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An Antisense Oligodeoxynucleotide to Lipocortin 1 Reverses the Inhibitory Actions of Dexamethasone on the Release of Adrenocorticotropin from Rat Pituitary Tissue in Vitro¹

Department of Pharmacology (A.D.T., H.C.C., J.C.B.), Charing Cross and Westminster Medical School, London, W6 8RF, United Kingdom; Department of Human Anatomy (J.F.M.), The University of Oxford, Oxford, OX1 3QX, United Kingdom; Department of Biochemical Pharmacology R.J.F.), The William Harvey Research Institute, St. Bartholomew’s, and the Royal London School of Medicine and Dentistry at Queen Mary and Westfield College, London, EC1M 6BQ, United Kingdom

Address all correspondence and requests for reprints to: Professor Julia Buckingham, Department of Pharmacology, Charing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF, United Kingdom. E-mail: j.buckingham{at}cxwms.ac.uk

	Abstract

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Our previous studies have demonstrated that lipocortin 1 (LC1, also called annexin 1) is an important mediator of glucocorticoid action in the neuroendocrine system, particularly with regard to the powerful inhibitory actions of the steroids on the secretion of ACTH and its hypothalamic releasing hormones. In the present study, we have used an antisense oligodeoxynucleotide (ODN) unique to LC1 to investigate further the role of this protein in the regulatory effects of dexamethasone on ACTH release in vitro from rat anterior pituitary cells. Pituitary cells dispersed with collagenase retained their functional and morphological integrity in vitro and sequestered ODNs in a time-dependent manner from the incubation medium. LC1 was readily detected in the cells by Western blot analysis or by immunoprecipitation/autoradiography after preloading with ³⁵S-methionine/cysteine; the bulk of the protein was contained within an intracellular pool but a small amount was attached to the outer cell surface (pericellular). Dexamethasone (100 nM, 2.5 h) initiated de novo synthesis of LC1; it also increased the amount of LC1 in the pericellular pool detected by either method and caused a concomitant decrease in intracellular LC1. The responses to the steroid were prevented by the inclusion in the medium of an LC1 antisense ODN (50 nM, 3.5 h) but the corresponding sense and scrambled ODN sequences were inert. None of the ODN sequences tested influence the expression of annexin 5 in the pituitary tissue. CRH-41 (100 pM-1 mM), forskolin (1 nM-1 mM) and an L-Ca²⁺-channel opener BAY K8644 (100 pM-1 µM) initiated concentration dependent increases in immunoreactive- (ir-) ACTH release from the pituitary cells that were reduced (P < 0.01) by preincubation with dexamethasone (100 nM, 2.5 h). The inhibitory effects of the steroid were reversed by the LC1 antisense ODN (50 nM, P < 0.01), whereas the LC1 sense and scrambled control sequences (50 nM) were both ineffective in this respect (P > 0.05). The results add further support to the view that the acute inhibitory effects of glucocorticoids on the secretion of ACTH by the pituitary gland are dependent on the generation of lipocortin 1.

	Introduction

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LIPOCORTIN 1 (LC1, also called annexin 1) is a well characterized member of a structurally related family of Ca²⁺ and phospholipid-binding proteins known collectively as the annexins. It was first described as a glucocorticoid-inducible protein with the capacity to block the activity of the enzyme phospholipase A₂ (PLA₂) and, hence, the generation of proinflammatory eicosanoids (1) and was therefore heralded as a potential second messenger protein for the antiinflammatory steroids. LC1 has since been shown to temper specific aspects of inflammatory responses in various experimental models, e.g. neutrophil migration in the mouse air pouch and glutamate-induced ischaemic brain damage (1, 2, 3). Work in our laboratory has focused on the potential role of LC1 as a mediator of glucocorticoid action in the neuroendocrine system. Our data have identified a key role for the protein in effecting the acute inhibitory actions of the steroids on the hypothalamo-pituitary adrenal (HPA) axis (4, 5, 6, 7, 8); in addition they have provided evidence that LC1 contributes to the regulatory actions of the glucocorticoids on the release of PRL, thyrotrophin, and GH (8, 9, 10, 11).

