Immune Netw.  2010 Dec;10(6):219-229. 10.4110/in.2010.10.6.219.

NDRG2-mediated Modulation of SOCS3 and STAT3 Activity Inhibits IL-10 Production

• Lee EB
• Kim A
• Kang K
• Kim H
• Lim JS

• KMID: 2150678
• DOI: http://doi.org/10.4110/in.2010.10.6.219

Abstract

BACKGROUND
N-myc downstream regulated gene 2 (NDRG2) is a member of the NDRG gene family. Our previous report indicated a possible role for NDRG2 in regulating the cytokine, interleukin-10 (IL-10), which is an important immunosuppressive cytokine. Several pathways, including p38-MAPK, NF-kappaB, and JAK/STAT, are used for IL-10 production, and the JAK/STAT pathway can be inhibited in a negative feedback loop by the inducible protein, SOCS3. In the present study, we investigated the effect of NDRG2 gene expression on IL-10 signaling pathway that is modulated via SOCS3 and STAT3.
METHODS
We generated NDRG2-overexpressing U937 cell line (U937-NDRG2) and treated the cells with PMA to investigate the role of NDRG2 in IL-10 production. U937 cells were also transfected with SOCS3- or NDRG2-specific siRNAs to examine whether the knockdown of SOCS3 or NDRG2 influenced IL-10 expression. Lastly, STAT3 and SOCS3 induction was measured to identify the signaling pathway that was associated with IL-10 production.
RESULTS
RT-PCR and ELISA assays showed that IL-10 was increased in U937-mock cells upon stimulation with PMA, but IL-10 was inhibited by overexpression NDRG2. After PMA treatment, STAT3 phosphorylation was decreased in a time-dependent manner in U937-mock cells, whereas it was maintained in U937-NDRG2 cells. SOCS3 was markedly reduced in U937-NDRG2 cells compared with U937-mock cells. IL-10 production after PMA stimulation was reduced in U937 cells when SOCS3 was inhibited, but this effect was less severe when NDRG2 was inhibited.
CONCLUSION
NDRG2 expression modulates SOCS3 and STAT3 activity, eventually leading to the inhibition of IL-10 production.

Keyword

NDRG2; IL-10; SOCS3; STAT3; U937

MeSH Terms

Enzyme-Linked Immunosorbent Assay
Gene Expression
Humans
Interleukin-10
NF-kappa B
Phosphorylation
RNA, Small Interfering
U937 Cells
Interleukin-10
NF-kappa B
RNA, Small Interfering

Figure

• 

  Figure 1 PMA stimulation of U937 cells induces IL-10 production. (A) Dose- and time-dependent increase in IL-10 mRNA after PMA stimulation. U937 cells were stimulated with PMA for different time periods and harvested for mRNA analysis. Transcript levels of IL-10 and β-actin (loading control) were determined by RT-PCR. (B) U937 cells were stimulated with different concentrations of PMA for 24 h. Cells were harvested, and IL-10 protein levels in the supernatants were determined by ELISA. Data represent the mean±SD of duplicates.

• 

  Figure 2 IL-10 production is reduced by NDRG2 expression. (A) U937, U937-mock and U937-NDRG2 cells were stimulated with PMA (50 ng/ml) for different time periods and harvested for mRNA analysis. Transcript levels of IL-10 and β-actin were determined by RT-PCR. (B) U937-mock and U937-NDRG2 cells (3×105 cells/ml) were stimulated with PMA (50 ng/ml) for 24 h. The levels of IL-10 in culture supernatants were determined by ELISA and corrected based on cell numbers after cell counting. Data represent the mean±SD of duplicates. (C) Cells (2×104 cells/well) were cultured in 96-well culture plates for 24 h and treated with MTT for 2 h. The cells were harvested and centrifuged at 1,200 rpm for 5 min. The pellets were dissolved in 200µl of DMSO, and absorbance was measured at 540 nm using an ELISA reader (upper). For cell counting by microscope, cells (5×105 cells/well) in 6-well culture plates were cultured for 1 to 4 days and centrifuged at 1,200 rpm for 5 min. Viable cells, which are visible by light microscopy, were enumerated using a hemocytometer. Data represent the mean±SD of triplicates.

• 

  Figure 3 IL-10 and SOCS3 mRNA levels after PMA stimulation are reduced by NDRG2 expression. (A) U937-mock and U937-NDRG2 cells were stimulated at various times with 50 ng/ml of PMA. Cells were harvested for analysis. Transcript levels of IL-10 and SOCS family members were determined by RT-PCR. (B) Cells were stimulated with PMA for 24 h and harvested for mRNA analysis. Transcript levels of IL-10 and SOCS genes were determined by RT-PCR.

• 

  Figure 4 NDRG2 expression inhibits induction of SOCS3 but promotes STAT3 phosphorylation after PMA stimulation. (A) U937-mock and U937-NDRG2 cells were stimulated with 50 ng/ml of PMA for 15 or 30 min. Cell lysates were harvested and equal amounts of lysates were subjected to electrophoresis on SDS-PAGE. Western blotting was performed using STAT3-, pSTAT3- and NDRG2-specific antibodies. (B) Cells were stimulated at various times with 50 ng/ml of PMA. Equal amounts of whole-cell lysates were subjected to electrophoresis on SDS-PAGE, and then western blotting was performed using specific antibodies.

• 

  Figure 5 Inhibition of STAT3 phosphorylation increases IL-10 production. (A) U937-mock cells were pretreated with cucurbitacin I for 2 h followed by PMA treatment for 48 h. Cells were harvested for mRNA analysis. Transcript levels of IL-10 were determined by RT-PCR. (B) U937-NDRG2 cells were pretreated with cucurbitacin I or AG490 for 2 h followed by PMA treatment for 48 h. Cells were harvested for mRNA analysis. Transcript levels of IL-10 were assessed by RT-PCR. (C) U937-NDRG2 cells were treated in the same manner as in (B). The levels of IL-10 in culture supernatants were determined by ELISA. Data represent the mean±SD of duplicates.

• 

  Figure 6 Proliferation of siRNA-transfected U937 cells. U937-mock and U937-NDRG2 cells were transfected with siRNA control, SOCS3- or NDRG2-specific siRNAs (100 nM) for 48 h. After transfection, cells (1×104) were plated in 60 mm dishes and counted daily for 4 days.

• 

  Figure 7 Effect of SOCS3 siRNA and NDRG2 siRNA on IL-10 mRNA or protein expression. (A) U937-mock and U937-NDRG2 cells were transfected with siRNA control, SOCS3- or NDRG2-specific siRNAs (100 nM) for 48 h. Cells were then stimulated with PMA and harvested for mRNA analysis. Transcript levels of IL-10, SOCS3, and NDRG2 genes were determined by RT-PCR. (B) After siRNA transfection for 48 h, U937-mock and U937-NDRG2 cells (3×105 cells/ml) were stimulated with PMA for 24 h. The levels of IL-10 in culture supernatants were determined by ELISA. Data represent the mean±SD of duplicates.

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