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Endocrinology Vol. 143, No. 8 3060-3070
Copyright © 2002 by The Endocrine Society

ARTICLE

Annexin 1-Dependent Actions of Glucocorticoids in the Anterior Pituitary Gland: Roles of the N-Terminal Domain and Protein Kinase C

Christopher John, Patricia Cover, Egle Solito, John Morris, Helen Christian, Roderick Flower and Julia Buckingham

Department of Neuroendocrinology (C.J., P.C., E.S., J.B.), Division of Neuroscience and Psychological Medicine, Faculty of Medicine, Imperial College of Science Technology and Medicine, Hammersmith Hospital Campus, London W12 ONN, United Kingdom; Department of Human Anatomy and Genetics (J.M., H.C.), The University of Oxford, Oxford OX1 3QX, United Kingdom; and Department of Biochemical Pharmacology (R.F.), The William Harvey Research Institute, St Bartholomew’s and the Royal London School of Medicine and Dentistry, London EC1M 6BQ, United Kingdom

Address all correspondence and requests for reprints to: Professor Julia Buckingham, Department of Neuroendocrinology, Faculty of Medicine, Imperial College of Science Technology and Medicine, Commonwealth Building, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, United Kingdom. E-mail: . j.buckingham{at}ic.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Annexin 1 (ANXA1) is an important mediator of glucocorticoid action in the neuroendocrine system. As the activity of this protein in other systems is modulated by phosphorylation of its N-terminal domain, we have explored the significance of this domain and its phosphorylation status to ANXA1 actions within the pituitary gland, using an established in vitro preparation. Two N-terminal peptides, ANXA1Ac2–26 and ANXA1Ac1–50, inhibited forskolin-evoked ACTH and prolactin release; however, they lacked the potency and full efficacy of the parent molecule (ANXA11–346), whereas other shorter N-terminal sequences were without effect. A chimeric protein comprising ANXA11–44 and the C-terminal core of ANXA5 (ANXA520–320) also produced a partial inhibition of peptide release. Protein kinase C (PKC) blockade (PKC19–36) abolished the inhibitory effects of dexamethasone on forskolin-evoked peptide release and attenuated the antisecretory actions of ANXA1Ac2–26. ANXA5, which sequesters PKC in other systems, produced similar effects. PKC19–36 also blocked the dexamethasone- induced translocation of a serine phosphorylated species of ANXA1 from the cytoplasm to the outer cell surface. These results suggest that 1) the N-terminal domain plays a fundamental role in effecting the inhibitory actions of ANXA1 on pituitary peptide release; 2) PKC-dependent mechanisms are essential for both the cellular exportation and the biological activity of ANXA1; and 3) ANXA1 exported from the cells is serine phosphorylated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
OUR PREVIOUS STUDIES have shown that the acute regulatory actions of glucocorticoids (GCs) on the secretion of ACTH and prolactin (PRL) are dependent in part upon the generation of a 37-kDa protein, annexin 1 (ANXA1, also known as lipocortin 1) (1). ANXA1 is a well characterized member of the annexin family of Ca2+ and phospholipid binding proteins. It is expressed in particular abundance in macrophages, neutrophils, and other cells within the host defense system where it was first identified as a potential mediator of the therapeutically important antiinflammatory actions of GCs. It is also strongly expressed in the neuroendocrine system (2, 3), particularly 1) in the anterior pituitary gland, where it is abundant in the S100-positive folliculo-stellate cells (4) and also expressed in endocrine cells (5), and 2) in specific loci in the hypothalamus, notably the median eminence and the paraventricular, arcuate and periventricular nuclei (2). In these tissues, as in the host-defense system, GCs regulate both the expression and the subcellular distribution of ANXA1; they thus augment the synthesis of ANXA1 and promote the translocation of the protein from within the cell (intracellular pool) to the outer cell surface (pericellular or extracellular pool) where it is retained by a Ca2+-dependent mechanism (3, 5, 6, 7). Binding and functional studies suggest that this process of externalization provides an important mechanism whereby ANXA1 gains access to binding sites (putative receptors) on the outer surface of cells and thereby exerts paracrine/autocrine regulatory actions on peptide release. Thus, high affinity ANXA1 binding sites have been identified on a variety of endocrine cells in the anterior pituitary gland, including corticotrophs and lactotrophs (8), and both human recombinant ANXA1 (hrANXA11–346) and a stable N-terminal ANXA1 protein (hrANXA11–188) have been shown to mimic the acute inhibitory effects of GCs on the release of ACTH and PRL from rat pituitary tissue induced in vitro by various secretagogues (3, 9). Furthermore, the regulatory actions of GCs in this preparation are specifically ablated by a monoclonal anti-ANXA1 antibody, which would be unlikely to penetrate cell membranes readily but would be expected to sequester ANXA1 at a pericellular site (3, 10). Further evidence of a role for ANXA1 in the regulation of ACTH and PRL secretion has been derived in vitro from studies using antisense probes directed against ANXA1 mRNA (10, 11) and in vivo by the use of immunoneutralization strategies (10, 12, 13) and ANXA1 peptides (6, 13).

