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Regulation of cell death in mitotic neural progenitor cells by asymmetric distribution of prostate apoptosis response 4 (PAR-4) and simultaneous elevation of endogenous ceramide.

Bieberich E, MacKinnon S, Silva J, Noggle S, Condie BG - J. Cell Biol. (2003)

Bottom Line: Morpholino oligonucleotide-mediated antisense knockdown of PAR-4 or inhibition of ceramide biosynthesis reduced stem cell apoptosis, whereas PAR-4 overexpression and treatment with ceramide analogs elevated apoptosis.In mitotic cells, asymmetric distribution of PAR-4 and nestin resulted in one nestin(-)/PAR-4(+) daughter cell, in which ceramide elevation induced apoptosis.Asymmetric distribution of PAR-4 and simultaneous elevation of endogenous ceramide provides a possible mechanism underlying asymmetric differentiation and apoptosis of neuronal stem cells in the developing brain.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, Room CB-2803, Augusta, GA 30912, USA. ebieberich@mail.mcg.edu

ABSTRACT
Cell death and survival of neural progenitor (NP) cells are determined by signals that are largely unknown. We have analyzed pro-apoptotic signaling in individual NP cells that have been derived from mouse embryonic stem cells. NP formation was concomitant with elevated apoptosis and increased expression of ceramide and prostate apoptosis response 4 (PAR-4). Morpholino oligonucleotide-mediated antisense knockdown of PAR-4 or inhibition of ceramide biosynthesis reduced stem cell apoptosis, whereas PAR-4 overexpression and treatment with ceramide analogs elevated apoptosis. Apoptotic cells also stained for proliferating cell nuclear antigen (a nuclear mitosis marker protein), but not for nestin (a marker for NP cells). In mitotic cells, asymmetric distribution of PAR-4 and nestin resulted in one nestin(-)/PAR-4(+) daughter cell, in which ceramide elevation induced apoptosis. The other cell was nestin(+), but PAR-4(-), and was not apoptotic. Asymmetric distribution of PAR-4 and simultaneous elevation of endogenous ceramide provides a possible mechanism underlying asymmetric differentiation and apoptosis of neuronal stem cells in the developing brain.

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Expression of differentiation markers and pro- or anti-apoptotic proteins in differentiating ES cells. (A) During in vitro neural differentiation of ES-J1 mouse ES cells, protein was extracted from the cells, separated by SDS-PAGE, and blotted onto nitrocellulose. Each lane shows the immunostaining corresponding to 35 μg cell protein. See Fig. 1 for definition of differentiation stages. The protein analysis was performed with five independent differentiation experiments. (B) RNA was isolated from differentiating ES-J1 cells (see Fig. 1 for definition of differentiation stages) and subjected to RT-PCR. SPT1/2, SPT subunit 1 and 2. Each experiment was repeated four times.
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fig6: Expression of differentiation markers and pro- or anti-apoptotic proteins in differentiating ES cells. (A) During in vitro neural differentiation of ES-J1 mouse ES cells, protein was extracted from the cells, separated by SDS-PAGE, and blotted onto nitrocellulose. Each lane shows the immunostaining corresponding to 35 μg cell protein. See Fig. 1 for definition of differentiation stages. The protein analysis was performed with five independent differentiation experiments. (B) RNA was isolated from differentiating ES-J1 cells (see Fig. 1 for definition of differentiation stages) and subjected to RT-PCR. SPT1/2, SPT subunit 1 and 2. Each experiment was repeated four times.

Mentions: To identify the genes involved in the regulation of apoptosis in differentiating ES cells, we have determined the temporal expression pattern of pro- or anti-apoptotic proteins and ceramide biosynthesis/metabolism during EB and NP formation. Fig. 6 A shows that PAR-4 expression was significantly elevated at the EB8 and NP2 stages (Fig. 6 A, lane 1 and lane 2). By the first day of neural differentiation (D1), the PAR-4 level dropped to <20% of that at NP2 (Fig. 6 A, lane 2 and lane 3). The peak time of PAR-4 expression at the NP2 stage was concurrent with caspase 3 activation and increased proliferating cell nuclear antigen (PCNA) levels (Fig. 6 A, lane 2). PCNA is a marker for mitotic cells (Chan et al., 2002), indicating that PAR-4 elevation and caspase 3 activation is predominant at stages with a large number of proliferating stem cells. The expression of PKCζ did not change during neuronal differentiation. PKCζ activity is inhibited by ceramide-enhanced binding of PAR-4, leading to an up-regulation of the caspase 9– and caspase 3–dependent apoptotic pathway (Diaz-Meco et al., 1996; Mattson, 2000). We confirmed the participation of caspase 9 in ceramide-induced apoptosis of differentiating ES cells by the observation that preincubation with the cell-permeable caspase 9 inhibitor peptide LEHD-CHO suppressed apoptosis that was inducible with S18 or natural ceramide isolated from ES cells. Upstream regulators of caspase 3, in particular, anti-apoptotic b-cell lymphoma 2 (Bcl-2) and pro-apoptotic Bcl-2 antagonist of death (Bad) were inversely regulated, suggesting activation of caspase 3 via the mitochondrial death pathway (Fig. 6 A). However, a high degree of Bad phosphorylation was detectable at NP2 (Fig. 6 A, lane 2) indicating the presence of both apoptotic and anti-apoptotic signaling at this stage. The expression of Bad dropped and that of Bcl-2 increased during D1 and D4, consistent with lower levels of caspase 3 activation and apoptosis at these differentiation stages (Fig. 6 A, lane 3 and lane 4).


