Laboratory of Oral Connective Tissue Biology

口腔结缔组织生物学实验室

• 批准号: 8750651
• 负责人: Martha Somerman
• 金额: $ 106.61万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 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/8750651
• 关键词: Affect; Agonist; Animals; Binding; Biology; Bone Resorption; Cell-Matrix Junction; Cells; Cementoblast; Cementogenesis; Cementum Formation; Clinical Trials; Collaborations; Collagen Fibril; Complex; Congenital Abnormality; Connective Tissue; Coupled; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA Sequence; Data; Defect; Degenerative polyarthritis; Dental; Dental Cementum; Dental Enamel; Dentin; Deposition; Development; Disease; Dose; Down-Regulation; Enzymes; Epithelial; Exhibits; Extracellular Matrix; Family; Forskolin; Future; Gene Expression; Gene Proteins; Genes; Goals; Growth; Healed; Histocytochemistry; Histology; Histone Deacetylase; Homeostasis; Human; Hydroxyapatites; In Vitro; Integrin Binding; Integrins; Knockout Mice; Knowledge; Laboratories; Maintenance; Maps; Mediating; Messenger RNA; Metabolism; Methods; Minerals; Modeling; Mus; National Institute of Dental and Craniofacial Research; Natural regeneration; Oral; Osteoclasts; Osteocytes; Outcome; Pathology; Pathway interactions; Pattern; Periodontal Diseases; Periodontal Ligament; Phenotype; Plant Roots; Play; Precipitation; Process; Property; Proteins; Proteoglycan; Reaction Time; Regulation; Research; Rodent Model; Role; Signal Transduction; Site; Staining method; Stains; Surface; TNFSF11 gene; Tendon structure; Testing; Therapeutic; Tissues; Tooth Exfoliation; Tooth Loss; Tooth structure; Transcript; Transforming Growth Factor beta; Vesicle; Vitamin D3 Receptor; Western Blotting; Wound Healing; alveolar bone; asporin; biglycan; bone morphogenetic protein receptors; bone sialoprotein; calcification; craniofacial; craniofacial complex; decorin; dentin matrix protein 1; fibromodulin; healing; in vivo; in vivo Model; inorganic phosphate; insight; lumican; mRNA Expression; mineralization; mouse model; parathyroid hormone (1-34); plasma cell membrane glycoprotein PC-1; polarized light; prevent; programs; promoter; protein expression; regenerative; repaired; small molecule; tissue repair; transcription factor; wound

A. We hypothesized that factors involved in controlling PPi/Pi levels, e.g., ANK, NPP1, PHOSPHO1, TNAP, would be involved in root formation, cementogenesis. a. Teeth obtained from Alpl KO mice, beyond cementum phenotype (no acellular cementum, altered PDL, exfoliation of teeth) exhibited dentin/enamel phenotype, mimicking humans. Enamel, dentin and cementum defects were corrected by TNAP enzyme replacement, mice and humans (latter by Millan's group independently) b. Healing, using a periodontal defect wound model, occurred more rapidly in Ank KO mice, demonstrating significance of decreasing PPi levels at local sites toward promoting periodontal tissue repair c. Teeth from AlplxPhospho1 dKO mice exhibited a more profound dentin phenotype than comparable tissues from KO of Alpl or Phospho1 demonstrating the function of PHOSPHO1 in controlling Pi levels in matrix vesicles (MVs), and TNAP and PHOSPHO1 act synergistically in regulating mineralization. Recent data, in collaboration with Millans group, suggest that PHOSPHO1 plays a role in cementum formation, an unexpected finding. Thus, either PHOSPHO1 has a role in regulating Pi/PPi beyond MVs and/or cementum formation involves MVs d. In vitro, ANK and NPP1 were shown to be mineralized-driven genes/protein and coupled with in vivo findings, demonstrated the importance of Pi/PPi modulation for cementogenesis and provide the rationale for ongoing studies using in vitro methods