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Latent KSHV Infected Endothelial Cells Are Glutamine Addicted and Require Glutaminolysis for Survival.

Sanchez EL, Carroll PA, Thalhofer AB, Lagunoff M - PLoS Pathog. (2015)

Bottom Line: Kaposi's Sarcoma-associated Herpesvirus (KSHV) is the etiologic agent of Kaposi's Sarcoma (KS).Knockdown of MondoA inhibits expression of both Mlx and SLC1A5 and induces a significant increase in cell death of only cells latently infected with KSHV, again, fully rescued by the supplementation of αKG.These findings expand our understanding of the required metabolic pathways that are activated during latent KSHV infection of endothelial cells, and demonstrate a novel role for the extended Myc-regulatory network, specifically MondoA, during latent KSHV infection.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology Program, University of Washington, Seattle, Washington, United States of America; Department of Microbiology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Kaposi's Sarcoma-associated Herpesvirus (KSHV) is the etiologic agent of Kaposi's Sarcoma (KS). KSHV establishes a predominantly latent infection in the main KS tumor cell type, the spindle cell, which is of endothelial cell origin. KSHV requires the induction of multiple metabolic pathways, including glycolysis and fatty acid synthesis, for the survival of latently infected endothelial cells. Here we demonstrate that latent KSHV infection leads to increased levels of intracellular glutamine and enhanced glutamine uptake. Depletion of glutamine from the culture media leads to a significant increase in apoptotic cell death in latently infected endothelial cells, but not in their mock-infected counterparts. In cancer cells, glutamine is often required for glutaminolysis to provide intermediates for the tri-carboxylic acid (TCA) cycle and support for the production of biosynthetic and bioenergetic precursors. In the absence of glutamine, the TCA cycle intermediates alpha-ketoglutarate (αKG) and pyruvate prevent the death of latently infected cells. Targeted drug inhibition of glutaminolysis also induces increased cell death in latently infected cells. KSHV infection of endothelial cells induces protein expression of the glutamine transporter, SLC1A5. Chemical inhibition of SLC1A5, or knockdown by siRNA, leads to similar cell death rates as glutamine deprivation and, similarly, can be rescued by αKG. KSHV also induces expression of the heterodimeric transcription factors c-Myc-Max and related heterodimer MondoA-Mlx. Knockdown of MondoA inhibits expression of both Mlx and SLC1A5 and induces a significant increase in cell death of only cells latently infected with KSHV, again, fully rescued by the supplementation of αKG. Therefore, during latent infection of endothelial cells, KSHV activates and requires the Myc/MondoA-network to upregulate the glutamine transporter, SLC1A5, leading to increased glutamine uptake for glutaminolysis. These findings expand our understanding of the required metabolic pathways that are activated during latent KSHV infection of endothelial cells, and demonstrate a novel role for the extended Myc-regulatory network, specifically MondoA, during latent KSHV infection.

No MeSH data available.


Related in: MedlinePlus

MondoA regulation of glutaminolysis is required for the survival of endothelial cells latently infected with KSHV.(A) TIME cells were transfected with siControl or siMondoA and then Mock- or KSHV-infected 24 hours post transfection. Whole-cell lysates were harvested at 48 hpi. Lysates were subjected to immunoblot analysis for MondoA, Mlx, SLC1A5 and TXNIP. KSHV infection elevates levels of all four proteins and loss of MondoA reduces the observed increase in protein expression. The standard γ-tubulin was included as a loading control. (B) Twenty-four hours post transfection of TIME cells with siControl or siMondoA, cells were Mock- or KSHV-infected and overlaid with replete media in the presence or absence of αKG. YOYO-1 or SytoGreen was added to the media to monitor relative fluorescence for cell death or total cells, respectively. Forty-eight hpi (72 hours post transfection), cells were scanned on the Typhoon. Data shown is the average fold change in relative fluorescence of KSHV over mock cells (YOYO-1 positive cells/SytoGreen24 positive cells) from two independent experiments. Error bars represent the SEM. A p < 0.05 is denoted by one asterisk and “ns” is shown where two averages are not significantly different.
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ppat.1005052.g007: MondoA regulation of glutaminolysis is required for the survival of endothelial cells latently infected with KSHV.(A) TIME cells were transfected with siControl or siMondoA and then Mock- or KSHV-infected 24 hours post transfection. Whole-cell lysates were harvested at 48 hpi. Lysates were subjected to immunoblot analysis for MondoA, Mlx, SLC1A5 and TXNIP. KSHV infection elevates levels of all four proteins and loss of MondoA reduces the observed increase in protein expression. The standard γ-tubulin was included as a loading control. (B) Twenty-four hours post transfection of TIME cells with siControl or siMondoA, cells were Mock- or KSHV-infected and overlaid with replete media in the presence or absence of αKG. YOYO-1 or SytoGreen was added to the media to monitor relative fluorescence for cell death or total cells, respectively. Forty-eight hpi (72 hours post transfection), cells were scanned on the Typhoon. Data shown is the average fold change in relative fluorescence of KSHV over mock cells (YOYO-1 positive cells/SytoGreen24 positive cells) from two independent experiments. Error bars represent the SEM. A p < 0.05 is denoted by one asterisk and “ns” is shown where two averages are not significantly different.

