Mary H. Bassett

The orphan nuclear receptor NGFIB regulates transcription of 3β-hydroxysteroid dehydrogenase: Implications for the control of adrenal functional zonation

3β-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 17α-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.

Transcriptional Regulation of Human 11β-Hydroxylase (hCYP11B1)

Steroid 11β-hydroxylase is a mitochondrial enzyme that catalyzes the conversion of deoxycortisol to cortisol. The gene encoding human 11β-hydroxylase (hCYP11B1) is expressed in the adrenal cortex under the control of circulating levels of ACTH. The current study was undertaken to define the cis-regulatory elements and transacting factors that regulate hCYP11B1 transcription. The hCYP11B1 5′-flanking DNA was studied using transient transfection of luciferase reporter constructs in NCI-H295R human adrenocortical cells. A cAMP analogue ((Bu)2cAMP) increased expression of a construct containing −1102 bp of hCYP11B1 5′-flanking DNA (pB1–1102). An element at position −71/−64 (TGACGTGA, previously termed Ad1) resembling a consensus cAMP response element (CRE) was required for maximal induction by cAMP. The Ad1 element bound several transcriptional factors in electrophoretic mobility shift assays, including CRE-binding protein, activating transcription factor-1 (ATF-1), and ATF-2, but only the ATF-2 complex migra...

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 3β‐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 17α‐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.

Regulation of human CYP11B2 and CYP11B1: comparing the role of the common CRE/Ad1 element.

In humans, the final steps in corticosteroid production results from the activity of aldosterone synthase in the glomerulosa and 11β-hydroxylase in the fasciculata. The regional expression of these isozymes is believed to result from transcriptional regulation of the aldosterone synthase (CYP11B2) and 11β-hydroxylase (CYP11B1) genes. Previous studies suggest that the primary cis-element needed for agonist enhanced transcription of the CYP11B genes shares high sequence similarity to a consensus cAMP Response Element (CRE). Here the role of the CRE/Adl was studied. Reporter constructs prepared with the 5′flanking DNA of hCYP11B2 and hCYP11B1 were transfected into NCI-H295R (H295R) adrenocortical tumor cells. Both hCYP11B2 and hCYP11B1 driven reporter constructs responded in a similar manner to treatment with angiotensin II, potassium, dbcAMP, or forskolin. Mutation of the hCYP11B1 CRE/Adl element decreased basal reporter expression and decreased response to agonist. Mutation of the hCYP11B2 CRE/Adl element caused a loss of basal expression but retained response to agonist suggesting a role for other cis-elements in hormonal regulation of hCYP11B2 In addition, both cis-elements were able to form complexes with in vitro prepared CRE binding (CREB) protein, activating transcription factor (ATF)-1 and ATF-2 in mobility shift assays. However, only the ATF-2 complex migrated similarly to a complex seen using H295R nuclear extract. Taken together these data suggest that the CRE/Adl element plays an important role in the transcriptional regulation of both hCYP11B genes but does not play an important role in the regional distribution of the two isozymes within the adrenal.

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.

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.