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Kaposin-B enhances the PROX1 mRNA stability during lymphatic reprogramming of vascular endothelial cells by Kaposi's sarcoma herpes virus.

Yoo J, Kang J, Lee HN, Aguilar B, Kafka D, Lee S, Choi I, Lee J, Ramu S, Haas J, Koh CJ, Hong YK - PLoS Pathog. (2010)

Bottom Line: Here, we demonstrate that the KSHV latent gene kaposin-B enhances the PROX1 mRNA stability and plays an important role in KSHV-mediated PROX1 upregulation.We found that PROX1 mRNA contains a canonical AU-rich element (ARE) in its 3'-untranslated region that promotes PROX1 mRNA turnover and that kaposin-B stimulates cytoplasmic accumulation of the ARE-binding protein HuR through activation of the p38/MK2 pathway.Together, our study demonstrates that kaposin-B plays a key role in PROX1 upregulation during lymphatic reprogramming of blood vascular endothelial cells by KSHV.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Kaposi's sarcoma (KS) is the most common cancer among HIV-positive patients. Histogenetic origin of KS has long been elusive due to a mixed expression of both blood and lymphatic endothelial markers in KS tumor cells. However, we and others discovered that Kaposi's sarcoma herpes virus (KSHV) induces lymphatic reprogramming of blood vascular endothelial cells by upregulating PROX1, which functions as the master regulator for lymphatic endothelial differentiation. Here, we demonstrate that the KSHV latent gene kaposin-B enhances the PROX1 mRNA stability and plays an important role in KSHV-mediated PROX1 upregulation. We found that PROX1 mRNA contains a canonical AU-rich element (ARE) in its 3'-untranslated region that promotes PROX1 mRNA turnover and that kaposin-B stimulates cytoplasmic accumulation of the ARE-binding protein HuR through activation of the p38/MK2 pathway. Moreover, HuR binds to and stabilizes PROX1 mRNA through its ARE and is necessary for KSHV-mediated PROX1 mRNA stabilization. Together, our study demonstrates that kaposin-B plays a key role in PROX1 upregulation during lymphatic reprogramming of blood vascular endothelial cells by KSHV.

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Kaposin-B upregulates PROX1 by promoting its mRNA stability through HuR.(A) PROX1 mRNA stability is increased by HuR and kaposin-B. LECs were transfected with a control (CTR), a HuR-expressing (HuR) or a kaposin B-expressing (kapB) vector for 16-hours and then treated with Actinomycin D (ActD) (2 µg/ml) for the indicated length of time. Total RNA was isolated and analyzed for PROX1 mRNA level by qRT-PCR analyses. (B,C) HuR is required for the kaposin-B-mediated PROX1 upregulation in LECs. LECs were transfected with a control (CTR) or a FLAG-tagged kaposin B-expressing (kapB) vector. After 16 hours, the control or kaposin-B-expressing cells were divided into two groups and then transfected again with siRNA against luciferase (siCTR) or HuR (siHuR). Total RNA and whole cell lysate was harvested from each group after 16-hours and subjected to qRT-PCR (B) or western (C) analyses. (D) Kaposin-B-mediated increase of PROX1 stability was abrogated by inhibition of HuR expression. LECs overexpressing kaposin-B were transfected with luciferase siRNA (kapB+siCTR) or HuR siRNA (kapB+siHuR) for 16-hours and then treated with Actinomycin D (ActD) (2 µg/ml). Total RNA was isolated at the indicated time points and analyzed for PROX1 mRNA level by qRT-PCR analyses. Similar results were obtained from three independent experiments and the error bars present standard deviations (SD) in a representative experiment. Asterisks in panels A &D present p-value less than 0.05.
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ppat-1001046-g005: Kaposin-B upregulates PROX1 by promoting its mRNA stability through HuR.(A) PROX1 mRNA stability is increased by HuR and kaposin-B. LECs were transfected with a control (CTR), a HuR-expressing (HuR) or a kaposin B-expressing (kapB) vector for 16-hours and then treated with Actinomycin D (ActD) (2 µg/ml) for the indicated length of time. Total RNA was isolated and analyzed for PROX1 mRNA level by qRT-PCR analyses. (B,C) HuR is required for the kaposin-B-mediated PROX1 upregulation in LECs. LECs were transfected with a control (CTR) or a FLAG-tagged kaposin B-expressing (kapB) vector. After 16 hours, the control or kaposin-B-expressing cells were divided into two groups and then transfected again with siRNA against luciferase (siCTR) or HuR (siHuR). Total RNA and whole cell lysate was harvested from each group after 16-hours and subjected to qRT-PCR (B) or western (C) analyses. (D) Kaposin-B-mediated increase of PROX1 stability was abrogated by inhibition of HuR expression. LECs overexpressing kaposin-B were transfected with luciferase siRNA (kapB+siCTR) or HuR siRNA (kapB+siHuR) for 16-hours and then treated with Actinomycin D (ActD) (2 µg/ml). Total RNA was isolated at the indicated time points and analyzed for PROX1 mRNA level by qRT-PCR analyses. Similar results were obtained from three independent experiments and the error bars present standard deviations (SD) in a representative experiment. Asterisks in panels A &D present p-value less than 0.05.

