摘要
胰岛素样生长因子1（Insulin like growth factor1，IGF1）是一种多功能肽，可以调节细胞生长、细胞分化，以及细胞外基质中多种蛋白表达。大量基础和临床研究证明，IGF1是骨形成和骨重建过程中的关键蛋白。但IGF1促进或抑制骨髓间充质干细胞（Bone marrow mesenchymal stemcells，BMSCs）向成骨细胞分化尚未定论。此外，有关IGF1对BMSCs向成骨细胞分化影响的分子机制和信号通路尚不清楚。
BMSCs是在骨髓中存在的一种具有多向分化潜能的干细胞，可以向脂肪细胞、成骨细胞、成软骨细胞或肌肉细胞等进行分化。正常情况下，BMSCs向成骨细胞和脂肪细胞两者的分化处于动态平衡，其分子调控机制具有非常重要的医学意义。具有盘状同源区域（PDZ）结合序列的转录共活化因子（Transcriptional coactivator with PDZ-binding motif，TAZ），在调节BMSCs成骨和成脂分化的平衡中发挥着非常重要的作用。相关研究证明，TAZ可以促进BMSCs向成骨细胞分化，同时阻止BMSCs向脂肪细胞分化。目前尚未清楚的是，IGF1对BMSCs向成骨细胞分化具有促进还是抑制作用？IGF1对BMSCs中TAZ的表达有何影响？IGF1主要通过哪些通路调控TAZ的表达？
已有研究证明，血清IGF1浓度影响骨代谢。另外，某些药物，如生长激素（Growth hormone，GH），可能会增加血清IGF1浓度。生长激素缺乏症（Growth hormone deficiency，GHD）已经被明确界定为一种综合征，GHD病人所出现的一系列临床与生化方面的异常表现也已知晓。与健康人群相比，成年GHD患者可以表现为骨密度（Bone mineral density，BMD）降低。对于接受GH治疗的GHD病人，其血清IGF1浓度如何变化？GH-IGF1轴活性增加后，对GHD病人的BMD有何影响？是目前关注热点之一。
本课题将动物细胞实验和临床荟萃分析相结合，从微观和宏观两个层面，研究IGF1对BMSCs向成骨细胞分化的影响和分子机制。研究内容包括三个部分：第一，采用全骨髓贴壁法和密度梯度离心法分离大鼠BMSCs，并进行原代培养，优选其中一种用于后续实验，并对BMSCs纯度和向成骨细胞分化能力进行鉴定；第二，研究IGF1对大鼠BMSCs向成骨细胞分化的影响及量效关系，确定TAZ的表达变化在其分子机制中占有的重要地位；第三，采用荟萃分析，研究GH治疗对GHD患者血清IGF1的影响，以及GH-IGF1轴活性增加对其BMD的影响。上述三部分研究，分别借助基础医学和循证医学两个视角，论证了IGF1在骨代谢中的重要意义，为临床治疗骨质疏松及其它骨矿盐疾病提供实验依据和理论基础。
第一部分大鼠BMSCs的原代培养和鉴定
目的：优选分离大鼠BMSCs的适宜方法，并对大鼠BMSCs的纯度和向成骨细胞分化能力进行鉴定。
方法：1采用全骨髓贴壁法和密度梯度离心法分离大鼠BMSCs，并对两种方法获得细胞的数量、形态、生长曲线和生长周期进行比较；2采用流式细胞术，对大鼠BMSCs表面抗原进行检测，以鉴定其纯度；3采用成骨培养基对大鼠BMSCs进行培养，观察向成骨细胞诱导后其生长曲线的变化；4对大鼠BMSCs进行成骨诱导后，检测细胞的碱性磷酸酶（Alkaline phosphatase，ALP）活性，并对其进行茜素红（Alizarin red，AR）染色，鉴定其向成骨细胞分化能力，并观察其向成骨细胞分化进程。
结果：
1两种分离方法获得BMSCs的比较
全骨髓贴壁法分离得到BMSCs的数量为密度梯度离心法的2倍以上，具有数量优势，两种分离方法得到的BMSCs在形态、生长曲线和生长周期方面没有明显差异。因此，选择全骨髓贴壁法用于后续实验。
2大鼠BMSCs表面抗原鉴定结果
采用流式细胞术对全骨髓贴壁法获得P3代细胞的表面抗原进行鉴定，结果发现，CD29/CD90共同表达的阳性率为91.41%；CD34/CD45共同表达的阳性率为1.24%。
3大鼠BMSCs向成骨细胞分化能力鉴定结果
经成骨诱导后，BMSCs的生长曲线出现明显变化，其增殖速度明显下降，而且对数生长期不明显；BMSCs经成骨诱导之后，ALP活性在不同时间点存在统计学差异，并存在时间递增效应，第3天、第7天和第14天的ALP活性高于第0天；第7天和第14天的ALP活性高于第3天；第7天和第14天的ALP活性没有统计学差异；BMSCs经成骨诱导之后，AR染色结果在不同时间点存在统计学差异，并存在时间递增效应，第3天和第7天的半定量结果没有统计学差异；第14天和第21天的半定量结果高于第3天和第7天；第21天的半定量结果高于第14天。
结论：全骨髓贴壁法分离大鼠BMSCs更适用于后续实验；经全骨髓贴壁法分离得到的BMSCs纯度较高，符合实验要求；经成骨诱导后，细胞分化能力增强，增殖能力降低，并逐渐表现出成骨细胞特征。
第二部分IGF1对大鼠BMSCs向成骨细胞分化影响的信号通路研究
目的：确定IGF1促进还是抑制大鼠BMSCs向成骨细胞分化及量效关系，初步探索其分子机制。
方法：1在成骨培养基中分别添加50、100和200ng/ml IGF1，观察不同浓度IGF1对细胞ALP活性和钙沉积量的影响，同时确定后续实验中IGF1的适宜浓度；2采用Real-time RT PCR和Western Blot方法，检测IGF1对TAZ，Runt相关转录因子2（Runt-related transcription factor2，RUNX2）和骨钙素（osteocalcin，OCN）的mRNA和蛋白水平的影响；3采用小干扰RNA（Small interfering RNA，SiRNA）技术阻断TAZ表达，观察SiTAZ能否抑制IGF1诱导的BMSCs向成骨细胞分化的增加；4应用UO126（MEK-ERK阻断剂）和LY294002（PI3K-Akt阻断剂），确定IGF1对TAZ表达产生影响的优势通路。
结果：
1IGF1促进BMSCs向成骨细胞分化
ALP活性检测和AR染色结果显示，IGF1可以增加大鼠BMSCs的ALP活性和钙沉积量，并存在剂量反应关系，其产生最大效应的浓度为100~200ng/ml。
2IGF1对TAZ，RUNX2和OCN表达的影响
Real-time RT-PCR和Western blot结果显示，IGF1可以在BMSCs向成骨细胞分化早期（第3~7天）上调TAZ和RUNX2的表达，而在BMSCs向成骨细胞分化的晚期（第7~14天）上调OCN的表达。
3SiTAZ转染抵消了IGF1对BMSCs向成骨细胞分化的促进作用
Real-time RT-PCR和Western blot结果显示，SiTAZ的转染可以成功降低TAZ的mRNA水平和蛋白表达。ALP活性检测和AR染色结果显示，IGF1+SiTAZ组的ALP活性和AR染色半定量结果明显低于IGF1+SiCON组；而且，ALP活性和AR染色半定量结果在SiCON组，SiTAZ组和IGF1+SiTAZ三组之间没有统计学差异。4IGF1主要通过MEK-ERK通路上调TAZ的表达
Real-time RT-PCR和Western blot结果显示，IGF1+UO126组TAZ的mRNA和蛋白水平低于IGF1组；但是，TAZ的mRNA和蛋白水平在IGF1+LY294002组和IGF1组之间没有统计学差异；此外，TAZ的mRNA和蛋白水平在对照组，UO126组，IGF1+UO126组和LY294002组之间没有统计学差异。
结论：IGF1增加BMSCs向成骨细胞分化过程中的ALP活性和钙沉积量，并具有剂量反应关系；IGF1在BMSCs向成骨细胞分化早期上调TAZ和RUNX2的表达，在BMSCs向成骨细胞分化晚期上调OCN的表达；通过SiRNA阻断TAZ表达后，IGF1对BMSCs向成骨细胞分化的促进作用被抵消；IGF对TAZ表达的影响主要是由MEK-ERK通路介导的。
第三部分GH-IGF1轴对成年GHD患者骨密度的影响：荟萃分析
目的：与健康人群相比，成年GHD患者往往表现为BMD降低，但是，对接受GH治疗的GHD患者而言，GH-IGF1轴对BMD的影响尚存争议。本研究旨在探索GH-IGF1轴活性增加对成年GHD患者的BMD是否存在积极意义。
方法：1对Medline，Embase和Cochrane Library数据库进行系统检索，获取相关文献；2利用标准化的电子表格对文献中的研究细节和研究数据进行提取；3建立随机效应模型进行荟萃分析，通过计算标准化均差（Standardized mean difference，SMD）及其95%可信区间（Confidenceinterval，CI），以及相应的P值来分析GH-IGF1轴对腰椎、股骨颈和全身BMD的影响；4采用Q检验评价纳入研究的异质性，采用Egger和Begg检验评价发表偏倚。
结果：
1纳入荟萃分析的文献
共有20篇文献，包括936名研究对象纳入本荟萃分析。
2GH治疗对血清IGF1的影响
在本荟萃分析所纳入的文献中，几乎所有的研究均证实，经GH治疗后的GHD患者，其血清IGF1水平显著提高。
3GH-IGF1轴对腰椎BMD的影响
GH-IGF1可以提高腰椎BMD（SMD=0.429，95%CI [0.263，0.594]，P <0.001；I2=50.0%，P=0.007for Q test）。亚组分析结果显示，在美洲人亚组中，GH-IGF1对腰椎BMD的影响并不明显。
4GH-IGF1轴对股骨颈BMD的影响
GH-IGF1可以提高股骨颈BMD（SMD=0.377，95%CI [0.158，0.595]，P=0.001；I2=67.8%，P <0.001for Q test）。亚组分析结果显示，在某些亚组人群中，GH-IGF1对股骨颈BMD的影响并不明显，包括：GH治疗时间≤2年亚组和美洲人亚组。
5GH-IGF1轴对全身BMD的影响
