Immune Netw.  2011 Dec;11(6):348-357. 10.4110/in.2011.11.6.348.

NDRG2 Promotes GATA-1 Expression through Regulation of the JAK2/STAT Pathway in PMA-stimulated U937 Cells

Affiliations
  • 1Department of Biological Science and the Research Center for Women's Disease, Sookmyung Women's University, Seoul 140-742, Korea. jslim@sookmyung.ac.kr

Abstract

BACKGROUND
N-myc downstream-regulated gene 2 (NDRG2), a member of a newly described family of differentiation-related genes, has been characterized as a regulator of dendritic cells. However, the role of NDRG2 on the expression and activation of transcription factors in blood cells remains poorly understood. In this study, we investigated the effects of NDRG2 overexpression on GATA-1 expression in PMA-stimulated U937 cells.
METHODS
We generated NDRG2-overexpressing U937 cell line (U937-NDRG2) and treated the cells with PMA to investigate the role of NDRG2 on GATA-1 expression.
RESULTS
NDRG2 overexpression in U937 cells significantly induced GATA-1 expression in response to PMA stimulation. Interestingly, JAK2/STAT and BMP-4/Smad pathways associated with the induction of GATA-1 were activated in PMA-stimulated U937-NDRG2 cells. We found that the inhibition of JAK2 activation, but not of BMP-4/Smad signaling, can elicit a decrease of PMA-induced GATA-1 expression in U937-NDRG2 cells.
CONCLUSION
The results reveal that NDRG2 promotes the expression of GATA-1 through activation of the JAK2/STAT pathway, but not through the regulation of the BMP-4/Smad pathway in U937 cells. Our findings further suggest that NDRG2 may play a role as a regulator of erythrocyte and megakaryocyte differentiation during hematopoiesis.

Keyword

GATA-1; NDRG2; U937; JAK2/STAT pathway

MeSH Terms

Blood Cells
Dendritic Cells
Erythrocytes
Hematopoiesis
Humans
Megakaryocytes
Transcription Factors
U937 Cells
Transcription Factors

Figure

  • Figure 1 Overexpression of NDRG2 in U937 cells. (A) Cell lysates were fractionated into cytoplasmic and nuclear components. Western blotting using an anti-NDRG2 antibody was performed for each fraction. Lamin A/C and α-actinin were used as control for protein loading as well as marker proteins for nuclear and cytoplasmic fraction, respectively. (B, C) U937-mock and U937-NDRG2 cells were treated with 0.1 µg/ml PMA for 48 h. Cell viability was determined using either the trypan blue exclusion assay (B) or the CCK-8 assay (C). The results represent the means±SD of duplicates. *p<0.05, **p<0.01. (D) PU.1, CSF-1R, and NDRG2 mRNA levels were measured by RT-PCR. β-actin was used as a loading control.

  • Figure 2 NDRG2 induces GATA-1 expression. (A) U937-mock and U937-NDRG2 cells were treated with 0.1µg/ml PMA for 48 h. mRNA levels for GATA-1 and NDRG2 were measured by RT-PCR. β-actin was used as a loading control. (B) Equal amounts of whole protein lysates were subjected to electrophoresis on SDS-PAGE gels, and Western blot analysis was performed using specific antibodies. Actin was used as an internal loading control. (C) Expression of genes promoting the differentiation of erythrocyte and megakaryocyte. U937-mock and U937-NDRG2 cells were treated with 0.1 µg/ml PMA for 48 h prior to RT-PCR analysis.

  • Figure 3 JAK2/STAT pathway is activated by NDRG2 expression. U937-mock and U937-NDRG2 cells were treated with 0.1µg/ml PMA for 48 h. Western blot analysis (A, B) and RT-PCR (C) were performed. α-actinin and β-actin were used as an internal loading control.

  • Figure 4 BMP-4/Smad signaling pathway is activated by NDRG2 expression. U937-mock and U937-NDRG2 cells were treated with 0.1µg/ml PMA for 48 h. (A) Equal amounts of whole cell lysates were subjected to electrophoresis on SDS-PAGE gels, and Western blot analysis was performed using specific antibodies. Actin was used as an internal loading control. (B, C) mRNA levels were measured by RT-PCR.

  • Figure 5 NDRG2 increases GATA-1 expression through regulation of JAK2/STAT pathway. U937-NDRG2 cells were pre-treated with 5µg/ml AG490 (A), 1µM dorsomorphin (DM) (B), or 5 ng/ml noggin (C) for 3 h and then, 0.1µg/ml PMA was added. After 48 h, Western blot analysis was performed.


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