P450c17 mRNA Research Articles

Developmental expression of PC3 gene is correlated with neuronal cell birthday

We examined the developmental expression of PC3, a nerve growth factor (NGF) early induced gene in PC12 cells, in the rat central nervous system (CNS) and we found that it represents a molecular marker of ongoing postmitotic neurons production. PC3 is initially expressed in the ventral quarter of the neural tube, at the level of the presumptive cervical spinal cord just where and when (10–11 days post coitum (dpc)) the motor neurons are arising. Subsequently, the appearance of PC3 expression follows a ventro-dorsal and a rosto-caudal gradient in the spinal cord and a caudo-rostal gradient across the brain vesicles that coincide, both spatially and temporally, with the gradients of neurogenesis described in the literature. As in PC12 cells, PC3 mRNA expression appears to be transient in vivo. In all regions of the CNS, it is restricted to the ventricular zone of the neuroepithelium, while neuronal precursors cease to express PC3 as they migrate to the mantle zone. Moreover, PC3 mRNA disappears from the various regions of the CNS as neurogenesis ceases.

Induction and genetic regulation of mouse hepatic cytochrome P450 by cannabidiol

Cannabidiol (CBD) has been shown to be a selective inactivator of cytochromes P450 (P450s) 2C and 3A in the mouse and, like many P450 inactivators, it can also induce P450s after repeated administration. The inductive effects of CBD on mouse hepatic P450s 2B, 3A, and 2C were determined using cDNA probes, polyclonal antibodies, and specific functional markers. P450 2B10mRNA was increased markedly after repeated CBD administration and correlated well with increased P450 2B immunoquantified content and functional activity. On the other hand, although the 2-fold increase in P450 3A mRNA detected after repeated CBD administration was consistent with the increased immunoquantified P450 3A protein content, the lack of an observable increase in P450 3A-specific functional activity suggested subsequent inactivation of the induced P450 3A. Repeated CBD treatment increased P450 2C mRNA content 2-fold, but did not increase either the P450 2C immunoquantified content or its functional activity. The effect of CBD treatment on the ability of tetrahydrocannabinol (THC) to induce P450 2B was also determined. A THC dose that did not induce P450 2B significantly was administered alone or in combination with a CBD dose that markedly inactivated P450s 2C- and 3A but submaximally increased P450 2B functional activity. The combination of THC and CBD did not increase P450 2B-catalyzed activity significantly over that observed after CBD treatment alone. Thus, prior CBD-mediated P450 inactivation does not appear to increase the ability of THC to induce P450 2B. To further characterize the relationship between P450 inactivation and induction, several structurally diverse CBD analogs with varying P450 inactivating potentials were tested for their ability to induce P450 2B. At least one CBD analog that is an effective P450 inactivator failed to induce P450 2B, while at least one CBD analog that is incapable of inactivating P450 was found to be a very good P450 2B inducer. It therefore appears that inherent structural features of the CBD molecule rather than its ability to inactivate P450 determine P450 2B inducibility. The complex effects of CBD treatment on P450 inactivation and induction have the potential to influence the pharmacological action of many clinically important drugs known to be metabolized by these various P450s. The mechanism of CBD-mediated P450 induction remains to be elucidated but does not appear to be related to CBD-mediated P450 inactivation.

Virilizing adrenal tumour mimicking congenital adrenal hyperplasia with P450c11 (11β-hydroxylase) deficiency

In a girl aged 5 years with a virilizing adrenal adenoma the urinary and plasma steroid findings suggested the diagnosis of congenital adrenal hyperplasia due to P450c11 (11 beta-hydroxylase) deficiency. After removal of the tumour clinical signs receded and the hormonal values normalized. RNA analysis of the tumour tissue revealed low amounts of P450c11 mRNA which indicates that P450c11 deficiency of the adenoma caused the steroid abnormalities in this girl.