Mentions: Our findings of kaposin-B-induced PROX1 upregulation and HuR-binding to PROX1-ARE directed us to ask whether HuR and/or kaposin-B upregulate PROX1 by enhancing PROX1 mRNA stability. Toward this question, we overexpressed HuR or kaposin-B in primary LECs and quantified the steady-state level of PROX1 mRNA by qRT-PCR. Indeed, the ectopic expression of HuR or kaposin-B delayed the turnover of PROX1 mRNA in LECs and increased the half-life of PROX1 mRNA from ∼60 minutes in the control LECs to ∼180 minutes in LECs overexpressing HuR or kaposin-B (Figure 5A). We then asked whether HuR is required for kaposin-B-mediated PROX1 upregulation by knockdown of HuR in kaposin-B-expressing LECs. We found that HuR-knockdown significantly inhibited kaposin-B-mediated upregulation of PROX1 mRNA and protein determined by qRT-PCR and western analyses, respectively (Figure 5B&C). Moreover, we confirmed that this reduction in kaposin-B-mediated PROX1 upregulation is due to a decrease in PROX1 mRNA stability upon knockdown of HuR (Figure 5D). Together, our data demonstrate that kaposin-B upregulates PROX1 by promoting its mRNA stability through HuR.


Kaposin-B enhances the PROX1 mRNA stability during lymphatic reprogramming of vascular endothelial cells by Kaposi's sarcoma herpes virus.

Yoo J, Kang J, Lee HN, Aguilar B, Kafka D, Lee S, Choi I, Lee J, Ramu S, Haas J, Koh CJ, Hong YK - PLoS Pathog. (2010)

Kaposin-B upregulates PROX1 by promoting its mRNA stability through HuR.(A) PROX1 mRNA stability is increased by HuR and kaposin-B. LECs were transfected with a control (CTR), a HuR-expressing (HuR) or a kaposin B-expressing (kapB) vector for 16-hours and then treated with Actinomycin D (ActD) (2 µg/ml) for the indicated length of time. Total RNA was isolated and analyzed for PROX1 mRNA level by qRT-PCR analyses. (B,C) HuR is required for the kaposin-B-mediated PROX1 upregulation in LECs. LECs were transfected with a control (CTR) or a FLAG-tagged kaposin B-expressing (kapB) vector. After 16 hours, the control or kaposin-B-expressing cells were divided into two groups and then transfected again with siRNA against luciferase (siCTR) or HuR (siHuR). Total RNA and whole cell lysate was harvested from each group after 16-hours and subjected to qRT-PCR (B) or western (C) analyses. (D) Kaposin-B-mediated increase of PROX1 stability was abrogated by inhibition of HuR expression. LECs overexpressing kaposin-B were transfected with luciferase siRNA (kapB+siCTR) or HuR siRNA (kapB+siHuR) for 16-hours and then treated with Actinomycin D (ActD) (2 µg/ml). Total RNA was isolated at the indicated time points and analyzed for PROX1 mRNA level by qRT-PCR analyses. Similar results were obtained from three independent experiments and the error bars present standard deviations (SD) in a representative experiment. Asterisks in panels A &D present p-value less than 0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2921153&req=5

