P-450 Messenger Research Articles

Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells

Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the rate-limiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol side-chain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL.

High endometrial aromatase P450 messenger RNA expression is associated with poor IVF outcome

Objective: The success of IVF treatment is thought to be dependent upon embryo quality and endometrial receptivity. Previous reports indicated that expression of aromatase P450 in the endometrium is restricted to women with proliferative reproductive tract disorders such as endometriosis, leiomyomas, and adenomyosis. The aim of this study was to determine if the level of endometrial aromatase P450 mRNA expression is a determinant of IVF outcome in infertile women.Design: A prospective multicenter case-control study.Setting: Eight European IVF units and a university center for reproductive research.Patients: A hundred twenty patients awaiting IVF treatment.Intervention(s): Endometrial biopsy prior to starting IVF treatment.Main Outcome Measures: Endometrial aromatase P450 and GAPDH transcripts were quantified by real-time RT-PCR using an ABI PRISM 7700 Sequence Detection System.Results: Aromatase P450 transcripts were detected in all endometria examined, although the levels varied considerably between samples ranging from 0.22 (arbitrary units, a.u.) to 486.6 a.u. (>2000 fold difference). The mean aromatase P450 mRNA level (±S.D.) in women who subsequently became pregnant after IVF treatment was 3.7 (±3.7) compared to 14.3 (±52.5) in patients with IVF failure (p > 0.05). The clinical pregnancy rate in women with high endometrial aromatase P450 mRNA levels (≥ 8.2 a.u.; n = 19) was 5.5% versus 22.8% in those patients with low expression levels (<8.2 a.u.; n = 101) (p < 0.05). The cycle day of the endometrial biopsy, cause of infertility, age, parity, number of oocytes collected, or number of embryos transferred did not differ between patients with high versus low endometrial aromatase P450 mRNA levels (p > 0.1).Conclusion: Quantitation of endometrial P450 mRNA levels is a potential diagnostic tool to identify women with high likelihood of IVF treatment failure. Objective: The success of IVF treatment is thought to be dependent upon embryo quality and endometrial receptivity. Previous reports indicated that expression of aromatase P450 in the endometrium is restricted to women with proliferative reproductive tract disorders such as endometriosis, leiomyomas, and adenomyosis. The aim of this study was to determine if the level of endometrial aromatase P450 mRNA expression is a determinant of IVF outcome in infertile women. Design: A prospective multicenter case-control study. Setting: Eight European IVF units and a university center for reproductive research. Patients: A hundred twenty patients awaiting IVF treatment. Intervention(s): Endometrial biopsy prior to starting IVF treatment. Main Outcome Measures: Endometrial aromatase P450 and GAPDH transcripts were quantified by real-time RT-PCR using an ABI PRISM 7700 Sequence Detection System. Results: Aromatase P450 transcripts were detected in all endometria examined, although the levels varied considerably between samples ranging from 0.22 (arbitrary units, a.u.) to 486.6 a.u. (>2000 fold difference). The mean aromatase P450 mRNA level (±S.D.) in women who subsequently became pregnant after IVF treatment was 3.7 (±3.7) compared to 14.3 (±52.5) in patients with IVF failure (p > 0.05). The clinical pregnancy rate in women with high endometrial aromatase P450 mRNA levels (≥ 8.2 a.u.; n = 19) was 5.5% versus 22.8% in those patients with low expression levels (<8.2 a.u.; n = 101) (p < 0.05). The cycle day of the endometrial biopsy, cause of infertility, age, parity, number of oocytes collected, or number of embryos transferred did not differ between patients with high versus low endometrial aromatase P450 mRNA levels (p > 0.1). Conclusion: Quantitation of endometrial P450 mRNA levels is a potential diagnostic tool to identify women with high likelihood of IVF treatment failure.

