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ARTICLE |
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.
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Introduction |
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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).
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Materials and Methods |
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Antisera
Primary antisera.
Total ANXA1 protein was detected using a polyclonal antiserum (pAb) raised in rabbit against human recombinant ANXA11–346; 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 ANXA115–31 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 ANXA11–188 (1 µg/lane) at the dilution used (1:1000); furthermore, whereas anti-tyr-P-ANXA1 pAb readily detected ANXA1Ac2–2621-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 ANXA1Ac2–2621-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 M
P9, 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), PKC19–31 (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.
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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.
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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.
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shows the time-dependent effects of dexamethasone (1 µM) on the expression and serine/tyrosine phosphorylation status of ANXA1 in the PDFS cells, as determined by Western blot analysis. Dexamethasone initiated a marked increase in ANXA1 mRNA (Fig. 2A) and protein (Fig. 2B) expression. The response emerged within 2 h of steroid contact, was maximal after 8 h (P < 0.01) and fell thereafter to a nadir at 16 h; an apparent secondary increase in ANXA1 mRNA expression at 24 h did not reach significance. In addition, dexamethasone caused a marked increase in the expression of serine phosphorylated species of ANXA1 (Ser-P-ANXA1) in the cells (Fig. 2C). Unlike the changes in ANXA1 mRNA and protein provoked by the steroid, the maximal levels of Ser-P-ANXA1 were evident within 30 min of contact with dexamethasone (P < 0.01) and declined with time over the ensuing 24 h. A tyrosine-phosphorylated species of ANXA1 was also detectable in the PDFS cells by Western blot analysis; the amount present was, however, only modest and was unaffected by exposure of the cells to dexamethasone (Fig. 2C). By contrast, none of the other steroids tested (testosterone, 17ß-estradiol, progesterone 1 µM, 30 min or 2 h) affected the expression, cellular disposition, or phosphorylation status of ANXA1 (data not shown).
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.
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illustrates the effects of the mRNA and protein synthesis inhibitors, actinomycin D (Act-D, 10 µg/ml) and cycloheximide (CHX, 5 µM), on de novo protein synthesis (Fig. 5A) and on the ability of dexamethasone (1 µM, 30 min and 2 h contact times) to cause the translocation of Ser-P-ANXA1 to the cell surface (Fig. 5, B and C). Dexamethasone (1 µM, 2 h) caused a significant increase in de novo protein synthesis as indexed by the incorporation of [14C]-lysine into protein (P < 0.001). This effect was abolished by actinomycin D and cycloheximide (P < 0.001), which themselves also reduced the basal rate of protein synthesis (P < 0.05, Fig. 5A). The relatively modest (
50%) inhibitory effect of cycloheximide and actinomycin D on basal protein synthesis reflects the fact that the cells were preloaded with 14C-lysine before the addition of the mRNA/protein synthesis inhibitors. Neither actinomycin D (Fig. 5B) or cycloheximide (Fig. 5C) influenced the translocation of Ser-P-ANXA1 to the cell surface, which was evident after a 30-min contact period with dexamethasone, a finding that suggests that this early action of the steroid is effected via a nongenomic mechanism. By contrast, cycloheximide (Fig. 5B) abolished (P < 0.005) and actinomycin D attenuated (Fig. 5C; P < 0.05) the effects of dexamethasone observed after a 2-h contact period, indicating that a later phase of the response required de novo mRNA/protein synthesis.
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, A and B, P < 0.001). A similar inhibitory effect was also observed in cells treated with a selective inhibitor of conventional Ca2+-dependent PKCs, GÖ6976 (25 nM, P < 0.05, Fig. 6B), or with the PI3-kinase inhibitor wortmannin (10 nM, Fig. 6C, P < 0.05). Blockade of MAPK also inhibited the dexamethasone-induced translocation of Ser-P-ANXA1 to the cell membrane of PDFS cells (Fig. 6C). The effects of the steroid were thus antagonized by an MEK-MAPK inhibitor PD98059 (5 µM; P < 0.005) and by a selective MEK1 and 2 inhibitor (UO126, 1 µM; P < 0.005), which itself also produced a small reduction in cell surface Ser-P-ANXA1. The specificity of the drugs was confirmed by the demonstration that phorbol 12-myristate-13-acetate and dexamethasone induce the expression of phospho-P44–42 (assessed by Western blot analysis), and their effects are blocked by PD98459 and U0126 (data not shown). Taken together, these data indicate that the nongenomic actions of dexamethasone that provoke the translocation of Ser-P-ANXA1 to the plasma membrane are effected via a signaling pathway that requires PKC and MEK kinases.