The mechanisms by which ANXA1 is exported from pituitary cells in response to a steroid challenge and acts on the endocrine cells to suppress peptide release are unknown. As a first step to addressing these issues, we are seeking to determine the regions within the ANXA1 molecule, which are essential for the biological actions of the protein in the pituitary gland and whether they are sensitive to posttranslational modification. Members of the annexin superfamily have a high degree of homology, each comprising a highly conserved C-terminal core of four (or eight) repeats of a 70- to 75-amino acid domain that confers the Ca2+ and phospholipid binding activity of the protein (14, 15, 16, 17) and an N-terminal tail (Fig. 1Go). The N-terminal domain of each annexin is unique in length and sequence and, in the case of ANXA1, includes potential sites for tyrosine and serine/threonine phosphorylation (18, 19), glycosylation and proteolysis (20, 21); the ANXA1 N-terminal is thus a substrate for a number of enzymes including protein kinase C (PKC) and epidermal growth factor receptor kinase (22, 23, 24, 25). Several lines of evidence suggest that the N-terminal region confers the biological specificity of individual members of the annexin family. In accord with this view, removal of the N-terminal domain destroys the antiinflammatory activity of ANXA1 in the rat paw edema model (26), whereas peptides derived from the N terminus mimic the actions of the full-length protein in various models of inflammation and cell growth. Phosphorylation of the tyrosine residue in position 21 appears to be essential for the regulatory actions of the protein on cell growth (27, 28) and also to decrease its Ca2+ requirement for phospholipid binding (29, 30). Other data advocate a role for phosphorylation of serine/threonine residues in the manifestation of other facets of ANXA1 action (31, 32). Interestingly, ANXA5, which is expressed in the anterior pituitary gland (2) and which possesses a very short N terminus (6 amino acids, Fig. 1Go) with no recognized phosphorylation sites, acts to sequester PKC and has been shown to inhibit in vitro phosphorylation of ANXA1 in a cell-free system (33, 34). The role of the N-terminal in mediating the actions of ANXA1 within the neuroendocrine system has not yet been explored; nor is there any information about the impact of kinases on the activity of ANXA1 despite the fact that PKC is implicated in the signaling mechanisms used by GCs in the anterior pituitary gland (35). Accordingly, in the present study, we have examined the influence of a series of ANXA1-derived proteins on the release in vitro of ACTH and PRL from rat pituitary tissue and sought to determine the potential role of PKC in the ANXA1-dependent inhibition of pituitary peptide release by GCs. Our results demonstrate a fundamental role for the N-terminal and suggest that PKC-dependent mechanisms are essential for both the cellular exportation of a serine-phosphorylated species of ANXA1 and for the biological activity of the protein in this in vitro system.



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  		Figure 1. Schematic diagram showing the basic structure of ANXA1 and ANXA5 and the synthetic and recombinant peptides used. Note that the four repeated units in ANXA1 and ANXA5 show approximately 80% homology. {blacksquare}, ANXA1 repeat unit; , ANXA5 repeat unit.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Animals
Adult male Sprague Dawley rats weighing approximately 200 ± 20 g were used. They were bred in-house from a closed, specific pathogen-free colony (coded CFY) and housed in groups of five per cage in a room with controlled lighting (lights on 0800–2000 h) in which the temperature was maintained at 21–23 C and food and water were available ad libitum. Experiments were always started between 0900 h and 1000 h to avoid changes associated with the circadian rhythm. All procedures were carried out under license in accord with the UK Animals (Scientific Procedures) Act, 1986.

Incubation of anterior pituitary tissue
The method used was a modification (3) of that described by Buckingham and Hodges (36). Briefly, anterior pituitary glands were removed from rats immediately after decapitation and divided into four pieces of approximately equal size. The segments were distributed randomly (1 segment per well) in the wells of modified 24-well tissue culture plates (Costar, Cambridge, MA) containing 1 ml Earle’s balanced salt solution (Sigma, Poole, UK) enriched with aprotonin (1%, Bayer plc, Newbury, UK). The plates were incubated for 2 h at 37 C in a humidified atmosphere saturated with 95% O2 / 5% CO2 gas with medium changes at 1 and 1.5 h. The segments were then transferred to fresh medium containing the adenylyl cyclase activator, forskolin (Sigma) or, in the case of controls, an equal volume (1.5 ml) of medium alone, and incubated for a further 1 h. Where appropriate, drugs (dexamethasone, recombinant annexin peptides, protein kinase inhibitors) were included throughout the preincubation and final incubation periods. The medium from the final incubation was collected and stored in aliquots at -20 C for subsequent immunoassay. The pituitary segments were weighed on a torsion balance and, if required, either stored at -80 C for measurement of 3'5'-cAMP or processed for analysis of cell surface (pericellular) ANXA1. For the latter, ANXA1 bound to the outer surface of the cell membranes was removed by washing the tissue for 20 min in 1 mM EDTA (Sigma) which, by chelating Ca2+, releases ANXA1 bound to the cell surface into the medium (37). Samples were frozen and stored at -80 C before detection of ANXA1.

