The human kidney is a progesterone-metabolizing and androgen-producing organ.

Progesterone (P) is a potent antagonist of the human mineralocorticoid receptor (MR) in vitro. We have previously demonstrated effective downstream metabolism of P in the kidney. This mechanism potentially protects the MR from P action. Here, we have investigated the expression and functional activity of steroidogenic enzymes in human kidney. RT-PCR analysis demonstrated the expression of 5 alpha-reductase type 1, 5 beta-reductase, aldo-keto-reductase (AKR) 1C1, AKR1C2, AKR1C3, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) type 2, and 17 alpha-hydroxylase/17,20-lyase (P450c17). The presence of 3 beta-HSD type 2 and P450c17 indicated that conversion of pregnenolone to dehydroepiandrosterone (DHEA) and to androstenedione may take place effectively in kidney. To investigate this further, we incubated kidney subcellular fractions with radiolabeled pregnenolone. This resulted in efficient formation of DHEA from pregnenolone, indicating both 17 alpha-hydroxylase and 17,20-lyase activities exerted by P450c17. Radiolabeled DHEA was converted via androstenedione, androstenediol, and testosterone, indicating both 3 beta-HSD type 2 activity and 17 beta-HSD activity. In addition, the conversion of testosterone to 5 alpha-dihydrotestosterone was detectable, indicating 5 alpha-reductase activity. In conclusion, we verified the expression and functional activity of several enzymes involved in downstream metabolism of P and androgen synthesis in human kidney. These findings may be critical to the understanding of water balance during the menstrual cycle and pregnancy and of sex differences in hypertension.

Expression of the progesterone receptor and progesterone- metabolising enzymes in the female and male human kidney.

Due to high binding affinity of progesterone to the human mineralocorticoid receptor (hMR), progesterone competes with the natural ligand aldosterone. In order to analyse how homeostasis can be maintained by mineralocorticoid function of aldosterone at the MR, especially in the presence of elevated progesterone concentrations during the luteal phase and pregnancy, we investigated protective mechanisms such as the decrease of free progesterone by additional binding sites and progesterone metabolism in renal cells. As a prerequisite for sequestration of progesterone by binding to the human progesterone receptor (hPR) we demonstrated the existence of hPR expression in female and male kidney cortex and medulla at the level of transcription and translation. We identified hPR RNA by sequencing the RT-PCR product and characterised the receptor by ligand binding and scatchard plot analysis. The localisation of renal hPR was shown predominantly in individual epithelial cells of distal tubules by immunohistology, and the isoform hPR-B was detected by Western blot analysis. As a precondition for renal progesterone metabolism, we investigated the expression of steroid-metabolising enzymes for conversion of progesterone to metabolites with lower affinity to the hMR. We identified the enzyme 17alpha-hydroxylase for renal 17alpha-hydroxylation of progesterone. For 20alpha-reduction, different hydroxysteroid dehydrogenases (HSDs) such as 20alpha-HSD, 17beta-HSD type 5 (3alpha-HSD type 2) and 3alpha-HSD type 3 were found. Further, we detected the expression of 3beta-HSD type 2 for 3beta-reduction, 5alpha-reductase (Red) type 1 for 5alpha-reduction, and 5beta-Red for 5beta-reduction of progesterone in the human kidney. Therefore metabolism of progesterone and/or binding to hPR could reduce competition with aldosterone at the MR and enable the mineralocorticoid function.

Agonistic and antagonistic properties of progesterone metabolites at the human mineralocorticoid receptor.

