Colin D. Clyne

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...

Ca2+-Regulated Expression of Aldosterone Synthase Is Mediated By Calmodulin and Calmodulin-Dependent Protein Kinases

The chronic maintenance of aldosterone production in the adrenal zona glomerulosa is associated with increased expression of aldosterone synthase (P450aldo), the enzyme responsible for the conversion of 11-deoxycorticosterone to aldosterone. The major physiologic regulators of aldosterone production are angiotensin II (ANG II) and (K+) which act in part through increasing intracellular calcium ([Ca2+]i). Recently we demonstrated that increased [Ca2+]i is associated with K+ induction of P450aldo expression. To determine whether Ca2+ regulation of P450aldo is mediated through calmodulin or calmodulin-dependent kinases (CaMK), we investigated the actions of calmidazolium (a calmodulin inhibitor) and KN93 (an inhibitor of CaMK) on expression of P450aldo in human adrenocortical H295R cell line. Treatment with either calmidazolium or KN93 completely inhibited K+-stimulated expression of P450aldo mRNA with little effect on ANG II or dibutyryl cyclic AMP-stimulated induction of this transcript. Cellular calcium l...

Potassium negatively regulates angiotensin II type 1 receptor expression in human adrenocortical H295R cells

We have previously shown that the human adrenocortical H295R cell line expresses the type 1 angiotensin II receptor (AT1-R) and that expression of this receptor is downregulated at the level of mRNA by forskolin or dibutyryl-cAMP as well as by angiotensin II (Ang II). In this study we examine the effects of K+ on both AT1-R mRNA and receptors, as monitored through 125I-Ang II binding in the presence of PD 123319. After treatment with a maximal stimulatory steroidogenic dose of K+ (14 mmol/L), H295R cells showed an increase in cytosolic free Ca2+ from 113 to 212 nmol/L. Unlike the effects of Ang II, this increase could be abolished by pretreatment with the Ca2+ channel antagonist nifedipine (1 mumol/L). AT1-R mRNA levels also fell in response to elevated extracellular K+ in a dose-dependent (Kd, 9 mmol/L; maximal fall in message at 12 mmol/L) and time-dependent (maximum 50% at 12 hours) manner. The change in AT1-R mRNA level was less rapid than that in response to activation of phosphoinositidase C by Ang II or adenylyl cyclase by forskolin or by dibutyryl-cAMP. Unlike the action of Ang II but similar to the action of forskolin or dibutyryl-cAMP, the action of K+ was sustained. Changes in mRNA level in response to treatment with K+, Ang II, or dibutyryl-cAMP were also paralleled by changes in 125I-Ang II binding in each case.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of transforming growth factor-β on steroidogenesis and expression of key steroidogenic enzymes with a human ovarian thecal-like tumor cell model

OBJECTIVE Our purpose was to determine the effects of transforming growth factor-beta on steroidogenesis and regulation of steroidogenic enzyme expression by use of a human ovarian thecal-like tumor cell culture system. STUDY DESIGN Human ovarian thecal-like tumor cells were treated in serum-free medium in the presence or absence of forskolin and transforming growth factor-beta 1. The accumulation of progesterone and androstenedione in the culture medium was evaluated by radioimmunoassay. The effects of forskolin with or without transforming growth factor-beta 1 on the enzymatic activity of P450c17 and 3 beta HSD, the expression of immunodetectable P450c17 protein, and the expression of messenger ribonucleic acid for P450scc, P450c17, and 3 beta HSD were determined. RESULTS Basal steroid secretion, steroidogenic enzyme activity, enzyme protein, and messenger ribonucleic acid expression were not affected by transforming growth factor-beta 1 alone. Forskolin treatment significantly stimulated steroid production and the enzymatic activity of P450c17 and 3 beta HSD up to 10-fold above basal levels. However, transforming growth factor-beta 1 inhibited forskolin-stimulated androstenedione production to near basal levels and increased progesterone 1.4- to 2-fold while suppressing P450c17 enzyme activity to near basal levels, but it did not affect 3 beta HSD activity. Forskolin-stimulated immunodetectable P450c17 alpha protein was markedly inhibited by transforming growth factor-beta 1. In addition, transforming growth factor-beta 1 markedly inhibited the forskolin-stimulation of P450c17 messenger ribonucleic acid, while not significantly altering P450scc or 3 beta HSD messenger ribonucleic acid expression. CONCLUSION Forskolin stimulated human ovarian thecal-like tumor cell steroidogenesis, P450c17 and 3 beta HSD activity, immunodetectable P450c17, and messenger ribonucleic acid content for P450scc, P450c17, and 3 beta HSD. Transforming growth factor-beta 1 inhibited forskolin stimulation of androstenedione production through the inhibition of P450c17 expression.

