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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

PROX1 mRNA has an unusually long 3′-untranslated region with a functional ARE that decreases its mRNA stability.(A) Alignment of 7-kb downstream genomic sequences of human PROX1 gene to human EST database identified numerous EST fragments (red lines) that are mapped to the regions. Locations of the stop-codon-containing exon 5, ARE and a poly-A signal sequence (pA) are marked. (B) Corresponding mouse Prox1 genomic sequence was also aligned against mouse EST database and the mapped ESTs are shown in read. Locations of the exon 5, ARE and a poly-A signal sequence (pA) are marked. (C) Cross-species sequence conservation analyses of the Prox1 3′-UTR from diverse primates, mammalian and vertebrates by using the University of California Santa Cruz (UCSC) Genome Brower revealed a high DNA sequence homology among various Prox1 3′-UTR. Thick black bar represents the conserved 3′-UTR of human PROX1. (D) Northern blot analyses showing human LEC PROX1 with an extended 3′-UTR. While lanes 1 and 2 were hybridized with a PROX1 open reading frame (ORF) probe, lanes 3, 4 and 5 respectively with the 3′-UTR probes P1, P2 and P3, of which locations are shown in panel A. (E) A 3′-RACE analysis for total RNA from primary human LECs demonstrates that human PROX1 mRNA terminates at 5,414-bp downstream from its stop codon. A putative poly-A signal sequence is marked with an asterisk. (F) Three highly conserved copies of the canonical AUUUA pentamer flanked by additional W (A or U) are present in the 3′-UTRs of dog, mouse, human, chicken and zebrafish Prox1 mRNAs. Asterisks mark the conserved bases. (G) A schematic diagram of the pTet-BBB vector constructs containing PROX1-ARE or c-fos-ARE in its 3′-UTR. Tet-promo, tetracycline-inducible promoter. (H) Northern blot analyses showing time-dependent decay of the β-globin mRNA containing no ARE (a negative control), PROX1-ARE or c-fos-ARE (a positive control) after tetracycline (doxycycline)-mediated shutdown of the transcription. β-globin DNA fragment was used as the probe and GAPDH mRNA was also shown as an internal loading control. (I) Intensity of the β-globin mRNA bands in panel H was quantified and normalized against GAPDH and the relative intensity was charted to show the stability of the β-globin mRNA with or without PROX1-ARE or c-fos-ARE.
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ppat-1001046-g003: PROX1 mRNA has an unusually long 3′-untranslated region with a functional ARE that decreases its mRNA stability.(A) Alignment of 7-kb downstream genomic sequences of human PROX1 gene to human EST database identified numerous EST fragments (red lines) that are mapped to the regions. Locations of the stop-codon-containing exon 5, ARE and a poly-A signal sequence (pA) are marked. (B) Corresponding mouse Prox1 genomic sequence was also aligned against mouse EST database and the mapped ESTs are shown in read. Locations of the exon 5, ARE and a poly-A signal sequence (pA) are marked. (C) Cross-species sequence conservation analyses of the Prox1 3′-UTR from diverse primates, mammalian and vertebrates by using the University of California Santa Cruz (UCSC) Genome Brower revealed a high DNA sequence homology among various Prox1 3′-UTR. Thick black bar represents the conserved 3′-UTR of human PROX1. (D) Northern blot analyses showing human LEC PROX1 with an extended 3′-UTR. While lanes 1 and 2 were hybridized with a PROX1 open reading frame (ORF) probe, lanes 3, 4 and 5 respectively with the 3′-UTR probes P1, P2 and P3, of which locations are shown in panel A. (E) A 3′-RACE analysis for total RNA from primary human LECs demonstrates that human PROX1 mRNA terminates at 5,414-bp downstream from its stop codon. A putative poly-A signal sequence is marked with an asterisk. (F) Three highly conserved copies of the canonical AUUUA pentamer flanked by additional W (A or U) are present in the 3′-UTRs of dog, mouse, human, chicken and zebrafish Prox1 mRNAs. Asterisks mark the conserved bases. (G) A schematic diagram of the pTet-BBB vector constructs containing PROX1-ARE or c-fos-ARE in its 3′-UTR. Tet-promo, tetracycline-inducible promoter. (H) Northern blot analyses showing time-dependent decay of the β-globin mRNA containing no ARE (a negative control), PROX1-ARE or c-fos-ARE (a positive control) after tetracycline (doxycycline)-mediated shutdown of the transcription. β-globin DNA fragment was used as the probe and GAPDH mRNA was also shown as an internal loading control. (I) Intensity of the β-globin mRNA bands in panel H was quantified and normalized against GAPDH and the relative intensity was charted to show the stability of the β-globin mRNA with or without PROX1-ARE or c-fos-ARE.