GH-IGF1可以提高全身BMD（SMD=0.242，95%CI [0.019，0.466]，P=0.034；I2=69.6%，P <0.001for Q test）。亚组分析结果显示，在某些亚组人群中，GH-IGF1对全身BMD的影响并不明显，包括：GH治疗时间≤2年亚组；GH治疗采用固定剂量亚组；采用Hologic Inc骨密度仪进行测量的亚组；采用GE-Lunar Inc骨密度仪进行测量的亚组；欧洲人亚组；美洲人亚组和大洋洲人亚组。
6异质性和发表偏倚
在对腰椎BMD、股骨颈BMD和全身BMD结果进行荟萃分析的过程中，均存在异质性；进行亚组分析时，异质性可以消除或降低。Egger’s和Begg检验结果显示，本研究不存在发表偏倚。
结论：GH-IGF1轴活性增加可以提高GHD患者BMD；但在某些亚组人群中，GH-IGF1对BMD的影响并不明显。
Insulin-like growth factor1(IGF1) is a multifunctional peptide thatregulates the cell growth, differentiation, and the expression of extracellularmatrix proteins. Numbers of basic and clinical researches have demonstratedthat IGF1is a key protein in bone formation and remodeling. However,whether IGF1inhibits or promotes osteogenic differentiation remainscontroversial. Moreover, biological mechanisms and signaling pathways bywhich IGF1affects osteogenic differentiation remain obscure.
Bone marrow mesenchymal stem cells (BMSCs) constitute a smallpopulation of pluripotent cells within the bone marrow, which differentiateinto adipocytes, osteoblasts, chondrocytes or myocytes under the influence ofparticular signaling pathways. The mechanisms that fine-tune the balancebetween the osteoblast and adipocyte differentiation of BMSCs are likely to beof medical importance. Transcriptional coactivator with PDZ-binding motif(TAZ) plays an important role in regulating the balance between the osteoblastand adipocyte differentiation of BMSCs. TAZ promotes the differentiation ofBMSCs into osteogenic lineages and blocks the differentiation of BMSCsfrom adipocyte lineages. Then, whether IGF1promotes or inhibits theosteogenic differentiation of BMSCs? How IGF1regulates the expression ofTAZ? And through which pathway does IGF1regulate TAZ expression?
Bone mass was known to be linked to circulating levels of IGF1. Inaddition, some medications, such as growth hormone (GH), might increasecirculating levels of IGF1. The condition of GH deficiency (GHD) has beenaccepted as a definite syndrome, and the clinical and biochemicalabnormalities in GHD patients are also well known. For example, adultpatients with childhood-onset or adult-onset GHD, exhibit reduced bonemineral density (BMD) compared with healthy controls. Then, how the circulating levels of IGF1change in the GHD patients receiving GH treatment?How the BMD changes as the increase of the activity of GH-IGF1axis?
To resolve the problems above, from the microcosmic and macroscopicaspects, we combined the methods of cell experiments and the methods ofclinical meta-analysis to study the effects of IGF1on the osteogenicdifferentiation and the mechanism. The whole study mainly includes threeparts: Firstly, we isolated rat BMSCs with the two methods below: the wholebone marrow adherent method and the density gradient centrifugation method,and cultured the primary cells. Then, we preferred one of the isolationmethods, and identified the purity and the osteogenic capacity of BMSCs.Secondly, we explored the effects of IGF1on the osteogenic differentiation ofBMSCs and the dose-effect relationship, and determined the important role ofTAZ expression in the molecular mechanism. Thirdly, with the meta-analysismethods, we explored the effects of GH treatment on the circulating levels ofIGF1of the GHD patients, and the effects of the increase of GH-IGF1axisactivity on the BMD. From the perspectives of basic and evidence-basedmedicine, we demonstrated the important significance of IGF1in the bonemetabolism in the three former parts, and provided the theoretical basis for theclinical treatment of osteoporosis and other bone diseases.
Part1Primary culture and identification of rat BMSCs
Objectives: To prefer the appropriate isolation method of rat BMSCs, andidentify the purity and the osteogenic capacity of rat BMSCs.
Methods:1We isolated rat BMSCs with the whole bone marrow adherentmethod and the density gradient centrifugation method, and then compared themorphologies, growth curves and cell cycles of the cells isolated with the twomethods;2We detected the surface antigens of rat BMSCs by flow cytometryto identify the purity;3We cultured rat BMSCs in osteogenic medium, andobserve the change of the growth curve after the osteogenic induction;4Wedetected the alkaline phosphatase (ALP) activities of the cells, and stained thecells with alizarin red (AR) to identify the osteogenic capacity of rat BMSCsand observe the osteogenic progress.
