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The transcriptional corepressor NAB2 inhibits NGF-induced differentiation of PC12 cells.

Qu Z, Wolfraim LA, Svaren J, Ehrengruber MU, Davidson N, Milbrandt J - J. Cell Biol. (1998)

Bottom Line: We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF.Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection.Cotransfection with p21(WAF1) restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The PC12 pheochromocytoma cell line responds to NGF by undergoing growth arrest and proceeding to differentiate toward a neuronal phenotype. Among the early genetic events triggered by NGF in PC12 cells are the rapid activation of the zinc finger transcription factor Egr1/NGFI-A, and a slightly delayed induction of NAB2, a corepressor that inhibits Egr1 transcriptional activity. We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF. Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection. Early events in the NGF signaling cascade, such as activation of MAP kinase and induction of immediate-early genes, were unaltered in the NAB2-overexpressing PC12 cell lines. However, induction of delayed NGF response genes such as TGF-beta1 and MMP-3 was inhibited. Furthermore, NAB2 overexpression led to downregulation of p21(WAF1), a molecule previously shown to play a pivotal role in the ability of PC12 cells to undergo growth arrest and commit to differentiation in response to NGF. Cotransfection with p21(WAF1) restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF.

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Overexpression of NAB2 in PC12 cell stable transfectants. (A) Total protein was isolated from wild-type PC12 cells,  cell lines stably transfected with NAB2 (N2-8, N2-61, N2-63, N2-64), and a cell line transfected with non-recombinant vector  (Neo4) grown in the presence or absence of NGF for 1 h. Protein  lysates were electrophoresed on an SDS–polyacrylamide gel,  blotted onto nitrocellulose, and then probed with an mAb directed against NAB2. To ensure equal loading of samples, the  blot was stripped and re-probed with a p44/42 MAPK antibody.  (B) Wild-type PC12 cells and a NAB2 stably transfected cell line,  N2-8, were grown on chamber slides. The cells were either  treated with NGF for 1 h or left untreated. Immunocytochemical  analysis was performed using an mAb directed against NAB2.
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Figure 1: Overexpression of NAB2 in PC12 cell stable transfectants. (A) Total protein was isolated from wild-type PC12 cells, cell lines stably transfected with NAB2 (N2-8, N2-61, N2-63, N2-64), and a cell line transfected with non-recombinant vector (Neo4) grown in the presence or absence of NGF for 1 h. Protein lysates were electrophoresed on an SDS–polyacrylamide gel, blotted onto nitrocellulose, and then probed with an mAb directed against NAB2. To ensure equal loading of samples, the blot was stripped and re-probed with a p44/42 MAPK antibody. (B) Wild-type PC12 cells and a NAB2 stably transfected cell line, N2-8, were grown on chamber slides. The cells were either treated with NGF for 1 h or left untreated. Immunocytochemical analysis was performed using an mAb directed against NAB2.

Mentions: The expression of the Egr1 transactivator is rapidly, but transiently, induced as PC12 cells are stimulated to differentiate in response to NGF. The corepressor NAB2, which inhibits Egr1 activity, is also transiently induced by NGF in these cells, although induction of NAB2 is slightly delayed relative to that of Egr1 (Svaren et al., 1996). To investigate the consequences of altering Egr1 activity on PC12 cell physiology, we attempted to generate PC12 clones that overexpress Egr1 but were unable to do so, presumably because Egr1 represses growth either by promoting apoptosis or by direct inhibition of proliferation (Muthukkumar et al., 1995; Liu et al., 1996). However, we were successful in generating PC12 cell lines that constitutively overexpress its corepressor, NAB2. Immunoblot analysis with a NAB2 mAb (Kirsch et al., 1996) showed that in wild-type PC12 cells, the level of NAB2 is dramatically increased within 1 h after NGF treatment. In contrast, PC12 cell lines stably transfected with the CMV-NAB2 expression vector (N2-8, N2-61, N2-63, N2-64) express high levels of NAB2 in the absence of NGF (Fig. 1 A). NAB2 migrates as a doublet, reflecting different levels of phosphorylation (Sevetson, B., and J. Milbrandt, unpublished data). Immunocytochemical analysis with the anti-NAB2 mAb revealed that NAB2-overexpressing cell lines exhibit intense nuclear staining without NGF stimulation. There is also a marked increase in nuclear staining 1 h after NGF addition, reflecting induced expression of the endogenous NAB2 gene (Fig. 1 B).


