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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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Related in: MedlinePlus

MK2 stimulated by kaposin-B activates the cytoplasmic accumulation of HuR protein and upregulates PROX1 mRNA stability.(A) Activation of p38 and MK2 by kaposin-B in LECs. LECs were transfected with a control (CTR) or a FLAG-tagged kaposin B-expressing vector (FLAG-kapB) for 16-hours and protein level of PROX1, FLAG-kaposin-B (FLAG), p38, phospho-p38 (P-p38), MK2, phospho-MK2 (P-MK2) and β-actin was determined by western blot analyses. (B) MK2 activation results in cytoplasmic accumulation of HuR protein. A control (CTR), a wild type-MK2 (WT), a constitutively active-MK2 (EE), or a dominant negative-MK2 (K76R) vector was transfected into LECs for 16 hours and the nuclear (N) and cytoplasmic (C) fractions were isolated and subjected to western blot analyses against HuR, lamin A/C (nuclear marker) and tubulin (cytoplasm marker). (C) HuR is required for MK2-mediated PROX1 upregulation. Expression of PROX1 protein was determined by western blot analyses in LECs that were transfected with a control (CTR), a wild type-MK2 (WT), a constitutively active-MK2 (EE) or a dominant negative-MK2 (K76R) vector. After 16 hours, transfected cells were divided into two groups and then transfected again with either luciferase siRNA (siCTR) or HuR siRNA (siHuR). Protein level of PROX1, Myc-tag, HuR and β-actin was determined after 16 hours. (D) Relative intensity of PROX1 bands in panel C was measured and charted in a graph. (E) MK2-mediated PROX1 upregulation is due to an increased expression of PROX1 mRNA. The steady-state level of PROX1 mRNA from the same set of experiment as panel (C) was determined by qRT-PCR. (F) MK2 activation increases Prox1 mRNA stability. A control (CTR) or a constitutive-MK2 (MK2 (EE)) vector was transfected into LECs for 16 hours and the steady-state level of PROX1 mRNA was determined by qRT-PCR analyses at the indicated time point (minutes) after Actinomycin-D (ActD) (2 µg/ml) treatment. (G) HuR is necessary for MK2-induced PROX1 stability. Constitutively active MK2 (EE) was overexpressed in LECs for 16 hours and the cells were then transfected again with luciferase siRNA (MK2 (EE)+siCTR) or HuR siRNA (MK2 (EE)+siHuR) for 16-hours, followed by Actinomycin-D (ActD) administration (2 µg/ml). PROX1 mRNA level at the indicated time points was determined by qRT-PCR analyses. Data are represented by mean and standard deviation (SD) and three independent experiments were performed to yield similar results. Asterisks in panels F and G present p-value less than 0.05.
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ppat-1001046-g007: MK2 stimulated by kaposin-B activates the cytoplasmic accumulation of HuR protein and upregulates PROX1 mRNA stability.(A) Activation of p38 and MK2 by kaposin-B in LECs. LECs were transfected with a control (CTR) or a FLAG-tagged kaposin B-expressing vector (FLAG-kapB) for 16-hours and protein level of PROX1, FLAG-kaposin-B (FLAG), p38, phospho-p38 (P-p38), MK2, phospho-MK2 (P-MK2) and β-actin was determined by western blot analyses. (B) MK2 activation results in cytoplasmic accumulation of HuR protein. A control (CTR), a wild type-MK2 (WT), a constitutively active-MK2 (EE), or a dominant negative-MK2 (K76R) vector was transfected into LECs for 16 hours and the nuclear (N) and cytoplasmic (C) fractions were isolated and subjected to western blot analyses against HuR, lamin A/C (nuclear marker) and tubulin (cytoplasm marker). (C) HuR is required for MK2-mediated PROX1 upregulation. Expression of PROX1 protein was determined by western blot analyses in LECs that were transfected with a control (CTR), a wild type-MK2 (WT), a constitutively active-MK2 (EE) or a dominant negative-MK2 (K76R) vector. After 16 hours, transfected cells were divided into two groups and then transfected again with either luciferase siRNA (siCTR) or HuR siRNA (siHuR). Protein level of PROX1, Myc-tag, HuR and β-actin was determined after 16 hours. (D) Relative intensity of PROX1 bands in panel C was measured and charted in a graph. (E) MK2-mediated PROX1 upregulation is due to an increased expression of PROX1 mRNA. The steady-state level of PROX1 mRNA from the same set of experiment as panel (C) was determined by qRT-PCR. (F) MK2 activation increases Prox1 mRNA stability. A control (CTR) or a constitutive-MK2 (MK2 (EE)) vector was transfected into LECs for 16 hours and the steady-state level of PROX1 mRNA was determined by qRT-PCR analyses at the indicated time point (minutes) after Actinomycin-D (ActD) (2 µg/ml) treatment. (G) HuR is necessary for MK2-induced PROX1 stability. Constitutively active MK2 (EE) was overexpressed in LECs for 16 hours and the cells were then transfected again with luciferase siRNA (MK2 (EE)+siCTR) or HuR siRNA (MK2 (EE)+siHuR) for 16-hours, followed by Actinomycin-D (ActD) administration (2 µg/ml). PROX1 mRNA level at the indicated time points was determined by qRT-PCR analyses. Data are represented by mean and standard deviation (SD) and three independent experiments were performed to yield similar results. Asterisks in panels F and G present p-value less than 0.05.

