Complete cDNA sequence of a cytochrome P-450 inducible by glucocorticoids in human liver.

Abstract

HLp is a human liver cytochrome P-450 that is immunochemically related to the glucocorticoid-inducible liver cytochrome P-450p in the rat and its homologue in the rabbit, P-450 LM3c. To investigate the structure and regulation of HLp, we used a monoclonal antibody that recognizes purified HLp to screen a human liver cDNA library in Agtil.We isolated and sequenced two overlapping cDNA clones that span the entire 2011 bases of an mRNA that codes for a protein of 504 amino acids. The predicted amino-terminal amino acid sequence of this protein is identical to the first 20 residues determined from purified HLp. HLp mRNA shares more than 70% sequence homology with related proteins from the rat and rabbit but less than 40% homology with other published cytochrome P-450 genes. Moreover, Southern blot analysis of human and rat genomic DNA revealed 50 and 60 kilobases of DNA, respectively, hybridizable to the HLp cDNAs. Blot analysis of human liver RNA from five patients revealed major (2.2 kilobase) and minor (3.0 kilobase) bands that hybridized to HLp cDNAs. The apparent concentration of these hybridizable mRNAs as well as the amounts of immunoreactive HLp protein in microsomes from the same liver were increased in a dose-dependent relationship in three patients who received dexamethasone, a potent glucocorticoid. Furthermore, in samples of RNA and of microsomes isolated from cultures of a human hepatoma cell line (Hep G2) incubated for 120 hr in medium containing dexamethasone, there was a 6-fold induction of the two mRNA species hybridizable to HLp cDNAs and a 3-fold induction ofimmunoreactive HLp protein as compared to the values for cultures incubated in steroid-free medium. We conclude that HLp is a human representative of a conserved glucocorticoid-inducible cytochrome P-450 gene family whose mechanism of induction involves accumulation of HLp mRNA.The liver cytochromes P-450 are a supergene family of microsomal hemoproteins that catalyze the oxidative biotransformation of numerous endogenous substrates, such as steroids, as well as xenobiotics, such as drugs. Extensive studies in many species of laboratory animals indicate that there are multiple polypeptide forms of these cytochromes that can be distinguished by differences in their structure, substrate specificity, or responses to various types of inducers (1-3). These hemoproteins appear to have been conserved in evolution. For example, in rats treated with pregnenolone 16a-carbonitrile, a "catatoxic" steroid that protects animals from stress and from the lethal (4) or carcinogenic (5) effects of many toxic substances, P-450p is the major form of liver cytochrome P-450 (6). We have found that there are forms of hepatic cytochrome P-450 in the rabbit (LM3c), the hamster, the gerbil, and the mouse that (i) cross-react with anti-P-450pantibodies; (ii) catalyze oxidation of the same model substtates; (iii) are inducible by glucocorticoids, phenobarbital, and macrolide antibiotics; (iv) are encoded by mRNA species inducible by a potent glucocorticoid, dexa...

Expression and Cyclic Variability of CYP3A4 and CYP3A7 Isoforms in Human Endometrium and Cervix During the Menstrual Cycle

“…The CYP3A family is known to be inducible, for example CYP3A mRNA increases in response to administration of synthetic and naturally occurring glucocorticoids (Scheutz et al, 1984;Molowa et al, 1986), macrolide antiobiotics , rifampin and rifabutin (Oesch et al, 1996), and environmental agents such as polychlorinated biphenyls and organochlorine pesticides (Scheutz et al, 1986). We observed an increase in CYP3A7 mRNA in the endometrium associated with the proliferative phase.…”

The increase in urinary excretion of 6 beta‐hydroxycortisol as a marker of human hepatic cytochrome P450IIIA induction.

“…The inducibility of P450IIIA3 and P450IIE1, in human, has only been recently demonstrated (Watkins et al, 1985;Molowa et al, 1986;Wrighton et al, 1986).…”

“…In human liver several forms of P450 have been characterized by protein purification and/or at the gene level by cloning the corresponding cDNA. They include P450IA1, P450IA2 (Jaiswal et al, 1985a,b;Kawajiri et al, 1986;Quattrochi et al, 1986;Distlerath et al, 1985), P450IIA3 (Philips et al, 1985), P450IIB6 (Miles et al, 1988), P450IIC8, IIC9, IIC10 (Okino et al, 1987;Ged et al, 1988;Shimada et al, 1986;Umbenhauer et al, 1987), P450IID6 (Distlerath et al, 1985;Gonzalez et al 1988), P4501IE1 (Wrighton et al, 1987;Song et al, 1986), P450IIIA3, IIIA4 (Watkins et al, 1985;Molowa et al, 1986;Guengerich et al, 1986;Beaune et al, 1986b).…”

Expression of human liver cytochrome P450 IIIA4 in yeast

“…Screening of a bacteriophage Agtll expression cDNA library using antibodies raised against P450 N F led to the isolation oftwo clones designated NF25 (Beaune et al, 1986) and NFlO (Bork et al, 1989). Two other cDNA clones coding for human liver P450 forms highly similar to P450 NF25 have been isolated: hPCNl (Gonzalez et al, 1988), encoding P450 hPCNl which differs from P450 NF25 by a single amino acid; and HLp (Molowa et al, 1986), encoding a protein of which the predicted sequence is 98% identical to that of P450 NF25 but contains an extra amino acid. hPCNl has been expressed in COS cells (Gonzalez et al, 1988) and HepG2 cells (Aoyama et al, 1989) and the resulting gene product was shown to oxidize several substrates including nifedipine.…”