The transcriptional corepressor NAB2 inhibits NGF-induced differentiation of PC12 cells.

Qu Z, Wolfraim LA, Svaren J, Ehrengruber MU, Davidson N, Milbrandt J - J. Cell Biol. (1998)

Overexpression of NAB2 in PC12 cell stable transfectants. (A) Total protein was isolated from wild-type PC12 cells,  cell lines stably transfected with NAB2 (N2-8, N2-61, N2-63, N2-64), and a cell line transfected with non-recombinant vector  (Neo4) grown in the presence or absence of NGF for 1 h. Protein  lysates were electrophoresed on an SDS–polyacrylamide gel,  blotted onto nitrocellulose, and then probed with an mAb directed against NAB2. To ensure equal loading of samples, the  blot was stripped and re-probed with a p44/42 MAPK antibody.  (B) Wild-type PC12 cells and a NAB2 stably transfected cell line,  N2-8, were grown on chamber slides. The cells were either  treated with NGF for 1 h or left untreated. Immunocytochemical  analysis was performed using an mAb directed against NAB2.
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Figure 1: Overexpression of NAB2 in PC12 cell stable transfectants. (A) Total protein was isolated from wild-type PC12 cells, cell lines stably transfected with NAB2 (N2-8, N2-61, N2-63, N2-64), and a cell line transfected with non-recombinant vector (Neo4) grown in the presence or absence of NGF for 1 h. Protein lysates were electrophoresed on an SDS–polyacrylamide gel, blotted onto nitrocellulose, and then probed with an mAb directed against NAB2. To ensure equal loading of samples, the blot was stripped and re-probed with a p44/42 MAPK antibody. (B) Wild-type PC12 cells and a NAB2 stably transfected cell line, N2-8, were grown on chamber slides. The cells were either treated with NGF for 1 h or left untreated. Immunocytochemical analysis was performed using an mAb directed against NAB2.
Mentions: The expression of the Egr1 transactivator is rapidly, but transiently, induced as PC12 cells are stimulated to differentiate in response to NGF. The corepressor NAB2, which inhibits Egr1 activity, is also transiently induced by NGF in these cells, although induction of NAB2 is slightly delayed relative to that of Egr1 (Svaren et al., 1996). To investigate the consequences of altering Egr1 activity on PC12 cell physiology, we attempted to generate PC12 clones that overexpress Egr1 but were unable to do so, presumably because Egr1 represses growth either by promoting apoptosis or by direct inhibition of proliferation (Muthukkumar et al., 1995; Liu et al., 1996). However, we were successful in generating PC12 cell lines that constitutively overexpress its corepressor, NAB2. Immunoblot analysis with a NAB2 mAb (Kirsch et al., 1996) showed that in wild-type PC12 cells, the level of NAB2 is dramatically increased within 1 h after NGF treatment. In contrast, PC12 cell lines stably transfected with the CMV-NAB2 expression vector (N2-8, N2-61, N2-63, N2-64) express high levels of NAB2 in the absence of NGF (Fig. 1 A). NAB2 migrates as a doublet, reflecting different levels of phosphorylation (Sevetson, B., and J. Milbrandt, unpublished data). Immunocytochemical analysis with the anti-NAB2 mAb revealed that NAB2-overexpressing cell lines exhibit intense nuclear staining without NGF stimulation. There is also a marked increase in nuclear staining 1 h after NGF addition, reflecting induced expression of the endogenous NAB2 gene (Fig. 1 B).

Bottom Line: We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF.Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection.Cotransfection with p21(WAF1) restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The PC12 pheochromocytoma cell line responds to NGF by undergoing growth arrest and proceeding to differentiate toward a neuronal phenotype. Among the early genetic events triggered by NGF in PC12 cells are the rapid activation of the zinc finger transcription factor Egr1/NGFI-A, and a slightly delayed induction of NAB2, a corepressor that inhibits Egr1 transcriptional activity. We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF. Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection. Early events in the NGF signaling cascade, such as activation of MAP kinase and induction of immediate-early genes, were unaltered in the NAB2-overexpressing PC12 cell lines. However, induction of delayed NGF response genes such as TGF-beta1 and MMP-3 was inhibited. Furthermore, NAB2 overexpression led to downregulation of p21(WAF1), a molecule previously shown to play a pivotal role in the ability of PC12 cells to undergo growth arrest and commit to differentiation in response to NGF. Cotransfection with p21(WAF1) restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF.

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Related in: MedlinePlus