LC1 is readily detectable in the neuroendocrine system by Western blot analysis, ELISA, and immunohistochemistry (reviewed in Ref. 8). It is particularly abundant in the anterior pituitary gland and the median eminence, but significant amounts are also present on other regions of the hypothalamus (e.g. paraventricular nucleus) and elsewhere in the brain (e.g. hippocampus, 12). In these tissues, as in peripheral immune/inflammatory cells (1, 2, 3), glucocorticoids regulate both the expression and subcellular distribution of LC1. They thus augment the synthesis of LC1 and promote the translocation of the protein from within the cell (intracellular pool) to the outer cell surface (pericellular or extracellular pool) where it is retained by a Ca²⁺-dependent mechanism (5, 6, 7, 8). Binding and functional studies performed in our laboratory suggest that this process of externalization provides an important mechanism whereby LC1 gains access to receptors or other sites on the outer surface of cells and thereby exerts paracrine/autocrine regulatory actions on peptide release (8, 13). Thus, using a combination of fluorescence-activated cell (FAC) analysis/sorting and electron microscopy we have demonstrated the presence of high affinity LC1 binding sites on a variety of pituitary cell types, including corticotrophs (13). In addition, we have shown that recombinant human LC1 (LC11–348) and a stable N-terminal LC1 fragment (LC11–188), neither of which would be expected to penetrate cell membranes readily, mimic the acute inhibitory effects of dexamethasone on the release in vitro of ACTH from rat anterior pituitary segments induced by various secretogogues (6). Moreover, the regulatory actions of the steroid in this preparation are specifically reversed by a monoclonal anti-LC1 antibody that would also be unlikely to penetrate the cells but could readily sequester LC1 at a pericellular site (6). Similarly, at the hypothalamic level LC11–346 and LC11–188 mimic and anti-LC1 antisera specifically reverse the ability of dexamethasone to suppress the release of corticotrophin releasing hormone (CRH-41) evoked in vitro by the interleukins (ILs) IL1, IL-1ß, IL-6, and IL-8 (4, 5, 7, 8). Furthermore, complementary in vivo experiments have shown that intracerebroventricular administration of LC11–348 inhibits the HPA responses to cytokine challenge (5). In addition, passive immunization of the animals with a polyclonal anti-LC1 antibody significantly reverses the inhibitory effects of dexamethasone on the increases in plasma ACTH and corticosterone evoked by ip administration of IL-1ß (7).

The use of antisense oligodeoxynucleotides (ODNs) designed to hybridize with and thus to neutralize specific sequences of messenger RNA (mRNA) has helped to clarify the role of many gene products in cellular function, both in vitro and in vivo, by blocking their production at source (for review see Ref. 14). In the present study, we have exploited this technology and designed an antisense ODN to a unique sequence of complementary DNA (cDNA) that encodes for amino acids in the N-terminal of LC1; we have used this probe together with appropriate control sequences to investigate further the role of endogenous LC1 in the inhibitory effects of glucocorticoids on ACTH release, using a well established in vitro system.

	Materials and Methods

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Animals
Adult male (200 g) rats obtained from a closed colony (coded CFY, derived from the Sprague Dawley strain) and bred in-house at Charing Cross and Westminster Medical School were used in all experiments. They were housed post weaning in groups of five per cage in a quiet room with controlled lighting (lights on 0800–2000 h) in which the temperature and humidity were maintained at 21–23 C and approximately 50%, respectively. Food and water were available ad libitum. All experiments were started between 0800–0900 h to avoid changes associated with the circadian rhythm.

Oligodeoxynucleotide preparations
A data base sequence search was performed to identify a unique sequence of bases that (a) coded for an amino acid sequence in the N-terminal of LC1 and (b) possessed a GC content of approximately 60%. We chose bases 83–98 inclusive (3'-G GTC CTG GTG GAA ACA-5'). This sequence, which codes for amino acids 29–33, fulfills both criteria and is thus unique and specific to LC1. From this sequence, a complementary antisense ODN (3'-TCT TTC CAC CAG GAC C-5') and a scrambled ODN sequence (3'-TTC CTC TAC GAC CGA G-5') were constructed together with the LC1 sense sequence (3'-G GTC CTG GTG GAA ACA-5') that also contains at least 60% GC content. The ODNs were modified by the addition of two phosphorothioate groups at both the 3' and the 5' end (100% efficiency at 10 µM, Oswel, University of Southampton, UK), a standard process that protects them from nuclease degradation (for review see Ref. 15). A fluorescein-labeled, phosphorothioate-modified antisense probe was also synthesised for imaging studies.

