Role of TGF-beta signaling pathway in salivary gland inflammation and fibrosis: The primary role of salivary glands in humans is to produce an exocrine secretion, saliva, which provides (A) most of the innate host defense for the upper GI tract, (B) specific protective and reparative functions for tooth enamel and the oral mucosa, and (C) essential molecules required for adequate mastication and processing of a food bolus. Any condition that results in decreased saliva production is undesirable and will have a negative impact on oral health and the patient's quality of life. There are 3 major causes of reduced saliva production: prescription medications, therapeutic radiation for head and neck cancer, and Sjogrens syndrome. The latter 2 conditions lead to irreversible damage to salivary gland secretory tissue, and both are resistant to current therapeutic interventions. TGF-beta plays a key role in the onset and resolution of autoimmune diseases, chronic inflammation, and fibrosis.
The aim of this study was to delineate the precise function of TGF-beta signaling in salivary gland inflammation. In collaboration with the NIDCR Gene Therapy and Therapeutics Branch, we impaired TGF-beta signaling in mouse salivary glands by conditionally inactivating expression of TGF-beta receptor I using either MMTV-Cre mice or by delivering adenoviral vector containing Cre to mouse salivary glands via retrograde infusion of the cannulated main excretory ducts of submandibular glands. Interestingly, among the TGF-beta receptor I conditional knockouts (TbetaRIcoko) generated with MMTV-Cre mice, only female mice developed lethal multifocal inflammation in salivary glands, mammary glands, and the heart. The female TbetaRIcoko mice also showed an increase in Th1 proinflammatory cytokines in salivary glands and exhibited an upregulation of peripheral T cells. In addition, these mice also displayed an atypical distribution of aquaporin-5 in their salivary glands, suggesting likely secretory impairment. Administration of an adenoviral vector encoding Cre recombinase to the salivary glands resulted in inflammatory foci only in the glands of female TGF-beta RI floxed mice, but not in wild-type male and female mice or male TbetaRI floxed mice. These results suggest that female mice are uniquely susceptible to developing inflammatory disorders because of impaired TGF-beta signaling in their salivary glands. Overexpression of TGF-beta1 has been implicated in many human disorders such as idiopathic pulmonary fibrosis, systemic sclerosis, and hepatic fibrosis. TGF-beta1 is also involved in the formation of scars and in radiation-induced fibrosis. To study the roles of TGF-beta in salivary gland fibrosis, we generated a mouse model that will allow temporal and spatial control of the expression of an active and HA-tagged version of TGF-beta1. We have established 8 mouse lines and bred 2 lines with MMTV-Cre. Preliminary results show perinatal lethality in the double-positive mice.? ? Molecular roles of TGF-beta signaling in head and neck squamous cell carcinogenesis (HNSCC): TGF-beta is believed to have a dual but context-dependent role in carcinogenesis, acting either as a tumor suppressor or a tumor promoter. Mutations and polymorphisms of TGF- receptor I (TbetaRI) have been associated with human HNSCC. However, the precise role of TGF-beta signaling in HNSCC has not been delineated.
The aims of our studies are to understand the molecular roles of the TbetaRI-mediated signaling pathway in the etiology of HNSCC and to test whether a disruption of TGF-beta signaling at the receptor level leads to spontaneous tumor formation or only increases the susceptibility. We have earlier reported the TGF-beta receptor I (TbetaRI) conditional knockout (COKO) mouse model generated by crossing TGF-beta receptor I (TbetaRI) floxed mice with neurofilament-H (NF-H) Cre mice. 35% of F1 COKO mice developed spontaneous squamous cell carcinomas (SCCs) in periorbital and/or perianal regions. Transplantation of these tumors into athymic nude mice resulted in 62% tumorigenicity. These mice can serve as a unique mouse model of SCC to evaluate the tumorigenicity and effect of anticancer therapeutics. We also developed an inducible oral epithelium-specific knockout system by crossing TbetaRI floxed mice with K14-CreERtam mice. By applying tamoxifen to the mouse oral cavity to induce Cre expression, we were able to conditionally delete TbetaRI in the oral epithelia. No spontaneous tumors in the head and neck epithelia of the TbetaRIf/f;K14-CreERtam (TbetaR1cKO) mice were noted after 11 months of observation. However, upon tumor induction with 7,12-dimethylbenzanthracene (DMBA), 20% of TbetaR1cKO mice developed HNSCC starting 16 weeks after treatment, while no tumors were observed in control littermates during the same time period. The molecular analysis of these tumors is now underway. We will utilize our mouse models to develop new strategies for targeting the TGF-beta signaling pathway in the therapeutic treatment of HNSCC.? ? Distribution of efforts between these two projects: Salivary Gland Disorders: 30%; HNSCC: 70%
