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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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NOTCH pathway plays a major role in KSHV-mediated PROX1 downregulation in LECs.(A) HEY1, an effector of NOTCH, was strongly upregulated by KSHV in BECs, HUVECs and LECs, as determined by qRT-PCR. (B,C) While activated NOTCH repressed PROX1 expression in LECs, the PROX1 expression level remained unchanged in BECs or HUVECs by NOTCH, based on qRT-PCR (B) and western blot (C) analyses. AdCTR, control adenovirus; AdNICD, NICD-expressing adenovirus. (D) Expression of PROX1 protein was strongly repressed by adenoviral overexpression of NICD and HEY1 in LECs based on western blot analyses. AdHey1: FLAG-HEY1-expressing adenovirus. (E) HEY1 ChIP assay against the human PROX1 promoter shows that HEY1 protein is associated with the PROX1 promoter. Primary LECs transduced with AdHey1 (expressing FLAG-tagged HEY1) were used to perform ChIP assays using an anti-FLAG antibody and two sets of primers against the human PROX1 promoter. Relative location of the primer sets is marked in panel F. (F) A diagram illustrating the relative location of the start codon (ATG), two PROX1 transcription start sites (TS1, TS2), the HEY1 ChIP primer sets and the boundaries of the PROX1 promoter fragments that were used to generate the luciferase reporter constructs. (G) Luciferase-reporter assays using PROX1-promoter constructs revealed that PROX1 repression by HEY1 can be mediated through a 1.8-kb promoter fragment (PROX1_P3). (H) Inhibition of HEY1 by siRNA abrogated the KSHV-mediated downregulation of PROX1 based on qRT-PCR and western blot analyses.
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ppat-1002770-g004: NOTCH pathway plays a major role in KSHV-mediated PROX1 downregulation in LECs.(A) HEY1, an effector of NOTCH, was strongly upregulated by KSHV in BECs, HUVECs and LECs, as determined by qRT-PCR. (B,C) While activated NOTCH repressed PROX1 expression in LECs, the PROX1 expression level remained unchanged in BECs or HUVECs by NOTCH, based on qRT-PCR (B) and western blot (C) analyses. AdCTR, control adenovirus; AdNICD, NICD-expressing adenovirus. (D) Expression of PROX1 protein was strongly repressed by adenoviral overexpression of NICD and HEY1 in LECs based on western blot analyses. AdHey1: FLAG-HEY1-expressing adenovirus. (E) HEY1 ChIP assay against the human PROX1 promoter shows that HEY1 protein is associated with the PROX1 promoter. Primary LECs transduced with AdHey1 (expressing FLAG-tagged HEY1) were used to perform ChIP assays using an anti-FLAG antibody and two sets of primers against the human PROX1 promoter. Relative location of the primer sets is marked in panel F. (F) A diagram illustrating the relative location of the start codon (ATG), two PROX1 transcription start sites (TS1, TS2), the HEY1 ChIP primer sets and the boundaries of the PROX1 promoter fragments that were used to generate the luciferase reporter constructs. (G) Luciferase-reporter assays using PROX1-promoter constructs revealed that PROX1 repression by HEY1 can be mediated through a 1.8-kb promoter fragment (PROX1_P3). (H) Inhibition of HEY1 by siRNA abrogated the KSHV-mediated downregulation of PROX1 based on qRT-PCR and western blot analyses.

Mentions: We next set out to investigate how KSHV-infection resulted in downregulation of PROX1 in LECs, despite the fact that KSHV upregulates PROX1 in BECs and HUVECs. Previous studies have shown an increased activity of the NOTCH pathway in KSHV-infected endothelial cells and KS-tumor cells in vivo[23], [25], [29], [30]. Moreover, we have recently reported that activated NOTCH represses PROX1 expression through HEY1 in LECs [22]. Accordingly, we came up with a hypothesis that KSHV-induced NOTCH activation may be involved in PROX1 downregulation in KSHV-infected LECs. Supporting this hypothesis, KSHV-infection of all three cell types, BECs, HUVECs and LECs, resulted in upregulation of HEY1 (Fig. 4A). Adenoviral overexpression of NOTCH intracellular domain (NICD) caused a significant downregulation of PROX1 in LECs, but not in BECs and HUVECs (Fig. 4B&C). In addition, Notch activation in LECs resulted in downregulation of additional lymphatic-signature genes such as podoplanin (PDPN) and CDKN1C, suggesting a suppressive role of Notch signaling in LEC phenotypes (Supplemental Fig. S2). Moreover, microarray-based analyses on the NICD-induced modulation of the transcriptional profiles in primary LECs (National Center for Biotechnology Information, Gene Expression Omnibus accession number: GSE20978) support the effect of Notch on LEC phenotype. Furthermore, we found that HEY1, like NICD, was able to strongly repress the expression of PROX1 protein, when overexpressed in LECs (Fig. 4D). Since HEY1 is known to repress target gene expression by binding to the promoter [31], we performed HEY1-ChIP assays against the PROX1 promoter in primary LECs and found that HEY1 was indeed physically associated with the PROX1 promoter around the two transcriptional start sites (Fig. 4E&F). We then generated a set of PROX1-promoter reporter constructs and found that a 1.8-kb proximal promoter region was sufficient to deliver the HEY1-mediated repression (Fig. 4G). Finally, inhibition of HEY1 expression by siRNA abrogated the KSHV-mediated downregulation of PROX1 mRNA and protein in LECs (Fig. 4H). Together, these findings demonstrate that NOTCH activation is responsible for the KSHV-mediated PROX1 downregulation in LECs, but not in BECs and HUVECs, and that the NOTCH effector HEY1 directly binds to the PROX1 promoter to downregulate its gene expression.


