An irreversible decrease 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 in certain rare genetic syndromes affecting fibroblast growth factor receptor 2b (FGFR2b) signaling and fibroblast growth factor 10 (FGF10). In humans, mutations in FGF10 result in ALSG syndrome, (Aplasia of Lacrimal and Salivary Glands), a condition where the major salivary glands fail to develop. The patients experience severe xerostomia, increased caries and dental erosion, periodontal disease and irritable eyes, eye infections and epiphoria (constant tearing). In addition, other mutations in FGFR2b or FGF10 result in the more severe LADD syndrome (Lacrimo-Auriculo-Dento-Digital syndrome), which affects lacrimal and salivary glands in a similar manner to ALSG, but also affects the development of teeth, ears, fingers and toes, kidneys, lungs, and genitalia. Thus, FGF10 and FGFR2b are critical for salivary gland development in humans. A similar situation exists in mice genetically engineered with no FGF10 and FGFR2b genes. These mice do not develop salivary glands while mice with one copy of the FGF10 gene have salivary gland hypoplasia (small glands). Understanding the mechanisms of FGF10 action during salivary gland development will provide targets for gland regeneration and/or tissue engineering approaches to restore glandular tissue and secretory function. The goal of the Matrix and Morphogenesis Unit is to identify the mechanisms of FGF/extracellular matrix interactions that regulate branching morphogenesis of salivary glands. Our primary model system to study salivary gland organogenesis is the developing mouse submandibular gland.? In the past year we have focused our studies on how heparan sulfate (HS) modulates the biological activity of FGF10 to regulate SMG branching morphogenesis. HS is attached to proteoglycans and glycolipids on the cell surface and in the extracellular matrix and binds to FGFs. Previously we identified a major role for FGFR2b signaling through its ligands FGF7 and FGF10 in the regulation of epithelial cell proliferation during development of the salivary epithelium. Our data suggest FGFR2b signaling involves an autocrine regulatory network of FGFR1b/FGF1/MMP2 expression that mediates budding and duct elongation during branching morphogenesis. The affinity of FGF binding to FGF receptors is increased by heparan sulfate. In vivo, the biological activity of heparan sulfate is regulated by heparanase, an endoglycosidase that degrades heparan sulfate. Addition of heparanase to SMG organ culture increases branching morphogenesis, while inhibition of heparanase activity decreases salivary gland branching suggesting heparan sulfate cleavage is required for FGF function during branching morphogenesis. Addition of exogenous FGF10 to epithelial rudiments induces cell proliferation at the tips of the ducts resulting in branching and duct elongation. This FGF10-mediated morphology is modified by heparin fragments containing specific sulfated sugars, suggesting the sulfation patterns of HS increases the receptor binding of FGF10, and increase cell proliferation. The enzymes that add the sulfates to HS are sulfotransferase enzymes, and they are differentially localized by in situ analysis in the SMG. We hypothesize that the sulfation pattern of HS in the mesenchyme may be important for localization and or storage of FGF10 while HS on the epithelium may be involved in signaling complexes with epithelial receptors. ? FGFs also regulate the expression of matrix metalloproteinases (MMPs) during branching morphogenesis. Chemical inhibition of MMP activity decreases branching morphogenesis in SMG organ cultures. RNAi knockdown of MMPs decreases branching morphogenesis in organ cultures. Salivary epithelium treated with either FGF7 or FGF10 develop with distinct morphologies, therefore we analyzed the changes of MMPs expression in epithelial cultures. FGF signaling regulates the expression and the activity of MMPs which are downstream effectors of branching morphogenesis; i.e. MMPs remodel ECM molecules as part of the morphogenic program of branching.? We have also begun to define the role of laminin alpha 5 during branching morphogenesis. FGF signaling also regulates gene expression and activity of extracellular matrix proteins and their receptors. Laminins are a family of heterotrimeric extracellular matrix proteins consisting of an alpha, beta, and gamma chain and are important during submandibular gland (SMG) development. Integrins are cell surface receptors for many extracellular matrix molecules including laminins. Many laminin and integrin isoforms and are localized in a cell-, tissue- or site-specific manner during development. We have analyzed the expression pattern of laminin isoforms during SMG development and in the laminin alpha 5 null mouse SMGs by real-time PCR. Laminin isoforms are developmentally regulated during SMG development. The major cell surface receptors for laminins are integrins. Inhibition of integrin function in SMG organ culture decreased branching morphogenesis and also decreased FGFR gene expression. Our findings suggest that laminin alpha 5/integrin receptor signaling regulates proliferation during branching morphogenesis by regulating FGFR expression.? 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 irreversible salivary hypofunction occurs.
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