Results:
1Comparison of the two cell isolation methods
The number of cells obtained with whole bone marrow adherent methodwas more than2times than the density gradient centrifugation method; themorphologies, growth curves and cell cycles of the cells isolated with the twomethods were not significantly different. Thus, we chose the whole bonemarrow adherent method for the follow-up experiments.
2Results of the identification of the surface antigens of rat BMSCs
The percentage of the double positive cells detected by anti-CD29andanti-CD90was91.41%; and the percentage of the double negative cellsdetected by anti-CD34and anti-CD45was1.24%.3Results of the identification of the osteogenic capacities of rat BMSCs
After the osteogenic induction, the growth curve of BMSCs wassignificantly changed: the proliferation rate significantly decreased, and thelogarithmic phase was not obvious. After the osteogenic induction, there wassignificant difference among the ALP activities at different points of time. TheALP activities at day3-14were higher than day0; the ALP activities at day7-14were higher than day3; and there was no significant difference betweenthe ALP activities at day7and day14. After the osteogenic induction, therewas significant difference among the AR staining results at different points oftime. There was no significant difference between the semi-quantitative resultsat day3and day7; the semi-quantitative results at day14and day21werehigher than day3and day7; and the semi-quantitative results at day21werehigher than day14.
Conclusions: The whole bone marrow adherent method for rat BMSCswere more appropriate for the follow-up experiments; the purity of BMSCsisolated with the whole bone marrow adherent method met the requirements ofour research; and after the osteogenic induction, the differentiative capacity ofcells increased, but the proliferation capacity of cells decreased, and cellsgradually exerted the features of osteoblasts.
Part2Effects of IGF1on the osteogenic differentiation of rat BMSCs andthe signal pathway
Objectives: To determine the effects of IGF1on the osteogenicdifferentiation of rat BMSCs and the dose-effect relationship, and initiallyexplore the molecular mechanism.
Methods:1We added50,100and200ng/ml IGF1into osteogenicmedium, and observed the effects of IGF1at different concentration on theALP activities and calcium depositions, and determined appropriateconcentration for the follow-up experiments;2With real-time RT PCR andWestern blot analysis, we detected the effects of IGF1on the mRNA andprotein levels of TAZ, Runt-related transcription factor2(RUNX2), andosteocalcin (OCN);3We use small interfering RNA (SiRNA) to inhibit theTAZ expression, and observed whether SiTAZ offset the IGF1inducedincrease of osteogenic differentiation;4We used UO126(MEK-ERK inhibitor)and LY294002(PI3K-Akt inhibitor) to determine the preponderant pathwaywhich mediated the effects of IGF1on TAZ expression.
Results:
1IGF1promotes the osteogenic differentiation of rat BMSCs
IGF1could dose dependently increased the ALP activities and calciumdepositions of rat BMSCs, the concentrations of IGF1which led to themaximum effects were100and200ng/ml.
2Effects of IGF1on TAZ, Runx2and OCN expression
Both Real-time RT-PCR and western blot analysis results suggested thatIGF1could increase TAZ and RUNX2expression at the early stage (day3-7)of osteogenic differentiation, but increase OCN expression at the late stage(day7-14).
3SiTAZ Transfection offset the effects of IGF1on osteogenic differentiation
Both Real-time RT-PCR and western blot analysis results suggested thatSiTAZ transfection significantly decreased TAZ expression. The ALPactivities and AR staining semi-quantitative results were significantly reducedin IGF1+SiTAZ treatment group compared with IGF1+SiCON treatment group, and there was no significant difference among SiCON, SiTAZ andIGF1+SiTAZ treatment group.
4IGF1increased TAZ expression mainly mediated by MEK-ERK pathway
Both Real-time RT-PCR and western blot analysis results revealed that theTAZ mRNA and protein levels were significantly reduced in IGF1+UO126treatment group compared with IGF1treatment group. However, there was nosignificant difference of the TAZ mRNA and protein levels betweenIGF1+LY294002and IGF1treatment group. Moreover, there was nosignificant difference of the TAZ mRNA and protein levels among control,UO126, IGF1+UO126and LY294002treatment group.
Conclusions: IGF1could dose dependently increased the ALP activitiesand calcium depositions of rat BMSCs in the progress of osteogenicdifferentiation; IGF1could increase TAZ and RUNX2expression at the earlystage of osteogenic differentiation, but increase OCN expression at the latestage; SiTAZ transfection offset the effects of IGF1on osteogenicdifferentiation; and IGF1increased TAZ expression mainly mediated byMEK-ERK pathway.
Part3Effects of GH-IGF1axis on the BMD of GHD adults: ameta-analysis
Objectives: GHD patients exhibited reduced BMD compared with healthycontrols, but previous researches demonstrated uncertainty about the effect ofGH replacement therapy on bone in GHD adults. The aim of this study was todetermine whether the increase of GH-IGF1axis activity could elevate BMDin GHD adults.
Methods:1Searches of Medline, Embase and Cochrane Library wereundertaken to identify studies in humans of the association between GHtreatment and BMD in GHD adults;2Study details and data were extracted toa standardized electronic form;3Random effects model was used for thismeta-analysis, a pooled standardized mean difference (SMD) with95%confidence intervals (CI) calculated using the final follow-up P values wereused to analyze the effects of GH-IGF1axis on spine, femoral neck and total body BMD;4Heterogeneity of the effect across studies was assessed by Qstatistics, Egger and Begg tests were performed to detected the publicationbias.
Results:
1Studies included in the meta-analysis
A total of20unique studies which included936subjects were availablefor this meta-analysis.
2Effects of GH treatment on the circulating levels of IGF1
Almost all of studies included in our meta-analysis revealed that thecirculating levels of IGF1increased after GH treatment.
3Effects of GH-IGF1axis on the BMD of spine
The results suggested significant association between GH-IGF1axis andincreased BMD of spine (SMD=0.429,95%CI [0.263,0.594], P <0.001; I2=50.0%, P=0.007for Q test). The results of subgroup analyses did not suggestsignificant association between GH-IGF1axis and BMD of spine in Americansubjects.
4Effects of GH-IGF1axis on the BMD of femoral neck
The results suggested significant association between GH-IGF1axis andincreased BMD of femoral neck (SMD=0.377,95%CI [0.158,0.595], P=0.001; I2=67.8%, P <0.001for Q test). The results of subgroup analyses didnot suggest significant association between GH-IGF1axis and BMD offemoral neck in subjects treated by GH for≤2yr and American subjects.
5Effects of GH-IGF1axis on the BMD of total body
The results suggested significant association between GH-IGF1axis andincreased BMD of total body (SMD=0.242,95%CI [0.019,0.466], P=0.034;I2=69.6%, P <0.001for Q test). The results of subgroup analyses did notsuggest significant association between GH-IGF1axis and BMD of total bodyin subjects whose treatment time≤2yr, subjects received fixed GH dosage,subjects whose BMD was measured by DXA scanner manufactured byHologic Inc, subjects whose BMD was measured by DXA scannermanufactured by GE-Lunar Inc, European subjects, American subjects and Oceanian subjects.
