Study Of The Role Of IGFBP4 During Chondrogenesis

Background:Obtaining phenotype-stable seeding cells was one of the challenges in cartilage tissue engineering research.The dynamic change of chondrocyte proliferation and differentiation was critical to the phenotype maintenance of cartilage.Insulin-like growth factors have been confirmed to play an important role in the proliferation and differentiation of chondrocytes through numbers of intracellular signaling pathways.Insulin-like growth factor-binding proteins have high affinity with insulin-like growth factors.It could regulate the action of IGFs to inhibit or promote chondrocyte proliferation and differentiation when combining with IGFs.In addition,researchers have identified a novel IGF-independent role for IGFBPs in the modulation of cartilage development.IGFBP4 was one of the members of the IGFBP family.A study has confirmed that knockout of the IGFBP4 gene in mice showed an embryonic growth and significant reduction in body size.In the previous study,we found the expression of IGFBP4 is higher in early chondrogenic differentiation stage than that in late stage.All these suggested a chondrogenic differentiation promoting effect of IGFBP4.Meanwhile,IGFBP4 was a negative regulator of IGF action and can block IGF-induced cell proliferation.Litter was known about the effects of IGFBP4 and its mechanism in chondrogenesis.In this study,we will firstly examine the gene expression profile of IGFBP4 during chondrogenesis and investigate the role and mechanism of IGFBP4 in maintaining phenotype of chondrocytes during cartilage differentiation based on the mesenchymal chondrogenic cell line RCJ3.1C5.18,which progresses spontaneously to differentiated growth plate chondrocytes.The result of the study may help us understanding the molecular mechanism in phenotypic stability of chondrocytes and provide research foundation for obtaining phenotype-stable seeding cells and constructing a stable tissue engineering cartilage.Objectives:1.To establish chondrogenic differentiation mode in vitro using the RCJ3.1C5.18 cell line and identify the cell differentiation and proliferation status at each stage of cartilage differentiation.2.To explore Insulin-like growth factor binding proteins gene expression profile at different stages of chondrogenesis.3.To clarify the role and mechanism of IGFBP4 in the process of chondrogenesis.Methods and results1.Identification of cell differentiation and proliferation status in the process of chondrogenesis.Methods:RCJ3.1C5.18 cells were grown in a-minimal essential medium supplemented with 10%heat-inactivated fetal bovine serum,10-7 M dexamethasone,and 2 mM sodium pyruvate.Cells were plated at a density of 6 × 104 cells/well in six-well dishes.After reaching confluence(4 days),fresh growth medium supplemented with 50?g/ml ascorbic acid and 10 mM?-glycerophosphate was added.Differentiating cells were fed again with supplemented medium at days 7 and 10 of culture.Cultures were monitored over a total period of 10 days.Then the cells were fixed and the histological stains of alcian blue,safranin O,and alizarin red staining were used to detect secretion of extracellular matrix at days 1?4?7 and 10 of culture respectively.At the same time,Total RNA was extracted and reversed transcript to cDNA.Real-time PCR was used to determine the expression of chondrogenic differentiation-related marker genes(Collagen type ?.SRY-Box 9 and Aggrecan),hypertrophic differentiation-related marker genes(type X collagen and Matrix Metallopeptidase 13)and osteogenic differentiation-related marker genes(Runt-related transcription factor 2 and Alkaline phosphatase).The status of cell viability,cycle and apoptosis was detected by MuserTM Cell Analyzer.Results:The cells.were adhered at 4h and grew well with a flat and polygonal shape.At day 4 of culture,the cells reached more than 90%confluence and showed.a cubic paving stone-like morphology;The result of histological staining showed that the levels of calcium nodule were upregulated at days 7-10 of culture,at this stage,RCJ3.1C5.18 cells viability decreased and gradually developed hypertrophic-like and osteogenic-like characteristics,which was assessed by gene expression of Alkaline phosphatase,Runt-related transcription factor 2,Matrix Metallopeptidase 13,and Collagen type X.The secretion of chondrocytes extracellular matrix paralleled the expression of chondrocytes marker genes like Collagen type ??SRY-Box 9 and Aggrecan,peaking during the