This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Solito, E. Articles by Buckingham, J. C. Search for Related Content PubMed PubMed Citation Articles by Solito, E. Articles by Buckingham, J. C. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB DEXAMETHASONE L-SERINE Medline Plus Health Information Steroids

Dexamethasone Induces Rapid Serine-Phosphorylation and Membrane Translocation of Annexin 1 in a Human Folliculostellate Cell Line via a Novel Nongenomic Mechanism Involving the Glucocorticoid Receptor, Protein Kinase C, Phosphatidylinositol 3-Kinase, and Mitogen-Activated Protein Kinase

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

Address all correspondence and requests for reprints to: Professor Julia Buckingham and Dr. Egle Solito, Department of Neuroendocrinology, Division of Neuroscience and Psychological Medicine, Faculty of Medicine, Imperial College London, Commonwealth Building, Hammersmith Hospital Campus, Du Cane Road, London W12 ONN, United Kingdom. E-mail: j.buckingham{at}imperial.ac.uk; e.solito{at}imperial.ac.uk.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our recent studies on rat pituitary tissue suggest that the annexin 1 (ANXA1)-dependent inhibitory actions of glucocorticoids on ACTH secretion are effected via a paracrine mechanism that involves protein kinase C (PKC)-dependent translocation of a serine-phosphorylated species of ANXA1 (Ser-P-ANXA1) to the plasma membrane of the nonsecretory folliculostellate cells. In the present study, we have used a human folliculostellate cell line (PDFS) to explore the signaling mechanisms that cause the translocation of Ser-P-ANXA1 to the membrane together with Western blot analysis and flow cytometry to detect the phosphorylated protein. Exposure of PDFS cells to dexamethasone caused time-dependent increases in the expression of ANXA1 mRNA and protein, which were first detected within 2 h of steroid contact. This genomic response was preceded by the appearance within 30 min of substantially increased amounts of Ser-P-ANXA1 and by translocation of the phosphorylated protein to the cell surface. The prompt membrane translocation of Ser-P-ANXA1 provoked by dexamethasone was inhibited by the glucocorticoid receptor, antagonist, mifepristone, but not by actinomycin D or cycloheximide, which effectively inhibit mRNA and protein synthesis respectively in our preparation. It was also inhibited by a nonselective PKC inhibitor (PKC_9–31), by a selective inhibitor of Ca²⁺-dependent PKCs (Go 6976) and by annexin 5 (which sequesters PKC in other systems). In addition, blockade of phosphatidylinositiol 3-kinase (wortmannin) or MAPK pathways with PD 98059 or UO 126 (selective for MAPK kinse 1 and 2) prevented the steroid-induced translocation of Ser-P-ANXA1 to the cell surface. These results suggest that glucocorticoids induce rapid serine phosphorylation and membrane translocation of ANXA1 via a novel nongenomic, glucocorticoid receptor-dependent mechanism that requires MAPK, phosphatidylinositiol 3-kinase, and Ca²⁺-dependent PKC pathways.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE ANNEXINS are a well-conserved family of structurally related proteins that bind Ca²⁺ and phospholipids. Members of the family are characterized by a highly homologous core domain of four (or eight in the case of annexin 6) internal repeats of a conserved 70- to 75-amino- acid sequence that extends to the C-terminal and permits Ca²⁺ and phospholipid binding (1). The N-terminal domain of each family member is unique in length and sequence and appears to determine the biological specificity of the individual proteins; in several cases (e.g. annexin 1), this domain includes potential sites for phosphorylation, glycosylation, and peptidase action (2). Annexins are implicated in the regulation of many physiological and pathophysiological functions, including cell growth and differentiation (3), inflammation (4), and neuroendocrine function (5). Our work has centered on annexin 1 (ANXA1), which we have shown to play a major role in the manifestation of the acute regulatory actions of glucocorticoids (GCs) in the rodent neuroendocrine system, acting at the levels of both the hypothalamus (6, 7, 8) and the anterior pituitary gland (9, 10, 11).

ANXA1 (previously known as lipocortin 1) is expressed in abundance in the rat and human neuroendocrine system (12, 13). Within the anterior pituitary gland, it is localized mainly to the S100-positive folliculostellate (FS) cells (13, 14, 15), but small amounts are also present in the endocrine cells (13, 15, 16). As in other tissues (17), GCs regulate both the expression and the subcellular distribution of ANXA1 in the anterior pituitary gland (7, 9, 10). Their initial effect is to cause translocation of ANXA1 from the cytoplasm to the outer cell surface where it is retained via a Ca²⁺-dependent mechanism (7, 9, 10). Subsequently, the steroids induce de novo ANXA1 synthesis, an action that appears to serve principally to replete the diminished stores of the protein (7). Binding and functional studies suggest that the GC-induced exportation of ANXA1 from pituitary cells is an important mechanism that enables the protein to gain access to binding sites (putative receptors) on the surface of the endocrine cells and thereby exert paracrine/autocrine regulatory actions on the release of ACTH and other pituitary hormones. In support of this hypothesis, we have identified high-affinity ANXA1 binding sites on the surface of all the main subtypes of endocrine cells in the rat anterior pituitary gland (18) and shown that recombinant ANXA1 (8, 9, 19) and peptides derived from the N-terminal of the protein (20) mimic the acute inhibitory actions of GCs on pituitary hormone release. Further evidence of a role for ANXA1 in mediating these early steroid actions has been derived in vitro from studies using antisense probes directed against ANXA1 mRNA (10, 11) and by the use of neutralizing anti-ANXA1 antisera in vivo and in vitro (8, 9, 11, 19, 21).

The precise role of the FS cells in the anterior pituitary gland is obscure. However, increasing evidence supports the contention that these stellate, agranular cells exert paracrine actions that influence both the growth and the secretory activity of the endocrine cells (22, 23). They may also protect the endocrine cells from toxins by acting as scavengers (24). In many respects, the FS cells resemble neuroglial cells and, indeed, they express two important markers of these cells, S-100 and glial fibrillary acidic protein (GFAP). In addition, they show similarities with the dendritic cells of the immune system and have the capacity to produce IL-6 and other cytokines (25), particularly in the face of an immune challenge (26). These and other data have led to suggestions that the FS cells may stem from an immunological lineage (27); they may therefore provide an important means of local cross-talk between the host-defense and neuroendocrine systems. Our finding that the FS cells are the principle source of ANXA1 within the anterior pituitary gland has led us to propose that ANXA1 serves as a paracrine mediator of GC action in the pituitary gland and that the FS cells are thus an important locus of GC action in this organ (5, 14, 18). Several other lines of evidence support this view. Firstly, the FS cells are rich in glucocorticoid receptors (GRs; Ref. 28). Secondly, on a temporal basis, the GC-induced cellular exportation of ANXA1 in the rodent anterior pituitary gland parallels the onset of the steroid inhibition of ACTH secretion, both emerging within 15 min and reaching a maximum within 2 h (9). Thirdly, drugs that inhibit the cellular exportation of ANXA1 also suppress the acute antisecretory actions of GCs (9, 10, 20). Fourthly, FS cell ANXA1 is found both in the cytoplasm and in association with the plasma membrane, particularly in the stellate projections that lie in close apposition with the endocrine cells (14). Finally, immunogold labeling suggests that GC treatment results in a marked increase in ANXA1 expression in the FS cells, most notably at the plasma membrane (29).