The family of subtilisin/kexin like pro-protein and pro-hormone convertases: Divergent or shared functions

Six mammalian processing enzymes were recently discovered which exhibit significant similarities to both yeast kexin and bacterial subtilisins. These subtilisin/kexin-like convertases were called furin/PACE, PC1/PC3, PC2, PACE4, PC4 and PC5/PC6. The analysis of the mRNA expression of these convertases in rat tissues and cell lines by Northern blot analysis demonstrated a unique pattern for each enzyme. Thus, although furin and PACE4 mRNA (4.4 kb each) exhibit a widespread tissue distribution only furin is ubiquitously expressed. PACE4 exhibits a major 4.4 kb mRNA form, and in some tissues a 3.9 kb form is detected. PC5 mRNA (3.8 kb major) is more restricted in its distribution than PACE4 and furin, and it exhibits the presence of multiple mRNA forms, resulting in variable lengths of the C-terminal Cys-rich domain. In addition, like furin and PACE4, PC5 is expressed in both regulated and constitutively secreting cells. In contrast, PC1 (3 and 5 kb) and PC2 (2.8 and 5 kb) are primarily expressed in tissues and cells containing secretory granules. Multiple mRNA forms are also detected, but as far as is known none affect their open reading frame and only result in a variable length of the 3′ non-coding sequence. Finally, PC4 mRNA (2.8 kb major and 1.9 kb minor) is only expressed in testicular germ cells. Biosynthetic analysis of the zymogen activation of PC1 and PC2 and their cleavage specificity following their cellular co-expression with a number of precursors, demonstrated that although pro-PC1 is rapidly activated to PC1 in the endoplasmic reticulum, pro-PC2 conversion into PC2 is rather slow. The cleavage of pro-PC2 into PC2 starts in the trans Golgi network and is regulated by an endogenous endocrine and neural precursor called 7B2. Although the genetic organization of the convertase genes is very similar, they exhibit unique promoter sequences and only furin and PACE4 genes are localized on the same chromosome.

Steroidogenic adrenocortical cell lines produced by genetically targeted tumorigenesis in transgenic mice.

Studies of adrenal steroidogenesis have been facilitated by the availability of immortalized mouse adrenocortical Y-1 cells. We sought to make new, alternative mouse steroidogenic cell lines by genetically targeted tumorigenesis. Transgenic mice were constructed expressing both the SV40 T-antigen and a bacterial neomycin-resistance gene under the control of the promoter for the human P450 cholesterol side-chain cleavage (P450scc) gene, which encodes the first and rate-limiting enzyme in steroidogenesis. Two female transgenic mice expressed T-antigen in various nonsteroidogenic tissues but generated tumors only in the adrenals, suggesting adrenal tumor formation was an early event. Ovarian tissues, which, unlike the adrenal, do not make steroids in fetal or early postnatal life, did not develop tumors. Cell lines derived from the adrenal tumors were resistant to the neomycin analog G418. Clonal sublines are stable, growing easily in monolayers with a doubling time of 24-60 h. The cell lines secrete progesterone and 11-deoxycorticosterone, indicating these cells express the P450scc system, 3 beta-hydroxysteroid dehydrogenase, and 21-hydroxylase activity. However the 21-hydroxylase activity was not mediated by P450c21, as the cells lacked P450c21 mRNA. The cells did not secrete any 11-hydroxylated steroids, although they contained P450c11 beta mRNA. Both the secretion of progesterone and the abundance of P450scc mRNA increase in response to 8-bromo-cAMP, but not to ACTH or angiotensin II. In addition to expression of steroidogenic enzyme mRNAs, one cell line also expresses mouse renin-1 mRNA, making these cells useful for studies of the role of adrenal renin in regulating adrenal steroidogenesis. These findings represent an approach in transgenic mice to develop highly differentiated adrenal cell lines.

Localization of cytochrome P450 cholesterol side-chain cleavage, cytochrome P450 17 alpha-hydroxylase/17, 20-lyase, and 3 beta-hydroxysteroid dehydrogenase isomerase steroidogenic enzymes in human and rhesus monkey fetal adrenal glands: reappraisal of functional zonation.

We examined the in situ localization of key steroidogenic enzymes in adrenal gland sections from midgestation (17-24 weeks) human fetuses and late gestation (130-142 days; term = 165 days) rhesus monkey fetuses. The rhesus monkey fetal adrenals were used as a model for the late gestation human fetal adrenal. The enzymes examined were cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17 alpha-hydroxylase/17,20-lyase (P450c17), and 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD). In human fetal adrenals, P450scc and P450c17 proteins and mRNAs were detected only in fetal zone (innermost cortical zone) and transitional zone (between the fetal and definitive zone) cells, not in definitive zone cells. Expression of 3 beta HSD was not detected in any cortical zone cells in midgestation human fetal adrenals. In rhesus monkey fetal adrenals, a similar pattern of P450scc and P450c17 expression was observed in the fetal and transitional zones. In the definitive zone cells of rhesus monkey fetal adrenals, expression of both P450scc and 3 beta HSD was detected. In addition, low levels of 3 beta HSD expression could be detected in some transitional zone cells. P450c17 expression was lacking in definitive zone cells from rhesus monkey fetal adrenals. These data suggest that early in gestation, cortisol is not produced by the human fetal adrenal cortex in vivo (because it does not express 3 beta HSD), whereas androgen production occurs in the transitional and fetal zones (which express P450scc and P450c17). Later in gestation, the definitive zone may produce minearlocorticoids (because it expresses P450scc and 3 beta HSD, but lacks P450c17), and the transitional zone may produce glucocorticoids (it expresses P450scc, P450c17, and 3 beta HSD), whereas the fetal zone continues to produce androgens. Thus, late in gestation the functional zonation of the human fetal adrenal cortex may be similar to that of the adult, with the definitive zone being analogous to the nascent zona glomerulosa, the transitional zone analogous to the zona fasciculata, and the fetal zone analogous to the zona reticularis.