RIA of ACTH, PRL, and cAMP
ACTH was determined in duplicate using a modification of the double antibody method described by Rees et al. (38) using an antibody raised in sheep against human ACTH1–24 (kindly donated by Professor P. J. Lowry, University of Reading, UK). The antibody was directed against the 10–24 region of the peptide and did not cross-react with corticotrophin-like intermediate lobe peptide, {alpha}-MSH, LH, or PRL. The reference preparation was synthetic human ACTH1–39 (National Institute for Biological Standards and Control, South Mimms, Hertsfordshire, UK) and the tracer 125I-labeled ACTH (a very generous, monthly gift from Professor Dame Lesley Rees, Department of Chemical Endocrinology, St. Bartholomew’s Hospital Medical College, UK). Separation of the bound and free peptide was achieved by sheep antirabbit IgG-coated beads (Amersham Pharmacia Biotech, Buckinghamshire, UK). Inter- and intraassay variation were 11.1% and 10.7%, respectively.

PRL was determined in duplicate by RIA (39) using reagents supplied by the National Hormone and Pituitary Program (Bethesda, MD). The PRL antibody (NIDDK-anti-rPRL-S-9) had negligible cross-reactivity with rat-GH, -TSH, -FSH, and -LH. The reference preparation and the 125I-labeled tracer were coded rat-PRL-RP-3 and PRL-I-9, respectively. Separation of the bound and free peptide was achieved using a mixture of antirabbit precipitating serum raised in donkey (Immunodiagnostic Systems, Boldon, UK) and nonimmune rabbit serum. Inter- and intraassay variation was 9.4% and 7.3%, respectively.

cAMP, extracted from pituitary tissue, was determined in duplicate by RIA, using a modification (40) of the protocol supplied by the National Hormone and Pituitary Program (Bethesda, MD). The cAMP antibody was coded CV-27 (NIDDK), and the cAMP standard and the 125I-labeled tracer (2.0'-mono succinyl camp tyrosine methyl ester) were supplied by Sigma. Separation of the bound and free peptide was achieved using the second antibody separation as described above for PRL. Inter- and intraassay variation were 10.9% and 8.2%, respectively.

Detection of cell surface annexin 1 by SDS-PAGE and Western blot analysis
The method employed is described in detail elsewhere (3). In essence, following protein estimation (41), the proteins contained within the EDTA washes were separated by electrophoresis following application to sodium dodecyl sulfate-polyacrylamide gels. In any given gel, an equal amount of protein (5–10 µg/lane) was applied to each lane in a volume of 20 µl. The separated proteins were transferred electrophoretically to nitrocellulose paper (Amersham Pharmacia Biotech) and incubated overnight at 4 C with a well characterized anti-ANXA1 polyclonal antibody [anti-ANXA1 polyclonal antibody (pAb), diluted 1:5000 in PBS-Tween], which was raised in-house in sheep against full-length human recombinant ANXA1 (42). Deposition of the antibody was visualized by a second antibody method using a peroxidase conjugated donkey antisheep antibody and diaminobenzidine (0.05% wt/vol both from Sigma). The molecular weights of the bands of immunoreactive (ir) ANXA1 were determined by comparison with the migration of molecular weight markers (Rainbow molecular weight markers, Amersham Pharmacia Biotech) and ANXA1 standard (human recombinant, Escherichia coli derived; Ref. 43). The blots were scanned using a Fujix-Bax 1500 imaging system with a low level light sensitive camera (Raytek, Germany).

Detection of serine-phosphorylated ANXA1
Following protein estimation (41), an equal amount of protein (200 µg) from within the EDTA washes was precipitated by incubation of the samples at 4 C with antiphosphoserine-agarose monoclonal antibody (10 µl, 24 h; Sigma). The suspension was centrifuged (3000 x g, 1 h, 4 C) and the supernatant fluid aspirated and discarded (11). The resultant pellets were resuspended in Tris-buffered saline [TBS; 20 mM Tris base (Sigma), 137 mM NaCl, 0.38% vol/vol HCl (Merck Eurolab Ltd., Poole, UK), pH 7.6, containing the protease inhibitors, 1 mM phenylmethylsulfonyl fluoride, 0.001% wt/vol pepstatin A, 0.001% wt/vol leupeptin and 0.001% aprotonin, the phosphatase inhibitors, 1 µM sodium orthovanadate, and 1 µM sodium fluoride]. The samples were then run on sodium dodecyl sulfate-polyacrylamide gels as described above, but with several modifications. Firstly, each cycle of washes consisted of five washes in TBS containing the peptidase and phosphatase inhibitors described previously and 0.1% Tween (TBS Tween). TBS Tween provided a more stringent wash than PBS Tween and minimized background. Secondly, the blot was incubated overnight at 4 C with an anti-ANXA1 polyclonal antibody (anti-ANXA1 pAb, Zymed Laboratories, Inc., San Francisco, CA) in 5% wt/vol milk powder in TBS Tween. Thirdly, deposition of the antibody was visualized by incubation at 4 C with a peroxidase conjugated sheep antirabbit antibody (Sigma, diluted 1:13,000 in TBS-Tween) and developed using the ECL immunodetection system (Amersham Pharmacia Biotech). The film was developed immediately and scanned as described previously.

Drugs
Human recombinant (hr) ANXA1, ANXA11–188, ANXA5 and a chimeric peptide comprising the core of ANXA5 (ANXA520–320) and the N- terminal sequence of ANXA1 (ANXA11–44) were prepared by expression in E. coli (43); endotoxin contamination of the product was less than 20 pg/ml as measured by Limulus amebocyte chromogenic assay and would therefore be unlikely to exert any direct effect on either basal or evoked pituitary peptide release (44). N-terminal ANXA1 peptides (corresponding rat sequence) were custom made in house by Dr. Ian Moss, Advanced Biotechnology Centre; purity of the product was verified by mass spectrometry and HPLC. A schematic diagram depicting the structures of the annexin peptides used is provided in Fig. 1Go.