ppat-1001046-g005: Kaposin-B upregulates PROX1 by promoting its mRNA stability through HuR.(A) PROX1 mRNA stability is increased by HuR and kaposin-B. LECs were transfected with a control (CTR), a HuR-expressing (HuR) or a kaposin B-expressing (kapB) vector for 16-hours and then treated with Actinomycin D (ActD) (2 µg/ml) for the indicated length of time. Total RNA was isolated and analyzed for PROX1 mRNA level by qRT-PCR analyses. (B,C) HuR is required for the kaposin-B-mediated PROX1 upregulation in LECs. LECs were transfected with a control (CTR) or a FLAG-tagged kaposin B-expressing (kapB) vector. After 16 hours, the control or kaposin-B-expressing cells were divided into two groups and then transfected again with siRNA against luciferase (siCTR) or HuR (siHuR). Total RNA and whole cell lysate was harvested from each group after 16-hours and subjected to qRT-PCR (B) or western (C) analyses. (D) Kaposin-B-mediated increase of PROX1 stability was abrogated by inhibition of HuR expression. LECs overexpressing kaposin-B were transfected with luciferase siRNA (kapB+siCTR) or HuR siRNA (kapB+siHuR) for 16-hours and then treated with Actinomycin D (ActD) (2 µg/ml). Total RNA was isolated at the indicated time points and analyzed for PROX1 mRNA level by qRT-PCR analyses. Similar results were obtained from three independent experiments and the error bars present standard deviations (SD) in a representative experiment. Asterisks in panels A &D present p-value less than 0.05.
Mentions: Our findings of kaposin-B-induced PROX1 upregulation and HuR-binding to PROX1-ARE directed us to ask whether HuR and/or kaposin-B upregulate PROX1 by enhancing PROX1 mRNA stability. Toward this question, we overexpressed HuR or kaposin-B in primary LECs and quantified the steady-state level of PROX1 mRNA by qRT-PCR. Indeed, the ectopic expression of HuR or kaposin-B delayed the turnover of PROX1 mRNA in LECs and increased the half-life of PROX1 mRNA from ∼60 minutes in the control LECs to ∼180 minutes in LECs overexpressing HuR or kaposin-B (Figure 5A). We then asked whether HuR is required for kaposin-B-mediated PROX1 upregulation by knockdown of HuR in kaposin-B-expressing LECs. We found that HuR-knockdown significantly inhibited kaposin-B-mediated upregulation of PROX1 mRNA and protein determined by qRT-PCR and western analyses, respectively (Figure 5B&C). Moreover, we confirmed that this reduction in kaposin-B-mediated PROX1 upregulation is due to a decrease in PROX1 mRNA stability upon knockdown of HuR (Figure 5D). Together, our data demonstrate that kaposin-B upregulates PROX1 by promoting its mRNA stability through HuR.

Bottom Line: Here, we demonstrate that the KSHV latent gene kaposin-B enhances the PROX1 mRNA stability and plays an important role in KSHV-mediated PROX1 upregulation.We found that PROX1 mRNA contains a canonical AU-rich element (ARE) in its 3'-untranslated region that promotes PROX1 mRNA turnover and that kaposin-B stimulates cytoplasmic accumulation of the ARE-binding protein HuR through activation of the p38/MK2 pathway.Together, our study demonstrates that kaposin-B plays a key role in PROX1 upregulation during lymphatic reprogramming of blood vascular endothelial cells by KSHV.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Kaposi's sarcoma (KS) is the most common cancer among HIV-positive patients. Histogenetic origin of KS has long been elusive due to a mixed expression of both blood and lymphatic endothelial markers in KS tumor cells. However, we and others discovered that Kaposi's sarcoma herpes virus (KSHV) induces lymphatic reprogramming of blood vascular endothelial cells by upregulating PROX1, which functions as the master regulator for lymphatic endothelial differentiation. Here, we demonstrate that the KSHV latent gene kaposin-B enhances the PROX1 mRNA stability and plays an important role in KSHV-mediated PROX1 upregulation. We found that PROX1 mRNA contains a canonical AU-rich element (ARE) in its 3'-untranslated region that promotes PROX1 mRNA turnover and that kaposin-B stimulates cytoplasmic accumulation of the ARE-binding protein HuR through activation of the p38/MK2 pathway. Moreover, HuR binds to and stabilizes PROX1 mRNA through its ARE and is necessary for KSHV-mediated PROX1 mRNA stabilization. Together, our study demonstrates that kaposin-B plays a key role in PROX1 upregulation during lymphatic reprogramming of blood vascular endothelial cells by KSHV.

Show MeSH
Related in: MedlinePlus