We have made advances understanding how miRNAs regulate genes involved in epithelial proliferation. The regulation of epithelial proliferation during organ morphogenesis is critical for normal development, as dysregulation is associated with tumor formation. Non-coding microRNAs, such as miR-200c, are post-transcriptional regulators of genes involved in cancer. However, the role of miR-200c during normal development is unknown. We screened miRNAs expressed in the developing submandibular gland (SMG), and found that miR-200c accumulates in the epithelial end buds. Using both loss- and gain-of-function, we demonstrated that miR-200c reduces epithelial proliferation during SMG morphogenesis. To identify the mechanism, we predicted miR-200c target genes and confirmed their expression during SMG development. We discovered that miR-200c targets the very-low density lipoprotein receptor (VLDLR) and its ligand reelin, which unexpectedly regulate FGFR-dependent epithelial proliferation. Thus, we demonstrate that miR-200c influences FGFR-mediated epithelial proliferation during branching morphogenesis via a VLDLR-dependent mechanism. miR-200c and VLDLR may be novel targets for controlling epithelial morphogenesis during glandular repair or regeneration. We continue to focus on the function of other miRNAs expressed in different cell types in the developing salivary glands. We are also investigating how heparan sulfate influences FGFR2b signaling in specific progenitor cell types in the epithelium. A major controversy remains in the HS field as to whether HS function requires specific patterns of sulfation or simply charge density. The reported function of 3-O-sulfated HS is to bind to antithrombin and the herpes simplex virus glycoprotein, gD1. We analyzed the expression of sulfotransferase enzymes in the SMG. We found that Hs3st-modified HS was present on the SMG epithelium and that RNAi knockdown of Hs3st reduces morphogenesis and proliferation. We are investigating how FGFR2b signaling controls HS production on the cell surface of epithelial end bud progenitor cells. We have also collaborated with other NIH investigators to identify viral vectors that are able to infect salivary glands and will be useful for gene manipulation both in vivo and ex vivo. Additionally we have collaborated to investigate how O-glycosylation influences secretion of the extracellular matrix.
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