甲状旁腺相关蛋白对鼠胚髁突软骨细胞增殖和分化的调控

目的 探讨甲状旁腺相关蛋白（PTHrP）对鼠胚髁突软骨细胞增殖和分化的影响。方法 在体外分离培养鼠胚髁突软骨细胞的基础上,观察PTHrP对其软骨结节形成、碱性磷酸酶（ALP）活性等的影响。结果 研究发现加入浓度分别为0.01 nmol/L、0.1 nmol/L、1 nmol/L、10 nmol/L的PTHrP后,0.01 nmol/L组与对照组相比,形成的软骨结节数量在统计学上无显著性差异（P>0.05）,而软骨结节数量从0.1 nmol/L组开始与对照组相比明显增加,统计学上
有显著性差异（P<0.05）。而ALP活性在0.1-10 nmol/L组与对照组相比明显升高,统计学上有显著性差异（P<0.05）。阻断其受体后,实验组和对照组软骨结节形成的数目明显减少（P<0.05）,而对其ALP活性的影响在实验组和对照组之间无显著性差异（P>0.05）。结论 PTHrP通过其受体具有促进髁突软骨细胞增殖和分化的作用,其调控机制与其在生长板软骨细胞及下颌膜内成骨中的作用相似。     

Distribution of parathyroid hormone-related protein (PTHrP) and type I parathyroid hormone (PTH) PTHrP receptor in developing mouse mandibular condylar cartilage.

The mandibular condylar cartilage undergoes endochondral bone formation and is an important growth site in the mandible. Parathyroid hormone-related protein (PTHrP) has received attention as a physiological regulator attenuating chondrocytic differentiation and preventing apoptotic cell death. In order to examine the localization of PTHrP and its receptor during fetal development of the condylar cartilage, an immunohistochemical study of PTHrP and the type I PTH/PTHrP receptor was carried out. At day 15 of gestation, the condylar cartilage was evident and some chondrocytes showed positive staining for PTHrP. At day 16, the cartilage was increasing in length and width, and PTHrP was localized in the flattened and hypertrophic cell layers. After day 17, when endochondral bone formation had already started, PTHrP was mainly observed in the flattened cell layer and in a few layers of the hypertrophic chondrocytes. The localization of the type I PTH/PTHrP receptor was similar to that of PTHrP on days 15 and 16, and was broadly distributed at day 18. Apoptotic chondrocytes were scarcely observed on days 15 and 16, and only a few cells were present in the erosion front at day 18. This temporal and spatial localization of PTHrP and the type I PTH/PTHrP receptor suggests that PTHrP is a possible autocrine/ paracrine factor regulating condylar chondrocytic differentiation during development.     

Immunohistochemical localization of parathyroid hormone-related protein in developing mouse Meckel''s cartilage and mandible

In order to clarify the role of parathyroid hormone-related protein (PTHrP) during Meckel's cartilage and mandibular development, an immunohistochemical study of PTHrP and its receptor, PTH/PTHrP receptor, was designed to examine their localization in the anterior region of Meckel's cartilage including the rostrum, which is known to contribute to the development of the mandible. Meckel's cartilage was first observed on day 13 of gestation and PTHrP was faintly localized in the chondrocytes. On day 16 of gestation, at the stage of elongation and initiation of endochondral ossificastitial in Meckel's cartilage, PTHrP was localized in the chondrocytes located in the area showing interstitial growth and in and around the nuclei of hypertrophic chondrocytes undergoing endochondral ossification. At day 18 of gestation, endochondral ossification was spread over the entire area proximal to the molar region in Meckel's cartilage, except in the mesial fusion site formed by immature chondrocytes. PTHrP was localized in the osteoblasts adjacent to the calcified matrix, but had disappeared from the chondrocytes forming Meckel's cartilage. The localization of PTH/PTHrP receptor was similar to that of PTHrP. These results show that localization of PTHrP is spatially and temporally related to the growth of Meckel's cartilage.     

甲状旁腺相关蛋白在鼠胚髁突外植体软骨内成骨中的作用

目的探讨甲状旁腺相关蛋白(PTHrP)对体外培养的鼠胚髁突软骨内成骨的影响。方法体外解剖分离鼠胚髁突,行外植体培养,通过组织学、免疫组化等方法观察PTHrP对体外培养的髁突外植体软骨内成骨的影响。结果髁突外植体在无血清半固态培养系统中能正常发育。加入人PTHrP(1-34)后,实验组髁突长度的增加较对照组明显,两组间差异有显著性(P<0.05);组织学及免疫组化染色显示:加入人PTHrP(1-34)后培养的髁突外植体增殖层和肥大软骨细胞层明显增厚,同时Ⅱ及Ⅹ型胶原在增殖层和肥大软骨细胞层中表达增强。结论PTHrP可刺激髁突外植体软骨增殖层和肥大层软骨细胞的增殖,促进髁突软骨内成骨的形成。?     

