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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 expression causes upregulation of p53 and its downstream target p21.A. The over-expression (O/E cell lysates) of the myc-tagged LacZ, MDM2-ALT1 or MDMX-ALT2 was confirmed using anti-myc tag antibody in the MCF7 cells that were transfected with the corresponding expression constructs (top panel). The level of p53 protein was examined in these samples using the anti-p53 antibody and an upregulation of p53 protein was observed upon MDM2-ALT1 or MDMX-ALT2 over-expression compared to LacZ expressing cells although the increase is more modest in MDMX-ALT2 over-expression (lanes 2 and 3 compared to lane 1). The positive control, UVC (50 J/m2) irradiated MCF7 cells show a strong upregulation of p53 protein levels in response to the stress when compared to untreated cells (lanes 4 and 5). β-actin was used as loading control. A minimum of three independent experiments was performed and representative gel images are shown. B.p21 expression at the mRNA level was examined using quantitative real-time PCR and GAPDH levels were used as the endogenous control. The ratio of p21 to GAPDH is represented graphically and the error bars represent standard deviations from at least 3 independent experiments. MCF7 cells over-expressing MDM2-ALT1 (2Alt1) show statistically significant increase in p21 transcript levels compared to LacZ expressing cells (p<0.01). The cells expressing MDMX-ALT2 (XAlt2) did not show statistically significant changes in p21 expression at the mRNA level. C. The levels of p21 protein in the MCF7 cells transfected with myc-tagged LacZ, MDM2-ALT1 or MDMX-ALT2 was examined using anti-p21 antibody. Both MDM2-ALT1 and MDMX-ALT2 over-expression lead to upregulation of p21 protein levels compared to LacZ over-expression (compare lanes 2 and 3 with lane 1). A minimum of three independent experiments was performed and consistent results observed. Representative images are shown here. Additionally, UVC-irradiated MCF7 cells were used as positive control and show an upregulation of p21 compared to untreated cells (lanes 4 and 5).
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pone-0104444-g002: MDM2-ALT1 and MDMX-ALT2 expression causes upregulation of p53 and its downstream target p21.A. The over-expression (O/E cell lysates) of the myc-tagged LacZ, MDM2-ALT1 or MDMX-ALT2 was confirmed using anti-myc tag antibody in the MCF7 cells that were transfected with the corresponding expression constructs (top panel). The level of p53 protein was examined in these samples using the anti-p53 antibody and an upregulation of p53 protein was observed upon MDM2-ALT1 or MDMX-ALT2 over-expression compared to LacZ expressing cells although the increase is more modest in MDMX-ALT2 over-expression (lanes 2 and 3 compared to lane 1). The positive control, UVC (50 J/m2) irradiated MCF7 cells show a strong upregulation of p53 protein levels in response to the stress when compared to untreated cells (lanes 4 and 5). β-actin was used as loading control. A minimum of three independent experiments was performed and representative gel images are shown. B.p21 expression at the mRNA level was examined using quantitative real-time PCR and GAPDH levels were used as the endogenous control. The ratio of p21 to GAPDH is represented graphically and the error bars represent standard deviations from at least 3 independent experiments. MCF7 cells over-expressing MDM2-ALT1 (2Alt1) show statistically significant increase in p21 transcript levels compared to LacZ expressing cells (p<0.01). The cells expressing MDMX-ALT2 (XAlt2) did not show statistically significant changes in p21 expression at the mRNA level. C. The levels of p21 protein in the MCF7 cells transfected with myc-tagged LacZ, MDM2-ALT1 or MDMX-ALT2 was examined using anti-p21 antibody. Both MDM2-ALT1 and MDMX-ALT2 over-expression lead to upregulation of p21 protein levels compared to LacZ over-expression (compare lanes 2 and 3 with lane 1). A minimum of three independent experiments was performed and consistent results observed. Representative images are shown here. Additionally, UVC-irradiated MCF7 cells were used as positive control and show an upregulation of p21 compared to untreated cells (lanes 4 and 5).

Mentions: Since MDM2-ALT1 and MDMX-ALT2 interact with full-length MDM2 and MDMX, it is possible that the formation of such complexes can impede the functions of these proteins. Moreover, MDM2-ALT1 expression has been shown previously to lead to the accumulation of p53 due to sequestration of MDM2 in the cytoplasm [29], [30], [32]. We hypothesized that MDMX-ALT2 expression could increase p53 levels in a similar manner. To test this, we examined the expression of p53 in MCF7 cells that were transfected with a negative control (LacZ), MDMX-ALT2 or MDM2-ALT1. As expected, MDM2-ALT1 expression caused a substantial increase in the levels of p53 protein compared to LacZ over-expression (Fig 2A p53 panel compare lanes 1 and 2). MDMX-ALT2 expression also resulted in an increase in the p53 protein levels compared to LacZ over-expression (Fig 2A p53 panel compare lanes 1 and 3). However, this effect was more moderate compared to the MDM2-ALT1-mediated upregulation of p53 protein (Fig 2A p53 panel). As a positive control for the accumulation of p53 protein, we compared p53 levels in whole cell protein lysates from untreated and UVC-irradiated (50 J/m2) MCF7 cells (Fig 2A, compare lanes 4 and 5).