Synthetic protein kinase fragments (Sigma) were used to inhibit protein kinase A (PKA6–22-amide) and PKC (PKC19–36), respectively. PKA6–22-amide is derived from the noncatalytic domain of the cAMP-dependent PKA and appears to target the catalytic domain of the enzyme. PKC19–36 serves as a pseudosubstrate for PKC and effectively blocks the various isoforms of the enzyme by binding to the active site (45). Although the mechanism by which each of these peptides crosses the plasma membrane is unknown, biological efficacy of these peptides on cellular activity has been demonstrated in a pituitary cell line (GH3, 46) and in several other cell preparations (47, 48).

Dexamethasone sodium phosphate (David Bull Laboratories, Inc., Warwick, UK), human recombinant ANXA1, ANXA11–188, ANXA5 and the ANXA1/ANXA5 chimeric peptide were diluted in medium immediately before use. Forskolin (Sigma) was first dissolved in small amounts of ethanol and then diluted in medium; the final concentration of ethanol never exceeded 0.1% and appropriate controls were included in all experiments. The ANXA1 N-terminal peptides and the protein kinase inhibitors were first dissolved in small amounts of 1 M ammonium bicarbonate and then diluted in medium; the final concentration of ammonium bicarbonate never exceeded 20 mM and appropriate controls were included in all experiments.

Data analysis
Preliminary analysis by the Shapiro and Wilks test showed that the data were normally distributed. Subsequent analysis was done by ANOVA with post hoc comparisons by Duncan’s multiple range tests. Differences were considered to be significant if P < 0.05. As the basal rate of anterior pituitary hormone release varied between experiments in vitro, statistical comparisons were made only within experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Preliminary studies
Initial studies showed that anterior pituitary tissue responded readily to the adenylate cyclase activator, forskolin (0.1–1000 µM) with significant (P < 0.01) concentration- dependent increases in immunoreactive (ir)-ACTH and ir-PRL release; these responses were overcome by preincubation of the tissue with dexamethasone (0.1 µM). We also showed that release of the two peptide hormones was unaffected by the diluent for forskolin, ethanol (0.05%, data not shown). As a result of these experiments, a submaximal ({approx}80%) concentration of forskolin (100 µM) was selected to elicit peptide release in subsequent studies; appropriate diluent controls were always run in parallel. In addition, the capacity of dexamethasone to block forskolin-induced peptide release was examined in all experiments as a positive control.

Effects of ANXA1- and ANXA5-derived peptides on pituitary peptide release
Figure 2Go demonstrates the effects of full-length hrANXA1 [0.48–480 pM, Fig. 2AGo (i) and (ii)] and two truncated ANXA1 peptides, ANXA1ac1–50 [0.04–37 µM, Fig. 2BGo (i) and (ii)], and ANXA1Ac2–26 [0.07–68 µM, Fig. 2CGo (i) and (ii)], on basal and forskolin-induced ir-ACTH and ir-PRL release from rat anterior pituitary tissue in vitro. In the concentrations tested, none of these substances influenced the basal release of either pituitary hormone. However, hrANXA1 produced a marked concentration dependent inhibition of the evoked release of both ir-ACTH and ir-PRL and, at the highest concentration tested (480 pM), completely abolished the secretory responses to forskolin [P < 0.01, Fig. 2AGo (i) and (ii)]. Similar effects were produced by hrANXA11–188 (0.27–270 pM; data not shown). The N- terminal ANXA1 peptides, ANXA1Ac1–50 [Fig. 2BGo (i) and (ii)] and ANXA1Ac2–26 [Fig. 2CGo (i) and (ii)], also inhibited the secretory responses to forskolin (P < 0.01). However, these peptides were effective only in the nanomolar to micromolar range and were therefore considerably less potent than hrANXA1 and hrANXA11–188. In addition, they lacked efficacy of hrANXA1 and hrANXA11–188 and produced at best 50–70% inhibition of forskolin evoked peptide release.



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  		Figure 2. The effects of graded concentrations of (A) full-length ANXA1 (hrANXA1), (B) ANXA1Ac1–50 and (C) ANXA1Ac2–26 on the spontaneous release of (i) ir-ACTH and (ii) ir-PRL from rat anterior pituitary segments in vitro (open squares) and on the secretion evoked by forskolin (100 µM, closed squares). The histograms represent the effect of dexamethasone on forskolin stimulated hormone release. Each point represents the mean ± SEM (n=6); {dagger}{dagger}, P < 0.01 vs. corresponding forskolin free control; **, P < 0.01 vs. corresponding ANXA1 protein or dexamethasone-free control (ANOVA and Duncan’s multiple range test).

 
The N-terminal ANXA1 fragments, ANXA1Ac7–12, ANXA1Ac1–12, and ANXA1Ac13–25 had no significant effect on the basal or pharmacologically evoked release of ir-ACTH or ir-PRL (data not shown), unlike with dexamethasone (0.1 µM, positive control), which effectively suppressed forskolin- stimulated peptide release.

