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Upregulation of xCT by KSHV-encoded microRNAs facilitates KSHV dissemination and persistence in an environment of oxidative stress.

Qin Z, Freitas E, Sullivan R, Mohan S, Bacelieri R, Branch D, Romano M, Kearney P, Oates J, Plaisance K, Renne R, Kaleeba J, Parsons C - PLoS Pathog. (2010)

Bottom Line: Upregulation of xCT, the inducible subunit of a membrane-bound amino acid transporter, replenishes intracellular glutathione stores to maintain cell viability in an environment of oxidative stress. xCT also serves as a fusion-entry receptor for the Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi's sarcoma (KS).Using gene transfer and whole virus infection experiments, we found that KSHV-encoded microRNAs (KSHV miRNAs) upregulate xCT expression by macrophages and endothelial cells, largely through miR-K12-11 suppression of BACH-1-a negative regulator of transcription recognizing antioxidant response elements within gene promoters.The clinical relevance of these findings is supported by our demonstration of increased xCT expression within more advanced human KS tumors containing a larger number of KSHV-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina, United States of America.

ABSTRACT
Upregulation of xCT, the inducible subunit of a membrane-bound amino acid transporter, replenishes intracellular glutathione stores to maintain cell viability in an environment of oxidative stress. xCT also serves as a fusion-entry receptor for the Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi's sarcoma (KS). Ongoing KSHV replication and infection of new cell targets is important for KS progression, but whether xCT regulation within the tumor microenvironment plays a role in KS pathogenesis has not been determined. Using gene transfer and whole virus infection experiments, we found that KSHV-encoded microRNAs (KSHV miRNAs) upregulate xCT expression by macrophages and endothelial cells, largely through miR-K12-11 suppression of BACH-1-a negative regulator of transcription recognizing antioxidant response elements within gene promoters. Correlative functional studies reveal that upregulation of xCT by KSHV miRNAs increases cell permissiveness for KSHV infection and protects infected cells from death induced by reactive nitrogen species (RNS). Interestingly, KSHV miRNAs simultaneously upregulate macrophage secretion of RNS, and biochemical inhibition of RNS secretion by macrophages significantly reduces their permissiveness for KSHV infection. The clinical relevance of these findings is supported by our demonstration of increased xCT expression within more advanced human KS tumors containing a larger number of KSHV-infected cells. Collectively, these data support a role for KSHV itself in promoting de novo KSHV infection and the survival of KSHV-infected, RNS-secreting cells in the tumor microenvironment through the induction of xCT.

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KSHV miRNAs enhance macrophage survival in an environment of oxidative stress through the upregulation of xCT.(A) RAW cells were treated with the indicated concentrations of SNAP or vehicle control for 12 h prior to nitrite quantification within culture supernatants. (B) Relative cell viability was determined for groups in (A) as described in Methods. (C) Cells were transfected with either control (n) or xCT-specific (x) siRNA and incubated with UV-K (KSHV−) or KSHV (KSHV+) 48 h later. (D) Cells were co-transfected with xCT siRNA and either control or miRNA-expressing vectors for 48 h, then incubated for an additional 12 h with SNAP prior to viability determinations. For all assays, error bars represent the S.E.M. for three independent experiments. * = p<0.05, * * = p<0.01.
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ppat-1000742-g007: KSHV miRNAs enhance macrophage survival in an environment of oxidative stress through the upregulation of xCT.(A) RAW cells were treated with the indicated concentrations of SNAP or vehicle control for 12 h prior to nitrite quantification within culture supernatants. (B) Relative cell viability was determined for groups in (A) as described in Methods. (C) Cells were transfected with either control (n) or xCT-specific (x) siRNA and incubated with UV-K (KSHV−) or KSHV (KSHV+) 48 h later. (D) Cells were co-transfected with xCT siRNA and either control or miRNA-expressing vectors for 48 h, then incubated for an additional 12 h with SNAP prior to viability determinations. For all assays, error bars represent the S.E.M. for three independent experiments. * = p<0.05, * * = p<0.01.

Mentions: To determine whether upregulation of xCT by KSHV miRNAs offers a protective mechanism for macrophages in an environment rich in RNS, we first established that provision of the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) [49] increased RNS concentrations within RAW cell culture supernatants and induced cell death in a dose-dependent manner (Fig. 7A and B). Subsequently, we found that either KSHV infection or overexpression of KSHV miRNAs significantly increased macrophage resistance to SNAP-induced cell death. Moreover, siRNA experiments confirmed that this effect was mediated primarily through the upregulation of xCT (Figs. 7C and D).