The mechanism by which GCs cause the translocation of ANXA1 from the cytoplasm to the cell membrane of FS cells is unknown. ANXA1 does not have a signal sequence and would therefore be unlikely to enter the endoplasmic reticulum-Golgi complex for vesicular packaging. In accord with this premise, the GC-induced cellular exportation of the protein in rodent pituitary tissue is unaffected by drugs that block the conventional exocytotic pathway (30). Our early studies (9) provided preliminary evidence that the steroids act via a novel nongenomic mechanism to export ANXA1. Our more recent work has identified a role for protein kinase C (PKC) in this regard and shown that the exported protein is serine-phosphorylated (20). In line with these findings, data from other systems suggest that nongenomic steroid actions fulfil a pivotal role in physiological processes as they regulate ion channels (31), second messengers and protein kinases, such as protein kinase A (32), PKC, and MAPKs (33, 34).

In the present study, we have used the first transformed human FS cell line (PDFS), derived from a gonadotroph adenoma of the pituitary (35), to examine further the mechanism by which glucocorticoids induce the translocation of annexin 1 to the cell membrane. Our data demonstrate clearly that the steroids induce the prompt appearance of a serine-phosphorylated species of ANXA1 (Ser-P-ANXA1) that is exported from the cell. They also reveal that the actions of the steroids are effected via a nongenomic GR-dependent signaling cascade that involves Ca²⁺-dependent PKC, phosphatidylinositol 3-kinase (PI3-K), and MAPK and that is antagonized by annexin 5 (ANXA5).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Drugs
Dexamethasone 21-phosphate disodium salt, testosterone, 17ß-estradiol, and progesterone were purchased from Sigma-Aldrich Corp. (Poole, UK). The inhibitors of PKC (PKC_19–31 and Gö 6976), MAPK (PD98059), or MAPK kinase (MEK; UO 126) were from Calbiochem (CN Bioscience, Beeston, Nottingham, UK). Mifepristone (RU-486) was synthesized by Roussel Uclaf (Romanville, France). Stock solutions of U0126, Gö6976, PD98059, and actinomycin D (Sigma-Aldrich Corp.) were prepared by dissolving the drugs in dimethylsulfoxide (Sigma-Aldrich Corp.). PKC_19–31 was dissolved initially in 5% acetic acid (1 mg/ml; VWR Int., Poole, UK), whereas cycloheximide (Sigma-Aldrich Corp.) and mifepristone were first dissolved in ethanol (VWR Int.). The drugs were diluted in incubation medium immediately before use; the final concentration of the diluents did not exceed 0.04%. Human recombinant ANXA5 was prepared as described previously (36). Endotoxin contamination was less than 20 pg/ml as measured by the Limulus amoebocyte chromogenic assay.

Antisera
Primary antisera.
Total ANXA1 protein was detected using a polyclonal antiserum (pAb) raised in rabbit against human recombinant ANXA1_1–346; this antiserum has been well characterized previously in studies on nonendocrine human tissue and cell lines (37, 38, 39). Phosphorylated forms of ANXA1 were detected using purified pAbs (HiTrap Protein G affinity column, Amersham Pharmacia Biotech, Buckinghamshire, UK) raised in rabbit against ANXA1_15–31 phosphorylated on either the serine residue at position 27 (anti-ser-P-ANXA1 pAb) or on the tyrosine residue at position 21 (anti-tyr-P-ANXA1 pAb), respectively (Neosystem, Strasbourg, France). Anti-ser-P-ANXA1 pAb was then affinity purified by incubation with the serine phosphorylated ANXA1 protein (27-Ser-P-ANXA1) linked to cyanogen bromide-activated Sepharose 4B (Sigma-Aldrich Corp.). Specificity of the antisera raised against phosphorylated ANXA1 proteins was assured by the findings in Western blot and dot blot analyses that neither antibody cross-reacted with the unphosphorylated protein human recombinant ANXA1_1–188 (1 µg/lane) at the dilution used (1:1000); furthermore, whereas anti-tyr-P-ANXA1 pAb readily detected ANXA1_Ac2–2621-Tyr-P, it failed to recognize a peptide in which the 21Tyr residue was replaced with phenylalanine. Conversely, at a dilution of 1:1000, the affinity purified anti-27-Ser-P-ANXA1 pAb showed no cross-reactivity with the tyrosine phosphorylated peptide ANXA1_Ac2–2621-Tyr-P or an unrelated phosphoserine peptide in a dot blot study but detected 10 ng of the serine phosphorylated peptide against which it was raised.

Antisera against human S100 (raised in rabbit), GFAP (monoclonal, clone G-A-5), human vimentin (monoclonal, clone VIM-13.2), and sea urchin -tubulin (monoclonal, clone B-5-1-2) were all purchased from Sigma-Aldrich Corp. A fluorescein isothiocyanate (FITC)-labeled anti-CD14 monoclonal antibody raised against human CD14 (clone MP9, Becton Dickinson, BD Biosciences, Oxford, UK) was also used.

Secondary antisera.
A peroxidase-conjugated goat antirabbit antiserum (Sigma-Aldrich Corp.) was used for Western blot analysis. FITC-coupled rabbit antimouse or goat antirabbit antisera (Chemicon International, Harrow, UK) were employed for fluorescence-activated cell scanning (FACS) analysis.

Cell culture
PDFS cells, which developed spontaneously from a clinically nonfunctioning pituitary macroadenoma, were a gift from Drs. Anne Klibansky and D. D. Danila (Harvard Medical School, Boston, MA; Ref. 35). The cells were cultured at 37 C and in an atmosphere of 95% O₂ and 5% CO₂ in DMEM-Glutamax (Invitrogen, Paisley, UK), pH 7.5, containing 10% fetal calf serum (PAA Laboratories, Teddington, Middlesex, UK), 1% nonessential amino acids, 0.1% transferrin, 0.1% selenium (Invitrogen), and 0.1% gentamycin (Sigma-Aldrich Corp.).

