Regulation of the Retinoblastoma-Dependent Mdm2 and E2F-1 Signaling Pathways during Neuronal Apoptosis

doi:10.1006/mcne.2000.0928

Molecular and Cellular Neuroscience

Volume 17, Issue 2, February 2001, Pages 342-353

https://doi.org/10.1006/mcne.2000.0928 Get rights and content

Abstract

We have previously demonstrated that the apoptotic signaling pathway in K⁺-deprived cerebellar granule neurons involves a caspase-dependent cleavage of the retinoblastoma protein (Rb). Here, we have further investigated the functional consequences of this cleavage on two Rb-binding partners: the oncoprotein Mdm2 and the transcription factor E2F-1. A K⁺ deprivation time course leads to a caspase inhibitor-sensitive degradation of Mdm2. Experimental blockade of Mdm2 expression with antisense oligodeoxynucleotides (ODN) results in neuronal death, suggesting an active role of Mdm2 in neuroprotection. By contrast, the E2F-1 protein accumulates in a caspase-independent manner following K⁺ withdrawal, a consequence of increased gene transcription. This is likely to result from the rapid cyclin-dependent kinase 4 activation observed in LK, that correlates with a transient Rb phosphorylation. Counteracting E2F-1 upregulation with antisense ODNs prevents neuronal loss. Taken together, these data demonstrate that Rb is a central player in regulating both caspase-dependent and -independent events leading to apoptosis.

References (70)

M.C. Athanasiou et al.
The transcription factor E2F-1 in SV40 T antigen-induced cerebellar Purkinje cell degeneration
Mol. Cell. Neurosci.
(1998)
M. Azuma-Hara et al.
Regulation and deregulation of E2F1 in postmitotic neurons differentiated from embryonal carcinoma P19 cells
Exp. Cell Res.
(1999)
L. Chen et al.
Proteolytic cleavage of the mdm2 oncoprotein during apoptosis
J. Biol. Chem.
(1997)
P. Erhardt et al.
Identification of the MDM2 oncoprotein as a substrate for CPP32-like apoptotic proteases
J. Biol. Chem.
(1997)
R.S. Freeman et al.
Analysis of cell cycle-related gene expression in postmitotic neurons: Selective induction of Cyclin D1 during programmed cell death
Neuron
(1994)
A. Giovanni et al.
E2F1 mediates death of B-amyloid-treated cortical neurons in a manner independent of p53 and dependent on bax and caspase 3 [In Process Citation]
J. Biol. Chem.
(2000)
J.F. Goetschy et al.
Fibronectin and collagens modulate the proliferation and morphology of astroglial cells in culture
Int. J. Dev. Neurosci.
(1987)
N. Heintz
Cell death and the cell cycle: A relationship between transformation and neurodegeneration?
Trends Biochem. Sci.
(1993)
R.U. Janicke et al.
Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis
J. Biol. Chem.
(1998)
C. Lemaire et al.
Inhibition of caspase activity induces a switch from apoptosis to necrosis
FEBS Lett.
(1998)

X. Liu et al.

DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis

Cell

(1997)

K. Macleod

pRb and E2f-1 in mouse development and tumorigenesis

Curr. Opin. Genet. Dev.

(1999)

J. Momand et al.

The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation

Cell

(1992)

M.J. O'Hare et al.

Induction and modulation of cerebellar granule neuron death by E2F-1

J. Biol. Chem.

(2000)

W.M. Ongkeko et al.

MDM2 and MDMX bind and stabilize the p53-related protein p73

Curr. Biol.

(1999)

C. Prives

Signaling to p53: Breaking the MDM2-p53 circuit

Cell

(1998)

G. Teoh et al.

MDM2 protein overexpression promotes proliferation and survival of multiple myeloma cells

Blood

(1997)

K.Y. Tsai et al.

Mutation of E2f-1 suppresses apoptosis and inappropriate S phase entry and extends survival of Rb-deficient mouse embryos

Mol. Cell

(1998)

E. Balint et al.

Mdm2 binds p73 alpha without targeting degradation

Oncogene

(1999)

O. Boussif et al.

A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine

Proc. Natl. Acad. Sci. USA

(1995)

A.L. Boutillier et al.

Depolarization regulates cyclin D1 degradation and neuronal apoptosis: A hypothesis about the role of the ubiquitin/proteasome signalling pathway

Eur. J. Neurosci.

(1999)

A.L. Boutillier et al.

Caspase-dependent cleavage of the retinoblastoma protein is an early step of neuronal apoptosis

Oncogene

(2000)

N. Canu et al.

Proteasome involvement and accumulation of ubiquitinated proteins in cerebellar granule neurons undergoing apoptosis

J. Neurosci.

(2000)

A. Copani et al.

Mitotic signaling by beta-amyloid causes neuronal death

Faseb J.

(1999)

S.P. Cregan et al.

Bax-dependent caspase-3 activation is a key determinant in p53-induced apoptosis in neurons

J. Neurosci.

(1999)

S.R. D'Mello et al.

A DEVD-inhibited caspase other than CPP32 is involved in the commitment of cerebellar granule neurons to apoptosis induced by K⁺ deprivation

J. Neurochem.

(1998)

S. de Rozieres et al.

The loss of mdm2 induces p53-mediated apoptosis

Oncogene

(2000)

J. DeGregori et al.

Distinct roles for E2F proteins in cell growth control and apoptosis

Proc. Natl. Acad. Sci. USA

(1997)

Q.P. Dou et al.

RB and apoptotic cell death

Front Biosci.