Expression of steroidogenic enzyme messenger ribonucleic acids and corticosteroid production in aldosterone-producing and "nonfunctioning" adrenal adenomas.

"Nonfunctioning" adrenal adenomas are often diagnosed in patients without recognizable clinical symptoms of adrenocortical hyperfunction. The objective of this study was to determine directly the steroidogenic activity of such adenomas (n = 12) and compare them histologically and functionally to normal human adrenals (n = 6) and aldosterone-producing adenomas (n = 15). The histological appearances of nonfunctioning and aldosterone-producing adenomas were surprisingly similar. Nonfunctioning adrenal adenomas expressed all mRNAs of P450scc, P450c17, P450c21, adrenodoxin, and adrenodoxin reductase with relative levels comparable to those found in normal adrenals. Consistent with their hormone-producing nature, these adenomas had cortisol and aldosterone contents as high as those in normal adrenal tissues, a significantly (P < 0.05) increased 17-hydroxyprogesterone content, and a disproportionally low expression of P450c21 mRNA compared to aldosterone-producing adenomas. Cells isolated from both aldosterone-producing and nonfunctioning adrenal adenomas exhibited highly ACTH-sensitive cortisol and aldosterone production, suggesting again the presence of both zona glomerulosa-like and zona fasciculata-like steroidogenesis in these adenoma tissues. These results indicate that so-called nonfunctioning adrenal adenomas are not without steroidogenic activity. Therefore, the assumption that adrenal adenomas are entirely nonfunctioning in the absence of recognizable hormonal hyperfunction may not be correct.

Regulation of human adrenal carcinoma cell (NCI-H295) production of C19 steroids.

The regulation of biosynthesis of the adrenal C19 steroids (the so-called adrenal androgens) remains unclear. Understanding adrenal production of C19 steroids is important when the benefits of these steroids are considered on processes and diseases associated with aging. In vitro studies defining the mechanisms that regulate the production of human adrenal C19 steroids have been limited because of the difficulties in obtaining adrenal tissue. A cell line that retains differentiated adrenal functions would greatly facilitate research in this area. Herein, we describe the use of the human adrenocortical tumor H295 cell line as a model to evaluate mechanisms controlling C19 and C21 steroid production. The cells were characterized with regard to ACTH, forskolin, and dibutyryl cAMP (dbcAMP) responsiveness, as measured by increased cAMP production, synthesis of steroids, and induction of 17 alpha-hydroxylase cytochrome P450 (P450c17). Forskolin and dbcAMP, which were more effective than ACTH, enhanced the production of cortisol, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), and androstenedione over a 48-h treatment period. Comparison of the relative amounts of measured steroid secreted under forskolin treatment indicated that the primary product was cortisol (70%), followed by androstenedione (14%), DHEA (9%), and DHEAS (7%). Cortisol was also demonstrated to be the major steroid product by examination of UV-detectable steroids after high performance liquid chromatographic separation. The increases in steroid production caused by ACTH, forskolin, and dbcAMP occurred in a concentration- and time-dependent manner. A key enzyme in the production of C19 steroids is P450c17. ACTH, forskolin, and dbcAMP increased the activity of 17 alpha-hydroxylase by approximately 2.5-, 10-, and 10-fold, respectively. These effects on enzyme activity occurred in a concentration-dependent manner and coincided with increased levels of P450c17 mRNA. In summary, H295 cells should provide a much-needed model to study mechanisms controlling the secretion of glucocorticoids and C19 steroids, because steroid production in these cells is hormonally controlled and associated with the induction of P450c17.

