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. 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. HS proteoglycans are essential for biological processes regulated by FGFs. HS regulates the activity of FGFs by acting as a coreceptor at the cell surface, enhancing FGF/FGFR affinity, and being a storage reservoir for FGFs in the extracellular matrix (ECM). Heparanase, an endoglycosidase, colocalized with perlecan, a major proteoglycan in the basement membrane and in epithelial clefts of SMGs. Inhibition of heparanase activity in organ culture decreased branching morphogenesis, and this inhibition was rescued specifically by FGF10 and not by other FGFs. In contrast, exogenous heparanase increased SMG branching and intracellular signaling, and when isolated epithelia were cultured in ECM with FGF10, it increased the number of lateral branches and end buds. In a solid-phase binding assays, an FGF10/FGFR2b complex was released from the ECM or purified perlecan HS by heparanase. We have used the FGF10/FGFR2b complex as a probe for HS in SMGs, and it colocalized with perlecan in the basement membrane and partly colocalized with syndecan-1 in the epithelium, and binding was reduced by treatment with heparanase. Thus heparanase releases FGF10 from perlecan HS in the basement membrane, increasing intracellular signaling, epithelial clefting, and lateral branch formation, which results in increased branching morphogenesis.? ? We have also studied the role of laminin alpha 5 during branching morphogenesis. FGF signaling 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 gammaa chain and are important during SMG development. Laminin isoforms are developmentally regulated during SMG development. The SMGs of mice lacking laminin alpha 5 have a striking phenotype: epithelial clefting is delayed, although proliferation occurs; there is decreased FGFR1b and FGFR2b, but no difference in laminin alpha 1 expression; later in development, epithelial cell organization and lumen formation are disrupted. In wild-type SMGs both laminin alpha 1 and alpha 5 are present in epithelial clefts but as branching begins, alpha 5 expression increases while alpha 1 decreases. Laminin alpha 5 siRNA also decreased branching, downstream signaling, and FGFR expression, and branching was rescued by FGF10. Reducing FGFR expression by siRNA also decreased laminin alpha 5 expression, suggesting that FGFR signaling provides positive feedback for laminin expression. The cell surface receptors for laminins are integrins and inhibiting integrin function decreases FGFR and laminin expression, suggesting that integrin signaling provides positive feedback for laminin and FGFR expression. Interestingly, mice lacking integrin alpha 3 and alpha 6, two important integrin receptors for laminin alpha 5, have SMGs with a similar phenotype to laminin alpha 5 null SMGs. Our findings suggest that laminin alpha 5 controls SMG epithelial morphogenesis through integrin signaling by regulating FGFR expression, which also reciprocally regulates the expression of laminin alpha 5. These data link changes in basement membrane composition during branching morphogenesis with FGFR expression and signaling.? ? 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.? ? 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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