As a first year Assistant Professor of Pediatrics, the applicant is interested in developmental genetics and refining the skills needed to achieve success as an independent researcher in academic medicine. The focus of this research proposal involves exploring the Hedgehog (Hh) pathway, a fundamental signaling cascade responsible for early embryonic patterning of nearly every organ system in the developing fetus. The Hh also pathway also plays a role in control of cell growth and proliferation in mature organisms, and mutations in this pathway cause cancer in susceptible tissues. Little is known about regulation of the subcellular localization, trafficking or function of the Hh receptor complex. Of particular interest is the fact that the Hh receptor Patched (Ptc), and the Hh ligand itself, seem to be modified, and potentially regulated not only by other proteins, but also by lipids, particularly cholesterol. This five-year award will provide enrichment of the applicant's research career development through investigating the regulation of Hedgehog receptor trafficking and function development through investigating the regulation of Hedgehog receptor trafficking and function by cholesterol and caveolin-l. Initially, confocal imaging studies will be employed to assess trafficking/subcellular localization of the receptor complex, including the ability of the receptor to internalize the Hh ligand. Mutant constructs of caveline-1 and cholesterol blocking agents will be used to identify the individual roles played by each of these components in Hh receptor trafficking and ligand internalization. A luciferase reporter construct, comprised of a Hh downstream target gene, Gli, will be used to assay function of the Hh receptor. This will help determine the regions of Ptc and caveolin that are critical for Hh receptor function and better define the role of cholesterol in this process. Caveolin-l null cell lines and caveolin-l adenoviral vectors will be generated for dynamic studies of the role of caveolin-l in trafficking and function. This work will be integrated with ongoing work in the mentor's lab on a caveolin-l knockout mouse, which will be examined for developmental defects and cancer formation. Dr. Timothy Thompson's lab, whose primary focus is the role of caveolin-l in control of cell growth, differentiation and tumorigenesis, is an excellent environment to aid in the development of this proposal. Because of the ubiquitous role of the Hh pathway, and its link to cancer predisposition, this project may provide clinical application to the understanding of aberrant human fetal development and control of cell growth and differentiation. Understanding the regulation of this pathway may suggest targets for gene therapy in the developing fetus or therapeutic agents for certain Hh-related cancers.
