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IL-32θ inhibits monocytic differentiation of leukemia cells by attenuating expression of transcription factor PU.1.

Kim MS, Kang JW, Park YS, Lee DH, Bak Y, Kwon T, Yoon DY - Oncotarget (2015)

Bottom Line: As a result, we concluded that these isoforms did not contribute to PMA-induced cell cycle arrest.IL-32θ was found to modulate expression of PU.1, a transcription factor necessary for myeloid lineage commitment.Additionally, transient expression of both CCAAT-enhancer-binding protein α (C/EBPα) and PU.1 in THP-1/IL-32θ cells exhibited synergistic effects in rescuing the differentiation defect.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul, Republic of Korea.

ABSTRACT
PU.1 is a key transcription factor regulating the myeloid differentiation. PU.1-induced monocytic differentiation into macrophage is also important for blood cancer development. Therefore, we chose THP-1 monocytic leukemia cells to investigate the function of a recently discovered IL-32θ. Genetic analyses identified differences in the sequences of IL-32θ and IL-32β. Using previously established cell lines that stably express IL-32θ and IL-32β and cell lines transiently expressing IL-32θ, we observed that expression of IL-32θ inhibited phorbol 12-myristate 13-acetate (PMA)-induced monocytic differentiation in both THP-1 and HL-60 cells. IL-32θ also suppressed expression of the macrophage cell surface markers, CD11b, CD18, and CD36. Interestingly, expression of IL-32β or IL-32θ had no effect on the expression levels of cell cycle related factors. As a result, we concluded that these isoforms did not contribute to PMA-induced cell cycle arrest. IL-32θ was found to modulate expression of PU.1, a transcription factor necessary for myeloid lineage commitment. Transient expression of PU.1 in THP-1/IL-32θ cells rescued the observed differentiation defect. Additionally, transient expression of both CCAAT-enhancer-binding protein α (C/EBPα) and PU.1 in THP-1/IL-32θ cells exhibited synergistic effects in rescuing the differentiation defect. These observations indicate that intracellular IL-32θ inhibits the differentiation of monocytes into macrophages by attenuating PU.1 expression.

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Effect of PU.1 and/or C/EBPα expression on monocyte differentiation in THP-1/IL-32θ cells(A) THP-1/IL-32θ cells were transfected with 1 μg of pcDNA3.1+ empty vector, pcDNA3.1+-PU.1 and/or C/EBPα expression vectors and incubated overnight. Cells were stimulated with 30 nM PMA for 72 h, and transfection efficiency was confirmed by RT-PCR and Western blot analyses. (B) Morphological changes were assessed by phase-contrast microscopy (100 ×). Scale bars represent 20 μm. After washing out non-adherent (undifferentiated) cells with PBS, the adherent cells were stained with Diff-Quick solution. At least 150 stained cells were counted in three randomly selected fields. To assess CD11b (C), CD18 (D), and CD36 (E) expression levels, cells were prepared as described above, and mRNA levels were measured by qRT-PCR. Data are presented as mean ± standard error of mean (n = 3). *p < 0.05. THP-1/IL-32θ cells versus cells transfected with PU.1 and/or C/EBPα, after PMA treatment.
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Figure 7: Effect of PU.1 and/or C/EBPα expression on monocyte differentiation in THP-1/IL-32θ cells(A) THP-1/IL-32θ cells were transfected with 1 μg of pcDNA3.1+ empty vector, pcDNA3.1+-PU.1 and/or C/EBPα expression vectors and incubated overnight. Cells were stimulated with 30 nM PMA for 72 h, and transfection efficiency was confirmed by RT-PCR and Western blot analyses. (B) Morphological changes were assessed by phase-contrast microscopy (100 ×). Scale bars represent 20 μm. After washing out non-adherent (undifferentiated) cells with PBS, the adherent cells were stained with Diff-Quick solution. At least 150 stained cells were counted in three randomly selected fields. To assess CD11b (C), CD18 (D), and CD36 (E) expression levels, cells were prepared as described above, and mRNA levels were measured by qRT-PCR. Data are presented as mean ± standard error of mean (n = 3). *p < 0.05. THP-1/IL-32θ cells versus cells transfected with PU.1 and/or C/EBPα, after PMA treatment.

Mentions: C/EBPα is a member of the Ets transcription factor family and is a key regulator during myeloid lineage development [40]. Although IL-32θ had no effect on the expression C/EBPα (data not shown), we predicted that increased expression of C/EBPα could support PU.1-mediated cell differentiation. To address this possibility, THP-1/IL-32θ cells were transfected with 1 μg of the C/EBPα and/or PU.1-expressing vectors, and transfection efficiency was confirmed by RT-PCR and Western blot analyses (Figure 7A). Morphological changes were monitored and differentiated cells were again quantified by enumerating adherent cells in culture dishes. The number of differentiated cells was significantly increased by co-transfection with PU.1 and C/EBPα compared to transfection with PU.1 or C/EBPα alone (Figure 7B). The expression levels of CD11b, CD18, and CD36 were also significantly increased in cells co-transfected with PU.1 and C/EBPα (Figure 7C–7E). Meanwhile, transfection with C/EBPα alone, resulted in a significant increase in CD18 expression levels. These findings indicate that overexpression of both PU.1 and C/EBPα resulted in a synergistic effect in the restoration of monocytic differentiation in IL-32θ-expressing cells.