to identify small molecules controlling mineralization and in vivo periodontal wound healing models to deliver factors controlling Pi/PPi, locally and systemically e. Intriguingly, examining periodontal tissues from Ank and Npp1 KO animals demonstrated a compensatory mechanism related to gene expression, ENPP1 protein levels increased in cementum in Ank KO tissues, while ANK protein levels increased in Npp1 KO tissues, yet the cementum phenotype was not altered, most likely due to the profound role of TNAP in providing Pi at local sites f. Cementum from of Npp1 or Ank KO mice exhibited a marked increase in protein expression for both OPN and DMP1, and further cementoblasts exposed to Pi in vitro also exhibited an increase in transcripts for these two genes. These data provide a unified hypothesis for how Pi/PPi ratio developmentally tunes cementum formation and properties, an insight that we plan to employ to stimulate cementum regeneration, using murine models. B. We hypothesized a relationship exists between the SIBLING family, e.g. BSP, OPN and DMP1, their associated integrins, and key regulators of phosphate metabolism and modulating SIBLINGS provides therapeutic strategy to regenerate cementum. a. Bone sialoprotein affects cell attachment and signaling through an RGD integrin-binding region, and acts as a positive regulator for mineral precipitation by nucleating hydroxyapatite crystals. To test our hypothesis we analyzed tooth development in a Bsp null (-/-) mouse model. Developmental analysis, in collaboration with Goldbergs group, by histology, histochemistry, and SEM revealed a marked reduction in acellular cementum formation on Bsp -/- teeth, and cementum deposited was hypomineralized.. Loss of BSP caused disorganized PDL and increased epithelial down-growth. Bsp -/- mice displayed extensive root and alveolar bone resorption, mediated by increased RANKL and osteoclasts. Increased staining for DMP1 in the region of acellular formation in tissues from KO mice vs. WT tissues was noted. Results suggest that BSP plays an essential and non-redundant role in acellular cementum formation, likely involved in initiating mineralization on the root surface. We are exploring the integrin/BSP relationship with Fisher at NIDCR b. In a related project with Young, NIDCR, we hypothesized that alterations in proteoglycans would compromise periodontal tissues and expression of key SIBLINGS. dKO for two SLRPs, fibromodulin and biglycan, acquire ectopic calcification of tendons and severe osteoarthritis. Polarized light studies demonstrated abnormal collagen fibrils and defective remodeling of alveolar bone of dKO mice. IHC revealed increased staining of SLRPs, asporin, lumican and decorin, in PDL of dKO mice, indicating compensatory mechanisms. DMP1, considered to be mechanosensory osteocyte marker, was increased markedly in dKO PDL. Disruption of homeostasis of dKO PDL was further supported by increased expression of RANKL and elevated numbers of TRAP positive osteoclasts. To elucidate underlying mechanisms, PDL tissues from dKO mice were analyzed by PCR array. Results indicated hyperactive TGF beta/BMP signaling in dKO tissues with significant increased expression of SMAD-4 and -5 genes, and elevated staining of phosphorylated SMAD5 in dKO PDL. Therefore we propose that Fmod and Bgn sequester BMP preventing binding to BMP receptors. The predicted outcome of deletion of Fmod and Bgn from ECM would be an elevated sensitivity to TGF beta/BMP which could affect BMPs signal transduction, causing an increased expression of phosphorylated SMAD5 in PDL. C. We hypothesized that the ability of PTH and 1,25D to modulate FGF23 expression involves an intermediate factor and specifically, DMP1. 