OBJECTIVE: Progesterone binds to the human mineralocorticoid receptor (hMR) with nearly the same affinity as do aldosterone and cortisol, but confers only low agonistic activity. It is still unclear how aldosterone can act as a mineralocorticoid in situations with high progesterone concentrations, e.g. pregnancy. One mechanism could be conversion of progesterone to inactive compounds in hMR target tissues. DESIGN: We analyzed the agonist and antagonist activities of 16 progesterone metabolites by their binding characteristics for hMR as well as functional studies assessing transactivation. METHODS: We studied binding affinity using hMR expressed in a T7-coupled rabbit reticulocyte lysate system. We used co-transfection of an hMR expression vector together with a luciferase reporter gene in CV-1 cells to investigate agonistic and antagonistic properties. RESULTS: Progesterone and 11beta-OH-progesterone (11beta-OH-P) showed a slightly higher binding affinity than cortisol, deoxycorticosterone and aldosterone. 20alpha-dihydro(DH)-P, 5alpha-DH-P and 17alpha-OH-P had a 3- to 10-fold lower binding potency. All other progesterone metabolites showed a weak affinity for hMR. 20alpha-DH-P exhibited the strongest agonistic potency among the metabolites tested, reaching 11.5% of aldosterone transactivation. The agonistic activity of 11beta-OH-P, 11alpha-OH-P and 17alpha-OH-P was 9, 5.1 and 4.1% respectively. At a concentration of 100 nmol/l, progesterone, 17alpha-OH-P and 20alpha-DH-P inhibit nearly 75, 40 and 35% of the transactivation by aldosterone respectively. All other progesterone metabolites tested demonstrate weaker affinity, and agonistic and antagonistic potency. CONCLUSIONS: The binding affinity for hMR and the agonistic and antagonistic activity diminish with increasing reduction of the progesterone molecule at C20, C17 and at ring A. We assume that progesterone metabolism to these compounds is a possible protective mechanism for hMR. 17alpha-OH-P is a strong hMR antagonist and could exacerbate mineralocorticoid deficiency in patients with congenital adrenal hyperplasia.

De novo expression of the Muc2 gene in pancreas carcinoma cells is triggered by promoter demethylation.

It has been established that mucin-producing variants of different subtypes of pancreatic carcinomas, including the intraductal papillary and ductal mucinous tumors, have usually a more favorable prognosis. Intraductal papillary and ductal mucinous tumors have also been shown to ectopically express the intestinal mucin gene MUC2. The mechanism of the de novo expression of this gene in tumors may have potential implications for the modulation of its behavior. We studied, therefore, the mechanism of the de novo expression of MUC2 in pancreas carcinoma cells in vitro. The MUC2 gene promoter is methylated in the nonexpressing pancreatic cell line PANC-1 and is not methylated in the expressing cell line BxPC-3. The promoter is silenced by methylation as shown by reporter expression assays. De novo expression of MUC2 in PANC-1 cells is triggered by treating the cells with a pharmacological inhibitor of DNA methylation (5-aza-2'-deoxycytidine). There was no decrease or loss of expression of the methyltransferase DNMT1 in the MUC2-producing cells. These data show that the de novo expression of the MUC2 gene in pancreas carcinoma cells is associated with promoter demethylation. They warrant further investigations on the relationship between MUC2 promoter demethylation in pancreatic cancer and the prognosis of carcinoma patients.

Regulation of the intestinal mucin MUC2 gene expression in vivo: evidence for the role of promoter methylation.

In the present work we investigated the in vivo regulation of the mucin gene MUC2, which is overexpressed in all mucinous colorectal carcinomas. The inhibition of methylation by 5-azadeoxycytidine induces de novo expression of MUC2 in the colon carcinoma cell line COLO 205. The expression is retained in xenograft tissue and the cells give rise to MUC2-expressing tumours in nude mice. The strong expression of MUC2 in the normal human goblet cells and in the tissue of human mucinous colorectal carcinomas is associated with the average methylation of about 50% at every investigated CpG site of the MUC2 promoter. In contrast, MUC2 promoter in the non-expressing normal columnar cells and in the non-mucinous carcinoma tissue is methylated to nearly 100%. These data show that (i) low methylation of MUC2 promoter is associated with MUC2 expression in vivo and (ii) the pattern of MUC2 promoter methylation in the normal goblet or columnar cells most closely resembles that in mucinous or non-mucinous colorectal carcinomas, respectively. They indicate that MUC2 expression in vivo is regulated by promoter methylation and support the hypothesis that cells with goblet-like differentiation give rise to mucinous colonic carcinomas.

Enzyme-mediated protection of the mineralocorticoid receptor against progesterone in the human kidney.

