Differential regulation of human CYP4A genes by peroxisome proliferators and dexamethasone

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Abstract

HepG2 cells that stably overexpress PPARα were used to examine the regulation of the two known human CYP4A genes by Wy14643. Specific PCR amplification across intron 5 and restriction endonuclease analysis indicated that HepG2 cells possess genes corresponding to both the CYP4A11 cDNA and a more recently characterized gene, CYP4A22, that exhibits 95% identity to CYP4A11 in the coding region. These are unlikely to represent alleles because both genes were present in DNA samples from 100 of 100 individuals. Quantitative real-time PCR determined that CYP4A22 mRNA is expressed at significantly lower levels than CYP4A11 mRNA in human liver samples. The PPARα agonist Wy14643 induced CYP4A11 mRNA in confluent cultures of HepG2 cells stably expressing the murine PPARα-E282G mutant. This mutant exhibits a significantly decreased ligand-independent trans-activation and can be activated by Wy14643 to a level similar to that of wild-type PPARα. Dexamethasone induced CYP4A11 mRNA in both control and PPARα- E282G-expressing HepG2 cells, indicating that the induction of CYP4A11 by dexamethasone is independent of elevated PPARα expression. Wy14643 or dexamethasone induction of CYP4A22 mRNA was not evident in either control or PPARα -E282G-expressing HepG2 cells. The results indicate that CYP4A11 expression can be induced by glucocorticoids and peroxisome proliferators.

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Materials and methods

Cell culture and treatments. The generation of the HepG2 cell lines expressing the murine PPARα-E282G mutant or human PPARα (ha1 and ha2) as well as vector-transfected controls has been described [21]. Cells were seeded in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (Summit, Ft. Collins, CO) and 500 μg/ml of Geneticin (G418) at a density that would reach 60% confluence after overnight culture. Cells were either untreated, treated with

Results

Kawashima et al. [24] characterized a human gene, currently annotated as CYP4A11 in the human genome project, that exhibits a coding sequence that is distinct but closely related to the reported human CYP4A11 cDNAs [22], [23]. The two sequences exhibit 95% identity leading the P450 Nomenclature Committee to confer a separate entry, CYP4A22, based on the low probability that the observed sequence differences would be seen in allelic genes. In order to test this hypothesis and to ascertain

Discussion

This study examines the potential for regulation of human CYP4A genes by PPARα and peroxisome proliferators. The increased expression of CYP4A genes in response to dietary fatty acids and peroxisome proliferators is regulated by PPARα in some species such as rabbits [13], rats [14], or mice [15], [16], but is not seen in guinea pigs [29], or nonhuman primates [30]. These species differences could reflect pharmacologic differences in receptor activation, differences in the expression of PPARα in

Acknowledgements

We thank Keith Griffin for critical reading of the manuscript. This work was supported by the United States Public Health Service Grant HD 04445.

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