(1998)

N. Dyson

The regulation of E2F by pRB-family proteins

Genes Dev.

(1998)

M. Enari et al.

A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD

Nature

(1998)

S.S. Fakharzadeh et al.

Tumorigenic potential associated with enhanced expression of a gene that is amplified in a mouse tumor cell line

EMBO J.

(1991)

C. Gaiddon et al.

Brain-derived neurotrophic factor exerts opposing effects on beta2-adrenergic receptor according to depolarization status of cerebellar neurons

J. Neurochem.

(1999)

J.S. Gill et al.

Cisplatin-induced apoptosis in rat dorsal root ganglion neurons is associated with attempted entry into the cell cycle

J. Clin. Invest.

(1998)

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Citation Excerpt :
Thus, in vivo data and in vitro data from neuronal cell cultures suggest that re-entry into the cell cycle is a potential step in the neuronal apoptotic cascade. It is widely known that cell division in mammalian cells is regulated by cyclin-dependent kinases (CDKs) (Smits et al., 2001; Nguyen et al., 2002; Verdaguer et al., 2005). In this process of cell cycle re-entry, the phosphorylation of Rb by CDKs is a key step, as unphosphorylated Rb can block cell cycle progression by binding to the transcription factor E2F-1 (Smith et al., 2000).
Recent studies have demonstrated that neuronal reentry in the cell cycle and specifically the expression of the transcription factor E2F-1, constitutes a pathway that may be involved in neuronal apoptosis after serum and potassium withdrawal. Other enzymes such as glycogen synthase kinase-3β (GSK-3β) are also involved in this apoptotic stimulus, and thus in the process of neuronal cell death. Primary cerebellar granule cells (CGNs) were used in this study to determine whether pharmacological inhibition of GSK-3β is involved in neuronal modulation of the cell cycle, and specifically in the regulation of E2F-1 and retinoblastoma protein (Rb). CGNs showed a dramatic increase in GSK-3β activity after 2 h of serum and potassium deprivation. Immunoblot and activity assays revealed that lithium and SB415286 inhibit fully the activation of GSK-3β and attenuate the expression of cyclin D, cyclin E, pRb phosphorylation and the transcription factor E2F-1. These data were confirmed using AR-014418, a selective GSK-3β inhibitor that prevents the expression of cell-cycle proteins. Our data indicate that GSK-3β inhibition regulates, in part, the cell cycle in CGNs by inhibiting Rb phosphorylation and thus inhibiting E2F-1 activity. However, the selective inhibition of GSK-3β with AR-A014418 had not effect on cell viability or apoptosis mediated by S/K withdrawal. Furthermore, our results suggest that selective GSK-3β inhibition is not sufficient to protect against apoptosis in this S/K withdrawal model, indicating that Li⁺ and SB415286 neuroprotective effects are mediated by the inhibition of additional targets to GSK3β. Therefore, there is a connection between cell cycle and GSK-3β activation and that these, along with other mechanisms, are involved in the molecular paths leading to the apoptotic process of rat CGNs triggered by S/K withdrawal.
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Regular ArticleRegulation of the Retinoblastoma-Dependent Mdm2 and E2F-1 Signaling Pathways during Neuronal Apoptosis

Abstract

Mol. Cell. Neurosci.

Exp. Cell Res.

J. Biol. Chem.

J. Biol. Chem.

Neuron

J. Biol. Chem.

Int. J. Dev. Neurosci.

Trends Biochem. Sci.

J. Biol. Chem.

FEBS Lett.

Cell

Curr. Opin. Genet. Dev.

Cell

J. Biol. Chem.

Curr. Biol.

Cell

Blood

Mol. Cell

Mdm2 binds p73 alpha without targeting degradation

Oncogene

A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine

Proc. Natl. Acad. Sci. USA

Depolarization regulates cyclin D1 degradation and neuronal apoptosis: A hypothesis about the role of the ubiquitin/proteasome signalling pathway

Eur. J. Neurosci.

Caspase-dependent cleavage of the retinoblastoma protein is an early step of neuronal apoptosis

Oncogene

Proteasome involvement and accumulation of ubiquitinated proteins in cerebellar granule neurons undergoing apoptosis

J. Neurosci.

Mitotic signaling by beta-amyloid causes neuronal death

Faseb J.

Bax-dependent caspase-3 activation is a key determinant in p53-induced apoptosis in neurons

J. Neurosci.

A DEVD-inhibited caspase other than CPP32 is involved in the commitment of cerebellar granule neurons to apoptosis induced by K+ deprivation

J. Neurochem.

The loss of mdm2 induces p53-mediated apoptosis

Oncogene

Distinct roles for E2F proteins in cell growth control and apoptosis

Proc. Natl. Acad. Sci. USA

RB and apoptotic cell death

Front Biosci.

The regulation of E2F by pRB-family proteins

Genes Dev.

A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD

Nature

Tumorigenic potential associated with enhanced expression of a gene that is amplified in a mouse tumor cell line

EMBO J.

Brain-derived neurotrophic factor exerts opposing effects on beta2-adrenergic receptor according to depolarization status of cerebellar neurons

J. Neurochem.

Cisplatin-induced apoptosis in rat dorsal root ganglion neurons is associated with attempted entry into the cell cycle

J. Clin. Invest.

Regular Article
Regulation of the Retinoblastoma-Dependent Mdm2 and E2F-1 Signaling Pathways during Neuronal Apoptosis

A DEVD-inhibited caspase other than CPP32 is involved in the commitment of cerebellar granule neurons to apoptosis induced by K⁺ deprivation