Changes in mRNA expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase in chickens

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and cholesterol 7 alpha-hydroxylase (CYP7A1), essential enzymes of cholesterol synthesis and excretion, respectively, were isolated from a chicken liver cDNA library. When their recombinant proteins were overexpressed in HNK293 cells, corresponding enzyme activities were observed. The complete open reading frames of MHGR and CYP7A1 contained (i) 2625 base pairs (bp), predicting a protein of 875 amino acids, and (ii) 1539 bp, predicting a protein of 513 amino acids, respectively. By Northern blot analysis, chicken HMGR mRNA expression was detected in most tissues examined, however, the highest levels were found in liver, brain and ileum. CYP7A1 mRNA was detected only in the liver. Changes in chicken HMGR and CYP7A1 mRNA expression with nutritional state were examined and were shown to respond to certain nutritional treatments, i.e. fast refeeding and cholesterol supplementation. HMGR and CYP7A1 mRNA levels were significantly increased with maturation (i.e. egg producing), when compared to immature chickens. However, these stimulations were not associated with estrogen, although this does enhance triacylglycerol and very low density lipoprotein secretion by the chicken liver. The present study is the first to report the molecular characterization of HMGR and CYP7A1, key enzymes of cholesterol metabolism in avian species.     

Cloning and expression analysis of a new member of the cytochrome P450, CYP2A15 from the Chinese hamster, encoding testosterone 7alpha-hydroxylase.

We cloned a new cytochrome P450 cDNA encoding testosterone 7alpha-hydroxylase in the Chinese hamster, designated CYP2A15 which shares significant amino acid sequence homology with members of the CYP2A subfamily. The CYP2A15 cDNA was isolated by screening a liver cDNA library and the sequence contains an open reading frame of 1482 nucleotides encoding a polypeptide of 493 amino acids with a calculated molecular mass of 56,295 Da. This is flanked by a 5'-untranslated region of 2 bp and a 3' untranslated region of 191 bp including the poly(A) tail. We determined the catalytic activity of CYP2A15 using microsomes obtained by transient expression of its cDNA in transfected COS-7 cells. The heterologously expressed CYP2A15 was found to hydroxylate testosterone at position 7alpha in a reconstituted system. RT-PCR experiments revealed that the mRNA of CYP2A15 was expressed in liver, but not detected in kidney, lung, or small intestine. The expression of CYP2A15 mRNA was slightly induced by treatment with either rifampicin or 3-methylcholanthrene.     

Cloning and functional expression of a first inducible avian cytochrome P450 of the CYP3A subfamily (CYP3A37)

CYP3As represent a family of cytochromes P450 involved in the metabolism of both endogenous and exogenous natural and synthetic compounds. Well described in mammals, none have yet been cloned and characterized in avian species. In this paper, we report the cloning and analysis of an avian CYP3A (CYP3A37). Using an RNA differential display approach, an 80-bp phenobarbital-inducible cDNA fragment was amplified from chicken embryo liver. Based on its homology with mammalian CYP3As, this fragment was used to clone a full-length cDNA consisting of 1638 bp encoding a putative protein of 509 amino acids. The sequence shares between 57.4 and 62% identity at the amino acid level with CYP3As of other species. This cDNA was designated CYP3A37 according to the current cytochrome P450 nomenclature. When expressed in COS1 cells, the CYP3A37 cDNA produced a protein of congruent with55 kDa, which was recognized by polyclonal anti-rat CYP3A1 antiserum. In a bacterial expression system, the CYP3A37 cDNA produced a protein capable of steroid 6beta-hydroxylation. At a substrate concentration of 100 microM, progesterone, testosterone, and androstenedione were found to be 6beta-hydroxylated at a rate of 15.4, 11.7, 12.2 nmol/min/nmol P450, respectively. Used as control, the human CYP3A4 gave similar hydroxylation rates. Finally, in both chicken embryo liver and chicken hepatoma cells (LMH), CYP3A37 mRNA was increased after treatment with typical CYP3A inducers, such as metyrapone, phenobarbital, dexamethasone, and pregnenolone 16alpha-carbonitrile, but not rifampicin. CYP2H1, a well-characterized inducible chicken cytochrome P450, also was induced by the same compounds, suggesting similar regulation of CYP3 and CYP2 genes in this species.     

Cloning and tissue distribution of a novel human cytochrome p450 of the CYP3A subfamily, CYP3A43

On the basis of the detection of an expressed sequence tag ('EST') similar to the human cytochrome P450 3A4 cDNA, we have identified a novel member of the human cytochrome P450 3A subfamily. The coding region is 1512-bp long and shares 84, 83, and 82% sequence identity on the cDNA level with CYP3A4, 3A5, and 3A7, respectively, with a corresponding amino acid identity of 76, 76, and 71%. Quantitative real time based mRNA analysis revealed CYP3A43 expression levels at about 0.1% of CYP3A4 and 2% of CYP3A5 in the liver, with significant expression in 70% of the livers examined. Gene specific PCR of cDNA from extrahepatic tissues showed, with the exception of the testis, only low levels of CYP3A43 expression. The CYP3A43 cDNA was heterologously expressed in yeast, COS-1 cells, mouse hepatic H2.35 cells and in human embryonic kidney (HEK) 293 cells, but in contrast to CYP3A4 which was formed in all cell types, no detectable CYP3A43 protein was produced. This indicates a nonfunctional protein or specific conditions required for proper folding. It is concluded that CYP3A43 mRNA is expressed mainly in liver and testis and that the protein would not contribute significantly to human drug metabolism.