For experiments, the cells were plated at a density of 1 million cells/well in poly-lysine coated (Sigma-Aldrich Corp.) 10-cm cell culture plates (Corning, Ltd., High Wycombe, UK) and incubated in the conditions described above until the cells attained 70% confluence (24–36 h). After careful washing in PBS (pH 7.4; Invitrogen), the cells were transferred to DMEM-Glutamax and incubated in the presence or absence (controls) of dexamethasone (1 µM) for a period ranging from 30 min to 24 h. Where appropriate, putative inhibitors of the steroid signaling pathway [mifepristone (1 µM), actinomycin D (10 µg/ml), cycloheximide (5 µM), PKC_19–31 (100 nM), GÖ 6976 (25 nM), PD 98059 (5 µM), UO 126 (1 µM), wortmannin (10 nM), and ANXA5 (1 µg/ml)] were present in the medium for 30 min before and throughout the period of contact with the steroid. Appropriate diluent controls were included in all experiments. At the end of the incubation period, the cells were processed for RT-PCR, Western blot, or FACS analyses. In further experiments, the specificity of dexamethasone action was assessed by comparison with testosterone (0.1–1 µM), 17ß-estradiol (0.1–1 µM), or progesterone (0.1–1 µM). In addition, the effectiveness of actinomycin D (10 µM) and cycloheximide (5 µM) in suppressing dexamethasone-induced mRNA and protein expression in the cells was assessed by measurement of the incorporation of [¹⁴C]-lysine into protein as described previously (8), preloading the cells for 30 min with the radiolabeled ligand before drug addition. The cells were used up to a maximum of 35 passages.

Determination of ANXA1 mRNA by RT-PCR
Expression of mRNA for ANXA1 was analyzed by RT-PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control. Details of the primers used are shown in Table 1. Total RNA was extracted from the samples according to the manufacturer’s instructions using the RNAeasy QIAGEN Kit (QIAGEN Ltd., Crawley UK). The samples were then analyzed for RNA concentration and purity by spectrophotometry and stored at -80 C until required. For first-strand cDNA synthesis, 3 µg total RNA from each sample was denatured with 3 µl oligo (deoxythymidine)₁₅ primer (Promega Corp., Southampton, UK) for 5 min at 65 C and reverse transcribed by incubation at 42 C for 2 h in a total volume of 40 µl buffer comprising 1.25 mM deoxy-NTPs (Amersham Biosciences, Buckinghamshire, UK), 4 U/ml ribonuclease inhibitor (RNAsine; Promega Corp.), and 9 U Moloney murine leukemia virus (Promega Corp.). For the PCR, an equivalent of 50–100 ng mRNA together with 50 mM MgCl₂, 10 mM deoxy-NTP, 50 mM KCl, 10 mM Tris-Cl (pH 8.3), 1 µl Taq polymerase (5 U/ml), and 0.5 pmol/µl of each of the specific primers (Table 1) was used in a total volume of 100 µl. The cycling parameters consisted of an initial denaturing step at 95 C for 5 min, followed by 30 cycles of denaturation (95 C, 1 min), primer specific annealing (1 min, see Table 1 for temperature) and extension (72 C, 1 min) and a final extension stage (15 min, 72 C). The PCR products (10 µl per lane) were separated by electrophoresis [100 V, 30 min in Tris acetate EDTA buffer (Invitrogen)] on 1% agarose gels (Invitrogen) and stained with ethidium bromide (Sigma-Aldrich Corp.). Analysis of the gels was performed under UV light using a camera image analysis system (Herolab GmBH Laborgerte, Wiesloch, Germany) and TINA software (version 2.10, Raytest Isotopenmessgeräte GmBH, Straubenhardt, Germany). Amplification of the correct sequence was confirmed by product length (DNA molecular weight marker III; Roche Molecular Biochemicals, Mannheim, Germany) and also by amplification of ANXA1, control plasmids (39). Preliminary studies confirmed that the degree of product amplification (ANXA1 and GAPDH) obtained under these assay conditions was within the exponential range of the curve, thereby permitting semiquantitative analysis of mRNA expression by comparison with GAPDH. Experiments were repeated three times.

FACS analysis
Quantification of cell surface ser-P-ANXA1 expression.
Aliquots of the cell suspension (1–3 x 10⁵ cells in 100 µl) were added to a 96-flat well plate in triplicate and incubated at 4 C for 1 h in the presence or absence (controls) of anti-ser-P ANXA1 pAb (1:300) in a buffer A; containing HEPES (25 mM), CaCl₂ (1 mM), and MgCl₂ (1 mM); nonspecific binding was minimized by the addition of human IgG (1.6 mg/ml, Roche Diagnostics, Lewes, UK). The cells were then washed in buffer A and incubated on ice for 30 min with F(ab')₂ fragments of goat antirabbit IgG FITC-conjugated secondary antibody (diluted 1:600). After further washing in buffer A, the cell surface fluorescence was analyzed immediately by flow cytometry. In the majority of experiments, cell membrane integrity was assessed by sequential incubation of the cells with anti-actin monoclonal antibody (diluted 1:20) and FITC-conjugated rabbit antimouse IgG (diluted 1:200). In some experiments, preimmune serum was employed as a further control.

Quantification of intracellular ser-P-ANXA1 expression.
The protocol was as above except that saponin (0.0025% wt/vol, Sigma-Aldrich Corp.) was included in the medium during the period of contact with both the primary and secondary antibodies so as to permeabilize the cells. Parallels measure of -actin were made to measure the effectiveness of the permeabilization procedure.

Detection of cell marker proteins.
Aliquots of PDFS cells were also incubated as described above (in saponin and buffer A) in the presence or absence of antisera against S100 (1:200), GFAP (1:200), vimentin (1:100), before a second incubation with FITC-conjugated rabbit antimouse or goat antirabbit IgG (1:1:200). Nonpermeabilized cells were also incubated with an antihuman CD14 antibody coupled to FITC (1:200) to measure the membrane expression of the protein.

Detection and analysis of fluorescence.
In all cases, a Becton Dickinson FACScan II analyser with air-cooled 100-mW argon ion laser tuned to 488 nm and Consort 32 computer running Lysis II software (Becton Dickinson and Co.) was used. At least 10,000 cells per sample were counted and characterized by dot plot according to their forward and side scatter characteristics. The data were analyzed as units of fluorescence measured in the FL1 channel (mean fluorescence intensity). Within each experiment, the data were normalized to background [i.e. fluorescence due to second antibody alone or, in the case of experiments in which the preimmune serum was also used, preimmune serum plus secondary antibody] and expressed as a percentage of control (i.e. unstimulated) cells. Where a vehicle was used, the data were further corrected for fluorescence due to vehicle.

Statistics
The optical density of bands of ANXA1 gene expression and immunoreactivity (arbitrary units) detected by RT-PCR and Western blot analyses respectively (see Figs. 2 and 3) was corrected for loading error by expression as a percentage of house keeping marker (GAPDH or -tubulin). Responses to the steroids were calculated as a percentage of the corresponding drug-free (i.e. basal) control and expressed as mean ± SEM (n = 3 gels); statistical comparison between groups were made using the Mann-Whitney U test. It must be noted that these measurements are essentially semiquantitative and give only a relative numerical guide to the ratios of the band intensities and their variance.