Aromatase P450 messenger RNA expression in eutopic endometrium is not a specific marker for pelvic endometriosis

Objective: To determine whether expression of aromatase P450 mRNA in eutopic endometrium is predictive of the presence of pelvic endometriosis. Design: A prospective, multicenter, observational study. Setting: Four tertiary centers for reproductive medicine. Patient(s): Sixty subjects of reproductive age undergoing laparoscopy for subfertility exploration, pain assessment, or sterilization. Intervention(s): Endometrial biopsy at time of laparoscopy. Main Outcome Measure(s): The expression of aromatase P450 mRNA in endometrial specimens was determined by single-tube reverse transcription-polymerase chain reaction (RT-PCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was amplified in parallel to exclude amplification failure. Result(s): The RT-PCR amplification was successful in 56 of the 60 biopsies (93%). Pelvic endometriosis was diagnosed in 34 patients (61%) and was strongly associated with aromatase P450 mRNA expression in eutopic endometrium. As a diagnostic marker for endometriosis, aromatase P450 mRNA expression yielded a sensitivity of 82%, a specificity of 59%, a positive predictive value of 76%, and a negative predictive value of 67%. If additional uterine pathology was taken in account, the sensitivity increased to 84%, the specificity to 72%, the positive predictive value to 87%, but the negative predictive value remained unchanged (67%). Conclusion(s): Although endometrial aromatase P450 gene expression is highly predictive of the presence of pelvic disease, the relative high incidence of false-negative results and lack of specificity is likely to impair clinical application.

Effects of dietary N-(4-hydroxyphenyl)retinamide on N-nitrosomethylbenzylamine metabolism and esophageal tumorigenesis in the Fischer 344 rat.

9-cis-Retinoic acid (9-cis-RA) and N-(4-hydroxyphenyl)retinamide (4-HPR) are effective chemopreventive agents against epithelial tumors in the oral cavity, breast, and prostate. We tested the inhibitory activity of these retinoids against N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in the rat esophagus. Male Fischer 344 rats were randomly assigned to receive diets either lacking or containing 9-cis-RA or 4-HPR for 1 week before tumor initiation with NMBA and then for the duration of the study. NMBA metabolism, O(6)-methylguanine adduct formation, and cytochrome P450 messenger RNA (mRNA) expression in the esophagi of the rats were studied to investigate the mechanisms by which dietary 4-HPR affects tumorigenesis. All statistical tests were two-sided. Dietary 4-HPR resulted in a dose-dependent and statistically significant enhancement (P<.05) of tumorigenesis in response to NMBA. In two different tumor bioassays, the mean tumor multiplicity for rats fed the highest concentration of dietary 4-HPR (0.8 g/kg diet) was increased by 5.9 tumors (95% confidence interval [CI] = 1.7 to 10.1 tumors) and 6.7 tumors (95% CI = 5.6 to 7.8 tumors) compared with the mean tumor multiplicity for rats that received the control diet lacking 4-HPR. Animals fed diets containing 9-cis-RA displayed no statistically significant increase in tumorigenesis. Compared with animals fed a diet lacking 4-HPR, animals fed 4-HPR had increased NMBA metabolism in esophageal explant cultures and had higher levels of O(6)-methylguanine DNA adducts and CYP2A3 mRNA in their esophagi. Dietary 4-HPR enhances tumorigenesis in response to NMBA in the rat esophagus by increasing tumor initiation events. Dietary 4-HPR may exert paradoxical effects at some sites, such as the aerodigestive tract, by modulating the bioactivation of carcinogens in target tissues.

Cellular observations and hormonal correlates of feedback control of luteinizing hormone secretion by testosterone in long-term castrated male rhesus monkeys.

Testosterone at physiological levels cannot exert negative feedback action on LH secretion in long-term castrated male monkeys. The cellular basis of this refractoriness is unknown. To study it, we compared two groups of male rhesus macaques: one group (group 1, n = 4) was castrated and immediately treated with testosterone for 30 days; the second group (group 2, n = 4) was castrated and treated with testosterone for 9 days beginning 21 days after castration. Feedback control of LH by testosterone in group 1 was normal, whereas insensitivity to its action was found in group 2. Using the endpoints of concentrations of aromatase activity (P450(AROM) messenger RNA [mRNA]) and androgen receptor mRNA in the medial preoptic anterior hypothalamus and in the medial basal hypothalamus, we found that aromatase activity in both of these tissues was significantly lower, P: < 0.01, in group 2 compared with group 1 males. P450(AROM) mRNA and androgen receptor mRNA did not differ, however. Our data suggest that the cellular basis of testosterone insensitivity after long-term castration may reside in the reduced capacity of specific brain areas to aromatize testosterone. Because P450(AROM) mRNA did not change in group 2 males, we hypothesize that an estrogen-dependent neural deficit, not involving the regulation of the P450(AROM) mRNA, occurs in long-term castrated monkeys.

