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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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p21WAF1 prevents the inhibition of differentiation mediated by NAB2 overexpression. PC12 cells were cotransfected  with a lacZ reporter plasmid and either NAB2, NAB2 +  p21WAF1, NAB2 + Egr4, or non-recombinant vector, as indicated.  The histogram shows percentages of lacZ-positive cells that remain undifferentiated after treatment with NGF for 3 d beginning 2 d after transfection. For each condition, >100 lacZ-positive cells were counted. Cells with neurites longer than two cell  body diameters were scored as morphologically differentiated.  The data is derived from four independent transfections. Error  bars indicate the standard deviation.
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Figure 8: p21WAF1 prevents the inhibition of differentiation mediated by NAB2 overexpression. PC12 cells were cotransfected with a lacZ reporter plasmid and either NAB2, NAB2 + p21WAF1, NAB2 + Egr4, or non-recombinant vector, as indicated. The histogram shows percentages of lacZ-positive cells that remain undifferentiated after treatment with NGF for 3 d beginning 2 d after transfection. For each condition, >100 lacZ-positive cells were counted. Cells with neurites longer than two cell body diameters were scored as morphologically differentiated. The data is derived from four independent transfections. Error bars indicate the standard deviation.

Mentions: To address the functional significance of p21WAF1 downregulation, PC12 cells were transiently transfected with NAB2 in the absence or presence of p21WAF1. As previously observed, transfection of NAB2 alone resulted in dramatic inhibition of NGF-mediated differentiation (71% undifferentiated cells), however cotransfection of a p21WAF1 expression construct along with NAB2 largely restored the ability to differentiate in response to NGF (24% undifferentiated cells) (Fig. 8). In comparison, cotransfection of Egr4 (which lacks an R1 domain) along with NAB2 was not able to restore the ability to differentiate in response to NGF (70% undifferentiated cells). Transfection with non-recombinant or p21WAF1 vector alone resulted in a minimal number of undifferentiated cells (10% and 9.8%, respectively). It has been shown that p21WAF1 induction by itself does not lead to PC12 cell differentiation in the absence of NGF (Yan and Ziff, 1995). These results clearly suggest that p21WAF1 prevents NAB2-mediated inhibition of PC12 cell differentiation, consistent with previous work indicating that NGF-induced expression of p21WAF1 is critical for exit from the cell cycle and subsequent differentiation of PC12 cells (Yan and Ziff, 1995; Billon, 1996; Erhardt and Pittman, 1998).


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)

p21WAF1 prevents the inhibition of differentiation mediated by NAB2 overexpression. PC12 cells were cotransfected  with a lacZ reporter plasmid and either NAB2, NAB2 +  p21WAF1, NAB2 + Egr4, or non-recombinant vector, as indicated.  The histogram shows percentages of lacZ-positive cells that remain undifferentiated after treatment with NGF for 3 d beginning 2 d after transfection. For each condition, >100 lacZ-positive cells were counted. Cells with neurites longer than two cell  body diameters were scored as morphologically differentiated.  The data is derived from four independent transfections. Error  bars indicate the standard deviation.
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Related In: Results  -  Collection

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Figure 8: p21WAF1 prevents the inhibition of differentiation mediated by NAB2 overexpression. PC12 cells were cotransfected with a lacZ reporter plasmid and either NAB2, NAB2 + p21WAF1, NAB2 + Egr4, or non-recombinant vector, as indicated. The histogram shows percentages of lacZ-positive cells that remain undifferentiated after treatment with NGF for 3 d beginning 2 d after transfection. For each condition, >100 lacZ-positive cells were counted. Cells with neurites longer than two cell body diameters were scored as morphologically differentiated. The data is derived from four independent transfections. Error bars indicate the standard deviation.
Mentions: To address the functional significance of p21WAF1 downregulation, PC12 cells were transiently transfected with NAB2 in the absence or presence of p21WAF1. As previously observed, transfection of NAB2 alone resulted in dramatic inhibition of NGF-mediated differentiation (71% undifferentiated cells), however cotransfection of a p21WAF1 expression construct along with NAB2 largely restored the ability to differentiate in response to NGF (24% undifferentiated cells) (Fig. 8). In comparison, cotransfection of Egr4 (which lacks an R1 domain) along with NAB2 was not able to restore the ability to differentiate in response to NGF (70% undifferentiated cells). Transfection with non-recombinant or p21WAF1 vector alone resulted in a minimal number of undifferentiated cells (10% and 9.8%, respectively). It has been shown that p21WAF1 induction by itself does not lead to PC12 cell differentiation in the absence of NGF (Yan and Ziff, 1995). These results clearly suggest that p21WAF1 prevents NAB2-mediated inhibition of PC12 cell differentiation, consistent with previous work indicating that NGF-induced expression of p21WAF1 is critical for exit from the cell cycle and subsequent differentiation of PC12 cells (Yan and Ziff, 1995; Billon, 1996; Erhardt and Pittman, 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.

Show MeSH
Related in: MedlinePlus