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

Abstract

The N-formyl peptide receptors (FPRs) are a family of G-protein coupled receptors that respond 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 A1 (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 corticotrophin (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-rs1, Fpr-rs2, Fpr-rs6, and Fpr-rs7. Functional studies confirmed that, like dexamethasone, ANXA1 and two ANXA1-derived peptides (ANXA1(1-188) and ANXA1(Ac2-26)) 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 ANXA1(Ac2-26). However, lipoxin A4 (LXA4, 0.02-2 microM, 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 microM) but not lower (10-100 nM) concentrations. Additionally, a nonselective FPR antagonist (Boc1, 100 microM) overcame the effects of dexamethasone, ANXA1(1-188), ANXA1(Ac2-26), fMLF, and LXA4 on ACTH release, although at a lower concentration (50 microM), 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.

Figure 1

Comparison of the effects of graded concentrations of the formyl peptide (A), fMLF (B), and lipoxin A4 (C) on the resting and forskolin-stimulated release of ACTH from rat pituitary tissue in vitro. Dexamethasone (100 nM) was included as a positive control in each experiment. Open columns, basal; solid columns, forskolin (100 μM). The data are the mean ± SEM (n=8). **P < 0.001 vs. basal; ^†P < 0.05; ^††P < 0.01 vs. forskolin alone (ANOVA plus Duncan’s multiple-range test).

Figure 2

Effects of an FPR antagonist, Boc1, on the ability of dexamethasone (A; 100 nM), ANXA1_1-188 (B; 27 pM), and ANXA1_Ac2-26 (C) (6.8 μM) to suppress the release of ACTH from rat pituitary tissue in vitro evoked by forskolin (100 μM). Open columns, Boc1-free; closed columns, Boc1 (50 μM); hatched columns, Boc1 (100 μM). **P < 0.01 vs. basal; ^††P < 0.001 vs. forskolin alone (ANOVA plus Duncan’s multiple range test, n=8).

Figure 3

Effects of the FPR antagonist, Boc1 (100 μM), in the presence and absence of the Fpr1 agonist, fMLF (10 μM) and the Fpr-rs1 agonist, LXA4 (0.2 μM) on the release of ACTH from rat pituitary tissue in vitro evoked by forskolin (100 μM). Open columns = control; closed columns = forskolin. **P < 0.01 vs. basal; ^††P < 0.001 vs. forskolin alone; (ANOVA plus Duncan’s multiple range test, n=6).

Figure 4

Effects of the FPR antagonist, Boc2 (100 μM), in the presence and absence of dexamethasone (100 nM) on the release of ACTH from rat pituitary tissue in vitro evoked by forskolin (100 μM). Open columns = control; closed columns = forskolin. **P < 0.01 vs. basal; ^†P < 0.05; ^††P < 0.001 vs. forskolin alone; ⁺P < 0.05 vs. corresponding Boc2-free group (ANOVA plus Duncan’s multiple range test, n=8).

Figure 5

Effects of dexamethasone (A; 100 nM) and ANXA1_Ac2-26 (B; 6.8 μM) on the resting and forskolin-stimulated the release of ACTH from Fpr1 knockout (FPR KO) and WT pituitary tissue in vitro. Open columns = control; closed columns = forskolin (100 μM). *P < 0.05 vs. basal; ^†P < 0.05, ^††P < 0.01 vs. forskolin alone; ⁺P < 0.05, ⁺⁺P < 0.01 vs. corresponding WT control (ANOVA plus Duncan’s multiple-range test, n=8).

Figure 6

RT-PCR analysis of the expression of mRNAs of FPR family members in mouse pituitary tissue using 30 cycles (A) and 40 cycles (B) amplification and mouse hypothalamic tissue using 30 cycles (C) and 40 cycles (D) amplification. Note that Fpr1 mRNA was not detectable in mouse pituitary tissue at an amplification of 30 PCR cycles. Comparable data were obtained using the other primers shown in Table 1.

Figure 7

Schematic diagram hypothesizing the role of FPR receptors in mediating the acute inhibitory actions on glucocorticoids on the regulation of ACTH release and, hence, the secretion of glucocorticoids. Annexin 1 (ANXA1, green spots) is normally abundant in the cytoplasm of folliculostellate (FS) cells, which are adjacent to corticotrophs. A) ACTH release is normally triggered by corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP), which synergistically act via specific receptors (CRH-R1 and V_1b-R) to increase intracellular 3′5′adenosine cyclic monophosphate (cAMP) and thereby activate protein kinase A (PKA). This triggers a sequence of events leading to Ca²⁺ entry via L-Ca²⁺ channels, vesicle fusion, and the release of ACTH by exocytosis. ACTH acts on the adrenal cortex to increase the synthesis and release of cortisol[b]. B) glucocorticoids (GCs) act via a glucocorticoid receptor (GR)-dependent mechanism in FS cells to activate a kinase cascade, which triggers the translocation of a serine-phosphorylate species of ANXA1 (ANXA1^SPO4) to the cell surface; the released protein acts on the adjacent corticotrophs via Fpr-rs1 and its associated heterotrimeric G-protein (Gi) to activate a signaling cascade, which triggers actin polymerization and thereby prevents vesicle fusion with the membrane and, hence, exocytosis. Note: Fpr-rs1 may also be a target for locally generated lipoxins, mitochondrially derived formylated peptides, and for microbial peptides, particularly formylated bacterial peptides and certain viral peptides. AC = adenylyl cyclase; PLC = phospholipase C; ATP = adenosine triphosphate; PIP₂ = phosphatidylinositol 4,5-bisphosphate; IP₃ = inositol trisphosphate; DAG = DAG; PKC = protein kinase A.