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Activation of p53-regulated pro-apoptotic signaling pathways in PrP-mediated myopathy.

Liang J, Parchaliuk D, Medina S, Sorensen G, Landry L, Huang S, Wang M, Kong Q, Booth SA - BMC Genomics (2009)

Bottom Line: These include the down-regulation of genes coding for the myofibrillar proteins and transcription factor MEF2c, and up-regulation of genes for lysosomal proteins that is concomitant with increased lysosomal activity in the skeletal muscles.Significantly, there was prominent up-regulation of p53 and p53-regulated genes involved in cell cycle arrest and promotion of apoptosis that paralleled the initiation and progression of the muscle pathology.The data provides the first in vivo evidence that directly links p53 to a wild type PrP-mediated disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA. jingjing.liang@case.edu

ABSTRACT

Background: We have reported that doxycycline-induced over-expression of wild type prion protein (PrP) in skeletal muscles of Tg(HQK) mice is sufficient to cause a primary myopathy with no signs of peripheral neuropathy. The preferential accumulation of the truncated PrP C1 fragment was closely correlated with these myopathic changes. In this study we use gene expression profiling to explore the temporal program of molecular changes underlying the PrP-mediated myopathy.

Results: We used DNA microarrays, and confirmatory real-time PCR and Western blot analysis to demonstrate deregulation of a large number of genes in the course of the progressive myopathy in the skeletal muscles of doxycycline-treated Tg(HQK) mice. These include the down-regulation of genes coding for the myofibrillar proteins and transcription factor MEF2c, and up-regulation of genes for lysosomal proteins that is concomitant with increased lysosomal activity in the skeletal muscles. Significantly, there was prominent up-regulation of p53 and p53-regulated genes involved in cell cycle arrest and promotion of apoptosis that paralleled the initiation and progression of the muscle pathology.

Conclusion: The data provides the first in vivo evidence that directly links p53 to a wild type PrP-mediated disease. It is evident that several mechanistic features contribute to the myopathy observed in PrP over-expressing mice and that p53-related apoptotic pathways appear to play a major role.

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Real-time PCR analysis of mdm2 and p53. qRT-PCR analysis of mdm2 (grey) and p53 (black) gene expression in RNA samples from Tg(HQK) PrP over-expressing mice relative to similarly treated wild-type control mice. Measurements of relative gene expression for 5 time points (over 4–60 days) in mice following treatment with 6 g Dox/kg food. Total RNA was extracted from skeletal muscles (quadriceps) from the hind legs and subjected qRT-PCR analysis. Results represent the mean ± s.e.m. of triplicate measurements performed. ** p < 0.01; *** p < 0.001.
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Figure 4: Real-time PCR analysis of mdm2 and p53. qRT-PCR analysis of mdm2 (grey) and p53 (black) gene expression in RNA samples from Tg(HQK) PrP over-expressing mice relative to similarly treated wild-type control mice. Measurements of relative gene expression for 5 time points (over 4–60 days) in mice following treatment with 6 g Dox/kg food. Total RNA was extracted from skeletal muscles (quadriceps) from the hind legs and subjected qRT-PCR analysis. Results represent the mean ± s.e.m. of triplicate measurements performed. ** p < 0.01; *** p < 0.001.

Mentions: The involvement of the p53 signaling pathways was of particular interest as mounting evidence suggests that over-expression of PrPC sensitizes cells to apoptotic stimuli through a p53-dependent pathway [5,20-22]. The p53 gene itself did not meet our selection criteria (a change of 3-fold or more in at least one time point) as significantly deregulated in the microarray analysis; however qRT-PCR showed it to be marginally up-regulated from day 7 following the onset of PrPC induction. This transient over-expression was low, approximately 1.5–2.5 fold, but statistically significant in all Tg(HQK) mice tested (Figure 4). However, regulation of p53 is known to take place mostly at the level of translation [42]. In accordance with this, immunoblot analysis of the levels of total p53 protein in the skeletal muscle (quadriceps) of Tg(HQK) mice, shown in Figure 5A and 5B, revealed a moderate but significant accumulation of p53 protein beginning at day 7 following the commencement of doxycycline treatment and rising to over 3-fold over age-matched WT controls 30–60 days post Dox induction. Activation of p53 is kept in check by its negative regulator MDM2 (mouse double minute 2) in a negative feedback regulatory loop since activated p53 induces expression of MDM2 [42]. We found that the levels of MDM2 were only marginally changed at early time points but were significantly up-regulated at the later time points (30 and 60 days), congruent with the accumulation of p53 protein (Figure 5). The moderate increase in p53 in the muscles of Dox-treated Tg(HQK) mice is consistent with the observed gradual and progressive muscle wasting.