Calcium ions positively modulate follicle-stimulating hormone- and exogenous cyclic 3',5'-adenosine monophosphate-driven transcription of the P450(scc) gene in porcine granulosa cells.

Given the evident modulation of FSH-induced steroidogenesis by Ca2+ in granulosa cells, we here test the hypothesis that Ca2+ controls expression of the enzymatically rate-limiting cytochrome P450(scc) (CYP11A) gene. To test this postulate, we quantitated the ability of Ca2+ to regulate: 1) transcriptional activity of a transiently transfected luciferase reporter gene driven by a 2.32-kb 5'-upstream fragment of the porcine P450(scc) gene promoter region; and 2) accumulation of endogenous P450(scc) transcripts in primary monolayer cultures of porcine granulosa cells. To this end, granulosa cells were stimulated for 4 h with FSH (15 ng/ml, NIDDK-oFSH-20) or 8-Bromo-cAMP (8 Br-cAMP, 1 mM) in serum-free medium containing either 1.8 mM Ca2- or no added Ca2+ with 100 microM EGTA or 100 microM CoCl2. In the presence of extracellular Ca2+, FSH and 8 Br-cAMP stimulated expression of the transfected P450(scc) promoter-reporter fusion construct by 5.6 +/- 1.1 and 3.6 +/- 0.67-fold, respectively over Ca2+-containing unstimulated control (P < or = 0.04, n = 5-6 experiments). The foregoing two agonists augmented 4-h progesterone production by cultured granulosa cells by 1.8 +/- 0.11 and 1.6 +/- 0.16-fold, respectively (P < or = 0.001 for FSH and P < or = 0.01 for 8 Br-cAMP). FSH and 8 Br-cAMP also significantly elevated endogenous P450(scc) transcript levels as measured by homologous solution-hybridization RNase protection assay; i.e. by 3.1 +/- 0.49 and 2.9 +/- 0.45-fold, respectively (P < or = 0.001). In Ca2+-free/EGTA-supplemented medium, basal luciferase reporter-gene activity and endogenous P450(scc) messenger RNA accumulation in granulosa cells declined to 34 +/- 12% and 78 +/- 12%, respectively, of corresponding values in control (unstimulated Ca2+-containing) cultures. Extracellular Ca2+ deprivation inhibited the stimulatory effect of FSH (and 8 Br-cAMP) on P450(scc) promoter-luciferase reporter expression to 58 +/- 30% (and 58 +/- 23%), and restrained endogenous P450(scc) message accumulation to 86 +/- 15% (and 96 +/- 18%) of the value in Ca2+-containing control. Extracellular Ca2+ withdrawal suppressed FSH (and 8 Br-cAMP)-driven progesterone production over 4 h to basal levels but did not alter FSH-stimulated cAMP accumulation by granulosa cells. Ca2+-deprived cells exposed to serum-containing media regained P450(scc) responsiveness to both agonists. Antagonism of cellular uptake of Ca2+ and other divalent cations via administration of cobalt chloride (100 microM) inhibited FSH and 8 Br-cAMP's stimulation of endogenous (but not exogenous promoter-driven) P450(scc) gene expression. In contrast, granulosa-cell concentrations of messenger RNA's encoding sterol-carrier protein-2 (SCP-2) and the low density lipoprotein receptor were not altered by Ca2+ withdrawal. In summary, uptake of extracellular Ca2+ by porcine granulosa cells significantly potentiates transactivation of the endogenously expressed and exogenously transfected P450(scc) gene by FSH and 8 Br-cAMP. The agonistic impact of Ca2+ on P450(scc) promoter activity is requisite downstream of FSH-induced cAMP second-messenger signaling.

