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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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miR-K12-11 suppresses BACH-1 expression and increases endothelial cell susceptibility to KSHV through upregulation of xCT.(A–G) HUVEC were incubated with vehicle control (DMSO), Msg, or Sul for 12 h followed by purified KSHV (K) using an MOI∼0.5–1. 16 h later, LANA IFA were performed as previously described. (A–D) Representative images from one of three independent experiments are shown. (H–K) HUVEC were transfected with control or miRNA-expressing vectors along with a 2'OMe RNA antagomir for miR-K12-11 or either control (n) or xCT-specific siRNA. (E,H) Relative LANA expression was determined as described in Methods. (F,I) qRT-PCR was used to determine relative xCT transcript expression. (G,J) qPCR was used to determine relative intracellular KSHV DNA content normalized to controls. (K) Western blots were used to identify BACH-1 protein expression and immunoreactivity quantified as previously described. For all assays, error bars represent the S.E.M. for three independent experiments. * = p<0.05, * * = p<0.01 (For Fig. H–J, comparisons are relative to either pc-miRNA or pc-miRNA+K).
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ppat-1000742-g005: miR-K12-11 suppresses BACH-1 expression and increases endothelial cell susceptibility to KSHV through upregulation of xCT.(A–G) HUVEC were incubated with vehicle control (DMSO), Msg, or Sul for 12 h followed by purified KSHV (K) using an MOI∼0.5–1. 16 h later, LANA IFA were performed as previously described. (A–D) Representative images from one of three independent experiments are shown. (H–K) HUVEC were transfected with control or miRNA-expressing vectors along with a 2'OMe RNA antagomir for miR-K12-11 or either control (n) or xCT-specific siRNA. (E,H) Relative LANA expression was determined as described in Methods. (F,I) qRT-PCR was used to determine relative xCT transcript expression. (G,J) qPCR was used to determine relative intracellular KSHV DNA content normalized to controls. (K) Western blots were used to identify BACH-1 protein expression and immunoreactivity quantified as previously described. For all assays, error bars represent the S.E.M. for three independent experiments. * = p<0.05, * * = p<0.01 (For Fig. H–J, comparisons are relative to either pc-miRNA or pc-miRNA+K).

Mentions: Published data have confirmed direct targeting of BACH-1 by miR-K12-11 in human cells [36]. To validate our observations and to determine their broader significance for human cells with known relevance to KS pathogenesis, we repeated our experiments using primary human umbilical vein endothelial cells (HUVEC). We found that Msg, Sul or KSHV miRNA transfection significantly increased xCT transcript expression and, based on IFA, KSHV episome copy number per cell following subsequent de novo infection (Fig. 5A–J). In contrast to what was observed for RAW cells, IFA indicated that the total number of infected HUVEC remained unchanged with these interventions (Fig. S1). In addition, either direct suppression of xCT by siRNA or concurrent inhibition of miR-K12-11 reduced xCT expression and intracellular viral load in KSHV miRNA transfectants (Fig. 5H–J). Collective expression of KSHV miRNAs also reduced BACH-1 expression in HUVEC, an effect suppressed with concurrent inhibition of miR-K12-11 (Fig. 5K).


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)

miR-K12-11 suppresses BACH-1 expression and increases endothelial cell susceptibility to KSHV through upregulation of xCT.(A–G) HUVEC were incubated with vehicle control (DMSO), Msg, or Sul for 12 h followed by purified KSHV (K) using an MOI∼0.5–1. 16 h later, LANA IFA were performed as previously described. (A–D) Representative images from one of three independent experiments are shown. (H–K) HUVEC were transfected with control or miRNA-expressing vectors along with a 2'OMe RNA antagomir for miR-K12-11 or either control (n) or xCT-specific siRNA. (E,H) Relative LANA expression was determined as described in Methods. (F,I) qRT-PCR was used to determine relative xCT transcript expression. (G,J) qPCR was used to determine relative intracellular KSHV DNA content normalized to controls. (K) Western blots were used to identify BACH-1 protein expression and immunoreactivity quantified as previously described. For all assays, error bars represent the S.E.M. for three independent experiments. * = p<0.05, * * = p<0.01 (For Fig. H–J, comparisons are relative to either pc-miRNA or pc-miRNA+K).
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Related In: Results  -  Collection

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ppat-1000742-g005: miR-K12-11 suppresses BACH-1 expression and increases endothelial cell susceptibility to KSHV through upregulation of xCT.(A–G) HUVEC were incubated with vehicle control (DMSO), Msg, or Sul for 12 h followed by purified KSHV (K) using an MOI∼0.5–1. 16 h later, LANA IFA were performed as previously described. (A–D) Representative images from one of three independent experiments are shown. (H–K) HUVEC were transfected with control or miRNA-expressing vectors along with a 2'OMe RNA antagomir for miR-K12-11 or either control (n) or xCT-specific siRNA. (E,H) Relative LANA expression was determined as described in Methods. (F,I) qRT-PCR was used to determine relative xCT transcript expression. (G,J) qPCR was used to determine relative intracellular KSHV DNA content normalized to controls. (K) Western blots were used to identify BACH-1 protein expression and immunoreactivity quantified as previously described. For all assays, error bars represent the S.E.M. for three independent experiments. * = p<0.05, * * = p<0.01 (For Fig. H–J, comparisons are relative to either pc-miRNA or pc-miRNA+K).
Mentions: Published data have confirmed direct targeting of BACH-1 by miR-K12-11 in human cells [36]. To validate our observations and to determine their broader significance for human cells with known relevance to KS pathogenesis, we repeated our experiments using primary human umbilical vein endothelial cells (HUVEC). We found that Msg, Sul or KSHV miRNA transfection significantly increased xCT transcript expression and, based on IFA, KSHV episome copy number per cell following subsequent de novo infection (Fig. 5A–J). In contrast to what was observed for RAW cells, IFA indicated that the total number of infected HUVEC remained unchanged with these interventions (Fig. S1). In addition, either direct suppression of xCT by siRNA or concurrent inhibition of miR-K12-11 reduced xCT expression and intracellular viral load in KSHV miRNA transfectants (Fig. 5H–J). Collective expression of KSHV miRNAs also reduced BACH-1 expression in HUVEC, an effect suppressed with concurrent inhibition of miR-K12-11 (Fig. 5K).

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