Molecular genetics of tooth development and disease

牙齿发育和疾病的分子遗传学

• 批准号: 7593379
• 负责人: Ashok B. KULKARNI
• 金额: $ 122.3万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593379
• 关键词: Accounting; Adenovirus Vector; Affect; Age; Aging; Ameloblasts; Amelogenesis Imperfecta; Attention; Autoimmune Diseases; Benign; Biology; Body Weight decreased; Bos taurus; Breast; Candidate Disease Gene; Cattle; Cell Adhesion Molecules; Cell Nucleus; Cell physiology; Cells; Cementoblast; Cessation of life; Conflict (Psychology); Crystal Formation; Cytoplasm; DNA; DNA Sequence; DSPP gene; Defect; Dental Cementum; Dental Enamel; Dental Enamel Hypoplasia; Dental Pulp; Dentin; Dentin Formation; Dentinogenesis Imperfecta; Development; Diagnosis; Diagnostic Neoplasm Staging; Disease; Ductal; Early Diagnosis; Enterobacteria phage P1 Cre recombinase; Epithelium; Etiology; Evaluation; Exhibits; Extracellular Matrix; Extracellular Matrix Proteins; Failure; Family member; Female; Fibrosis; Future; Gene Expression; Gene Mutation; Genes; Goals; Growth Factor; Head and Neck Cancer; Head and Neck Squamous Cell Carcinoma; Head and neck structure; Heart; Hereditary Disease; Human; Immune response; Immunohistochemistry; Infiltration; Inflammation; Inflammatory; Kidney; Knock-out; Knockout Mice; Labyrinth; Lead; Left; Lesion; Link; Localized; Lung; Lymphocyte; Lymphoepithelial Lesion; Malignant Neoplasms; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Mammary gland; Metabolic; Microscopy; Minerals; Molecular; Molecular Genetics; Mus; Mutation; Numbers; Odontoblasts; Oral mucous membrane structure; Osteocalcin; Patients; Pattern; Periodontal Ligament; Phenotype; Physiological Processes; Plant Roots; Play; Positioning Attribute; Process; Proline; Protein Isoforms; Protein Overexpression; Proteins; RNA Splicing; Rickets; Role; Salivary Glands; Signal Pathway; Signal Transduction; Site; Sjogren' s Syndrome; Squamous cell carcinoma; Stomach; Structure; Survival Rate; System; T-Lymphocyte; TGF Beta Signaling Pathway; Testing; Threonine; Tissues; Tooth Components; Tooth root structure; Tooth structure; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; Transforming Growth Factors; Transgenes; Transgenic Mice; Tumor Promoters; Tumor Suppressor Proteins; United States; Variant; Vitamin D3 Receptor; Week; Wound Healing; amelogenin; biglycan; biomineralization; bone; carcinogenesis; case finding; craniofacial; decorin; functional genomics; homologous recombination; human TGFB1 protein; improved; knockout gene; leucine-rich amelogenin peptide; male; migration; mortality; mouse model; osteoclastogenesis; osteopontin; promoter; receptor; receptor expression; recombinase; research study; spatiotemporal; transcription factor; tumorigenesis

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.