View larger version (17K): [in this window] [in a new window]   Figure 2. Time-dependent effects of dexamethasone (1 µM) on the expression of A, ANXA1 mRNA (normalized to GAPDH); B, total ANXA1 protein (normalized to -tubulin); and C, serine-phosphorylated ANXA1 (intact line) and tyrosine-phosphorylated ANXA1 (dotted line), normalized to -tubulin. A, ANXA1 mRNA analysis of the band [expressed as optical density (% of steroid free control) and corrected for GAPDH]. The data in panel B (mean ± SEM, n = 3 experiments) were derived by Western blot analysis and show ANXA1 protein [expressed as optical density (% of steroid free control) and normalized to -tubulin]; **, P < 0.01 and *, P < 0.025 vs. untreated controls (Mann-Whitney U test).

View larger version (14K): [in this window] [in a new window]   Figure 3. Effects of dexamethasone (1 µM) on the expression of membrane-bound and cytosolic serine-phosphorylated ANXA1 (Ser-P-ANXA1) as determined by Western blot analysis (A) and FACS analysis (B and C). The data in panel A (mean ± SE, n = 3 experiments) are expressed as optical density (normalized to -tubulin and expressed as a percentage of the corresponding dexamethasone-free control); intact line, cytosolic ANXA1; dotted line, membrane bound ANXA1; **, P < 0.01; *, P < 0.025 vs. untreated controls (Mann-Whitney U test). B, Representative profile showing fluorescence intensity of cells stained for membrane-bound Ser-P-ANXA1 following incubation of the cells for 30 min in the presence or absence of dexamethasone (1 µM). Note the low background fluorescence due to the second antibody (IIAb) and the lack of actin fluorescence, confirming the integrity of the cell membrane. C, Mean data (expressed as mean of intensity of fluorescence FL1) from three experiments in which the cells were exposed to dexamethasone (1 µM) for 30 min; open columns, cytosolic Ser-P-ANXA1; closed columns, membrane-bound Ser-P-ANXA1. Data are expressed as mean ± SEM (n = 3); *, P < 0.05; P < 0.001 vs. untreated controls (ANOVA and Bonferroni test).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of the PDFS cells
In initial experiments, we used FACS analysis to examine the expression of three well-defined FS cell markers, S-100, GFAP, and vimentin, by the PDFS cells. Figure 1A shows clearly that the cells were positive for all three markers. As S100 and GFAP are also markers of neuroglial cells, these findings suggest that, like normal FS cells, the PDFS cells show some characteristics of neuroglial cells. Further studies revealed that, like microglia (41), the PDFS cells also express the monocyte/macrophage marker CD14 (Fig. 1B) as too does the murine FS cell line, TtT/GF (26). Taken together, the data raise the possibility that the PDFS cells are a potential pituitary monocyte/macrophage cell line with glia-like characteristics.

View larger version (23K): [in this window] [in a new window]   Figure 1. Detection of cell makers in PDFS cells by flow cytometry. Representative FACS profiles demonstrating increased fluorescence intensity in PDFS cells immmunostained for (A) S100, GFAP, and vimentin or (B) CD14. IIAb, Fluorescence intensity due to second antibody alone. Actin positivity (A) or negativity (B) is a control of membrane permeabilization.

Dexamethasone-induced translocation of Ser-P-ANXA1 to the cell surface
Figure 3 illustrates the effects of dexamethasone (1 µM) on the distribution of Ser-P-ANXA1 between the cytoplasm (intracellular) and the plasma membrane (cell surface ANXA1) of PDFS cells, as determined by Western blot (Fig. 3A) and FACS (Fig. 3, B and C) analyses. In the absence of steroid, small amounts of Ser-P-ANXA1 were detectable by both methods both within the cells and on the cell membrane. Exposure of the cells to dexamethasone for up to 8 h caused a prompt and persistent increase in cell surface Ser-P-ANXA1, with maximal levels occurring at 30 min (P < 0.01 Fig. 3A); an increase in intracellular Ser-P-ANXA1 was also observed after 2 h contact with the steroid (P < 0.05). Similarly, when Ser-P-ANXA1 was measured by FACS analysis after a 30-min exposure to dexamethasone (1 µM), substantially increased amounts of the protein were observed on the cell surface (P < 0.001, Fig. 3, B and C); in contrast, a decrease in cytoplasmic Ser-P-ANXA1 was evident at this time (P < 0.05, Fig. 3C). No changes in the expression or cellular disposition of Ser-P-ANXA1 were detectable with shorter periods of steroid contact (5 min, data not shown). Inclusion of the GR antagonist, mifepristone (1 µM), in the medium (30 min before and during the period of steroid contact) prevented the appearance of Ser-P-ANXA1 on the cell surface induced by exposure to dexamethasone for 30 min (P < 0.005) or 2 h (P < 0.005), suggesting that these actions of the steroid are mediated via the GR (Fig. 4). Mifepristone alone had no effect on membrane Ser-P-ANXA1 during the shorter contact time (i.e. 30-min preincubation plus 30-min incubation period). However, when in contact with the cells for a total of 2.5 h (30-min preincubation plus 2-h incubation period), it caused a small but significant increase (P < 0.005) in membrane Ser-P-ANXA1. This may reflect the partial agonist activity of the drug at the GR [Fig. 4B, (42)], although such a mechanism would not explain the powerful combined actions of mifepristone with dexamethasone.

View larger version (24K): [in this window] [in a new window]   Figure 4. Effects of the GR antagonist mifepristone (RU-486, 1 µM) on the translocation of Ser-P-ANXA1 to the membrane [determined by Western blot (A) and FACS analysis (B and C)] induced by exposure of PDFS cells to dexamethasone for 30 min or 2 h. B, Representative scans showing fluorescence intensity of cells stained for membrane-bound Ser-P-ANXA1 following incubation of the cells for 30 min in the presence or absence of dexamethasone (1 µM) and or mifepristone. Note the low background fluorescence due to the second antibody (IIAb) and that mifepristone alone had no effect on the membrane expression of Ser-P-ANXA1 (A), whereas it is blocking the response to the steroid (B). C, Mean data ± SEM [corrected for background fluorescence and expressed as a percentage of the steroid-free control (MIF = 16.63 ± 0.5)] from three experiments in which the cells were exposed to dexamethasone (1 µM) and or mifepristone (1 µM) for 30 min (open columns) or 2 h (closed columns). **, P < 0.005 and ***, P < 0.001 vs. untreated controls; ##, P < 0.005; vs. dexamethasone alone; 2 + P < 0.005 vs. mifepristone alone (ANOVA and Bonferroni test).

