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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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PROX1 upregulates HEY1 in primary LECs.(A) Knockdown of PROX1, but not COUP-TFII, resulted in downregulation of HEY1 in LECs. qRT-PCR analyses were performed to determine the expression of HEY1, PROX1 and COUP-TFII in primary LECs that were transfected for 48 hours with siRNAs targeting the firefly luciferase (siCTR), PROX1 (siProx1), COUP-TFII (siCOUP) or both PROX1 and COUP-TFII (siBOTH). (B) Adenoviral expression of PROX1, but not of COUP-TFII, increased the expression of HEY1 mRNA in LECs. qRT-PCR analyses were performed to determine the level of HEY1 mRNA in LECs that were transduced with a control (AdCTR), PROX1 (AdProx1), COUP-TFII (AdCOUP) adenovirus or with both PROX1 and COUP-TFII (AdBOTH) for 48 hours. (C) Luciferase reporter constructs of the promoters of HEY1, Hey2 or HeyL were transiently transfected into HEK293 cells along with a control vector (Empty Vector), PROX1 wild type (Prox1_WT) or PROX1 DNA-binding mutant (Prox1_Mut). After 48 hours, luciferase activity was measured and each value was normalized by total cell lysate amounts. (D) PROX1 also upregulated HEY1 in aortic endothelial cells (human umbilical aortic endothelial cells, HUAEC). Relative expression was determined for HEY1, Hey2 and PROX1 in primary HUAECs that were transduced with a control or PROX1-adenovirus for 48 hours. (E) Chromatin immunoprecipitation (ChIP) assay demonstrating a physical association between PROX1 protein and the HEY1 promoter in LECs. Primary LECs were transfected with a control (LEC/CTR) or a PROX1-expressing vector (LEC/Prox1) for 48 hours and then subjected to ChIP analyses using a normal IgG (IgG) or anti-PROX1 (α-Prox1) antibodies. (F) A ∼0.6-kb HEY1 proximal promoter was sufficient to deliver the PROX1-mediated repression of HEY1 expression. Reporter constructs of the HEY1 promoter were transfected with a control or PROX1-expressing vector into HEK293 cells for 48 hours. (G) Ectopic expression of PROX1 in LECs resulted in downregulation of endogenous PROX1. LECs were transduced with a control (AdCTR) or PROX1 (AdProx1) adenovirus. After 48 hours, qRT-PCR was performed by using two sets of probes that target the 3′ untranslated region (UTR) of endogenous PROX1. All data were shown as a relative average expression ± standard deviation (SD).
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ppat-1002770-g005: PROX1 upregulates HEY1 in primary LECs.(A) Knockdown of PROX1, but not COUP-TFII, resulted in downregulation of HEY1 in LECs. qRT-PCR analyses were performed to determine the expression of HEY1, PROX1 and COUP-TFII in primary LECs that were transfected for 48 hours with siRNAs targeting the firefly luciferase (siCTR), PROX1 (siProx1), COUP-TFII (siCOUP) or both PROX1 and COUP-TFII (siBOTH). (B) Adenoviral expression of PROX1, but not of COUP-TFII, increased the expression of HEY1 mRNA in LECs. qRT-PCR analyses were performed to determine the level of HEY1 mRNA in LECs that were transduced with a control (AdCTR), PROX1 (AdProx1), COUP-TFII (AdCOUP) adenovirus or with both PROX1 and COUP-TFII (AdBOTH) for 48 hours. (C) Luciferase reporter constructs of the promoters of HEY1, Hey2 or HeyL were transiently transfected into HEK293 cells along with a control vector (Empty Vector), PROX1 wild type (Prox1_WT) or PROX1 DNA-binding mutant (Prox1_Mut). After 48 hours, luciferase activity was measured and each value was normalized by total cell lysate amounts. (D) PROX1 also upregulated HEY1 in aortic endothelial cells (human umbilical aortic endothelial cells, HUAEC). Relative expression was determined for HEY1, Hey2 and PROX1 in primary HUAECs that were transduced with a control or PROX1-adenovirus for 48 hours. (E) Chromatin immunoprecipitation (ChIP) assay demonstrating a physical association between PROX1 protein and the HEY1 promoter in LECs. Primary LECs were transfected with a control (LEC/CTR) or a PROX1-expressing vector (LEC/Prox1) for 48 hours and then subjected to ChIP analyses using a normal IgG (IgG) or anti-PROX1 (α-Prox1) antibodies. (F) A ∼0.6-kb HEY1 proximal promoter was sufficient to deliver the PROX1-mediated repression of HEY1 expression. Reporter constructs of the HEY1 promoter were transfected with a control or PROX1-expressing vector into HEK293 cells for 48 hours. (G) Ectopic expression of PROX1 in LECs resulted in downregulation of endogenous PROX1. LECs were transduced with a control (AdCTR) or PROX1 (AdProx1) adenovirus. After 48 hours, qRT-PCR was performed by using two sets of probes that target the 3′ untranslated region (UTR) of endogenous PROX1. All data were shown as a relative average expression ± standard deviation (SD).