ameleogeny Imperfecta：我们较早产生的amelogentin-Null小鼠，该小鼠表现出典型的X-link-inn-null null小鼠（AI）表型（AI）表型，其特征在于白牙齿牙齿，牙釉质低质，缺乏棱镜晶体和cuspal ratiention。此外，我们通过极化显微镜分析了其牙釉质ECM的超分子结构，并发现搪瓷ECM的有序组织显着异常。 我们的研究确定，氨基蛋白蛋白对于组织晶体模式和搪瓷发育至关重要，但对于矿物质晶体形成而不是必需。随后，我们与过表达牛叶丁素肽（LRAP）的转基因小鼠（LRAP）（交替剪接的蛋白酶之一）越过了这些无效小鼠，以评估其对杏仁素无效表型的影响。 这些双重转基因小鼠未能挽救在氨基蛋白酶无小鼠中看到的牙齿缺陷，这表明在氨基蛋白酶剪接变体中功能差异的重要性。 除了它们的牙釉质特异性作用外，氨基蛋白蛋白还与根胶质的形成有关。 我们的研究清楚地表明，氨基蛋白蛋白的剪接变体在牙齿的非烯酰胺成分中表达，即牙根根，从而暗示氨基蛋白酶的其他作用。我们检测到衰老无效小鼠的骨骼中的渐进缺陷。 最近，我们表征了蛋白质生物学中氨基蛋白蛋白同工型的功能，尤其是在破骨细胞生成中，以及胶质细胞/牙周韧带（CM/PDL）细胞的增殖和迁移中。 我们还测试了一个假设，即单个脯氨酸到硫酸（P70T）的变化将导致类似于AI的牙釉质缺陷。我们产生的转基因小鼠在氨基蛋白酶启动子的控制下用p70t突变表达了氨基蛋白蛋白转基因。这些小鼠表现出AI表型，证实了这种突变参与这种搪瓷疾病。 牙齿发生不完美：在DSPP基因中已经发现了牙抑制性不完美的患者（DGI）的几个突变。 DSPP主要在产生牙本质的Odontobasts中表达。 在其他几个组织中也发现了低水平的水平，例如骨骼，内耳，唾液腺，肾脏等。但是，与DSPP基因表达相关的功能尚未清楚地了解。 为了表征此类功能，我们通过同源重组产生了DSPP基因基因敲除小鼠，发现这些小鼠会形成DGI-III样表型：增大的predentin，扩大的​​牙髓腔，球状牙本质和次矿化。 有趣的是，该表型也类似于有鼠患者的牙齿。 在DSPP无效的小鼠和人rick鼠的患者的牙齿中，我们发现了大型群和装饰的水平升高。 在这些牙齿中，通过免疫组织化学分析了维生素D受体（VDR）表达。 VDR蛋白异常定位在细胞质中，而不是细胞核中，并被发现在功能上有缺陷，如VDR调节基因的表达（例如Biglycan，DecorIn，bosteocalcalcin和stopepopontin）所证明的那样。 为了理解这种表型的分子机制，我们首先检查了大型群体和装饰水平升高是导致因素还是升高。 为了实现这一目标，我们生成了2个鼠标模型，它们是DSPP的双重敲除； Biglycan或DSPP； Decorin。对这两种小鼠模型的详细分析表明，Decorin的缺乏，但没有大会挽救DSPP-null小鼠的predentin表型的扩大。 唾液腺炎症：TGF-betas普遍表达，并用小鼠进行具有各种TGF-β家族成员的突变和TGF-beta信号途径的成分的突变，显示了TGF-beta在多个生理过程中的至关重要的作用。 TGF-BETA1-NULL小鼠主要在肺，心脏和唾液腺中出现严重的多灶性炎症，并在3-4周的年龄到三个星期内屈服于多机器人衰竭。 TGF-BETA1-NULL小鼠中的唾液腺病变类似于Sjogren综合征（SS）患者，其特征是局灶性淋巴细胞浸润。 在初级SS患者以及患有自身免疫性疾病的患者中，TGF-β同工型的导管表达水平改变了，产生良性淋巴上皮病变（BLEL）。 然而，免疫组织化学研究对SS患者的唾液腺上皮中的TGF-β水平产生了矛盾的结果。 TGF-β信号传导的确切作用尚未在影响唾液腺（如SS和BLEL）的自身免疫性疾病的病因中表征。 我们通过使用CRE-LOX系统主要在小鼠唾液腺中损害了TGF-β受体I（RI）的表达来评估这一作用。在该系统中，来自噬菌体P1的CRE重组酶会分解位于位于同一线性DNA片段上的2个单向LOX位点之间的中间DNA序列，将LOX位点留在后面，在我们目前的研究中，这会导致TGF-Beta Ri序列的删除，而TGF-beta ri序列在CRE中的组织中表达了表达。 仅在4-5周的女性TGF-beta RI有条件敲除（TBRICOKO）小鼠中观察到体重减轻和早期死亡率。 雌性Tbricoko小鼠的组织病理学分析显示唾液腺，乳腺和心脏中多灶性炎症。 此外，流式细胞仪分析表明，这些小鼠的唾液腺中T细胞浸润类似于SS患者中看到的浸润。有趣的是，在唾液腺中编码CRE重物组合酶的腺病毒载体导致雌性Tri Flox小鼠的炎症灶，但在野生型雄性和雌性小鼠或雄性TBRI Floxed小鼠中却没有，这表明雌性小鼠对Imbimune Disorders funderimenders fummaired tgffffeta themime of AutoMune Disorders the trimpeiale to tgfffbeta themementa foci foci。 头部和颈部鳞状细胞癌：每年有500,000多例病例在全球范围内被头颈鳞状细胞癌（HNSCC）诊断。仅在美国，每年就会发现40,000个新案件。它占所有新癌症病例的3.2％，占癌症相关死亡的2.1％。 HNSCC患者的5年生存率约为50％，在20多年中没有提高。更好地了解HNSCC的分子肿瘤发生，可以允许早期检测，保证金评估，预测和开发新的治疗策略。 TGF-β在癌变中起重要作用。它在癌症发展的早期阶段充当有效的肿瘤抑制剂，而后来TGF-beta可以用作肿瘤启动子。在包括HNSCC在内的各种类型的人类癌症中发现了TGF-β信号通路中成分的改变。 1型TG​​F-β受体（TBRI）突变导致对TGF-β的反应性较小，并且与HNSCC，乳腺癌和卵巢癌以及胰腺癌有关。 NF-H-CRE/TBR1 KO小鼠中有35％在头部和颈部，胃和毛层区域中出现自发的鳞状细胞癌。与TGF-BRII相比，迄今为止，TGF-BR1受到的关注较少。目前尚不清楚两个受体中突变的组合是否增强了共同的效果，或者2个成分的改变是否具有2种不同的影响。在某些情况下，TGF-BRI可能独立于TGF-BRII的功能，并且可能与其他受体结合使用一些其他效果。