Progesterone (P) is a mineralocorticoid (MC)-antagonist in vitro. During pregnancy, plasma P concentrations exceed aldosterone concentrations at least 50-fold, but plasma aldosterone increases only 4-8-fold in a compensatory manner. Since the in vivo anti-MC activity of P seems to be only moderate, we hypothesized that P is metabolized by enzymes of MC target tissue similar to the way cortisol is metabolized by 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2 in order to protect the MC receptor. We, therefore, examined P metabolism using 4-(14)C-P in subcellular fractions of human postmenopausal and male kidneys, and in homogenates of one premenopausal kidney. We found that P is converted effectively, even at high P concentrations (10(-6) mol/l), to various metabolites: 20alpha-dihydro(DH)-P; 17alpha-OH-P; 17alpha-OH,20alpha-DH-P; 5alpha-DH-P; 3beta,5alpha-tetrahydro(TH)-P; and 20alpha-DH,5alpha-DH-P. Homogenates of premenopausal kidney also showed conversion to 3alpha- and 5beta-reduced P metabolites. These results confirm the existence of an efficient renal enzyme system as a possible mechanism of an enzyme-mediated MC receptor selectivity.

Hypothalamic modulation of growth hormone secretion in the rhesus monkey: evidence from intracerebroventricular infusions of glucose, free fatty acid, and ketone bodies.

To evaluate the hypothalamus as a possible site of metabolic modulation of GH secretion, we studied the GH response to insulin hypoglycemia (IHG) and nicotinic acid (NA)-induced FFA depression in the absence and presence of third ventricular (ivt) infusions of glucose, oleic acid (Ol-Ac), or beta-hydroxybutyrate (beta OHB). Four rhesus monkeys had been prepared for chronic remote iv and ivt infusions as well as blood sampling from the adjacent room. Statistical evaluation used a two-way analysis of variance and individual comparisons with Tukey's Studentized range test. The GH response (area under the curve +/- SE) to IHG was significantly reduced by a concomitant ivt glucose infusion (control, 1.0 +/- 0.1; IHG, 12.1 +/- 3.3; IHG plus ivt glucose, 7.0 +/- 1.2 microgram/L.120 min). The GH response to FFA depression was significantly reduced by ivt Ol-Ac or beta OHB infusion (control, 6.0 +/- 1.0; NA, 51.5 +/- 4.1; Na plus Ol-Ac, 81.2 +/- 1.3; NA plus beta OHB, 38.6 +/- 3.5 microgram/L.300 min). Introcerebroventricular infusions of glucose, Ol-Ac, or beta OHB alone had no effect on plasma GH, glucose, FFA, or beta OHB concentrations. These results provide evidence for a hypothalamic site of metabolic modulation of GH secretion in the rhesus monkey. This does not exclude an additional effect directly at the pituitary gland.

SV40 chromatin structure is not essential for viral gene expression.

The biological activity and the fate of SV40 DNA (minichromosomes, DNA I, DNA II, DNA III) were tested in culture cells by immunofluorescence staining and blot analysis. Following microinjection of 2-4 circular SV40 molecules (minichromosomes, DNA I, DNA II) into the cytoplasm or the nuclei of monkey and rat cells, T- and V-antigen synthesis was demonstrable in nearly every recipient cell. Only linear DNA induced T-antigen synthesis with a very low efficiency after cytoplasmic injection. This low activity correlates with a rapid degradation of DNA III in the recipient cells. Further modifications observed immediately after injection are relaxation of superhelical molecules and formation of high-Mr DNA. Assembly of the injected DNA into SV40 chromatin-like structure, however, occurred only late after early viral gene expression.

The second large simian virus 40 T-antigen exon contains the information for maximal cell transformation.

Microinjection of early simian virus 40 (SV40) DNA fragments has shown that maximal transformation of rat cells (Ref 52) is a property of the second SV40 T-antigen exon. Expression of this particular T-antigen region was obtained by coinjection of the Taq/Bam DNA fragment with the early promoter/enhancer HpaII/BglI fragment. Microinjection of the DNA fragment mixture induced two categories of transformants; namely, maximally and minimally transformed cells. The maximally transformed cells synthesize two Taq/Bam-specific polypeptides, and the minimally transformed cells only the lower molecular weight form. Both types of transformants contain the cellular p52 protein at high concentrations. Furthermore, maximal transformation of Ref 52 cells requires the carboxy terminus of the T-antigen. Cells transformed by microinjection of the SV40 Pst A-fragment display different parameters of maximally transformed cells but not anchorage-independent growth.

DNA-mediated gene transfer in Friend leukemia cells by cotransfection of simian virus 40 DNA with herpes simplex virus thymidine kinase DNA.