Treatment of Severe Postmenopausal Endometriosis With an Aromatase Inhibitor

Objective: To treat an unusually aggressive case of recurrent postmenopausal endometriosis. Design: Case report. Setting: University of Texas Southwestern Medical Center (Dallas, Texas). Patient(s): A 57-year-old woman who presented with recurrent severe endometriosis after hysterectomy and bilateral salpingo-oophorectomy. Intervention(s): Oral administration of anastrozole (an aromatase inhibitor) (1 mg/d) and elemental calcium (1.5 g/d) for 9 months. Alendronate (a nonestrogenic inhibitor of bone resorption), 10 mg/d, was added to this regimen. Main Outcome Measure(s): Reduction in size of endometriotic lesion, pain relief, tissue levels of aromatase P450 messenger RNA, bone density. Result(s): Circulating levels of estradiol-17β were reduced to approximately 50% of the baseline value after the onset of treatment with anastrozole. Pain rapidly decreased and completely disappeared after the 2nd month of treatment. The 30 × 30 × 20-mm bright red polypoid vaginal lesion was reduced to a 3-mm gray tissue by the end of 9 months of treatment. Markedly high pretreatment levels of aromatase P450 messenger RNA in the endometriotic tissue became undetectable in a specimen obtained from a repeated biopsy after 6 months of treatment. Bone density of lumbar spine decreased by 6.2% after 9 months of treatment. Conclusion(s): This is the first description of the use of an aromatase inhibitor in the treatment of endometriosis. The short-term results were extraordinarily successful in elimination of pain and near-complete eradication of implants associated with severe endometriosis not responsive to other therapy. We conclude that the recently developed potent aromatase inhibitors are candidate drugs in the treatment of endometriosis that is resistant to standard regimens.

Basal steroidogenic activity of adrenocortical cells is increased 10-fold by coculture with chromaffin cells.

Historically, catecholamine-producing chromaffin cells and steroid-producing adrenocortical cells have been regarded as two independent endocrine systems that are united under a common capsule to form the adrenal gland. There is increasing evidence for bidirectional interactions, with regulatory influences of adrenocortical secretory products on adrenomedullary functions and vice versa. However, the direct involvement of chromaffin cells on the regulation and maintenance of cortical function has not yet been demonstrated. Therefore, we analyzed glucocorticoid secretion and P450 messenger RNA (mRNA) expression in bovine adrenocortical cells in cocultures with chromaffin cells compared with those in pure cortical cell cultures. Cortisol release from cortical cells in coculture with chromaffin cells was 10 times as high (mean +/- SEM, 1035 +/- 119%) as that from the same number of isolated cortical cells (100 +/- 11%). By a [3H]thymidine incorporation assay, it was demonstrated that this effect was not due to a higher proliferation rate. Northern analysis revealed an increasing expression of P450(17alpha) mRNA in the coculture from days 1-5, whereas in isolated cortical cells, P450(17alpha) mRNA decreased, leading to a 6-fold difference on day 5. Inhibitors of protein (cycloheximide) or RNA (actinomycin D) synthesis completely annulled the observed increase in cortisol release, indicating that de novo protein synthesis is required for this activation of adrenocortical steroidogenesis. Addition of the cyclooxygenase inhibitor indomethacin reduced the stimulatory effect, suggesting that this stimulation is in part mediated by PGs. Locally produced ACTH, catecholamines, and interleukin-1 accounted for 43% of the effect. Secretory products of chromaffin cells that act in concert are believed to be responsible for the stimulation of steroidogenesis in the coculture. The coculture system is an in vitro model that corresponds to the in vivo situation in the intact adrenal gland, where both endocrine cell systems are in close contact. Our data demonstrate the requirement of intraadrenal cellular communication for the full strength of the adrenocortical hormonal response.

Basal Steroidogenic Activity of Adrenocortical Cells Is Increased 10-Fold by Coculture with Chromaffin Cells

Historically, catecholamine-producing chromaffin cells and steroid-producing adrenocortical cells have been regarded as two independent endocrine systems that are united under a common capsule to form the adrenal gland. There is increasing evidence for bidirectional interactions, with regulatory influences of adrenocortical secretory products on adrenomedullary functions and vice versa. However, the direct involvement of chromaffin cells on the regulation and maintenance of cortical function has not yet been demonstrated. Therefore, we analyzed glucocorticoid secretion and P450 messenger RNA (mRNA) expression in bovine adrenocortical cells in cocultures with chromaffin cells compared with those in pure cortical cell cultures. Cortisol release from cortical cells in coculture with chromaffin cells was 10 times as high (mean ± sem, 1035 ± 119%) as that from the same number of isolated cortical cells (100 ± 11%). By a [3H]thymidine incorporation assay, it was demonstrated that this effect was not due to a higher proliferation rate. Northern analysis revealed an increasing expression of P45017α mRNA in the coculture from days 1–5, whereas in isolated cortical cells, P45017α mRNA decreased, leading to a 6-fold difference on day 5. Inhibitors of protein (cycloheximide) or RNA (actinomycin D) synthesis completely annulled the observed increase in cortisol release, indicating that de novo protein synthesis is required for this activation of adrenocortical steroidogenesis. Addition of the cyclooxygenase inhibitor indomethacin reduced the stimulatory effect, suggesting that this stimulation is in part mediated by PGs. Locally produced ACTH, catecholamines, and interleukin-1 accounted for 43% of the effect. Secretory products of chromaffin cells that act in concert are believed to be responsible for the stimulation of steroidogenesis in the coculture. The coculture system is an in vitro model that corresponds to the in vivo situation in the intact adrenal gland, where both endocrine cell systems are in close contact. Our data demonstrate the requirement of intraadrenal cellular communication for the full strength of the adrenocortical hormonal response.

