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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
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Similar concentration-response curves were obtained for forskolin (100
pM–1 µM) and BAY K 8644 (1
nM–10 µM, data not shown). On the basis of
these experiments, submaximal concentrations of the secretagogues were
selected for further study, namely CRH-41 (10 nM),
forskolin (100 nM) and BAY K 8644 (100 nM).
Further experiments showed that the secretory responses evoked by these
agents were prevented in a concentration dependent manner by
preincubation (2.5 h) of the cells with dexamethasone (10
nM–1 µM, data not shown); a concentration of
dexamethasone (100 nM) that reproducibly produced an
80–90% inhibition of secretagogue induced ir-ACTH release was used
subsequently. Examination at the electron microscope level demonstrated
that the dispersed pituitary cells were intact and that their
ultrastructural morphology was well preserved [see Fig. 1b that
illustrates a transmission electron micrograph (x6000) of a pituitary
cell; note the moderately electron dense granules (150–300
nM in diameter) at the perimeter of the cell that are
characteristic of a corticotroph, 24]. All of the main secretory cell
types were present in the cell suspension in proportions comparable to
those observed in the intact gland. However, the bulk of the
nonsecretory cells (i.e. macrophages, folliculostellate, and
dendritic cells) was lost during the dispersal procedure. Further
ultrastructural studies demonstrated that the pituitary cells retained
their morphological integrity after exposure in vitro to LC1
antisense, sense, scrambled ODN sequences (data not shown); moreover,
cell viability, as assessed by the trypan blue exclusion test was well
maintained (>97%).
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approximately 90% of the cells visualised exhibited strong
fluorescence. Inclusion of dexamethasone (100 nM) in the
medium did not affect either the uptake or the cellular localization of
the LC1 antisense (data not shown). Parallel measurements of the ODN
content of the medium also indicated that the nucleotides were readily
taken up by the cells (Fig. 2b). This process was time dependent; thus,
the amounts of antisense, sense, and scrambled nucleotide present in
the medium were already reduced within 0.5 h of contact with the
pituitary cells and declined progressively to reach a nadir at 2.5
h (data not shown).
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shows a typical autoradiograph
illustrating the effects of dexamethasone on the synthesis of LC1 by
pituitary cells (as indexed by 35S-labeled ir-LC1) in the
presence and absence of LC1 antisense, scrambled or sense ODNs;
corresponding measurements of band density (n = 3 experiments) are
shown in Table 1. A pronounced band of LC1
immunoreactivity with a mol wt of approximately 37 kDa was observed in
all samples (Fig. 3, lanes 1–8); a band of higher mol wt (58 kDa) was
also observed in most instances. Exposure of the cells to dexamethasone
(100 nM) caused a marked increase in the tissue content of
35S-labeled ir-LC1 (Fig. 3, lane 2 vs. lane 1,
Table 1). In the absence of dexamethasone none of the nucleotides
sequences produced any obvious effect on the the expression of
35S-labeled ir-LC1 (Fig. 3a, lanes 3, 5 and 7
vs. lane 1, Table 1). However, the LC1 antisense ODN
prevented the rise in 35S-labeled ir-LC1 induced by
dexamethasone (Fig. 3a, lane 4 vs. lane 3, Table 1); by
contrast the LC1 scrambled and sense sequences were inert in this
regard (Fig. 3a, lane 6 vs. lane 5 and lane 8 vs.
lane 7, Table 1). Parallel measurements of 35S-labeled
ir-annexin 5 (Fig. 3b, Table 1) demonstrated that the de
novo synthesis of this closely related protein was unaffected by
dexamethasone and/or the LC1 antisense, scrambled or sense ODN
sequences.
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, a and b, shows two typical Western blots
that illustrate ir-LC1 contained respectively within the pericellular
(a) and intracellular (b) pools of dispersed of pituitary cells after
treatment with dexamethasone and/or ODNs; corresponding measurements of
band densities (n = 3 experiments) are detailed in Table 2. Two species of ir-LC1 with mol wts (judged by
comparison with the mol wt markers, lane M) of approximately 37K
(native species) and 32K were readily detected in both the pericellular
and intracellular pools, irrespective of the pharmacological treatments
applied (lanes 1–8). Inclusion of dexamethasone (0.1 µM)
in the medium caused a marked increase in the amount of ir-LC1 detected
in the pericellular pool (Fig. 4a, lane 2 vs. lane 1, Table 2) and a concomitant decrease in the ir-LC1 content of the cells (Fig. 4b, lanes 2 vs. lane 1, Table 2). LC1 antisense did not
influence the amount of LC1 detected in the pericellular pool (Fig. 4a, lane 3 vs. lane 1, Table 2) but appeared to decrease the
amount of LC1 contained within the intracellular pool (Fig. 4b, lane 3
vs. lane 1, Table 2). The antisense ODN also prevented the
exportation of LC1 from the cells normally induced by dexamethasone;
thus, in the presence of the antisense, dexamethasone had no obvious
effect on the amounts of LC1 contained within either the pericellular
(Fig. 4a, lane 3 vs. lane 4, Table 2) or the intracellular
(Fig. 4b, lane 3 vs. lane 4, Table 2) pools. By contrast,
the responses to dexamethasone were unaffected by exposure of the cells
to either the scrambled ODN control (Fig. 4, a and b, lanes 5 and 6) or
the LC1 sense sequence (50 nM; Fig. 4, a and b, lanes 7 and
8); thus, in these groups dexamethasone induced increases in ir-LC1 in
the pericellular pool and concomitant reductions in the ir-LC1 content
of the intracellular pool that were qualitatively similar to those
observed in the nucleotide free control groups (lanes 1 and 2, Table 2). Annexin 5 was not detectable in the pericellular compartment by
western blot analysis but was present in abundance within the pituitary
cells (Fig. 4c, Table 2). The amount of annexin 5 contained within the
intracellular compartment was unaffected by dexamethasone and/or the
LC1 antisense, scrambled and sense ODN sequences (Fig. 4c, Table 2).
