Effects Of IGF1on The Osteogenic Differentiation Of BMSCs And The Mechanism

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 BMSCsObjectives: 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 methodsThe 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 BMSCsThe 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 BMSCsAfter 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 pathwayObjectives: 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 BMSCsIGF1could 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 expressionBoth 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 differentiationBoth 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 pathwayBoth 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-analysisObjectives: 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-analysisA total of20unique studies which included936subjects were availablefor this meta-analysis.2Effects of GH treatment on the circulating levels of IGF1Almost 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 spineThe 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 neckThe 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 bodyThe 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 biasSignificant 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.