Association between omega-3 fatty acids and serum prostate-specific antigen.

We examined the association between omega-3 fatty acids (O3FAs) and prostate-specific antigen (PSA) in a cross-sectional analysis of 6219 men examined at the Cooper Clinic from 2009 to 2013. We assayed O3FAs from red blood cell membranes and measured PSA levels in study participants. Multiple logistic regression was used to examine the association between O3FAs and PSA. The mean age of study participants was 55.5 years (SD = 9.8) with a mean PSA level of 1.31 ng/mL (SD = 1.5). Unadjusted analyses indicated that there was a slight, direct association with PSA and each of the O3FAs tested. However, after adjusting for age and body mass index (BMI), the associations were reversed but nonsignificant [odds ratio (OR) for PSA > 4 ng/mL: total omega-3 OR = 0.98 per each 1% of total fatty acids, 95% confidence interval (CI) = 0.93-1.03; docosahexaenoic acid OR = 1.01, 95% CI = 0.92-1.11; omega-3 index OR = 0.99, 95% CI = 0.93-1.05). Similar results were obtained after age and BMI adjustment when the omega-3 index was divided into undesirable (0.01-3.99%), intermediate (4.0-7.99%), and desirable ranges ( ≥ 8.0%). Given that the study had >80% power to detect an odds ratio <0.9 or >1.1, we conclude that associations between O3FAs and PSA levels are either nonexistent or quite weak in the population that this healthy sample represents.

The AP-1 family member FOS blocks transcriptional activity of the nuclear receptor steroidogenic factor 1.

Steroid production in the adrenal zona glomerulosa is under the control of angiotensin II (Ang II), which, upon binding to its receptor, activates protein kinase C (PKC) within these cells. PKC is a potent inhibitor of the steroidogenic enzyme CYP17. We have demonstrated that, in the ovary, PKC activates expression of FOS, a member of the AP-1 family, and increased expression of this gene is linked to CYP17 downregulation. However, the pathway and the molecular mechanism responsible for the inhibitory effect of PKC on CYP17 expression are not defined. Herein, we demonstrated that Ang II inhibited CYP17 through PKC and ERK1/2-activated FOS and that blocking FOS expression decreased PKC-mediated inhibition. Although CYP17 transcription was activated by the nuclear receptor SF-1, expression of FOS resulted in a decrease in SF-1-mediated gene transcription. FOS physically interacted with the hinge region of SF-1 and modulated its transactivity, thus preventing binding of cofactors such as SRC1 and CBP, which were necessary to fully activate CYP17 transcription. Collectively, these results indicate a new regulatory mechanism for SF-1 transcriptional activity that might influence adrenal zone-specific expression of CYP17, a mechanism that can potentially be applied to other steroidogenic tissues.

Insulin-like growth factor-I and fibroblast growth factor, but not growth hormone, affect growth plate chondrocyte proliferation.

Of the many factors that regulate linear growth, IGF-I has a central role in epiphyseal chondrocyte development. Whether IGF-I is solely of systemic or also of local origin is uncertain, as is how other growth factors interact with IGF-I at the growth plate. We studied the proliferative effects of IGF-I on juvenile bovine epiphyseal chondrocytes fractionated by density gradient centrifugation. Cell density correlated with size, glycogen content, and gene expression patterns. There was a gradient of response to IGF-I, with the greatest proliferative response in high-density cells corresponding to the reserve zone, as measured by [3H]thymidine uptake. Low-density (hypertrophic zone) cells proliferated only when exposed to IGF-I and basic fibroblast growth factor (FGF). The gradient of IGF-I response correlated with [125I]IGF-I binding as determined by Scatchard analysis: IGF-I receptor number was 10-fold greater in reserve zone cells than in hypertrophic cells. When exposed to basic FGF for 24 hours, IGF-I binding in hypertrophic cells increased 3-fold. In contrast, no specific binding of GH was demonstrated in juvenile bovine chondrocytes. GH produced neither signal transducer and activator of transcription phosphorylation, increased proliferation, nor increased IGF-I mRNA levels in any chondrocyte fraction. IGF-I mRNA levels were extremely low at 800-1100 copies/microg 18S RNA in bovine chondrocytes. These results suggest that the major regulator of chondrocyte proliferation is systemic IGF-I; FGFs may influence the actions of IGF-I at the growth plate by altering its receptor number in chondrocytes.

