There is a fundamental gap in understanding how the post-Golgi transport and secretion of Wnt proteins are controlled in mammalian epithelial cells in order to sustain tissue homeostasis. Continued existence of this knowledge gap prevents the complete understanding of Wnt biology and its contribution to human diseases such as colorectal cancers. The long-term goal is to elucidate the molecular mechanisms of key intracellular trafficking machinery that deploys and coordinates the signaling events essential for intesti- nal stem cell renewal and epithelial homeostasis. The objective of this application is to determine the molecular function of Rab8- and Rab11-regulated exocytotic and recycling endosome compartments in intestinal crypt homeostasis and stem cell division. The central hypothesis is that Rab8- and Rab11- mediated vesicle budding and trafficking events execute the post-Golgi transport of Wnt/Wntless to plasma membrane for secretion. This hypothesis has been formulated on the basis of preliminary data generated in the applicant's laboratory. The rationale for the proposed research is that understanding the regulatory mechanisms of intracellular Wnt transport and secretion will yield translational insights into novel strategies fr manipulating Wnt production. Guided by strong in vitro and in vivo preliminary data, this hypothesis will be tested by pursuing two specific aims: (1) To define the role of Rab8a in Wnt secre- tion and crypt homeostasis;and (2) To identify the contribution of Rab11a to Wnt secretion and crypt homeostasis. We will determine the regulation of Wnt transport and release by Rab8 vesicle trafficking machinery in mouse intestines and primary cell lines in the first aim. The impact of Rab11a deficiency on intestinal epithelial morphogenesis and stem cell division will be determined under the second aim. Exist- ing and newly-derived knockout mouse lines, as well as genetically engineered primary mouse embry- onic cell lines that are already on hand will be used. This proposal is innovative, because (1) it utilizes new knockout mouse model and cel lines, which represent a substantive departure from the existing methodologies for studying these small GTPases;and (2) it explores the regulation of Wnt secretion in vivo, which is conceptually novel from studies focused on Wnt downstream signaling events. This project is significant because it is a critical step in a continuum of research towards the better understanding of Wnt ligand release and reception in stem cell and cancer biology. Such knowledge will ultimately trans- late into improved therapeutic strategies for the treatment of Wnt-related human diseases. In addition, insights gained from mouse intestinal epithelium will have a broader impact on the mechanism of the morphogen gradient formation and maintenance in other mammalian tissues and diseases.
This project uses knockout mouse models and genetically engineered cell lines to explore the regulation of Wnt traffic and secretion. The cell division and homeostasis of the smal intestinal crypt compartment will be the major focus of this grant.
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