Wnt2 acts as a cell type-specific, autocrine growth factor in rat hepatic sinusoidal endothelial cells cross-stimulating the VEGF pathway.
The mechanisms regulating the growth and differentiation of hepatic sinusoidal endothelial cells (HSECs) are not well defined. Because Wnt signaling has become increasingly important in developmental processes such as vascular and hepatic differentiation, we analyzed HSEC-specific Wnt signaling in detail. Using highly pure HSECs isolated by a newly developed protocol selecting against nonsinusoidal hepatic endothelial cells, we comparatively screened the multiple components of the Wnt pathway for differential expression in HSECs and lung microvascular endothelial cells (LMECs) via reverse-transcription polymerase chain reaction (RT-PCR). As confirmed via quantitative RT-PCR and northern and western blotting experiments, Wnt2 (and less so Wnt transporter wls/evi) and Wnt coreceptor Ryk were overexpressed by HSECs, whereas Wnt inhibitory factor (WIF) was strongly overexpressed by LMECs. Exogenous Wnt2 superinduced proliferation of HSECs (P < 0.05). The Wnt inhibitor secreted frizzled-related protein 1 (sFRP1) (P < 0.005) and transfection of HSECs with Wnt2 small interfering RNA (siRNA) reduced proliferation of HSECs. These effects were rescued by exogenous Wnt2. Tube formation of HSECs on matrigel was strongly inhibited by Wnt inhibitors sFRP1 and WIF (P < 0.0005). Wnt signaling in HSECs activated the canonical pathway inducing nuclear translocation of beta-catenin. GST (glutathione transferase) pull-down and co-immunoprecipitation assays showed Fzd4 to be a novel Wnt2 receptor in HSECs. Gene profiling identified vascular endothelial growth factor receptor-2 (VEGFR-2) as a target of Wnt2 signaling in HSECs. Inhibition of Wnt signaling down-regulated VEGFR-2 messenger RNA and protein. Wnt2 siRNA knock-down confirmed Wnt2 specificity of VEGFR-2 regulation in HSECs.


CONCLUSIONS
Wnt2 is an autocrine growth and differentiation factor specific for HSECs that synergizes with the VEGF signaling pathway to exert its effects.
Klein
Diana
D
Department of Dermatology, Venereology, and Allergology, University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany. diana.klein.@haut.ma.uni-heidelberg.de
Demory
Alexandra
A
Peyre
Francis
F
Kroll
Jens
J
Augustin
Hellmut G
HG
Helfrich
Wijnand
W
Kzhyshkowska
Julia
J
Schledzewski
Kai
K
Arnold
Bernd
B
Goerdt
Sergij
S
D000818:Animals; D049109:Cell Proliferation; D042783:Endothelial Cells / Q000378:metabolism*; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism*; D009024:Morphogenesis / Q000235:genetics*; D051381:Rats; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D051154:Wnt2 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D036341:Intercellular Signaling Peptides and Proteins; D042461:Vascular Endothelial Growth Factor A; D051154:Wnt2 Protein; D040301:Vascular Endothelial Growth Factor Receptor-2