Regulation Of Skeletal Growth

骨骼生长的调节

• 批准号: 7594144
• 负责人: Jeffrey Baron
• 金额: $ 363.68万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/7594144
• 关键词: Achondroplasia; Age; Bone Growth; Bone Morphogenetic Proteins; Cartilage; Cell Proliferation; Cell physiology; Cells; Childhood; Chondrocytes; Chondrogenesis; Complex; Conflict (Psychology); Data; Development; Development, Other; Developmental Biology; Differentiation and Growth; Epiphysial cartilage; FGFR1 gene; FGFR2 gene; FGFR3 gene; FGFR4 gene; Fibroblast Growth Factor; Fibroblast Growth Factor Receptor 2; Fibroblast Growth Factor Receptors; Gene Expression; Gene Expression Regulation; Genes; Goals; Growth; Growth Disorders; Growth and Development function; Human; IGF Type 2 Receptor; Immunohistochemistry; In Situ Hybridization; Individual; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Insulin-Like Growth-Factor Binding Protein 1; Insulin-Like Growth-Factor-Binding Proteins; Laboratories; Life; Ligands; Liver; Medical; Messenger RNA; Methods; Microarray Analysis; Microdissection; Molecular; Muscle; Mutation; Numbers; Osteogenesis; Other Finding; Pathway interactions; Pattern; Polymerase Chain Reaction; Protein Isoforms; Proteins; RNA; Rate; Rattus; Regulation; Rest; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Transduction; Skeletal system; Somatomedins; Staging; Structure; System; Thinking; Time; Tissues; Week; Work; angiogenesis; bone; bone morphogenetic protein 2; bone morphogenetic protein 6; cell motility; concept; growth differentiation factor 10; human GHR protein; human IGF2R protein; improved; inhibitor/antagonist; mRNA Expression; osteogenin; postnatal; programs; receptor; receptor expression; senescence; size; skeletal dysplasia; stem

Longitudinal bone growth occurs at the growth plate, which consists of three principal layers: the resting zone, the proliferative zone, and the hypertrophic zone. Studies in our laboratory indicate that stem-like cells in the resting zone differentiate into rapidly dividing chondrocytes of the proliferative zone. The proliferative chondocytes then terminally differentiate into the nondividing chondrocytes of the hypertrophic zone. To explore the molecular switches responsible for this two-step differentiation program, we developed a microdissection method to isolate RNA from the resting, proliferative, and hypertrophic zones of growing rats. Microarray analysis followed by real-time PCR analysis identified genes whose expression changed dramatically during the differentiation program, including multiple genes functionally related to bone morphogenetic proteins (BMPs). BMP-2 and BMP-6 were found to be upregulated in hypertrophic zone compared with resting zone and proliferative zone. In contrast, BMP signaling inhibitors, including BMP-3, gremlin, and growth differentiation factor-10, were expressed early in the differentiation pathway, in the resting and proliferative zones. Our findings suggest a BMP signaling gradient across the growth plate, which is established by differential expression of multiple BMPs and BMP inhibitors in specific zones. We have previously shown evidence that BMPs can stimulate both proliferation and hypertrophic differentiation of growth plate chondrocytes. Therefore, taken together, our findings suggest that low levels of BMP signaling in the resting zone may help maintain these cells in a quiescent state. In the lower resting zone, greater BMP signaling may help induce differentiation to proliferative chondrocytes. Farther down the growth plate, even greater BMP signaling may help induce hypertrophic differentiation. Thus, BMP signaling gradients may be a key mechanism responsible for spatial regulation of chondrocyte proliferation and differentiation in growth plate cartilage. Fibroblast growth factor (FGF) signaling is also essential for endochondral bone formation. Mutations in FGF receptors cause skeletal dysplasias including achondroplasia, the most common human skeletal dysplasia. To explore the role of FGF signaling in the postnatal growth plate, we quantitatively analyzed expression of FGFs and FGF receptors (FGFRs). Rat proximal tibial growth plates and surrounding tissues were microdissected, and specific mRNAs were quantitated by real-time RT-PCR. To assess the FGF system without bias, we first screened for expression of all known FGFs and major FGFR isoforms. Perichondrium expressed FGFs 1, 2, 6, 7, 9, and 18 and, at lower levels, FGFs 21 and 22. Growth plate expressed FGFs 2, 7, 18, and 22. Perichondrial expression was generally greater than growth plate