Preparation and incubation of dispersed anterior pituitary cells
Suspensions of dissociated anterior pituitary cells were prepared using a modification of the method described by Cowell et al. (16). Anterior pituitary glands were obtained post mortem from rats immediately after decapitation and rinsed in Earle’s balanced salt solution (EBSS; Sigma Chemical Co. Ltd., Poole, UK). Tissues from 30 animals were pooled and cut into blocks of approximately 1 mm³ using a scalpel blade. The cells were then dissociated by incubation for 1h at 37 C in collagenase (0.2% wt/vol. Boehringer Mannheim, Sussex, UK) and deoxyribonuclease (DNAase, 0.05% wt/vol, Sigma) in 20 ml EBSS enriched with BSA (0.4%; Sigma); the dispersion was aided by gentle trituration (30 sec every 10 min). The resulting cell suspension was then centrifuged (300 x g, 10 min), the pellet resuspended in 5 ml of BSA-enriched EBSS and the suspension filtered through 20 µm nylon mesh to remove any large debris clumps. The filtrate was then centrifuged (300 x g, 10 min) and the pellet resuspended in 5 ml incubation medium [1% aprotinin vol/vol (Bayer, Saffron Walden, UK), 1% penicillin streptomycin vol/vol (Sigma) in EBSS, pH 7.4]. The cells were examined at the light microscope level to verify the effectiveness of the dispersion and counted using a haemocytometer. Cell viability was assessed by the trypan blue exclusion test and always found to be >97%.

The pituitary cells were then plated out at a density of 2.5 x 10⁵ cells/ml medium/well in 24-well cell culture plates (Costar, High Wycombe, UK) and incubated for 2.5 h at 37 C in a humidified atmosphere saturated with 95%O₂/5%CO₂ gas. They were then challenged for 1 h with CRH-41 (rat CRH-41, Bachem Ltd., Saffron Walden, UK), forskolin (Sigma) or an L-Ca²⁺ channel opener, BAY K8644; controls were incubated an equal volume of incubation medium alone. Where appropriate, dexamethasone (100 nM, David Bull Laboratories, Slough, UK) was included throughout both the preincubation and final incubation periods. LC1 antisense, sense, or scrambled ODNs (50 nM) were included in the medium at the beginning of the experiment and replenished at 1.5 h and 2.5 h. After the 1-h stimulation period, the culture plates were centrifuged (600 x g, 10 min); the supernatant fluid was collected and either assayed immediately for ACTH or stored in aliquots (300 µl) at -20 C for subsequent peptide measurement. The viability of the remaining cells was tested by the trypan blue exclusion and always found to be >97%. In some experiments, the cells were retained for LC1 and annexin 5 (control protein) measurement or for histology (see below). For studies involving the measurement of newly synthesised LC1 or annexin 5 (control protein) by immunoprecipitation and autoradiography, the pituitary cells were preincubated with ³⁵S-labeled cysteine/methionine (specific activity >1000 Ci/nmol, Pro-mix, Amersham International plc, Little Chalfont, UK) before the addition of dexamethasone and/or the ODNs.

Determination of ACTH
ACTH was determined in duplicate using a modification of the double antibody RIA described by Rees et al. (17) and a primary antibody raised in the rabbit against human ACTH1–39 (negligible cross-reactivity with -MSH and CLIP; National Hormone and Pituitary Program, Bethesda, MD). The reference preparation was synthetic human ACTH1–39 (National Institute for Biological Standards and Control, South Mimms, Herts, UK) and tracer ¹²⁵I-ACTH1–39. Separation of the bound and free ACTH was achieved by goat antirabbit IgG coated beads (Pharmacia, Uppsala, Sweden). The sensitivity of the assay was 10 pg/ml and the inter- and intraassay coefficients of variation were 10.0% and 5.2%, respectively. Dilution curves of the samples were parallel with those of the standard ACTH. Specificity studies confirmed that none of the drugs or ODNs employed in the experiments interfered with the assay.

Detection of lipocortin 1 and annexin 5
Lipocortin 1 and annexin 5 (control) contained within the intracellular and pericellular pools were detected either by SDS-PAGE and Western blot analysis or, for cells preincubated in ³⁵S-labeled cysteine/methionine, by immunoprecipitation and autoradiography. For studies measuring "total" LC1 or annexin 5 (see Fig. 3), the pituitary tissue was extracted in 1 ml 10 mM EDTA/1% Triton vol/vol (BDH Chemicals Ltd., Lutterworth, UK) in PBS. For measurement of the two proteins in the pericellular and intracellular compartments (see Figs. 4 and 5), the cells were washed initially for 1 min in a Ca²⁺ chelating agent [EDTA 250 µl, 1 mM (Sigma) in PBS (Oxoid Chemicals Ltd., Basingstoke, UK)] that releases LC1/annexin 5 bound by a Ca²⁺-dependent process to the outer surface of the cell membranes into the medium (2, 3, 6). The remaining tissue was then extracted as described above. The washes and tissue extracts were either analysed immediately or frozen and stored at -80 C (6).

