Interleukin-6 inhibits adrenal androgen release from bovine adrenal zona reticularis cells by inhibiting the expression of steroidogenic proteins.

Interleukin-6 (IL-6) is secreted by adrenocortical cells and modifies cortisol secretion. In this study, the effects of IL-6 on adrenal androgen release were investigated. The zona reticularis (ZR) was generally isolated from bovine adrenal glands by dissection. In select experiments, the intact adrenal cortex (ie, all 3 adrenocortical zones) was dissected from the adrenal glands. For androgen release experiments, ZR and intact adrenocortical cubes were dispersed into isolated cells, the cells cultured and exposed to IL-6 and/or adrenocorticotropic hormone (ACTH), and androgen release determined by radioimmunoassay. Basal and ACTH-stimulated androgen release from the ZR was inhibited by IL-6 in a concentration-dependent (10-1000 pg/mL) and time-dependent (4-24 h) manner (P < 0.01 by 1-way analysis of variance and the Bonferroni test). In contrast, IL-6 increased basal and ACTH-stimulated androgen release from mixed adrenocortical cells (P < 0.01). The mechanism of IL-6 inhibition of androgen release was investigated by exposing ZR strips to IL-6 and measuring the expression of the messenger RNA (mRNA) and protein of steroidogenic factors. Basal and ACTH-stimulated expression of the mRNA and protein for steroidogenic acute regulatory protein, cholesterol side chain cleavage enzyme, 3-ß-hydroxysteroid dehydrogenase type 2, steroid 17-α-hydroxylase/17,20 lyase/17,20 desmolase, and the nuclear factor steroidogenic factor 1 (SF-1), that stimulates steroidogenesis, were decreased by IL-6 (P < 0.01). In contrast IL-6 increased the mRNA and protein for dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1), a nuclear factor that inhibits steroidogenesis (P < 0.01). In summary, IL-6 decreased androgen release and the expression of steroidogenic factors in the ZR, and this decrease may be mediated in part through increasing DAX-1 and decreasing SF-1.

Bovine adrenal cells secrete interleukin-6 and tumor necrosis factor in vitro.

Interleukin-6 (IL-6) and tumor necrosis factor (TNF) are secreted and/or synthesized by the rat and human adrenal cortex. In this study, the release of IL-6 and TNF from bovine adrenal cells was determined. Bovine adrenal glands were collected from an abattoir and dissected into the zona glomerulosa (ZG), zona fasciculata (ZF), zona reticularis (ZR), and medulla. The tissues were enzymatically dispersed to single cells and cultured for 4-6 days. The cells were then exposed (4 h) to angiotensin II (AII), adrenocorticotrophic hormone (ACTH), phorbol dibutyrate (PDB), interleukin-1beta (IL-1beta), interleukin-1alpha (IL-1alpha), and endotoxin (LPS). The IL-6 and TNF content of the incubation medium was determined by bioassays. The release of IL-6 and TNF from the ZG, ZF, ZR, and medulla was increased by PDB, IL-1alpha, IL-1beta, and LPS. In contrast, ACTH and AII increased IL-6 release from the ZG, ZF, and ZR but had no effect on IL-6 release from the medulla. ACTH decreased TNF release from all adrenal cortical zones but had no effect on TNF release from the medulla. Immunohistochemistry utilizing antibodies against TNFalpha demonstrated TNFalpha-containing cells throughout the adrenal gland. The majority of the cells of the ZG, ZF, and ZR contained TNFalpha. However, the cells of the ZG contained more TNFalpha than the cells of the ZR or ZF. Small patches of TNFalpha-containing cells were also found in the adrenal medulla and capsule. These findings support the hypothesis that IL-6 and TNF may have autocrine/paracrine effects on the adrenal gland.

Possible function of IL-6 and TNF as intraadrenal factors in the regulation of adrenal steroid secretion.

Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF alpha) and their mRNAs are present in the human, rat, and bovine adrenal cortex. The release of these cytokines from adrenal cells is regulated by factors that alter adrenal function (e.g., ACTH, angiotensin II, interleukin-1). IL-6 and TNF type 1 receptors are also present on adrenocortical cells. Exposure to IL-6 increases cortisol or corticosterone release from human, bovine, and rat adrenal cells. IL-6 increases basal and ACTH-stimulated aldosterone release, but inhibits angiotensin II-stimulated aldosterone secretion from bovine adrenal cells. IL-6 increases dehydroepiandrosterone (DHEA) release from human cells, but decreases DHEA secretion from bovine cells. TNF alpha inhibits corticosterone release from normal rat adrenal cells or fragments, but increases corticosterone release from cholestatic rat adrenal slices. TNF alpha decreases cortisol release from bovine and fetal human adrenal cells, but increases cortisol release from adult human adrenal cells. TNF alpha inhibits aldosterone secretion from rat and bovine adrenocortical cells. TNF alpha does not affect DHEA secretion from fetal human adrenocortical cells, but inhibits basal and ACTH-stimulated DHEA release from bovine adrenal cell. Because IL-6 and TNF alpha are produced in the adrenal gland and modify adrenal steroid secretion, these cytokines may function as intraadrenal factors in the regulation of adrenal steroid secretion.

Stimulation by interleukin-6 and inhibition by tumor necrosis factor of cortisol release from bovine adrenal zona fasciculata cells through their receptors.

Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are synthesized and released from adrenal cells. Therefore, the effects of TNF-alpha and IL-6 on cortisol release from bovine zona fasciculata (ZF) cells were investigated. IL-6 (10-1000 pg/mL) significantly increased basal and adrenocorticotropic hormone (ACTH)-stimulated cortisol release in a concentration-dependent manner. This stimulatory effect of IL-6 became apparent at intervals as short as 4 h and continued through 24 h. IL-6 also potentiated the cortisol release stimulated by the adenylyl cyclase activator forskolin. By contrast, TNF-alpha (0.1-10 ng) inhibited basal and ACTH-stimulated cortisol release in a concentration-dependent manner. The inhibitory effects of TNF-alpha on cortisol release were significant at time intervals as short as 4 h and continued through 24 h. TNF-alpha inhibited forskolin-stimulated cortisol release. Binding studies demonstrated that ZF cells have IL-6 receptors (100 receptors/cell, Kd of 7.5 x 10(-11)) and TNF receptors (200 receptors/cell, Kd of 2.4 x 10(-9) M). Immunohistochemical analysis provided evidence that the majority of ZF cells have IL-6 receptors, TNF type 1 receptors, and TNF type 2 receptors. Because IL-6 and TNF-alpha are released from the adrenal cortex and these cytokines modify the release of cortisol from the ZF, IL-6 and TNF-alpha may play a paracrine or autocrine role in the regulation of adrenal function.

Gonadotropin-releasing hormone activates the equine luteinizing hormone beta promoter through a protein kinase C/mitogen-activated protein kinase pathway.

GnRH regulation of LH secretion is well understood and involves Ca(2+) mobilization. However, the mechanism by which GnRH activates transcription of the LHbeta gene is controversial. GnRH is known to elevate intracellular calcium and activate the protein kinase C (PKC) pathway. The present study evaluated the pathway(s) involved in GnRH induction of LHbeta transcription. We have previously reported that the equine LHbeta (eLHbeta -448/+60) promoter is active in alphaT3-1 cells. Therefore, we created a clonal, stably transfected alphaT3-1 gonadotroph cell line harboring the eLHbeta promoter (-448/+60) fused to the luciferase reporter gene. Administration of a GnRH agonist resulted in induction of promoter activity that was completely inhibited by the antagonist antide. Various calcium-affecting drugs had no effect on the promoter. Administration of phorbol 12-myristate 13-acetate (PMA) elicited an activation similar to, albeit lower than, that with GnRH. Down-regulation or pharmacological inhibition of PKC completely blocked PMA's induction of the promoter, while GnRH induction was only partly attenuated. Treatment with the mitogen-activated protein kinase (MAPK) kinase inhibitor, PD98059, completely inhibited the activation of eLHbeta by PMA but only partly diminished GnRH's induction. Expression of the transcription factor, early growth response protein 1 (Egr1), correlated completely with activation of MAPK, suggesting that Egr1 is the factor through which PKC/MAPK acts. Our data suggest that GnRH induces activity of the eLHbeta promoter by activating a signal transduction cascade involving PKC-MAPK-Egr1 but that has no significant requirement for calcium.