View larger version (14K): [in this window] [in a new window]   Figure 5. Effects of cycloheximide (CHX, 5 µM) and actinomycin D (Act-D, 10 µg/ml) on (A) the incorporation of 14C-lysine into protein (open columns, control; closed columns, dexamethasone) and (B and C) the translocation of Ser-P-ANXA1 to the plasma membrane induced by exposure of the PDFS cells to dexamethasone for 30 min (open columns) or 2 h (closed columns). The data in A are expressed as the mean ± SEM (n = 3). *, P < 0.025; **, P < 0.01; P < 0.001 vs. steroid-free control; open columns, controls; black columns, dexamethasone (1 µM). 2++, P < 0.001 vs. dexamethasone alone. The data shown in B and C are derived from Western blot (top panels) and FACS (histograms) analyses; the latter are corrected for background fluorescence and expressed as a percentage of the steroid-free control (MIF = 16.63 ± 0.5). The data are expressed as the mean ± SEM (n = 3 experiments); *, P < 0.05; vs. untreated controls; ##, P < 0.005 vs. dexamethasone alone (ANOVA and Bonferroni test).

View larger version (16K): [in this window] [in a new window]   Figure 6. Effects of (A and B) the PKC inhibitors PKC19–31 (100 nM) and Gö 6976 (25 nM, selective for Ca2+-dependent PKCs) and (C) the MAPK inhibitor (PD 98059, 5 µM), the selective MEK 1 and 2 inhibitor (UO 126, 1 µM) and the PI3-kinase inhibitor wortmannin (10 nM) on the translocation of Ser-P-ANXA1 (determined by FACS analysis) to the plasma membrane induced by exposure of the PDFS cells to dexamethasone (1 µM, 30 min). A, Representative scan and shows the fluorescence intensity of cells stained for membrane-bound Ser-P- ANXA1. Note 1) the low background fluorescence due to the second antibody (IIAb) alone and 2) that actin (an intracellular protein) was barely detectable, indicating that the membrane has retained its integrity. PKC9–31 alone had no effect on the membrane expression of Ser-P-ANXA1, but it blocked the response to the steroid. B and C, Mean data ± SEM from three experiments [corrected for background fluorescence and calculated as a percentage of the steroid-free control (MIF = 17 ± 0.7 and MIF = 17.9 ± 1, respectively)]; *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. untreated controls; #, P < 0.05; ##, P < 0.01; ###, P < 0.001; vs. dexamethasone alone (ANOVA and Bonferroni test).

View larger version (19K): [in this window] [in a new window]   Figure 7. Effects of ANXA5 (30 nM) on the translocation of Ser-P-ANXA1 (determined by FACS analysis) to the plasma membrane induced by exposure of the PDFS cells to dexamethasone (1 µM, 30 min). A, Representative scan and shows the fluorescence intensity of cells stained for membrane-bound Ser-P-ANXA1. Note that the low background fluorescence due to the second antibody (IIAb) and that, whereas ANXA5 alone had no effect on the membrane expression of Ser-P-ANXA1, it reduced the response to the steroid. B, Mean data ± SEM from three experiments (corrected for background fluorescence and calculated as a percentage of the steroid-free control (MIF = 74.04 ± 2.3). *, P < 0.05 vs. untreated controls; #, P < 0.05; vs. dexamethasone alone (ANOVA and Bonferroni test).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In recent years, increasing evidence has accumulated to support the premise that GCs and other steroid hormones exert rapid effects that cannot be accommodated within the classical framework of steroid action, i.e. through alterations in transcription brought about by interactions of the activated ligand-receptor complex with DNA, transcription factors, or coactivators/corepressors (45). The nongenomic actions, which emerge in minutes rather than hours, as is usually the case for genomic actions, have been most widely studied in neuronal tissues (46), but there is also substantive evidence for such actions in the endocrine system and elsewhere (29). With respect to feedback actions of GCs within the hypothalamo-pituitary-adrenal axis, nongenomic actions have been associated principally with the initial rapid phase of feedback, which is evident within 2–3 min of steroid contact and persists for up to 15 min (47). As reviewed briefly in the introduction, our work suggests that ANXA1 is concerned with the manifestation of the second phase of GC inhibition within this axis, i.e. the early delayed phase, which emerges within 15–30 min of a steroid challenge and persists for up to 24 h (48), and that its actions in the pituitary gland are mediated via a paracrine mechanism involving translocation of the protein to the surface of FS cells (9, 10, 11). It has frequently been suggested that the early delayed feedback actions of GCs are effected via classical genomic actions (5). In accord with this view, the present data demonstrate de novo expression of ANXA1 mRNA and protein in PDFS cells treated for 2 h with dexamethasone. In addition, they show that the concomitant steroid-induced translocation of Ser-P-ANXA1 to the cell membrane observed at this time point is susceptible to inhibition by mifepristone, actinomycin D, and cycloheximide and is thus dependent upon GR-mediated mRNA and protein synthesis. In contrast, however, the cellular exportation of Ser-P-ANXA1 that occurred during a shorter period of steroid contact (30 min) was unaffected by actinomycin D or cycloheximide. These novel findings suggest that in the first instance the steroid-induced exportation of Ser-P-ANXA1 is independent of transcription/translation and is, thus, effected via a nongenomic GR-dependent mechanism. Taken together, these findings raise the possibility that the early-delayed feedback actions of GCs within the hypothalamo-pituitary-adrenal axis involve both genomic and nongenomic mechanisms, as was also mooted by Taylor et al. (9).

The mechanisms of nongenomic steroid actions are a focus of much current research. Particular attention has focused on the allosteric actions of neurosteroids on membrane-bound neurotransmitter receptors within the brain, for example the GABA_A receptor (46, 49). However, increasing evidence points to the existence of specific membrane-bound steroid receptors (50), which may be coupled either to PKC- or protein kinase A-dependent signaling pathways via G proteins (51, 52) or to other signaling systems. For example, in MCF-7 cells (mammary gland tumor cells) ligand activation of the estrogen receptor triggers the tysosine-kinase/p21^ras/MAPK pathway (53), whereas 17ß-estradiol activates MAPK pathways in rat cardiomyocytes (33). Further nongenomic steroid actions leading, for example, to activation of MAPK and other signaling cascades may also be brought about through stimulation of the classical intracellular receptors (54). Our data show that the early actions of dexamethasone within the PDFS cells, which cause the translocation of serine-phosphorylated ANXA1 to the cell membrane, are blocked by mifepristone. They thus suggest that the actions of the steroid are mediated by GR, although extensive pharmacological studies with more selective compounds are required to verify this. Although we did not examine the effects of mifepristone and other drugs on the intracellular pool of Ser-P-ANXA1, the fact that we could not detect changes in total or membrane Ser-P-ANXA1 with dexamethasone contact times of less than 30 min (data not shown) and that after 30-min exposure to the steroid much of the phosphorylated protein was found in association with the cell membrane suggests that phosphorylation of ANXA1 is an important early step in this GR-dependent, nongenomic signaling cascade. Direct measures of steroid-dependent ANXA1 phosphorylation following exposure to mifepristone and RNA/protein synthesis inhibitors are now underway to address this issue.

