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Opposing regulation of PROX1 by interleukin-3 receptor and NOTCH directs differential host cell fate reprogramming by Kaposi sarcoma herpes virus.

Yoo J, Lee HN, Choi I, Choi D, Chung HK, Kim KE, Lee S, Aguilar B, Kang J, Park E, Lee YS, Maeng YS, Kim NY, Koh CJ, Hong YK - PLoS Pathog. (2012)

Bottom Line: Moreover, PROX1 was found to be required to maintain HEY1 expression in LECs, establishing a reciprocal regulation between PROX1 and HEY1.Upon co-activation of IL3Rα and NOTCH, PROX1 was upregulated in BECs, but downregulated in LECs.Together, our study provides the molecular mechanism underlying the cell type-specific endothelial fate reprogramming by KSHV.

View Article: PubMed Central - PubMed

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

ABSTRACT
Lymphatic endothelial cells (LECs) are differentiated from blood vascular endothelial cells (BECs) during embryogenesis and this physiological cell fate specification is controlled by PROX1, the master regulator for lymphatic development. When Kaposi sarcoma herpes virus (KSHV) infects host cells, it activates the otherwise silenced embryonic endothelial differentiation program and reprograms their cell fates. Interestingly, previous studies demonstrated that KSHV drives BECs to acquire a partial lymphatic phenotype by upregulating PROX1 (forward reprogramming), but stimulates LECs to regain some BEC-signature genes by downregulating PROX1 (reverse reprogramming). Despite the significance of this KSHV-induced bidirectional cell fate reprogramming in KS pathogenesis, its underlying molecular mechanism remains undefined. Here, we report that IL3 receptor alpha (IL3Rα) and NOTCH play integral roles in the host cell type-specific regulation of PROX1 by KSHV. In BECs, KSHV upregulates IL3Rα and phosphorylates STAT5, which binds and activates the PROX1 promoter. In LECs, however, PROX1 was rather downregulated by KSHV-induced NOTCH signal via HEY1, which binds and represses the PROX1 promoter. Moreover, PROX1 was found to be required to maintain HEY1 expression in LECs, establishing a reciprocal regulation between PROX1 and HEY1. Upon co-activation of IL3Rα and NOTCH, PROX1 was upregulated in BECs, but downregulated in LECs. Together, our study provides the molecular mechanism underlying the cell type-specific endothelial fate reprogramming by KSHV.

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IL3Rα plays a key role in KSHV-mediated PROX1 upregulation.(A) Adenoviral expression of IL3Rα increased the expression of PROX1 mRNA and protein in BECs and HUVECs, but not in LECs based on qRT-PCR and western blot analyses. AdCTR, control adenovirus; AdIL3Rα, IL3Rα-expressing adenovirus. (B) The expression of various LEC-associated genes such as PDPN, VEGFR3, FGFR3, LYVE1, CDKN1C, ITGA1, PPL and SLC was determined by qRT-PCR in BECs or HUVECs transduced by a control (AdCTR) versus IL3Rα- (AdIL3Rα) adenovirus for 48 hours. The graph shows fold changes in expression of each gene by IL3Rα-expressing adenovirus over the control virus. (C) Inhibition of IL3Rα by siRNA or a neutralizing antibody partially abrogated the KSHV-mediated PROX1 upregulation in BECs and HUVECs, as determined by qRT-PCR (BECs and HUVECs) and western blot (BEC only) analyses. siCTR, siRNA for the firefly luciferase; siIL3Rα, siRNA for IL3Rα; anti-IL3Rα, neutralizing antibody against IL3Rα.
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ppat-1002770-g002: IL3Rα plays a key role in KSHV-mediated PROX1 upregulation.(A) Adenoviral expression of IL3Rα increased the expression of PROX1 mRNA and protein in BECs and HUVECs, but not in LECs based on qRT-PCR and western blot analyses. AdCTR, control adenovirus; AdIL3Rα, IL3Rα-expressing adenovirus. (B) The expression of various LEC-associated genes such as PDPN, VEGFR3, FGFR3, LYVE1, CDKN1C, ITGA1, PPL and SLC was determined by qRT-PCR in BECs or HUVECs transduced by a control (AdCTR) versus IL3Rα- (AdIL3Rα) adenovirus for 48 hours. The graph shows fold changes in expression of each gene by IL3Rα-expressing adenovirus over the control virus. (C) Inhibition of IL3Rα by siRNA or a neutralizing antibody partially abrogated the KSHV-mediated PROX1 upregulation in BECs and HUVECs, as determined by qRT-PCR (BECs and HUVECs) and western blot (BEC only) analyses. siCTR, siRNA for the firefly luciferase; siIL3Rα, siRNA for IL3Rα; anti-IL3Rα, neutralizing antibody against IL3Rα.