While the N-terminal domain differs in sequence and length between various members of the annexin family, the carboxy core domain of each family member comprises four (or eight in the case of annexin 6) structurally related tandem repeat domains of 70–75 amino acids. Thus, to provide more insight to the respective roles of the N-terminal and the core domains of ANXA1 in inhibiting pituitary peptide release, we tested a chimeric annexin protein ANXA1/5 and examined the activity of hrANXA5 as a control. The results of these studies are shown (see Fig. 4Go). ANXA5 (0.48–480 pM) had no significant effect on the basal release of ir-ACTH [Fig. 3AGo (i)] or ir-PRL [Fig. 3AGo (ii)] from the pituitary tissue. It also failed to modify forskolin-evoked ir-ACTH release [P > 0.05, Fig. 3AGo (i)] although, at concentrations of 0.48 pM, 48 pM, and 480 pM, it had a weak inhibitory effect on the ir-PRL response to forskolin [P < 0.05, Fig. 3AGo (ii)]. In the same concentration range, the chimeric protein (ANXA1/5, 0.48–480 pM) had no effect on the resting or evoked release of ir-ACTH or ir-PRL (data not shown). However, at higher concentrations (5.2–520 nM) it caused a partial inhibition of the ir-ACTH [Fig. 3BGo (i)] and ir-PRL [Fig. 3BGo (ii)] responses to forskolin without affecting basal peptide release [Fig. 3BGo (i) and (ii)]; thus, at a concentration of 52 nM the chimeric peptide reduced the forskolin-induced increments in ir-ACTH and ir-PRL release by approximately 65.5% (P < 0.01) and 40% (P < 0.01), respectively [Fig. 3BGo (i) and (ii)].



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  		Figure 4. The effects of the PKA inhibitor, PKA6–22-amide (5 µM), on (A) the inhibitory actions of the ANXA1 N-terminal peptide, ANX-1Ac2–26 (2–26; 6.8 µM) and dexamethasone (Dex.; 0.1 µM) on basal and forskolin-stimulated i) ir-ACTH and ii) ir-PRL release by rat anterior pituitary segments in vitro and (B) the concomitant accumulation of cyclic AMP. Open columns, Controls; closed columns, PKA6–22-amide (5 µM). Each value represents the mean ± SEM (n = 6); {dagger}{dagger}, P < 0.01 vs. forskolin alone group; **, P < 0.01 vs. PKA6–22-amide free group (ANOVA and Duncan’s multiple range test).

 


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  		Figure 3. The effects of graded concentrations of (A) full-length ANXA5 (hrANXA1) and (B) a chimeric annexin peptide (ANXA1/5) on the spontaneous release of i) ir-ACTH and ii) ir-PRL from rat anterior pituitary segments in vitro (open squares and on the secretion evoked by forskolin (100 µM, closed squares). The histograms represent the effect of dexamethasone on forskolin-stimulated hormone release. Each point represents the mean ± SEM (n=6); {dagger}{dagger}, P < 0.01 vs. corresponding forskolin free control; *, P < 0.05; **P < 0.01 vs. corresponding annexin protein or dexamethasone-free control (ANOVA and Duncan’s multiple range test).

 
Effects of kinase inhibitors on the regulatory actions of ANXA peptides and dexamethasone on pituitary peptide release
Figure 4Go demonstrates the effects of the PKA inhibitor PKA6–22-amide (5 µM) on the forskolin-induced release of ir-ACTH and ir-PRL [Fig. 4AGo (i) and (ii)] and on the associated accumulation of cAMP (Fig. 4BGo). As anticipated, PKA6–22-amide abolished 1) the increases in pituitary peptide release induced by forskolin alone and 2) the increases in peptide release initiated by forskolin in the presence of ANXA1Ac2–26 (6.8 µM). The data also confirmed that blockade of PKA has no effect on the forskolin-stimulated accumulation of cAMP in rat anterior pituitary tissue in vitro and showed that the accumulation of the nucleotide induced by forskolin is not affected by either dexamethasone (0.1 µM) or ANXA1Ac2–26 (6.8 µM).

Blockade of PKC with PKC19–36 (5 µM) suppressed the inhibitory actions of dexamethasone on anterior pituitary hormone release evoked by forskolin [Fig. 5AGo (i) and (ii), ir-ACTH, P < 0.05; ir-PRL, P < 0.01)]. PKC19–36 (5 µM) also attenuated the inhibitory actions of ANXA1Ac2–26 (6.8 µM) on forskolin stimulated anterior pituitary hormone release [Fig. 5AGo (i) and (ii)]. Interestingly, ANXA5 (48 pM), which has been shown to sequester PKC in other tissues (33, 34, 49, 50), produced effects similar to those evoked by the PKC inhibitor, PKC19–36. It thus, suppressed the inhibitory actions of dexamethasone (0.1 µM) on anterior pituitary hormone release evoked by forskolin [Fig. 5BGo (i) and (ii), ir-ACTH, P < 0.05; ir-PRL, P < 0.01] and attenuated the inhibitory actions of ANXA1Ac2–26 (6.8 µM) on forskolin stimulated anterior pituitary hormone release [Fig. 5BGo (i) and (ii)].



