Investigation on the intranuclear localization of Wnt3a and its extracellular interaction with fibronectin

Abstract

Wnt signaling is known to play an important role in development, embryogenesis, axis patterning, and cell fate determination. For instance, Wnt3a, one of the 19 Wnt ligands found in human, can play a crucial role in regulating cell differentiation, proliferation and migration. Previous study shows that a novel subpopulation of Wnt3a is detected in the cell nuclear fraction. However, no further work has substantiated this finding. The nuclear localization of Wnt has been studied in Wnt2b, where it can directly enhance Wnt signaling, changing our understanding that Wnt proteins need to be secreted to induce Wnt signaling. Thus, such unconventional localization of Wnt to the nucleus deserves further investigation so as to understand their signaling mechanism and to better alter cell fate. These literature reviews are summarized in chapter 1.

Here in chapter 2, we report that Wnt3a can localize within the nucleus of the cell, independent of animal species and immortalization, with the exception of mouse L-Wnt3a cells which are commonly used to make Wnt3a conditioned medium. Super-resolution and subcellular fractionation of HeLa cells and human mesenchymal stem cells (hMSCs) both confirm the presence of intranuclear Wnt3a. Further investigations are being carried out to determine its intranuclear function(s) through its nuclear binding partner(s).

Cells such as hMSCs are known to deposit a network of extracellular matrix (ECM) proteins called cell-derived matrix (CDM), which can enhance cell proliferation, migration and multilineage differentiation. Moreover, CDM has been shown to sequester growth factors, where it can act as storage for these extracellular growth factors. Growth factors are enhanced by the ECM by being concentrated closer to the cells instead of freely floating in the extracellular space. Meanwhile, Wnt3a has not only been reported in the extracellular space but also in human ECM, prompting us to investigate how Wnt3a can be tethered to the ECM to alter cell fate.

In chapter 3, we report that Wnt3a protein is colocalized with the fibronectin meshwork of CDM. In situ proximity ligation assay demonstrates interaction between them while Co-IP demonstrates that Wnt3a binds directly to fibronectin. Wnt3a-fibronectin binding can affect canonical Wnt/β-catenin signaling. Such binding did not prevent the Wnt3a from activating β-catenin and can stabilize Wnt3a in serum-free condition, implying that fibronectin can act as a reservoir for Wnt3a. Consequently, we demonstrate that CDM-bound Wnt3a can promote both canonical Wnt signaling and cell proliferation. In recent years, Wnt3a exosome delivery has been used for therapeutic purposes such as cartilage repair. This shows the potential of using CDM as an alternative way to immobilize Wnt3a for tissue engineering applications.