Expression profiles for steroidogenic enzymes in adrenocortical disease.

CONTEXT: Excess production of aldosterone or cortisol has profound effects on cardiovascular function and impacts other major organ systems. The mechanisms leading to the autonomous hypersecretion of aldosterone or cortisol in aldosterone-producing adenoma (APA) or cortisol-producing adenoma (CPA) are unknown. OBJECTIVE: The objective of this study was to compare the expression profiles of several steroid-metabolizing enzymes and transcription factors from normal adrenal (NA), APAs, and CPAs. DESIGN: RNA from NAs, APAs, and CPAs were analyzed by microarray and real-time RT-PCR. SETTING: This study was performed at academic research laboratories. PATIENTS: At least nine normal controls and 12 patients with APA or CPA were studied. INTERVENTION: There was no intervention procedure. MAIN OUTCOME MEASURE: The main outcome measure was the expression of steroidogenic enzymes in adrenocortical disease. RESULTS: A microarray indicated a greater than 3-fold increase in the expression of CYP11B2 (aldosterone synthase) in APA, whereas 11beta-hydroxysteroid dehydrogenase type 2 (HSD11B2) and HSD17B1 had greater than 3-fold increases in expression in CPA compared with NA. Real-time RT-PCR showed that APAs produced higher levels of HSD3B2, CYP21 (21-hydroxylase), and CYP11B2 mRNA, whereas CPAs produced higher levels of CYP11A (cholesterol side-chain cleavage), CYP17 (17alpha-hydroxylase/17-20 lyase), HSD3B2, and CYP11B1 (11beta-hydroxylase) mRNA compared with normal adrenal. Steroidogenic factor-1, DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome gene 1), and GATA-6 were expressed at higher levels in APAs and CPAs, whereas NURR1 was expressed at higher levels in APAs than in CPAs or NAs. CONCLUSION: Elevated production of aldosterone in APAs and of cortisol in CPAs is associated with increased expression of enzymes needed for corticosteroid production along with alterations in transcription factors that enhance the expression of steroid-metabolizing enzymes.

The orphan nuclear receptors NURR1 and NGFIB regulate adrenal aldosterone production.

Aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex is regulated by transcription of CYP11B2 (encoding aldosterone synthase). The effects of nerve growth factor-induced clone B (NGFIB) (NR4A1), Nur-related factor 1 (NURR1) (NR4A2), and steroidogenic factor-1 (SF-1) (NR5A1) on transcription of human CYP11B2 (hCYP11B2) and hCYP11B1 (11 beta-hydroxylase) were compared in human H295R adrenocortical cells. hCYP11B2 expression was increased by NGFIB and NURR1. Although hCYP11B1 was activated by SF-1, cotransfection with SF-1 inhibited activation of hCYP11B2 by NGFIB and NURR1. NGFIB and NURR1 transcript and protein levels were strongly induced by angiotensin (Ang) II, the major regulator of hCYP11B2 expression in vivo. Sequential deletion and mutagenesis of the hCYP11B2 promoter identified two functional NGFIB response elements (NBREs), one located at -766/-759 (NBRE-1) and the previously studied Ad5 element at -129/-114. EMSAs suggested that both elements bound NGFIB and NURR1. In human adrenals, NURR1 immunoreactivity was preferentially localized in the zona glomerulosa and to a lesser degree in the zona fasciculata, whereas NGFIB was detected in both zones. The calmodulin kinase inhibitor KN93 partially blocked K(+)-stimulated transcription of NGFIB and NURR1. KN93 partially inhibited the effect of Ang II on NURR1 mRNA levels but did not modify the effect on expression of NGFIB. Mutation of the NBRE-1, Ad5, and Ad1/cAMP response element (CRE) cis-elements reduced both basal and Ang II-induced levels of hCYP11B2, demonstrating that all three elements are important for maximal transcriptional activity. Our results suggest that NGFIB and NURR1 are key regulators of hCYP11B2 expression and may partially mediate the regulation of hCYP11B2 by Ang II.

