TRANS-ACTING FACTORS MEDIATING THE DIFFERENTIATION AND HORMONAL CONTROL OF THE SCD1 AND 422(aP2) GENES IN 3T3-L1 CELLS
Creators: name: Geiman, Deborah E.
Publisher: name: Maryland Shared Open Access Repository
Year: 1994

description: The 3T3-L1 cell culture system is a model used to study the regulation of adipocyte differentiation. 3T3-L1 cells grow initially as fibroblastic cells until they reach confluence. After the addition of three agents, isobutylmethylxanthine (MIX), dexamethasone (DEX) and insulin, the cells begin to differentiate into adipocytes. During the differentiation of 3T3-L1 preadipocytes into adipocytes, there is an increase in the expression of many genes characteristic of adipose tissue. The hormonal and differential regulation of two of these genes, 422(aP2) and SCD1, was examined in this work. Both genes contain a consensus C/EBP binding site and both have been shown to be transactivated by C/EBP­­­α. C/EBP­­­α is a transcription factor shown by Northern analysis to follow the expression of other adipocyte-specific genes, such as 422(aP2), during the differentiation of 3T3-L1 cells. The expression of two other members of the C/EBP family of transcription factors, C/EBP­­­β and C/EBP­­­δ, increase early during the differentiation program. Their expression increases rapidly upon initiation of the differentiation of 3T3-L1 cells and then decreases as C/EBP­­­α expression increases and predominates through the differentiated state. The sequential expression of the C/EBP isoforms may be important in the coordinate expression of adipocyte-specific genes such as 422(aP2) and SCD1. To sort out the mechanism of transcriptional activation of adipocyte-specific genes, the effect of the agents used to initiate the differentiation 3T3-L1 cells was looked at separately. 8-Br-cAMP, which may be used in place of MIX, was found to be the most effective component in promoting differentiation. The addition of cAMP to 3T3-L1 preadipocytes induced an increase in the mRNA and protein level of 422(aP2) between 8-16 hours after addition. Previous work had found the SCD1 mRNA to increase within 6 hours after addition of cAMP, however, this study found the protein level of SCD1 to be unchanged after cAMP treatment. DNase I footprinting analysis using nuclear extracts from preadipocytes treated with 8-Br-cAMP showed enhanced protection at the AP-1 and C/EBP sites of 422(aP2), but did not affect the binding at the SCD1/BP and C/EBP site of the SCD1 gene promoter. In the case of 422(aP2) this would indicate an increase in the binding of the C/EBP family of transcription factors to the C/EBP site, as well as Fos and Jun to the AP-1 site. Electrophoresis mobility shift analysis revealed there was enhanced binding at the C/EBP site of both 422(aP2) and SCD1 after the addition of cAMP. The predominate factors binding were C/EBP­­­β and C/EBP­­­δ with very little C/EBP­­­α involved. This is not unexpected since C/EBP­­­β and C/EBP­­­δ are known to respond to agents such as cAMP. To determine the importance of the C/EBP and AP-1 sites in the 422(aP2) and the SCD1/BP and C/EBP sites in the SCD1 gene promoters, base mutations were made in those sites by site-directed mutagenesis. DNase I footprint analysis revealed that the mutations in the C/EBP site of both genes eliminated the binding of nuclear factors to that site. The mutations were also cloned into a CAT expression vector to determine the effect on the regulation of the 422(aP2) and SCD1 genes. When cotransfected into preadipocytes with an expression vector for C/EBP­­­α, all of the SCD1 mutant CAT chimeras, with the exception of one made in the C/EBP site, eliminated transactivation by C/EBPoc. In addition the same mutations lowered the CAT activity of the SCD1 gene when transiently transfected into adipocytes. All the mutations in the C/EBP and AP-1 sites of 422(aP2) eliminated transactivation by C/EBP­­­α in preadipocytes and substantially lowered the expression of 422(aP2) when transfected into 3T3-L1 adipocytes. These results would indicate the C/EBP sites in both genes, the AP-1 site in the 422(aP2) gene and the SCD1/BP site in SCD1 are important for the regulation of these genes during differentiation. However, while the DNA binding sites studied in this work are important, other elements further upstream in the promoters of these genes may also be involved in their regulation during adipocyte differentiation.