Mentions: We have previously reported that PROX1 physically and functionally interacts with the orphan nuclear receptor COUP-TFII to specify the cell fate of LECs [32]. We also performed a genome-wide search for PROX1 target genes using microarray analyses and identified a list of genes, whose expression was altered by PROX1 knockdown in LECs [32]. Interestingly, the microarray analyses revealed that the expression of HEY1 was significantly downregulated in LECs by PROX1 knockdown (GEO accession: GSE12846). This unexpected regulation of HEY1 by PROX1 was further confirmed using qRT-PCR of total RNAs isolated from LECs that were transfected with siRNA against PROX1 and/or COUP-TFII (Fig. 5A). Notably, knockdown of COUP-TFII, a PROX1-interacting protein, did not alter the HEY1 expression. On the contrary, adenoviral overexpression of PROX1, but not COUP-TFII, in LECs resulted in a strong upregulation of HEY1 (Fig. 5B). We then asked whether PROX1 could activate the proximal promoter of HEY1 and thus performed a series of luciferase reporter assays using promoter constructs of HEY1 and two other HEY family members, HEY2 and HEYL. Indeed, the HEY1 promoter was found to be activated by PROX1 wild type, but not by a PROX1 mutant lacking DNA-binding activity [33] (Fig. 5C). Moreover, adenoviral expression of PROX1 in human umbilical aortic endothelial cells (HUAEC) also resulted in upregulation of HEY1 (Fig. 5D). We then performed PROX1 ChIP assays against the HEY1 promoter and found that PROX1 protein is physically associated with the HEY1 promoter (Fig. 5E). Subsequently, a set of HEY1 promoter reporter constructs was generated and used to further study the PROX1 regulation of HEY1. Notably, a ∼0.7 kb-long HEY1 promoter (pHey1C) was sufficient to deliver the PROX1-mediated activation of the HEY1 promoter (Fig. 5F). Therefore, we concluded that PROX1 positively regulates the expression of HEY1 by directly binding to its promoter. Together with the findings above (Fig. 4), these data established a reciprocal regulation between PROX1 and HEY1: HEY1 functions as a repressor of PROX1 and PROX1 is required to upregulate or maintain HEY1 expression. According to this reciprocal feedback regulation, PROX1 could negatively regulate its own gene expression. To confirm this auto-regulation, we ectopically overexpressed PROX1 in LECs using adenovirus that harbors the PROX1 open reading frame (ORF) only and then determined the expression level of the endogenous PROX1 by two qRT-PCR probes detecting the PROX1 3′-untranslated region (UTR), which is not present in the adenovirus. Indeed, ectopic expression of PROX1 resulted in a significant downregulation of the endogenous PROX1 (Fig. 5G). Taken together, our data uncovered an intricate auto-regulatory mechanism for the PROX1 gene expression that utilizes the HEY1 repressor, a component of NOTCH signal pathway.