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shows two typical autoradiographs that
illustrate 35S-labeled ir-LC1 (i.e. newly
synthesised) in the pericellular (a) and intracellular (b) compartments
of pituitary cells exposed to dexamethasone (100 nM,
3.5 h) and/or ODNs (50 nM, 3.5 h); complementary
estimates on band density are shown in Table 3. A
pronounced band of ir-LC1 immunoreactivity with a mol wt of
approximately 37K was observed in all samples (Fig. 5, a and b, lanes
1–8); a band of higher mol wt (58K) was also observed in most
instances. Exposure of the cells to dexamethasone (100 nM)
caused a marked increase in the amount of 35S-labeled
ir-LC1 (37K) detected in the pericellular pool (Fig. 5, a, lane 2
vs. lane 1, Table 3) and a concomitant decrease in the
amount of newly synthesised ir-LC1 contained within the cells (Fig. 4b, lane 2 vs. lane 1, Table 3). LC1 antisense did not influence
the amounts of 35S-labeled ir-LC1 detected in either the
pericellular (Fig. 5a, lane 3 vs. lane 1, Table 3) or
intracellular (Fig. 5b, lane 3 vs. lane 1, Table 3) pool.
However, the antisense ODN prevented the appearance of newly
synthesised ir-LC1 on the outer cell surface normally induced by
dexamethasone; thus, in the presence of the antisense, dexamethasone
has no obvious effect on the amounts of 35S-labeled ir-LC1
contained within either the pericellular (Fig. 5a, lane 3
vs. lane 4, Table 3) or the intracellular (Fig. 5b, lane 3
vs. lane 4, Table 3) pools. By contrast, the responses to
dexamethasone were unaffected by exposure of the cells to either the
scrambled ODN control (Fig. 5, a and b, lanes 5 and 6) or the LC1 sense
sequence (Fig. 5, a and b, lanes 7 and 8); thus, in these groups
dexamethasone induced increases in 35S-labeled ir-LC1 in
the pericellular pool and concomitant reductions in the
35S-labeled ir-LC1 content of the intracellular pool
analogous to those observed in the nucleotide free control groups (Fig. 5, a and b, lanes 1 and 2, Table 3). In accord with the data shown in
Fig. 4, 35S-labeled ir-annexin 5 was detected in the
intracellular but not the pericellular compartment. De novo
synthesis of this protein was unaffected by dexamethasone and/or the
LC1 antisense, scrambled or sense ODN sequences (Fig. 5c, Table 3).
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demonstrates the ability of the LC1
antisense ODN to reverse specifically the inhibitory actions of
dexamethasone on the release of ir-ACTH induced by submaximal
concentrations of CRH-41 (10 nM, Fig. 6a), forskolin (100
nM, Fig. 6b), and the L-Ca2+ channel opener BAY
K8644 (100 nM, Fig. 6c). In the absence of exogenous
nucleotides, all three secretagogues produced significant
(P < 0.01) increases in ir-ACTH release that were
significantly (P < 0.01) overcome by preincubation of
the cells with dexamethasone (100 nM, 3.5 h). In the
absence of dexamethasone, none of the nucleotides [LC1 antisense (50
nM), LC1 sense (50 nM) or the scrambled ODN
sequence (50 nM)] influenced either the basal release of
ir-ACTH (P > 0.01) or the rises in ir-ACTH release
induced by CRH-41 (Fig. 6a, P > 0.01), forskolin (Fig. 6b, P > 0.01), or BAY K8644 (Fig. 6c, P > 0.01). In addition, all three ODNs failed to
influence the resting ir-ACTH release in the presence of dexamethasone
(100 nM, P > 0.01). However, the LC1
antisense ODN (50 nM) fully reversed the inhibitory effects
of dexamethasone (100 nM) on the release of ir-ACTH evoked
by CRH-41 (10 nM, Fig. 6a), forskolin (100 nM,
Fig. 6b), and the L-Ca2+ channel opener BAY K8644 (100
nM, Fig. 6c). In complete contrast, the LC1 sense and the
scrambled ODNs were inert in this respect and, thus, the highly
significant (P < 0.01) inhibitory effects of
dexamethasone on the release of ACTH initiated by all three
secretagogues persisted. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 (Kd
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.
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Footnotes |
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Received December 30, 1996.
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receptors. Biochem Biophys Res Commun 192:351–358[CrossRef][Medline]
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, 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; 