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  		Figure 5. The effects of (A) the PKC inhibitor, PKC19–36 (5 µM), and (B) ANXA5 (48 pM) on the inhibitory actions of the ANXA1 N-terminal peptide ANXA1Ac2–26 (2–26; 6.8 µM) and dexamethasone (Dex; 0.1 µM) on forskolin stimulated i) ir-ACTH and ii) ir-PRL release by rat anterior pituitary segments in vitro. Open columns, Controls; closed columns, PKC19–36 (5 µM); hatched columns, ANXA5 (48 pM). ); {dagger}, P < 0.05, {dagger}{dagger}, P < 0.01 vs. forskolin alone group; *, P < 0.05; **, P < 0.01 vs. PKC19–36 free group (ANOVA and Duncan’s multiple range test).

 
Western blot analysis
Figure 6AGo illustrates the effects of dexamethasone (0.1 µM), in the presence and absence of the PKC inhibitor, PKC19–36 (5 µM) on the expression of ANXA1 on the surface of anterior pituitary cells in vitro. In accord with our earlier studies (3, 7), two species of ANXA1 with molecular masses of 37 kDa and 34 kDa were detected on the cell surface; these corresponded to the full-length protein and an N-terminally clipped metabolite (3, 7). Inclusion of dexamethasone (0.1 µM) in the medium caused a marked increase in the amount of ANXA1 detected in the pericellular pool (Figure 6AGo, lanes 3 and 4 vs. lanes 1 and 2). PKC19–36 did not influence the amount of ANXA1 detected in the pericellular pool (lanes 5 and 6 vs. lanes 1 and 2) but prevented the exportation of ANXA1 from the cells induced by dexamethasone; thus, in the presence of the PKC inhibitor, PKC19–36, dexamethasone failed to influence the amount of ANXA1 contained within the pericellular pool (lanes 7 and 8 vs. lanes 3 and 4).



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  		Figure 6. Western blots demonstrating the effects of dexamethasone (Dex; 0.1 µM) in the presence and absence of the PKC inhibitor PKC19–36 (5 µM) on the expression of (A) ANXA1 and (B) serine-phosphorylated ANXA1 on the surface of rat anterior pituitary tissue in vitro. S1, 37-kDa ANXA1 standard; S2, 34-kDa ANXA1 standard.

 
Figure 6BGo demonstrates the effects of dexamethasone (0.1 µM), in the presence and absence of the PKC inhibitor, PKC19–36 (5 µM) on the expression of serine phosphorylated ANXA1 on the surface of pituitary cells. Two species of serine phosphorylated ANXA1 were readily detected on the cell surface of the untreated cells with molecular masses of approximately 37 kDa and 34 kDa (lanes 1 and 2). Dexamethasone caused a marked increase in the amount of serine phosphorylated ANXA1 on the cell surface (lanes 3 and 4 vs. lanes 1 and 2). By contrast, the PKC inhibitor, PKC19–36, reduced cell surface serine-phosphorylated ANXA1 (lanes 5 and 6 vs. lanes 1 and 2) and prevented the dexamethasone-induced exportation of the serine-phosphorylated protein (lanes 7 and 8 vs. lanes 3 and 4).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
The present study aimed to advance our understanding of the mechanisms by which ANXA1 inhibits the release of ACTH and PRL from the rat anterior pituitary gland, using a well established in vitro system as a model (3, 10). Our principal strategy was to compare the activity of a series of truncated ANXA1 proteins/peptides with those of the full-length protein and to determine whether their activity, or that of dexamethasone, could be modified by drugs that influence the activity of PKC. In addition, we explored the role of PKC in effecting the dexamethasone-induced exportation of ANXA1 by pituitary cells. The results demonstrate a fundamental role for the N-terminal domain in effecting the inhibitory actions of ANXA1 on peptide release. In addition, they provide important novel evidence to suggest 1) that PKC-dependent mechanisms are essential for both the cellular exportation and the biological activity of ANXA1 and 2) that the exported protein is serine phosphorylated.

Peptide analogs
The results confirm our previous finding (3, 9) that the forskolin-evoked release of ACTH and PRL is readily inhibited, in a concentration-dependent manner, by hrANXA11–346 and ANXA11–188. The two proteins were equipotent in this regard and, at concentrations in the region of 500 pM, both produced a maximal inhibition of stimulated peptide release analogous to that induced by dexamethasone (0.1 µM) and by other GCs (10, 51). We conclude from these findings that the third and fourth C- terminal repeat domains (which are deleted in ANXA11–188) do not contribute to the inhibitory actions of ANXA1 on pituitary peptide release. Data from studies in other biological systems (e.g. models of inflammation and cell growth) suggest that the N-terminal domain plays a crucial role in the manifestation of the actions of the protein (26, 42, 52) and that a core sequence (residues 18–22, EQEYV) is particularly important with regard to cell growth (27). Our finding that the N-terminal peptides ANXA1Ac1–50 (which includes the first 6 amino acids of the first repeat domain) and ANXA1Ac2–26 suppress the forskolin-evoked release of ACTH and PRL is consistent with this view. However, these N-terminal ANXA1 peptides lacked the potency and the efficacy of the full length parent protein in inhibiting pituitary peptide release. They were thus effective only at relatively high concentrations (0.5–50 µM) and appeared to act as partial agonists, producing at best 40–70% inhibition of forskolin-induced ACTH and PRL release. Low potency is also a feature of the activity profile of ANXA1Ac2–26 and other N-terminal ANXA1 peptides in models of inflammation (26, 42, 52) and cell growth (27, 53). However, in contrast to its actions in the pituitary gland, ANXA1Ac2–26 retains the efficacy of the parent protein in these biological systems and thus acts as a full agonist (52, 53). These findings raise the possibility that the mechanism effecting the inhibitory actions of ANXA1 on pituitary peptide release differ from those responsible for the anti-inflammatory and antiproliferative actions of the protein. Further evidence to this effect is provided by our finding that ANXA1Ac2–12 and ANXA1Ac13–25, which respectively mimic the ability of hrANXA1 to promote neutrophil detachment (43) and to inhibit the growth and proliferation of A549 cells (53), are inert in our system. Moreover, the difference in potency between ANXA11–346 and ANXA1Ac2–26 in our pituitary system is more pronounced than it is in various models of inflammation (52) and of cell growth and proliferation (27). Furthermore, in these systems, unlike the pituitary gland, ANXA11–188 is less active than the parent molecule (27, 52).