甲状旁腺相关蛋白及其受体PTH1R及Ihh信号在鼠胚髁突软骨中的分布特征

目的探讨甲状旁腺相关蛋白(PTHrP)及其受体PTH1R及Ihh信号在髁突软骨发育过程中的分布特征及意义。方法取E14~18 d鼠胚,以免疫组化及原位杂交染色方法,检测PTH1R抗体及PTHrPI、hh mRNA在下颌髁突软骨发生过程中的分布特征。结果在鼠胚下颌髁突软骨发育过程中,PTHrP、PTH1R主要表达于软骨膜间质细胞、软骨细胞及大部分肥大软骨细胞中,Ihh主要位于肥大软骨细胞上层及其与扁平细胞层交界处,PTHrP及Ihh信号可能通过自分泌和(或)旁分泌作用调控髁突软骨细胞的增殖和分化。结论鼠胚下颌髁突软骨发育过程中,PTHrP、PTH1RI、hh的分布特征与其在生长板分布不同,提示PTHrP可能对髁突全层软骨细胞均具有增殖促进作用。     

单侧前牙反牙合对大鼠下颌髁突软骨内CaR、PTHrP表达的影响研究

目的　探索单侧前牙反牙合（unilateral anterior crossbite,UAC）对大鼠下颌髁突软骨内钙离子敏感受体（calcium-sensing receptor,CaR）、甲状旁腺激素相关蛋白（parathyroid hormone-related protein,PTHrP）分子表达以及软骨细胞增殖和发育的影响。方法　选择6周龄雌性SD大鼠48只,根据实验时间点（2、4、8周）随机分为3大组,每大组再随机分为2组（对照组、UAC实验组）,每组动物数量8只。对SD大鼠施加UAC刺激,于2、4、8周后分别处死各组大鼠,对颞下颌关节软骨进行CaR、PTHrP、PTHrP 受体（PPR）分子的免疫组化染色,提取软骨内mRNA进行增殖标志分子、软骨细胞表型分子和终末分化相关分子的Real-time PCR检测。结果　UAC实验组大鼠髁突软骨2周时即开始出现CaR的表达增高,4周时继发有PTHrP的表达上调,但是其惟一受体PPR的表达从2周时即显著降低;增殖标志分子和软骨细胞表型分子的表达从2周时开始显著降低,终末分化相关分子的表达从4周开始显著增高。结论　UAC可导致下颌髁突软骨内CaR、PTHrP表达改变,进而影响软骨细胞的增殖和分化状态。     

Cyclic mechanical strain regulates the PTHrP expression in cultured chondrocytes via activation of the Ca2+ channel

The association between mechanical stimulation and chondrocyte homeostasis has been reported. However, the participation of PTHrP (parathyroid-hormone-related protein) in the mechano-regulation of chondrocyte metabolism remains unclear. We determined whether mechanical stimulation of chondrocytes induces the expression of PTHrP and, further, whether the mechano-modulation of PTHrP is dependent on the maturational status of chondrocytes. Cyclic mechanical strain was applied to rat growth plate chondrocytes at the proliferating, matrix-forming, and hypertrophic stages at 30 cycles/min. Cyclic mechanical strain significantly increased PTHrP mRNA levels in chondrocytes at the proliferating and matrix-forming stages only. The induction of PTHrP was dependent on loading magnitude at the proliferating stage. Using specific ion channel blockers, we determined that mechano-induction of PTHrP was inhibited by nifedipine, a Ca2+ channel blocker. These results suggest that mechanical induction of PTHrP possibly provides the environment for greater chondrocyte replication and matrix formation that would subsequently affect cartilage formation.     

The cranial base in craniofacial development: a gene therapy study

The etiology of midface retrusion remains largely unclear. We hypothesized that the cranial base synchondroses play a key role in the development of the craniofacial skeleton in the Sandhoff mouse model. We observed that developmental abnormalities of the cranial base synchondroses involving proliferative chondrocytes are important in craniofacial growth and development. Neonatal restitution of beta-hexosaminidase in mutant mice by gene therapy successfully ameliorated the attendant skeletal defects and restored craniofacial morphology in vivo, suggesting this as a critical temporal window in craniofacial development. Analysis of our data implicates parathyroid-related peptide (PTHrP) and cyclo-oxygenase-2 (COX-2) as possible factors underlying the development of the aforementioned skeletal defects. Hence, timely restitution of a genetic deficiency or, alternatively, the restoration of PTHrP or cyclo-oxygenase activity by the administration of PTH and/or non-steroidal anti-inflammatory drugs or COX-2 selective inhibitors to affected individuals may prove beneficial in the management of midface retrusion.     

