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.

牙釉质生成不全：我们之前培育了牙釉蛋白无效的小鼠，它们表现出典型的 X 连锁牙釉质生成不全 (AI) 表型，其特征是白垩色牙齿、牙釉质发育不全、缺乏棱柱状晶体和牙尖磨损。此外，我们通过偏光显微镜分析了其牙釉质ECM的超分子结构，发现牙釉质ECM的有序组织明显异常。 我们的研究表明，牙釉蛋白对于晶体图案的组织和牙釉质的发育至关重要，但对于矿物晶体形成的起始不是必需的。随后，我们将这些无效小鼠与过度表达牛富含亮氨酸的牙釉蛋白肽（LRAP）（交替剪接的牙釉蛋白之一）的转基因小鼠杂交，以评估其对牙釉蛋白无效表型的影响。 这些双转基因小鼠未能挽救在牙釉蛋白无效小鼠中观察到的牙齿缺陷，这表明牙釉蛋白剪接变体中功能差异的重要性。 除了牙釉质特有的作用外，牙釉蛋白还参与牙骨质的形成。 我们的研究清楚地表明，牙釉蛋白剪接变体在牙齿的非釉质成分（即牙根）中表达，从而暗示牙釉蛋白具有额外的作用。我们检测到衰老无效小鼠牙骨质的进行性缺陷。 最近，我们表征了牙釉质异构体在牙骨质生物学中的功能，特别是在破骨细胞生成以及成牙骨质细胞/牙周膜（CM/PDL）细胞的增殖和迁移中的功能。 我们还测试了一个假设，即单个脯氨酸到苏氨酸 (P70T) 的变化会导致类似于 AI 的牙釉质缺陷。我们生成了在牙釉蛋白启动子控制下表达具有 P70T 突变的牙釉蛋白转基因的转基因小鼠。这些小鼠表现出 AI 表型，证实了这种突变与牙釉质疾病有关。 牙本质发生不全症：在牙本质发生不全症 (DGI) 患者的 DSPP 基因中发现了一些突变。 DSPP 主要在产生牙本质的成牙本质细胞中表达。 在骨、内耳、唾液腺、肾脏等其他一些组织中也检测到了低水平。然而，与 DSPP 基因表达相关的功能尚不清楚。 为了表征这些功能，我们通过同源重组产生了 DSPP 基因敲除小鼠，发现这些小鼠出现了类似 DGI-III 的表型：前牙本质增大、牙髓腔变宽、牙本质呈球状和矿化不足。 有趣的是，这种表型也类似于佝偻病患者的牙齿。 我们发现 DSPP 缺失小鼠和人类佝偻病患者受影响的牙齿中双糖链蛋白聚糖和核心蛋白聚糖水平升高。 通过免疫组织化学分析这些牙齿中维生素 D 受体 (VDR) 的表达。 VDR 蛋白异常定位于细胞质而不是细胞核，并且被发现存在功能缺陷，VDR 调节基因（如双糖链蛋白聚糖、核心蛋白聚糖、骨钙蛋白和骨桥蛋白）的表达证明了这一点。 为了了解这种表型背后的分子机制，我们首先检查双糖链蛋白聚糖和核心蛋白聚糖水平的升高是致病因素还是结果升高。 为了实现这一目标，我们生成了 2 个小鼠模型，它们是 DSPP;biglycan 或 DSPP;decorin 的双敲除。对这两种小鼠模型的详细分析表明，核心蛋白聚糖的缺乏而非双糖链蛋白聚糖的缺乏可以挽救 DSPP 缺失小鼠的扩大的前牙本质表型。 唾液腺炎症：TGF-β 普遍表达，对具有各种 TGF-β 家族成员和 TGF-β 信号通路成分突变的小鼠进行的实验表明，TGF-β 在多种生理过程中发挥着至关重要的作用。 TGF-β1缺失的小鼠主要在肺、心脏和唾液腺中出现严重的多灶性炎症，并在3-4周龄时死于多器官衰竭。 TGF-β1缺失小鼠的唾液腺病变类似于干燥综合征（SS）患者的唾液腺病变，其特征是局灶性淋巴细胞浸润。 在原发性 SS 患者和患有产生良性淋巴上皮病变 (BLEL) 的自身免疫性疾病患者中观察到 TGF-β 同种型的导管表达水平发生变化。 然而，免疫组织化学研究对 SS 患者唾液腺上皮细胞中 TGF-β 水平产生了相互矛盾的结果。 TGF-β 信号传导的确切作用尚未在影响唾液腺的自身免疫性疾病（如 SS 和 BLEL）的病因学中得到表征。 我们通过使用 Cre-lox 系统主要损害小鼠唾液腺中的 TGF-β 受体 I (RI) 表达来评估这种作用。在这个系统中，来自噬菌体 P1 的 Cre 重组酶切除了位于同一线性 DNA 片段上 2 个单向 lox 位点之间的插入 DNA 序列，留下了 lox 位点，在我们目前的研究中，这导致了 TGF- Cre 表达组织中的 beta RI 序列。 仅在 4-5 周龄的雌性 TGF-β RI 条件性敲除 (TBRIcoko) 小鼠中观察到体重减轻和早期死亡。 对雌性 TBRicoko 小鼠的组织病理学分析显示，其唾液腺、乳腺和心脏存在多灶性炎症。 此外，流式细胞术分析显示这些小鼠的唾液腺中存在 T 细胞浸润，这与 SS 患者中观察到的浸润类似。有趣的是，在唾液腺中施用编码Cre重组酶的腺病毒载体会导致雌性TRI floxed小鼠出现炎症灶，但在野生型雄性和雌性小鼠或雄性TBRI floxed小鼠中不会出现炎症灶，这表明雌性小鼠更容易患自身免疫性疾病由于 TGF-β 信号传导受损。 头颈鳞状细胞癌：全世界每年诊断出超过 500,000 例头颈鳞状细胞癌 (HNSCC)。仅在美国，每年就会发现 4 万例新病例。它占所有新发癌症病例的 3.2% 和癌症相关死亡的 2.1%。 HNSCC患者的5年生存率约为50%，并且20多年来没有改善。更好地了解 HNSCC 的分子肿瘤发生可能有助于早期检测、边缘评估、预测和开发新的治疗策略。 TGF-β在癌发生过程中发挥着重要作用。它在癌症发展的早期阶段充当有效的肿瘤抑制因子，而后期 TGF-β 可以充当肿瘤促进剂。在包括 HNSCC 在内的各种类型的人类癌症中都发现了 TGF-β 信号通路成分的改变。 1 型 TGF-β 受体 (TBRI) 突变导致对 TGF-β 的反应性降低，并与 HNSCC、乳腺癌、卵巢癌以及胰腺癌相关。 35% 的 NF-H-Cre/TBR1 KO 小鼠在头颈、胃和肛周区域发生自发性鳞状细胞癌。与 TGF-BRII 相比，TGF-BR1 迄今为止受到的关注较少。目前尚不清楚两种受体的突变组合是否会增强共同效应，或者两种成分的改变是否会产生两种不同的效应。 TGF-BRI 在某些情况下可能独立于 TGF-BRII 发挥作用，并且可能与其他受体结合产生一些额外的作用。