Characterization of rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase cDNAs and differential tissue-specific expression of the corresponding mRNAs in steroidogenic and peripheral tissues.
The conversion of 3 beta-hydroxy-5-ene steroids by the enzyme complex 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) is an essential step in the biosynthesis of all classes of hormonal steroids. We report the characterization of two types of cDNA clones encoding rat 3 beta-HSD isolated from a rat ovary lambda gt11 cDNA library with a human 3 beta-HSD cDNA probe. Both type I and type II cDNAs encode proteins of 372 amino acids having 94% homology. Transient expression of the type I and the type II 3 beta-HSD cDNAs in HeLa human cervical carcinoma cells reveals that both proteins possess 3 beta-hydroxysteroid dehydrogenase as well as delta 5-delta 4 isomerase activities for both delta 5-pregnene and delta 5-androstene precursors, although the type I 3 beta-HSD protein is more active than the type II. RNA blot analysis using type I 3 beta-HSD cDNA identifies major mRNA transcripts of 1.7 kilobase in rat ovary, testis, and adrenal poly(A)+ RNA. RNase protection assay using type I- and type II-specific cRNA probes revealed the existence of the two corresponding mRNAs in male and female rat adrenals and gonads as well as in female adipose tissue while only type I mRNA is present in male and female kidney. Moreover, in situ hybridization performed using type-specific labeled 24-mer oligonucleotides confirms that type I is the major mRNA species in the ovary and further indicates that both mRNA species have a similar cellular distribution in the ovarian tissue with the highest level of expression found in corpora lutea. Immunoblot analysis using polyclonal antibodies raised against purified human placental 3 beta-HSD identified a single 42-kDa band in rat ovary, testis, and adrenal, which agrees with the calculated molecular masses of 41,911 and 42,150 daltons for the type I and II proteins, respectively. Determination of 3 beta-HSD enzymatic activity using [14C]pregnenolone and [14C]dehydroepiandrosterone as substrates shows that 3 beta-HSD activity is present not only in the gonads and adrenals of animals of both sexes, but also in many peripheral tissues including adipose tissue, mammary gland, kidney, liver, prostate, seminal vesicle, uterus, skin, brain, heart, thymus, pancreas, lung, and spleen. The present data indicate the existence of two mRNAs encoding rat 3 beta-HSD and their differential tissular distribution in both steroidogenic and peripheral tissues.(ABSTRACT TRUNCATED AT 400 WORDS)
Zhao
H F
HF
Medical Research Council Group in Molecular Endocrinology, CHUL Research Centre, Quebec, Canada.
Labrie
C
C
Simard
J
J
de Launoit
Y
Y
Trudel
C
C
Martel
C
C
Rhéaume
E
E
Dupont
E
E
Luu-The
V
V
Pelletier
G
G
D000273:Adipose Tissue / Q000201:enzymology; D000311:Adrenal Glands / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D005260:Female; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D007668:Kidney / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D015345:Oligonucleotide Probes; D009928:Organ Specificity; D010053:Ovary / Q000201:enzymology; D011375:Progesterone Reductase / Q000235:genetics*; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains; D015183:Restriction Mapping; D013251:Steroid Isomerases / Q000235:genetics*; D013737:Testis / Q000201:enzymology; D014162:Transfection
C021425:3 beta-hydroxysteroid oxidoreductase-delta(5) 3-ketosteroid isomerase; D007527:Isoenzymes; D009097:Multienzyme Complexes; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D004247:DNA; D011375:Progesterone Reductase; D013251:Steroid Isomerases