The role of tumor necrosis factor-alpha in the regulation of mouse Leydig cell steroidogenesis.

Tumor necrosis factor-alpha (TNF alpha), a cytokine secreted by activated macrophages, has been shown to modulate Leydig cell steroidogenesis. The present study examined the regulation of mouse Leydig cell function by TNF alpha at the molecular level. The effects of TNF alpha on both basal and 8-Br-cAMP-stimulated testosterone production, as well as cholesterol side-chain cleavage enzyme (P450scc) and 17 alpha-hydroxylase/C17,20 lyase (P450c17), were investigated. Treatment of Leydig cells with 0.1, 1.0, and 10.0 ng/ml TNF alpha inhibited basal testosterone secretion by 20 +/- 5.0%, 61.1 +/- 6.6%, and 60.7 +/- 5.8% of control, respectively, but had no effects on basal P450scc messenger RNA (mRNA) or protein levels. Treatment of Leydig cells with 8-Br-cAMP caused a 150.7 +/- 32.9-fold increase in testosterone production and marked stimulation of P450scc and P450c17 mRNA and protein accumulation. TNF alpha caused a significant and dose-dependent inhibition of 8-Br-cAMP-stimulated testosterone secretion by 35.9 +/- 9.9%, 90.9 +/- 1.7%, and 96.9 +/- 1.4% with 0.1, 1.0, and 10.0 ng/ml TNF alpha, respectively. TNF alpha also caused a decrease in P450scc and P450c17 mRNA and protein. Treatment with 0.1, 1.0, and 10.0 ng/ml TNF alpha decreased 8-Br-cAMP-stimulated P450scc mRNA by 11.5 +/- 6.9%, 29.3 +/- 2.7%, and 59.2 +/- 8.7%, and decreased 8-Br-cAMP-induced P450c17 mRNA 41.9 +/- 13.5%, 95.7 +/- 2.3%, and 98.5 +/- 1.2%, respectively. The inhibitory effects of TNF alpha on 8-Br-cAMP-stimulated P450 enzyme protein accumulation were also dose dependent, 35.6 +/- 11.4%, 52.9 +/- 14.1%, and 56.0 +/- 7.9% inhibition of P450scc protein levels, and 65.8 +/- 9.4%, 95.5 +/- 1.9%, and 96.9 +/- 2.1% suppression on P450c17 protein levels were observed with 0.1, 1.0, and 10.0 ng/ml TNF alpha, respectively. The inhibitory effect of TNF alpha on 8-Br-cAMP-induced P450c17 mRNA expression was reversible. Within 48 h after the removal of TNF alpha from culture, P450c17 mRNA was restored to 80.6 +/- 3.1% of the level in cultures treated with 8-Br-cAMP alone for 4 days. TNF alpha-mediated inhibition of 8-Br-cAMP-stimulated testosterone secretion from Leydig cells was also reversible. In addition, no significant cell mortality was noted in TNF alpha-treated cells. These data demonstrate that TNF alpha inhibits both basal and 8-Br-cAMP-stimulated testosterone secretion from Leydig cells in a dose-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Ontogeny of the prohormone convertases PC1 and PC2 in the mouse hypophysis and their colocalization with corticotropin and alpha-melanotropin.

In the adult pituitary, anterior lobe corticotrophs and intermediate lobe melanotrophs differentially process proopiomelanocortin (POMC). Within the corticotrophs, POMC is processed mainly to corticotropin (ACTH) and beta-lipotropin, while alpha-melanotropin (alpha MSH) and beta-endorphin are the major end products in the melanotrophs. The observed transient presence of alpha MSH-like immunoreactivity during ontogeny suggested an age-dependent variation in POMC processing in the adenohypophysis. In this tissue, cell-specific POMC products are likely the result of differential expression of the two known prohormone convertases PC1 and PC2. In the present ontogeny study done in the mouse intermediate and anterior pituitary, we examined how the expression pattern of PC1 and PC2 mRNA transcripts correlates with that of ACTH and alpha MSH-like immunoreactivities. Our data demonstrated that both PC1 and PC2 transcripts can be detected in the presumptive adenohypophysis starting on embryonic day 15 (E15). In the intermediate lobe, PC1 and PC2 mRNAs appear on E18 and E16, respectively, and their levels increased during ontogeny, reaching maximal expression in the adult. Similarly, PC1 expression in the anterior pituitary increased from E15 to adulthood. However, PC2 mRNA expression peaked between postnatal days 1 (P1) and 14 (P14) and then decreased to adult levels. The distribution of PC1 and PC2 immunoreactivity is nicely correlated with the in situ hybridization data. In the anterior lobe, during the P1-P14 postnatal period, PC2 immunoreactivity was detected within cells synthesizing an alpha MSH-like peptide(s). This observation substantiates our earlier biochemical data suggesting that PC2 is the important convertase in the processing of POMC into alpha MSH. Furthermore, the demonstrated variation in the relative ratio of PC1/PC2 expression during ontogeny rationalizes the observed plasticity of POMC processing in the adenohypophysis. It is expected that beta-endorphin processing will follow that of alpha MSH.