Ihh-PTHrP信号轴介导髁突软骨细胞对压力微环境改变的调控研究

目的 探究Ihh-PTHrP负反馈信号通路与静压力下髁突软骨细胞生物学响应的关系。方法 体外分离培养4周龄健康雌性新西兰大白兔髁突软骨细胞,100 kPa静压力条件下,分别对P2代髁突软骨细胞进行0、1、2、3、4 h的加压处理;采用Western印迹的方法比较分析髁突软骨细胞COL2A1、Ihh和PTHrP的蛋白表达变化;通过RT-qPCR检测分析静压力对Ihh和PTHrP基因水平表达的影响;选取0 h和3 h组,使用扫描电子显微镜扫描分析静压力对髁突软骨细胞形态的影响;采用SPSS 19.0软件包对数据进行统计学分析。结果 Western印迹实验发现：压力加载3 h后,COL2A1出现明显高表达;Ihh蛋白表达在0～2 h内逐渐升高,在2～4 h内却逐渐降低;PTHrP在0～2 h内蛋白表达逐渐升高,并在2～4 h内维持着一个较高水平的表达。RT-qPCR检测发现Ihh在压力加载1 h时出现明显高表达,而PTHrP则在2 h时出现明显高表达。100 kPa静压力加载3 h后,髁突软骨细胞突起增多、变长。结论 兔髁突软骨细胞对压力微环境的改变具有一定的适应能力;适宜的静压力可以提高髁突软骨细胞的成软骨能力;Ihh-PTHrP负反馈信号通路参与髁突软骨细胞对压力微环境改变的适应性改建过程。     

Involvement of PI3K/Akt pathway in rat condylar chondrocytes regulated by PTHrP treatment

Objective

The aim of this study was to explore the mutual communication of the parathyroid hormone-related peptide (PTHrP) and phosphatidylinositol 3-kinase/threonine protein kinase (PI3K/Akt) pathway on the proliferation and differentiation of condylar chondrocytes from Sprague-Dawley (SD) rats.

Methods

Condylar chondrocytes from the condylar cartilage were cultured and an organ culture system of mandibular condyles was employed. The distribution of PI3K, phospho-Akt (p-Akt), and PTHrP in condylar cartilage was detected by either immunohistochemistry or immunofluorescence. The second passage chondrocytes and condyle specimens in the organ culture system were treated with PTHrP, LY294002, PTHrP and LY294002 in combination, or dimethyl sulfoxide (DMSO), separately. The mRNA and protein levels of type II (Col II) and type X collagen (Col X) were investigated by real-time polymerase chain reaction (PCR) and Western blot analysis. The condyle growth in organ culture system was analysed by haematoxylin–eosin staining.

Results

PTHrP, PI3K, and p-Akt were mainly located in the proliferative and hypertrophic zones. PTHrP promoted the proliferation of condylar chondrocytes, while LY294002 limited this effect. The mRNA and protein levels of Col II and Col X in these cells were reduced by PTHrP and enhanced by LY294002. Organ culture showed a significant enhancement of condyle elongation with PTHrP treatment or a combination of PTHrP and LY294002 treatment. After treatment with LY294002, the length of condyles was reduced compared with the samples treated with DMSO.

Conclusions

We conclude that the PI3K/Akt pathway plays an essential role in the proliferation and differentiation of condylar chondrocytes and is a potential target for PTHrP in regulating chondrocyte differentiation at condylar cartilage.     

核心结合因子-α_1和甲状旁腺素相关蛋白在软骨内成骨中的作用

在软骨细胞不断分化并最终肥大化过程中,核心结合因子（Cbf）-α1起到促进软骨细胞肥大的作用,而甲状旁腺素相关蛋白（PTHrP）能通过Cbf-α1依赖性和非依赖性途径来抑制软骨细胞的肥大化。下面就Cbf-α1对软骨细胞产生的作用、PTHrP在软骨内成骨中的作用、PTHrP延缓软骨细胞成熟过程和PTHrP阻止cbf-α1表达的机制等研究进展作一综述。     

Developmental role of PTHrP in murine molars

Although PTHrP is produced in multiple fetal tissues, its precise physiological functions have yet to be clearly elucidated. The present study was undertaken to elucidate the biological role of PTHrP in the development of tooth. In rat tooth germs, the PTHrP and its receptor genes were expressed in the enamel organ and dental mesenchyme, respectively. When mouse tooth explants were cultured with antisense oligodeoxyribonucleotides (ODN) against mouse PTHrP mRNA in serum-free medium, an invasion of bone tissue was observed in the tooth germs. On the other hand, the explants cultured without ODN or with sense ODN showed normal histological structures similar to those observed in vivo. These results suggest that PTHrP is essential for tooth development and for the protection of tooth germs from the invasion of bone tissue.     