IL-32θ inhibits monocytic differentiation of leukemia cells by attenuating expression of transcription factor PU.1.

Kim MS, Kang JW, Park YS, Lee DH, Bak Y, Kwon T, Yoon DY - Oncotarget (2015)

Effect of PU.1 and/or C/EBPα expression on monocyte differentiation in THP-1/IL-32θ cells(A) THP-1/IL-32θ cells were transfected with 1 μg of pcDNA3.1+ empty vector, pcDNA3.1+-PU.1 and/or C/EBPα expression vectors and incubated overnight. Cells were stimulated with 30 nM PMA for 72 h, and transfection efficiency was confirmed by RT-PCR and Western blot analyses. (B) Morphological changes were assessed by phase-contrast microscopy (100 ×). Scale bars represent 20 μm. After washing out non-adherent (undifferentiated) cells with PBS, the adherent cells were stained with Diff-Quick solution. At least 150 stained cells were counted in three randomly selected fields. To assess CD11b (C), CD18 (D), and CD36 (E) expression levels, cells were prepared as described above, and mRNA levels were measured by qRT-PCR. Data are presented as mean ± standard error of mean (n = 3). *p < 0.05. THP-1/IL-32θ cells versus cells transfected with PU.1 and/or C/EBPα, after PMA treatment.
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Figure 7: Effect of PU.1 and/or C/EBPα expression on monocyte differentiation in THP-1/IL-32θ cells(A) THP-1/IL-32θ cells were transfected with 1 μg of pcDNA3.1+ empty vector, pcDNA3.1+-PU.1 and/or C/EBPα expression vectors and incubated overnight. Cells were stimulated with 30 nM PMA for 72 h, and transfection efficiency was confirmed by RT-PCR and Western blot analyses. (B) Morphological changes were assessed by phase-contrast microscopy (100 ×). Scale bars represent 20 μm. After washing out non-adherent (undifferentiated) cells with PBS, the adherent cells were stained with Diff-Quick solution. At least 150 stained cells were counted in three randomly selected fields. To assess CD11b (C), CD18 (D), and CD36 (E) expression levels, cells were prepared as described above, and mRNA levels were measured by qRT-PCR. Data are presented as mean ± standard error of mean (n = 3). *p < 0.05. THP-1/IL-32θ cells versus cells transfected with PU.1 and/or C/EBPα, after PMA treatment.
Mentions: C/EBPα is a member of the Ets transcription factor family and is a key regulator during myeloid lineage development [40]. Although IL-32θ had no effect on the expression C/EBPα (data not shown), we predicted that increased expression of C/EBPα could support PU.1-mediated cell differentiation. To address this possibility, THP-1/IL-32θ cells were transfected with 1 μg of the C/EBPα and/or PU.1-expressing vectors, and transfection efficiency was confirmed by RT-PCR and Western blot analyses (Figure 7A). Morphological changes were monitored and differentiated cells were again quantified by enumerating adherent cells in culture dishes. The number of differentiated cells was significantly increased by co-transfection with PU.1 and C/EBPα compared to transfection with PU.1 or C/EBPα alone (Figure 7B). The expression levels of CD11b, CD18, and CD36 were also significantly increased in cells co-transfected with PU.1 and C/EBPα (Figure 7C–7E). Meanwhile, transfection with C/EBPα alone, resulted in a significant increase in CD18 expression levels. These findings indicate that overexpression of both PU.1 and C/EBPα resulted in a synergistic effect in the restoration of monocytic differentiation in IL-32θ-expressing cells.

Bottom Line: As a result, we concluded that these isoforms did not contribute to PMA-induced cell cycle arrest.IL-32θ was found to modulate expression of PU.1, a transcription factor necessary for myeloid lineage commitment.Additionally, transient expression of both CCAAT-enhancer-binding protein α (C/EBPα) and PU.1 in THP-1/IL-32θ cells exhibited synergistic effects in rescuing the differentiation defect.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul, Republic of Korea.

ABSTRACT
PU.1 is a key transcription factor regulating the myeloid differentiation. PU.1-induced monocytic differentiation into macrophage is also important for blood cancer development. Therefore, we chose THP-1 monocytic leukemia cells to investigate the function of a recently discovered IL-32θ. Genetic analyses identified differences in the sequences of IL-32θ and IL-32β. Using previously established cell lines that stably express IL-32θ and IL-32β and cell lines transiently expressing IL-32θ, we observed that expression of IL-32θ inhibited phorbol 12-myristate 13-acetate (PMA)-induced monocytic differentiation in both THP-1 and HL-60 cells. IL-32θ also suppressed expression of the macrophage cell surface markers, CD11b, CD18, and CD36. Interestingly, expression of IL-32β or IL-32θ had no effect on the expression levels of cell cycle related factors. As a result, we concluded that these isoforms did not contribute to PMA-induced cell cycle arrest. IL-32θ was found to modulate expression of PU.1, a transcription factor necessary for myeloid lineage commitment. Transient expression of PU.1 in THP-1/IL-32θ cells rescued the observed differentiation defect. Additionally, transient expression of both CCAAT-enhancer-binding protein α (C/EBPα) and PU.1 in THP-1/IL-32θ cells exhibited synergistic effects in rescuing the differentiation defect. These observations indicate that intracellular IL-32θ inhibits the differentiation of monocytes into macrophages by attenuating PU.1 expression.

Show MeSH
Related in: MedlinePlus