Mentions: We then set out to investigate the molecular mechanism underlying kaposin-B-induced PROX1 upregulation. Kaposin-B has been demonstrated to upregulate various cytokine genes by stabilizing their mRNAs through AREs located in their 3′-UTRs [57]. We thus examined the mRNA structure of the PROX1 gene. Although the open reading frame (ORF) of human or mouse PROX1 gene is about 2.2 kb long and encodes a 737-amino acid-long protein, reported northern blot analyses revealed that PROX1 transcript was as large as 8-kb in various tissues [60], [61], [62], suggesting that the PROX1 transcript has a long UTR at the 5′- and/or 3′ ends. In fact, we found that an 8-kb PROX1 transcript harboring an extended 3′-UTR has been annotated in a public genome database (ENST00000366958). However, the corresponding Prox1 transcript from mouse has not been annotated in the same public database. To further confirm the presence of PROX1 transcript with an extended 3′-UTR in both human and mouse, we aligned 7-kb downstream genomic sequences of human or mouse PROX1 against human or mouse expressed sequence tag (EST) databases and found that numerous EST sequences were mapped to the downstream of both human PROX1 and mouse Prox1 genes (Figure 3A&B), indicating that this region is indeed transcribed as a part of the fifth exon of PROX1 mRNA in both species. We then investigated sequence conservation of this 3′-UTR among Prox1 genes of other species. Analyses using a genome browser revealed a high DNA sequence homology in this extended 3′-UTR, especially in the second half, of the Prox1 genes from primates, placental mammals or vertebrates covering 48-speices [63] (Figure 3C).


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)

PROX1 mRNA has an unusually long 3′-untranslated region with a functional ARE that decreases its mRNA stability.(A) Alignment of 7-kb downstream genomic sequences of human PROX1 gene to human EST database identified numerous EST fragments (red lines) that are mapped to the regions. Locations of the stop-codon-containing exon 5, ARE and a poly-A signal sequence (pA) are marked. (B) Corresponding mouse Prox1 genomic sequence was also aligned against mouse EST database and the mapped ESTs are shown in read. Locations of the exon 5, ARE and a poly-A signal sequence (pA) are marked. (C) Cross-species sequence conservation analyses of the Prox1 3′-UTR from diverse primates, mammalian and vertebrates by using the University of California Santa Cruz (UCSC) Genome Brower revealed a high DNA sequence homology among various Prox1 3′-UTR. Thick black bar represents the conserved 3′-UTR of human PROX1. (D) Northern blot analyses showing human LEC PROX1 with an extended 3′-UTR. While lanes 1 and 2 were hybridized with a PROX1 open reading frame (ORF) probe, lanes 3, 4 and 5 respectively with the 3′-UTR probes P1, P2 and P3, of which locations are shown in panel A. (E) A 3′-RACE analysis for total RNA from primary human LECs demonstrates that human PROX1 mRNA terminates at 5,414-bp downstream from its stop codon. A putative poly-A signal sequence is marked with an asterisk. (F) Three highly conserved copies of the canonical AUUUA pentamer flanked by additional W (A or U) are present in the 3′-UTRs of dog, mouse, human, chicken and zebrafish Prox1 mRNAs. Asterisks mark the conserved bases. (G) A schematic diagram of the pTet-BBB vector constructs containing PROX1-ARE or c-fos-ARE in its 3′-UTR. Tet-promo, tetracycline-inducible promoter. (H) Northern blot analyses showing time-dependent decay of the β-globin mRNA containing no ARE (a negative control), PROX1-ARE or c-fos-ARE (a positive control) after tetracycline (doxycycline)-mediated shutdown of the transcription. β-globin DNA fragment was used as the probe and GAPDH mRNA was also shown as an internal loading control. (I) Intensity of the β-globin mRNA bands in panel H was quantified and normalized against GAPDH and the relative intensity was charted to show the stability of the β-globin mRNA with or without PROX1-ARE or c-fos-ARE.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2921153&req=5