The effects of KN62, A Ca2+/Calmodulin-dependent protein kinase II inhibitor, on adrenocortical cell aldosterone production

The effects of KN62 on aldosterone secretion have been studied using an angiotensin II (AII)- and K(+)-responsive human adrenocortical tumor cell line (H295R). Basal aldosterone secretion (measured by RIA) was 0.57 +/- 0.22 pmol/mg protein.h. The physiologicial agonists AII (10 nM) and K+ (14 mM) increased aldosterone secretion by 6.9- and 5.0-fold, respectively. Aldosterone secretion was also stimulated by dibutyryl cyclic AMP (dbcAMP, 1 mM, 10.3-fold over basal). Nifedipine dose-dependently inhibited K(+)- and AII-stimulated aldosterone secretion. In contrast, dbcAMP-stimulated secretion was relatively insensitive to this agent (26.8% inhibition at 1 microM nifedipine). K(+)- and AII-stimulated aldosterone production was also dose-dependently inhibited by KN62, which produced 93.9% and 82.3% inhibition at 10 microM KN62 (both p < 0.01). In order to test the specificity of KN62 in H295R cells, its effects on various other steroidogenic agonists were assessed. KN62 dose-dependently inhibited aldosterone secretion stimulated by dbcAMP, 22-hydroxycholesterol and pregnenolone. In addition, KNO4, a derivative of KN62 which is not a potent inhibitor of CaM Kinase II, exhibited a similar pattern of inhibition. These data confirm the requirement for extracellular Ca2+ in the stimulation of human adrenocortical cell aldosterone secretion by AII and K+. However, the non-specific inhibitory effects of KN62 in H295R cells limit the usefulness of this agent as a tool for investigations of the involvement of CaM kinase II in adrenocortical steroidogenesis.

The M3 muscarinic receptor mediates acetylcholine-induced cortisol secretion from bovine adrenocortical zona fasciculata/reticularis cells.

In order to characterize the receptor subtype mediating acetylcholine (ACh)-induced cortisol secretion from purified bovine adrenocortical zona fasciculata/reticularis cells in primary culture, the potencies of a range of selective muscarinic antagonists of ACh-induced steroidogenesis were assessed by Schild analysis. Basal secretion of cortisol was 10.2 +/- 1.4 pmol/well/30 min. ACh stimulated a dose-dependent increase in cortisol secretion and was maximally effective at 10(-5) M, at which concentration cortisol secretion was 143.4 +/- 12.9 pmol/well/30 min. Hexahydro-sila-difenidol and para-fluoro-hexa-hydro-sila-difenidol were potent competitive antagonists of ACh-stimulated cortisol secretion, with pA2 values of 8.68 +/- 0.28 and 7.96 +/- 0.29, respectively. Pirenzepine (pA2 = 6.95 +/- 0.28) and methoctramine (pA2 = 6.06 +/- 0.27) were relatively weak competitive antagonists. The pA2 values determined in this study are characteristic of the M3 muscarinic receptor, and we conclude that this receptor subtype mediates ACh-induced cortisol secretion from bovine zona fasciculata/reticularis cells.

Studies of hormone-sensitive and -insensitive pools of phosphoinositides in cultured bovine zona fasciculata/reticularis cells: Evidence that acetylcholine and angiotensin II stimulate the breakdown of a common pool of phosphoinositides

The effects of acetylcholine (ACh) and manganese pre-incubation on angiotensin II (AII)-stimulated incorporation of [3H]inositol into phosphoinositide, phosphoinositol and free inositol fractions of adrenocortical cells isolated from the bovine zona fasciculata/reticularis (zfr) were investigated. In cells pre-labelled for 6 hr with [3H]inositol, ACh and AII stimulated the incorporation of cytosolic [3H]inositol into a common hormone-sensitive pool of phosphoinositides, which was distinct from the non-hormone-sensitive pool labelled in the presence of manganese. Regression analysis of the cortisol versus [3H]inositol headgroup responses for both AII (10(-11)-10(-7) M) and ACh (10(-9)-10(-3) M) showed that the gradients of these responses were not significantly different. These data provide strong evidence that in cultured bovine zfr cells, ACh and AII stimulate the breakdown and resynthesis of a common pool of phosphoinositides.