Regulation of 11?? -hydroxylase cytochrome P450 expression by cholesterol in spontaneously hypertensive rats

To investigate whether hypercholesterolaemia interferes with the expression of the enzymes involved in steroid biosynthesis in the adrenal cortex. Twenty-four 5-week-old male spontaneously hypertensive rats (SHR) were randomly assigned to a high (1%) cholesterol diet (n = 8) or to a matched cholesterol-free diet (n = 8) for 6 weeks. A third group (n = 8) was studied after 2 weeks of washout from the high-cholesterol intake. A cohort of age- and sex-matched normotensive Wistar-Kyoto (WKY) rats (n = 24) underwent the same treatments and was used as a control. In SHR cholesterol feeding reduced urinary sodium excretion (0.8 +/- 0.1 versus 1.4 +/- 0.1 mmol/24 h in the cholesterol-free group), increased plasma aldosterone levels (299 +/- 60 versus 154 +/- 24) and reduced plasma corticosterone levels (142 +/- 21 versus 278 +/- 35 ng/ml). Those responses were associated with a reduction of 11 beta-hydroxylase cytochrome P450 messenger RNA (mRNA) in the adrenal cortex (-52.3 +/- 3.4%) whereas aldosterone synthase mRNA remained unchanged. That effect and the changes in electrolyte excretion and steroid levels were no longer detectable after withdrawal of the diet. In WKY rats high-cholesterol diet induced no significant changes in urinary electrolyte excretion, steroid levels and expression of 11 beta-hydroxylase cytochrome P450 and aldosterone synthase in the adrenals. The present results indicate that in SHR hypercholesterolaemia selectively interferes with the adrenal steroid biosynthetic pathway by reducing the expression of 11 beta-hydroxylase, leading to accumulation of mineralocorticoids and sodium retention.

Regulation by retinoids of luteinizing hormone/chorionic gonadotropin receptor, cholesterol side-chain cleavage cytochrome P-450, 3β-hydroxysteroid dehydrogenase/Δ 5−4-isomerase and 17α-hydroxylase/C 17–20 lyase cytochrome P-450 messenger ribonucleic acid levels in the K9 mouse Leydig cell line

Vitamin A is a potent regulator of testicular function. We have reported that retinol (R) and retinoic acid (RA) induced a down regulation of luteinizing hormone/human chorionic gonadotropin (LH/CG) binding sites in K9 Leydig cells. In the present study we evaluated the effect of R and RA on LH/CG receptors, cholesterol side-chain cleavage cytochrome P-450 (P-450 scc), 17α-hydroxylase/C 17–20 lyase (P-450 17α) and 3β-hydroxysteroid dehydrogenase (3β HSD) mRNA levels in K9 mouse Leydig cells. To validate K9 cells as a model for studying Leydig cell steroidogenesis at the molecular level, we first investigated the effect of hCG on mRNA levels of the steroidogenic enzymes. P-450 scc, 3β HSD and P-450 17α were expressed constitutively. The addition of 10 ng/ml hCG enhanced mRNA levels for the three genes within 2 h. Maximal accumulation of P-450 scc, P-450 17α and 3β HSD mRNA in treated cells represents a 2.5-, 8.5- and 4-fold increase over control values, respectively. P-450 17α expression reached a maximum by 4 h and then declined rapidly to return to control value by 24 h. The pattern of LH/CG receptor mRNAs in K9 cells was very similar to that of MA10 Leydig cells and showed six transcripts of 1.1, 1.6, 1.9, 2.6, 4.2 and 7.0kb. Treatment of cells with R or RA resulted in a time- and dose-dependent decrease in all six species. Comparing the time course of the reduction in mRNA levels and in binding activity, we postulate that retinoids down regulate LH/CG receptors primarily by reducing their mRNAs. Addition of R or RA greatly enhanced P-450 17α mRNA levels in a time- and dose-dependent manner with maximal induction at 12 h (about a 5-fold increase) using 10 −6 M R or RA. The level of P-450 17α mRNA remained high throughout the treatment. Under the same conditions, 3β HSD mRNA levels were repressed but the effect was long term (70% decrease after 8 days with 10 −6 M R or RA). On the contrary, P-450 scc expression was not affected.