proliferative phase(Days 4)and decreasing thereafter,while changes of cell apoptosis were in the opposite trend simultaneously.Compared with days 4/7/10 of culture,the result of cell cycle showed that the proportion of cells in the G0/G1 phase was lower and the proportion in the S phase was higher at culture of the first day.2.The expression profile of Insulin-like growth factor binding proteins gene in theprocess of chondrogenesis.Methods:Real-time PCR was used to detect the changes of IGFBP family members(IGFBP1-6)at day s 1,4,7and 10 of culture.The correlation between expression of IGFBPs and specific marker gene at different stages of cartilage differentiation was analyzed by SPSS.Results:The expression of IGFBP1 was low in the early stage of cell culture(1d and 4d),then increased significantly and peaked in the middle-late period(7d);The change of IGFBP2 was not statistically significant in the whole period of culture.Compared with the early period(1d and 4d)of cell culture,the expression levels of IGFBP3 and IGFBP5 were significantly increased in the later culture period(7d and 10d)while the expression of IGFBP4 and IGFBP6 showed an opposite trend.The result of SPSS showed that the expression of IGFBP2/4/6,especially IGFBP4,was highly positive correlated with cartilage differentiation-related genes(Col2,Sox9,Acan)and negatively correlated with hypertrophic differentiation-related genes(Co110,mp13)and osteogenic differentiation-related genes(Runx2,Alp).In contrast,the expression of IGFBP3/5 was negatively correlated with the chondrogenic differentiation gene and positively correlated with the expression of hypertrophic and osteoblastic differentiation genes.3.The role and mechanism of IGFBP4 in the process of chondrogenesisMethods:The lentivirus-based IGFBP4 knockdown and overexpression recombinant plasmid was harvested and then infected RCJ3.1C5.18 cells.Cell Counting Kit-8(CCK-8)was used to assess the proliferation of cells.Cell cycle and Apoptosis was detected by Muse Cell Analyzer.The effect of knockdown and overexpression of IGFBP4 on chondrogenic differentiation marker gene was detected by Real-time PCR.The level of phosphorylation of key proteins in various pathways was detected by protein chip after 1GFBP4 overexpression at day 7 of culture.Results:Overexpression of IGFBP4 significantly inhibited the proliferation of hypertrophic-like chondrocytes and promoted apoptosis remarkably While knockdown of IGFBP4 at day 4 of culture have litter effect on proliferation and apoptosis of cells.In addition,Overexpression of IGFBP4 promoted the upregulation of chondrogenic differentiation-related marker genes(Collagen type II and SRY-Box 9)in hypertrophic chondrocytes and inhibited the expression of hypertrophic differentiation-related marker genes(type X collagen and Matrix Metallopeptidase 13).However,chondrogenic differentiation-related marker genes showed an opposite result when we knockdown IGFBP4 at day 4 of culture.After overexpression of IGFBP4,proteins with obviously different phosphorylation levels were mainly enriched in MAPK and PI3K-AKT signaling pathways.Conclusions:1.RCJ3.1C5.18 cells are able to progress through chondrogenic differentiation,hypertrophic differentiation and osteogenic differentiation over a total culture period of 10 days,and the cells in different stages of differentiation showed a different status of proliferation,cycle and apoptosis.2.The expression profiles of different IGFBP genes in different stages of chondrogenesis are different,suggesting the role of IGFBP gene family members may be different during chondrocyte proliferation and differentiation.3.Overexpression of.IGFBP4 can significantly inhibit the proliferation of hypertrophic-like chondrocytes,induce cells staying in G0/G1 phase and decrease the ratio of cells in S and G2/M phase.And overexpression of IGFBP4 could Promote apoptosis through up-regulating Bax and Ciaspase-3.4.Overexpression of IGFBP4 can down-regulate the expression of hypertrophy differentiation marker genes Col 10 and Mmp13 and increase the expression of cartilage differentiation marker genes Sox9 and Col2.And chondrogenic differentiation-related marker genes showed an opposite result when we knockdown IGFBP4 at day 4 of culture.All these suggest that IGFBP4 can promote phenotypic stability of chondrocytes,as well as inhibit hypertrophic differentiation of chondrocytes5.IGFBP4 may promote the expression of chondrogenic phenotype through MAPK and PI3K-AKT signaling pathways.