Analysis of WNT protein secretion and solubility

WNT proteins are critical developmental signaling molecules with a wide range of activities, including stem cell self-renewal, expansion and differentiation. The diverse effects of WNT signaling are explained by their ability to stimulate multiple and distinct signaling cascades, including the so-called canonical and non-canonical WNT signaling pathways. However, despite the vast literature describing WNT functions, relatively little is known about their biochemical properties. Interestingly, WNT proteins carry a covalently attached lipid moiety, an essential modification that renders the secreted WNT protein highly hydrophobic. This unique property of WNT presents the question of how these secreted signaling molecules exist in the extracellular environment. The goal of my research project seeks to gain a better understanding of how various WNT proteins are processed and secreted from cells engineered to overexpress WNT genes. To this end I will employ a cell culture system of Chinese Hamster Ovary (CHO) cells carrying inducible WNT transgenes. Currently we have cells overexpressing 7 WNT genes (WNT1, 3A, 4, 5A, 7A, 10B and 16). Of these, only WNT3A and 5A are efficiently secreted in a biologically active form. The remaining WNTs are extremely poorly secreted and remain tightly associated with the cell surface and/or the extracellular matrix. In my experiments, I will take several approaches to increase levels of the poorly secreted WNT proteins. The long-term goal of these experiments is to gain a better understanding of WNT secretion, which will advance the application of these potent stem cell growth factors in tissue engineering and cell based therapies.