C. D. John

Cytokines: regulation of the hypothalamo–pituitary–adrenocortical axis

Many of the pro-inflammatory cytokines that are released in response to immune/inflammatory insults exert marked stimulatory influences on the hypothalamo-pituitary-adrenocortical axis. Thus, they provoke the release of glucocorticoids that, in turn, temper the ensuing immune/inflammatory response, and thereby complete a homeostatic neuroendocrine loop. The mechanisms by which cytokines cause glucocorticoid release are complex and can be affected by repeated or sustained cytokine exposure, gender and age, or counter-regulatory mechanisms.

Annexin 1, Glucocorticoids, and the Neuroendocrine–Immune Interface

Abstract:  Annexin 1 (ANXA1) was originally identified as a mediator of the anti‐inflammatory actions of glucocorticoids (GCs) in the host defense system. Subsequent work confirmed and extended these findings and also showed that the protein fulfills a wider brief and serves as a signaling intermediate in a number of systems. ANXA1 thus contributes to the regulation of processes as diverse as cell migration, cell growth and differentiation, apoptosis, vesicle fusion, lipid metabolism, and cytokine expression. Here we consider the role of ANXA1 in the neuroendocrine system, particularly the hypothalamo‐pituitary‐adrenocortical (HPA) axis. Evidence is presented that ANXA1 plays a critical role in effecting the negative feedback effects of GCs on the release of corticotrophin (ACTH) and its hypothalamic‐releasing hormones and that it is particularly pertinent to the early‐onset actions of the steroids that are mediated via a nongenomic mechanism. The paracrine/juxtacrine mode of ANXA1 action is discussed in detail, with particular reference to the significance of the secondary processing of ANXA1, the processes that control the intracellular and transmembrane trafficking of the protein of the molecule and the mechanism of ANXA1 action on its target cells. In addition, the role of ANXA1 in the perinatal programming of the HPA axis is discussed.

Annexin 1 and the regulation of endocrine function

Annexin 1 (ANXA1) was first identified as a mediator of the anti-inflammatory actions of glucocorticoids in the host defence system. Subsequent work revealed that this protein fulfils a wider brief and it is now recognized as an important signalling intermediate in a variety of other systems. Here, we consider the role of ANXA1 in the endocrine system, placing particular emphasis on new insights into the mechanisms and functional significance of the secondary processing of ANXA1, the processes that control the intracellular and transmembrane trafficking of the molecule and the molecular mechanisms of ANXA1 action that have identified a novel role for the protein as a paracrine/juxtacrine mediator of the non-genomic actions of glucocorticoids in the neuroendocrine system.

Annexin A1 and the formyl peptide receptor family: neuroendocrine and metabolic aspects

Annexin A1 (ANXA1, formerly termed lipocortin 1 or macrocortin) is an important protein mediator of the feedback actions of glucocorticoids within the hypothalamo-pituitary-adrenocortical (HPA) axis. Here we consider the mechanisms by which ANXA1 exerts these actions, with particular reference to the potential role of the formyl peptide receptors (FPRs), a family of G-protein-coupled receptors which has only very recently been implicated in the regulation of neuroendocrine function. In addition, we discuss evidence that ANXA1 contributes to the regulation of other aspects of endocrine and metabolic function and to the aetiology of sexual dimorphisms.

Kinase‐Dependent Regulation of the Secretion of Thyrotrophin and Luteinizing Hormone By Glucocorticoids and Annexin 1 Peptides

Our previous studies have identified a role for annexin 1 (ANXA1), a protein produced by the pituitary folliculostellate cells, as a paracrine/juxtacrine mediator of the acute regulatory effects of glucocorticoids on the release of adrenocorticotropic hormone and other pituitary hormones. In the present study, we focused on the secretion of thyroid stimulating hormone (TSH) and luteinizing hormone (LH) and used a battery of ANXA1‐derived peptides to identify the key domains in the ANXA1 molecule that are critical to the inhibition of peptide release. In addition, as ANXA1 is a substrate for protein kinase C (PKC) and tyrosine kinase, we examined the roles of these kinases in the manifestation of the ANXA1‐dependent inhibitory actions of dexamethasone on TSH and LH release. Dexamethasone suppressed the forskolin‐induced release of TSH and LH from rat anterior pituitary tissue in vitro. Its effects were mimicked by human recombinant ANXA1 (hrANXA1) and a truncated protein, ANXA11‐188. ANXA1Ac2−26, also suppressed stimulated peptide release but it lacked both the potency and the efficacy of the parent protein. Shorter N‐terminal ANXA1 sequences were without effect. The PKC inhibitor PKC19‐36 abolished the inhibitory actions of dexamethasone on the forskolin‐evoked release of TSH and LH; it also attenuated the inhibitory actions of ANXA1Ac2−26. Similar effects were produced by annexin 5 (ANXA5) which sequesters PKC in other systems. By contrast, the tyrosine kinase inhibitors, p60v‐src (137–157) and genistein, had no effect on the secretion of TSH or LH alone or in the presence of forskolin and/or dexamethasone. Dexamethasone caused the translocation of a tyrosine‐phosphorylated species of ANXA1 to the surface of pituitary cells. The total amount of ANXA1 exported from the cells in response to the steroid was unaffected by tyrosine kinase blockade. However, the degree of tyrosine‐phosphorylation of the exported protein was markedly reduced by genistein. These results suggest that (i) the ANXA1‐dependent inhibitory actions of dexamethasone on the release of TSH and LH require PKC and sequences in the N‐terminal domain of ANXA1, but are independent of tyrosine kinase, and (ii) while dexamethasone induces the cellular exportation of a tyrosine‐phosphorylated species of ANXA1, tyrosine phosphorylation per se is not critical to the steroid‐induced passage of ANXA1 across the membrane.

