TY - JOUR
T1 - CCAAT/enhancer-binding protein-α-dependent transactivation of CYP2C12 in rat hepatocytes
AU - Tollet, Petra
AU - Lahuna, Olivier
AU - Ahlgren, Rannhild
AU - Mode, Agneta
AU - Gustafsson, Jan Åke
PY - 1995/12
Y1 - 1995/12
N2 - Expression of the rat CYP2C12 gene is liver specific and is induced by GH at the transcriptional level. In primary cultures of rat hepatocytes, GH inducibility of CYP2C12 and the presence of C/EBP α protein were demonstrated to be equally dependent on attachment of the cells to an extracellular matrix gel (Matrigel). Transient transfection of a C/EBP α expression vector into hepatocytes, cultured without Matrigel, increased the cellular P4502C12 messenger RNA levels 10-fold. Cotransfection studies using deletion constructs of the CYP2C12 promoter fused to the luciferase reporter gene localized the C/EBP α response to the region -250 to -180. Sequence comparisons and deoxyribonuclease I footprinting using rat liver nuclear extracts indicated two potential C/EBP binding sites in this region. Mutagenesis of the most upstream element (-229 to -207) abolished transactivation by C/EBP α. Using gel mobility supershift assays, this element was demonstrated to bind C/EBP α and C/EBP β in liver nuclear extracts and in lysates from hepatocytes cultured on Matrigel. GH treatment of the cells did not alter the C/EBP protein levels or the C/EBP-binding activity to this element. Neither did GH increase the expression of CYP2C12 reporter gene constructs regardless of the presence of different amounts of cotransfected C/EBP α. We conclude that C/EBP α is a potent transactivator of the CYP2C12 gene and most likely contributes to its liver-specific expression. Although the results presented here do not exclude the possibility of a GH-enhanced transactivating ability of C/EBP α, the mechanism of GH-induced levels of P4502C12 is not through increased levels of C/EBP α or via enhanced DNA-binding activity of this transcription factor.
AB - Expression of the rat CYP2C12 gene is liver specific and is induced by GH at the transcriptional level. In primary cultures of rat hepatocytes, GH inducibility of CYP2C12 and the presence of C/EBP α protein were demonstrated to be equally dependent on attachment of the cells to an extracellular matrix gel (Matrigel). Transient transfection of a C/EBP α expression vector into hepatocytes, cultured without Matrigel, increased the cellular P4502C12 messenger RNA levels 10-fold. Cotransfection studies using deletion constructs of the CYP2C12 promoter fused to the luciferase reporter gene localized the C/EBP α response to the region -250 to -180. Sequence comparisons and deoxyribonuclease I footprinting using rat liver nuclear extracts indicated two potential C/EBP binding sites in this region. Mutagenesis of the most upstream element (-229 to -207) abolished transactivation by C/EBP α. Using gel mobility supershift assays, this element was demonstrated to bind C/EBP α and C/EBP β in liver nuclear extracts and in lysates from hepatocytes cultured on Matrigel. GH treatment of the cells did not alter the C/EBP protein levels or the C/EBP-binding activity to this element. Neither did GH increase the expression of CYP2C12 reporter gene constructs regardless of the presence of different amounts of cotransfected C/EBP α. We conclude that C/EBP α is a potent transactivator of the CYP2C12 gene and most likely contributes to its liver-specific expression. Although the results presented here do not exclude the possibility of a GH-enhanced transactivating ability of C/EBP α, the mechanism of GH-induced levels of P4502C12 is not through increased levels of C/EBP α or via enhanced DNA-binding activity of this transcription factor.
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U2 - 10.1210/mend.9.12.8614413
DO - 10.1210/mend.9.12.8614413
M3 - Article
C2 - 8614413
AN - SCOPUS:0028971180
SN - 0888-8809
VL - 9
SP - 1771
EP - 1781
JO - Molecular Endocrinology
JF - Molecular Endocrinology
IS - 12
ER -