Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor

Abstract

Lymphatic vessels are essential for fluid homeostasis, immune surveillance and fat adsorption, and also serve as a major route for tumor metastasis in many types of cancer. We found that isolated human primary lymphatic and blood vascular endothelial cells (LECs and BECs, respectively) show interesting differences in gene expression relevant for their distinct functions in vivo. Although these phenotypes are stable in vitro and in vivo, overexpression of the homeobox transcription factor Prox-1 in the BECs was capable of inducing LEC-specific gene transcription in the BECs, and, surprisingly, Prox-1 suppressed the expression of approximately 40% of the BEC-specific genes. Prox-1 did not have global effects on the expression of LEC-specific genes in other cell types, except that it up-regulated cyclin E1 and E2 mRNAs and activated the cyclin e promoter in various cell types. These data suggest that Prox-1 acts as a cell proliferation inducer and a fate determination factor for the LECs. Furthermore, the data provide insights into the phenotypic diversity of endothelial cells and into the possibility of transcriptional reprogramming of differentiated endothelial cells.

Fig. 1. Examples of differentially expressed genes in LECs and BECs. (A) Northern blotting and hybridization for the indicated transcripts. Equal loading was verified by probing with GAPDH. For the microarray analyses, RNA was extracted from LECs that were cultured in the presence of VEGF-C (+C). When validating the array results, RNA was also extracted as a control from cultures of LECs in which VEGF-C was not added (–C). STAT6 is expressed specifically in BECs but not in LECs. Western blotting for STAT6 and STAT5b (B), and immunofluorescence double-staining for STAT6 (red) and LEC- specific marker podoplanin (green) (C). The nuclei were stained with Hoechst fluorochrome (blue).

Fig. 2. Cytoskeletal structures, integrin α9 and NRP-1 expression in BECs and LECs. Mixed cultures of LECs and BECs were double-stained for β-catenin (A), plakoglobin (C), F-actin (E) and integrin α9 (G), and for the LEC-specific marker podoplanin (green; B, D, F and H). Expression of integrin α9 in the lymphatic (arrow) but not in blood vessel endothelia (arrowhead). Adjacent sections of human skin were stained with antibodies against integrin α9 (I), VEGFR-3 (J) or blood vessel endothelial antigen PAL-E (K). Expression of NRP-1 in a subset of blood vessels (arrowhead) but not lymphatic vessels (arrow). Adjacent sections of mouse skin were stained with antibodies against NRP-1 (L), the pan-endothelial marker PECAM-1 (M) or lymphatic endothelial marker LYVE-1 (N).

Fig. 3. AdProx-1 expression induces LEC-specific transcripts in the BECs. (A) Prox-1 mRNA is expressed in LECs but not BECs. LECs were cultured in the absence (–C) or in the presence (+C) of VEGF-C. (B and C) Human dermal microvascular endothelial cells were double-stained with monoclonal 2E11D11 (anti-VEGFR-3) and polyclonal Prox-1 antiserum. Note nuclear staining for Prox-1 (green) only in the VEGFR-3-positive LECs. (D) Examples of Prox-1-regulated genes. Northern blotting and hybridization of RNA from AdLacZ- or AdProx-1-infected BECs and LECs for the indicated transcripts. APC and GAPDH were used as controls for equal loading.

Fig. 4. Prox-1 induces expression of cyclins E1 and E2, and transactivates the cyclin e promoter. (A) Total RNA was extracted from AdLacZ- or AdProx-1-infected BECs and used for northern blotting (cyclin E1) or RT–PCR (cyclin E2). (B) BECs infected with AdLacZ or AdProx-1 were stained for PCNA (red), Prox-1 (green) or DNA (blue). (C) Prox-1 transactivates the cyclin e promoter. Promoter activity is presented as fold induction of luciferase activity (mean ± SD) in cells transfected with the expression plasmid encoding wild-type or mutant Prox-1 and with the promoter-luc constructs. C, empty expression vector pAMC; WT, mycProx-1/pAMC; mut, mycProx- 1mut/pAMC. pGL2-luc was used as a reporter control. (D) Prox-1-dependent transactivation of 6xE2F-luc reporter is abolished by co-expression of p27/Kip1a and p16.

Fig. 5. Prox-1 induces expression of cyclin E1 in different cell types, while the induction of VEGFR-3 is endothelial cell specific. Total RNA was extracted from AdProx-1- or AdLacZ-infected primary SAVECs, CAECs and HUACs, and used for northern blotting and hybridization with the indicated probes. Equal loading was verified by probing with GAPDH.