Formyl peptide receptors and the regulation of ACTH secretion: targets for annexin A1, lipoxins, and bacterial peptides

The N‐formyl peptide receptors (FPRs) are a family of G‐protein coupled receptors that re‐spond to proinflammatory N‐formylated bacterial peptides (e.g., formyl‐Met‐Leu‐Phe, fMLF) and, thus, contribute to the host response to bacterial infection. Paradoxically, a growing body of evidence suggests that some members of this receptor family may also be targets for certain anti‐inflammatory molecules, including annexin Al (ANXA1), which is an important mediator of glucocorticoid (GC) action. To explore further the potential role of FPRs in mediating ANXA1 actions, we have focused on the pituitary gland, where ANXA1 has a well‐defined role as a cell‐cell mediator of the inhibitory effects of GCs on the secretion of cortico‐trophin (ACTH), and used molecular, genetic, and pharmacological approaches to address the question in well‐established rodent models. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis identified mRNAs for four FPR family members in the mouse anterior pituitary gland, Fpr‐rsl, Fpr‐rs2, Fpr‐rs6, and Fpr‐rs7. Functional studies confirmed that, like dexa‐methasone, ANXA1 and two ANXAl‐derived peptides (ANXA11_188 and ANXA1Ac2_26) inhibit the evoked release of ACTH from rodent anterior pituitary tissue in vitro. Fpr1 gene deletion failed to modify the pituitary responses to dexamethasone or ANXA1Ac2_26. However, lipoxin A4 (LXA4, 0.02–2μM, a lipid mediator with high affinity for Fpr‐rs1) mimicked the inhibitory effects of ANXA1 on ACTH release as also did fMLF in high (1–100 μM) but not lower (10–100 nM) concentrations. Additionally, a nonselective FPR antagonist (Boc1, 100 μM) overcame the effects of dexametha‐sone, ANXA11_188, ANXA1Ac2_26, fMLF, and LXA4 on ACTH release, although at a lower concentration (50 μM), it was without effect. Together, the results suggest that the actions of ANXA1 in the pituitary gland are independent of Fpr1 but may involve other FPR family members, in particular, Fpr‐rs1 or a closely related receptor. They thus provide the first evidence for a role of the FPR family in the regulation of neuroendocrine function.—John, C. D., Sahni, V., Mehet, D., Morris, J. F., Christian, H. C., Perretti, M., Flower, R. J., Solito E., Buckingham J. C. Formyl peptide receptors and the regulation of ACTH secretion: targets for annexin A1, lipoxins, and bacterial peptides. FASEB J. 21, 1037–1046 (2007)

Annexin 1 (lipocortin 1) mimics inhibitory effects of glucocorticoids on testosterone secretion and enhances effects of interleukin-1β

Annexin 1 is an important mediator of glucocorticoid action in the hypothalamo-pituitary axis; however, little is known of its role in mediating glucocorticoid actions in the peripheral endocrine organs. Accordingly, we have carried out a preliminary study to investigate the effects of annexin 1 in vitro on the testicular secretion of testosterone, a process inhibitied by both glucocorticoids and interleukin-1β (IL-1β). Luteinizing hormone (LH) and forskolin stimulated the release of testosterone from dispersed murine testicular cells in vitro. Their effects were reduced in cells from mice pretreated with dexamethasone (DEX). Similarly, preincubation of testicular cells from untreated mice with DEX, corticosterone, or 11-dehydrocorticosterone reduced LH-stimulated testosterone release, as did the 11β-hydroxysteroid dehydrogenase inhibitors, glycyrrhetinic acid and carbenoxolone. The inhibitory actions of the steroids were mimicked by annexin 11–188 (ANXA11–188) (a stable annexin 1 analog). IL-1β produced a marked decrease in the response to LH, which was blocked by indomethacin, a nonselective cyclooxygenase inhibitor and an additive effect with DEX and ANXA11–188. These results confirm reports that glucocorticoids and IL-1β inhibit LH-stimulated testosterone release from mouse testicular cells. They also show, for the first time, that the effects of the steroids are mimicked by annexin 1 and that, in contrast to their mutually antagonistic effects in the neuroendocrine system, IL-1β and annexin 1 exert additive actions in the testis.

