PrPC Overexpression Research Articles

The C-terminal Products of Cellular Prion Protein Processing, C1 and C2, Exert Distinct Influence on p53-dependent Staurosporine-induced Caspase-3 Activation

The cellular prion protein (PrP(c)) undergoes various endopro-teolytic attacks within its N-terminal domain, leading to the production of C-terminal fragments (C) tethered to the plasma membrane and soluble N-terminal peptides (N). One of these cleavages occurs at position 110/111, thereby generating C1 and N1 products. We have reported that disintegrins ADAM-10, -9, and -17 participate either directly or indirectly to this proteolytic event. An alternative proteolytic event taking place around residue 90 yields C2 and N2 fragments. The putative function of these proteolytic fragments remained to be established. We have set up two novel human embryonic kidney 293 cell lines stably overexpressing either C1 or C2. We show that C1 potentiates staurosporine-induced caspase-3 activation through a p53-dependent mechanism. Thus, C1 positively controls p53 transcription and mRNA levels and increases p53-like immunoreactivity and activity. C1-induced caspase-3 activation remained unaffected by the blockade of endocytosis in HEK 293 cells and was abolished in p53-deficient fibroblasts. Conversely, overexpression of the C2 fragment did not significantly sensitize HEK 293 cells to apoptotic stimuli and did not modify p53 mRNA levels or activity. Therefore, the nature of the proteolytic cleavage taking place on PrP(c) yielded C-terminal catabolites with distinct function and could be seen as a switch mechanism controlling the function of the PrP(c) in cell survival.

Overexpression of Cellular Prion Protein Induces an Antioxidant Environment Altering T Cell Development in the Thymus

Cellular prion protein (PrP(C)) is an ubiquitously expressed glycoprotein whose roles are still widely discussed, particularly in the field of immunology. Using TgA20- and Tg33-transgenic mice overexpressing PrP(C), we investigated the consequences of this overexpression on T cell development. In both models, overexpression of PrP(C) induces strong alterations at different steps of T cell maturation. On TgA20 mice, we observed that these alterations are cell autonomous and lead to a decrease of alphabeta T cells and a concomitant increase of gammadelta T cell numbers. PrP(C) has been shown to bind and chelate copper and, interestingly, under a copper supplementation diet, TgA20 mice presented a partial restoration of the alphabeta T cell development, suggesting that PrP(C) overexpression, by chelating copper, generates an antioxidant context differentially impacting on alphabeta and gammadelta T cell lineage.

Prion protein prevents human breast carcinoma cell line from tumor necrosis factor alpha-induced cell death.

To define genetic determinants of tumor cell resistance to the cytotoxic action of tumor necrosis factor alpha (TNF), we have applied cDNA microarrays to a human breast carcinoma TNF-sensitive MCF7 cell line and its established TNF-resistant clone. Of a total of 5760 samples of cDNA examined, 3.6% were found to be differentially expressed in TNF-resistant 1001 cells as compared with TNF-sensitive MCF7 cells. On the basis of available literature data, the striking finding is the association of some differentially expressed genes involved in the phosphatidylinositol-3-kinase/Akt signaling pathway. More notably, we found that the PRNP gene coding for the cellular prion protein (PrP(c)), was 17-fold overexpressed in the 1001 cell line as compared with the MCF7 cell line. This differential expression was confirmed at the cell surface by immunostaining that indicated that PrP(c) is overexpressed at both mRNA and protein levels in the TNF-resistant derivative. Using recombinant adenoviruses expressing the human PrP(c,) our data demonstrate that PrP(c) overexpression converted TNF-sensitive MCF7 cells into TNF-resistant cells, at least in part, by a mechanism involving alteration of cytochrome c release from mitochondria and nuclear condensation.

Primary Cultured Neurons Devoid of Cellular Prion Display Lower Responsiveness to Staurosporine through the Control of p53 at Both Transcriptional and Post-transcriptional Levels

We assessed the contribution of the cellular prion protein (PrPc) in the control of neuronal apoptosis by examining cell death in both human cells and murine primary cultured neurons. We first confirmed our previous finding that staurosporine-induced caspase activation is increased by PrPc overexpression in HEK293 cells. We show here that this phenotype is fully dependent on p53 and that the control of p53 activity by PrPc occurs at both transcriptional and post-transcriptional levels in human cells. Of most interest, we demonstrate that neuronal endogenous PrPc also controls a p53-dependent pro-apoptotic phenotype. Thus, DNA fragmentation and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling)-positive cells were lower in primary cultured neurons derived from Zrch-1 mice embryos in which PrPc has been abrogated than in wild-type neurons. PrPc knock-out neurons also displayed drastically diminished caspase-3-like activity and immunoreactivity together with reduced p53 expression and transcriptional activity, a phenotype complemented in part by PrPc transfection. Interestingly, p53 expression was also reduced in the brain of adult Prnp-/- mice. Neuronal PrPc likely controls p53 at a post-transcriptional level because the deletion of cellular prion protein is accompanied by a higher Mdm2-like immunoreactivity and reduced phosphorylated p38 MAPK expression. We therefore propose that the physiological function of endogenous cellular prion could be to regulate p53-dependent caspase-3-mediated neuronal cell death. This phenotype likely occurs through up-regulation of p53 promoter transactivation as well as downstream by controlling p53 stability via Mdm2 expression.

Overexpression of PrPc triggers caspase 3 activation: potentiation by proteasome inhibitors and blockade by anti-PrP antibodies.

We examined the influence of cellular prion protein (PrPc) in the control of cell death in stably transfected HEK293 cell line and in the PrPc-inducible Rov9 cells. PrPc expression in stably transfected HEK293 human cells did not modify basal apoptotic tonus but drastically potentiated staurosporine-stimulated cellular toxicity and DNA fragmentation as well as caspase 3-like activity and immunoreactivity. An identical staurosporine-induced caspase 3 activation was observed after doxycycline in the PrPc-inducible Rov9 cell line. Interestingly, proteasome inhibitors increase PrPc-like immunoreactivity and unmasked a basal caspase 3 activation. Conversely, we show that anti-PrPc antibodies sequestrate PrPc at the cell surface and drastically lower PrPc-dependent caspase activation. We suggest that intracellular PrPc could sensitize human cells to pro-apoptotic phenotype and that blockade of PrPc internalization could be a track to prevent intracellular toxicity associated with PrPc overexpression.