Loss of salivary gland function in humans often occurs after removal of salivary tumors, after therapeutic radiation of head and neck tumors, as a result of Sjogrens Syndrome, and as a side effect of medications. Loss of salivary gland function impairs the oral health of patients. Understanding how salivary glands form during development will provide targets for gland regeneration and/or tissue engineering approaches to restore secretory function. The goal of the Matrix and Morphogenesis Unit is to identify the mechanisms of growth factor/extracellular matrix interactions that regulate branching morphogenesis of salivary glands. In addition, we are interested in discovering novel genes required for submandibular gland initiation and early branching morphogenesis. Our primary model system to study salivary gland organogenesis is the developing mouse submandibular gland. In the past year we have reported (1) that branching of submandibular glands decreases when either Fgfr2 expression is down-regulated or soluble recombinant FGFR2b competes out the endogenous growth factors. However, a combination of neutralizing FGF antibodies was required to inhibit branching in the intact gland, suggesting multiple FGFs are required for branching. We have identified a major role for FGFR2 in the regulation of epithelial cell proliferation during development of the salivary epithelium. Apoptosis does not appear to play a major role in regulating early epithelial morphogenesis. We have focused on the roles of FGF7 and FGF10, the ligands for FGFR2b, and how they influence the early morphogenesis of the epithelium. FGF7 induces epithelial budding, whereas FGF10 induces duct elongation, and both ERK1/2 and PI 3-kinase signaling pathways are downstream of these FGFs. FGFR1b and FGFR2b are present throughout the epithelium although FGFR1b is more highly expressed around the periphery of the buds and the duct tips. Using real time PCR we showed that FGF7 signaling increases FGFR1b and Fgf1 expression and MMP2 activity compared with FGF10, resulting in increased cell proliferation and expansion of the epithelial bud, while FGF10 stimulates localized proliferation at the tip of the duct. FGF7-and FGF10-mediated morphogenesis is inhibited by an MMP inhibitor and a neutralizing antibody to FGF1, suggesting both FGF1 and MMP activity are essential downstream mediators of epithelial morphogenesis. Our data suggests FGFR2b signaling involves a regulatory network of FGFR1b/FGF1/MMP2 expression that mediates budding and duct elongation during branching morphogenesis. FGFs bind to heparan sulfate, a negatively charged sugar that is attached to proteoglycans and glycolipids on the cell surface and in the extracellular matrix. Our continuing studies are focusing on how heparan sulfate regulates FGF biological activity during gland morphogenesis. In the past year we have used siRNA to decrease FGFR gene expression in salivary organ culture. The knockdown of FGFR1 in epithelial culture decreases gene expression of FGFR2, laminin isoforms and Fgf1. The knockdown of FGFR2 expression decreases FGF10-mediated epithelial morphogenesis and also decreases gene expression of FGFR1, laminin isoforms and Fgf1. Our findings suggest that FGFR signaling regulates both FGF and FGFR gene expression in an autocrine manner, and that it also regulates expression of extracellular matrix molecules during morphogenesis. We continue to focus on the role of individual laminin isoforms in the salivary gland basement membrane. The lab also focuses on analyzing genes identified by microarray analysis to be more highly expressed at early stages of salivary gland development. This work led to a collaboration with researchers at the University of Virginia to analyze gene expression of ion channels during a time course of early inner ear development (2). We are also profiling protease gene expression during development and during organ culture. We are currently using time-lapse confocal analysis of salivary epithelial rudiments to localize protease function during epithelial morphogenesis. Protease activity during FGF induced morphogenesis was localized using quenched fluorescent collagen added to the laminin matrix. The sites of most active matrix remodeling are the epithelial rudiment surface particularly at cleft sites, and along the ductal structures. In conclusion, our studies on the basic biologic mechanisms that result in branching morphogenesis provide a rationale for a biologically-based therapeutic approach for regeneration of salivary gland tissue. Understanding the cellular processes involved in tissue morphogenesis including proliferation, differentiation, apoptosis, and migration is critical to regenerating tissue. The regeneration of salivary glands could be a potential therapeutic option in cases where pathological gland destruction, surgical removal, or salivary hypofunction occurs.
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