The Effect Of GILZ On Adipocytes Differentiation And Its Regulative Mechanisms

Adipocytes is a type of functional mammalian cells which play a key role in maintaining lipid and energy metabolism balance. Adipocytes differentiation is a very complex process which many transcription factors,cell factors and hormones involves in regulating it from pre-adipocytes to mature adipocytes.Dysfunction of adipocytes differentiation may lead to many diseases, such as obesity, type 2 diabetes mellitus, fatty liver,hyperlipidemia and mammary cancer,et al.Investigation of pre-adipocytes proliferation and differentiation is the core of obesities mechanism,and it is beneficial to look for the drug of body weight reduction. So, the research on adipocytes differentiation in molecular and cellular levels has a theoretical and practical significance on studing the above life and diseases process,as well as curing and preventing these diseases.Glucocorticoid (GC) induced leucine zipper (GILZ) has been identified as a glucocorticoids-induced protein, has many important biological functions, such as inhibiting inflammation and protecting T cells from apoptosis et al. GC regulates a spectrum of cell functions, including growth, differentiation, and apoptosis. It is also among the best known anti-inflammatory and immune suppressive agents and is widely used for treating diseases such as rheumatoid arthritis, pulmonary disease, asthma and in organ transplantation to prevent rejection. Importantly, long term GC treatment can accelerate bone loss and result in osteoporosis, as well as replace of marrow cell populations with adipose tissues (fatty marrow). Primary reports showed that GILZ antagonizes GC's function by inhibiting GC induced adipogenesis and suggested that GILZ functions as an antagonist in the GC negative-feedback circuit. GILZ belongs to the leucine-zipper family of transcription factors.Several studies suggested GILZ as a transcriptional repressor to inhibit gene expression through direct association with nuclear factor-KB and activating protein. It also was found to involve in signal transduction of Fas/FasL,NF-κB,Ap1,Raf21 signal passways.During Adipocytes come into being, The peroxisome proliferator-activated receptor gamma (PPARy) family and C/EBPs play an important role as the target transcription factors in relative cell signal passways. PPARy belongs to the nuclear hormone receptor subfamily of transcription factors that can bind to specific DNA response elements in the regulatory regions of target genes and there are three protein products, PPARγ1, PPARγ2 and PPARγ3, which are isoforms transcribed from the same gene with different promoter usage.PPARy2 is predominantly expressed in adipose tissue. Of these three isoforms, PPARy2 is a key regulator of adipogenesis and is expressed at an early stage of the adipogenesis program. PPARy2 can activate a battery of genes necessary for lipid metabolism, including lipoprotein lipase (LPL), phosphoenolpyruvate carboxykinase, fatty acid-binding and transport proteins, and stearoyl-CoA desaturase-1 (SCD-1). Functional PPAR response elements (PPREs) have been identified in the promoter regions of these genes. Another important transcription factor family of adipocytes differentiation C/EBPs, which have three protein isoforms, C/EBPa, C/EBPβand c/EBPδ. All of them can promote adipocytes differentiation. C/EBPa maybe mediates the effects of GILZ on adipocytes differentiation and it can active transcription with PPARy2 each other to further initiate adipocytes differentiation.GILZ acts as an important kind of signal molecule and nuclear transcription factor, However, it is still unclear whether GILZ involves in adipocytes differentiation, what about its regulatory mechanisms,and whether C/EBPaand PPARy2 mediate the effect of GILZ. Therefore,To elucidate the effects of GILZ on adipocytes differentiation and its regulatory mechanism has an important signality for curing and prevention of obesity, type-2 diabetes mellitus.This thesis is divided into three parts to investigate the effect of GILZ on adipocytes differentiation and its regulatory mechanisms:1)Generally construct HA-GILZ/PcDNA3 recombinant mammalian expression vector and identify its expression and