Nur-related factor 1 and nerve growth factor-induced clone B in human adrenal cortex and its disorders.

Nerve growth factor-induced clone B (NGFI-B; NR4A1) and Nur-related factor 1 (Nurr1; NR4A2) are members of NGFI-B family of orphan receptors. We recently demonstrated induction of CYP11B2 (aldosterone synthase) by Nurr1 and NGFI-B, suggesting possible important roles of these transcriptional factors in the regulation of adrenocortical steroidogenesis. Therefore, we immunolocalized Nurr1 and NGFI-B in various human adrenal specimens to study their biological significance. In nonpathological adrenal glands (n = 25), Nurr1 and NGFI-B immunoreactivities were detected at high levels in the fetal definitive zone or postnatal zona glomerulosa. NGFI-B immunoreactivity was increased according to development in the zona fasciculata, reaching a level similar to that in the zona glomerulosa in adult adrenal cortex. In adrenocortical neoplasms (n = 44), Nurr1 immunoreactivity was higher in aldosteronoma than in Cushing's adenoma or adrenocortical carcinoma. NGFI-B immunoreactivity was also higher in aldosteronoma than in adrenocortical carcinoma, but was not significantly different among the types of adenoma. Both Nurr1 and NGFI-B mRNA expressions were correlated with their immunoreactivities in adrenocortical neoplasms (n = 23), and mRNA expression of Nurr1 was significantly (P < 0.0001) associated with that of CYP11B2. These results suggest that the expression of Nurr1 and NGFI-B plays an important role in human adrenal cortex and its neoplasms, including possible regulation of steroidogenesis.

The regulation of aldosterone synthase expression.

Aldosterone, the primary human mineralocorticoid, is a major regulator of intravascular volume and blood pressure. The capacity of the adrenal gland to produce aldosterone is controlled, in large part, by the regulated transcription of CYP11B2, the gene encoding aldosterone synthase. Aldosterone synthase is responsible for the conversion of 11-deoxycorticosterone to aldosterone and is expressed only within the zona glomerulosa of the adrenal cortex. The development of new systems for in vitro studies of expression has helped define molecular mechanisms that regulate this enzyme and thus the capacity of the adrenal gland to produce aldosterone. Both potassium and angiotensin II (ANG II) increase intracellular calcium levels, which regulate expression of CYP11B2 through transcription factors that interact with defined sites in the 5'-flanking region of the gene.

SCF, BDNF, and Gas6 are regulators of growth plate chondrocyte proliferation and differentiation.

We previously demonstrated that bovine epiphyseal chondrocytes separated by density gradient centrifugation differ in proliferative response to IGF-I and IGF-I receptor number. To identify novel modifiers of IGF-I action at the growth plate, we used microarray analyses to compare bovine hypertrophic and reserve zones and identified several receptors differentially expressed across the growth plate: NTRK2 [receptor for brain-derived neurotrophic factor (BDNF)], KIT [receptor for stem cell factor (SCF)], and MER and AXL [two receptors for growth arrest-specific 6 (Gas6)]. The corresponding ligands were tested for their ability to stimulate either proliferation of isolated chondrocytes or differentiation in ATDC5 cells. Each factor inhibited IGF-I-mediated proliferation in isolated chondrocytes by attenuating ERK1/2 activation. SCF, BDNF, Gas6, and C-type natriuretic peptide promoted differentiation in ATDC5 cells, each factor producing different expression patterns for collagen X, collagen 2, aggrecan, and lysyl oxidase. Whereas multiple factors stimulated ATDC5 differentiation, only IGF-I and high-dose insulin, out of several factors implicated in chondrocyte maturation, stimulated proliferation of isolated chondrocytes. IGF-I appears to be the primary proliferative signal in growth plate chondrocytes, whereas multiple factors including SCF, BDNF, and Gas6 regulate the pace of differentiation at the growth plate.

A role for the NGFI-B family in adrenal zonation and adrenocortical disease.