6Heterogeneity and publication bias
Significant heterogeneity was separately observed among the availablestudies on BMD of spine, femoral neck and total body. Significantheterogeneity was removed or decreased in some subgroups, but still existedin other subgroups. Egger’s and Begg methods didn’t show publication bias.
Conclusions: The activation of GH-IGF1axis could increase BMD ofGHD adults; but in some subject populations, the influence was not evident.
引文
1Bi Y, Gong M, Zhang X, et al. Pre-activation of retinoid signalingfacilitates neuronal differentiation of mesenchymal stem cells. DevGrowth Differ,2010,52(5):419-31
2Shi ZB, Wang KZ. Effects of recombinant adeno-associated viral vectorson angiopoiesis and osteogenesis in cultured rabbit bone marrow stemcells via co-expressing hVEGF and hBMP genes: a preliminary study invitro. Tissue Cell,2010,42(5):314-21
3Saulnier N, Lattanzi W, Puglisi MA, et al. Mesenchymal stromal cellsmultipotency and plasticity: induction toward the hepatic lineage. Eur RevMed Pharmacol Sci,2009,13Suppl1:71-8
4Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adulthuman mesenchymal stem cells. Science,1999,284(5411):143-7
5Sotiropoulou PA, Perez SA, Salagianni M, et al. Characterization of theoptimal culture conditions for clinical scale production of humanmesenchymal stem cells. Stem Cells,2006,24(2):462-71
6Li Y, Chopp M. Marrow stromal cell transplantation in stroke andtraumatic brain injury. Neurosci Lett,2009,456(3):120-3
7Ding DC, Shyu WC, Lin SZ. Mesenchymal stem cells. Cell Transplant,2011,20(1):5-14
8Eibes G, dos Santos F, Andrade PZ, et al. Maximizing the ex vivoexpansion of human mesenchymal stem cells using a microcarrier-basedstirred culture system. J Biotechnol,2010,146(4):194-7
9Abdallah BM, Kassem M. Human mesenchymal stem cells: from basicbiology to clinical applications. Gene Ther,2008,15(2):109-16
10Wang X, Hisha H, Mizokami T, et al. Mouse mesenchymal stem cells cansupport human hematopoiesis both in vitro and in vivo: the crucial role ofneural cell adhesion molecule. Haematologica,2010,95(6):884-91
11Guo KT, SchAfer R, Paul A, et al. A new technique for the isolation andsurface immobilization of mesenchymal stem cells from whole bonemarrow using high-specific DNA aptamers. Stem Cells,2006,24(10):2220-31
12Oswald J, Boxberger S, Jorgensen B, et al. Mesenchymal stem cells can bedifferentiated into endothelial cells in vitro. Stem Cells,2004,22(3):377-84
13Johnson K, Pritzker K, Goding J, et al. The nucleoside triphosphatepyrophosphohydrolase isozyme PC-1directly promotes cartilagecalcification through chondrocyte apoptosis and increased calciumprecipitation by mineralizing vesicles. J Rheumatol,2001,28(12):2681-91
14Delorme B, Chateauvieux S, Charbord P. The concept of mesenchymalstem cells. Regen Med,2006,1(4):497-509
15Geng S, Guo Y, Wang Q, et al. Cancer stem-like cells enriched with CD29and CD44markers exhibit molecular characteristics withepithelial-mesenchymal transition in squamous cell carcinoma. ArchDermatol Res,2013,305(1):35-47
16Vassilopoulos A, Chisholm C, Lahusen T, et al. A critical role of CD29andCD49f in mediating metastasis for cancer-initiating cells isolated from aBrca1-associated mouse model of breast cancer. Oncogene,2013
17Zucchini A, Del Zotto G, Brando B, et al. Cd90. J Biol Regul HomeostAgents,2001,15(1):82-5
18Kitayama J, Emoto S, Yamaguchi H, et al. CD90(+) Mesothelial-LikeCells in Peritoneal Fluid Promote Peritoneal Metastasis by Forming aTumor Permissive Microenvironment. PLoS One,2014,9(1): e86516
19Koller MR, Papoutsakis ET. Cell adhesion in animal cell culture:physiological and fluid-mechanical implications. Bioprocess Technol,1995,20:61-110
20Cheng SL, Yang JW, Rifas L, et al. Differentiation of human bone marrowosteogenic stromal cells in vitro: induction of the osteoblast phenotype bydexamethasone. Endocrinology,1994,134(1):277-86
21McQuillan DJ, Richardson MD, Bateman JF. Matrix deposition by acalcifying human osteogenic sarcoma cell line (SAOS-2). Bone,1995,16(4):415-26
22Coelho MJ, Fernandes MH. Human bone cell cultures in biocompatibilitytesting. Part II: effect of ascorbic acid, beta-glycerophosphate anddexamethasone on osteoblastic differentiation. Biomaterials,2000,21(11):1095-102
23Maniatopoulos C, Sodek J, Melcher AH. Bone formation in vitro bystromal cells obtained from bone marrow of young adult rats. Cell TissueRes,1988,254(2):317-30
1Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adulthuman mesenchymal stem cells. Science,1999,284(5411):143-7
2Nuttall ME, Gimble JM. Is there a therapeutic opportunity to eitherprevent or treat osteopenic disorders by inhibiting marrow adipogenesis?Bone,2000,27(2):177-84
3Hong JH, Yaffe MB. TAZ: a beta-catenin-like molecule that regulatesmesenchymal stem cell differentiation. Cell Cycle,2006,5(2):176-9
4Hong JH, Hwang ES, McManus MT, et al. TAZ, a transcriptionalmodulator of mesenchymal stem cell differentiation. Science,2005,309(5737):1074-8
5Jeong H, Bae S, An SY, et al. TAZ as a novel enhancer of MyoD-mediatedmyogenic differentiation. FASEB J,2010,24(9):3310-20
6Sheng MH, Lau KH, Baylink DJ. Role of Osteocyte-derived Insulin-LikeGrowth Factor I in Developmental Growth, Modeling, Remodeling, andRegeneration of the Bone. J Bone Metab,2014,21(1):41-54
7Romanelli RJ, LeBeau AP, Fulmer CG, et al. Insulin-like growth factortype-I receptor internalization and recycling mediate the sustainedphosphorylation of Akt. J Biol Chem,2007,282(31):22513-24
8Li Y, Song YH, Mohler J, et al. ANG II induces apoptosis of humanvascular smooth muscle via extrinsic pathway involving inhibition of Aktphosphorylation and increased FasL expression. Am J Physiol Heart CircPhysiol,2006,290(5): H2116-23
9Song YH, Li Y, Du J, et al. Muscle-specific expression of IGF-1blocksangiotensin II-induced skeletal muscle wasting. J Clin Invest,2005,115(2):451-8
10Delafontaine P, Song YH, Li Y. Expression, regulation, and function ofIGF-1, IGF-1R, and IGF-1binding proteins in blood vessels. ArteriosclerThromb Vasc Biol,2004,24(3):435-44
11Kofidis T, de Bruin JL, Yamane T, et al. Insulin-like growth factorpromotes engraftment, differentiation, and functional improvement aftertransfer of embryonic stem cells for myocardial restoration. Stem Cells,2004,22(7):1239-45
12Li Y, Higashi Y, Itabe H, et al. Insulin-like growth factor-1receptoractivation inhibits oxidized LDL-induced cytochrome C release andapoptosis via the phosphatidylinositol3kinase/Akt signaling pathway.Arterioscler Thromb Vasc Biol,2003,23(12):2178-84
