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Stress-induced alternative splice forms of MDM2 and MDMX modulate the p53-pathway in distinct ways.

Jacob AG, Singh RK, Comiskey DF, Rouhier MF, Mohammad F, Bebee TW, Chandler DS - PLoS ONE (2014)

Bottom Line: We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2.Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21.In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Childhood Cancer at the Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America; The Department of Pediatrics, and Molecular, Cellular and Developmental Biology (MCDB) program, The Ohio State University, Columbus, Ohio, United States of America; Center for RNA Biology, Wexner Medical Center, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
MDM2 and MDMX are the chief negative regulators of the tumor-suppressor protein p53 and are essential for maintaining homeostasis within the cell. In response to genotoxic stress and also in several cancer types, MDM2 and MDMX are alternatively spliced. The splice variants MDM2-ALT1 and MDMX-ALT2 lack the p53-binding domain and are incapable of negatively regulating p53. However, they retain the RING domain that facilitates dimerization of the full-length MDM proteins. Concordantly, MDM2-ALT1 has been shown to lead to the stabilization of p53 through its interaction with and inactivation of full-length MDM2. The impact of MDM2-ALT1 expression on the p53 pathway and the nature of its interaction with MDMX remain unclear. Also, the role of the architecturally similar MDMX-ALT2 and its influence of the MDM2-MDMX-p53 axis are yet to be elucidated. We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2. Additionally, we demonstrate that MDMX-ALT2 is able to dimerize with both full-length MDMX and MDM2 and that the expression of MDM2-ALT1 and MDMX-ALT2 leads to the upregulation of p53 protein, and also of its downstream target p21. Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21. Finally we present evidence that MDM2-ALT1 and MDMX-ALT2 expression can activate subtly distinct subsets of p53-transcriptional targets implying that these splice variants can modulate the p53 tumor suppressor pathway in unique ways. In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

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MDM2-ALT1 and MDMX-ALT2 lead to the activation of subtly different subsets of p53 transcriptional targets.A. MCF7 cells transfected with myc-tagged LacZ, or MDM2-ALT1 (Alt1) or MDMX-ALT2 (XAlt2) were examined for mRNA levels of a panel of 9 p53 transcriptional targets using quantitative real-time PCR and were normalized to LacZ-expressing cells. The bar graphs represent the relative quantification (2−ΔΔCt) values from at least 3 independent experiments and each consisting of 3 technical replicates. The error bars represent standard error means (SEM). B. qRT-PCR was used to compare the relative levels (2−ΔΔCt) of miRNA targets of p53 between cells over-expressing LacZ or MDM2-ALT1 or MDMX-ALT2. Here also a minimum of 3 independent trials was performed with 3 technical replicates each. The error bars represent standard error means (SEM). * represents p<0.05 and ** represents p<0.01 in all cases.
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pone-0104444-g004: MDM2-ALT1 and MDMX-ALT2 lead to the activation of subtly different subsets of p53 transcriptional targets.A. MCF7 cells transfected with myc-tagged LacZ, or MDM2-ALT1 (Alt1) or MDMX-ALT2 (XAlt2) were examined for mRNA levels of a panel of 9 p53 transcriptional targets using quantitative real-time PCR and were normalized to LacZ-expressing cells. The bar graphs represent the relative quantification (2−ΔΔCt) values from at least 3 independent experiments and each consisting of 3 technical replicates. The error bars represent standard error means (SEM). B. qRT-PCR was used to compare the relative levels (2−ΔΔCt) of miRNA targets of p53 between cells over-expressing LacZ or MDM2-ALT1 or MDMX-ALT2. Here also a minimum of 3 independent trials was performed with 3 technical replicates each. The error bars represent standard error means (SEM). * represents p<0.05 and ** represents p<0.01 in all cases.

Mentions: Since the over-expression of MDM2-ALT1 and MDMX-ALT2 lead to the upregulation of p53-target p21, we hypothesized that other transcriptional targets of p53 could also be upregulated under these circumstances. We assessed the transcript levels of candidate genes involved in the p53 tumor suppressor pathway and known to play roles in either cell-cycle control and/or DNA damage repair (p21, GADD45A, WIP1, PCNA, Cyclin D1 and 14-3-3σ) or apoptosis (Bax, Fas1, PUMA, Noxa) [54]–[66]. Of the 9 additional p53-target genes whose expression was assayed, only 3 (Bax, Cyclin D1, and Fas1) showed a significant increase in response to MDM2-ALT1 or MDMX-ALT2 over-expression. Bax transcript levels were significantly upregulated in response to the over-expression of MDM2-ALT1 as well as MDMX-ALT2 (Fig 4A). However, Cyclin D1 levels showed significant increase only upon MDMX-ALT2 expression and Fas1 transcripts were significantly higher only in cells over-expressing MDM2-ALT1. In all cases the transcript levels of the p53-target genes upon MDM2-ALT1 or MDMX-ALT2 over-expression were normalized to corresponding transcript levels in LacZ expressing cells and GAPDH mRNA was used as the endogenous control (Fig 4A).