As a first step toward the characterization of the signaling mechanisms effecting the prompt, steroid-induced translocation of Ser-P-ANXA1 to the cell surface in PDFS cells, we explored the potential roles of PKCs and MAPK, both of which have been implicated in nongenomic steroid signaling in other systems (33, 34). Our findings suggest that both enzyme systems have an essential part to play. With respect to PKC, the results demonstrate that the dexamethasone-induced translocation of the phosphorylated protein to the cell membrane is effectively blocked by the nonselective PKC inhibitor PKC_19–36; they also provide evidence that the PKC isoform responsible is Ca²⁺-dependent and therefore likely to be of the , ß₁, ß₁₁, or subtype. These findings accord with our recent findings in rodent pituitary tissue that dexamethasone acts via a PKC-dependent mechanism to induce the cellular exportation of Ser-P-ANXA1 (20) and with data from our own and other groups that suggest that PKC plays an essential role in the manifestation of the early delayed feedback actions of GCs on the pituitary gland (20, 55). Whether the Ca²⁺-dependent PKC itself causes serine-phosphorylation of ANXA1 in the PDFS cells, as it does in other systems (56), or acts at a different point in the steroid-signaling cascade to facilitate the translocation of the phosphorylated protein to the cell membrane remains to be determined. In addition to identifying a role for PKC, this study also demonstrated that the translocation of Ser-P-ANXA1 to the cell membrane provoked by dexamethasone is inhibited by wortmannin, PD98059 and U0126 and is thus dependent on PI3-kinase, MAPK, and MEK. Data from other systems have provided evidence that PKC can be activated by PI3-kinase, raising the possibility that ANXA1 is serine phosphorylated as a consequence of sequential PI3-kinase-PKC activation. For example, as PKC is activated by PtIns(3,4)P2 and PtIns(3,4,5)P3 (57, 58, 59) and the phosphorylation of the PKC substrate pleckstrin is inhibited by wortmannin (60), Ser-P-ANXA1 could be phosphorylated as a consequence of PI3-K-PKC activation. Further work is now required to investigate this possibility and to explore the role of the MAPK system, which is increasingly being implicated in signaling systems within the pituitary gland (61).

A further interesting observation reported here is the ability of ANXA5 to suppress the steroid-induced appearance of Ser-P-ANXA1 on the cell surface. How this occurs is unclear but, if a similar phenomenon occurs in primary pituitary tissue, it may go some way to explain our recent finding that ANXA5 opposes the inhibitory effects of dexamethasone on the secretion of ACTH and other pituitary hormones (20). In a previous study (18), we described specific ANXA5 binding sites on the surface of rat pituitary cells that might serve either as receptors, and therefore be coupled to a signaling system, or to transport the protein across the cell membrane. Interestingly, in a number of cell-free systems, ANXA5 has been shown to inhibit the PKC-dependent substrate phosphorylation (43, 56, 62) via a mechanism that involves direct inhibition of the kinase (63), rather than phosphorylation of ANXA5 as an alternative substrate (64). Certainly in the light of the data reported here, if ANXA5 enters the cells, blockade of PKC would provide an effective means of opposing the regulatory actions of the steroids on the translocation of Ser-P-ANXA1 to the membrane.

Finally, the question remains, what is the biological relevance of the serine phosphorylation of ANXA1? One possibility is that serine phosphorylation is essential for the translocation of the protein across the cell membrane which our recent data suggest is effected via an ATP binding cassette transporter(s) (29). Alternatively, the inhibitory actions of ANXA1 on the secretion of ACTH and other hormones from the endocrine cells may require serine phosphorylation, a finding that is supported by our recent observation that the biological actions of exogenous ANXA1 in the pituitary are dependent upon PKC (20) and by reports that Ser-P-ANXA1 prevents membrane aggregation (65). Serine phosphorylation may thus be a significant factor in determining the biological activity of ANXA1 in the pituitary gland as tyrosine phosphorylation has been shown to be in other systems (66).

	Acknowledgments

 
We are grateful to the Wellcome Trust (Grant No. 051887/Z/97/A) for generous financial support, to the Medical Research Council for the studentship held by Abeda Mulla, to Drs. A. Klibansky and D. D. Danila (Harvard Medical School, Boston, MA) for the gift of PDFS cell line, and to Mr. Colin Rantle for expert technical assistance.

	Footnotes

 
Abbreviations: ANXA1, Annexin 1; ANXA5, annexin 5; FACS, fluorescence-activated cell scanning; FITC, fluorescein isothiocyanate; FS, folliculostellate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, glucocorticoid; GFAP, glial fibrillary acidic protein; GR, GC receptor; MEK, MAPK kinase; pAb, polyclonal antiserum; PDFS, a human FS cell line; PI3-K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PKC_9–31, nonselective PKC inhibitor; Ser-P-ANXA1, serine-phosphorylated species of ANXA1.

Received June 6, 2002.