13Goto M, Iwase A, Harata T, et al. IGF1-induced AKT phosphorylation andcell proliferation are suppressed with the increase in PTEN duringluteinization in human granulosa cells. Reproduction,2009,137(5):835-42
14Minuto F, Palermo C, Arvigo M, et al. The IGF system and bone. JEndocrinol Invest,2005,28(8Suppl):8-10
15Rosen CJ. Insulin-like growth factor I and bone mineral density:experience from animal models and human observational studies. BestPract Res Clin Endocrinol Metab,2004,18(3):423-35
16Huang Z, Ren PG, Ma T, et al. Modulating osteogenesis of mesenchymalstem cells by modifying growth factor availability. Cytokine,2010,51(3):305-10
17Ueland T. GH/IGF-I and bone resorption in vivo and in vitro. Eur JEndocrinol,2005,152(3):327-32
18Srouji S, Blumenfeld I, Rachmiel A, et al. Bone defect repair in rat tibia byTGF-beta1and IGF-1released from hydrogel scaffold. Cell Tissue Bank,2004,5(4):223-30
19Blumenfeld I, Srouji S, Lanir Y, et al. Enhancement of bone defect healingin old rats by TGF-beta and IGF-1. Exp Gerontol,2002,37(4):553-65
20Wang S, Mu J, Fan Z, et al. Insulin-like growth factor1can promote theosteogenic differentiation and osteogenesis of stem cells from apicalpapilla. Stem Cell Res,2012,8(3):346-56
21Yu Y, Mu J, Fan Z, et al. Insulin-like growth factor1enhances theproliferation and osteogenic differentiation of human periodontal ligamentstem cells via ERK and JNK MAPK pathways. Histochem Cell Biol,2012,137(4):513-25
22Osyczka AM, Leboy PS. Bone morphogenetic protein regulation of earlyosteoblast genes in human marrow stromal cells is mediated byextracellular signal-regulated kinase and phosphatidylinositol3-kinasesignaling. Endocrinology,2005,146(8):3428-37
23Johnson K, Pritzker K, Goding J, et al. The nucleoside triphosphatepyrophosphohydrolase isozyme PC-1directly promotes cartilagecalcification through chondrocyte apoptosis and increased calciumprecipitation by mineralizing vesicles. J Rheumatol,2001,28(12):2681-91
24Livak KJ, Schmittgen TD. Analysis of relative gene expression data usingreal-time quantitative PCR and the2(-Delta Delta C(T)) Method. Methods,2001,25(4):402-8
25Bustin SA. Absolute quantification of mRNA using real-time reversetranscription polymerase chain reaction assays. J Mol Endocrinol,2000,25(2):169-93
26Rubin R, Arzumanyan A, Soliera AR, et al. Insulin receptor substrate(IRS)-1regulates murine embryonic stem (mES) cells self-renewal. J CellPhysiol,2007,213(2):445-53
27Cho HH, Shin KK, Kim YJ, et al. NF-kappaB activation stimulatesosteogenic differentiation of mesenchymal stem cells derived from humanadipose tissue by increasing TAZ expression. J Cell Physiol,2010,223(1):168-77
28Hong D, Chen HX, Xue Y, et al. Osteoblastogenic effects ofdexamethasone through upregulation of TAZ expression in ratmesenchymal stem cells. J Steroid Biochem Mol Biol,2009,116(1-2):86-92
29Byun MR, Kim AR, Hwang JH, et al. Phorbaketal A stimulates osteoblastdifferentiation through TAZ mediated Runx2activation. FEBS Lett,2012,586(8):1086-92
30Takeda S, Bonnamy JP, Owen MJ, et al. Continuous expression of Cbfa1in nonhypertrophic chondrocytes uncovers its ability to inducehypertrophic chondrocyte differentiation and partially rescuesCbfa1-deficient mice. Genes Dev,2001,15(4):467-81
31Karsenty G, Wagner EF. Reaching a genetic and molecular understandingof skeletal development. Dev Cell,2002,2(4):389-406
32Yamaguchi A, Komori T, Suda T. Regulation of osteoblast differentiationmediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. EndocrRev,2000,21(4):393-411
33Li J, Zhao Q, Wang E, et al. Transplantation of Cbfa1-overexpressingadipose stem cells together with vascularized periosteal flaps repairsegmental bone defects. J Surg Res,2012,176(1): e13-20
34Komori T, Yagi H, Nomura S, et al. Targeted disruption of Cbfa1results ina complete lack of bone formation owing to maturational arrest ofosteoblasts. Cell,1997,89(5):755-64
35Komori T. Regulation of osteoblast differentiation by transcription factors.J Cell Biochem,2006,99(5):1233-9
36Kobayashi I, Kiyoshima T, Wada H, et al. Type II/III Runx2/Cbfa1isrequired for tooth germ development. Bone,2006,38(6):836-44
37Liu TM, Lee EH. Transcriptional Regulatory Cascades in Runx2Dependent Bone Development. Tissue Eng Part B Rev,2013,19(3):254-63
38Neve A, Corrado A, Cantatore FP. Osteocalcin: Skeletal and extra-skeletaleffects. J Cell Physiol,2013,228(6):1149-53
39Carvallo L, Henriquez B, Paredes R, et al.1alpha,25-dihydroxy vitaminD3-enhanced expression of the osteocalcin gene involves increasedpromoter occupancy of basal transcription regulators and gradualrecruitment of the1alpha,25-dihydroxy vitamin D3receptor-SRC-1coactivator complex. J Cell Physiol,2008,214(3):740-9
40Martinez ME, del Campo MT, Medina S, et al. Influence of skeletal site oforigin and donor age on osteoblastic cell growth and differentiation. CalcifTissue Int,1999,64(4):280-6
41Ozono K, Liao J, Kerner SA, et al. The vitamin D-responsive element inthe human osteocalcin gene. Association with a nuclear proto-oncogeneenhancer. J Biol Chem,1990,265(35):21881-8
42Paredes R, Arriagada G, Cruzat F, et al. Bone-specific transcription factorRunx2interacts with the1alpha,25-dihydroxyvitamin D3receptor toup-regulate rat osteocalcin gene expression in osteoblastic cells. Mol CellBiol,2004,24(20):8847-61
43Javed A, Gutierrez S, Montecino M, et al. Multiple Cbfa/AML sites in therat osteocalcin promoter are required for basal and vitamin D-responsivetranscription and contribute to chromatin organization. Mol Cell Biol,1999,19(11):7491-500
44Turjanski AG, Vaque JP, Gutkind JS. MAP kinases and the control ofnuclear events. Oncogene,2007,26(22):3240-53
45Celil AB, Campbell PG. BMP-2and insulin-like growth factor-I mediateOsterix (Osx) expression in human mesenchymal stem cells via the MAPKand protein kinase D signaling pathways. J Biol Chem,2005,280(36):31353-9
46Wu Y, Zhang X, Zhang P, et al. Intermittent traction stretch promotes theosteoblastic differentiation of bone mesenchymal stem cells by theERK1/2-activated Cbfa1pathway. Connect Tissue Res,2012,53(6):451-9
1Shimon I. Growth hormone replacement for adult growth hormonedeficiency. Expert Opin Pharmacother,2003,4(11):1977-83
2Ernst M, Froesch ER. Growth hormone dependent stimulation ofosteoblast-like cells in serum-free cultures via local synthesis ofinsulin-like growth factor I. Biochem Biophys Res Commun,1988,151(1):142-7
