Extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by a benign testicular leydig cell tumor

We present an unusual case with bilateral testicular Leydig cell tumors displaying extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase. Histological examination of a 38-yr-old man infertile due to azoospermia showed him to have bilateral testicular Leydig cell tumors. The in vitro steroidogenic potential of the tumors and their adjacent testicular tissue was evaluated using organ culture. Tumor tissue was found to secrete deoxycorticosterone (DOC), corticosterone (B) and cortisol, which are not produced in normal adult testis, into the medium, while testicular tissue adjacent to the tumors secreted a small amount of DOC and B. Northern blot analysis with cytochrome P-450_C21 complementary DNA (cDNA) and P-450_11β cDNA as probes revealed that the tumor contained a considerable amount of mRNA for P-450_C21 and P-450_11β, while the mRNAs were not detected in the testicular tissues adjacent to the tumors. It is suggested that the high local levels of estrogen and/or progesterone within the Leydig cell tumors and their adjacent testicular tissues induced extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by the tumors and their adjacent testicular tissues.     

Identification and characterization of two upstream elements that regulate adrenocortical expression of steroid 11 beta-hydroxylase

We have studied the mechanisms that regulate the expression of the mouse gene encoding steroid 11 beta-hydroxylase (11 beta-OHase), a steroidogenic cytochrome P450 enzyme that is expressed only in the adrenal cortex. DNase I footprinting and gel-mobility shift analyses revealed potential regulatory elements at -370 and -310 in the 11 beta-OHase promoter region. To determine the contributions of these elements to expression, we altered their sequences by site-selected mutagenesis and studied promoter activity after transfection into Y1 mouse adrenocortical tumor cells. Mutation of either element markedly decreased basal promoter activity but did not affect the response to treatment with 8-bromo cAMP. These experiments thus document the functional roles of these elements, within the context of the intact promoter, in constitutive expression of 11 beta-OHase. Moreover, addition of either of these elements to p-40GH, a 5'-deletion plasmid containing 11 beta-OHase sequences from -40 to +8 upstream of a growth hormone reporter gene, significantly increased promoter activity but did not confer cAMP responsiveness. Finally, increased expression was seen after transfection of Y1 derivatives deficient in cAMP-dependent protein kinase, indicating that neither element required cAMP-dependent protein kinase activity. These studies thus define two regulatory elements that play important roles in 11 beta-OHase expression.     

The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA

The nucleotide sequence of a cloned 5.3 kilobase cDNA for the human low density lipoprotein receptor revealed five domains in the 839 amino acid protein: 322 NH2-terminal amino acids, extremely rich in disulfide-bonded cysteine residues (15%) and including an 8-fold repeat of 40 residues that may contain the LDL binding site; 350 residues homologous to the precursor of mouse epidermal growth factor; a region immediately outside the plasma membrane, rich in serine and threonine and the site of O-linked glycosylation; 22 hydrophobic amino acids, spanning the plasma membrane; and 50 COOH-terminal amino acids, projecting into the cytoplasm. The mRNA for the receptor contains a 3' untranslated region of 2.5 kilobases that includes multiple copies of the Alu family of repetitive DNAs. Transfection of simian COS cells with the human LDL receptor cDNA linked to the SV40 early promoter resulted in expression of functional cell surface receptors.     

Decreased levels of steroid 21-hydroxylase [P450(c21)] and its mRNA in an adrenocortical adenoma associated with 21-hydroxylase deficiency

Adrenocortical adenoma incidentally found in a 37-yr-old female patient, with simple virilizing form of 21-hydroxylase deficiency, was studied. Cultured adenoma cells revealed excessive secretion of 17 alpha-hydroxyprogesterone in response to 10(-8) M ACTH, compared with those of 11-deoxycortisol and cortisol, which indicated impaired activity of the 21-hydroxylase. To elucidate the molecular mechanisms of this defective 21-hydroxylase in the adenoma, we analyzed the gene encoding specific cytochrome P450 (P450c21) for steroid 21-hydroxylation and its expression. DNA and RNA were extracted from the adrenal adenoma and were hybridized with a probe of human P450c21 gene, by Southern and Northern blot analysis. In Southern blot analysis with Taq I, Bgl II or Bam HI, there was no difference between the pattern of restriction fragments in DNA from the adenoma and normal peripheral leucocytes. Northern blot analysis of the adenoma showed the same size of P450c21 mRNA as in the normal adrenal gland, but the amount was low--about a half that of the normal adrenal. In Western blot analysis with polyclonal antibody to P450c21, only a small amount of P450c21 protein was detected in the adenoma, although it was found to be of the same molecular weight as that in the normal adrenal gland. In view of these findings it is conceivable as one of possibilities that a mild and small mutation in the structural or promotor region of the P450c21 gene may cause the decreased 21-hydroxylase activity in this adenoma.     

Molecular and endocrine characterization of a mutation involving a recombination between the steroid 21-hydroxylase functional gene and pseudogene

The gene encoding steroid 21-hydroxylase activity, P450c21B, is located in the major histocompatibility complex (MHC) class III region, in close proximity to a highly homologous pseudogene, P450c21A. Recombinations between P450c21B and P450c21A have been shown to result in deficiency of 21-hydroxylase activity, the usual cause of congenital adrenal hyperplasia (CAH). A mutant P450c21 gene from a patient with simple virilizing CAH was identified and shown to be consistent with a recombination between P450c21A and P450c21B. Sequence analysis of the mutant gene showed the recombination site to be located between the first exon and the second intron. The mutant gene encodes a leucine instead of the normal proline at codon 31. This mutation resides on a chromosome bearing the HLA-B44 serotype. A comparison of mutation associated with HLA-B44 and that normally found with the HLA-Bw47 serotype suggests that the HLA-B44 mutations are of more ancient origin. The patient's homologous chromosome has a deletion of P450c21B. Endocrinological testing therefore allows for testing of the mutant gene in genetic isolation. Such testing demonstrated that the patient was capable of producing aldosterone and retaining sodium in response to a low-sodium diet, indicating that the mutant gene encodes an enzyme with partial 21-hydroxylase activity.