location in cell lines.3T3-L1 preadipocytes differentiation is induced with obutylmethylxanthine, insulin and dexamethasone,and set up the cell model of adipocytes differentiation. After 3T3-L1 cells were transfected with Lipofeactamine 2000 liposome and screened with G418, HA-GILZ stable and high expressed cell lines is cloned. It mainly aimed to provide gene and cell tools for studying the biology functions of GILZ.2)Compare the effect of GILZ on preadipocytes proliferation, special color of lipid in differentiation adipocytes and the relative mark gene PPARγ2, C/EBPα, LPL and FAS mRNA expression. Study the biology functions of GILZ and its regulatory mechanism at cell and mRNA expression.3)Primary analysis of the effect C/EBP and AP-1 cis-elements on GILZ inhibiting PPARy2 promotor transcript activities.Study the effect mechanisms of GILZ in molecular regulatory level of DNA transcription cis-element.Part 1:Construction of HA-GILZ mammalian expression vector and identified its expression and location in the cell lines, cloning GILZ stable expressed 3T3-L1 cell lines AIM:To provide the tools for studying the function and location of glucocorticoid induced leucine zipper (GILZ) in adipocytes differentiation, we constructed HA-GILZ expressive vector and observe the expression and location of HA-GILZ in cell lines, set up the differentiation cells and GILZ high stable expressed 3T3-L1 cell line models.METHODS:1.GILZ cDNA codon domain was amplified by RT-PCR from C3H10T1/2 cell line and recombined into PcDNA3 plasmid which was signed with HA-Tag. The recombinant vector HA-GILZ/PcDNA3 was identified by the assay of two restrictional enzymes BamH I/Xho I and sequencing.2.The recombinant vector HA-GILZ/PcDNA3 was instantaneously transfected into C3H10T1/2 cells by Lipofeactamine 2000 liposome and instantaneously transfected into 293T cells.3.The recombinant vector HA-GILZ/PcDNA3 was transfected into 3T3-L1 cells by Lipofeactamine 2000 liposome and the stable expressed cells were screened with G418.4.After 48h transfection, expression and location of GILZ in C3H10T1/2 and 3T3-L1 cells were detected with immunofluorescence,expression and molecular weight of GILZ in 293T cells and HA-GILZ expressed 3T3-L1 cells determinated with western bolt.5.3T3-L1 cell differentiation was induced with adipocyte induction medium (MID) containing isobutylmethylxanthine (0.5mM), insulin (10μg/ml) and dexamethasone (0.25μM). Lipid special staining and relative content of triglyceride were determined by Oil-O staining.RESULTS:1.Constructed recombinant vector HA-GILZ/PcDNA3 was confirmed successfully with the identifications of restrictional enzyme assay and sequencing analysis. Immunofluorescence further showed HA-GILZ fusion protein expressed in both cytoplasm and nuclear, however, it mainly expressed in the cytoplasm of cells. Western blot showed high-efficiently expressed in 3T3-L1 and 293T cell lines treansfected with recombinant vector HA-GILZ/PcDNA3 and has a special band of protein molecular weight about 20kD.2.After 3T3-L1 pre-adipocytes were induced 3 days with MID, some small lipid droplets were observed in cells and more and more cells appeared lipid droplets with the differentiation development. On the 9th days with MID, lots of round lipid droplets were seen in adipocytes cytoplasm, while cells grown in normal growth media had no lipid accumulation. Oil-Red O staining showed the lipid droplets in differentiation adipocyte is orange-red color. Compare with uninduced control, the relative content of triglyceride significantly increase (0.139±0.022 vs 0.356±0.014, t=14.521, p=0.000) in induced cells.3.Transfected 3T3-L1 cells were transfected with Lipofeactamine 2000 liposome and screened by G418, immunofluorescence and western blot showed GILZ is high expressed. Look like C3H10T1/2 cells, GILZ protein was also observed to mainly expressed in the cytoplasm of 3T3-L1 cells.CONCLUSIONS:1.HA-GILZ fusion gene vector was successfully constructed and the protein expressed in cytoplasm of C3H10T1/2 cells. It provides a stable gene tool for investigating the biologic function of GILZ.2.GILZ protein was observed to mainly express in the cytoplasm of C3H10T1/2 and 3T3-L1 cells.3.3T3-L1 pre-adipocytes differentiation