Regulation of cell death in mitotic neural progenitor cells by asymmetric distribution of prostate apoptosis response 4 (PAR-4) and simultaneous elevation of endogenous ceramide.

Bieberich E, MacKinnon S, Silva J, Noggle S, Condie BG - J. Cell Biol. (2003)

Expression of differentiation markers and pro- or anti-apoptotic proteins in differentiating ES cells. (A) During in vitro neural differentiation of ES-J1 mouse ES cells, protein was extracted from the cells, separated by SDS-PAGE, and blotted onto nitrocellulose. Each lane shows the immunostaining corresponding to 35 μg cell protein. See Fig. 1 for definition of differentiation stages. The protein analysis was performed with five independent differentiation experiments. (B) RNA was isolated from differentiating ES-J1 cells (see Fig. 1 for definition of differentiation stages) and subjected to RT-PCR. SPT1/2, SPT subunit 1 and 2. Each experiment was repeated four times.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172704&req=5

fig6: Expression of differentiation markers and pro- or anti-apoptotic proteins in differentiating ES cells. (A) During in vitro neural differentiation of ES-J1 mouse ES cells, protein was extracted from the cells, separated by SDS-PAGE, and blotted onto nitrocellulose. Each lane shows the immunostaining corresponding to 35 μg cell protein. See Fig. 1 for definition of differentiation stages. The protein analysis was performed with five independent differentiation experiments. (B) RNA was isolated from differentiating ES-J1 cells (see Fig. 1 for definition of differentiation stages) and subjected to RT-PCR. SPT1/2, SPT subunit 1 and 2. Each experiment was repeated four times.
Mentions: To identify the genes involved in the regulation of apoptosis in differentiating ES cells, we have determined the temporal expression pattern of pro- or anti-apoptotic proteins and ceramide biosynthesis/metabolism during EB and NP formation. Fig. 6 A shows that PAR-4 expression was significantly elevated at the EB8 and NP2 stages (Fig. 6 A, lane 1 and lane 2). By the first day of neural differentiation (D1), the PAR-4 level dropped to <20% of that at NP2 (Fig. 6 A, lane 2 and lane 3). The peak time of PAR-4 expression at the NP2 stage was concurrent with caspase 3 activation and increased proliferating cell nuclear antigen (PCNA) levels (Fig. 6 A, lane 2). PCNA is a marker for mitotic cells (Chan et al., 2002), indicating that PAR-4 elevation and caspase 3 activation is predominant at stages with a large number of proliferating stem cells. The expression of PKCζ did not change during neuronal differentiation. PKCζ activity is inhibited by ceramide-enhanced binding of PAR-4, leading to an up-regulation of the caspase 9– and caspase 3–dependent apoptotic pathway (Diaz-Meco et al., 1996; Mattson, 2000). We confirmed the participation of caspase 9 in ceramide-induced apoptosis of differentiating ES cells by the observation that preincubation with the cell-permeable caspase 9 inhibitor peptide LEHD-CHO suppressed apoptosis that was inducible with S18 or natural ceramide isolated from ES cells. Upstream regulators of caspase 3, in particular, anti-apoptotic b-cell lymphoma 2 (Bcl-2) and pro-apoptotic Bcl-2 antagonist of death (Bad) were inversely regulated, suggesting activation of caspase 3 via the mitochondrial death pathway (Fig. 6 A). However, a high degree of Bad phosphorylation was detectable at NP2 (Fig. 6 A, lane 2) indicating the presence of both apoptotic and anti-apoptotic signaling at this stage. The expression of Bad dropped and that of Bcl-2 increased during D1 and D4, consistent with lower levels of caspase 3 activation and apoptosis at these differentiation stages (Fig. 6 A, lane 3 and lane 4).

Bottom Line: Morpholino oligonucleotide-mediated antisense knockdown of PAR-4 or inhibition of ceramide biosynthesis reduced stem cell apoptosis, whereas PAR-4 overexpression and treatment with ceramide analogs elevated apoptosis.In mitotic cells, asymmetric distribution of PAR-4 and nestin resulted in one nestin(-)/PAR-4(+) daughter cell, in which ceramide elevation induced apoptosis.Asymmetric distribution of PAR-4 and simultaneous elevation of endogenous ceramide provides a possible mechanism underlying asymmetric differentiation and apoptosis of neuronal stem cells in the developing brain.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, Room CB-2803, Augusta, GA 30912, USA. ebieberich@mail.mcg.edu

ABSTRACT
Cell death and survival of neural progenitor (NP) cells are determined by signals that are largely unknown. We have analyzed pro-apoptotic signaling in individual NP cells that have been derived from mouse embryonic stem cells. NP formation was concomitant with elevated apoptosis and increased expression of ceramide and prostate apoptosis response 4 (PAR-4). Morpholino oligonucleotide-mediated antisense knockdown of PAR-4 or inhibition of ceramide biosynthesis reduced stem cell apoptosis, whereas PAR-4 overexpression and treatment with ceramide analogs elevated apoptosis. Apoptotic cells also stained for proliferating cell nuclear antigen (a nuclear mitosis marker protein), but not for nestin (a marker for NP cells). In mitotic cells, asymmetric distribution of PAR-4 and nestin resulted in one nestin(-)/PAR-4(+) daughter cell, in which ceramide elevation induced apoptosis. The other cell was nestin(+), but PAR-4(-), and was not apoptotic. Asymmetric distribution of PAR-4 and simultaneous elevation of endogenous ceramide provides a possible mechanism underlying asymmetric differentiation and apoptosis of neuronal stem cells in the developing brain.

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