View larger version (100K): [in this window] [in a new window]   Figure 3. Typical autoradiographs from three replicate experiments demonstrating the effects of dexamethasone (100 nM) in the presence and absence of LC1 antisense or control oligonucleotides (50 nM) on the synthesis of (a) lipocortin 1 and (b) annexin 5 (expressed as 35S-cysteine/methionine-labeled protein separated by immunoprecipitation and SDS-PAGE) by anterior pituitary cells in vitro. Lane 1 = basal; lane 2 = dexamethasone; lane 3 = LC1 antisense; lane 4 = LC1 antisense + dexamethasone; lane 5 = scrambled control sequence; lane 6 = scrambled control sequence + dexamethasone; lane 7 = LC1 sense; lane 8 = LC1 sense + dexamethasone. For quantification of the labeled bands see Table 1, a and b.

View larger version (74K): [in this window] [in a new window]   Figure 4. Typical Western blots from three to four replicate experiments demonstrating the effects of dexamethasone (100 nM) in the presence and absence of LC1 antisense or control oligonucleotides (50 nM) on (a and b) the expression of lipocortin 1 (37K and the 32K metabolite) in anterior pituitary cells in vitro and the distribution of the protein between (a) pericellular and (b) intracellular pools; a complementary blot showing the effects of the treatments on the expression of annexin 5 in the intracellular compartment in shown in (c). Lane M = Rainbow mol wt markers [Amersham International plc, UK; myosin (200K); phosphorylase b (92.5K); BSA (69K); ovalbumin (46K); carbonic anhydrase (30K); trysin inhibitor (21.5K); lysozyme (4.3K)[; lane 1, basal; lane 2, dexamethasone; lane 3, LC1 antisense; lane 4, LC1 antisense + dexamethasone; lane 5, scrambled control sequence; lane 6, scrambled control sequence + dexamethasone; lane 7 = LC1 sense; lane 8, LC1 sense + dexamethasone. For quantification of the immunoreactive bands see Table 2, a–c.

View larger version (92K): [in this window] [in a new window]   Figure 5. Typical autoradiographs for three replicate experiments demonstrating the effects of dexamethasone (100 nM) in the presence and absence of LC1 antisense or control oligonucleotides (50 nM) on the expression of newly synthesized lipocortin 1 (i.e.35S-cysteine/methionine-labeled LC1 separated by immunoprecipitation and SDS-PAGE) in anterior pituitary cells and the distribution of the protein between (a) pericellular and (b) intracellular pools. Parallel measurements of intracellular 35S-cysteine/methionine-labeled-annexin 5 are shown in c. Lane 1, basal; lane 2, dexamethasone; lane 3, LC1 antisense; lane 4, LC1 antisense + dexamethasone; lane 5, scrambled control sequence; lane 6, scrambled control sequence + dexamethasone; lane 7, LC1 sense; lane 8, LC1 sense + dexamethasone. For quantification of the labeled bands see Table 3, a–c.

Immunoprecipitation and autoradiography
Proteins contained within the intracellular or pericellular pools of pituitary cells that had been preincubated with ³⁵S-labeled cysteine/methionine were extracted in EDTA-containing medium as described above and analyzed immediately. LC1 and annexin 5 within the samples were each precipitated specifically by a double antibody method (19). Briefly, anti-LC1 pAb or antiannexin 5 pAb (10 µl, diluted 1:200 in 1 ml PBS)were added to each tube. The tubes were incubated for 24 h at 4 C after which an immunoprecipitating antibody [50 µl, donkey antisheep (IDS, UK) diluted 1:10 in PBS] was added and the resultant suspension vortexed and incubated for a further 2 h. The suspension was centrifuged (4000 rpm, 1 h, 4 C) and the supernatant fluid aspirated and discarded. The pellets from the pericellular and intracellular samples were resuspended in 50 µl or 250 µl PBS, respectively, and their protein contents determined (17). The proteins [4 µg/channel (washes) and 40 µg/channel (extracts) in a volume of 20 µl] were run on SDS polyacrylamide gels as described above. The gels were then wrapped in cling film to prevent them from drying out and ³⁵S-labeled cysteine/methionine-labeled LC1 was detected by exposing them to x-ray film (Kodak, Deeside, UK) for at least 2 days. The film was developed using conventional techniques and reagents (all Kodak).