Early growth response protein 1 binds to the luteinizing hormone-beta promoter and mediates gonadotropin-releasing hormone-stimulated gene expression.

The hypothalamic neuropeptide, GnRH, regulates the synthesis and secretion of LH from pituitary gonadotropes. Furthermore, it has been shown that the LH beta-subunit gene is regulated by the transcription factors steroidogenic factor-1 (SF-1) and early growth response protein 1 (Egr1) in vitro and in vivo. The present study investigated the roles played by Egr1 and SF-1 in regulating activity of the equine LH beta-subunit promoter in the gonadotrope cell line, alpha T3-1, and the importance of these factors and cis-acting elements in regulation of the promoter by GnRH. All four members of the Egr family were found to induce activity of the equine promoter. The region responsible for induction by Egr was localized to the proximal 185 bp of the promoter, which contained two Egr response elements. Coexpression of Egr1 and SF-1 led to a synergistic activation of the equine (e)LH beta promoter. Mutation of any of the Egr or SF-1 response elements attenuated this synergism. Endogenous expression of Egr1 in alpha T3-1 cells was not detectable under basal conditions, but was rapidly induced after GnRH stimulation. Reexamination of the promoter constructs harboring mutant Egr or SF-1 sites indicated that these sites were required for GnRH induction. In fact, mutation of both Egr sites within the eLH beta promoter completely attenuated its induction by GnRH. Thus, GnRH induces expression of Egr1, which subsequently activates the eLH beta promoter. Finally, GnRH not only induced expression of Egr1, but also its corepressor, NGFI-A (Egr1) binding protein (Nab1), which can repress Egr1- induced transcription of the eLH beta promoter.

Phosphorylation of rat muscle acetyl-CoA carboxylase by AMP-activated protein kinase and protein kinase A.

This study was designed to compare functional effects of phosphorylation of muscle acetyl-CoA carboxylase (ACC) by adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) and by AMP-activated protein kinase (AMPK). Muscle ACC (272 kDa) was phosphorylated and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. Functional effects of phosphorylation were determined by measuring ACC activity at different concentrations of each of the substrates and of citrate, an activator of the enzyme. The maximal velocity (Vmax) and the Michaelis constants (Km) for ATP, acetyl-CoA, and bicarbonate were unaffected by phosphorylation by PKA. Phosphorylation by AMPK increased the Km for ATP and acetyl-CoA. Sequential phosphorylation by PKA and AMPK, first without label and second with label, appeared to reduce the extent of label incorporation, regardless of the order. The activation constant (Ka) for citrate activation was increased to the same extent by AMPK phosphorylation, regardless of previous or subsequent phosphorylation by PKA. Thus muscle ACC can be phosphorylated by PKA but with no apparent functional effects on the enzyme. AMPK appears to be the more important regulator of muscle ACC.

Role of the cytokines in the hypothalamic-pituitary-adrenal and gonadal axes.

Cytokines are soluble mediators of immune function that also regulate several endocrine systems. Interleukin-1 (IL-1), IL-6 and tumor necrosis factor-alpha (TNF alpha) each mediate certain aspects of inflammation. In addition, these agents regulate hormone secretion from and cellular proliferation within endocrine tissues. Thus, IL-1 and IL-6 each affect hormone release from anterior pituitary cells (e.g., growth hormone) and inhibit the proliferation of these cells. Cytokines are also localized within discrete nuclei of the hypothalamus (e.g., IL-1 in the paraventricular nucleus), where they may affect production of neuropeptides and biogenic amines (e.g., corticotropin-releasing hormone). Similarly, IL-1 and TNF alpha affect granulosa cell steroidogenesis and IL-6 production. Follicular atresia may either be augmented or inhibited by cytokines depending on their ability to regulate cellular apoptosis. Compartmentation of cytokines within adrenal tissue (e.g., IL-6 in the zona glomerulosa) allows localized effects of these factors on glucocorticoid secretion. Thus, cytokines affect via paracrine or autocrine pathways both hormone secretion from, and possibly cellular differentiation within, endocrine tissues.