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)

NOTCH pathway plays a major role in KSHV-mediated PROX1 downregulation in LECs.(A) HEY1, an effector of NOTCH, was strongly upregulated by KSHV in BECs, HUVECs and LECs, as determined by qRT-PCR. (B,C) While activated NOTCH repressed PROX1 expression in LECs, the PROX1 expression level remained unchanged in BECs or HUVECs by NOTCH, based on qRT-PCR (B) and western blot (C) analyses. AdCTR, control adenovirus; AdNICD, NICD-expressing adenovirus. (D) Expression of PROX1 protein was strongly repressed by adenoviral overexpression of NICD and HEY1 in LECs based on western blot analyses. AdHey1: FLAG-HEY1-expressing adenovirus. (E) HEY1 ChIP assay against the human PROX1 promoter shows that HEY1 protein is associated with the PROX1 promoter. Primary LECs transduced with AdHey1 (expressing FLAG-tagged HEY1) were used to perform ChIP assays using an anti-FLAG antibody and two sets of primers against the human PROX1 promoter. Relative location of the primer sets is marked in panel F. (F) A diagram illustrating the relative location of the start codon (ATG), two PROX1 transcription start sites (TS1, TS2), the HEY1 ChIP primer sets and the boundaries of the PROX1 promoter fragments that were used to generate the luciferase reporter constructs. (G) Luciferase-reporter assays using PROX1-promoter constructs revealed that PROX1 repression by HEY1 can be mediated through a 1.8-kb promoter fragment (PROX1_P3). (H) Inhibition of HEY1 by siRNA abrogated the KSHV-mediated downregulation of PROX1 based on qRT-PCR and western blot analyses.
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ppat-1002770-g004: NOTCH pathway plays a major role in KSHV-mediated PROX1 downregulation in LECs.(A) HEY1, an effector of NOTCH, was strongly upregulated by KSHV in BECs, HUVECs and LECs, as determined by qRT-PCR. (B,C) While activated NOTCH repressed PROX1 expression in LECs, the PROX1 expression level remained unchanged in BECs or HUVECs by NOTCH, based on qRT-PCR (B) and western blot (C) analyses. AdCTR, control adenovirus; AdNICD, NICD-expressing adenovirus. (D) Expression of PROX1 protein was strongly repressed by adenoviral overexpression of NICD and HEY1 in LECs based on western blot analyses. AdHey1: FLAG-HEY1-expressing adenovirus. (E) HEY1 ChIP assay against the human PROX1 promoter shows that HEY1 protein is associated with the PROX1 promoter. Primary LECs transduced with AdHey1 (expressing FLAG-tagged HEY1) were used to perform ChIP assays using an anti-FLAG antibody and two sets of primers against the human PROX1 promoter. Relative location of the primer sets is marked in panel F. (F) A diagram illustrating the relative location of the start codon (ATG), two PROX1 transcription start sites (TS1, TS2), the HEY1 ChIP primer sets and the boundaries of the PROX1 promoter fragments that were used to generate the luciferase reporter constructs. (G) Luciferase-reporter assays using PROX1-promoter constructs revealed that PROX1 repression by HEY1 can be mediated through a 1.8-kb promoter fragment (PROX1_P3). (H) Inhibition of HEY1 by siRNA abrogated the KSHV-mediated downregulation of PROX1 based on qRT-PCR and western blot analyses.
Mentions: We next set out to investigate how KSHV-infection resulted in downregulation of PROX1 in LECs, despite the fact that KSHV upregulates PROX1 in BECs and HUVECs. Previous studies have shown an increased activity of the NOTCH pathway in KSHV-infected endothelial cells and KS-tumor cells in vivo[23], [25], [29], [30]. Moreover, we have recently reported that activated NOTCH represses PROX1 expression through HEY1 in LECs [22]. Accordingly, we came up with a hypothesis that KSHV-induced NOTCH activation may be involved in PROX1 downregulation in KSHV-infected LECs. Supporting this hypothesis, KSHV-infection of all three cell types, BECs, HUVECs and LECs, resulted in upregulation of HEY1 (Fig. 4A). Adenoviral overexpression of NOTCH intracellular domain (NICD) caused a significant downregulation of PROX1 in LECs, but not in BECs and HUVECs (Fig. 4B&C). In addition, Notch activation in LECs resulted in downregulation of additional lymphatic-signature genes such as podoplanin (PDPN) and CDKN1C, suggesting a suppressive role of Notch signaling in LEC phenotypes (Supplemental Fig. S2). Moreover, microarray-based analyses on the NICD-induced modulation of the transcriptional profiles in primary LECs (National Center for Biotechnology Information, Gene Expression Omnibus accession number: GSE20978) support the effect of Notch on LEC phenotype. Furthermore, we found that HEY1, like NICD, was able to strongly repress the expression of PROX1 protein, when overexpressed in LECs (Fig. 4D). Since HEY1 is known to repress target gene expression by binding to the promoter [31], we performed HEY1-ChIP assays against the PROX1 promoter in primary LECs and found that HEY1 was indeed physically associated with the PROX1 promoter around the two transcriptional start sites (Fig. 4E&F). We then generated a set of PROX1-promoter reporter constructs and found that a 1.8-kb proximal promoter region was sufficient to deliver the HEY1-mediated repression (Fig. 4G). Finally, inhibition of HEY1 expression by siRNA abrogated the KSHV-mediated downregulation of PROX1 mRNA and protein in LECs (Fig. 4H). Together, these findings demonstrate that NOTCH activation is responsible for the KSHV-mediated PROX1 downregulation in LECs, but not in BECs and HUVECs, and that the NOTCH effector HEY1 directly binds to the PROX1 promoter to downregulate its gene expression.

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