Pre- and postnatal ontogeny of aromatase cytochrome P450 messenger ribonucleic acid expression in the male rat brain studied by in situ hybridization.

A central step in the development and sexual differentiation of the brain is the intraneuronal conversion of testosterone to estrogen. This conversion is catalyzed by an enzyme complex comprised of cytochrome P450 aromatase and the ubiquitous NADPH-dependent cytochrome P450 reductase. Brain aromatase activity is developmentally regulated and expressed only in specific hypothalamic and limbic structures. To determine whether developmental change in aromatase activity are the result of differential regulation of aromatase gene expression, we designed two specific oligonucleotides complementary to a 5'-region or to the heme-binding region of the rat aromatase complementary DNA. Relative amounts of messenger RNA (mRNA) encoding aromatase cytochrome P450 were examined on cryostat sections from male fetuses and offspring of time-pregnant Long-Evans rats using the in situ hybridization technique. On gestational day (GD) 16 (GD 1 = 24 h after mating), aromatase-specific mRNA was detected in the preoptic/hypothalamic area. On GD18 and GD20, aromatase mRNA expression was more abundant and more widespread. High levels of mRNA were detected in the medical preoptic nucleus, the sexually dimorphic nucleus of the preoptic area, the bed nucleus of stria terminalis, and the medial amygdala. Lower levels were found in the periventricular preoptic nucleus and the ventromedial hypothalamic nucleus. On postnatal day (PN) 2 and throughout further development (PN6, PN15, and adult), aromatase mRNA decreased to much lower levels in the preoptic area, but remained high in the bed nucleus of stria terminalis and amygdala. An intermediate situation, i.e. transient expression with a peak around birth, was observed for aromatase mRNA levels in the ventromedial hypothalamic nucleus. Interestingly, during early postnatal development, specific aromatase mRNA was also detected in the hippocampal formation as well as in the subfornical organ. In summary, these results demonstrate that the low quantities of aromatase transcripts in the developing rat brain can be detected by means of in situ hybridization. Depending on the particular region examined, aromatase expression appears to be subjected to different developmental programs. As the expression of aromatase mRNA parallels the results obtained from activity assays, it is likely that developmental changes in aromatase activity are the result of regulation of aromatase gene expression.

Pre- and postnatal ontogeny of aromatase cytochrome P450 messenger ribonucleic acid expression in the male rat brain studied by in situ hybridization

Pre- and postnatal ontogeny of aromatase cytochrome P450 messenger ribonucleic acid expression in the male rat brain studied by in situ hybridization

Involvement of Cytochrome P450 in Conferring Chloroquine Resistance to the Malarial Parasite, Plasmodium falciparum

The higher levels of cytochrone P-450 dependent enzyme activities reported earlier are traced to higher levels of cytochrome P-450 (CYPIIB1/B2 like) messenger RNA in the chloroquine resistant than the sensitive strains. The messenger RNA is also induced by phenobarbitone in the sensitive strain. Pretreatment with phenobarbitone affords partial protection to chloroquine toxicity in the sensitive strain and this is not due to a differential accumulation of the drug.

Effect of insulin-like growth factor-I on cholesterol side-chain cleavage cytochrome P450 messenger ribonucleic acid expression in ovarian theca-interstitial cells stimulated to differentiate in vitro