Lack of Annexin 1 Results in an Increase in Corticotroph Number in Male but not Female Mice

Annexin 1 (ANXA1) is a member of the annexin family of phospholipid‐ and calcium‐binding proteins with a well demonstrated role in early delayed (30 min to 3 h) inhibitory feedback of glucocorticoids in the pituitary. We have examined corticotrophs in wild‐type and ANXA1 knockout mice to determine the effects of lack of ANXA1 in male and female animals. Anterior pituitary tissue from ANXA1 wild‐type, heterozygote and null mice was fixed and examined (i) by confocal immunocytochemistry to determine the number of corticotrophs and (ii) by electron microscopy to examine the size, secretory granule population and secretory machinery of corticotrophs. No differences in these parameters were detected in female mice. In male ANXA1 null mice, there were approximately four‐fold more corticotrophs than in wild‐type animals. However, the corticotrophs in ANXA1 null mice were smaller and had reduced numbers of secretory granules (the reduction in granules paralleled the reduction in cell size). No differences in the numerical density of folliculo‐stellate, gonadotroph, lactotroph or somatotroph cells were detected in male ANXA1 null mice. Plasma corticosterone, adrenocorticotrophic hormone (ACTH) and pituitary pro‐opiomelanocortin mRNA were unchanged but pituitary ACTH content was increased in male ANXA1 null mice. Interleukin (IL)‐6 pituitary content was significantly elevated in male and reduced in female ANXA1 null mice compared to wild‐type. In conclusion, these data indicate that ANXA1 deficiency is associated with gender‐specific changes in corticotroph number and structure, via direct actions of ANXA1 and/or indirect changes in factors such as IL‐6.

In vitro and in vivo studies on CCR10 regulation by Annexin A1

The mode of action of annexin A1 (ANXA1) is poorly understood. By using rapid subtraction hybridization we studied the effects of human recombinant ANXA1 and the N‐terminal ANXA1 peptide on gene expression in a human larynx cell line. Three genes showed strong downregulation after treatment with ANXA1. In contrast, expression of CCR10, a seven transmembrane G‐protein coupled receptor for chemokine CCL27 involved in mucosal immunity, was increased. Moreover the reduction in CCR10 expression induced by ANXA1 gene deletion was rescued by intravenous treatment with low doses of ANXA1. These findings provide new evidence that ANXA1 modulates gene expression.

Evidence From In Vitro and In Vivo Studies Showing that Nuclear Factor-Kappa B Within the Pituitary Folliculostellate Cells and Corticotrophs Regulates Adrenocorticotrophic Hormone Secretion in Experimental Endotoxaemia

The hypothalamic‐pituitary‐adrenocortical (HPA) responses to bacterial infection are mediated, in part, by the actions of lipopolysaccharide (LPS) on pituitary folliculostellate (FS) cells that release pro‐inflammatory cytokines [e.g. interleukin (IL)‐6] and thereby facilitate adrenocorticotrophic hormone (ACTH) release from neighbouring corticotrophs. In the present study, two murine pituitary cell lines [TtT/GF (FS cells) and AtT20 D16:16 (corticotrophs)], alone and in co‐culture, and an in vivo model of endotoxaemia were used to examine the potential role of nuclear factor‐kappa B (NF‐κB) in mediating LPS‐induced ACTH secretion. Both cell lines expressed mRNAs for the key components of the LPS signalling system. LPS stimulated IL‐6 release from TtT/GF cells via a glucocorticoid‐sensitive, NF‐κB‐dependent mechanism; it also activated NF‐κB in AtT20 cells, as did corticotrophin‐releasing hormone (CRH). IL‐6 potentiated (but LPS reduced) the stimulatory effects of CRH on ACTH release from AtT20 cells, whereas blockade of NF‐κB (SC‐514) increased the ACTH release induced by CRH in the presence or absence of LPS. In co‐cultures, CRH and LPS acted synergistically to induce release of both IL‐6 and ACTH. However, although SC‐514 suppressed the release of IL‐6 evoked by CRH and LPS, it potentiated the concomitant increase in ACTH release. In vivo both immunological (LPS) and psychological (restraint) stress increased intrapituitary NF‐κB, whereas an NF‐κB inhibitor (PHA781535E) attenuated the LPS‐induced release of ACTH and abolished the HPA response to restraint stress. The results obtained in the present study support the premise that NF‐κB plays an important role in mediating LPS signalling in the anterior pituitary gland, particularly in relation to IL‐6 and ACTH secretion, and provide novel evidence that NF‐κB blockade in vivo compromises stress‐induced ACTH release.