Accepted for publication December 12, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Raynal P, Pollard HB 1994 Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins. Biochim Biophys Acta 1197:63–93[Medline]
2. Wallner BP, Mattaliano RJ, Hession C, Cate RL, Tizard R, Sinclair LK, Foeller C, Chow EP, Browning JL, Ramachandran KL, Pepinsky RB 1986 Cloning and expression of human lipocortin, a phospholipase A₂ inhibitor with potential anti-inflammatory activity. Nature 320:77–81[CrossRef][Medline]
3. Violette SM, King I, Browning JL, Pepunsky RB, Wallner BP, Sartorelli AC 1990 Role of lipocortin 1 in the glucocorticoid induction of the terminal differentiation of a human squamous carcinoma. J Cell Physiol 142:70–72[CrossRef][Medline]
4. Flower RJ, Blackwell GJ 1979 Anti-inflammatory steroids induce biosynthesis of a phospholipase A₂ inhibitor which prevents prostaglandin generation. Nature 278:456–459[CrossRef][Medline]
5. Buckingham JC, Loxley HD, Christian HC, Philip JG 1996 Activation of the HPA axis by immune insults: roles and interactions of cytokines, eicosanoids, glucocorticoids. Pharmacol Biochem Behav 54:285–298[CrossRef][Medline]
6. Loxley H, Cowell A-M, Flower R, Buckingham J 1993 Modulation of the hypothalamo-pituitary-adrenocortical responses to cytokines in the rat by lipocortin 1 and glucocorticoids: a role for lipocortin 1 in the feedback inhibition of CRF-41 release? Neuroendocrinology 57:801–814[Medline]
7. Philip JG, Flower RJ, Buckingham JC 1997 Glucocorticoids modulate the cellular disposition of lipocortin 1 in the rat brain in vivo and in vitro. Neuroreport 8:1871–1876[Medline]
8. Taylor AD, Cowell AM, Flower RJ, Buckingham JC 1995 Dexamethasone suppresses the release of prolactin from the rat anterior pituitary gland by lipocortin 1 dependent and independent mechanisms. Neuroendocrinology 62:530–542[Medline]
9. Taylor AD, Cowell AM, Flower J, Buckingham JC 1993 Lipocortin 1 mediates an early inhibitory action of glucocorticoids on the secretion of ACTH by the rat anterior pituitary gland in vitro. Neuroendocrinology 58:430–439[Medline]
10. Taylor AD, Christian HC, Morris JF, Flower RJ, Buckingham JC 1997 An antisense oligodeoxynucleotide to lipocortin 1 reverses the inhibitory actions of dexamethasone on the release of adrenocorticotropin from rat pituitary tissue in vitro. Endocrinology 138:2909–2918[Abstract/Free Full Text]
11. Taylor AD, Christian HC, Morris JF, Flower RJ, Buckingham JC 2000 Evidence from immunoneutralization and antisense studies that the inhibitory actions of glucocorticoids on growth hormone release in vitro require annexin 1 (lipocortin 1). Br J Pharmacol 131:1309–1316[CrossRef][Medline]
12. Smith CW 1993 Endothelial adhesion molecules and their role in inflammation. Can J Physiol Pharmacol 71:76–87[Medline]
13. Johnson M, Kamso-Pratt J, Pepinsky R, Whetsell WJ 1989 Lipocortin-1 immunoreactivity in central and peripheral nervous system glial tumors. Hum Pathol 20:772–776[CrossRef][Medline]
14. Traverso V, Christian HC, Morris JF, Buckingham JC 1999 Lipocortin 1 (annexin 1): a candidate paracrine agent localized in pituitary folliculo-stellate cells. Endocrinology 140:4311–4319[Abstract/Free Full Text]
15. Mulla A, Christian H, Morris J, Mendoza N, Solito E, Buckingham J 2000 Expression of Annexin 1 (lipocortin 1) and Annexin 5 in human pituitary tumors. J Endocr 167 (Abstract suppl OC14)
16. Christian HC, Flower RJ, Morris JF, Buckingham JC 1999 Localisation and semi-quantitative measurement of lipocortin 1 in rat anterior pituitary cells by fluorescence-activated cell analysis/sorting and electron microscopy. J Neuroendocrinol 11:707–714[CrossRef][Medline]
17. Buckingham JC, Flower RJ 1997 Lipocortin 1: a second messenger of glucocorticoid action in the hypothalamo-pituitary-adrenocortical axis. Mol Med Today 3:296–302[CrossRef][Medline]
18. Christian HC, Taylor AD, Flower RJ, Morris JF, Buckingham JC 1997 Characterization and localization of lipocortin 1-binding sites on rat anterior pituitary cells by fluorescence-activated cell analysis/sorting and electron microscopy. Endocrinology 138:5341–5351[Abstract/Free Full Text]
19. Taylor AD, Flower RJ, Buckingham JC 1995 Dexamethasone inhibits the release of TSH from the rat anterior pituitary gland in vitro by mechanisms dependent on de novo protein synthesis and lipocortin 1. J Endocrinol 147:533–544[Abstract]
20. John C, Cover P, Solito E, Morris J, Christian H, Flower J, Buckingham J 2002 Annexin1-dependent actions of glucocorticoids in the anterior pituitary gland: roles of the N terminal domain and protein kinase C. Endocrinology 143:3060–3070[Abstract/Free Full Text]
21. Philip JG, John CD, Cover PO, Morris JF, Christian HC, Flower RJ, Buckingham JC 2001 Opposing influences of glucocorticoids and interleukin-1beta on the secretion of growth hormone and ACTH in the rat in vivo: role of hypothalamic annexin 1. Br J Pharmacol 134:887–895[CrossRef][Medline]
22. Baes M, Denef C 1987 Evidence that stimulation of growth hormone release by epinephrine and vasoactive intestinal peptide is based on cell-to-cell communication in the pituitary. Endocrinology 120:280–290[Abstract]
23. Soji T, Mabuchi Y, Kurono C, Herbert DC 1997 Folliculo-stellate cells and intercellular communication within the rat anterior pituitary gland. Microsc Res Tech 39:138–149[CrossRef][Medline]
24. Inoue K, Couch EF, Takano K, Ogawa S 1999 The structure and function of folliculo-stellate cells in the anterior pituitary gland. Arch Histol Cytol 62:205–218[CrossRef][Medline]
25. Vankelecom H, Matthys P, Van Damme J, Heremans H, Billiau A, Denef C 1993 Immunocytochemical evidence that S-100-positive cells of the mouse anterior pituitary contain interleukin-6 immunoreactivity. J Histochem Cytochem 41:151–156[Abstract]
26. Lohrer P, Gloddek J, Nagashima AC, Korali Z, Hopfner U, Pereda MP, Arzt E, Stalla GK, Renner U 2000 Lipopolysaccharide directly stimulates the intrapituitary interleukin-6 production by folliculostellate cells via specific receptors and the p38 mitogen-activated protein kinase/nuclear factor-B pathway. Endocrinology 141:4457–4465[Abstract/Free Full Text]
27. Giometto B, Miotto D, Botteri M, Alessio L, Scanarini M, An SF, Tavolato B 1997 Folliculo-stellate cells of human pituitary adenomas: immunohistochemical study of the monocyte/macrophage phenotype expression. Neuroendocrinology 65:47–52[CrossRef][Medline]