3Joung YH, Lim EJ, Darvin P, et al. MSM Enhances GH Signaling via theJak2/STAT5b Pathway in Osteoblast-Like Cells and OsteoblastDifferentiation through the Activation of STAT5b in MSCs. PLoS One,2012,7(10): e47477
4Carroll PV, Christ ER, Bengtsson BA, et al. Growth hormone deficiency inadulthood and the effects of growth hormone replacement: a review.Growth Hormone Research Society Scientific Committee. J ClinEndocrinol Metab,1998,83(2):382-95
5Geisler A, Lass N, Reinsch N, et al. Quality of life in children andadolescents with growth hormone deficiency: association with growthhormone treatment. Horm Res Paediatr,2012,78(2):94-9
6Kann P, Piepkorn B, Schehler B, et al. Effect of long-term treatment withGH on bone metabolism, bone mineral density and bone elasticity inGH-deficient adults. Clin Endocrinol (Oxf),1998,48(5):561-8
7Tritos NA, Hamrahian AH, King D, et al. A longer interval without GHreplacement and female gender are associated with lower bone mineraldensity in adults with childhood-onset GH deficiency: a KIMS databaseanalysis. Eur J Endocrinol,2012,167(3):343-51
8Wuster C, Abs R, Bengtsson BA, et al. The influence of growth hormonedeficiency, growth hormone replacement therapy, and other aspects ofhypopituitarism on fracture rate and bone mineral density. J Bone MinerRes,2001,16(2):398-405
9Rosen T, Wilhelmsen L, Landin-Wilhelmsen K, et al. Increased fracturefrequency in adult patients with hypopituitarism and GH deficiency. Eur JEndocrinol,1997,137(3):240-5
10Leung DW, Spencer SA, Cachianes G, et al. Growth hormone receptor andserum binding protein: purification, cloning and expression. Nature,1987,330(6148):537-43
11Andreassen M, Frystyk J, Faber J, et al. GH activity and markers ofinflammation: a crossover study in healthy volunteers treated with GH anda GH receptor antagonist. Eur J Endocrinol,2012,166(5):811-9
12Mathews LS, Hammer RE, Brinster RL, et al. Expression of insulin-likegrowth factor I in transgenic mice with elevated levels of growth hormoneis correlated with growth. Endocrinology,1988,123(1):433-7
13Becker NS, Verdu P, Georges M, et al. The role of GHR and IGF1genes inthe genetic determination of African pygmies' short stature. Eur J HumGenet,2013,21(6):653-8
14Barner C, Petersson M, Eden Engstrom B, et al. Effects on insulinsensitivity and body composition of combination therapy with GH andIGF1in GH deficient adults with type2diabetes. Eur J Endocrinol,2012,167(5):697-703
15Holly JM, Wass JA. Insulin-like growth factors; autocrine, paracrine orendocrine? New perspectives of the somatomedin hypothesis in the lightof recent developments. J Endocrinol,1989,122(3):611-8
16Arnaldez FI, Helman LJ. Targeting the insulin growth factor receptor1.Hematol Oncol Clin North Am,2012,26(3):527-42, vii-viii
17Yakar S, Rosen CJ, Beamer WG, et al. Circulating levels of IGF-1directlyregulate bone growth and density. J Clin Invest,2002,110(6):771-81
18Fideleff HL, Boquete HR, Stalldecker G, et al. Comparative results of a4-year study on cardiovascular parameters, lipid metabolism, bodycomposition and bone mass between untreated and treated adult growthhormone deficient patients. Growth Horm IGF Res,2008,18(4):318-24
19Van den Heijkant S, Hoorweg-Nijman G, Huisman J, et al. Effects ofgrowth hormone therapy on bone mass, metabolic balance, and well-beingin young adult survivors of childhood acute lymphoblastic leukemia. JPediatr Hematol Oncol,2011,33(6): e231-8
20Suganuma N, Furuhashi M, Hirooka T, et al. Bone mineral density in adultpatients with Turner's syndrome: analyses of the effectiveness of GH andovarian steroid hormone replacement therapies. Endocr J,2003,50(3):263-9
21Hansen TB, Brixen K, Vahl N, et al. Effects of12months of growthhormone (GH) treatment on calciotropic hormones, calcium homeostasis,and bone metabolism in adults with acquired GH deficiency: a doubleblind, randomized, placebo-controlled study. J Clin Endocrinol Metab,1996,81(9):3352-9
22Johannsson G, Rosen T, Bosaeus I, et al. Two years of growth hormone(GH) treatment increases bone mineral content and density inhypopituitary patients with adult-onset GH deficiency. J Clin EndocrinolMetab,1996,81(8):2865-73
23Rodriguez-Arnao J, James I, Jabbar A, et al. Serum collagen crosslinks asmarkers of bone turn-over during GH replacement therapy in growthhormone deficient adults. Clin Endocrinol (Oxf),1998,48(4):455-62
24Cuneo RC, Judd S, Wallace JD, et al. The Australian Multicenter Trial ofGrowth Hormone (GH) Treatment in GH-Deficient Adults. J ClinEndocrinol Metab,1998,83(1):107-16
25Biller BM, Sesmilo G, Baum HB, et al. Withdrawal of long-termphysiological growth hormone (GH) administration: differential effects onbone density and body composition in men with adult-onset GH deficiency.J Clin Endocrinol Metab,2000,85(3):970-6
26Koranyi J, Svensson J, Gotherstrom G, et al. Baseline characteristics andthe effects of five years of GH replacement therapy in adults with GHdeficiency of childhood or adulthood onset: a comparative, prospectivestudy. J Clin Endocrinol Metab,2001,86(10):4693-9
27Lanzi R, Losa M, Villa I, et al. GH replacement therapy increases plasmaosteoprotegerin levels in GH-deficient adults. Eur J Endocrinol,2003,148(2):185-91
28Underwood LE, Attie KM, Baptista J. Growth hormone (GH)dose-response in young adults with childhood-onset GH deficiency: atwo-year, multicenter, multiple-dose, placebo-controlled study. J ClinEndocrinol Metab,2003,88(11):5273-80
29Hubina E, Kovacs L, Szabolcs I, et al. The effect of gender and age ongrowth hormone replacement in growth hormone-deficient patients. HormMetab Res,2004,36(4):247-53
30Bravenboer N, Holzmann PJ, ter Maaten JC, et al. Effect of long-termgrowth hormone treatment on bone mass and bone metabolism in growthhormone-deficient men. J Bone Miner Res,2005,20(10):1778-84
31Arwert LI, Roos JC, Lips P, et al. Effects of10years of growth hormone(GH) replacement therapy in adult GH-deficient men. Clin Endocrinol(Oxf),2005,63(3):310-6
32Boguszewski CL, Meister LH, Zaninelli DC, et al. One year of GHreplacement therapy with a fixed low-dose regimen improves bodycomposition, bone mineral density and lipid profile of GH-deficient adults.Eur J Endocrinol,2005,152(1):67-75