Currently available evidence supports the hypothesis that insulin-like growth factor-I (IGF-I) may play a role in stimulating ovarian theca-interstitial cell (TIC) differentiation in preantral follicles. The purpose of the present studies was to examine the potential role of IGF-I in TIC differentiation by determining the effects of IGF-I on cholesterol side-chain cleavage cytochrome P450 (P450 SCC) mRNA expression in TIC stimulated to differentiate in vitro. TIC were isolated from the ovaries of hypophysectomized immature rats by Percoll gradient centrifugation and cultured in the presence and absence of LH and IGF-I up to 6 days. At various times cytoplasmic RNA was extracted from the TIC and P450 SCC mRNA was measured by specific assay using reverse transcription followed by the polymerase chain reaction. Increasing concentrations of LH (0–1 μg/ml) stimulated a dose-related increase in P450 SCC mRNA (ED 50 = 36.2 ± 5.5 ng/ml) which reached maximal levels at 100 ng/ml of LH. Addition of IGF-I (30 ng/ml) caused a small increase in P450 SCC mRNA over TIC treated with LH alone but did not alter the ED 50 for LH stimulation. IGF-I alone also stimulated an increase in P450 SCC mRNA which reached approximately 3-fold over unstimulated levels at 100 ng/ml. In the presence of LH, IGF-I stimulated a dose-related increase in P450 SCC mRNA (ED 50 = 1.2 ± 0.05 ng/ml). Time-course studies revealed that expression of P450 SCC mRNA was greatest at 2 days in TIC treated with IGF-I alone, LH alone or LH plus IGF-I and then declined at 4 and 6 days. Detailed studies demonstrated that LH stimulated a rapid increase in progesterone production after an approximately 2 h lag. Progesterone levels reached maximal levels at 10 h and were maintained until approximately 20 h after which they began to decline. Although IGF-I alone did not alter progesterone production, concomitant treatment with LH and IGF-I caused progesterone to increase in the culture medium until 18 h when the levels were approximately 2-fold higher than in TIC treated with LH alone. After 20 h progesterone levels began to decrease. P450 SCC mRNA expression followed a pattern very similar to the pattern of progesterone production with two exceptions. First, IGF-I alone stimulated an approximately 3-fold increase in P450 SCC mRNA at 24 h of treatment which was sustained throughout the culture period. Second, maximal levels of P450 SCC mRNA in TIC treated with LH plus IGF-I were maintained until 30 h before a decline was observed. The results of our studies demonstrate that IGF-I stimulates the expression of P450 SCC mRNA in ovarian TIC and support the hypothesis that IGF-I may play a role in stimulating TIC differentiation in developing follicles.

Effects of 1,25-dihydroxyvitamin D3 and phorbol ester on 25- hydroxyvitamin D3 24-hydroxylase cytochrome P450 messenger ribonucleic acid levels in primary cultures of rat renal cells

The renal 25-hydroxyvitamin D3 24-hydroxylase enzyme, which may be the starting point in the catabolic pathway for vitamin D metabolism, is markedly induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the hormonal form of vitamin D. The purpose of this study was to investigate the regulation of the cytochrome P450 component of this enzyme (P450cc24) by 1,25-(OH)2D3 and phorbol 12-myristate 13-acetate (TPA). P450cc24 messenger RNA (mRNA) levels were measured using the full-length rat complementary DNA probe (p108). In primary cultures of rat renal tubular cells, 1,25-(OH)2D3 produced a 26-fold increase in P450cc24 mRNA which was detectable at 4 h, maximal at 24 h, and returned almost to baseline by 48 h. The induction was inhibited by actinomycin D, 5,6-dichloro-1-b-D-ribofuranosyl benzimidazole (DRB), and cycloheximide, and it was specific for vitamin D compounds containing a 1-hydroxyl group. TPA alone had no effect, but TPA in the presence of 1,25-(OH)2D3 produced an increase in P450cc24 mRNA within 30 min, and this increase peaked at 2 h. TPA also shifted the dose-response curve of 1,25-(OH)2D3 to the left, so that 1,25-(OH)2D3 was effective at a concentration as low as 1 nM. In the same experiments, TPA increased c-fos mRNA levels, and this increase was accelerated by 1,25-(OH)2D3. These studies suggest that the induction of P450cc24 mRNA by 1,25-(OH)2D3 is a receptor-mediated genomic event and that this induction may account for the stimulation of 24-hydroxylase enzyme activity by 1,25-(OH)2D3. In addition, TPA accelerates the effect of 1,25-(OH)2D3 by a mechanism which may involve protein kinase C.

Effects of 1,25-dihydroxyvitamin D3 and phorbol ester on 25-hydroxyvitamin D3 24-hydroxylase cytochrome P450 messenger ribonucleic acid levels in primary cultures of rat renal cells.