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 expression causes upregulation of p53 and its downstream target p21.A. The over-expression (O/E cell lysates) of the myc-tagged LacZ, MDM2-ALT1 or MDMX-ALT2 was confirmed using anti-myc tag antibody in the MCF7 cells that were transfected with the corresponding expression constructs (top panel). The level of p53 protein was examined in these samples using the anti-p53 antibody and an upregulation of p53 protein was observed upon MDM2-ALT1 or MDMX-ALT2 over-expression compared to LacZ expressing cells although the increase is more modest in MDMX-ALT2 over-expression (lanes 2 and 3 compared to lane 1). The positive control, UVC (50 J/m2) irradiated MCF7 cells show a strong upregulation of p53 protein levels in response to the stress when compared to untreated cells (lanes 4 and 5). β-actin was used as loading control. A minimum of three independent experiments was performed and representative gel images are shown. B.p21 expression at the mRNA level was examined using quantitative real-time PCR and GAPDH levels were used as the endogenous control. The ratio of p21 to GAPDH is represented graphically and the error bars represent standard deviations from at least 3 independent experiments. MCF7 cells over-expressing MDM2-ALT1 (2Alt1) show statistically significant increase in p21 transcript levels compared to LacZ expressing cells (p<0.01). The cells expressing MDMX-ALT2 (XAlt2) did not show statistically significant changes in p21 expression at the mRNA level. C. The levels of p21 protein in the MCF7 cells transfected with myc-tagged LacZ, MDM2-ALT1 or MDMX-ALT2 was examined using anti-p21 antibody. Both MDM2-ALT1 and MDMX-ALT2 over-expression lead to upregulation of p21 protein levels compared to LacZ over-expression (compare lanes 2 and 3 with lane 1). A minimum of three independent experiments was performed and consistent results observed. Representative images are shown here. Additionally, UVC-irradiated MCF7 cells were used as positive control and show an upregulation of p21 compared to untreated cells (lanes 4 and 5).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126728&req=5

pone-0104444-g002: MDM2-ALT1 and MDMX-ALT2 expression causes upregulation of p53 and its downstream target p21.A. The over-expression (O/E cell lysates) of the myc-tagged LacZ, MDM2-ALT1 or MDMX-ALT2 was confirmed using anti-myc tag antibody in the MCF7 cells that were transfected with the corresponding expression constructs (top panel). The level of p53 protein was examined in these samples using the anti-p53 antibody and an upregulation of p53 protein was observed upon MDM2-ALT1 or MDMX-ALT2 over-expression compared to LacZ expressing cells although the increase is more modest in MDMX-ALT2 over-expression (lanes 2 and 3 compared to lane 1). The positive control, UVC (50 J/m2) irradiated MCF7 cells show a strong upregulation of p53 protein levels in response to the stress when compared to untreated cells (lanes 4 and 5). β-actin was used as loading control. A minimum of three independent experiments was performed and representative gel images are shown. B.p21 expression at the mRNA level was examined using quantitative real-time PCR and GAPDH levels were used as the endogenous control. The ratio of p21 to GAPDH is represented graphically and the error bars represent standard deviations from at least 3 independent experiments. MCF7 cells over-expressing MDM2-ALT1 (2Alt1) show statistically significant increase in p21 transcript levels compared to LacZ expressing cells (p<0.01). The cells expressing MDMX-ALT2 (XAlt2) did not show statistically significant changes in p21 expression at the mRNA level. C. The levels of p21 protein in the MCF7 cells transfected with myc-tagged LacZ, MDM2-ALT1 or MDMX-ALT2 was examined using anti-p21 antibody. Both MDM2-ALT1 and MDMX-ALT2 over-expression lead to upregulation of p21 protein levels compared to LacZ over-expression (compare lanes 2 and 3 with lane 1). A minimum of three independent experiments was performed and consistent results observed. Representative images are shown here. Additionally, UVC-irradiated MCF7 cells were used as positive control and show an upregulation of p21 compared to untreated cells (lanes 4 and 5).
Mentions: Since MDM2-ALT1 and MDMX-ALT2 interact with full-length MDM2 and MDMX, it is possible that the formation of such complexes can impede the functions of these proteins. Moreover, MDM2-ALT1 expression has been shown previously to lead to the accumulation of p53 due to sequestration of MDM2 in the cytoplasm [29], [30], [32]. We hypothesized that MDMX-ALT2 expression could increase p53 levels in a similar manner. To test this, we examined the expression of p53 in MCF7 cells that were transfected with a negative control (LacZ), MDMX-ALT2 or MDM2-ALT1. As expected, MDM2-ALT1 expression caused a substantial increase in the levels of p53 protein compared to LacZ over-expression (Fig 2A p53 panel compare lanes 1 and 2). MDMX-ALT2 expression also resulted in an increase in the p53 protein levels compared to LacZ over-expression (Fig 2A p53 panel compare lanes 1 and 3). However, this effect was more moderate compared to the MDM2-ALT1-mediated upregulation of p53 protein (Fig 2A p53 panel). As a positive control for the accumulation of p53 protein, we compared p53 levels in whole cell protein lysates from untreated and UVC-irradiated (50 J/m2) MCF7 cells (Fig 2A, compare lanes 4 and 5).

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