The finding that ANXA1Ac2–26 and ANXA1Ac1–50 lack the potency and efficacy of the full-length protein in the pituitary gland suggests that other sequences within the core are essential for the manifestation of the full biological activity of the ANXA1 molecule. Such sequences could serve to anchor the protein to the cell surface and align it appropriately for biological action or possibly for posttranslational modification (e.g. phosphorylation), which may in turn influence biological activity. To obtain some insight to this question, we took advantage of the considerable degree of sequence homology between the core domains of ANXA1 and ANXA5 and examined the activity of a chimeric protein (ANXA1/5), which comprised the ANXA1 N-terminal (residues 1–44) and the ANXA5 core (Fig. 1Go). The data showed that ANXA1/5 is some 100-fold more potent than ANXA1Ac2–26 and ANXA1Ac1–50 in suppressing peptide release but that like the N-terminal peptides it shows only partial agonist activity. They thus suggested that the core is instrumental in the manifestation of potency but not efficacy. The failure of ANXA1/5 to display the activity of ANXA11–346 and ANXA11–188, may reflect critical sequence differences in the first two repeat units of the core domains of ANXA1 and ANXA5, which influence the binding and folding of the protein. Interpretation of these data are, however, further complicated by our finding that ANXA5 itself suppresses the evoked release of PRL, an observation that accords with reports that the protein suppresses TRH-stimulated PRL release from rat anterior pituitary cells (54). In addition, ANXA5 effectively opposed the inhibitory effects of dexamethasone, and to a lesser extent ANXA1Ac2–26 on the evoked release of ir-ACTH and ir-PRL; this action may relate to the ability of ANXA5 to oppose the actions of PKC (34, 55 ; see below).

Role of PKC
The present data point to a critical role for PKC in the manifestation of the regulatory effects of GCs on pituitary peptide release and thus concur with the earlier reports of Shipston and colleagues (35). Blockade of PKC with PKC19–36 effectively abolished the inhibitory actions of dexamethasone on forskolin stimulated ACTH and PRL release in our system and also attenuated the activity of ANXA1Ac2–26. In addition, PKC blockade prevented the dexamethasone- induced translocation of ANXA1 to the cell surface, a step we believe is critical to the putative paracrine action of ANXA1 in the anterior pituitary gland (3, 4, 11). The latter finding raised two possibilities. First, that the steroid-induced activation of PKC causes serine/threonine phosphorylation of ANXA1 and that the phosphorylated protein is a substrate for the transporter system; it is thus translocated to the pericellular compartment where it exerts its regulatory actions on peptide release. Alternatively, PKC may phosphorylate a component of the ANXA1 transport machinery and thereby trigger the cellular exportation of the protein and its consequent delivery to its site of action. While our data clearly demonstrate that ANXA1 exported from the pituitary cells is serine phosphorylated, we cannot exclude the possibility that the transport system, which may involve ATP-binding cassette transporter proteins (56), is regulated by PKC. Indeed, our observation that the PKC inhibitor reduces the exportation of serine phosphorylated ANXA1 in the absence of steroids supports this view. Other evidence however points strongly to ANXA1 as a key target for PKC-dependent phosphorylation. Importantly, the protein is known to serve as a substrate for PKC in several other cellular systems; moreover, its phosphorylation sites are well conserved from sponges to humans and therefore likely to fulfil biologically important functions (19, 57). Furthermore, we have recently demonstrated GC-induced PKC-dependent serine-phosphorylation of ANXA1 in a human folliculo-stellate cell line (PDFS, 58). Our novel finding that ANXA5 significantly attenuates the inhibitory actions of dexamethasone on ir-ACTH and ir-PRL release may also be explained by inhibition of PKC activity as ANXA5 has been shown to inhibit PKC-dependent ANXA1 phosphorylation in a number of in vitro systems (49, 51), apparently by sequestering the enzyme rather than serving as an alternative substrate (19, 57, 59). Indeed, PKC-dependent ANXA1 phosphorylation is undetectable in cells that co-express ANXA1 and ANXA5, such as endothelial cells (33). The physiological significance of the effects of ANXA5 observed in our in vitro system remains to be determined. However, because ANXA5 and ANXA1 are both expressed in the anterior pituitary gland (2), the data raise the intriguing possibility that ANXA5 may serve as a physiological antagonist of GC action in this tissue.