Parathyroid hormone-related protein (PTHrP) as a regulating factor of endochondral bone formation

Parathyroid hormone-related protein (PTHrP) was first identified as a pathogenetic factor for the hypercalcemia of malignancy. Recently PTHrP is focused as a physiological paracrine factor regulating cell proliferation and differentiation in many tissues during fetal and postnatal growth. Evidence for the skeletal origin of PTHrP comes from several sources and targeted disruption of the PTHrP gene in mice has resulted in a phenotype with accelerated endochondral bone formation, suggesting PTHrP as a factor regulating chondrocyte differentiation. Indian hedgehog, one of the conserved family of hedgehog regulating segmentation of Drosophila , is found to be an upstream factor of PTHrP in a regulating pathway of chondrocyte differentiation. Moreover, Bcl-2, a protein that controls programmed cell death in several cell types, is suggested to lie downstream of PTHrP in this pathway. A point mutation of PTH/PTHrP receptor is identified in a patient with Jansen-type metaphyseal chondrodysplasia and constitutive, ligand independent activation is indicated in this mutant receptor.     

PTHrP regulates chondrocyte maturation in condylar cartilage

PTHrP is a key factor regulating the pace of endochondral ossification during skeletal development. Mandibular advancement solicits a cascade of molecular responses in condylar cartilage. However, the pace of cellular maturation and its effects on condylar growth are still unknown. The purpose of this study was to evaluate the pattern of expression of PTHrP and correlate it to cellular dynamics of chondrocytes in condylar cartilage during natural growth and mandibular advancement. We fitted 35-day-old Sprague-Dawley rats with functional appliances. Experimental animals with matched controls were labeled with bromodeoxyuridine 3 days before their death, so that mesenchymal cell differentiation could be traced. Mandibular advancement increased the number of differentiated chondroblasts and subsequently increased the cartilage volume. Higher levels of PTHrP expression in experimental animals coincided with the slowing of chondrocyte hypertrophy. Thus, mandibular advancement promoted mesenchymal cell differentiation and triggered PTHrP expression, which retarded their further maturation to allow for more growth.     

The role of parathyroid hormone-related protein in the regulation of osteoclastogenesis by cementoblasts

BACKGROUND: Parathyroid hormone-related protein (PTHrP) promotes osteoclastogenesis by inhibiting expression of osteoprotegerin (OPG), a decoy receptor for the receptor activator of nuclear factor kappa B (RANK), and by enhancing production of RANK ligand (RANKL) by osteoblasts. However, little is known regarding the role of PTHrP in regulating cementoblast-mediated osteoclastogenesis. METHODS: This study determined the impact of PTHrP on osteoclastogenesis using: 1) OCCM-30 (immortalized murine cementoblasts), 2) RAW 264.7 cells (murine myeloid cells), or 3) OCCM-30 plus RAW 264.7 cells. Cells were treated with PTHrP (1-34), RANKL, or PTHrP and RANKL combined. Enzyme-linked immunosorbent assays (ELISAs) for OPG and RANKL were performed on media and cell lysates, and tartrate-resistant acid phosphatase (TRAP) and mRNA detection for the osteoclast associated receptor (OSCAR) were performed. RESULTS: The highest numbers of TRAP-positive cells and cells expressing OSCAR were found in the RAW cell group treated with either RANKL alone or RANKL and PTHrP. TRAP-positive cells were fewer when OCCM cells were co-cultured with RAW, but the greatest numbers were still with both PTHrP and RANKL. OPG levels were highest from OCCM cells and PTHrP decreased these levels. In contrast, RANKL levels were low in OCCM cell lysates and PTHrP increased RANKL. In vivo studies also revealed high osteoclastic activity surrounding developing teeth in mice administered PTH. CONCLUSIONS: These results demonstrate that PTHrP influences the balance of OPG and RANKL production by cementoblasts, and further indicate that this effect, in the context of surrounding cells, might have a significant impact on osteoclastogenesis, root resorption, and tooth eruption.