Detection of oligodeoxynucleotides in the incubation medium
Samples of incubation medium (50 µl) containing oligonucleotides were run on 20 x 20 cm polyacrylamide gels comprising 100 ml Tris borate buffer (1 M, pH 7.5), 15% wt/vol polyacrilamide, 0.1% N, N, N¹, N¹ tetramethyethylene diamine (TEMED 0.04% wt/vol, ammonium persulphate (0.04% wt/vol). The gel was run for 3 h at 200 mV. The gels were then gently shaken in 300 ml 10% ethanol for 5 min before transfer to 300 ml nitric acid (1% in distilled water) for 10 min to allow oxidation of the nucleotides. They were then rinsed briefly in 300 ml distilled water and placed in 300 ml 0.012 M silver nitrate (BDH Chemicals) solution in distilled water. Twenty minutes later, the solution was carefully decanted and the gels reduced by repeated transfer to solutions containing 0.28 M NaHCO₃ and 0.19% formalin (BDH Chemicals) in distilled water so as to visualize the nucleotides (22). Once the image developed to an appropriate intensity, the process was stopped by placing the gel in 300 ml 10% glacial acetic acid in distilled water. After 5 min, the gel was rinsed thoroughly in distilled water and allowed to dry slightly before photographing.

Visualization of the 5'-fluorescein-labeled LC1 antisense in pituitary cells
Cells were dispersed and incubated for 3.5 h as described above in the presence or absence of fluorescein 5'-labeled LC1 antisense oligonucleotide (50 nM) and/or dexamethasone (100 nM). The antisense was included at the beginning of the experiment and replenished after 1.5 h and 2.5 h. At the end of the incubation, the cell the suspensions were centrifuged (600 g, 5 min), washed in 10 ml PBS, centrifuged again, and resuspended in 500 µl PBS. The cells were fixed in an equal volume of paraformaldehyde [Sigma, (2% wt/vol in PBS)] and smeared onto gelatinised microscope slides (20 µl/slide) and mounted in the commercial mountant Vectorshield (Vector Laboratories, Peterborough, UK). Slides were stored (4 C in the dark) and examined within 1 week with a confocal microscope (Bio-Rad MRC-500 laser scanning device).

Electron microscopy
Cells were prepared for electron microscopy as previously described (23). Briefly, cells were postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer, stained in 2% uranyl acetate in distilled water, dehydrated through a graded series of increasing ethanol concentrations and embedded in Spurr’s resin. Ultra-thin (50–80 nm thickness) sections were cut using a Reichart-Jung Ultracut ultramicrotome and mounted onto formvar-coated 200-mesh nickel grids. Sections were double stained at room temperature, first in an aqueous solution of uranyl acetate (2% wt/vol) for 10 min and second with lead citrate for 10 min in a CO₂-depleted environment. Sections were viewed by use of a JOEL transmission microscope (JEM-100S).

Data analysis
The optical density of bands of LC1 and annexin 5 immunoreactivity (arbitrary units) detected on the autoradiographs (see Figs. 3 and 5) and Western blots (see Fig. 4) was measured using a Fujix Bas 1500 imaging system with a low level light sensitive camera (Raytek, Sheffield, UK) and TINA software (Sheffield, UK); the responses to dexamethasone challenge and/ODNs were calculated as a percentage of the corresponding drug free (i.e. basal) control and expressed as the mean ± pscap]sd (n = 3). 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; statistical comparisons of these data were made by the Mann Whitney U test. Preliminary analysis by the Shapiro and Wilks test for normality for small n values demonstrated that the data from the functional studies (see Fig. 6) were normally distributed. Hence, all subsequent analysis was done by parametric methods using ANOVA with post hoc comparisons by Duncan’s multiple range test. Differences were considered significant if P < 0.05. Statistical comparisons were made only from data within experiments. Each of the studies was repeated several times (for specific details see legends) and in all instances a similar profile of data was seen.

View larger version (30K): [in this window] [in a new window]   Figure 6. Neutralization by the lipocortin 1 antisense but not by either the scrambled oligonucleotide sequence or LC1 sense of the inhibitory effects of dexamethasone (100 nM) on the release of ir-ACTH from freshly dispersed rat anterior pituitary tissue in vitro induced by (a) corticotrophin releasing hormone (CRH-41, 10 nM), forskolin (100 nM, b) and BAY K8644 (100 nM, c). , secretagogue alone; , secretagogue + antisense (50 nM); , secretagogue + sense (50 nM); = secretagogue + scrambled sequence (50 nM). The open areas at the base of each column represent peptide release in corresponding groups in the absence of secretagogue. Each column represents mean ± SEM (n = 6); **P < 0.01 vs. corresponding secretagogue-free control; P < 0.01 vs. corresponding dexamethasone-free control, ANOVA plus Duncan’s multiple range test. Typical data from three to four replicate experiments.