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)

PROX1 upregulates HEY1 in primary LECs.(A) Knockdown of PROX1, but not COUP-TFII, resulted in downregulation of HEY1 in LECs. qRT-PCR analyses were performed to determine the expression of HEY1, PROX1 and COUP-TFII in primary LECs that were transfected for 48 hours with siRNAs targeting the firefly luciferase (siCTR), PROX1 (siProx1), COUP-TFII (siCOUP) or both PROX1 and COUP-TFII (siBOTH). (B) Adenoviral expression of PROX1, but not of COUP-TFII, increased the expression of HEY1 mRNA in LECs. qRT-PCR analyses were performed to determine the level of HEY1 mRNA in LECs that were transduced with a control (AdCTR), PROX1 (AdProx1), COUP-TFII (AdCOUP) adenovirus or with both PROX1 and COUP-TFII (AdBOTH) for 48 hours. (C) Luciferase reporter constructs of the promoters of HEY1, Hey2 or HeyL were transiently transfected into HEK293 cells along with a control vector (Empty Vector), PROX1 wild type (Prox1_WT) or PROX1 DNA-binding mutant (Prox1_Mut). After 48 hours, luciferase activity was measured and each value was normalized by total cell lysate amounts. (D) PROX1 also upregulated HEY1 in aortic endothelial cells (human umbilical aortic endothelial cells, HUAEC). Relative expression was determined for HEY1, Hey2 and PROX1 in primary HUAECs that were transduced with a control or PROX1-adenovirus for 48 hours. (E) Chromatin immunoprecipitation (ChIP) assay demonstrating a physical association between PROX1 protein and the HEY1 promoter in LECs. Primary LECs were transfected with a control (LEC/CTR) or a PROX1-expressing vector (LEC/Prox1) for 48 hours and then subjected to ChIP analyses using a normal IgG (IgG) or anti-PROX1 (α-Prox1) antibodies. (F) A ∼0.6-kb HEY1 proximal promoter was sufficient to deliver the PROX1-mediated repression of HEY1 expression. Reporter constructs of the HEY1 promoter were transfected with a control or PROX1-expressing vector into HEK293 cells for 48 hours. (G) Ectopic expression of PROX1 in LECs resulted in downregulation of endogenous PROX1. LECs were transduced with a control (AdCTR) or PROX1 (AdProx1) adenovirus. After 48 hours, qRT-PCR was performed by using two sets of probes that target the 3′ untranslated region (UTR) of endogenous PROX1. All data were shown as a relative average expression ± standard deviation (SD).
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ppat-1002770-g005: PROX1 upregulates HEY1 in primary LECs.(A) Knockdown of PROX1, but not COUP-TFII, resulted in downregulation of HEY1 in LECs. qRT-PCR analyses were performed to determine the expression of HEY1, PROX1 and COUP-TFII in primary LECs that were transfected for 48 hours with siRNAs targeting the firefly luciferase (siCTR), PROX1 (siProx1), COUP-TFII (siCOUP) or both PROX1 and COUP-TFII (siBOTH). (B) Adenoviral expression of PROX1, but not of COUP-TFII, increased the expression of HEY1 mRNA in LECs. qRT-PCR analyses were performed to determine the level of HEY1 mRNA in LECs that were transduced with a control (AdCTR), PROX1 (AdProx1), COUP-TFII (AdCOUP) adenovirus or with both PROX1 and COUP-TFII (AdBOTH) for 48 hours. (C) Luciferase reporter constructs of the promoters of HEY1, Hey2 or HeyL were transiently transfected into HEK293 cells along with a control vector (Empty Vector), PROX1 wild type (Prox1_WT) or PROX1 DNA-binding mutant (Prox1_Mut). After 48 hours, luciferase activity was measured and each value was normalized by total cell lysate amounts. (D) PROX1 also upregulated HEY1 in aortic endothelial cells (human umbilical aortic endothelial cells, HUAEC). Relative expression was determined for HEY1, Hey2 and PROX1 in primary HUAECs that were transduced with a control or PROX1-adenovirus for 48 hours. (E) Chromatin immunoprecipitation (ChIP) assay demonstrating a physical association between PROX1 protein and the HEY1 promoter in LECs. Primary LECs were transfected with a control (LEC/CTR) or a PROX1-expressing vector (LEC/Prox1) for 48 hours and then subjected to ChIP analyses using a normal IgG (IgG) or anti-PROX1 (α-Prox1) antibodies. (F) A ∼0.6-kb HEY1 proximal promoter was sufficient to deliver the PROX1-mediated repression of HEY1 expression. Reporter constructs of the HEY1 promoter were transfected with a control or PROX1-expressing vector into HEK293 cells for 48 hours. (G) Ectopic expression of PROX1 in LECs resulted in downregulation of endogenous PROX1. LECs were transduced with a control (AdCTR) or PROX1 (AdProx1) adenovirus. After 48 hours, qRT-PCR was performed by using two sets of probes that target the 3′ untranslated region (UTR) of endogenous PROX1. All data were shown as a relative average expression ± standard deviation (SD).
