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Mouse vas deferens protein (MVDP) is an aldose reductase-like protein that is highly expressed in the vas deferens and adrenal glands and whose physiological functions were unknown. We hereby describe the enzymatic characteristics of MVDP and its role in murine adrenocortical Y1 cells. The murine aldose reductase (AR) and MVDP cDNAs were expressed in bacteria to obtain recombinant proteins and to compare their enzymatic activities. Recombinant MVDP was functional and displayed kinetic properties distinct from those of murine AR toward various substrates, a preference for NADH, and insensitivity to AR inhibitors. For MVDP, isocaproaldehyde, a product of side-chain cleavage of cholesterol generated during steroidogenesis, is the best natural substrate identified so far. In Y1 cells, we found that NADH-linked isocaproaldehyde reductase (ICR) activity was much higher than NADPH-linked ICR activity and was not abolished by AR inhibitors. We demonstrate that in Y1 cells, forskolin-induced MVDP expression enhanced NADH-linked ICR activity by 5-6-fold, whereas no variation in ICR-linked NADPH activity was observed in the same experiment. In cells stably transfected with MVDP antisense cDNA, NADH-linked ICR activity was abolished even in the presence of forskolin, and the isocaproaldehyde toxicity was increased compared with that of intact Y1 cells, as measured by isocaproaldehyde LD 50 . In Y1 cells transfected with MVDP antisense cDNA, forskolin-induced toxicity was abolished by aminoglutethimide. These results indicate that in adrenocortical cells, MVDP is responsible for detoxifying isocaproaldehyde generated by steroidogenesis.

The human aldose reductase, AKR1B1, participates in glucose metabolism and osmoregulation and is supposed to play a protective role against toxic aldehydes derived from lipid peroxidation and steroidogenesis that could affect cell growth/differentiation when accumulated. Adrenal gland is a major site of expression of AKR1B1, and we asked whether changes in its expression could be associated with adrenal disorders. Therefore, we examined AKR1B1 gene expression in human fetal adrenals, adrenocortical cell line, and tumors and compared the results with the expression of steroidogenic genes (StAR and CYP11A) and regulators of adrenal cortex development [steroidogenic factor-1 (SF-1) and dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1)]. Using specific antibodies, Northern blotting, and enzymatic assays, we present evidences that AKR1B1 detectable in 15-wk-old fetal glands is regulated by cAMP in NCI-H295 cells and thus that AKR1B1 is functionally related to the ACTH-responsive murine akr1b7/mvdp gene rather than to its direct ortholog, the mouse aldose reductase akr1b3 gene. Although low DAX1 expression in aldosterone-producing adenomas (n = 5) was confirmed (P < 0.05), no correlation was found between the expression of all other genes and the tumors endocrine activity. In contrast, relative abundance of AKR1B1 mRNA was decreased in adrenocortical carcinomas (n = 5; mean +/- sem, 0.95 +/- 0.2) when compared with adenomas (n = 12; 9.29 +/- 3.05; P < 0.001). Most (seven of eight) adrenocortical carcinomas (19.0 +/- 5.4) had very low relative AKR1B1 protein levels when compared with benign tumors (cortisol-producing adenomas, n = 5, 63.0 +/- 9.8; nonfunctional adenomas, n = 5, 58.0 +/- 10.4; aldosterone-producing adenomas, n = 4, 65.3 +/- 7.7; P < 0.001), Cushing's hyperplasia (n = 5, 54.6 +/- 5.3; P < 0.01), or normal adrenals (n = 4; 37.1 +/- 5.3; P < 0.001). These properties provide the first evidence that expression of cAMP-regulated AKR1B1 is decreased in adrenocortical cancer. This might take part in adrenal tumorigenesis and could be investigated as a marker of malignancy for the diagnosis of adrenal tumors.