33Benedini S, Dalle Carbonare L, Albiger N, et al. Effect of short-termtherapy with recombinant human growth hormone (GH) on metabolicparameters and preclinical atherosclerotic markers in hypopituitarypatients with growth hormone deficiency. Horm Metab Res,2006,38(1):16-21
34Snyder PJ, Biller BM, Zagar A, et al. Effect of growth hormonereplacement on BMD in adult-onset growth hormone deficiency. J BoneMiner Res,2007,22(5):762-70
35Gotherstrom G, Bengtsson BA, Bosaeus I, et al. Ten-year GH replacementincreases bone mineral density in hypopituitary patients with adult onsetGH deficiency. Eur J Endocrinol,2007,156(1):55-64
36Rota F, Savanelli MC, Tauchmanova L, et al. Bone density and turnover inyoung adult patients with growth hormone deficiency after2-year growthhormone replacement according with gender. J Endocrinol Invest,2008,31(2):94-102
37Conway GS, Szarras-Czapnik M, Racz K, et al. Treatment for24monthswith recombinant human GH has a beneficial effect on bone mineraldensity in young adults with childhood-onset GH deficiency. Eur JEndocrinol,2009,160(6):899-907
38Elbornsson M, Gotherstrom G, Bosaeus I, et al. Fifteen years of GHreplacement increases bone mineral density in hypopituitary patients withadult-onset GH deficiency. Eur J Endocrinol,2012,166(5):787-95
39Kassem M, Mosekilde L, Eriksen EF. Growth hormone stimulatesproliferation of normal human bone marrow stromal osteoblast precursorcells in vitro. Growth Regul,1994,4(3):131-5
40Wit JM, Camacho-Hubner C. Endocrine regulation of longitudinal bonegrowth. Endocr Dev,2011,21:30-41
41Baroncelli GI, Bertelloni S, Ceccarelli C, et al. Dynamics of bone turnoverin children with GH deficiency treated with GH until final height. Eur JEndocrinol,2000,142(6):549-56
42Klefter O, Feldt-Rasmussen U. Is increase in bone mineral content causedby increase in skeletal muscle mass/strength in adult patients withGH-treated GH deficiency? A systematic literature analysis. Eur JEndocrinol,2009,161(2):213-21
43Proctor DN, Melton LJ, Khosla S, et al. Relative influence of physicalactivity, muscle mass and strength on bone density. Osteoporos Int,2000,11(11):944-52
44Frost HM. The Utah paradigm of skeletal physiology: an overview of itsinsights for bone, cartilage and collagenous tissue organs. J Bone MinerMetab,2000,18(6):305-16
45Giustina A, Mazziotti G, Canalis E. Growth hormone, insulin-like growthfactors, and the skeleton. Endocr Rev,2008,29(5):535-59
46Kaur A, Phadke SR. Analysis of Short Stature Cases Referred for GeneticEvaluation. Indian J Pediatr,2012,79(12):1597-600
47Murray RD, Skillicorn CJ, Howell SJ, et al. Dose titration and patientselection increases the efficacy of GH replacement in severely GHdeficient adults. Clin Endocrinol (Oxf),1999,50(6):749-57
48Pocock NA, Noakes KA, Griffiths M, et al. A comparison of longitudinalmeasurements in the spine and proximal femur using lunar and hologicinstruments. J Bone Miner Res,1997,12(12):2113-8
49Drake WM, Rodriguez-Arnao J, Weaver JU, et al. The influence of genderon the short and long-term effects of growth hormone replacement on bonemetabolism and bone mineral density in hypopituitary adults: a5-yearstudy. Clin Endocrinol (Oxf),2001,54(4):525-32
50Olson LE, Ohlsson C, Mohan S. The role of GH/IGF-I-mediatedmechanisms in sex differences in cortical bone size in mice. Calcif TissueInt,2011,88(1):1-8
51Carroll PV, Drake WM, Maher KT, et al. Comparison of continuation orcessation of growth hormone (GH) therapy on body composition andmetabolic status in adolescents with severe GH deficiency at completionof linear growth. J Clin Endocrinol Metab,2004,89(8):3890-5
52Prabhakar VK, Shalet SM. Aetiology, diagnosis, and management ofhypopituitarism in adult life. Postgrad Med J,2006,82(966):259-66
53Woodhouse LJ, Mukherjee A, Shalet SM, et al. The influence of growthhormone status on physical impairments, functional limitations, andhealth-related quality of life in adults. Endocr Rev,2006,27(3):287-317
54Hazem A, Elamin MB, Bancos I, et al. Body composition and quality oflife in adults treated with GH therapy: a systematic review andmeta-analysis. Eur J Endocrinol,2012,166(1):13-20
55Deijen JB, Arwert LI, Witlox J, et al. Differential effect sizes of growthhormone replacement on Quality of Life, well-being and health status ingrowth hormone deficient patients: a meta-analysis. Health Qual LifeOutcomes,2005,3:63
56Renehan AG, Zwahlen M, Minder C, et al. Insulin-like growth factor(IGF)-I, IGF binding protein-3, and cancer risk: systematic review andmeta-regression analysis. Lancet,2004,363(9418):1346-53
1Komm BS, Terpening CM, Benz DJ, et al. Estrogen binding, receptormRNA, and biologic response in osteoblast-like osteosarcoma cells.Science,1988,241(4861):81-4
2Zhao JW, Gao ZL, Mei H, et al. Differentiation of human mesenchymalstem cells: the potential mechanism for estrogen-induced preferentialosteoblast versus adipocyte differentiation. Am J Med Sci,2011,341(6):460-8
3Chen FP, Hu CH, Wang KC. Estrogen modulates osteogenic activity andestrogen receptor mRNA in mesenchymal stem cells of women.Climacteric,2013,16(1):154-60
4Hiyama S, Sugiyama T, Kusuhara S, et al. Evidence for estrogen receptorexpression during medullary bone formation and resorption inestrogen-treated male Japanese quails (Coturnix coturnix japonica). J VetSci,2012,13(3):223-7
5Mamalis A, Markopoulou C, Lagou A, et al. Oestrogen regulatesproliferation, osteoblastic differentiation, collagen synthesis and periostingene expression in human periodontal ligament cells through oestrogenreceptor beta. Arch Oral Biol,2011,56(5):446-55
6Wang Y, Li LZ, Zhang YL, et al. LC, a novel estrone-rhein hybridcompound, promotes proliferation and differentiation and protects againstcell death in human osteoblastic MG-63cells. Mol Cell Endocrinol,2011,344(1-2):59-68
7Krum SA. Direct transcriptional targets of sex steroid hormones in bone. JCell Biochem,2011,112(2):401-8
8Rudnik V, Sanyal A, Syed FA, et al. Loss of ERE binding activity byestrogen receptor-alpha alters basal and estrogen-stimulated bone-relatedgene expression by osteoblastic cells. J Cell Biochem,2008,103(3):896-907
9Almeida M, Martin-Millan M, Ambrogini E, et al. Estrogens attenuateoxidative stress and the differentiation and apoptosis of osteoblasts byDNA-binding-independent actions of the ERalpha. J Bone Miner Res,2010,25(4):769-81
10Moriarty K, Kim KH, Bender JR. Minireview: estrogen receptor-mediatedrapid signaling. Endocrinology,2006,147(12):5557-63