Mentions: SLC1A5 is directly regulated by the nutrient-sensing Myc extended network member MondoA in many human cancer cells[18]. To determine if MondoA controls SLC1A5 expression during latent KSHV infection of endothelial cells, we examined the expression of SLC1A5 upon siRNA knockdown of MondoA in mock- and KSHV-infected endothelial cells. MondoA protein expression was significantly reduced in both mock and KSHV-infected TIME cells transfected with a mix of four siRNAs specific for MondoA (siMondoA), as compared to cells transfected with a scrambled non-target control (siControl) (Fig 7A). While SLC1A5 protein levels are elevated by KSHV infection, loss of MondoA results in a reduction in detected SLC1A5 in all samples. Additionally, protein levels of Mlx, a co-stabilized MondoA binding partner[18], and TXNIP, a known downstream target of MondoA/Mlx regulation, are also reduced upon loss of MondoA. These data support the hypothesis that MondoA is directly regulating SLC1A5, the major glutamine transporter, during latent KSHV infection of endothelial cells.


Latent KSHV Infected Endothelial Cells Are Glutamine Addicted and Require Glutaminolysis for Survival.

Sanchez EL, Carroll PA, Thalhofer AB, Lagunoff M - PLoS Pathog. (2015)

MondoA regulation of glutaminolysis is required for the survival of endothelial cells latently infected with KSHV.(A) TIME cells were transfected with siControl or siMondoA and then Mock- or KSHV-infected 24 hours post transfection. Whole-cell lysates were harvested at 48 hpi. Lysates were subjected to immunoblot analysis for MondoA, Mlx, SLC1A5 and TXNIP. KSHV infection elevates levels of all four proteins and loss of MondoA reduces the observed increase in protein expression. The standard γ-tubulin was included as a loading control. (B) Twenty-four hours post transfection of TIME cells with siControl or siMondoA, cells were Mock- or KSHV-infected and overlaid with replete media in the presence or absence of αKG. YOYO-1 or SytoGreen was added to the media to monitor relative fluorescence for cell death or total cells, respectively. Forty-eight hpi (72 hours post transfection), cells were scanned on the Typhoon. Data shown is the average fold change in relative fluorescence of KSHV over mock cells (YOYO-1 positive cells/SytoGreen24 positive cells) from two independent experiments. Error bars represent the SEM. A p < 0.05 is denoted by one asterisk and “ns” is shown where two averages are not significantly different.
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ppat.1005052.g007: MondoA regulation of glutaminolysis is required for the survival of endothelial cells latently infected with KSHV.(A) TIME cells were transfected with siControl or siMondoA and then Mock- or KSHV-infected 24 hours post transfection. Whole-cell lysates were harvested at 48 hpi. Lysates were subjected to immunoblot analysis for MondoA, Mlx, SLC1A5 and TXNIP. KSHV infection elevates levels of all four proteins and loss of MondoA reduces the observed increase in protein expression. The standard γ-tubulin was included as a loading control. (B) Twenty-four hours post transfection of TIME cells with siControl or siMondoA, cells were Mock- or KSHV-infected and overlaid with replete media in the presence or absence of αKG. YOYO-1 or SytoGreen was added to the media to monitor relative fluorescence for cell death or total cells, respectively. Forty-eight hpi (72 hours post transfection), cells were scanned on the Typhoon. Data shown is the average fold change in relative fluorescence of KSHV over mock cells (YOYO-1 positive cells/SytoGreen24 positive cells) from two independent experiments. Error bars represent the SEM. A p < 0.05 is denoted by one asterisk and “ns” is shown where two averages are not significantly different.
Mentions: SLC1A5 is directly regulated by the nutrient-sensing Myc extended network member MondoA in many human cancer cells[18]. To determine if MondoA controls SLC1A5 expression during latent KSHV infection of endothelial cells, we examined the expression of SLC1A5 upon siRNA knockdown of MondoA in mock- and KSHV-infected endothelial cells. MondoA protein expression was significantly reduced in both mock and KSHV-infected TIME cells transfected with a mix of four siRNAs specific for MondoA (siMondoA), as compared to cells transfected with a scrambled non-target control (siControl) (Fig 7A). While SLC1A5 protein levels are elevated by KSHV infection, loss of MondoA results in a reduction in detected SLC1A5 in all samples. Additionally, protein levels of Mlx, a co-stabilized MondoA binding partner[18], and TXNIP, a known downstream target of MondoA/Mlx regulation, are also reduced upon loss of MondoA. These data support the hypothesis that MondoA is directly regulating SLC1A5, the major glutamine transporter, during latent KSHV infection of endothelial cells.