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, A and B, P < 0.05).
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Discussion |
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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 GABAA 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/p21ras/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 PKC19–36; 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).
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Acknowledgments |
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Footnotes |
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Received June 6, 2002.
Accepted for publication December 12, 2002.
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mitogen-activated protein kinase/nuclear factor-
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isozyme of protein kinase C by phosphatidylinositol 3, 4, 5-trisphosphate. J Biol Chem 268:13–16This article has been cited by other articles:
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 M.-L. Kaljusto, K.-O. Stenslokken, T. Mori, A. Panchenko, M.-L. Frantzen, G. Valen, and J. Vaage Preconditioning effects of steroids and hyperoxia on cardiac ischemia-reperfusion injury and vascular reactivity Eur. J. Cardiothorac. Surg., March 1, 2008; 33(3): 355 - 363. [Abstract] [Full Text] [PDF]
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 K. M. Breen, T. L. Davis, L. C. Doro, T. M. Nett, A. E. Oakley, V. Padmanabhan, L. A. Rispoli, E. R. Wagenmaker, and F. J. Karsch Insight into the Neuroendocrine Site and Cellular Mechanism by which Cortisol Suppresses Pituitary Responsiveness to Gonadotropin-Releasing Hormone Endocrinology, February 1, 2008; 149(2): 767 - 773. [Abstract] [Full Text] [PDF]
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 A. Y. Meliton, N. M. Munoz, X. Zhu, and A. R. Leff Attenuated translocation of group IVa phospholipase A2 and up-regulated annexin-1 synthesis by glucocorticoid blocks {beta}2-integrin adhesion in neutrophils J. Leukoc. Biol., February 1, 2008; 83(2): 344 - 351. [Abstract] [Full Text] [PDF]
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 T. Tateno, H. Izumiyama, M. Doi, T. Yoshimoto, M. Shichiri, N. Inoshita, K. Oyama, S. Yamada, and Y. Hirata Differential gene expression in ACTH -secreting and non-functioning pituitary tumors Eur. J. Endocrinol., December 1, 2007; 157(6): 717 - 724. [Abstract] [Full Text] [PDF]
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 S. R. Hammes and E. R. Levin Extranuclear Steroid Receptors: Nature and Actions Endocr. Rev., December 1, 2007; 28(7): 726 - 741. [Abstract] [Full Text] [PDF]
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 S. D. Reynolds, P. R. Reynolds, J. C. Snyder, F. Whyte, K. J. Paavola, and B. R. Stripp CCSP regulates cross talk between secretory cells and both ciliated cells and macrophages of the conducting airway Am J Physiol Lung Cell Mol Physiol, July 1, 2007; 293(1): L114 - L123. [Abstract] [Full Text] [PDF]
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 L. H. K. Lim and S. Pervaiz Annexin 1: the new face of an old molecule FASEB J, April 1, 2007; 21(4): 968 - 975. [Abstract] [Full Text] [PDF]
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C. D. John, V. Sahni, D. Mehet, J. F. Morris, H. C. Christian, M. Perretti, R. J. Flower, E. Solito, and J. C. Buckingham Formyl peptide receptors and the regulation of ACTH secretion: targets for annexin A1, lipoxins, and bacterial peptides FASEB J, April 1, 2007; 21(4): 1037 - 1046. [Abstract] [Full Text] [PDF]
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E. Davies, S. Omer, J. C. Buckingham, J. F. Morris, and H. C. Christian Expression and Externalization of Annexin 1 in the Adrenal Gland: Structure and Function of the Adrenal Gland in Annexin 1-Null Mutant Mice Endocrinology, March 1, 2007; 148(3): 1030 - 1038. [Abstract] [Full Text] [PDF]