Mentions: We have previously reported that PROX1 physically and functionally interacts with the orphan nuclear receptor COUP-TFII to specify the cell fate of LECs [32]. We also performed a genome-wide search for PROX1 target genes using microarray analyses and identified a list of genes, whose expression was altered by PROX1 knockdown in LECs [32]. Interestingly, the microarray analyses revealed that the expression of HEY1 was significantly downregulated in LECs by PROX1 knockdown (GEO accession: GSE12846). This unexpected regulation of HEY1 by PROX1 was further confirmed using qRT-PCR of total RNAs isolated from LECs that were transfected with siRNA against PROX1 and/or COUP-TFII (Fig. 5A). Notably, knockdown of COUP-TFII, a PROX1-interacting protein, did not alter the HEY1 expression. On the contrary, adenoviral overexpression of PROX1, but not COUP-TFII, in LECs resulted in a strong upregulation of HEY1 (Fig. 5B). We then asked whether PROX1 could activate the proximal promoter of HEY1 and thus performed a series of luciferase reporter assays using promoter constructs of HEY1 and two other HEY family members, HEY2 and HEYL. Indeed, the HEY1 promoter was found to be activated by PROX1 wild type, but not by a PROX1 mutant lacking DNA-binding activity [33] (Fig. 5C). Moreover, adenoviral expression of PROX1 in human umbilical aortic endothelial cells (HUAEC) also resulted in upregulation of HEY1 (Fig. 5D). We then performed PROX1 ChIP assays against the HEY1 promoter and found that PROX1 protein is physically associated with the HEY1 promoter (Fig. 5E). Subsequently, a set of HEY1 promoter reporter constructs was generated and used to further study the PROX1 regulation of HEY1. Notably, a ∼0.7 kb-long HEY1 promoter (pHey1C) was sufficient to deliver the PROX1-mediated activation of the HEY1 promoter (Fig. 5F). Therefore, we concluded that PROX1 positively regulates the expression of HEY1 by directly binding to its promoter. Together with the findings above (Fig. 4), these data established a reciprocal regulation between PROX1 and HEY1: HEY1 functions as a repressor of PROX1 and PROX1 is required to upregulate or maintain HEY1 expression. According to this reciprocal feedback regulation, PROX1 could negatively regulate its own gene expression. To confirm this auto-regulation, we ectopically overexpressed PROX1 in LECs using adenovirus that harbors the PROX1 open reading frame (ORF) only and then determined the expression level of the endogenous PROX1 by two qRT-PCR probes detecting the PROX1 3′-untranslated region (UTR), which is not present in the adenovirus. Indeed, ectopic expression of PROX1 resulted in a significant downregulation of the endogenous PROX1 (Fig. 5G). Taken together, our data uncovered an intricate auto-regulatory mechanism for the PROX1 gene expression that utilizes the HEY1 repressor, a component of NOTCH signal pathway.

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