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Cell Biology International (2005) 29, 177179 (Printed in Great Britain)
Differential expression of murine CGI-105 gene in 3T3-L1 cells by adrenocorticotropic hormones
Hyuck Choia, Myunggi Baikb and Kyuho Myunga*
aDepartment of Animal Science, Chonnam National University, Gwangju 500-757, Republic of Korea
bDepartment of Applied Bioscience and Biotechnology, Chonnam National University, Gwangju 500-757, Republic of Korea
The effects of adrenocorticotropic hormones on murine CGI-105 gene expression were investigated in 3T3-L1 cells. Expression was markedly increased in differentiated cells and it was up-regulated 2-fold in cells induced to differentiate with dexamethasone.
Keywords: CGI-105, Differentiation, 3T3-L1.
*Corresponding author. Tel.: +82 62 530 2122; fax: +82 62 530 0431.
The fumarylacetate hydrolase gene, CGI-105 in the comparative gene identification system, is responsible for the human genetic disorder heritable tyrosinemia, which is associated with fumarylacetate hydrolase deficiency and liver and kidney dysfunction (Klebig et al., 1992). It has recently been also found that this gene may be involved in intramuscular fat deposition in cattle skeletal muscle (Shin et al., 2001).
The murine 3T3-L1 fibroblast converts to an adipocyte-like cell under certain culture conditions and has often been used as a model for differentiation (Aratani and Kitagawa, 1988). These cells become more sensitive to adrenocorticotropic hormones and acquire an increased number of insulin receptors during differentiation (Green and Meuth, 1974). As 3T3-L1 cells are a reliable system for analysing adipocyte development, we used this system to study whether the CGI-105 gene is differently expressed at different growth stages and/or after treatment with adipose inducing or inhibiting substances.
2 Materials and methods
2.1 Cell culture and treatment
3T3-L1 fibroblasts were cultured and induced to differentiate as described by Chen et al. (1997). Briefly, the cells were placed in culture (d −6) and grown to confluence (d −2) before induction (d 0). Terminal differentiation occurred by d 10. In order to investigate the effects of adrenocorticotropic hormones on CGI-105 expression, the cells were induced to differentiate into adipocytes in the presence of DMEM-F12 (Gibco BRL, USA), 10% heat-inactivated fetal calf serum and 5
2.2 Glycerol-3-phosphate dehydrogenase (GPDH) activity measurement
GPDH (EC 188.8.131.52) was assayed spectrophotometrically by the disappearance of NADH during the reduction of dihydroxyacetone phosphate under zero-order conditions as modified by Wise and Green (1979).
2.3 RNA isolation and real-time reverse transcriptase-PCR (real-time RT-PCR)
Cells were cultured after the aforementioned treatments in 75
Forward and reverse primers for real-time PCR for CGI-105 and 18S mRNAs
Values are means
CGI-105 expressions at d −4, d −2, d 5 and d 10 were 11, 16, 210 and 230 arbitrary units (AUs), respectively. Clearly, expression of this gene increases very significantly (P
Expression of the CGI-105 gene in 3T3-L1 cells throughout the growth and differentiation periods, determined by the real-time reverse transcriptase-PCR (real-time RT-PCR) assay. CGI-105 and 18S mRNA levels were measured at d −4 (50% confluence), d −2 (100% confluence), d 5 (50% differentiation) and d 10 (100% differentiation). CGI-105/18S mRNA ratios were calculated. The bar graph indicates the mean
Three hundred and ninety nanograms per milliliter of D significantly increased GPDH activity, while 115
Glycerol-3-phosphate dehydrogenase (GPDH) activity of 3T3-L1 cells induced with various adrenocorticotropic hormones. The activities were measured in cultures induced with 5
Expression of the CGI-105 gene in 3T3-L1 cells induced with various adrenocorticotropic hormones, examined by the real-time reverse transcriptase-PCR (real-time RT-PCR) assay. CGI-105 and 18S mRNA levels were measured from cultures induced with 5
This study has shown that the CGI-105 gene is characteristic of the transformation of mouse 3T3-L1 cells into adipocytes: its rate of expression increases 200 times during preadipocyte differentiation. This may also be true in other species, but only Shin et al. (2001) have provided suggestive evidence, showing the involvement of the gene in fat deposition in cattle.
When 3T3 cells grow to confluence they convert to adipose cells. In the course of this conversion the activities of many lipogenic enzymes increase, including GPDH, which must play an important role in the conversion process (Wise and Green, 1979). The cells become more sensitive to adrenocorticotropic hormones and acquire more insulin receptors (Rubin et al., 1977). These hormones double the total adenylate cyclase activity; adipocyte induction appears to be mediated by increased intracellular cyclic AMP levels (Russell and Ho, 1976). However, the I
In conclusion, our findings reveal that the CGI-105 gene is characteristic of adipocytes and is up-regulated by adrenocorticotropic hormones during differentiation.
The authors thank the Basic Research Program of the Korea Science & Engineering Foundation for financial support by grant No. R01-2002-00013-0 in 2002.
Aratani Y, Kitagawa, Y. Enhanced synthesis and secretion of type IV collagen and entactin during adipose conversion of 3T3-L1 cells and production of unorthodox laminin complex. J Biol Chem 1988:263:16163-9
Chen C, Brodle, AE, Hu, CY. CCAAT/enhancer-binding protein β cannot overcome TCDD inhibition of 3T3-L1 preadipocyte differentiation. Obesity Res 1997:5:146-52
Klebig ML, Russell, LB, Rinchik, EM. Murine fumarylacetoacetate hydrolase (Fah) gene is disrupted by a neonatally lethal albino deletion that defines the hepatocyte-specific developmental regulation 1 (hsdr-1) locus. Proc Natl Acad Sci U S A 1992:89:1363-7
Russell TR, Ho, R. Conversion of 3T3 fibroblasts into adipose cells: triggering of differentiation by prostaglandin F2a and 1-methyl-3-isobutyl xanthine. Proc Natl Acad Sci U S A 1976:73:4516-20
Received 19 April 2004/28 October 2004; accepted 11 November 2004doi:10.1016/j.cellbi.2004.11.023