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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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NAB2 overexpression results in decreased levels of  p21WAF1 protein. Wild-type PC12 cells, NAB2-overexpressing  cell lines (N2-61, N2-63, N2-64, and N2-8), and a cell line transfected with non-recombinant vector (Neo4) were incubated in the  absence or presence of NGF for 48 h. Protein lysates were electrophoresed on a SDS–polyacrylamide gel, blotted onto nitrocellulose, and then probed with an anti-p21WAF1 antibody (top  panel). The same blot was stripped and re-probed with antibodies  against cyclin D1 (middle panel) or p27Kip1 (bottom panel).
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Figure 7: NAB2 overexpression results in decreased levels of p21WAF1 protein. Wild-type PC12 cells, NAB2-overexpressing cell lines (N2-61, N2-63, N2-64, and N2-8), and a cell line transfected with non-recombinant vector (Neo4) were incubated in the absence or presence of NGF for 48 h. Protein lysates were electrophoresed on a SDS–polyacrylamide gel, blotted onto nitrocellulose, and then probed with an anti-p21WAF1 antibody (top panel). The same blot was stripped and re-probed with antibodies against cyclin D1 (middle panel) or p27Kip1 (bottom panel).

Mentions: Immunoblot analyses of p21WAF1 levels before and after NGF treatment in wild-type PC12 cells clearly showed an increase after 48 h. However, in NAB2-overexpressing cell lines, p21WAF1 levels were dramatically downregulated (Fig. 7). Not only the induced level of p21WAF1 expression, but also the basal level (in the absence of NGF) was strongly decreased in NAB2-overexpressing cell lines. This effect on p21WAF1 levels was specific, as the levels of another cdi, p27Kip1, and cyclin D1 were unaffected.


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)

NAB2 overexpression results in decreased levels of  p21WAF1 protein. Wild-type PC12 cells, NAB2-overexpressing  cell lines (N2-61, N2-63, N2-64, and N2-8), and a cell line transfected with non-recombinant vector (Neo4) were incubated in the  absence or presence of NGF for 48 h. Protein lysates were electrophoresed on a SDS–polyacrylamide gel, blotted onto nitrocellulose, and then probed with an anti-p21WAF1 antibody (top  panel). The same blot was stripped and re-probed with antibodies  against cyclin D1 (middle panel) or p27Kip1 (bottom panel).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132876&req=5

Figure 7: NAB2 overexpression results in decreased levels of p21WAF1 protein. Wild-type PC12 cells, NAB2-overexpressing cell lines (N2-61, N2-63, N2-64, and N2-8), and a cell line transfected with non-recombinant vector (Neo4) were incubated in the absence or presence of NGF for 48 h. Protein lysates were electrophoresed on a SDS–polyacrylamide gel, blotted onto nitrocellulose, and then probed with an anti-p21WAF1 antibody (top panel). The same blot was stripped and re-probed with antibodies against cyclin D1 (middle panel) or p27Kip1 (bottom panel).
Mentions: Immunoblot analyses of p21WAF1 levels before and after NGF treatment in wild-type PC12 cells clearly showed an increase after 48 h. However, in NAB2-overexpressing cell lines, p21WAF1 levels were dramatically downregulated (Fig. 7). Not only the induced level of p21WAF1 expression, but also the basal level (in the absence of NGF) was strongly decreased in NAB2-overexpressing cell lines. This effect on p21WAF1 levels was specific, as the levels of another cdi, p27Kip1, and cyclin D1 were unaffected.

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.

Show MeSH
Related in: MedlinePlus