expression, supporting the concept that perichondrial FGFs regulate growth plate chondrogenesis. Nevertheless, FGFs synthesized by growth plate chondrocytes may be physiologically important because of their proximity to target receptors. In growth plate, we found expression of FGFRs 1, 2, and 3, primarily, but not exclusively, the c isoforms. FGFRs 1 and 3, thought to negatively regulate chondrogenesis, were expressed at greater levels and at later stages of chondrocyte differentiation, with FGFR1 upregulated in the hypertrophic zone and FGFR3 upregulated in both proliferative and hypertrophic zones. In contrast, FGFRs 2 and 4, putative positive regulators, were expressed at earlier stages of differentiation, with FGFR2 upregulated in the resting zone and FGFR4 in the resting and proliferative zones. Thus, this analysis identified ligands and receptors not previously known to be expressed in growth plate and revealed a complex pattern of spatial regulation of FGFs and FGFRs in the different zones of the growth plate. Previous studies of the insulin-like growth factor (IGF) system gene expression in growth plate using immunohistochemistry and in situ hybridization have yielded conflicting results. We therefore studied the spatial patterns of mRNA expression of the IGF system in the rat proximal tibial growth plate quantitatively. IGF-I mRNA expression was minimal in growth plate compared with perichondrium, metaphyseal bone, muscle, and liver. In contrast, IGF-II mRNA was expressed at higher levels than in bone and liver. IGF-II expression was higher in the proliferative and resting zones compared with the hypertrophic zone. GH receptor and type 1 and 2 IGF receptors were expressed throughout the growth plate. Expression of IGF-binding proteins (IGFBPs) -1 through -6 mRNA was low throughout the growth plate compared with perichondrium and bone. These data suggest that regulation primarily depends on IGF-II produced by chondrocytes, and IGF-I produced by surrounding structures. With age, growth plate chondrocyte proliferation slows down, causing longitudinal bone growth to slow and eventually stop. This functional change in the growth plate is accompanied by structural changes; with age, the number of resting, proliferative, and hypertrophic chondrocytes decreases as does the size of the individual hypertrophic cells. The chondrocyte columns also become more widely spaced. We have termed this developmental program, growth plate senescence. Growth plate senescence appears to be caused by a mechanism intrinsic to the growth plate. To explore the molecular mechanisms responsible for growth plate senescence, we analyzed how gene expression patterns change in the growth plate during postnatal life, as the rate of longitudinal bone growth decreases. In particular we analyzed the insulin-like growth factor (IGF) system because IGFs are capable of potently regulating growth plate chondrocyte proliferation and differentiation. With increasing age (3-, 6-, 9-, and 12-week rats), IGF-I mRNA levels increased in the proliferative zone but remained at least tenfold lower than levels in perichondrium and bone. IGF-II mRNA decreased dramatically, 780-fold, in proliferative zone whereas, type 2 IGF receptor and IGF binding proteins (IGFBPs)-1, -2, - 3, and -4 increased significantly with age in growth plate and/or surrounding perichondrium and bone. These findings suggest that growth plate senescence, including the decrease in growth velocity that occurs with age, may be caused, in part, by decreasing expression of IGF-II and increasing expression of type 2 IGF receptor and multiple IGFBPs. We also analyzed temporal changes in fibroblast growth factor (FGF) expression in the growth plate. We identified several changes in FGF and FGFR expression that may contribute to growth plate senescence. In the growth plate, FGFRs 2 and 4, both implicated as positive regulators of growth, undergo a decline in expression with age. In perichondrium, we observed increases in FGFs 1, 7, 18, and 22 mRNA with age. Increasing levels of these ligands, interacting with constant levels of FGFR3 in growth plate might contribute to growth plate senescence. These studies have begun to elucidate the regulation of gene expression that is responsible for the complex spatial organization of the growth plate and for the temporal changes of growth plate senescence. Combined with previous functional studies performed in our lab and by others, the findings indicate a highly complex system involving BMPs, FGFs, and IGFs, their receptors and other interacting proteins.