	Results

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View larger version (64K): [in this window] [in a new window]   Figure 1. a, Effects of graded concentrations of CRH-41 (0.1–1000 nM) on the release of ir-ACTH from dispersed anterior pituitary cell in vitro. Values represent the mean ± SEM (n = 6). **, P < 0.01 vs. control (ANOVA and Duncan’s multiple range test). b, Electronmicrograph (magnification x6000) showing a typical corticotroph in a population of dispersed pituitary cells; note the moderately electron dense granules evident (150–300 nM diameter) at the perimeter of the cell that are characteristic of a corticotroph (24 ). Typical data from three replicate experiments.

View larger version (86K): [in this window] [in a new window]   Figure 2. a, Confocal micrograph (magnification, x800) showing the uptake and concentration in the nucleus of 5' fluorescein-labeled LC1 antisense oligonucleotide in a typical population of dispersed pituitary cells. b, Residual oligonucleotide content of the medium after incubation for 3.5 h in the presence (lanes 4–9) or absence (lanes 1–3) of anterior pituitary cells (2.5 x 105 cells/ml). Lanes 1, 4, and 5 = LC1 antisense oligonucleotide; lanes 2, 6, and 7 = scrambled oligonucleotide sequence; lanes 3, 8, and 9 = LC1 sense oligonucleotide. The initial concentration of nucleotide in the medium was 50 nM. Typical data from three replicate experiments.

	Discussion

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The results presented confirm our previous reports that the acute inhibitory effects of dexamethasone on secretagogue-induced ir-ACTH release are accompanied by translocation of the glucocorticoid-inducible protein, LC1, from an intracellular compartment to the outer surface of the cell (6, 8). They also demonstrate for the first time that both the inhibition of ir-ACTH release and the synthesis and exportation of LC1 from the cells induced by dexamethasone are reversed specifically by an LC1 antisense oligonucleotide. They thus add further support to the body of evidence that suggests that LC1 plays a key role as mediator of the acute inhibitory effects of glucocorticoids on the secretion of ACTH (6, 7, 8, 13).

Much of our previous in vitro work on LC1 has been based on the use of static incubates of pituitary pieces as an experimental model. While this system has several advantages, concerns about the ability of ODNs to penetrate tissue pieces led us to exploit a single cell preparation in the present study. The system we selected, in which pituitary cells were dispersed by mild enzymatic treatment and trituration, has been used widely to examine the mechanisms controlling the secretion of several pituitary hormones; its advantages and disadvantages have recently been reviewed (25). Our preliminary studies revealed that the dispersed cells are well preserved morphologically and that, like other pituitary preparations (6), they respond to CRH-41, forskolin, and BAY K8644 with concentration-dependent increases in the release of ir-ACTH that are readily reversed by preincubation of the cells with dexamethasone. In addition, the ultrastructural features of the cells were well maintained after exposure to LC1 antisense, scrambled, and sense nucleotide sequences as also was the cell viability, as indexed by trypan blue exclusion. Moreover, the ODN-treated cells responded readily to ACTH secretagogues with marked increases in peptide release, analogous to those exhibited by untreated cells.

LC1 was readily detectable in the pituitary cells by Western blot analysis, with a major band at 37K that corresponds to the biologically active species and a second band at 32K that is reported to represent a metabolite (5, 6, 26). In accord with our previous studies based on Western blot analysis and ELISA (12), treatment of the cells with dexamethasone resulted in a marked increase in the de novo synthesis of LC1, as indexed by incorporation of ³⁵S-labeled amino acids into ir-LC1; by contrast, the expression of annexin 5 was unaffected by the steroid treatment. In cells not exposed to dexamethasone, the bulk of LC1 was contained within the intracellular pool. However, as reported previously (6), exposure of the pituitary cells to dexamethasone resulted in a marked increase in the amount of LC1 associated with the outer surface of the pituitary cells and a concomitant reduction in the intracellular pool. Our studies using ³⁵S-methionine/cysteine as a tracer showed for the first time that a proportion of the LC1 exported from the cell in response to dexamethasone is newly synthesized. Indeed, quantitative comparisons between the autoradiographs of pericellular (Fig. 5a) and intracellular (Fig. 5b) ³⁵S-labeled LC1, which represent applications of 4 µg and 40 µg protein per lane respectively, strongly suggest that the bulk of the LC1 synthesized in response to a dexamethasone challenge is promptly translocated to the cell surface. The newly synthesized protein had a molecular mass of approximately 37K, although a weak band of higher mol wt (58K), which may represent an asymmetrically clipped dimer (12), was also apparent; the lower mol wt species detected by Western blot analysis was not observed in the newly synthesized pool.