1,25D: Murine cementoblasts and osteocyte-like cells were used. Dmp1 mRNA levels decreased by 50% in the presence of 1,25D, while use of a VDR agonist and antagonist confirmed that vitamin D receptor pathway activation was required. Further analysis showed that histone deacetylase (HDAC) recruitment was necessary. PTH: Cementoblasts and cementocytes exposed to PTH (1-34) exhibited a decrease in expression of Dmp1 mRNA in a dose-response and time-dependent manner. Western blot detected a 30% down-regulation of DMP1 with PTH treatment.Treatment with PTH increased intracellular cAMP levels by 10-fold compared to control, and forskolin, a cAMP/PKA pathway agonist, also decreased Dmp1 mRNA expression, implicating the cAMP/PKA pathway. Our results suggest that PTH may modulate FGF23 via several pathways e.g. SOST as well as DMP-1 and further that PTH and 1,25D-mediated effects on FGF23 may share some common mechanistic aspects. Future studies using RNAseq approaches will help determine the specific DNA sequences and transcription factor(s) involved in Dmp1 regulation. Research related to the DMP1 promoter has been in collaboration with Collins at NIDCR and Presland at UW.

答：我们假设控制 PPi/Pi 水平的因素，例如 ANK、NPP1、PHOSPHO1、TNAP，将参与牙根形成、牙骨质形成。 一个。从 Alpl KO 小鼠获得的牙齿，除了牙骨质表型（无无细胞牙骨质、PDL 改变、牙齿脱落）外，还表现出模仿人类的牙本质/牙釉质表型。小鼠和人类（后者由 Millan 小组独立）通过 TNAP 酶替代品纠正了牙釉质、牙本质和牙骨质缺陷 b．使用牙周缺损伤口模型，Ank KO 小鼠的愈合速度更快，这表明降低局部部位的 PPi 水平对于促进牙周组织修复具有重要意义。 AlplxPhospho1 dKO 小鼠的牙齿比 Alpl 或 Phospho1 KO 的同类组织表现出更深刻的牙本质表型，证明 PHOSPHO1 在控制基质囊泡 (MV) 中 Pi 水平方面的功能，并且 TNAP 和 PHOSPHO1 在调节矿化方面发挥协同作用。与 Millans 小组合作的最新数据表明 PHOSPHO1 在牙骨质形成中发挥作用，这是一个意外的发现。 因此，PHOSPHO1 在调节 MV 之外的 Pi/PPi 中发挥作用，和/或牙骨质形成涉及 MV d。在体外，ANK 和 NPP1 被证明是矿化驱动的基因/蛋白质，并与体内研究结果相结合，证明了 Pi/PPi 调节对牙骨质形成的重要性，并为正在进行的使用体外方法识别控制矿化的小分子的研究提供了理论依据以及体内牙周伤口愈合模型，以提供局部和系统控制 Pi/PPi 的因子。有趣的是，检查Ank和Npp1 KO动物的牙周组织表明了与基因表达相关的补偿机制，Ank KO组织中牙骨质中的ENPP1蛋白水平增加，而Npp1 KO组织中的ANK蛋白水平增加，但牙骨质表型没有改变，大多数可能是由于 TNAP 在本地站点提供 Pi 方面发挥的深远作用 f． Npp1 或 Ank KO 小鼠的牙骨质中 OPN 和 DMP1 的蛋白表达均显着增加，并且体外暴露于 Pi 的成牙骨质细胞也表现出这两个基因转录物的增加。 这些数据为 Pi/PPi 比率如何发育地调节牙骨质形成和特性提供了统一的假设，我们计划利用小鼠模型来刺激牙骨质再生。 B. 我们假设 SIBLING 家族之间存在关系，例如BSP、OPN 和 DMP1、它们相关的整合素以及磷酸盐代谢的关键调节因子和调节 SIBLINGS 提供了牙骨质再生的治疗策略。 一个。骨唾液酸蛋白通过 RGD 整合素结合区域影响细胞附着和信号传导，并通过使羟基磷灰石晶体成核作为矿物质沉淀的正调节剂。为了检验我们的假设，我们分析了 Bsp null (-/-) 小鼠模型中的牙齿发育。与 Goldbergs 小组合作，通过组织学、组织化学和 SEM 进行的发育分析显示，Bsp -/- 牙齿上的脱细胞牙骨质形成显着减少，并且沉积的牙骨质矿化不足。BSP 的损失导致 PDL 紊乱和上皮向下生长增加。 Bsp -/- 小鼠表现出广泛的牙根和牙槽骨吸收，这是由 RANKL 和破骨细胞增加介导的。与 WT 组织相比，KO 小鼠组织中脱细胞形成区域的 DMP1 染色增加。结果表明，BSP 在脱细胞牙骨质形成中起着重要且非多余的作用，可能参与根表面矿化的启动。我们正在与 NIDCR b 的 Fisher 一起探索整合素/BSP 的关系。在与 Young、NIDCR 的一个相关项目中，我们假设蛋白多糖的改变会损害牙周组织和关键 SIBLINGS 的表达。两种 SLRP（纤维调节蛋白和双糖链蛋白聚糖）的 dKO 会导致肌腱异位钙化和严重的骨关​​节炎。偏振光研究表明 dKO 小鼠存在异常的胶原纤维和牙槽骨重塑缺陷。 IHC 显示 dKO 小鼠 PDL 中 SLRP、阿孢菌素、lumican 和核心蛋白聚糖的染色增加，表明补偿机制。 DMP1 被认为是机械感觉骨细胞标记物，在 dKO PDL 中显着增加。 RANKL 表达增加和 TRAP 阳性破骨细胞数量增加进一步支持了 dKO PDL 稳态的破坏。为了阐明潜在机制，通过 PCR 阵列分析了 dKO 小鼠的 PDL 组织。结果表明 dKO 组织中 TGF beta/BMP 信号传导过度活跃，SMAD-4 和 -5 基因的表达显着增加，并且 dKO PDL 中磷酸化 SMAD5 的染色升高。因此，我们建议 Fmod 和 Bgn 隔离 BMP，防止与 BMP 受体结合。从 ECM 中删除 Fmod 和 Bgn 的预测结果将是对 TGF beta/BMP 的敏感性升高，这可能会影响 BMP 信号转导，导致 PDL 中磷酸化 SMAD5 的表达增加。 C. 我们假设 PTH 和 1,25D 调节 FGF23 表达的能力涉及一个中间因子，特别是 DMP1。 1,25D：使用鼠成牙骨质细胞和骨细胞样细胞。在 1,25D 存在下，Dmp1 mRNA 水平降低了 50%，而使用 VDR 激动剂和拮抗剂证实需要激活维生素 D 受体途径。 进一步的分析表明组蛋白脱乙酰酶（HDAC）招募是必要的。 PTH：暴露于 PTH (1-34) 的成牙骨质细胞和牙骨质细胞表现出 Dmp1 mRNA 表达以剂量反应和时间依赖性方式降低。 Western blot 检测到 PTH 处理后 DMP1 下调 30%。与对照相比，PTH 处理使细胞内 cAMP 水平增加 10 倍，而 cAMP/PKA 通路激动剂毛喉素 (forskolin) 也降低了 Dmp1 mRNA 表达，这表明 cAMP/ PKA途径。我们的结果表明 PTH 可能通过多种途径调节 FGF23，例如SOST 以及 DMP-1 以及 PTH 和 1,25D 介导的对 FGF23 的影响可能具有一些共同的机制方面。未来使用 RNAseq 方法的研究将有助于确定参与 Dmp1 调控的特定 DNA 序列和转录因子。与 DMP1 启动子相关的研究是与 NIDCR 的 Collins 和华盛顿大学的 Presland 合作进行的。