Mentions: Because IL-3 activates PROX1 expression in BECs and HUVECs, but not in LECs [18], we investigated whether overexpression of its receptor, IL3Rα, could upregulate PROX1 in each cell type. We found that adenoviral expression of IL3Rα resulted in a strong upregulation of PROX1 mRNA and protein in BECs and HUVECs, but not in LECs (Fig. 2A), suggesting that the PROX1-upregulating signal by IL3Rα is operative only in PROX1-deficient BECs and HUVECs and does not affect the already abundant expression of PROX1 in LECs. Furthermore, ectopic expression of IL3Rα in BECs and HUVECs resulted in upregulation of various LEC-signature genes such as podoplanin (PDPN), VEGFR3, FGFR3, LYVE1, CDKN1C (p57Kip2), ITGA1 (integrin α1), PPL (periplakin) and SLC (secondary lymphoid chemokine) in both BECs and HUVECs (Fig. 2B), indicating that IL3Rα may play an important role in lymphatic reprogramming of KSHV-infected BECs and HUVECs.


Opposing regulation of PROX1 by interleukin-3 receptor and NOTCH directs differential host cell fate reprogramming by Kaposi sarcoma herpes virus.

Yoo J, Lee HN, Choi I, Choi D, Chung HK, Kim KE, Lee S, Aguilar B, Kang J, Park E, Lee YS, Maeng YS, Kim NY, Koh CJ, Hong YK - PLoS Pathog. (2012)

IL3Rα plays a key role in KSHV-mediated PROX1 upregulation.(A) Adenoviral expression of IL3Rα increased the expression of PROX1 mRNA and protein in BECs and HUVECs, but not in LECs based on qRT-PCR and western blot analyses. AdCTR, control adenovirus; AdIL3Rα, IL3Rα-expressing adenovirus. (B) The expression of various LEC-associated genes such as PDPN, VEGFR3, FGFR3, LYVE1, CDKN1C, ITGA1, PPL and SLC was determined by qRT-PCR in BECs or HUVECs transduced by a control (AdCTR) versus IL3Rα- (AdIL3Rα) adenovirus for 48 hours. The graph shows fold changes in expression of each gene by IL3Rα-expressing adenovirus over the control virus. (C) Inhibition of IL3Rα by siRNA or a neutralizing antibody partially abrogated the KSHV-mediated PROX1 upregulation in BECs and HUVECs, as determined by qRT-PCR (BECs and HUVECs) and western blot (BEC only) analyses. siCTR, siRNA for the firefly luciferase; siIL3Rα, siRNA for IL3Rα; anti-IL3Rα, neutralizing antibody against IL3Rα.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375311&req=5