Characterization of pro-opiomelanocortin processing in heterologous neuronal cells that express PC2 mRNA

We have investigated processing of monkey pro-opiomelanocortin (POMC) following transfection into heterologous neuronal Neuro 2A (N2A) cells. In several separately transfected stable cell lines (termed N2A POMC2 -like ; n = 4), POMC was processed to βE only, by direct cleavage from the precursor. Thus, these cell lines did not produce βE in the orderly manner observed in the pituitary, that is, via the intermediate peptide βLPH. Analysis of one representative N2A POMC2 cell line revealed that the extent of processing to βE appeared to be negatively correlated with precursor expression level, suggesting that the processing enzyme(s) in these cells was present in limiting amounts. Northern analysis of PC1 and PC2, two recently cloned processing enzymes, showed that N2A POMC2 cells expressed low levels of PC2 mRNA, but no detectable PC1 mRNA. These data suggest that (1) the order of processing observed in the pituitary is not exclusively determined by tertiary folding of the precursor, but rather by the complement of processing enzymes in a particular cell, and (2) if PC2 is responsible for POMC processing in N2A POMC2 cells, this enzyme, expressed in limiting amounts, appeared to show selectivity for the βE amino terminal processing site.

Regulation of steroidogenesis in NCI-H295 cells: a cellular model of the human fetal adrenal.

NCI-H295 is a recently described human adrenocortical carcinoma cell line that makes a variety of steroid hormones. We sought to determine if steroidogenesis in these cells employs the same enzymes as those used in normal adrenal steroidogenesis, and if the genes encoding those enzymes exhibit characteristic responsiveness to activators of the protein kinase-A and -C pathways of intracellular second messengers. Northern blots show that NCI-H295 cells contain abundant mRNAs for three key steroidogenic enzymes, cytochrome P450scc, cytochrome P450c17, and cytochrome P450c21. These mRNAs accumulated in a time- and dose-dependent fashion in response to 8-bromo-cAMP (8Br-cAMP), forskolin, cholera toxin, and 3-isobutyl-1-methylxanthine, all activators of the protein kinase-A pathway. Nuclear run-on assays and actinomycin-D transcriptional inhibition experiments show that cAMP regulates the expression of all three genes primarily at the transcriptional level. Inhibition of protein synthesis with cycloheximide did not prevent the cAMP-induced accumulation of P450scc or P450c17 mRNAs, but did inhibit accumulation of P450c21 mRNA, suggesting that cAMP is acting through a mechanism dependent on protein synthesis to promote accumulation of P450c21 mRNA. Stimulation of the protein kinase-C pathway with phorbol ester decreased P450scc and P450c17 mRNAs, but stimulated the accumulation of P450c21 mRNA. RNase protection experiments, Northern blot hybridizations, and reverse transcription-polymerase chain reaction show that NCI-H295 cells express both the 11 beta-hydroxylase (P450c11 beta) encoded by the P450c11B1 gene and the aldosterone synthetase (P450c11AS) encoded by the P450c11B2 gene. 8Br-cAMP increased the abundance of both of these mRNAs with similar kinetics, with maximal accumulation of both after about 24 h. NCI-H295 cells also contain the mRNAs for aromatase and insulin-like growth factor-II. 8Br-cAMP increased the abundance of aromatase mRNA and decreased the abundance of IGF-II mRNA. These studies show that NCI-H295 cells express most of the enzymes needed for human adrenal steroidogenesis, and that the genes encoding these enzymes respond to stimulation of second messenger pathways in a manner similar to that of human adrenals. NCI-H295 cells appear to be a good model for studying the molecular regulation of human adrenal steroidogenesis.

Angiotensin-II stimulates an increase in cAMP and expression of 17 alpha-hydroxylase cytochrome P450 in fetal bovine adrenocortical cells.