was successfully induced and 3T3-L1 cell lines of stable expressed of GILZ was set up. It provides a stable cell tool for investigating the biologic function of GILZ.Part 2:The effect of GILZ on preadipocytes proliferation and adipocytes differentiation and relative gene expressions.AIM:To observe the effect of GILZ on 3T3-L1 pre-adipocytes proliferation, study the effects on mRNA expression of the relative marker genes PPARy2, C/EBPα,LPL and FAS of adipocytes differentiation and investigate the adipocyte differentiation mechanism of regulation of GILZ.METHODS:1. From day 1 to day 11, cell proliferation of 3T3-L1 cell clones of stable expression of GILZ was determinated every two days with MTT method.2.The effect of GILZ on adipocyte differentiation was analyzed with Oil-0 staining. It was divided into four groups:1, cells transfected with PcDNA3, un-induced with MID; 2, cells transfected with HA-GILZ/PcDNA3, un-induced with MID; 3, cells transfected with PcDNA3, induced with MID for 9 days; 4, cells transfected with HA-GILZ/PcDNA3, induced with MID for 9 days.3.Generally constructed GILZ-shRNA/pLentiLox 3.7 (short for GILZ-shRNA) recombinant Lentivirus vector, sequenced, transfected with Lipofeactamine 2000 liposome, coated with 293T cells, infected into 3T3-L1 cell clones of stable expression of GILZ, GILZ mRNA expression examined with Real-time PCR method.4.The mRNA expression of the relative marker genes PPARy2, C/EBPa, LPL and FAS of adipocytes differentiation were measured with Real-time PCR. For GILZ overexpression, it was divided into two groups:transfected PcDNA3 or HA-GILZ/PcDNA3; For GILZ silence, it was also divided into two groups:infected pLentiLox 3.7 and GILZ-shRNA/pLentiLox 3.7 reapectively.RESULTS:1. MTT method showed that, for 3T3-L1 control cells proliferation at the day lst,3rd,5th,7th,9th and day 11th, the ODs value were 0.090±0.008,0.154±0.004, 0.273±0.017,0.471±0.008,0.609±0.013,0.610±0.018 respectively; while for 3T3-L1 cells of stable expression of GILZ, the ODs value 0.083±0.002, 0.164±0.012,0.294±0.031,0.473±0.008,0.605±0.005,0.583±0.007respectively. Compared with control, GILZ overexpression can not obviously promote pre-adipocytes proliferation (F=2.460, P=0.068). That is to say, GILZ has no obviously effect on adipocytes proliferation.2.The sequencing result of GILZ-shRNA/pLentiLox 3.7 was consisting with design. After 3T3-L1 cells were infected with coated GILZ-shRNA for 48h, cells more than 95% were observed green under fluorescent microscopy. After 48h, Real-time PCR detected GILZ mRNA expression in 3T3-L1 cells of stable expression of GILZ. Compared with the control, GILZ mRNA expression significantly decreased in GILZ-shRNA Lentivirus silence group (0.191±0.027 vs 0.125±0.018, t=3.459, p=0.026) 3.After cell confluent for 48h,3T3-L1 cells were treated with MID for 48h, then changed into culture medium containing 10μg/ml insulin (the other day changed medium) for 9 days. Oil-Red O staining showed, compared with the control, orange-red cells in GILZ overexpression group greatly decreased. The Oil-Red O stained cells were dissolved with 100% isopropyl alcohol, and the relative content of triglyceride at the day 9th of adipocyte differentiation was measured by OD500. The relative content of triglyceride significantly also decreased (0.365±0.012 vs 0.181±0.014, F=80.023, P=0.000),while GILZ expression of SiRNA silence can decrease this inhibition. Compared with the control, the relative content of triglyceride significantly increased (0.184±0.027 vs 0.335±0.029, F=27.189, P=0.000).4.After induction with MID, Real-time PCR showed PPARγ2, C/EBPa,LPL and FAS mRNA expression significantly decreased (at cell differentiation 9 days, 11.447±0.831 vs 1.173±0.290,17.700±0.915 vs 1.557±0.384,67.057±5.288 vs 9.467±3.406,40.946±3.968 vs 4.967±1.091, P=0.000), while ShRNA silence can significantly increase these gene expression (1.056±0.275 vs 6.162±0.432, 1.500±0.644 vs 9.147±0.958,8.967±1.046 vs 45.127±2.585,4.518±1.143 vs 27.974±1.634, P=0.000).CONCLUSIONS:1.Our study indicated that GILZ has no effect on preadipocyte proliferation.2.Prepared GILZ-shRNA Lentivirus can successfully silence GILZ expression.3.GILZ overexpression or silence can significantly inhibits or increases