Mentions: Kaposin-B has been shown to activate the p38/MK2 pathway and stabilize various cytokine mRNAs [57], [58]. We further examined this previous observation in LECs and found that the ectopic expression of kaposin-B activated phosphorylation of p38 and MK2 proteins (Figure 7A). We then investigated whether activation of the p38/MK2 pathway is required for kaposin-B-mediated PROX1 upregulation. Notably, previous studies have shown that p38 MAPK promotes cytoplasmic accumulation of HuR in different cell types [70], [71], [72]. Therefore, we asked if activated MK2 can promote cytoplasmic localization of HuR protein in LECs and transfected LECs with vectors expressing a wild type, constitutively active (EE) or dominant negative (K76R) form of MK2 protein [73]. We found that whereas the constitutively active (EE) MK2 protein stimulated cytoplasmic accumulation of HuR, wild type or dominant negative MK2 protein did not (Figure 7B). Moreover, the expression of PROX1 was upregulated by constitutively active (EE) MK2 protein, but not by wild type or dominant negative MK2 protein, in LECs determined by western analyses (Figure 7C,D). Importantly, this upregulation of PROX1 by MK2 (EE) protein was abrogated by siRNA-mediated knockdown of HuR (Figure 7C,D), indicating that HuR is required for the kaposin-B/p38/MK2 pathway-mediated PROX1 upregulation. We also confirmed these findings by using quantitative qRT-PCR measuring PROX1 mRNA level (Figure 7E). Interestingly, we found that HuR knockdown slightly decreases PROX1 expression compared to control siRNA (Figure 7D,E). We think that this is because HuR protein is present at a basal level in the cytoplasm of LECs (Figure 6B) and may stabilize PROX1 mRNA under the normal condition and thus knockdown of HuR resulted in decrease of PROX1 expression. This speculation is supported by our endogenous HuR co-immunoprecipitation data (Figure 4D) demonstrating a stable complex formation between HuR protein and PROX1 mRNA in normal primary LECs.


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)