No abstract

We established the first adrenocortical tumor cell lines with complete zona fasciculata (ZF) cell phenotype from tumors induced in transgenic mice by large T-antigen of simian virus 40 under the control of the aldose reductase-like akr1b7 gene promoter. Adrenocortical tumor cell lines produced high amounts of corticosterone and were responsive to ACTH. All genes that are supportive for glucocorticoid synthesis including cyp21a1 and cyp11b1 were expressed, and most of them were transiently up-regulated by ACTH at transcriptional level: stimulation culminated after 3-6 h and returned to basal levels after 24 h. Taking advantage of these cells, we have examined the effect of ACTH on DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on X-chromosome, gene 1) and SF-1 (steroidogenic factor 1), two transcription factors known to respectively repress and activate adrenocortical steroidogenesis by acting on common target genes. According to their antagonistic activities, DAX-1 mRNA and protein levels were transiently down-regulated by ACTH, whereas those of SF-1 were stimulated, with kinetics paralleling those of steroidogenic genes expression, notably of two known SF-1 target genes, star and akr1b7. This suggests an essential role of SF-1/DAX-1 proteins ratio to achieve proper ACTH control of steroidogenic gene expression in cells derived from ZF. This was confirmed in mice adrenals, where repression of dax-1 gene and concomitant up-regulation of sf-1, star, and akr1b7 genes were observed in response to ACTH stimulation. In conclusion, using both unique differentiated cell lines and in vivo approaches, we provide the first evidence that hormonally induced changes in SF-1/DAX-1 ratio are part of the molecular arsenal of ZF cells to fine tune ACTH responsiveness.

Abstract. Ovine corticotrophin-releasing factor (oCRF) (1 μg/kg) and arginine vasopressin (AVP) (1 μg/kg) were injected iv in sheep, both separately and in combination. Plasma levels of immunoreactive ACTH (IRACTH), cortisol, and aldosterone were measured for 3 h after the injections. Mean levels before injections were 8 ± 4 pmol/l for ACTH, 7 ± 3 nmol/l for cortisol, and 28 ± 9 pmol/l for aldosterone. CRF caused a rapid rise in IR-ACTH and a peak level of 125 ± 52 pmol/l was obtained 15 min after injection. Highest values for cortisol and aldosterone levels were 40 ± 9 nmol/l and 64 ± 13 pmol/l, respectively, 30 min after injection. AVP also increased IR-ACTH (maximum level: 202 ± 77 pmol/l at 5 min) and aldosterone (128 ± 36 pmol/l at 15 min), whereas the cortisol increase was lower than after CRF. Simultaneous injection of CRF and AVP produced an addition of the IR-ACTH response (295 ± 82 pmol/l at 15 min), but the changes in cortisol levels were similar to those obtained after CRF alone and those in aldosterone levels resembled those induced by AVP alone. Plasma Na and K, osmolality, and plasma renin activity (PRA) were not modified by either CRF or AVP. It is suggested that the increase in aldosterone levels after CRF could be mediated by ACTH and that after AVP by an IR-ACTH peptide with less effect on cortisol secretion.

The polypeptide pattern of the plasma membrane from tobacco was studied by two-dimensional gel electrophoresis. When using classical carrier ampholyte isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) approximately 400 polypeptide spots were detected after silver staining and computer analysis using the QUEST software. This resolution was sufficient to assess physiological effects such as changes in a phytohormone concentration. By using pH 4-8 immobilized pH gradient (IPG)-IEF and 10%T SDS-PAGE gels, approximately 600 polypeptides, corresponding to ca. 80% of the total population expected, were resolved. This cross-section of the plasma membrane polypeptide population was mainly constituted by low or intermediate molecular mass (25 to 45 kDa) and acidic (5.2 < pI < 6.1) polypeptides. After sample application by in-gel rehydration, large amounts of plasma membrane protein (between 5 mg and 10 mg protein) were analyzed using IPG-IEF, and N-terminal protein sequencing was performed for polypeptides collected from one gel. Internal protein sequences were also obtained. Nearly all protein sequences corresponded to unidentified proteins but several of them matched translated sequences from unidentified plant expressed sequence tags (ESTs). It is concluded that the combined use of IPG-IEF gels and in-gel rehydration allows, in the case of plant membrane protein, both analytical and micropreparative separations with an efficiency comparable to that demonstrated for soluble proteins. Finally, it is suggested that a systematic investigation of plant plasma membrane polypeptides is feasible and would constitute a source of new and plant-specific genes.

Aldose reductase (AR; EC 1.1.1.21) is an oxidoreductase that catalyzes the NADPH-dependent conversion of glucose to sorbitol, the first step of the polyol pathway. AR is of great interest due to its implication in the etiology of diabetic complications. In renal medullary cells, AR also plays an osmoregulatory role by accumulating sorbitol to maintain the intracellular osmotic balance during antidiuresis. We have previously cloned the AR cDNA from mouse kidney, and we report here the isolation of the mouse AR gene promoter. Transient transfection of chloramphenicol acetyltransferase reporter constructs containing various 5-flanking regions of the mouse AR gene in CV1 cells led to the identification of a sequence spanning base pairs ؊1053 to ؊1040, required for an enhancer activity in hypertonic compared with isotonic cell culture conditions. This sequence is similar to the tonicity-responsive element first characterized in the betaine-␥-aminobutyric acid transporter promoter.