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

Kang K, Jung H, Nam S, Lim JS - Immune Netw (2011)

Bottom Line: In this study, we investigated the effects of NDRG2 overexpression on GATA-1 expression in PMA-stimulated U937 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and the Research Center for Women's Disease, Sookmyung Women's University, Seoul 140-742, Korea.

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.

No MeSH data available.


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.
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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.

Mentions: U937 cells differentiate into monocyte-like cells upon treatment with phorbol 12-myristate 13-acetate (PMA), and their growth is arrested by enhanced expression of PU.1-dependent genes (22). To identify the effect of NDRG2 expression during PMA-induced differentiation in U937 cells, U937-mock and U937-NDRG2 cells were treated with PMA for 48 h. Ectopic expression of NDRG2 was detected only in the cytoplasm of U937 cells (Fig. 1A). After treatment with PMA for 48 h, U937-mock cells attached to culture plate, but U937-NDRG2 cells showed comparatively reduced attachment (data not shown). A trypan blue exclusion assay revealed that both U937-mock and U937-NDRG2 cells showed no significant differences in cell proliferation. However, U937-mock cells did not proliferate more in response to PMA. Interestingly, U937-NDRG2 cells continued to proliferate during PMA-induced differentiation into macrophages (Fig. 1B). In addition, it was also confirmed using a CCK-8 assay that U937-NDRG2 cells maintained a higher level of proliferation than U937-mock cells during PMA-induced differentiation (Fig. 1C). Interestingly, NDRG2 expression significantly inhibited PU.1 expression in U937 cells although PU.1 levels were also weakly increased in U937-NDRG2 cells after treatment with PMA (Fig. 1D). Consistent with these results, CSF-1R expression, which is regulated by PU.1, was not observed in U937-NDRG2 cells after treatment with PMA. Accordingly, these data demonstrate that NDRG2 hinders the differentiation of U937 premonocytes into macrophages by PMA stimulation and promotes continued cell growth through the suppression of PU.1 expression.


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

Kang K, Jung H, Nam S, Lim JS - Immune Netw (2011)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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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.
Mentions: U937 cells differentiate into monocyte-like cells upon treatment with phorbol 12-myristate 13-acetate (PMA), and their growth is arrested by enhanced expression of PU.1-dependent genes (22). To identify the effect of NDRG2 expression during PMA-induced differentiation in U937 cells, U937-mock and U937-NDRG2 cells were treated with PMA for 48 h. Ectopic expression of NDRG2 was detected only in the cytoplasm of U937 cells (Fig. 1A). After treatment with PMA for 48 h, U937-mock cells attached to culture plate, but U937-NDRG2 cells showed comparatively reduced attachment (data not shown). A trypan blue exclusion assay revealed that both U937-mock and U937-NDRG2 cells showed no significant differences in cell proliferation. However, U937-mock cells did not proliferate more in response to PMA. Interestingly, U937-NDRG2 cells continued to proliferate during PMA-induced differentiation into macrophages (Fig. 1B). In addition, it was also confirmed using a CCK-8 assay that U937-NDRG2 cells maintained a higher level of proliferation than U937-mock cells during PMA-induced differentiation (Fig. 1C). Interestingly, NDRG2 expression significantly inhibited PU.1 expression in U937 cells although PU.1 levels were also weakly increased in U937-NDRG2 cells after treatment with PMA (Fig. 1D). Consistent with these results, CSF-1R expression, which is regulated by PU.1, was not observed in U937-NDRG2 cells after treatment with PMA. Accordingly, these data demonstrate that NDRG2 hinders the differentiation of U937 premonocytes into macrophages by PMA stimulation and promotes continued cell growth through the suppression of PU.1 expression.

Bottom Line: In this study, we investigated the effects of NDRG2 overexpression on GATA-1 expression in PMA-stimulated U937 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and the Research Center for Women's Disease, Sookmyung Women's University, Seoul 140-742, Korea.

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.

No MeSH data available.