纵向骨生长发生在生长板处，生长板由三个主要层组成：静止区、增殖区和肥大区。我们实验室的研究表明，静止区的干细胞样细胞分化为增殖区的快速分裂的软骨细胞。然后增殖的软骨细胞最终分化成肥大区的非分裂软骨细胞。 为了探索负责这种两步分化程序的分子开关，我们开发了一种显微切割方法，从生长大鼠的静息区、增殖区和肥大区中分离 RNA。微阵列分析和实时 PCR 分析确定了在分化过程中表达发生显着变化的基因，包括与骨形态发生蛋白 (BMP) 功能相关的多个基因。与静止区和增殖区相比，肥厚区的 BMP-2 和 BMP-6 表达上调。相比之下，BMP 信号传导抑制剂，包括 BMP-3、gremlin 和生长分化因子 10，在分化途径的早期、静止区和增殖区表达。我们的研究结果表明，整个生长板存在 BMP 信号梯度，该梯度是通过特定区域中多种 BMP 和 BMP 抑制剂的差异表达而建立的。我们之前的证据表明，BMP 可以刺激生长板软骨细胞的增殖和肥大分化。因此，综上所述，我们的研究结果表明，静息区中低水平的 BMP 信号传导可能有助于维持这些细胞处于静止状态。在较低的休息区，更强的 BMP 信号传导可能有助于诱导分化为增殖性软骨细胞。在生长板的更深处，更强的 BMP 信号可能有助于诱导肥大分化。因此，BMP信号梯度可能是负责生长板软骨中软骨细胞增殖和分化空间调节的关键机制。 成纤维细胞生长因子（FGF）信号对于软骨内骨形成也至关重要。 FGF 受体突变会导致骨骼发育不良，包括软骨发育不全（最常见的人类骨骼发育不良）。为了探讨 FGF 信号在出生后生长板中的作用，我们定量分析了 FGF 和 FGF 受体 (FGFR) 的表达。对大鼠胫骨近端生长板和周围组织进行显微解剖，并通过实时 RT-PCR 定量特定 mRNA。为了无偏见地评估 FGF 系统，我们首先筛选所有已知 FGF 和主要 FGFR 亚型的表达。软骨膜表达 FGF 1、2、6、7、9 和 18，以及较低水平的 FGF 21 和 22。生长板表达 FGF 2、7、18 和 22。软骨膜表达通常大于生长板表达，支持软骨膜 FGF 调节生长板软骨形成的概念。然而，生长板软骨细胞合成的 FGF 由于靠近靶受体，因此可能具有重要的生理意义。在生长板中，我们发现 FGFR 1、2 和 3 的表达，主要但不限于 c 亚型。 FGFR 1 和 3 被认为对软骨形成负调节，在软骨细胞分化的后期以更高水平表达，其中 FGFR1 在肥大区上调，FGFR3 在增殖区和肥大区均上调。相比之下，FGFR 2 和 4（假定的正调节因子）在分化的早期阶段表达，其中 FGFR2 在静息区上调，FGFR4 在静息区和增殖区上调。 因此，该分析鉴定了以前未知的在生长板中表达的配体和受体，并揭示了生长板不同区域中 FGF 和 FGFR 的空间调节的复杂模式。 先前使用免疫组织化学和原位杂交对生长板中胰岛素样生长因子（IGF）系统基因表达的研究产生了相互矛盾的结果。因此，我们定量研究了大鼠胫骨近端生长板中 IGF 系统 mRNA 表达的空间模式。与软骨膜、干骺端骨、肌肉和肝脏相比，IGF-I mRNA 表达在生长板中最低。相反，IGF-II mRNA 的表达水平高于骨骼和肝脏。与肥大区相比，增殖区和静止区的 IGF-II 表达较高。 GH 受体以及 1 型和 2 型 IGF 受体在整个生长板中表达。与软骨膜和骨相比，整个生长板中 IGF 结合蛋白 (IGFBP) -1 至 -6 mRNA 的表达较低。这些数据表明，调节主要取决于软骨细胞产生的 IGF-II 和周围结构产生的 IGF-I。 随着年龄的增长，生长板软骨细胞增殖减慢，导致纵向骨生长减慢并最终停止。生长板的这种功能变化伴随着结构变化；随着年龄的增长，静息、增殖和肥大软骨细胞的数量会减少，单个肥大细胞的大小也会减少。软骨细胞柱的间距也变得更宽。我们将这种发育程序称为生长板衰老。生长板衰老似乎是由生长板固有的机制引起的。 为了探索生长板衰老的分子机制，我们分析了出生后随着纵向骨生长速率的降低，生长板中的基因表达模式如何变化。 我们特别分析了胰岛素样生长因子（IGF）系统，因为 IGF 能够有效调节生长板软骨细胞的增殖和分化。随着年龄的增加（3周、6周、9周和12周的大鼠），增殖区的IGF-I mRNA水平增加，但仍比软骨膜和骨中的水平低至少十倍。在增殖区，IGF-II mRNA 急剧减少 780 倍，而在生长板和/或周围软骨膜和骨中，2 型 IGF 受体和 IGF 结合蛋白 (IGFBP)-1、-2、-3 和 -4 随着年龄的增长显着增加。这些发现表明，生长板衰老，包括随年龄增长而发生的生长速度下降，部分原因可能是 IGF-II 表达减少和 2 型 IGF 受体和多种 IGFBP 表达增加所致。 我们还分析了生长板中成纤维细胞生长因子（FGF）表达的时间变化。 我们发现 FGF 和 FGFR 表达的一些变化可能导致生长板衰老。在生长板中，FGFR 2 和 4 均作为生长的正调节因子，随着年龄的增长，其表达量会下降。在软骨膜中，我们观察到 FGF 1、7、18 和 22 mRNA 随着年龄的增长而增加。增加这些配体的水平，与生长板中恒定水平的 FGFR3 相互作用可能会导致生长板衰老。 这些研究已开始阐明基因表达的调节，该调节负责生长板的复杂空间组织和生长板衰老的时间变化。结合我们实验室和其他实验室之前进行的功能研究，研究结果表明，这是一个高度复杂的系统，涉及 BMP、FGF 和 IGF、它们的受体和其他相互作用的蛋白质。