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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 blocks the induction of NGF-responsive late genes TGF-β1 and MMP-3. RNA was isolated  from wild-type PC12 cells and a NAB2 stably transfected cell line  (N2-8) after stimulation with NGF for the indicated length of  time. RNA blot analysis was performed using 10-μg samples of  total RNA per lane and a 32P-labeled TGF-β1 or MMP-3 probe  as indicated. The 18S ribosomal RNA band visualized with ethidium bromide is shown below to demonstrate equal loading.
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Figure 6: Overexpression of NAB2 blocks the induction of NGF-responsive late genes TGF-β1 and MMP-3. RNA was isolated from wild-type PC12 cells and a NAB2 stably transfected cell line (N2-8) after stimulation with NGF for the indicated length of time. RNA blot analysis was performed using 10-μg samples of total RNA per lane and a 32P-labeled TGF-β1 or MMP-3 probe as indicated. The 18S ribosomal RNA band visualized with ethidium bromide is shown below to demonstrate equal loading.

Mentions: In contrast to the immediate-early gene Egr1, there are a number of genes whose induction by NGF is dependent on de novo protein synthesis. NGF activates these genes, which include TGF-β1 and MMP-3 (stromelysin, transin), with a more extended time course. Furthermore, TGF-β1 has been shown to be regulated by Egr1 (Kim et al., 1994; Liu et al., 1996), and MMP-3 has multiple Egr1 consensus binding elements in its promoter region (Yee et al., 1997). To determine whether the expression of these genes was altered by NAB2 overexpression, we stimulated wild-type and NAB2-overexpressing PC12 cells with NGF and compared the induction of these genes by RNA blot analysis (Fig. 6). In accord with previous reports, both of these genes were induced in wild-type PC12 cells, but their induction was abolished in NAB2-overexpressing cells. These results suggest that NAB2 overexpression is inhibiting Egr1-mediated gene activation.


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 blocks the induction of NGF-responsive late genes TGF-β1 and MMP-3. RNA was isolated  from wild-type PC12 cells and a NAB2 stably transfected cell line  (N2-8) after stimulation with NGF for the indicated length of  time. RNA blot analysis was performed using 10-μg samples of  total RNA per lane and a 32P-labeled TGF-β1 or MMP-3 probe  as indicated. The 18S ribosomal RNA band visualized with ethidium bromide is shown below to demonstrate equal loading.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Overexpression of NAB2 blocks the induction of NGF-responsive late genes TGF-β1 and MMP-3. RNA was isolated from wild-type PC12 cells and a NAB2 stably transfected cell line (N2-8) after stimulation with NGF for the indicated length of time. RNA blot analysis was performed using 10-μg samples of total RNA per lane and a 32P-labeled TGF-β1 or MMP-3 probe as indicated. The 18S ribosomal RNA band visualized with ethidium bromide is shown below to demonstrate equal loading.
Mentions: In contrast to the immediate-early gene Egr1, there are a number of genes whose induction by NGF is dependent on de novo protein synthesis. NGF activates these genes, which include TGF-β1 and MMP-3 (stromelysin, transin), with a more extended time course. Furthermore, TGF-β1 has been shown to be regulated by Egr1 (Kim et al., 1994; Liu et al., 1996), and MMP-3 has multiple Egr1 consensus binding elements in its promoter region (Yee et al., 1997). To determine whether the expression of these genes was altered by NAB2 overexpression, we stimulated wild-type and NAB2-overexpressing PC12 cells with NGF and compared the induction of these genes by RNA blot analysis (Fig. 6). In accord with previous reports, both of these genes were induced in wild-type PC12 cells, but their induction was abolished in NAB2-overexpressing cells. These results suggest that NAB2 overexpression is inhibiting Egr1-mediated gene activation.

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