In recent years, the use of antisense strategies has helped to clarify the role of many gene products in cellular function (for reviews see Refs. 14, 27, 28). However, there is increasing controversy in the literature regarding the molecular basis of antisense action and several workers have questioned the ability of ODNs to cross cell membranes. Our confocal studies using fluorescein-labeled probe demonstrated that our LC1 antisense sequence readily passed into the pituitary cells and moved into the nucleus where it appeared to be concentrated. Further evidence that the ODNs were taken into the cells emerged from the finding that the nucleotide content of the medium bathing the pituitary cells declined progressively with time and that, at the end of the 3.5-h incubation period, only very small amounts of the antisense, sense and scrambled sequences remained in the medium. It may be argued that at least some of the nucleotides were metabolized during this period; however, it seems unlikely that this would happen to any great degree as the sequences were protected from nuclease degradation by the addition of phosphorothioate groups (15); moreover, as Fig. 2b illustrates, all three nucleotides migrated as a single band when subjected to electrophoresis. The mechanism by which the nucleotides enter the cells awaits definition although there is evidence from other studies that that it may involve fluid phase pinocytosis or absorptive endocytosis (15).

Our functional studies showed clearly that exposure of the dispersed pituitary cells to the LC1 antisense ODN effectively reversed the marked inhibitory effects of dexamethasone on the release of ACTH induced by CRH-41, forskolin, or BAY K 8644. In addition, the antisense overcame the ability of the steroid to augment the synthesis and subsequent exportation of LC1 from the cells. These responses appeared to be specific as both the sense and the scrambled control sequences were inert in all of our experiments. Furthermore, in designing the ODNs a data base search ensured that the antisense was directed against a DNA sequence unique to rat LC1; the antisense should not therefore recognize sequences that code for any other known protein. Additional evidence of the specificity of the responses derives from our finding that the antisense probe did not influence the expression of annexin 5, a Ca²⁺- and phospholipid-binding protein with a strong structural similarity to LC1 (29). The molecular mechanism underlying the powerful biological actions of the antisense are unclear. In principle, the probe was designed to hybridise with LC1 mRNA and hence to block the generation of LC1. Such a mechanism is consistent with our finding that the antisense overcame the ability of dexamethasone to induce the synthesis of a substantial pool of LC1 (i.e.³²S-labeled), much of that was destined to be translocated promptly to the outer cell surface of the cells. It also accords with our previous observations that the ability of glucocorticoids to suppress ACTH release and to promote the exportation of LC1 from pituitary cells is dependent on protein synthesis (6). Nonetheless, other modes of action have been attributed to antisense oligonucleotides and these may have contributed to the response. These include the formation of DNA/DNA hybrids or triplex DNA structures, interactions with RNase H or other factors that reduce the stability of the target mRNA and interactions with various proteins (reviewed in Refs. 14, 15, 27, 28). Indeed, mounting evidence suggests that the phosphothioate-ODNs specifically enhance the activity of RNase H and thereby facilitate the degradation of mRNA bound to the ODN sequence (14, 15). Interestingly, the concentrations of oligonucleotide that proved effective in our system were lower than those required to block mRNA or protein expression in several other systems (30) or to modulate other aspects of neuroendocrine function (31). The reasons for this are unclear. In a recent review on the efficacy of antisense oligonucleotides, Wahlstedt (32) identified several important criteria that increase the likelihood of success; these include not only the base sequence but also the length of the nucleotide, the relative abundance of various bases and the contact time. In accordance with these recommendations, we exploited a sequence of 16 bases with a guanine/cytosine content of approximately 55%. Because the pharmacokinetics and potency of the nucleotides are likely to reflect the system employed, the nucleotide concentration (50 nM) used in the present study was selected on the basis of preliminary concentration/time response studies that, in view of the potential toxicity of phosphorothioate derivatives (14, 27), aimed to determine the minimum concentration and contact time necessary to produce a near maximal effects in our functional study. Our experiments using ³⁵S-cysteine/methionine as a tracer also revealed that this ODN concentration effectively blocked the increase in LC1 synthesis induced by dexamethasone although, in the absence of the steroid, it permitted a low level of LC1 synthesis to persist. The reasons for this are obscure. Our confocal micrographs indicated clearly that the fluoroscein-labeled anti-LC1 antisense ODN concentrated mainly in the nucleus rather than the cytoplasm. While we cannot exclude the possibility that the fluoroscein marker may itself influence the subcellular distribution of the nucleotide (15), this finding raises the possibility that the probe acts primarily in the nucleus, possibly targeting the primary transcript rather than binding to mature mRNA already in the cytoplasm.