ppat-1002770-g002: IL3Rα plays a key role in KSHV-mediated PROX1 upregulation.(A) Adenoviral expression of IL3Rα increased the expression of PROX1 mRNA and protein in BECs and HUVECs, but not in LECs based on qRT-PCR and western blot analyses. AdCTR, control adenovirus; AdIL3Rα, IL3Rα-expressing adenovirus. (B) The expression of various LEC-associated genes such as PDPN, VEGFR3, FGFR3, LYVE1, CDKN1C, ITGA1, PPL and SLC was determined by qRT-PCR in BECs or HUVECs transduced by a control (AdCTR) versus IL3Rα- (AdIL3Rα) adenovirus for 48 hours. The graph shows fold changes in expression of each gene by IL3Rα-expressing adenovirus over the control virus. (C) Inhibition of IL3Rα by siRNA or a neutralizing antibody partially abrogated the KSHV-mediated PROX1 upregulation in BECs and HUVECs, as determined by qRT-PCR (BECs and HUVECs) and western blot (BEC only) analyses. siCTR, siRNA for the firefly luciferase; siIL3Rα, siRNA for IL3Rα; anti-IL3Rα, neutralizing antibody against IL3Rα.
Mentions: Because IL-3 activates PROX1 expression in BECs and HUVECs, but not in LECs [18], we investigated whether overexpression of its receptor, IL3Rα, could upregulate PROX1 in each cell type. We found that adenoviral expression of IL3Rα resulted in a strong upregulation of PROX1 mRNA and protein in BECs and HUVECs, but not in LECs (Fig. 2A), suggesting that the PROX1-upregulating signal by IL3Rα is operative only in PROX1-deficient BECs and HUVECs and does not affect the already abundant expression of PROX1 in LECs. Furthermore, ectopic expression of IL3Rα in BECs and HUVECs resulted in upregulation of various LEC-signature genes such as podoplanin (PDPN), VEGFR3, FGFR3, LYVE1, CDKN1C (p57Kip2), ITGA1 (integrin α1), PPL (periplakin) and SLC (secondary lymphoid chemokine) in both BECs and HUVECs (Fig. 2B), indicating that IL3Rα may play an important role in lymphatic reprogramming of KSHV-infected BECs and HUVECs.

Bottom Line: Moreover, PROX1 was found to be required to maintain HEY1 expression in LECs, establishing a reciprocal regulation between PROX1 and HEY1.Upon co-activation of IL3Rα and NOTCH, PROX1 was upregulated in BECs, but downregulated in LECs.Together, our study provides the molecular mechanism underlying the cell type-specific endothelial fate reprogramming by KSHV.

View Article: PubMed Central - PubMed

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

ABSTRACT
Lymphatic endothelial cells (LECs) are differentiated from blood vascular endothelial cells (BECs) during embryogenesis and this physiological cell fate specification is controlled by PROX1, the master regulator for lymphatic development. When Kaposi sarcoma herpes virus (KSHV) infects host cells, it activates the otherwise silenced embryonic endothelial differentiation program and reprograms their cell fates. Interestingly, previous studies demonstrated that KSHV drives BECs to acquire a partial lymphatic phenotype by upregulating PROX1 (forward reprogramming), but stimulates LECs to regain some BEC-signature genes by downregulating PROX1 (reverse reprogramming). Despite the significance of this KSHV-induced bidirectional cell fate reprogramming in KS pathogenesis, its underlying molecular mechanism remains undefined. Here, we report that IL3 receptor alpha (IL3Rα) and NOTCH play integral roles in the host cell type-specific regulation of PROX1 by KSHV. In BECs, KSHV upregulates IL3Rα and phosphorylates STAT5, which binds and activates the PROX1 promoter. In LECs, however, PROX1 was rather downregulated by KSHV-induced NOTCH signal via HEY1, which binds and represses the PROX1 promoter. Moreover, PROX1 was found to be required to maintain HEY1 expression in LECs, establishing a reciprocal regulation between PROX1 and HEY1. Upon co-activation of IL3Rα and NOTCH, PROX1 was upregulated in BECs, but downregulated in LECs. Together, our study provides the molecular mechanism underlying the cell type-specific endothelial fate reprogramming by KSHV.

Show MeSH
Related in: MedlinePlus