Thymidine kinase-negative Friend leukemia cells were cotransfected with simian virus 40 (SV40) DNA and thymidine kinase gene DNA of herpes simplex virus type 1. The transfected thymidine kinase-positive cells were selected in HAT medium, and SV40 T-antigen expression was observed over many months in cells cultured under selective conditions, and after adaptation to normal growth medium under nonselective conditions. It was shown by Southern blot hybridization that SV40 DNA was integrated in multiple copies in the chromosomal DNA of several clones. All SV40 DNA-containing Friend leukemia cell clones analyzed were able to undergo induced erythroid differentiation. Induced cultures still expressed SV40 T-antigen to the same extent that untreated control cultures did.

Pattern of plasma cortisol during the 24-hour sleep/wake cycle in the rhesus monkey.

The 24-h pattern of plasma cortisol and changes induced by alterations of the sleep/wake cycle were studied in 12 male rhesus monkeys. The chair-living animals were chronically prepared with a right atrial catheter and electroencephalogram electrodes. Hormone (blood samples every 15 min) and continuous activity/electroencephalogram profiles were obtained from the adjacent room for 96 h (4 animals), 24 h or various shorter periods of time. Plasma cortisol showed a circadian rhythm with a late evening minimum (1900-2100 h; approximately 60 micrograms/liter) and an early morning maximum (0400-0700 h; approximately 160 micrograms/liter). Superimposed were episodic fluctuations for which powerspectral analysis showed a weakly expressed 30- to 60-min periodicity in 24 of 27 24-h profiles. Cross-correlation analysis indicated no relation between cortisol on the one hand and daytime activity-arousal, nocturnal waking, slow wave sleep (SWS) or rapid eye movement sleep (REM), respectively. Five-hour total sleep deprivation, specific SWS-deprivation, and severe disruption of the REM-pattern provided no evidence for an immediate effect of sleep onset or sleep stages on the cortisol pattern. Cortisol rose significantly after termination of the 5-h deprivation, but the mechanism of this elevation remains to be determined. Cross-correlation analysis between the cortisol time series and those of GH, PRL, and TSH from already published data gave no evidence for a regular temporal relationship between the episodic patterns of these hormones.

Twenty-four-hour pattern of growth hormone secretion in the rhesus monkey: studies including alterations of the sleep/wake and sleep stage cycles.

The 24-h pattern of GH secretion and its possible relation to the sleep/wake cycle and to sleep stages were studied in 12 male rhesus monkeys. Blood samples were drawn every 15 min for 96 h, 24 h, or shorter periods of time through chronic right atrial catheters which extended through the wall into the adjacent room. In addition, activity rating (daytime) and determination of sleep stages from electroencephalogram recordings (nighttime) were done. GH profiles were obtained during undisturbed conditions and during deprivation of nap, 5 h total sleep, slow wave sleep (SWS), and rapid eye movement (REM) sleep. GH secretion was episodic, with peak concentrations often exceeding 20 ngeq/ml and nadirs mostly below 1 ngeq/ml. Autocorrelation analysis demonstrated a circadian and an ultradian rhythm during undisturbed conditions. However, the cycle length of the ultradian rhythm showed large inter- and intraindividual variations (from 3--6 h). Neither cross-correlation analysis between hormonal and activity/electroencephalogram sleep stage time series nor results of deprivation experiments produced evidence for a link between nap phases, the sleep/wake cycle, or the SWS/REM sleep stage cycle on the one hand and the GH secretory pattern on the other hand. However, while SWS deprivation was highly effective, REM deprivation did not substantially reduce total REM sleep time due to frequent entries into abortive REM sleep epochs. During the daytime, there was no significant correlation between activity/arousal and GH, but during the night, there was a significant positive correlation between stage waking and GH. A direct or indirect synchronizing effect of the matutinal light change is suggested by the pattern of the 24-h curve of mean GH concentrations during undisturbed conditions: a steep increase from very low concentrations at light onset, followed by a succession of nadirs and peaks at approximately 4.5-h intervals. However, the nadirs became progressively more shallow until there was no apparent periodicity during the night due to the loss of synchronization. It is concluded that GH in the rhesus monkey shows a circadian and an ultradian periodicity. However, in contrast to man, sleep and SWS are not important determinators of the 24-h GH pattern.