ppat-1001046-g003: PROX1 mRNA has an unusually long 3′-untranslated region with a functional ARE that decreases its mRNA stability.(A) Alignment of 7-kb downstream genomic sequences of human PROX1 gene to human EST database identified numerous EST fragments (red lines) that are mapped to the regions. Locations of the stop-codon-containing exon 5, ARE and a poly-A signal sequence (pA) are marked. (B) Corresponding mouse Prox1 genomic sequence was also aligned against mouse EST database and the mapped ESTs are shown in read. Locations of the exon 5, ARE and a poly-A signal sequence (pA) are marked. (C) Cross-species sequence conservation analyses of the Prox1 3′-UTR from diverse primates, mammalian and vertebrates by using the University of California Santa Cruz (UCSC) Genome Brower revealed a high DNA sequence homology among various Prox1 3′-UTR. Thick black bar represents the conserved 3′-UTR of human PROX1. (D) Northern blot analyses showing human LEC PROX1 with an extended 3′-UTR. While lanes 1 and 2 were hybridized with a PROX1 open reading frame (ORF) probe, lanes 3, 4 and 5 respectively with the 3′-UTR probes P1, P2 and P3, of which locations are shown in panel A. (E) A 3′-RACE analysis for total RNA from primary human LECs demonstrates that human PROX1 mRNA terminates at 5,414-bp downstream from its stop codon. A putative poly-A signal sequence is marked with an asterisk. (F) Three highly conserved copies of the canonical AUUUA pentamer flanked by additional W (A or U) are present in the 3′-UTRs of dog, mouse, human, chicken and zebrafish Prox1 mRNAs. Asterisks mark the conserved bases. (G) A schematic diagram of the pTet-BBB vector constructs containing PROX1-ARE or c-fos-ARE in its 3′-UTR. Tet-promo, tetracycline-inducible promoter. (H) Northern blot analyses showing time-dependent decay of the β-globin mRNA containing no ARE (a negative control), PROX1-ARE or c-fos-ARE (a positive control) after tetracycline (doxycycline)-mediated shutdown of the transcription. β-globin DNA fragment was used as the probe and GAPDH mRNA was also shown as an internal loading control. (I) Intensity of the β-globin mRNA bands in panel H was quantified and normalized against GAPDH and the relative intensity was charted to show the stability of the β-globin mRNA with or without PROX1-ARE or c-fos-ARE.
Mentions: We then set out to investigate the molecular mechanism underlying kaposin-B-induced PROX1 upregulation. Kaposin-B has been demonstrated to upregulate various cytokine genes by stabilizing their mRNAs through AREs located in their 3′-UTRs [57]. We thus examined the mRNA structure of the PROX1 gene. Although the open reading frame (ORF) of human or mouse PROX1 gene is about 2.2 kb long and encodes a 737-amino acid-long protein, reported northern blot analyses revealed that PROX1 transcript was as large as 8-kb in various tissues [60], [61], [62], suggesting that the PROX1 transcript has a long UTR at the 5′- and/or 3′ ends. In fact, we found that an 8-kb PROX1 transcript harboring an extended 3′-UTR has been annotated in a public genome database (ENST00000366958). However, the corresponding Prox1 transcript from mouse has not been annotated in the same public database. To further confirm the presence of PROX1 transcript with an extended 3′-UTR in both human and mouse, we aligned 7-kb downstream genomic sequences of human or mouse PROX1 against human or mouse expressed sequence tag (EST) databases and found that numerous EST sequences were mapped to the downstream of both human PROX1 and mouse Prox1 genes (Figure 3A&B), indicating that this region is indeed transcribed as a part of the fifth exon of PROX1 mRNA in both species. We then investigated sequence conservation of this 3′-UTR among Prox1 genes of other species. Analyses using a genome browser revealed a high DNA sequence homology in this extended 3′-UTR, especially in the second half, of the Prox1 genes from primates, placental mammals or vertebrates covering 48-speices [63] (Figure 3C).

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