11Matsumoto Y, Otsuka F, Takano-Narazaki M, et al. Estrogen facilitatesosteoblast differentiation by upregulating bone morphogenetic protein-4signaling. Steroids,2013,78(5):513-20
12Syed FA, Fraser DG, Monroe DG, et al. Distinct effects of loss of classicalestrogen receptor signaling versus complete deletion of estrogen receptoralpha on bone. Bone,2011,49(2):208-16
13Matsumoto Y, Otsuka F, Takano M, et al. Estrogen and glucocorticoidregulate osteoblast differentiation through the interaction of bonemorphogenetic protein-2and tumor necrosis factor-alpha in C2C12cells.Mol Cell Endocrinol,2010,325(1-2):118-27
14Nakashima K, de Crombrugghe B. Transcriptional mechanisms inosteoblast differentiation and bone formation. Trends Genet,2003,19(8):458-66
15Kumar A, Ruan M, Clifton K, et al. TGF-beta mediates suppression ofadipogenesis by estradiol through connective tissue growth factorinduction. Endocrinology,2012,153(1):254-63
16Li B, Wang Y, Liu Y, et al. Altered gene expression involved in insulinsignaling pathway in type II diabetic osteoporosis rats model. Endocrine,2013,43(1):136-46
17Bu YH, Peng D, Zhou HD, et al. Insulin receptor substrate2playsimportant roles in17beta-estradiol-induced bone formation. J EndocrinolInvest,2009,32(8):682-9
18Sunters A, Armstrong VJ, Zaman G, et al. Mechano-transduction inosteoblastic cells involves strain-regulated estrogen receptoralpha-mediated control of insulin-like growth factor (IGF) I receptorsensitivity to Ambient IGF, leading to phosphatidylinositol3-kinase/AKT-dependent Wnt/LRP5receptor-independent activation ofbeta-catenin signaling. J Biol Chem,2010,285(12):8743-58
19Kubota T, Michigami T, Ozono K. Wnt signaling in bone metabolism. JBone Miner Metab,2009,27(3):265-71
20Shi YC, Worton L, Esteban L, et al. Effects of continuous activation ofvitamin D and Wnt response pathways on osteoblastic proliferation anddifferentiation. Bone,2007,41(1):87-96
21Rossini M, Gatti D, Adami S. Involvement of WNT/beta-catenin signalingin the treatment of osteoporosis. Calcif Tissue Int,2013,93(2):121-32
22Modder UI, Clowes JA, Hoey K, et al. Regulation of circulating sclerostinlevels by sex steroids in women and in men. J Bone Miner Res,2011,26(1):27-34
23Kousteni S, Han L, Chen JR, et al. Kinase-mediated regulation of commontranscription factors accounts for the bone-protective effects of sexsteroids. J Clin Invest,2003,111(11):1651-64
24Yang YH, Chen K, Li B, et al. Estradiol inhibits osteoblast apoptosis viapromotion of autophagy through the ER-ERK-mTOR pathway. Apoptosis,2013,18(11):1363-75
25Liu LJ, Liu LQ, Bo T, et al. Puerarin Suppress Apoptosis of HumanOsteoblasts via ERK Signaling Pathway. Int J Endocrinol,2013,2013:786574
26Bradford PG, Gerace KV, Roland RL, et al. Estrogen regulation ofapoptosis in osteoblasts. Physiol Behav,2010,99(2):181-5
27Marathe N, Rangaswami H, Zhuang S, et al. Pro-survival effects of17beta-estradiol on osteocytes are mediated by nitric oxide/cGMP viadifferential actions of cGMP-dependent protein kinases I and II. J BiolChem,2012,287(2):978-88
28Cooper LF, Tiffee JC, Griffin JP, et al. Estrogen-induced resistance toosteoblast apoptosis is associated with increased hsp27expression. J CellPhysiol,2000,185(3):401-7
29Kovacic N, Grcevic D, Katavic V, et al. Fas receptor is required forestrogen deficiency-induced bone loss in mice. Lab Invest,2010,90(3):402-13
1Hey PJ, Twells RC, Phillips MS, et al. Cloning of a novel member of thelow-density lipoprotein receptor family. Gene,1998,216(1):103-11
2Burgers TA, Williams BO. Regulation of Wnt/beta-catenin signalingwithin and from osteocytes. Bone,2013,54(2):244-9
3Gong Y, Slee RB, Fukai N, et al. LDL receptor-related protein5(LRP5)affects bone accrual and eye development. Cell,2001,107(4):513-23
4Maeda K, Takahashi N, Kobayashi Y. Roles of Wnt signals in boneresorption during physiological and pathological states. J Mol Med (Berl),2013,91(1):15-23
5Moon RT, Kohn AD, De Ferrari GV, et al. WNT and beta-cateninsignalling: diseases and therapies. Nat Rev Genet,2004,5(9):691-701
6Glass DA,2nd, Bialek P, Ahn JD, et al. Canonical Wnt signaling indifferentiated osteoblasts controls osteoclast differentiation. Dev Cell,2005,8(5):751-64
7Ducy P, Karsenty G. The two faces of serotonin in bone biology. J CellBiol,2010,191(1):7-13
8Yadav VK, Arantes HP, Barros ER, et al. Genetic analysis of Lrp5function in osteoblast progenitors. Calcif Tissue Int,2010,86(5):382-8
9Boyden LM, Mao J, Belsky J, et al. High bone density due to a mutation inLDL-receptor-related protein5. N Engl J Med,2002,346(20):1513-21
10Zuo C, Huang Y, Bajis R, et al. Osteoblastogenesis regulation signals inbone remodeling. Osteoporos Int,2012,23(6):1653-63
11Cui Y, Niziolek PJ, MacDonald BT, et al. Lrp5functions in bone toregulate bone mass. Nat Med,2011,17(6):684-91
12Yadav VK, Ryu JH, Suda N, et al. Lrp5controls bone formation byinhibiting serotonin synthesis in the duodenum. Cell,2008,135(5):825-37
13Babij P, Zhao W, Small C, et al. High bone mass in mice expressing amutant LRP5gene. J Bone Miner Res,2003,18(6):960-74
14Warden SJ, Robling AG, Sanders MS, et al. Inhibition of the serotonin(5-hydroxytryptamine) transporter reduces bone accrual during growth.Endocrinology,2005,146(2):685-93
15Holmen SL, Zylstra CR, Mukherjee A, et al. Essential role of beta-cateninin postnatal bone acquisition. J Biol Chem,2005,280(22):21162-8
16Joiner DM, Ke J, Zhong Z, et al. LRP5and LRP6in development anddisease. Trends Endocrinol Metab,2013,24(1):31-9
17Goltzman D. LRP5, serotonin, and bone: complexity, contradictions, andconundrums. J Bone Miner Res,2011,26(9):1997-2001
18Warden SJ, Robling AG, Haney EM, et al. The emerging role of serotonin(5-hydroxytryptamine) in the skeleton and its mediation of the skeletaleffects of low-density lipoprotein receptor-related protein5(LRP5). Bone,2010,46(1):4-12
19Monroe DG, McGee-Lawrence ME, Oursler MJ, et al. Update on Wntsignaling in bone cell biology and bone disease. Gene,2012,492(1):1-18
20Zhang W, Drake MT. Potential role for therapies targeting DKK1, LRP5,and serotonin in the treatment of osteoporosis. Curr Osteoporos Rep,2012,10(1):93-100
21Korvala J, Juppner H, Makitie O, et al. Mutations in LRP5cause primaryosteoporosis without features of OI by reducing Wnt signaling activity.BMC Med Genet,2012,13:26
22Boudin E, Jennes K, de Freitas F, et al. No mutations in the serotoninrelated TPH1and HTR1B genes in patients with monogenic sclerosingbone disorders. Bone,2013,55(1):52-6