While known to be a potent activator of phosphoinositidase C, angiotensin II (A-II) also causes a small but significant increase in cAMP production through the type 1 A-II (AT1) receptor in bovine adrenocortical cells (Mol Cell Endocrinol 81:33-41, 1991). We have carried out studies on primary cultures of fetal bovine adrenocortical cells to examine the effects of A-II on the expression of cytochrome P450 17 alpha-hydroxylase (P450c17), which is known to be regulated in a cAMP-dependent fashion. Prolonged treatment (48 h) of cells with A-II (10(-7) M) did not give rise to a detectable increase in P450c17 as measured by immunoblotting, although both A-II and the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA) attenuated the large increase in P450c17 induced by ACTH (10(-8) M). A-II alone (10(-7) M) however, caused a time-dependent increase in cAMP secretion, reaching 8-fold within 3 h. Prolonged treatment of cells with A-II also resulted in a 3-fold increase in P450c17 mRNA within 12 h (10(-7) M), and a dose-dependent increase in 17 alpha-hydroxylase activity within 48 h (16.4-fold max at 10(-7) M). The stimulatory actions of A-II alone (10(-7) M) on cAMP levels, P450c17 mRNA, and 17 alpha-hydroxylase activity were much smaller than in response to ACTH (10(-8) M), but were largely reproduced by TPA (10(-7) M), suggesting a role for protein kinase C in mediating these responses to A-II. These findings indirectly support the hypothesis that A-II alone can stimulate an increase in cAMP in adrenocortical cells. Such a stimulation of cAMP may then result in increased expression of steroidogenic enzymes, as we have shown is the case for P450c17 expression. However, A-II in the presence of ACTH appears to attenuate the ACTH-stimulated expression of P450c17.

&lt;b&gt;CHARACTERIZATION OF PREPROTACHYKININ B PROCESSING IN HETEROLOGOUS ENDOCRINE CELL &lt;/b&gt;&lt;b&gt;LINES &lt;/b&gt;

We have investigated endoproteolytic processing of protachykinin B and regulated release of neurokinin B following transfection into heterologous endocrine cell lines, AtT-20 and MIN6. Both of the transfected cell lines produced mature neurokinin B and secreted it in response to a secretagogue, 8-Br-cAMP. Northern blot analysis of PC3 and PC2, two recently cloned Kex2-like processing endoprotease, showed that AtT-20 cells expressed PC3 mRNA, while MIN6 cells expressed both PC2 and PC3 mRNAs. These data suggest that protachykinin B is processed to neurokinin B by at least PC3 within the regulated secretory pathway

Regulation of steroid 21-hydroxylation by 17 beta-estradiol in rat liver: in vivo and in vitro study.

In various extraadrenal organs, progesterone (P4) is converted to deoxycorticosterone (DOC) by steroid 21-hydroxylation. To investigate the regulation of extraadrenal 21-hydroxylase activity by 17 beta-estradiol (E2), the following two experiments were performed. Exp. 1). Three-week-old male SD rats were testectomized (TX) and injected with E2 (1 mg) or sesame-oil s.c. Sham rats were treated with sesame-oil. In these groups the serum concentration of DOC and corticosterone (B), microsomal 21-Hydroxylase activity and the expression of P450c21 mRNA of the liver and the adrenals were analyzed. Exp. 2). Isolated rat hepatocytes were cultured and stimulated by E2, 10(-9) approximately 10(-5) M. 21-Hydroxylase activity of these cells was analysed by the rate of production of DOC in the medium containing P4. The results of experiment 1 showed that both serum DOC concentration and 21-hydroxylase activity in the liver microsomal fraction were increased by E2 injection, but the expression of P450c21 mRNA was not detected in the liver even after E2 injection. In experiment 2, the activity of steroid 21-hydroxylase in isolated rat hepatocytes was stimulated by E2 in a dose dependent manner. These data provided evidence that: in rats the liver was one sites of the extraadrenal steroid 21-hydroxylase activity which had been stimulated by E2. The results also suggested that this hepatic enzyme was a different enzyme from the steroid 21-hydroxylase P450c21 expressed in the adrenal gland.

