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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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NGF-mediated MAP kinase activation and immediate-early gene induction are intact in NAB2-overexpressing PC12  cells. (A) Total protein was collected from wild-type PC12 cells  and two NAB2 stably transfected cell lines, N2-8 and N2-64,  which had been treated with NGF for the indicated times. The  samples were blotted with an antibody against phosphorylated  MAP kinase (α-P-MAPK). The blots were stripped and re-probed with anti–MAP kinase antibody (α-MAPK) to show that  equal amounts of protein were loaded in each lane. (B) 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 Egr1 probe. The  18S ribosomal RNA band visualized with ethidium bromide is  shown below to demonstrate equal loading.
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Figure 5: NGF-mediated MAP kinase activation and immediate-early gene induction are intact in NAB2-overexpressing PC12 cells. (A) Total protein was collected from wild-type PC12 cells and two NAB2 stably transfected cell lines, N2-8 and N2-64, which had been treated with NGF for the indicated times. The samples were blotted with an antibody against phosphorylated MAP kinase (α-P-MAPK). The blots were stripped and re-probed with anti–MAP kinase antibody (α-MAPK) to show that equal amounts of protein were loaded in each lane. (B) 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 Egr1 probe. The 18S ribosomal RNA band visualized with ethidium bromide is shown below to demonstrate equal loading.

Mentions: Some of the earliest responses of PC12 cells to NGF include dimerization and autophosphorylation of the Trk A receptor and sustained activation of Ras-dependent MAPK (Szeberenyi, 1996). To determine whether the signal transduction pathway itself might be blocked by NAB2 overexpression, we first examined MAPK activation in NAB2-overexpressing cell lines after exposure to NGF. A Western blot with a phospho-specific MAP kinase antibody showed that the sustained activation of MAPK normally observed in NGF-treated PC12 cells is unaltered in the NAB2-overexpressing lines (Fig. 5 A). We also examined the NGF activation of the immediate-early gene Egr1 in these cells and found that Egr1 mRNA levels were elevated with the same time course in wild-type PC12 cells and those overexpressing NAB2 (Fig. 5 B). Together these data indicate that NAB2 interferes with NGF-induced PC12 cell differentiation at a step downstream of signal transduction and immediate-early gene induction.


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)

NGF-mediated MAP kinase activation and immediate-early gene induction are intact in NAB2-overexpressing PC12  cells. (A) Total protein was collected from wild-type PC12 cells  and two NAB2 stably transfected cell lines, N2-8 and N2-64,  which had been treated with NGF for the indicated times. The  samples were blotted with an antibody against phosphorylated  MAP kinase (α-P-MAPK). The blots were stripped and re-probed with anti–MAP kinase antibody (α-MAPK) to show that  equal amounts of protein were loaded in each lane. (B) 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 Egr1 probe. The  18S ribosomal RNA band visualized with ethidium bromide is  shown below to demonstrate equal loading.
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Related In: Results  -  Collection

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Figure 5: NGF-mediated MAP kinase activation and immediate-early gene induction are intact in NAB2-overexpressing PC12 cells. (A) Total protein was collected from wild-type PC12 cells and two NAB2 stably transfected cell lines, N2-8 and N2-64, which had been treated with NGF for the indicated times. The samples were blotted with an antibody against phosphorylated MAP kinase (α-P-MAPK). The blots were stripped and re-probed with anti–MAP kinase antibody (α-MAPK) to show that equal amounts of protein were loaded in each lane. (B) 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 Egr1 probe. The 18S ribosomal RNA band visualized with ethidium bromide is shown below to demonstrate equal loading.
Mentions: Some of the earliest responses of PC12 cells to NGF include dimerization and autophosphorylation of the Trk A receptor and sustained activation of Ras-dependent MAPK (Szeberenyi, 1996). To determine whether the signal transduction pathway itself might be blocked by NAB2 overexpression, we first examined MAPK activation in NAB2-overexpressing cell lines after exposure to NGF. A Western blot with a phospho-specific MAP kinase antibody showed that the sustained activation of MAPK normally observed in NGF-treated PC12 cells is unaltered in the NAB2-overexpressing lines (Fig. 5 A). We also examined the NGF activation of the immediate-early gene Egr1 in these cells and found that Egr1 mRNA levels were elevated with the same time course in wild-type PC12 cells and those overexpressing NAB2 (Fig. 5 B). Together these data indicate that NAB2 interferes with NGF-induced PC12 cell differentiation at a step downstream of signal transduction and immediate-early gene induction.

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