MK2 stimulated by kaposin-B activates the cytoplasmic accumulation of HuR protein and upregulates PROX1 mRNA stability.(A) Activation of p38 and MK2 by kaposin-B in LECs. LECs were transfected with a control (CTR) or a FLAG-tagged kaposin B-expressing vector (FLAG-kapB) for 16-hours and protein level of PROX1, FLAG-kaposin-B (FLAG), p38, phospho-p38 (P-p38), MK2, phospho-MK2 (P-MK2) and β-actin was determined by western blot analyses. (B) MK2 activation results in cytoplasmic accumulation of HuR protein. A control (CTR), a wild type-MK2 (WT), a constitutively active-MK2 (EE), or a dominant negative-MK2 (K76R) vector was transfected into LECs for 16 hours and the nuclear (N) and cytoplasmic (C) fractions were isolated and subjected to western blot analyses against HuR, lamin A/C (nuclear marker) and tubulin (cytoplasm marker). (C) HuR is required for MK2-mediated PROX1 upregulation. Expression of PROX1 protein was determined by western blot analyses in LECs that were transfected with a control (CTR), a wild type-MK2 (WT), a constitutively active-MK2 (EE) or a dominant negative-MK2 (K76R) vector. After 16 hours, transfected cells were divided into two groups and then transfected again with either luciferase siRNA (siCTR) or HuR siRNA (siHuR). Protein level of PROX1, Myc-tag, HuR and β-actin was determined after 16 hours. (D) Relative intensity of PROX1 bands in panel C was measured and charted in a graph. (E) MK2-mediated PROX1 upregulation is due to an increased expression of PROX1 mRNA. The steady-state level of PROX1 mRNA from the same set of experiment as panel (C) was determined by qRT-PCR. (F) MK2 activation increases Prox1 mRNA stability. A control (CTR) or a constitutive-MK2 (MK2 (EE)) vector was transfected into LECs for 16 hours and the steady-state level of PROX1 mRNA was determined by qRT-PCR analyses at the indicated time point (minutes) after Actinomycin-D (ActD) (2 µg/ml) treatment. (G) HuR is necessary for MK2-induced PROX1 stability. Constitutively active MK2 (EE) was overexpressed in LECs for 16 hours and the cells were then transfected again with luciferase siRNA (MK2 (EE)+siCTR) or HuR siRNA (MK2 (EE)+siHuR) for 16-hours, followed by Actinomycin-D (ActD) administration (2 µg/ml). PROX1 mRNA level at the indicated time points was determined by qRT-PCR analyses. Data are represented by mean and standard deviation (SD) and three independent experiments were performed to yield similar results. Asterisks in panels F and G 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-g007: MK2 stimulated by kaposin-B activates the cytoplasmic accumulation of HuR protein and upregulates PROX1 mRNA stability.(A) Activation of p38 and MK2 by kaposin-B in LECs. LECs were transfected with a control (CTR) or a FLAG-tagged kaposin B-expressing vector (FLAG-kapB) for 16-hours and protein level of PROX1, FLAG-kaposin-B (FLAG), p38, phospho-p38 (P-p38), MK2, phospho-MK2 (P-MK2) and β-actin was determined by western blot analyses. (B) MK2 activation results in cytoplasmic accumulation of HuR protein. A control (CTR), a wild type-MK2 (WT), a constitutively active-MK2 (EE), or a dominant negative-MK2 (K76R) vector was transfected into LECs for 16 hours and the nuclear (N) and cytoplasmic (C) fractions were isolated and subjected to western blot analyses against HuR, lamin A/C (nuclear marker) and tubulin (cytoplasm marker). (C) HuR is required for MK2-mediated PROX1 upregulation. Expression of PROX1 protein was determined by western blot analyses in LECs that were transfected with a control (CTR), a wild type-MK2 (WT), a constitutively active-MK2 (EE) or a dominant negative-MK2 (K76R) vector. After 16 hours, transfected cells were divided into two groups and then transfected again with either luciferase siRNA (siCTR) or HuR siRNA (siHuR). Protein level of PROX1, Myc-tag, HuR and β-actin was determined after 16 hours. (D) Relative intensity of PROX1 bands in panel C was measured and charted in a graph. (E) MK2-mediated PROX1 upregulation is due to an increased expression of PROX1 mRNA. The steady-state level of PROX1 mRNA from the same set of experiment as panel (C) was determined by qRT-PCR. (F) MK2 activation increases Prox1 mRNA stability. A control (CTR) or a constitutive-MK2 (MK2 (EE)) vector was transfected into LECs for 16 hours and the steady-state level of PROX1 mRNA was determined by qRT-PCR analyses at the indicated time point (minutes) after Actinomycin-D (ActD) (2 µg/ml) treatment. (G) HuR is necessary for MK2-induced PROX1 stability. Constitutively active MK2 (EE) was overexpressed in LECs for 16 hours and the cells were then transfected again with luciferase siRNA (MK2 (EE)+siCTR) or HuR siRNA (MK2 (EE)+siHuR) for 16-hours, followed by Actinomycin-D (ActD) administration (2 µg/ml). PROX1 mRNA level at the indicated time points was determined by qRT-PCR analyses. Data are represented by mean and standard deviation (SD) and three independent experiments were performed to yield similar results. Asterisks in panels F and G present p-value less than 0.05.
Mentions: Kaposin-B has been shown to activate the p38/MK2 pathway and stabilize various cytokine mRNAs [57], [58]. We further examined this previous observation in LECs and found that the ectopic expression of kaposin-B activated phosphorylation of p38 and MK2 proteins (Figure 7A). We then investigated whether activation of the p38/MK2 pathway is required for kaposin-B-mediated PROX1 upregulation. Notably, previous studies have shown that p38 MAPK promotes cytoplasmic accumulation of HuR in different cell types [70], [71], [72]. Therefore, we asked if activated MK2 can promote cytoplasmic localization of HuR protein in LECs and transfected LECs with vectors expressing a wild type, constitutively active (EE) or dominant negative (K76R) form of MK2 protein [73]. We found that whereas the constitutively active (EE) MK2 protein stimulated cytoplasmic accumulation of HuR, wild type or dominant negative MK2 protein did not (Figure 7B). Moreover, the expression of PROX1 was upregulated by constitutively active (EE) MK2 protein, but not by wild type or dominant negative MK2 protein, in LECs determined by western analyses (Figure 7C,D). Importantly, this upregulation of PROX1 by MK2 (EE) protein was abrogated by siRNA-mediated knockdown of HuR (Figure 7C,D), indicating that HuR is required for the kaposin-B/p38/MK2 pathway-mediated PROX1 upregulation. We also confirmed these findings by using quantitative qRT-PCR measuring PROX1 mRNA level (Figure 7E). Interestingly, we found that HuR knockdown slightly decreases PROX1 expression compared to control siRNA (Figure 7D,E). We think that this is because HuR protein is present at a basal level in the cytoplasm of LECs (Figure 6B) and may stabilize PROX1 mRNA under the normal condition and thus knockdown of HuR resulted in decrease of PROX1 expression. This speculation is supported by our endogenous HuR co-immunoprecipitation data (Figure 4D) demonstrating a stable complex formation between HuR protein and PROX1 mRNA in normal primary LECs.

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