Bottom Line: Kaposi's Sarcoma-associated Herpesvirus (KSHV) is the etiologic agent of Kaposi's Sarcoma (KS).Knockdown of MondoA inhibits expression of both Mlx and SLC1A5 and induces a significant increase in cell death of only cells latently infected with KSHV, again, fully rescued by the supplementation of αKG.These findings expand our understanding of the required metabolic pathways that are activated during latent KSHV infection of endothelial cells, and demonstrate a novel role for the extended Myc-regulatory network, specifically MondoA, during latent KSHV infection.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology Program, University of Washington, Seattle, Washington, United States of America; Department of Microbiology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Kaposi's Sarcoma-associated Herpesvirus (KSHV) is the etiologic agent of Kaposi's Sarcoma (KS). KSHV establishes a predominantly latent infection in the main KS tumor cell type, the spindle cell, which is of endothelial cell origin. KSHV requires the induction of multiple metabolic pathways, including glycolysis and fatty acid synthesis, for the survival of latently infected endothelial cells. Here we demonstrate that latent KSHV infection leads to increased levels of intracellular glutamine and enhanced glutamine uptake. Depletion of glutamine from the culture media leads to a significant increase in apoptotic cell death in latently infected endothelial cells, but not in their mock-infected counterparts. In cancer cells, glutamine is often required for glutaminolysis to provide intermediates for the tri-carboxylic acid (TCA) cycle and support for the production of biosynthetic and bioenergetic precursors. In the absence of glutamine, the TCA cycle intermediates alpha-ketoglutarate (αKG) and pyruvate prevent the death of latently infected cells. Targeted drug inhibition of glutaminolysis also induces increased cell death in latently infected cells. KSHV infection of endothelial cells induces protein expression of the glutamine transporter, SLC1A5. Chemical inhibition of SLC1A5, or knockdown by siRNA, leads to similar cell death rates as glutamine deprivation and, similarly, can be rescued by αKG. KSHV also induces expression of the heterodimeric transcription factors c-Myc-Max and related heterodimer MondoA-Mlx. Knockdown of MondoA inhibits expression of both Mlx and SLC1A5 and induces a significant increase in cell death of only cells latently infected with KSHV, again, fully rescued by the supplementation of αKG. Therefore, during latent infection of endothelial cells, KSHV activates and requires the Myc/MondoA-network to upregulate the glutamine transporter, SLC1A5, leading to increased glutamine uptake for glutaminolysis. These findings expand our understanding of the required metabolic pathways that are activated during latent KSHV infection of endothelial cells, and demonstrate a novel role for the extended Myc-regulatory network, specifically MondoA, during latent KSHV infection.

No MeSH data available.


Related in: MedlinePlus