PPARγ2, C/EBPa, LPL and FAS mRNA expression, which indicated that down-regulation of adipocytes differentiation transcript factors PPARy2 and C/EBPa inhibits adipocytes special gene LPL and FAS expression, so as to further inhibit adipocyte differentiation.4. GILZ can inhibit C/EBPa expression. Part 3:The effect of C/EBP and AP-1 cis-elements on GILZ inhibiting PPARy2 promotor transcript activities.AIM:To further study the regulatory mechanism of GILZ inhibiting adipocytes differentiation, the effect of C/EBP and AP-1 cis-elements on GILZ inhibiting PPARy2 promotor transcript activities were analyzed.METHODS:The effect of C/EBP and AP-1 cis-elements on GILZ inhibiting PPARy2 promotor transcript activities was quantified with luciferase dual reporter gene.1. After 3T3-L1 cells were transfected with 0.1μg-615/-265-Luc reporter gene (containing C/EBP cis-element of PPARy2 promotor region) or-265/-1-Luc reporter gene (containing AP-1 cis-element of PPARy2 promotor region), together with 0.01μg pRL-SV40 (reporter gene ascalibration), PcDNA3 (contorl) and HA-GILZ/PcDNA3 plasmid in different proportion for 48h, luciferase activities were determinated. Based on the different proportion of PcDNA3and HA-GILZ/PcDNA3 plasmid, it is divided into four groups:1, contorl,0.45 [μg PcDNA3; 2,0.30μg PcDNA3+0.15μg HA-GILZ/PcDNA3; 3,0.15μg PcDNA3+0.30μg HA-GILZ/PcDNA3; 4,0.45μgHA-GILZ/PcDNA3.2. After 3T3-L1 cells were transfected with 0.1μg-615/-1-Luc reporter gene (containing both of C/EBP and AP-1 cis-elements of PPARy2 promotor region),-615/-265-Luc reporter gene,-265/-1-Luc reporter gene respectively, together with 0.01μg pRL-SV40 (reporter gene ascalibration), PcDNA3 (contorl) and HA-GILZ/PcDNA3 plasmid in different proportion for 48h, luciferase activities were determinated. Based on the different reporter genes, it is divided into four groups:1, contorl,0.45μg PcDNA3+0.1μ-615/-1-Luc; 2,0.45μg HA-GILZ/PcDNA3+0.1μg-615/-265-Luc; 3,0.45μg HA-GILZ/PcDNA3+0.1μg-265/-1-Luc; 4,0.45μg HA-GILZ/PcDNA3+0. 1μg-615/-1-Luc.RESULTS: 1. Luciferase assay showed that GILZ can significantly decreased transcript activities of -615/-265-Luc reporter gene containing C/EBP cis-element (46.353±3.267 vs 22.68±1.59, F=44.394, P=0.000)and the inhibiting effect enhenced along with GILZ content increased.2. Luciferase assay showed that GILZ can significantly decreased transcript activities of-265/-1-Luc reporter gene containing AP-1 cis-element (46.476±1.859 vs 29.039±1.639, F=18.293, P=0.001) and the inhibiting effect enhenced along with GILZ content increased.3. Luciferase assay showed that the inhibitory effect of HA-GILZ on the transcript activitie of reporter gene containing C/EBP cis-element is significantly stronger than that of containing AP-1 cis-element (22.567±1.653 vs 29.121±0.934, F=211.567, P＜0.05). The transcript activitie of-615/-1-Luc reporter gene containing both C/EBP and AP-1 cis-elements is significantly lower than those of containing separate C/EBP or AP-1 cis-element (14.157±0.527, P＜0.05)CONCLUSIONS:1. Both C/EBP and AP-1 cis-elements GILZ play obvious effect on GILZ inhibiting PPARy2 promotor transcript activities.2. When C/EBP and AP-1 cis-elements involved in the inhibtory effects of GILZ on transcript activities of PPARy2 promotor, the inhibitory effect of C/EBP cis-element is higher than that of AP-1.3. When C/EBP and AP-1 cis-elements involved in the inhibtory effects of GILZ on transcript activitie of PPARy2 promotor, they can cooperate each other.SUMMARY:We summarized the three-part tests as following:1. Successfully constructed recombinant vector of HA-GILZ/PcDNA3 and set up 3T3-L1 cell differentiation model of stable expression of GILZ.2.Immunofluorescence showed GILZ protein expressed in both cytoplasm and nuclear of C3H10T1/2 and 3T3-L1 cells. However, it mainly expressed in the cytoplasm of cells.3.Our study indicated that GILZ has no effect on preadipocyte proliferation, but it can inhibit 3T3-L1 pre-adipocyte differentiation. We suggested that one of the probably inbiting mechanisms is down-regulation of adipocytes differentiation relative genes PPARy2, C/EBPa, LPL and FAS expression via C/EBP and AP-1 cis-element of PPARy2 promotor region involving in GILZ inhibiting PPARy2 transcription.