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Cell Biology International (2005) 29, 177–179 (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


Abstract

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.


1 Introduction

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 5μg/ml insulin (I) with or without 390ng/ml dexamethasone (D) and/or 115μg/ml 3-isobutyl-1-methylxanthine (M).

2.2 Glycerol-3-phosphate dehydrogenase (GPDH) activity measurement

GPDH (EC 1.1.1.8) 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 75cm3 flasks and total RNA was extracted by the acid/guanidium thiocyanate/phenol chloroform method (Chomozynski and Sacchi, 1987). Real-time RT-PCR was used to measure the quantities of CGI-105 (accession no. AK 005134) mRNA relative to the quantity of 18S (accession no. S56974) mRNA. Measurement of the relative quantities of cDNA was conducted using a SYBER Green real-time RT-PCR Master Mix (Qiagen, USA), appropriate forward and reverse primers (0.5μM) (Table 1), and 0.2μg RNA. Assays were performed in the Rotor-Gene 2000 Real-Time Cycler using appropriate analysis software (Corbett Research, Sydney, Australia) and the thermal cycling parameters recommended by the manufacturers (40 cycles of 15s at 94°C and 30s at 55°C). Titration of CGI-105 and 18S (0.5μM) forward and reverse primers against increasing amounts of cDNA gave linear responses with slopes of −0.24.


Table 1.

Forward and reverse primers for real-time PCR for CGI-105 and 18S mRNAs

ItemPrimer
CGI-105
 Forward5′-AAGTTGACTGGGAGGTCGAGATGG-3′
 Reverse5′-ACTAGCAGAGGCGGGATCCAGAAC-3′
18S
 Forward5′-GATCCATTGGAGGGCAAGTCTGG-3′
 Reverse5′-TACCCACTGAGCCATCTCACCAGC-3′


2.4 Statistics

Values are means±SEM of at least three independent experiments. Differences between groups were analysed by Duncan's multiple range test using the SAS System (SAS Inst. Inc, USA).

3 Results

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<0.01), around 200-fold, as differentiation progresses (Fig. 1).


Fig. 1

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±SEM of three independent analyses. a,bMeans above the columns followed by different letters differ significantly (P<0.01).


Three hundred and ninety nanograms per milliliter of D significantly increased GPDH activity, while 115ng/ml M affected no significant increase. Simultaneous addition of D and M to the medium had almost the same effect as D alone (Fig. 2). When 3T3-L1 cells were induced to differentiate with 390ng/ml D, CGI-105 expression was up-regulated from 126 to 270AU (P<0.05). When M and D were added simultaneously, the up-regulation increased to 367AU. Addition of M alone increased CGI-105 expression from 126 to 153AU, but this was not statistically significant compared to the I treatment (Fig. 3).


Fig. 2

Glycerol-3-phosphate dehydrogenase (GPDH) activity of 3T3-L1 cells induced with various adrenocorticotropic hormones. The activities were measured in cultures induced with 5μg/ml insulin (I), I+390ng/ml dexamethasone (D), I+115μg/ml 3-isobutyl-1-methylxanthine (M) and I+D+M. The bar graph indicates the mean±SEM of three independent analyses. a,bMeans above the columns followed by different letters differ significantly (P<0.05).


Fig. 3

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 5μg/ml insulin (I), I+390ng/ml dexamethasone (D), I+115μg/ml 3-isobutyl-1-methylxanthine (M) and I+D+M. CGI-105/18S mRNA ratios were calculated. The bar graph indicates the mean±SEM of three independent analyses. a,bMeans above the columns followed by different letters differ significantly (P<0.05).



4 Discussion

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+M-induced cells did not show increased GPDH activity, while this activity almost doubled in I+D cells compared to the I cells (Fig. 2). D seems to be a more potent trigger of cell differentiation than M. The mode of CGI-105 gene expression was quite similar to that of GPDH when the cells were induced with various adrenocorticotropic hormones (Fig. 3). The CGI-105 gene might be involved with traditional adipogenic transcription factors such as members of the C/EBP and PPAR families (Cowherd et al., 1999) after such induction, because the gene is persistently up-regulated throughout the differentiation period (Figs. 1 and 3).

In conclusion, our findings reveal that the CGI-105 gene is characteristic of adipocytes and is up-regulated by adrenocorticotropic hormones during differentiation.

Acknowledgements

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.

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Received 19 April 2004/28 October 2004; accepted 11 November 2004

doi:10.1016/j.cellbi.2004.11.023


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)