Korean J Physiol Pharmacol.  2020 May;24(3):203-212. 10.4196/kjpp.2020.24.3.203.

Interaction of promyelocytic leukemia/p53 affects signal transducer and activator of transcription-3 activity in response to oncostatin M

• Lim J
• Choi JH
• Park EM
• Choi YH

• KMID: 2502502
• DOI: http://doi.org/10.4196/kjpp.2020.24.3.203

Abstract

Promyelocytic leukemia (PML) gene, through alternative splicing of its Cterminal region, generates several PML isoforms that interact with specific partners and perform distinct functions. The PML protein is a tumor suppressor that plays an important role by interacting with various proteins. Herein, we investigated the effect of the PML isoforms on oncostatin M (OSM)-induced signal transducer and activator of transcription-3 (STAT-3) transcriptional activity. PML influenced OSMinduced STAT-3 activity in a cell type-specific manner, which was dependent on the p53 status of the cells but regardless of PML isoform. Interestingly, overexpression of PML exerted opposite effects on OSM-induced STAT-3 activity in p53 wild-type and mutant cells. Specifically, overexpression of PML in the cell lines bearing wild-type p53 (NIH3T3 and U87-MG cells) decreased OSM-induced STAT-3 transcriptional activity, whereas overexpression of PML increased OSM-induced STAT-3 transcriptional activity in mutant p53-bearing cell lines (HEK293T and U251-MG cells). When wild-type p53 cells were co-transfected with PML-IV and R273H-p53 mutant, OSM-mediated STAT-3 transcriptional activity was significantly enhanced, compared to that of cells which were transfected with PML-IV alone; however, when cells bearing mutant p53 were co-transfected with PML-IV and wild-type p53, OSM-induced STAT-3 transcriptional activity was significantly decreased, compared to that of transfected cells with PML-IV alone. In conclusion, PML acts together with wild-type or mutant p53 and influences OSM-mediated STAT-3 activity in a negative or positive manner, resulting in the aberrant activation of STAT-3 in cancer cells bearing mutant p53 probably might occur through the interaction of mutant p53 with PML.

Keyword

Promyelocytic leukemia; p53; Signal transducer and activator of transcription-3; Transcriptional activity

Figure

• 

  Fig. 1 PML affects differently OSM-induced STAT-3 transcri­ptional activity in immortalized cells and glioma cells. (A) NIH3T3, (B) HEK293T, (C) U87-MG, and (D) U251-MG cells were co-transfected with the STAT-3-Luc reporter and either empty vector or PML isoform expression vectors. At 24 h after transfection, cells were either untreated or treated with OSM (10 ng/ml) for 24 h and then assayed for luciferase activity. The pCMV-β-galactosidase vector was included to normalize transfection efficiency. Data are presented as fold increase in relative luciferase activity (RLA) compared with RLA in the absence of OSM. PML protein expression and OSM-induced STAT-3 phosphorylation were verified for each assay by immunoblotting. The results are representative of three independent experiments. mOSM, murine OSM; hOSM, human OSM. **p < 0.01, ***p < 0.001 vs. untreated control; #p < 0.05, ###p < 0.001 between cells were transfected with PML expression vectors and cells were transfected with mock vector in the presence of OSM.

• 

  Fig. 2 The status of p53 and differential protein expression level. (A) The table indicates the p53 status of cell lines used in this study. p53 status was determined through the TP53 web site (http://p53.fr). (B, upper) NIH3T3, HEK293T, U87-MG, and U251-MG cells were either untreated or treated with OSM (10 ng/ml) for 24 h, and the lysed in RIPA buffer. Whole cell lysates were analyzed by immunoblotting using anti-PML, anti-p53, anti-p-STAT-3, anti-STAT-3, anti-α-tubulin antibodies. (B, lower) The graph represents the normalized intensities of p-STAT-3 against those of STAT-3 determined from three independent experiments. WT, wild-type. **p < 0.01, ***p < 0.001 vs. untreated control.

• 

  Fig. 3 The opposite effects of PML-IV on OSM-induced STAT-3 activity in p53-status dependent manner. (A) NIH3T3 and (B) U87-MG cells were transfected with either PML-IV or R273H-p53 or combination PML-IV and R273H-p53. (C) HEK293T and (D) U251-MG cells were transfected with either PML-IV or WT-p53 or combination PML-IV and WT-p53. At 24 h after transfection, cells were either untreated or treated with OSM (10 ng/ml) for 24 h and then assayed for luciferase activity. Data are presented as fold increase in relative luciferase activity (RLA) compared with RLA in the absence of OSM. PML and p53 protein expression and OSM-induced STAT-3 phosphorylation were verified for each assay by immunoblotting. The results are representative of three independent experiments. mOSM, murine OSM; hOSM, human OSM; WT, wild-type. *p < 0.05, **p < 0.01, ***p < 0.001 vs. untreated control; #p < 0.05, ###p < 0.001 between cells were transfected with PML-IV and cells were transfected with either combination PML-IV and R273H-p53 mutant or combination PML-IV and WT-p53 in the presence of OSM.

• 

  Fig. 4 The effects of PML-IV and its deletion constructs on OSM-mediated STAT-3 activity. (A) Depiction of the PML-IV deletion constructs generated in this study. (B) NIH3T3, (C) U87-MG, (D) HEK293T, (E) U251-MG cells were co-transfected with the STAT-3-Luc reporter and empty vector, PML-IV, and PML-IV deletion plasmids. At 24 h after transfection, cells were either untreated or treated with OSM (10 ng/ml) for 24 h and then assayed for luciferase activity. The pCMV-β-galactosidase vector was included to normalize transfection efficiency. Data are presented as fold increase in relative luciferase activity (RLA) compared with RLA in the absence of OSM. PML protein expression and OSM-induced STAT-3 phosphorylation were verified for each assay by immunoblotting. The results are representative of three independent experiments. mOSM, murine OSM; hOSM, human OSM. *p < 0.05, ***p < 0.001 vs. untreated control; ##p < 0.01. ###p < 0.001 between cells were transfected with either PML-IV or PML-IV deletion vectors and cells were transfected with mock vector in the presence of OSM.

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