Study of gene expression in thyrotropin-stimulated thyroid cells by cDNA expression array: ID3 transcription modulating factor as an early response protein and tumor marker in thyroid carcinomas.

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific induction or repression of a large number of genes. To identify genes differentially regulated by the cAMP-dependent transduction pathway, which is poorly characterized so far, we used the cDNA expression array technology. Hybridizations of Atlas human cDNA expression arrays with (32)P-labeled cDNA probes derived from control or thyrotropin (TSH)-stimulated dog thyrocytes in primary culture generated expression profiles of hundreds of genes simultaneously. Among the genes that displayed modified expression, we selected the transcription factor ID3, whose expression was increased by a cAMP-dependent stimulus. ID3 overexpression after TSH stimulation was first verified by Northern blotting analysis, and its mRNA regulation was then investigated in response to a variety of agents acting on thyrocyte proliferation and/or differentiation. We show that: (1) ID3 mRNA induction was stronger after stimulation of the cAMP cascade, but was not restricted to this signaling pathway, as phorbol myristate ester (TPA) and insulin also stimulated mRNA accumulation; (2) in contrast, powerful mitogens for thyroid cells, epidermal growth factor and hepatocyte growth factor, did not significantly modify ID3 mRNA levels; (3) ID3 protein levels closely parallelled mRNA levels, as revealed by immunofluorescence experiments showing a nuclear signal regulated by TSH; (4) in papillary thyroid carcinomas, ID3 mRNA was downregulated. Our results suggest that ID3 expression might be more related to the differentiating process induced by TSH than to the proliferative action of this hormone.

Characterization of human 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase gene and its expression in mammalian cells

Three β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3β-HSD) catalyze the oxidative conversion of Δ5-3β-hydroxysteroids to the Δ4-3-keto configuration and is therefore essential for the biosynthesis of all classes of hormonal steroids, namely progesterone, glucocorticoids, mineralocorticoids, androgens, and estrogens. Using human 3β-HSD cDNA as probe, a human 3β-HSD gene was isolated from a λ-EMBL3 library of leucocyte genomic DNA. A fragment of 3β-HSD genomic DNA was also obtained by amplification of genomic DNA using the polymerase chain reaction. The 3β-HSD gene contains a 5′-untranslated exon of 53 base pairs (bp) and three successive translated exons of 232, 165, and 1218 bp, respectively, separated by introns of 129, 3883, and 2162 bp. The transcription start site is situated 267 nucleotides upstream from the ATG initiating codon. DNA sequence analysis of the 5′-flanking region reveals the existence of a putative TATA box (ATAAA) situated 28 nucleotides upstream from the transcription start site while a putative CAAT binding sequence is located 57 nucleotides upstream from the TATA box. Expression of a cDNA insert containing the coding region of 3β-HSD in nonsteroidogenic cells shows that the gene encodes a single 42-kDa protein containing both 3β-hydroxysteroid dehydrogenase and Δ5-Δ4-isomerase activities. Moreover, all natural steroid substrates tested are transformed with comparable efficiency by the enzyme. In addition to its importance for studies of the regulation of expression of 3β-HSD in gonadal as well as peripheral tissues, knowledge of the structure of the human 3β-HSD gene should permit investigation of the molecular defects responsible for 3β-HSD deficiency, the second most common cause of adrenal hyperplasia in children.

Expression of liver-specific member of the 3β-hydroxysteroid dehydrogenase family, an isoform possessing an almost exclusive 3-ketosteroid reductase activity

We have recently characterized two types of rat 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) isoenzymes expressed in adrenals and gonads. In addition, we have cloned a third type of cDNA encoding a predicted type III 3β-HSD protein specifically expressed in the male rat liver which shares 80% similarity with the two other isoenzymes. Transient expression in human HeLa cells of the cDNAs reveals that the type III 3β-HSD protein does not display oxidative activity for the classical substrates of 3β-HSD, in contrast to the type I 3β-HSD isoenzyme. However, in the presence of NADH, type III isoenzyme, in common with the type I isoform, converts 5α-androstane-3,17-dione (A-dione) and 5α-dihydrotestosterone (DHT) to the corresponding 3β-hydroxysteroids. In fact, the type I and the type III isoenzymes have the same affinity for DHT with K(m) values of 5.05 and 6.16 μM, respectively. When NADPH is used as cofactor, the affinity for DHT of the type III isoform becomes higher than that of the type I isoform with K(m) values of 0.12 and 1.18 μM, respectively. The type III isoform is thus a 3-ketoreductase using NADPH as preferred cofactor which is responsible for the conversion of 3-keto-saturated steroids such as DHT and A-dione into less active steroids.