28. Ozawa H, Ito T, Ochiai I, Kawata M 1999 Cellular localization and distribution of glucocorticoid receptor immunoreactivity and the expression of glucocorticoid receptor messenger RNA in rat pituitary gland. A combined double immunohistochemistry study and in situ hybridization histochemical analysis. Cell Tissue Res 295:207–214[CrossRef][Medline]
29. Morris JF, Christian HC, Chapman LP, Epton MJ, Buckingham JC, Ozawa H, Nishi M, Kawata M 2002 Steroid effects on secretion from subsets of lactotrophs: role of folliculo-stellate cells and annexin 1. Arch Physiol Biochem 110:54–61[CrossRef][Medline]
30. Philip JG, Flower RJ, Buckingham JC 1998 Blockade of the classical pathway of protein secretion does not affect the cellular exportation of lipocortin 1. Regul Pept 73:133–139[CrossRef][Medline]
31. Pearce D 2001 The role of SGK1 in hormone-regulated sodium transport. Trends Endocrinol Metab 12:341–347[CrossRef][Medline]
32. Dina OA, Aley KO, Isenberg W, Messing RO, Levine JD 2001 Sex hormones regulate the contribution of PKC epsilon and PKA signalling in inflammatory pain in the rat. Eur J Neurosci 13:2227–2233[CrossRef][Medline]
33. Nuedling S, Kahlert S, Loebbert K, Meyer R, Vetter H, Grohe C 1999 Differential effects of 17ß-estradiol on mitogen-activated protein kinase pathways in rat cardiomyocytes. FEBS Lett 454:271–276[CrossRef][Medline]
34. Li X, Qiu J, Wang J, Zhong Y, Zhu J, Chen Y 2001 Corticosterone-induced rapid phosphorylation of p38 and JNK mitogen-activated protein kinases in PC12 cells. FEBS Lett 492:210–214[CrossRef][Medline]
35. Danila DC, Zhang X, Zhou Y, Dickersin GR, Fletcher JA, Hedley-Whyte ET, Selig MK, Johnson SR, Klibanski A 2000 A human pituitary tumor-derived folliculostellate cell line. J Clin Endocrinol Metab 85:1180–1187[Abstract/Free Full Text]
36. Lim LH, Solito E, Russo-Marie F, Flower RJ, Perretti M 1998 Promoting detachment of neutrophils adherent to murine postcapillary venules to control inflammation: effect of lipocortin 1. Proc Natl Acad Sci USA 95:14535–14539[Abstract/Free Full Text]
37. Solito E, de Coupade C, Parente L, Flower R, Russo Marie F 1998 IL-6 stimulates Annexin 1 expression and translocation and suggests a new biological role as class II acute phase protein. Cytokine 10:514–521[CrossRef][Medline]
38. Solito E, de Coupade C, Parente L, Flower RJ, Russo-Marie F 1998 Human annexin 1 is highly expressed during the differentiation of the human epithelial cell line A 549. Involvement of NF-IL6 in phorbol ester induction of annexin 1. Cell Growth Differ 9:327–336[Abstract]
39. Solito E, Raguenes-Nicol C, de Coupade C, Bisagni-Faure A, Russo-Marie F 1998 U 937 cells deprived of annexin 1 demonstrate an increased PLA₂ activity. Br J Pharmacol 124:1675–1683[CrossRef][Medline]
40. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
41. Becher B, Fedorowicz V, Antel JP 1996 Regulation of CD14 expression on human adult central nervous system-derived microglia. J Neurosci Res 45:375–381[CrossRef][Medline]
42. Schulz M, Eggert M, Baniahmad A, Dostert A, Heinzel T, Renkawitz R 2002 RU486-induced glucocorticoid receptor agonism is controlled by the receptor N terminus and by corepressor binding. J Biol Chem 277:26238–26243[Abstract/Free Full Text]
43. Raynal P, Hullin F, Ragab TJ, Fauvel J, Chap H 1993 Annexin 5 as a potential regulator of annexin 1 phosphorylation by protein kinase C. In vitro inhibition compared with quantitative data on annexin distribution in human endothelial cells. Biochem J 92:759–765
44. Croxtall JD, van Hal PT, Choudhury Q, Gilroy DW, Flower RJ 2002 Different glucocorticoids vary in their genomic and non-genomic mechanism of action in A549 cells. Br J Pharmacol 135:511–519[CrossRef][Medline]
45. Valverde MA, Parker MG 2002 Classical and novel steroid actions: a unified but complex view. Trends Biochem Sci 27:172–173[CrossRef][Medline]
46. Baulieu EE, Robel P, Schumacher M 2001 Neurosteroids: beginning of the story. Int Rev Neurobiol 46:1–32[Medline]
47. Jones MT, Brush FR, Neame RL 1972 Characteristics of fast feedback control of corticotrophin release by corticosteroids. J Endocrinol 55:489–497[Medline]
48. Buckingham JC 1979 The influence of corticosteroids on the secretion of corticotrophin and its hypothalamic releasing hormone. J Physiol 286:331–342[Abstract/Free Full Text]
49. Lambert JJ, Harney SC, Belelli D, Peters JA 2001 Neurosteroid modulation of recombinant and synaptic GABAA receptors. Int Rev Neurobiol 46:177–205[Medline]
50. Gametchu B 1987 Glucocorticoid receptor-like antigen in lymphoma cell membranes: correlation to cell lysis. Science 236:456–461[Abstract/Free Full Text]
51. Yaswen P, Goyette M, Shank P, Fausto N 1987 Expression of c-Ki ras, c-Ha-ras, and c-myc in specific cell types during hepatocarcinogenesis. Mol Cell Biol 5:780–786
52. Chen YZ, Qiu J 1999 Pleiotropic signaling pathways in rapid, nongenomic action of glucocorticoid. Mol Cell Biol Res Commun 2:145–149[CrossRef][Medline]
53. Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo P, Nola E, Auricchio F 1996 Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells. EMBO J 15:1292–300[Medline]
54. Croxtall JD, Choudhury Q, Flower RJ 2000 Glucocorticoids act within minutes to inhibit recruitment of signalling factors to activated EGF receptors through a receptor-dependent, transcription-independent mechanism. Br J Pharmacol 130:289–298[CrossRef][Medline]
55. Tian L, Philp JA, Shipston MJ 1999 Glucocorticoid block of protein kinase C signalling in mouse pituitary corticotroph AtT20 D16:16 cells. J Physiol 516(Part 3):757–768
56. Rothhut B, Dubois T, Feliers D, Russo-Marie F, Oudinet JP 1995 Inhibitory effect of annexin V on protein kinase C activity in mesangial cell lysates. Eur J Biochem 232:865–872[Medline]
57. Palmer RH, Dekker LV, Woscholski R, Le Good JA, Gigg R, Parker PJ 1995 Activation of PRK1 by phosphatidylinositol 4, 5-bisphosphate and phosphatidylinositol 3, 4, 5-trisphosphate. A comparison with protein kinase C isotypes. J Biol Chem 270:224122241–122246
58. Toker A, Meyer M, Reddy KK, Falck JR, Aneja R, Aneja S, Parra A, Burns DJ, Ballas LM, Cantley LC 1994 Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3, 4-P2 and PtdIns-3, 4, 5-P3. J Biol Chem 269:32358–32367[Abstract/Free Full Text]
59. Nakanishi H, Brewer KA, Exton JH 1993 Activation of the isozyme of protein kinase C by phosphatidylinositol 3, 4, 5-trisphosphate. J Biol Chem 268:13–16[Abstract/Free Full Text]
60. Toker A, Bachelot C, Chen CS, Falck JR, Hartwig JH, Cantley LC, Kovacsovics TJ 1995 Phosphorylation of the plate