The renal 25-hydroxyvitamin D3 24-hydroxylase enzyme, which may be the starting point in the catabolic pathway for vitamin D metabolism, is markedly induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the hormonal form of vitamin D. The purpose of this study was to investigate the regulation of the cytochrome P450 component of this enzyme (P450cc24) by 1,25-(OH)2D3 and phorbol 12-myristate 13-acetate (TPA). P450cc24 messenger RNA (mRNA) levels were measured using the full-length rat complementary DNA probe (p108). In primary cultures of rat renal tubular cells, 1,25-(OH)2D3 produced a 26-fold increase in P450cc24 mRNA which was detectable at 4 h, maximal at 24 h, and returned almost to baseline by 48 h. The induction was inhibited by actinomycin D, 5,6-dichloro-1-b-D-ribofuranosyl benzimidazole (DRB), and cycloheximide, and it was specific for vitamin D compounds containing a 1-hydroxyl group. TPA alone had no effect, but TPA in the presence of 1,25-(OH)2D3 produced an increase in P450cc24 mRNA within 30 min, and this increase peaked at 2 h. TPA also shifted the dose-response curve of 1,25-(OH)2D3 to the left, so that 1,25-(OH)2D3 was effective at a concentration as low as 1 nM. In the same experiments, TPA increased c-fos mRNA levels, and this increase was accelerated by 1,25-(OH)2D3. These studies suggest that the induction of P450cc24 mRNA by 1,25-(OH)2D3 is a receptor-mediated genomic event and that this induction may account for the stimulation of 24-hydroxylase enzyme activity by 1,25-(OH)2D3. In addition, TPA accelerates the effect of 1,25-(OH)2D3 by a mechanism which may involve protein kinase C.

Brain aromatase cytochrome P-450 messenger RNA levels and enzyme activity during prenatal and perinatal development in the rat

Aromatase cytochrome P-450 (P-450 AROM) enzyme activity catalyzes the conversion of androgens to estrogens in specific brain areas. During development local estrogen formation is thought to influence the sexual differentiation of neural structures (i.e. increase neurite growth and establish neural circuitry) and modulate reproductive functions. This study was undertaken to investigate the ontogeny of the (P-450 AROM) enzyme and its messenger RNA (mRNA) in medial basal hypothalamic (MBH) and preoptic area (POA) tissue during late fetal and perinatal development of the rat. Aromatase activity in the MBH-POA was negligible before gestational day (GD) 16 (<0.1 pmol/h/mg protein), increased over 10-fold at GD 17 and continued to increase (over 5-fold) to peak levels at GD 19 (> 5.0 pmol/h/mg protein), and then declined to low levels at GD 22 and 2 days post-birth (≈ 1 pmol/h/mg protein). The profile of P-450 AROM mRNA in the MBH-POA tissue was characterized by a predominant 2.7 kilobase (kb) mRNA species, similar in size to the largest functional P-450 AROM mRNA observed in adult rat ovarian tissue. At GD 15, the P-450 AROM mRNA was undetectable; low but detectable levels were seen at GD 17, the abundance increased at later time points and remained at peak levels on GDs 18 through 20, decreased slightly by GD 22, and then declined further by 2 days post-birth. The developmental increase in P-450 AROM mRNA levels correlated with the ascending pattern of enzyme activity before GD 19, but the marked decrease in enzyme activity seen after GD 19 was not accompanied by a corresponding decline in mRNA levels. These findings provide evidence for the presence of P-450 AROM mRNA in neural tissue and suggest that increases in P-450 AROM mRNA levels determine, at least in part, enzymatic activity during the period of development where brain aromatase is increasing. In contrast, the perinatal decline in enzyme activity appears to be regulated by factors that operate via mechanisms other than inhibition of P-450 AROM gene expression.

Determination of aromatase cytochrome P450 messenger ribonucleic acid in human breast tissue by competitive polymerase chain reaction amplification

Determination of aromatase cytochrome P450 messenger ribonucleic acid in human breast tissue by competitive polymerase chain reaction amplification

Dietary potassium supplementation and sodium restriction stimulate aldosterone synthase but not 11 beta-hydroxylase P-450 messenger ribonucleic acid accumulation in rat adrenals and require angiotensin II production

Dietary potassium supplementation and sodium restriction stimulate aldosterone synthase but not 11 beta-hydroxylase P-450 messenger ribonucleic acid accumulation in rat adrenals and require angiotensin II production