative GSH levels.