Amelogenesis imperfecta: We had earlier generated amelogenin-null mice, which displayed a typical X-linked amelogenesis imperfecta (AI) phenotype characterized by chalky white teeth, enamel hypoplasia, a lack of prismatic crystals, and cuspal attrition. Additionally, we analyzed the supramolecular structure of their enamel ECM by polarizing microscopy and found that ordered organization of enamel ECM was significantly abnormal. Our studies established that amelogenins are essential for the organization of the crystal pattern and enamel development but are not required for initiation of mineral crystal formation. Subsequently, we crossed these null mice with transgenic mice overexpressing bovine leucine-rich amelogenin peptide (LRAP), one of the alternately spliced amelogenins, to assess its effects on the amelogenin-null phenotype. These double-transgenic mice failed to rescue the tooth defects seen in the amelogenin-null mice, indicating the importance of functional differences in amelogenin splice variants. In addition to their enamel-specific roles, amelogenins are also implicated in the formation of root cementum. Our studies clearly demonstrated that the amelogenin splice variants are expressed in a nonenamel component of the tooth, namely tooth roots, thereby implying additional roles for amelogenins. We detected progressive defects in the cementum of aging null mice. Recently, we characterized functions of the amelogenin isoforms in cementum biology, particularly in osteoclastogenesis, and in the proliferation and migration of cementoblast/periodontal ligament (CM/PDL) cells. We also tested the hypothesis that a single proline-to-threonine (P70T) change would lead to an enamel defect similar to AI. We generated transgenic mice that expressed the amelogenin transgene with the P70T mutation under the control of an amelogenin promoter. These mice exhibited the AI phenotype, confirming involvement of this mutation in this enamel disease. ? ? Dentinogenesis imperfecta: Several mutations have been identified in the DSPP gene in patients with dentinogenesis imperfecta (DGI). DSPP is predominantly expressed in dentin-producing odontoblasts. Low levels have also been detected in several other tissues such as bone, inner ear, salivary gland, kidney, etc. However, the functions associated with DSPP gene expression are not clearly understood. To characterize such functions, we generated DSPP gene knockout mice by homologous recombination and found that these mice developed a DGI-III-like phenotype: enlarged predentin, a widened pulp cavity, globular dentin, and hypomineralization. Interestingly, the phenotype also resembles the teeth from patients with rickets. We found elevated levels of biglycan and decorin in both the affected teeth of DSPP-null mice and human rickets patients. In these teeth, vitamin D receptor (VDR) expression was analyzed by immunohistochemistry. VDR protein was abnormally localized in the cytoplasm instead of the nucleus and was found to be functionally defective as evidenced by the expression of VDR-regulated genes such as biglycan, decorin, osteocalcin, and osteopontin. In order to understand the molecular mechanism underlying this phenotype, we first examined whether the elevated levels of biglycan and decorin were causative factors or elevated as a consequence. Towards this goal, we have generated 2 mouse models that are double knockouts for either DSPP;biglycan or DSPP;decorin. Detailed analysis of these 2 mouse models indicates that the deficiency of decorin but not biglycan rescues the enlarged predentin phenotype of DSPP-null mice. ? ? Salivary gland inflammation: TGF-betas are ubiquitously expressed, and experiments with mice with mutations of various TGF-beta family members and components of the TGF-beta signaling pathway have shown crucial roles of TGF-beta in multiple physiological processes. TGF-beta1-null mice develop severe multifocal inflammation primarily in the lungs, heart, and salivary glands and succumb to multiorgan failure by the age of 3-4 weeks. The salivary gland lesions in the TGF-beta1-null mice resemble those seen in patients with Sjogren's syndrome (SS), which are characterized by focal lymphocytic infiltrations. Altered levels of ductal expression of TGF-beta isoforms were observed in patients with primary SS and in those with an autoimmune disorder that produces benign lymphoepithelial lesions (BLEL). However, immunohistochemical studies have yielded conflicting results on TGF-beta levels in salivary gland epithelia from SS patients. The precise role of TGF-beta signaling has not been characterized in the etiology of autoimmune disorders that affect salivary glands such as SS and BLEL. We have evaluated this role by impairing TGF-beta receptor I (RI) expression primarily in mouse salivary glands by using the Cre-lox system. In this system, the Cre recombinase from bacteriophage P1 excises the intervening DNA sequence located between 2 unidirectional lox sites positioned on the same linear DNA segment, leaving the lox site behind, and in our present studies, this results in the deletion of the TGF-beta RI sequence in tissues where Cre is expressed. Body weight loss and early mortality were observed only in the female TGF-beta RI conditional knockout (TBRIcoko) mice at the age of 4-5 weeks. Histopathological analysis of female TBRIcoko mice showed multifocal inflammation in the salivary glands, mammary glands, and heart. Moreover, flow cytometric analysis revealed T-cell infiltration in salivary glands of these mice, which resembles the infiltration seen in SS patients. Interestingly, administration of an adenoviral vector encoding Cre recombinase in the salivary glands resulted in inflammatory foci in female TRI floxed mice, but not in wild-type male and female mice or male TBRI floxed mice, indicating that female mice are more susceptible to autoimmune disorders due to impaired TGF-beta signaling. ? ? Head and neck squamous cell carcinoma: Each year more than 500,000 cases are diagnosed worldwide with head and neck squamous cell carcinoma (HNSCC). In the United States alone, 40,000 new cases are found every year. It accounts for 3.2% of all new cancer cases and 2.1% of cancer-related deaths. The 5-year survival rate of HNSCC patients is about 50% and has not improved in more than 2 decades. A better understanding of molecular tumorigenesis of HNSCC may allow for early detection, margin evaluation, prognostication, and development of new strategies for treatment. TGF-beta plays an important role in carcinogenesis. It acts as a potent tumor suppressor in the early stage of cancer development, while later TGF-beta can function as a tumor promoter. Alterations of components in the TGF-beta signaling pathway have been found in various types of human cancer, including HNSCC. Type 1 TGF-beta receptor (TBRI) mutations result in less responsiveness to TGF-beta and have been associated with HNSCC, breast and ovarian cancer, and pancreatic cancer. Thirty-five percent of NF-H-Cre/TBR1 KO mice developed spontaneous squamous cell carcinoma in the head and neck, stomach, and perianal regions. Compared to TGF-BRII, TGF-BR1 has received less attention to date. It is not clear if the combination of mutations in both receptors strengthens a common effect or if alteration of the 2 components has 2 distinct effects. It is possible that TGF-BRI might in some circumstance function independently of TGF-BRII and have some additional effects, perhaps in conjunction with other receptors.
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