Stress-induced alternative splice forms of MDM2 and MDMX modulate the p53-pathway in distinct ways.

Jacob AG, Singh RK, Comiskey DF, Rouhier MF, Mohammad F, Bebee TW, Chandler DS - PLoS ONE (2014)

MDM2-ALT1 and MDMX-ALT2 lead to the activation of subtly different subsets of p53 transcriptional targets.A. MCF7 cells transfected with myc-tagged LacZ, or MDM2-ALT1 (Alt1) or MDMX-ALT2 (XAlt2) were examined for mRNA levels of a panel of 9 p53 transcriptional targets using quantitative real-time PCR and were normalized to LacZ-expressing cells. The bar graphs represent the relative quantification (2−ΔΔCt) values from at least 3 independent experiments and each consisting of 3 technical replicates. The error bars represent standard error means (SEM). B. qRT-PCR was used to compare the relative levels (2−ΔΔCt) of miRNA targets of p53 between cells over-expressing LacZ or MDM2-ALT1 or MDMX-ALT2. Here also a minimum of 3 independent trials was performed with 3 technical replicates each. The error bars represent standard error means (SEM). * represents p<0.05 and ** represents p<0.01 in all cases.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126728&req=5

pone-0104444-g004: MDM2-ALT1 and MDMX-ALT2 lead to the activation of subtly different subsets of p53 transcriptional targets.A. MCF7 cells transfected with myc-tagged LacZ, or MDM2-ALT1 (Alt1) or MDMX-ALT2 (XAlt2) were examined for mRNA levels of a panel of 9 p53 transcriptional targets using quantitative real-time PCR and were normalized to LacZ-expressing cells. The bar graphs represent the relative quantification (2−ΔΔCt) values from at least 3 independent experiments and each consisting of 3 technical replicates. The error bars represent standard error means (SEM). B. qRT-PCR was used to compare the relative levels (2−ΔΔCt) of miRNA targets of p53 between cells over-expressing LacZ or MDM2-ALT1 or MDMX-ALT2. Here also a minimum of 3 independent trials was performed with 3 technical replicates each. The error bars represent standard error means (SEM). * represents p<0.05 and ** represents p<0.01 in all cases.
Mentions: Since the over-expression of MDM2-ALT1 and MDMX-ALT2 lead to the upregulation of p53-target p21, we hypothesized that other transcriptional targets of p53 could also be upregulated under these circumstances. We assessed the transcript levels of candidate genes involved in the p53 tumor suppressor pathway and known to play roles in either cell-cycle control and/or DNA damage repair (p21, GADD45A, WIP1, PCNA, Cyclin D1 and 14-3-3σ) or apoptosis (Bax, Fas1, PUMA, Noxa) [54]–[66]. Of the 9 additional p53-target genes whose expression was assayed, only 3 (Bax, Cyclin D1, and Fas1) showed a significant increase in response to MDM2-ALT1 or MDMX-ALT2 over-expression. Bax transcript levels were significantly upregulated in response to the over-expression of MDM2-ALT1 as well as MDMX-ALT2 (Fig 4A). However, Cyclin D1 levels showed significant increase only upon MDMX-ALT2 expression and Fas1 transcripts were significantly higher only in cells over-expressing MDM2-ALT1. In all cases the transcript levels of the p53-target genes upon MDM2-ALT1 or MDMX-ALT2 over-expression were normalized to corresponding transcript levels in LacZ expressing cells and GAPDH mRNA was used as the endogenous control (Fig 4A).

Bottom Line: We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2.Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21.In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Childhood Cancer at the Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America; The Department of Pediatrics, and Molecular, Cellular and Developmental Biology (MCDB) program, The Ohio State University, Columbus, Ohio, United States of America; Center for RNA Biology, Wexner Medical Center, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
MDM2 and MDMX are the chief negative regulators of the tumor-suppressor protein p53 and are essential for maintaining homeostasis within the cell. In response to genotoxic stress and also in several cancer types, MDM2 and MDMX are alternatively spliced. The splice variants MDM2-ALT1 and MDMX-ALT2 lack the p53-binding domain and are incapable of negatively regulating p53. However, they retain the RING domain that facilitates dimerization of the full-length MDM proteins. Concordantly, MDM2-ALT1 has been shown to lead to the stabilization of p53 through its interaction with and inactivation of full-length MDM2. The impact of MDM2-ALT1 expression on the p53 pathway and the nature of its interaction with MDMX remain unclear. Also, the role of the architecturally similar MDMX-ALT2 and its influence of the MDM2-MDMX-p53 axis are yet to be elucidated. We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2. Additionally, we demonstrate that MDMX-ALT2 is able to dimerize with both full-length MDMX and MDM2 and that the expression of MDM2-ALT1 and MDMX-ALT2 leads to the upregulation of p53 protein, and also of its downstream target p21. Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21. Finally we present evidence that MDM2-ALT1 and MDMX-ALT2 expression can activate subtly distinct subsets of p53-transcriptional targets implying that these splice variants can modulate the p53 tumor suppressor pathway in unique ways. In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

Show MeSH
Related in: MedlinePlus