Activation of p53-regulated pro-apoptotic signaling pathways in PrP-mediated myopathy.

Liang J, Parchaliuk D, Medina S, Sorensen G, Landry L, Huang S, Wang M, Kong Q, Booth SA - BMC Genomics (2009)

Real-time PCR analysis of mdm2 and p53. qRT-PCR analysis of mdm2 (grey) and p53 (black) gene expression in RNA samples from Tg(HQK) PrP over-expressing mice relative to similarly treated wild-type control mice. Measurements of relative gene expression for 5 time points (over 4–60 days) in mice following treatment with 6 g Dox/kg food. Total RNA was extracted from skeletal muscles (quadriceps) from the hind legs and subjected qRT-PCR analysis. Results represent the mean ± s.e.m. of triplicate measurements performed. ** p < 0.01; *** p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683871&req=5

Figure 4: Real-time PCR analysis of mdm2 and p53. qRT-PCR analysis of mdm2 (grey) and p53 (black) gene expression in RNA samples from Tg(HQK) PrP over-expressing mice relative to similarly treated wild-type control mice. Measurements of relative gene expression for 5 time points (over 4–60 days) in mice following treatment with 6 g Dox/kg food. Total RNA was extracted from skeletal muscles (quadriceps) from the hind legs and subjected qRT-PCR analysis. Results represent the mean ± s.e.m. of triplicate measurements performed. ** p < 0.01; *** p < 0.001.
Mentions: The involvement of the p53 signaling pathways was of particular interest as mounting evidence suggests that over-expression of PrPC sensitizes cells to apoptotic stimuli through a p53-dependent pathway [5,20-22]. The p53 gene itself did not meet our selection criteria (a change of 3-fold or more in at least one time point) as significantly deregulated in the microarray analysis; however qRT-PCR showed it to be marginally up-regulated from day 7 following the onset of PrPC induction. This transient over-expression was low, approximately 1.5–2.5 fold, but statistically significant in all Tg(HQK) mice tested (Figure 4). However, regulation of p53 is known to take place mostly at the level of translation [42]. In accordance with this, immunoblot analysis of the levels of total p53 protein in the skeletal muscle (quadriceps) of Tg(HQK) mice, shown in Figure 5A and 5B, revealed a moderate but significant accumulation of p53 protein beginning at day 7 following the commencement of doxycycline treatment and rising to over 3-fold over age-matched WT controls 30–60 days post Dox induction. Activation of p53 is kept in check by its negative regulator MDM2 (mouse double minute 2) in a negative feedback regulatory loop since activated p53 induces expression of MDM2 [42]. We found that the levels of MDM2 were only marginally changed at early time points but were significantly up-regulated at the later time points (30 and 60 days), congruent with the accumulation of p53 protein (Figure 5). The moderate increase in p53 in the muscles of Dox-treated Tg(HQK) mice is consistent with the observed gradual and progressive muscle wasting.

Bottom Line: These include the down-regulation of genes coding for the myofibrillar proteins and transcription factor MEF2c, and up-regulation of genes for lysosomal proteins that is concomitant with increased lysosomal activity in the skeletal muscles.Significantly, there was prominent up-regulation of p53 and p53-regulated genes involved in cell cycle arrest and promotion of apoptosis that paralleled the initiation and progression of the muscle pathology.The data provides the first in vivo evidence that directly links p53 to a wild type PrP-mediated disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA. jingjing.liang@case.edu

ABSTRACT

Background: We have reported that doxycycline-induced over-expression of wild type prion protein (PrP) in skeletal muscles of Tg(HQK) mice is sufficient to cause a primary myopathy with no signs of peripheral neuropathy. The preferential accumulation of the truncated PrP C1 fragment was closely correlated with these myopathic changes. In this study we use gene expression profiling to explore the temporal program of molecular changes underlying the PrP-mediated myopathy.

Results: We used DNA microarrays, and confirmatory real-time PCR and Western blot analysis to demonstrate deregulation of a large number of genes in the course of the progressive myopathy in the skeletal muscles of doxycycline-treated Tg(HQK) mice. These include the down-regulation of genes coding for the myofibrillar proteins and transcription factor MEF2c, and up-regulation of genes for lysosomal proteins that is concomitant with increased lysosomal activity in the skeletal muscles. Significantly, there was prominent up-regulation of p53 and p53-regulated genes involved in cell cycle arrest and promotion of apoptosis that paralleled the initiation and progression of the muscle pathology.

Conclusion: The data provides the first in vivo evidence that directly links p53 to a wild type PrP-mediated disease. It is evident that several mechanistic features contribute to the myopathy observed in PrP over-expressing mice and that p53-related apoptotic pathways appear to play a major role.

Show MeSH
Related in: MedlinePlus