Upregulation of xCT by KSHV-encoded microRNAs facilitates KSHV dissemination and persistence in an environment of oxidative stress.

Qin Z, Freitas E, Sullivan R, Mohan S, Bacelieri R, Branch D, Romano M, Kearney P, Oates J, Plaisance K, Renne R, Kaleeba J, Parsons C - PLoS Pathog. (2010)

KSHV miRNAs enhance macrophage survival in an environment of oxidative stress through the upregulation of xCT.(A) RAW cells were treated with the indicated concentrations of SNAP or vehicle control for 12 h prior to nitrite quantification within culture supernatants. (B) Relative cell viability was determined for groups in (A) as described in Methods. (C) Cells were transfected with either control (n) or xCT-specific (x) siRNA and incubated with UV-K (KSHV−) or KSHV (KSHV+) 48 h later. (D) Cells were co-transfected with xCT siRNA and either control or miRNA-expressing vectors for 48 h, then incubated for an additional 12 h with SNAP prior to viability determinations. For all assays, error bars represent the S.E.M. for three independent experiments. * = p<0.05, * * = p<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2813276&req=5

ppat-1000742-g007: KSHV miRNAs enhance macrophage survival in an environment of oxidative stress through the upregulation of xCT.(A) RAW cells were treated with the indicated concentrations of SNAP or vehicle control for 12 h prior to nitrite quantification within culture supernatants. (B) Relative cell viability was determined for groups in (A) as described in Methods. (C) Cells were transfected with either control (n) or xCT-specific (x) siRNA and incubated with UV-K (KSHV−) or KSHV (KSHV+) 48 h later. (D) Cells were co-transfected with xCT siRNA and either control or miRNA-expressing vectors for 48 h, then incubated for an additional 12 h with SNAP prior to viability determinations. For all assays, error bars represent the S.E.M. for three independent experiments. * = p<0.05, * * = p<0.01.
Mentions: To determine whether upregulation of xCT by KSHV miRNAs offers a protective mechanism for macrophages in an environment rich in RNS, we first established that provision of the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) [49] increased RNS concentrations within RAW cell culture supernatants and induced cell death in a dose-dependent manner (Fig. 7A and B). Subsequently, we found that either KSHV infection or overexpression of KSHV miRNAs significantly increased macrophage resistance to SNAP-induced cell death. Moreover, siRNA experiments confirmed that this effect was mediated primarily through the upregulation of xCT (Figs. 7C and D).

Bottom Line: Upregulation of xCT, the inducible subunit of a membrane-bound amino acid transporter, replenishes intracellular glutathione stores to maintain cell viability in an environment of oxidative stress. xCT also serves as a fusion-entry receptor for the Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi's sarcoma (KS).Using gene transfer and whole virus infection experiments, we found that KSHV-encoded microRNAs (KSHV miRNAs) upregulate xCT expression by macrophages and endothelial cells, largely through miR-K12-11 suppression of BACH-1-a negative regulator of transcription recognizing antioxidant response elements within gene promoters.The clinical relevance of these findings is supported by our demonstration of increased xCT expression within more advanced human KS tumors containing a larger number of KSHV-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina, United States of America.

ABSTRACT
Upregulation of xCT, the inducible subunit of a membrane-bound amino acid transporter, replenishes intracellular glutathione stores to maintain cell viability in an environment of oxidative stress. xCT also serves as a fusion-entry receptor for the Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi's sarcoma (KS). Ongoing KSHV replication and infection of new cell targets is important for KS progression, but whether xCT regulation within the tumor microenvironment plays a role in KS pathogenesis has not been determined. Using gene transfer and whole virus infection experiments, we found that KSHV-encoded microRNAs (KSHV miRNAs) upregulate xCT expression by macrophages and endothelial cells, largely through miR-K12-11 suppression of BACH-1-a negative regulator of transcription recognizing antioxidant response elements within gene promoters. Correlative functional studies reveal that upregulation of xCT by KSHV miRNAs increases cell permissiveness for KSHV infection and protects infected cells from death induced by reactive nitrogen species (RNS). Interestingly, KSHV miRNAs simultaneously upregulate macrophage secretion of RNS, and biochemical inhibition of RNS secretion by macrophages significantly reduces their permissiveness for KSHV infection. The clinical relevance of these findings is supported by our demonstration of increased xCT expression within more advanced human KS tumors containing a larger number of KSHV-infected cells. Collectively, these data support a role for KSHV itself in promoting de novo KSHV infection and the survival of KSHV-infected, RNS-secreting cells in the tumor microenvironment through the induction of xCT.

Show MeSH
Related in: MedlinePlus