Abstract
Wnt/β-catenin signaling is important for blood-brain barrier (BBB) development and is implicated in BBB breakdown under various pathophysiological conditions. In the present study, a comprehensive characterization of the relevant genes, transport and permeability processes influenced by both the autocrine and external activation of Wnt signaling in human brain endothelial cells was examined using hCMEC/D3 culture model. The hCMEC/D3 expressed a full complement of Wnt ligands and receptors. Preventing Wnt ligand release from hCMEC/D3 produced minimal changes in brain endothelial function, while inhibition of intrinsic/autocrine Wnt/β-catenin activity through blocking β-catenin binding to Wnt transcription factor caused more modest changes. In contrast, activation of Wnt signaling using exogenous Wnt ligand (Wnt3a) or LiCl (GSK3 inhibitor) improved the BBB phenotypes of the hCMEC/D3 culture model, resulting in reduced paracellular permeability, and increased P-glycoprotein (P-gp) and breast cancer resistance associated protein (BCRP) efflux transporter activity. Further, Wnt3a reduced plasmalemma vesicle associated protein (PLVAP) and vesicular transport activity in hCMEC/D3. Our data suggest that this in vitro model of the BBB has a more robust response to exogenous activation of Wnt/β-catenin signaling compared to autocrine activation, suggesting that BBB regulation may be more dependent on external activation of Wnt signaling within the brain microvasculature.
Highlights
Wnt/β-catenin signaling is important for blood-brain barrier (BBB) development and is implicated in BBB breakdown under various pathophysiological conditions
It should be noted that public database analysis confirms that many intracellular transporters and intercellular junction proteins important in maintaining a BBB phenotype have a binding site for TCF4, TCF7 and LEF-1, that are known transcripton factors in the Wnt/β-catenin pathway
Genes for Pgp, breast cancer resistance associated protein (BCRP), MRP4, Glucose transporter 1 (Glut-1), claudin-1, claudin-3 and plasmalemma vesicle associated protein (PLVAP) have a regulatory element that interacts with TCF-4
Summary
Expression of Wnt receptors, ligands and modulators in hCMEC/D3. Using PCR and qPCR, the various Wnt receptors, activators and modulators were profiled in hCMEC/D3 monolayers. Such increases were similar in magnitude to those observed following treatment with the Pgp transport inhibitor, GF120918 or GF (Fig. 4g) Together these studies suggest that inhibition of intrinsic Wnt/β-catenin activity with ICRT-3 has minimal impact on paracellular barrier properties, but significantly diminished Pgp transporter function in hCMEC/D3 monolayers. The increased expression of BBB relevant transporters and adhesion molecules observed at the mRNA level following extrinsic pharmacological activation of Wnt/β-catenin were observed at the protein level Both Wnt3a and LiCl treatments resulted in significant increases in cellular Pgp protein levels by ~2.0 and ~1.6 fold respectively (p < 0.05, Fig. 5f). The effects of LiCl on both Rhodamine 123 and mitoxantrone accumulation were abolished by ICRT-3 (Fig. 6c,d) These studies suggested that improvements in paracellular barrier, P-glycoprotein and BCRP function observed with LiCl treatment were mediated by the binding of β-catenin to TCF-4. A similar reduction was observed following treatment with the vesicular transport inhibitor, genestein, (200 μM), suggesting a role of Wnt/β-catenin signaling in regulating vesicular transport in the hCMEC/D3 brain endothelial cell model
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