Further studies are now required to determine the serine residues within the ANXA1 molecule that are phosphorylated in the pituitary gland and their respective significance to peptide release. Our studies in human pituitary cells suggest that Ser-27–28 is an important target (58), although this site is poorly conserved with the rat expressing Tyr at residue 28. Other potentially important sites include serines 5, 34, 37, 45–46 (conserved between human and rat) and serine/threonines 24 and 41 (differences in amino acid sequence between human and rat). While phosphorylation sites in both the N-terminal and the core of ANXA1 may contribute to the exportation of ANXA1, our finding that the PKC inhibitor exerted an inhibitory effect, albeit relatively modest, on the suppressive effects of ANXA1Ac2–26 on the forskolin-evoked release of ir-ACTH and ir-PRL suggests that phosphorylation sites in the N-terminal peptide (i.e. Ser-5 or Thr 24) may be important; how such peptides would be phosphorylated in our in vitro system is unclear. Alternatively, PKC may act at a site within the signaling cascade that effects the blockade of peptide release by ANXA1. It is evident from the data reported here and elsewhere (60, 61, 62) that forskolin-evoked pituitary hormone release involves the cAMP signal transduction pathway in the anterior pituitary gland and is dependent upon the activation of PKA (60, 61, 62). Our previous studies have suggested that the inhibitory actions of ANXA1 and GCs on forskolin-induced pituitary hormone release are exerted at a point distal to the formation of cAMP (9, 10); in accord with these findings we report here that while ANXA1Ac2–26 and dexamethasone readily block forskolin-stimulated peptide release, neither drug interferes with the evoked accumulation of cAMP, an observation that concurs data from an earlier study on GCs in primary anterior pituitary cell culture (63).

Mechanisms of ANXA1 action
ANXA1 has been implicated in the process of membrane fusion and exocytosis in a variety of other systems (22, 23, 64, 65, 66). Paradoxically, however, and in stark contrast to our findings in the hypothalamo-pituitary system, the majority of data suggest that it facilitates secretory processes, possibly by promoting Ca2+-dependent membrane aggregation (49, 64, 65, 66). However, such actions may be concentration dependent; whereas low concentrations of ANXA1 promote exocytotic membrane fusion in human neutrophils, higher concentrations (e.g. induced for example by dexamethasone) effectively inhibit Ca2+-dependent fusion (64). Other data suggest that changes in phosphorylation status of the protein may also be crucial in this regard. Thus, tyrosine-kinase-dependent phosphorylation of residue 21 (Tyr-21) reduces the amount of Ca2+ required for ANXA1 binding to phospholipid vesicles in a cell free system (29). On the other hand, when phosphorylated by PKC at an undefined locus in the N-terminal, ANXA1 has been shown to inhibit granule aggregation at low Ca2+ levels (32). These findings, together with the present data, raise the possibility that PKC-mediated phosphorylation is essential for the manifestation of the powerful antisecretory properties of the protein reported by us here and elsewhere (3, 6, 9, 10, 11, 12). The apparent lack of prosecretory ANXA1 activity in the anterior pituitary is interesting and clearly contrasts with other systems. It may be explained at least in part by the fact that ANXA1 is expressed predominantly in the nonsecretory folliculo-stellate cells (4, 7), where it undergoes serine phosphorylation before externalization, not in the endocrine cells where the machinery normally effecting exocytosis would almost certainly be located. Alternatively, the mechanisms for tyrosine phosphorylation of ANXA1, which appears to facilitate the prosecretory actions in other tissues (29) may be lacking in normal pituitary tissue, a possibility we are currently exploring.


   Conclusion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
The results show for the first time that amino acid residues 2–26 are important in mediating the inhibitory effects of ANXA1 on the release of ACTH and PRL from the anterior pituitary gland, but that sequences within the first two repeat units of the core domain are also required for the manifestation of the full efficacy and potency of the molecule. They also provide new evidence that the ANXA1-dependent regulatory actions of dexamethasone on pituitary peptide release are dependent upon PKC, which serves to facilitate the steroid-dependent exportation of a serine-phosphorylated species of ANXA1 from the pituitary cells, and possibly also for the inhibitory actions of ANXA1 on the secretory cells.


   Acknowledgments
 
We are grateful to Dr. Jamie Croxtall (William Harvey Research Institute), Professor Phil Lowry (University of Reading, UK), and Professor Dame Lesley Rees, respectively, for the generous gifts of anti-ANXA1 pAb, anti-ACTH pAb and 125I-labeled ACTH.


   Footnotes
 
We are grateful to the Wellcome Trust (Grant No. 051887/C/97/Z/MW/NP/JF), Hammersmith Hospital Trustees and Chemodyne (Geneva) for generous financial support.

Abbreviations: ANXA1, Annexin 1; GC, glucocorticoid; hr, human recombinant; ir, immunoreactive; pAB, polyclonal antibody; PKC, protein kinase C; PRL, prolactin; TBS, Tris-buffered saline.

Received March 6, 2002.

Accepted for publication April 29, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

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