Expression of nucleotide pyrophosphatase and alkaline phosphodiesterase I activities of PC-1, the murine plasma cell antigen

Nucleotide pyrophosphatase (EC 3.6.1.9) is a membrane enzyme purified from a number of mammalian sources that may have alkaline phosphodiesterase I (EC 3.1.4.1) activity as well. The mol. wt and subunit structure of this membrane glycoprotein are similar to that of the murine plasma cell alloantigen, PC-1. The PC-1 protein is a disulfide-bonded dimer of identical 115kDa polypeptides that is selectively expressed on B lineage cells that have reached the degree of maturation associated with immunoglobulin secretion. It also has restricted expression in certain non-lymphoid tissues. In this report, we show that alkaline phosphodiesterase I activity parallels PC-1 mRNA expression in a number of B lineage cell lines at different stages of differentiation. Furthermore, we demonstrate increases in both nucleotide pyrophosphatase and alkaline phosphodiesterase I enzymatic activities in transiently transfected COS-7 cells expressing a cloned PC-1 cDNA construction. These results extend our previous immunological and correlative studies and directly ascribe an enzymatic activity to this cell surface differentiation antigen. These experiments also demonstrate that a single protein is responsible for both alkaline phosphodiesterase I and nucleotide pyrophosphatase activities.

The role of cytokines in the regulation of Leydig cell P450c17 gene expression

Cytokines produced by immune-activated testicular interstitial macrophages (TIMs) may play a fundamental role in the local control mechanisms of testosterone biosynthesis in Leydig cells. We investigated whether in vivo immune-activation of TIMs can modulate Leydig cell steroidogenesis. To immune activate TIMs in vivo, mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS, 6 mg/kg). TIMs and Leydig cells were purified for RNA analysis. LPS treatment resulted in a 47-fold increase in interleukin-1β (IL-1β) mRNA in TIMs. P450c17 mRNA levels in the Leydig cells from the same animals, decreased to less than 10% compared to control. The effect of LPS on IL-1β and P450c17 mRNA levels was reversible on both TIMs and Leydig cells, respectively. To determine if the effect of LPS on P450c17 was mediated by a possible decrease in pituitary LH secretion, mice were co-injected with LPS and hCG. Treatment with hCG did not change the effect observed with LPS alone, in TIMs or in Leydig cells. In vitro, LPS treatment of TIMs resulted in marked induction of IL-1β mRNA expression. In parallel, in vitro treatment of Leydig cells with recombinant IL-1 resulted in a dose-dependent inhibition of P450c17 mRNA expression and testosterone production. These data demonstrate that LPS treatment, in vivo and in vitro, induced IL-1 gene expression in TIMs, and that IL-1 inhibits P450c17 mRNA in vitro. Therefore, we suggest that immune-activation of TIMs might have caused the observed inhibition of P450c17 gene expression in Leydig cells in vivo.

Molecular cloning of monkey liver cytochrome P-450 cDNAs : Similarity of the primary sequences to human cytochromes P-450

Three cDNAs coding for monkey cytochrome P-450 (P450) 2C, 2E and 3A (MKmp13, MKj1 and MKnf2, respectively) were isolated from a λgt11 cDNA library of a liver from a 3-methylcholanthrene (3MC)-treated crab-eating monkey, using cDNA fragments for human P450 2C, 2E and 3A as respective probes. MKmp13 and MKnf2 were 1901 and 2032 bp long, containing entire coding regions for polypeptides of 490 and 503 residues, respectively. The deduced N-terminal amino acid sequences of MKmp13 and MKnf2 were identical with those of P450-MK1 and P450-MK2, which had been purified from liver microsomes of untreated and polychlorinated biphenyl (PCB)-treated crab-eating monkeys, respectively. MKj1 was 1508 bp long, encoding a polypeptide of 449 residues, which is presumed to lack N-terminal 45 residues as compared with the sequence for human P450 2E1. Northern blot analysis indicated that monkey P450 2C, 2E and 3A mRNAs were expressed constitutively in monkey livers. P450 2E and 3A mRNAs were induced by both 3MC and PCB, while P450 2C mRNA was induced only by PCB. The deduced amino acid sequences of four monkey cytochrome P-450 cDNAs, including P450 1A1 (MKah1) which we isolated previously, were more than 92% identical with those of corresponding human cytochrome P-450 cDNAs

In vivo regulation of gene expression of enzymes controlling aldosterone synthesis in rat adrenal

