Wnt3a regulates proliferation and migration of HUVEC via canonical and non-canonical Wnt signaling pathways

https://doi.org/10.1016/j.bbrc.2009.06.033Get rights and content

Abstract

Untangling the signaling pathways involved in endothelial cell biology is of central interest for the development of antiangiogenesis based therapies. Here we report that Wnt3a induces the proliferation and migration of HUVECs, but does not affect their survival. Wnt3a-induced proliferation was VEGFR signaling independent, but reduced upon CamKII inhibition. In a search for the downstream mediators of Wnt3a’s effects on HUVEC biology, we found that Wnt3a treatment leads to phosphorylation of DVL3 and stabilization of β-catenin. Moreover, under the same conditions we observed an upregulation in c-MYC, TIE-2 and GJA1 mRNA transcripts. Although treatment of HUVECs with Wnt5a induced DVL3 phosphorylation, we did not observe any of the other effects seen upon Wnt3a stimulation. Taken together, our data indicate that Wnt3a induces canonical and non-canonical Wnt signaling in HUVECs, and stimulates their proliferation and migration.

Introduction

Wnts are a family of secreted glycoproteins that bind to transmembrane Frizzled (Fzd) receptors and initiate signaling cascades with indispensable roles during development, cell proliferation, migration and survival [1]. Several pathways downstream of Wnt binding to Frizzleds are known, Wnt–β-catenin (or Wnt ‘canonical’ pathway) being the most studied. Activation of the canonical Wnt pathway results in recruitment of the cytoplasmic mediator Dishevelled (Dvl) and subsequent stabilization of the transcription co-factor β-catenin. Stabilized β-catenin accumulates in the cytoplasm, enters the nucleus and acts as a co-activator for the T-cell factor/lymphoid enhancer factor-1 (TCF/LEF) transcription factors, thus initiating transcription of their target genes. Several ‘non-canonical’ Wnt pathways have also been described, the Wnt–planar cell polarity (PCP) and Wnt–calcium pathways being the best understood [2]. Both pathways activate Dvl, but diverge further downstream to activate either the Rho/Rac GTPases, or calcium activated protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase II (CamKII), respectively. Although usually described as individual pathways, reported crosstalk and shared components between the Wnt signaling branches suggest that Wnts act through a complex intracellular signaling network [3].

Angiogenesis is the process of blood vessel formation from pre-existing vasculature [4]. Accumulating data suggest the importance of Wnt signaling in endothelial cell proliferation, migration and survival [5]. In this study, we show that Wnt3a induces DVL3 phosphorylation and β-catenin stabilization in cultured human umbilical vein endothelial cells (HUVECs). We report that Wnt3a promotes HUVEC proliferation and migration, but has no effect on their survival. Finally, we provide evidence that the cellular proliferation induced by Wnt3a is VEGFR signaling independent, but reduced upon CamKII blockade.

Section snippets

Materials and methods

Cell culture and reagents. HUVECs were obtained from Promo Cell and grown on plates coated with 1.5% gelatin either in Endothelial Cell Growth Medium (Promo Cell, C-22010) supplemented with 2.5% Fetal Bovine Serum (FBS, Sigma, St. Louis, MO, USA) or when indicated, in Endothelial Cell Basal Medium (Promo Cell, C-90210) supplemented with 0.5% or 1% FBS, or 0.5% bovine serum albumin (BSA). For the experiments, cells with no more than six passages were used. Recombinant Wnt3a, Wnt5a and

Human umbilical vein endothelial cells express FZD4 and FZD6

It has already been shown that Frizzled receptors and other components of the Wnt pathway are expressed by HUVECs [6]. To analyze the expression of Frizzled receptors in the HUVECs used in our experiments we performed a microarray analysis. The results indicate that FZD4 and FZD6 are the only FZD receptors expressed at significant levels in HUVECs (Fig. 1A). The expression of both receptors was confirmed by PCR (Fig. 1B).

Wnt3a and Wnt5a induce DVL3 phosphorylation in HUVEC

The signaling cascades stimulated by the Wnt ligands are diverse and

Discussion

Angiogenesis is a complex process requiring the interplay between several signaling pathways. While the inductive role of VEGFs and FGFs in endothelial cell biology is well documented, much less is known about the contribution of Wnt family members to this process. Here we report that Wnt3a is able to induce canonical and non-canonical Wnt signaling in HUVECs and their in vitro proliferation and migration.

Our analysis of the expression of Frizzleds, the conventional Wnt receptors, in HUVECs

Acknowledgments

We would like to thank Dr. Aaron Ponti for the help with migration assay data analysis. HUVEC microarray data were generated at Novartis Pharma AG. Dr. Thomas Schlange was supported by a grant from the Swiss Cancer League (CCROCS-01445-12-2003). The laboratory of Dr. Nancy E. Hynes is supported by the Friedrich Miescher Institute for Biomedical Research (Basel, CH) part of the Novartis Research Foundation.

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