In theory, all classes of lipoproteins have the capability to carry lipid-modified proteins in the phospholipid monolayer surrounding the fat core. A possible role for lipoprotein receptors is to bring lipoprotein particles in close vicinity to the exoplasmic leaflet of cell membranes, which contain Wnt. Then, Wnt can be transferred to the lipoprotein particle, either by passive diffusion or by an active, but yet unidentified, protein machinery. In our experiments, Wnt is released from cells only to HDL but not to LDL. However, once associated to lipoproteins, Wnt could freely exchange between lipoprotein particles, similar to other proteins, like the small apolipoproteins apoE and apoC (39–41). From ldlA cells, the release of Wnt was facilitated by the SR-BI/II receptor, and several other lipoprotein receptors tested did not show similar effects. In contrast, knockdown of SR-BI/II in L-Wnt cells resulted in an increase of Wnt release. The SR-BI/II knockout mouse does not show phenotypes related to impaired Wnt signaling (42), which would be in line with our results in L-cells. Both cell lines, ldlA and L-cells, are not very well characterized in terms of lipoprotein receptor expression. For ldlA cells, it is known that they have a defect in LDL receptor expression (29,43) and express only low amounts of HDL binding/selective uptake activity (30); therefore, these cells probably adapted to a situation with impaired lipid uptake from lipoproteins. It is difficult to predict, however, whether upon knockdown of SR-BI/II in L-cells, other lipoprotein receptors would be upregulated and also facilitate Wnt release. Furthermore, it is known that membrane structure is dependent on SR-BI/II expression, as shown in Sf9 cells and for the formation and organization of microvilli in adrenal gland cells (44–46). Hereby, the lack of SR-BI/II leads to a decrease in membrane thickness, possibly because of a decrease in membrane cholesterol (45). It is possible that extraction of Wnt from the plasma membrane is facilitated under these conditions. Zhai et al. showed that Drosophila Wnt1 is present in cholesterol-rich, detergent-resistant membranes (9), which indicates that Wnt is organized in specialized membrane domains. Therefore, interference with membrane lipid composition may result in different membrane anchoring.
We find that transfection of ldlA cells with SR-BII leads to more efficient Wnt release than that with SR-BI. Hereby, sorting in the endocytic system, which was found to be essential for Wnt release (47–51), could be responsible for this effect. SR-BII preferentially localized to endocytic compartments and the plasma membrane (52), suggesting a role in HDL recycling. Possibly, Wnt can also be released on recycling lipoprotein particles other than HDL (53,54). Whereas LDL particles and their associated proteins are taken up through the LDL receptor and targeted for degradation to the lysosome, HDL can be recycled by SR-BI/II or ABCA1 (52,55,56). Thus, if Wnt would have been released onto LDL, both proteins would have been targeted to lysosomal degradation.
Certainly, the cell culture system we used is poorly understood. Therefore, it would have to be tested whether other lipoprotein receptors are also involved in Wnt release, for example from the large family of LDL receptor-related proteins or other scavenger receptors. Furthermore, it is important to decipher the mechanism, how Wnt is transferred to lipoprotein particles. Possibly, a change in membrane environment is required, for example, during recycling. Finally, the release should be studied in vivo. Perhaps, the use of inducible reporter genes for Wnt signaling in combination with inducible, tissue-specific knockdowns of lipoproteins and their receptors will give more insight in Wnt transport in vivo.