We studied the effect of alterations in the intake of sodium and potassium as well as changes in circulating adrenocorticotropin (ACTH) on the expression of the two rate-limiting systems of aldosterone formation in the rat. Low sodium and high potassium intake promoted time-dependent increases in the zona glomerulosa cytochrome P450scc ( P450scc) and cytochrome P450c11 ( P450c11) protein and mRNA levels, but no changes were found in the zona fasciculata-reticularis. In addition, these responses were associated with markedly elevated transcriptional activities. To further define the contribution of P450c11 and P450c18 (aldosterone synthase) in response to these differing intakes, we evaluated their mRNA levels using gene-specific oligonucleotide probes. P450c18 mRNA was restricted to the zona glomerulosa, whereas P450c11 mRNA was detected in both zona glomerulosa and zona fasciculata-reticularis. Furthermore, only P450c18 mRNA was induced by both low sodium or high potassium intake, as P450c11 mRNA levels remained unchanged. Captopril, an inhibitor of angiotensin-I converting enzyme, abolished the enhancing effects of the low sodium regimen on P450scc and P450c18 mRNA levels. Captopril also suppressed the augmentation of P450c18 mRNA observed with potassium supplementation but had no effect on P450scc mRNA levels. When the hypocholesterolemic drug 4-aminopyrazolopyrimidine (4-APP) was administered to rats for 3 consecurive days, both the level of plasma ACTH and the adrenal content of mRNA encoding P450scc increased 24 h post final injection. The coadministration of dexamethasone with 4-APP prevented these increases. In contrast, the mRNA content of P450c11 remained at control levels. In conclusion, this work demonstrates that variations in the intake of sodium and potassium act on the expression of the CYP11B2 gene, but not on that of the CYP11B1 gene. Moreover angiotensin-II (A-II) is an important factor in this mechanism of action. Both ions also enhance the expression of the CYP11A1 gene. A-II appears to participate in the mechanism of action of the low sodium intake at this level. Another mechanism is postulated for the action of potassium supplementation since captopril did not prevent the increased expression of the CYP11A1 gene. In addition, the fact that 4-APP enhanced the mRNA level of P450scc but not that of P450c, also demonstrates different regulation of the P450s involved at the early and final steps of aldosteroone formation in the rat adrenal zona glomerulosa in vivo.

Interleukin-1 inhibits Leydig cell steroidogenesis primarily by decreasing 17 alpha-hydroxylase/C17-20 lyase cytochrome P450 expression.

Macrophage-secreted cytokines, such as interleukin-1 (IL-1), have been shown to modulate Leydig cell function. The present study examined the effect of recombinant murine IL-1 alpha on Leydig cell steroidogenesis and its mechanism of action. Addition of IL-1 to macrophage-depleted primary cultures of mouse Leydig cells caused a dose-dependent decrease in cAMP-dependent testosterone production and 17 alpha-hydroxylase/C17-20 lyase (P450c17) mRNA levels. Chronic treatment (48 h) of Leydig cells in culture with 50 microM 8-bromo-cAMP (8-Br-cAMP) resulted in a 60-fold increase in testosterone production. Treatment with 8-Br-cAMP plus 0.2 and 2 U/ml IL-1 decreased testosterone production to 63 +/- 14% and 41 +/- 19%, respectively, while 5, 10, and 20 U/ml IL-1 decreased testosterone production to less than 5% that of cells treated with 8-Br-cAMP alone. Chronic treatment with IL-1 plus 8-Br-cAMP caused a shift in steroid production from testosterone to progesterone, but total steroid production (the sum of testosterone plus progesterone) was unaffected by IL-1 treatment. Treatment with 8-Br-cAMP alone caused a marked increase in P450c17 mRNA levels compared to that in control cultures, where P450c17 mRNA was undetectable. IL-1 caused a dose-dependent decrease in 8-Br-cAMP-stimulated P450c17 levels (0.2 U/ml by 31 +/- 9%, 2 U/ml by 82 +/- 12%, and 10 or 20 U/ml by 100% compared to that in cells treated with 8-Br-cAMP alone). In contrast to the effect on P450c17 mRNA, only the highest concentrations of IL-1 (10 and 20 U/ml) had any effect on cholesterol side-chain cleavage enzyme (P450scc) mRNA levels (53 +/- 16% and 38 +/- 20% decreases, respectively). The inhibitory effect of IL-1 on 8-Br-cAMP-stimulated P450c17 expression was reversible. Within 12 h after the removal of IL-1, P450c17 mRNA was restored to 24%; after 24 h, to 36%; after 36 h, to 65%; and after 48 h, to 84% of that with 8-Br-cAMP alone. P450c17 expression was more sensitive to IL-1-mediated inhibition than P450scc; therefore, inhibition of P450c17 is most likely primarily responsible for the observed inhibitory effects of IL-1 on Leydig cell testosterone production.