The striking ability of the LC1 antisense oligonucleotide to reverse specifically the actions of dexamethasone in the dispersed pituitary cell preparation accords with previous studies on A549 cells (human lung adenocarcinoma, 21) and adds further support to our hypothesis that LC1 plays a key role in effecting the negative feedback actions of glucocorticoids on the HPA axis in the rat. We have previously suggested that the cellular exportation of LC1 is critical to LC1 action as it provides a means by which the protein gains access to receptors on the outer surface of the cells. This concept is supported by evidence that treatments that block the exportation of the protein (LC1 antisense or protein synthesis inhibitors) also inhibit the regulatory actions of the steroids on ir-ACTH release and by observations that the generation and exportation of the protein develops in parallel with the inhibition of hormone release (6). Moreover, antisera to LC1 that would not be expected to penetrate cell membranes but could sequester LC1 at a pericellular site specifically reverse the inhibitory actions of glucocorticoids on ir-ACTH release from dispersed pituitary cells (13) or pituitary segments in vitro (6) and in vivo (7). Similarly, LC11–346 and LC11–188, which would also be unlikely to enter cells easily, readily depress ir-ACTH release (6). For technical reasons, it has not been possible to use conventional ligand binding or autoradiographic methods to detect the putative binding sites. However, using a combination of computerized FAC analysis/sorting and electron microscopy we have recently demonstrated the presence of high affinity (K_d 13 nM), saturable LC1 binding sites on the surface of several pituitary cell types, including corticotrophs. These sites, which are essential for the biological actions of LC1 (13), appear to be proteinaceous in nature (13) and to resemble those identified in human peripheral leukocytes (33, 34). Our preliminary data from immunohistochemical studies (Traverso, Buckingham, Flower and Morris, unpublished) and from experiments in which intracellular LC1 was detected in permeabilized cells by FAC analysis (35) suggest that LC1 is produced by both secretory and nonsecretory cells in the pituitary gland. As the in vitro preparations we have used in this and other studies represent a heterogeneous cell population, we cannot determine at this stage whether LC1 is externalized by the corticotrophs in response to a steroid challenge and acts in an autocrine manner or whether it originates from adjacent cells and thereby exerts a paracrine influence. The fact that both antisense and immunoneutralization strategies are effective in attenuating steroid action in the dispersed cell preparation suggests, however, that juxtaposition of the cells is not critical to the response. Nonetheless, the possibility of a paracrine influence cannot be dismissed; indeed, such an influence may form a novel and important route of immune-neuroendocrine communication in conditions of inflammation or other immune insults providing a means whereby not only resident but also infiltrating steroid sensitive cells (e.g. macrophages that are rich in LC1) may temper the secretion of ACTH and indeed other hormones that themselves, directly or indirectly, may modulate the process of the inflammatory response.

In conclusion, the application of antisense technology to a well established in vitro preparation has provided further important insight to the role of lipocortin 1 as a mediator of the acute inhibitory actions of the glucocorticoids on ACTH secretion. Although the molecular basis of our antisense probe requires further clarification, our data show clearly that it gains ready access to its target, that it effectively inhibits the synthesis, the exportation and the actions of the target protein and that its actions are specific. Further studies based on the exploitation of this nucleotides in this and other in vitro models and in several in vivo systems are currently underway with preliminary data that look promising.

	Acknowledgments

 
Our thanks are due to Professor Tom Brown (Oswel, University of Southampton, UK) for advice in the design and synthesis of the oligonucleotides, Keith Foster, Charing Cross and Westminster Medical School, London, UK) for help with the measurement of oligonucleotides, Dr. Jamie Croxtall (The William Harvey Research Institute, London, UK) and Dr. J. Browning (Biogen Co. Inc., Cambridge, MA) for the polyclonal anti-LC1 and antiannexin 5 antisera, to The National Hormone and Pituitary Program (Bethesda, MD), National Institute for Biological Standards (South Mimms, Herts, UK) and Professor Lesley Rees (St. Bartholomew’s Hospital, London, UK) fo reagents for the ACTH assay.

	Footnotes

 
¹ We are grateful to the Wellcome Trust (Grant No. 041943/Z/94/Z/MP/JF) for financial support.

Received December 30, 1996.

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