The three zones of the human adrenal cortex are functionally distinct with the glomerulosa producing aldosterone, the fasciculata producing cortisol, and the reticularis producing DHEA/DHEAS. This functional zonation is largely due to the zone-specific expression of steroidogenic enzymes. Recent evidence suggests a role for the NGFI-B family of orphan nuclear receptors (particularly NURR1 and NGFI-B) in the zone-specific expression of two key steroidogenic enzymes, aldosterone synthase (CYP11B2) and 3beta-hydroxysteroid dehydrogenase (HSD3B2). Herein we discuss the evidence that suggests a role for NURR1 (NR4A2) in the expression of CYP11B2 in the glomerulosa as well as in the dysregulation of CYP11B2 gene expression as is seen in aldosterone-producing adenoma (APA), a major cause of endocrine hypertension. NURR1 appears to be important for CYP11B2 transcription and is found at higher levels in glomerulosa and in APA. Its expression in adrenal cells is also readily increased by angiotensin II treatment. HSD3B2 is a steroid-metabolizing enzyme that is essential for adrenal production of mineralocorticoids and glucocorticoids. Thus, HSD3B2 is expressed at high levels in the glomerulosa and fasciculata where these steroids are produced but at low levels in the adrenal reticularis, which produces mainly DHEA. We recently demonstrated that NGFI-B (nur77 or NR4A1) plays an important role in the regulation of HSD3B2 transcription and may play an important role in the functional zonation of the adrenal gland. Immunohistochemistry confirmed that, within adult and fetal adrenal gland, NGFI-B expression paralleled expression of HSD3B2. Transient transfections demonstrated that NGFI-B family members enhanced HSD3B2 reporter activity but had no effect on a 17alpha-hydroxylase (CYP17) promoter construct. Taken together these results suggest that the NGFI-B family of transcription factors plays a role in establishing the functional zonation of the human adrenal by regulating CYP11B2 and HSD3B2 gene transcription.

The orphan nuclear receptor NGFIB regulates transcription of 3beta-hydroxysteroid dehydrogenase. implications for the control of adrenal functional zonation.

3beta-Hydroxysteroid dehydrogenase type 2 (HSD3B2) is a steroid-metabolizing enzyme that is essential for adrenal production of mineralocorticoids and glucocorticoids. Thus, HSD3B2 is expressed at high levels in the glomerulosa and fasciculata, where these steroids are produced. In contrast, the production of dehydroepiandrosterone (DHEA) and DHEA sulfate in the adrenal reticularis is inversely correlated with the expression of HSD3B2. The reasons for the zonal expression of HSD3B2 are not known but represent an important aspect in the biochemical zonation of the adrenal. Using microarray, real time reverse transcriptase-PCR, immunohistochemistry, and HSD3B2 promoter analysis, we demonstrate that the NGFIB family of nuclear hormone receptors plays a critical part in the regulation of HSD3B2 transcription and may play an important role in the functional zonation of the adrenal gland. Microarray analysis of cortisol- versus DHEA sulfate-producing adrenal tissue demonstrated that NGIFB paralleled expression of HSD3B2 with expression much higher in cortisol-producing adrenal tissue; this observation was also demonstrated using real time reverse transcriptase-PCR analysis. In addition, immunohistochemistry confirmed that within adult and fetal adrenal gland NGFIB expression paralleled expression of HSD3B2. Transient transfections into H295R adrenal cells demonstrated that NGFIB family members enhanced HSD3B2 reporter activity but had no effect on a 17alpha-hydroxylase (CYP17) promoter construct. Deletion and mutational analyses of the 5'-flanking region of the HSD3B2 gene identified a consensus NGFIB response element that bound NGFIB in mobility shift assays. Infection of cultured human adrenal cells with adenovirus-containing NGFIB increased cortisol production by 8-fold and increased expression of HSD3B2 mRNA 26-fold over that observed in mock-infected cells. In primary cultures of adrenal cells, ACTH, an activator of HSD3B2, rapidly induced expression of NGFIB. These results suggest that NGFIB plays a crucial role in adrenal zonation by regulating HSD3B2 gene transcription.