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 R. Gros, Q. Ding, S. Armstrong, C. O'Neil, J. G. Pickering, and R. D. Feldman Rapid effects of aldosterone on clonal human vascular smooth muscle cells Am J Physiol Cell Physiol, February 1, 2007; 292(2): C788 - C794. [Abstract] [Full Text] [PDF]
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S. Omer, D. Meredith, J. F. Morris, and H. C. Christian Evidence for the Role of Adenosine 5'-Triphosphate-Binding Cassette (ABC)-A1 in the Externalization of Annexin 1 from Pituitary Folliculostellate Cells and ABCA1-Transfected Cell Models Endocrinology, July 1, 2006; 147(7): 3219 - 3227. [Abstract] [Full Text] [PDF]
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E. Solito, H. C. Christian, M. Festa, A. Mulla, T. Tierney, R. J. Flower, and J. C. Buckingham Post-translational modification plays an essential role in the translocation of annexin A1 from the cytoplasm to the cell surface FASEB J, July 1, 2006; 20(9): 1498 - 1500. [Abstract] [Full Text] [PDF]
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E. Theogaraj, C. D. John, H. C. Christian, J. F. Morris, S. F. Smith, and J. C. Buckingham Perinatal Glucocorticoid Treatment Produces Molecular, Functional, and Morphological Changes in the Anterior Pituitary Gland of the Adult Male Rat Endocrinology, November 1, 2005; 146(11): 4804 - 4813. [Abstract] [Full Text] [PDF]
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V. A. Verriere, D. Hynes, S. Faherty, J. Devaney, J. Bousquet, B. J. Harvey, and V. Urbach Rapid Effects of Dexamethasone on Intracellular pH and Na+/H+ Exchanger Activity in Human Bronchial Epithelial Cells J. Biol. Chem., October 28, 2005; 280(43): 35807 - 35814. [Abstract] [Full Text] [PDF]
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 L.-L. Wang, C.-C. Ou, and J. Y.H. Chan Receptor-Independent Activation of GABAergic Neurotransmission and Receptor-Dependent Nontranscriptional Activation of Phosphatidylinositol 3-kinase/Protein Kinase Akt Pathway in Short-Term Cardiovascular Actions of Dexamethasone at the Nucleus Tractus Solitarii of the Rat Mol. Pharmacol., February 1, 2005; 67(2): 489 - 498. [Abstract] [Full Text] [PDF]
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 A. Mulla, C. LeRoux, E. Solito, and J. C. Buckingham Correlation between the Antiinflammatory Protein Annexin 1 (Lipocortin 1) and Serum Cortisol in Subjects with Normal and Dysregulated Adrenal Function J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 557 - 562. [Abstract] [Full Text] [PDF]
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K. M. Breen, C. A. Stackpole, I. J. Clarke, A. V. Pytiak, A. J. Tilbrook, E. R. Wagenmaker, E. A. Young, and F. J. Karsch Does the Type II Glucocorticoid Receptor Mediate Cortisol-Induced Suppression in Pituitary Responsiveness to Gonadotropin-Releasing Hormone? Endocrinology, June 1, 2004; 145(6): 2739 - 2746. [Abstract] [Full Text] [PDF]
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 H.-Y. Fan, L.-J. Huo, D.-Y. Chen, H. Schatten, and Q.-Y. Sun Protein Kinase C and Mitogen-Activated Protein Kinase Cascade in Mouse Cumulus Cells: Cross Talk and Effect on Meiotic Resumption of Oocyte Biol Reprod, April 1, 2004; 70(4): 1178 - 1187. [Abstract] [Full Text] [PDF]
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K. M. Breen and F. J. Karsch Does Cortisol Inhibit Pulsatile Luteinizing Hormone Secretion at the Hypothalamic or Pituitary Level? Endocrinology, February 1, 2004; 145(2): 692 - 698. [Abstract] [Full Text] [PDF]
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B. BARTHOLOME, C. M. SPIES, T. GABER, S. SCHUCHMANN, T. BERKI, D. KUNKEL, M. BIENERT, A. RADBRUCH, G.-R. BURMESTER, R. LAUSTER, et al. Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis FASEB J, January 1, 2004; 18(1): 70 - 80. [Abstract] [Full Text] [PDF]
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