Ligand-regulated secretion of recombinant annexin V from
cultured thyroid epithelial cells
Xiuqiong
Wang1,
Marcia A.
Kaetzel1,
Sung E.
Yoo2,
Paul S.
Kim2, and
John R.
Dedman1
1 Department of Molecular and Cellular Physiology
and 2 Division of Endocrinology and Metabolism,
Department of Internal Medicine, University of Cincinnati, College
of Medicine, Cincinnati, Ohio 45267
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ABSTRACT |
The exposure of anionic phospholipids
on the external surface of injured endothelial cells and activated
platelets is a primary biological signal to initiate blood coagulation.
Disease conditions that promote the formation of ectopic thrombi result
in tissue ischemia. Annexins, Ca2+-dependent
anionic phospholipid binding proteins, are potential therapeutic agents
for the inhibition of coagulation. We have designed a transgene that
targets secretion of annexin V from cultured thyroid cells under the
control of doxycycline. Our results indicate that annexin V in the
endoplasmic reticulum (ER)/Golgi lumen does not affect the synthesis,
processing, and secretion of thyroglobulin. ER luminal Ca2+
was moderately increased and can be released by inositol
1,4,5-trisphosphate. Our study demonstrates that targeting and
secretion of annexin V through the secretory pathway of mammalian cells
does not adversely affect cellular function. Regulated synthesis and
release of annexin V may exert anticoagulatory and anti-inflammatory
effects systemically and may prove useful in further developing
therapeutic strategies for conditions including antiphospholipid syndrome.
antiphospholipid syndrome; coagulation; thyroglobulin
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INTRODUCTION |
ANNEXIN V is an
intracellular Ca2+-dependent phospholipid binding protein.
Annexin V inhibits early stages of coagulation and inflammation by
competing for anionic membrane surfaces that initiate both processes.
These extracellular effects of exogenous annexin V have been reported
both in vitro and in whole animal models (1-3, 5, 6, 11, 27,
29-31, 36, 37, 41). A limitation of the use of annexin V as
a potential therapeutic agent for hypercoagulable and inflammatory
conditions is that annexin V is rapidly cleared when infused into the
circulation (32). Continuous, regulated delivery of
annexin V into the circulation will circumvent this problem.
One approach to evaluate the extracellular therapeutic potential of
annexin V is to target the protein to the endoplasmic reticulum
(ER)/Golgi secretory pathway for inducible release into the
circulation. We have demonstrated that endogenous annexin V, consistent
with the fact that it does not contain a signal peptide, is not
secreted (40). The signal sequence of secretory proteins
targets the nascent protein through the ER membrane into the lumen,
where exportable proteins undergo posttranslational modifications. The
ER is also the site of synthesis of individual phospholipids and the
development of asymmetry of the plasma membrane lipid-bilayer. Because
a single annexin V molecule binds as many as 12-15
Ca2+ ions (34) and covers 30-40 anionic
phospholipid head groups, we evaluated whether the presence of annexin
V in the ER/Golgi secretory pathway would affect Ca2+
homeostasis, protein processing, and cell proliferation. In this study,
an annexin V transgene was engineered to target the protein to the
secretory pathway of a differentiated rat thyroid epithelial cell line
with high secretory capacity (33). In these thyrocytes, an
elevated level of all ER molecular chaperones is associated with
efficient synthesis, processing, and export of large amounts of
thyroglobulin (Tg) (16). In addition, to avoid the
potential effect of chronic expression of annexin V on cellular
functions, the thyrocytes were stably transfected with an inducible
transgene whose transcription is regulated by the exogenous ligand
doxycycline (12). Our findings demonstrate that targeting
of annexin V to the secretory pathway does not alter cellular function
and may prove useful in developing therapeutic approaches to treat
hypercoagulable disorders.
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MATERIALS AND METHODS |
Cell culture.
PCrTTA7 cells are rat thyroid epithelial cells that are stably
transfected with reverse tetracycline transactivator (rTTA) and the
pUHG72-neo gene and, therefore, express rTTA and are G418 resistant
(19). These cells were maintained in F-12 Coon's
modification nutrition mix (with 285 mg/l L-glutamine and
0.863 mg/l zinc sulfate; Sigma), supplemented with 5% fetal bovine
serum (FBS), 1 mIU/ml thyroid stimulating hormone (TSH), 10 µg/ml
insulin, 5 µg/ml transferrin, and 10 nM hydrocortisone.
Construction of (SP)-FLAG-tagged annexin V for targeting the
protein to the secretory pathway.
Full-length rat annexin V cDNA was amplified by polymerase chain
reaction (PCR) and subcloned into the pCMV-FLAG-1 mammalian expression
vector (Eastman Kodak) that contains a signal peptide (SP) derived from
preprotrypsin, MSALLILALVGAAVA, and an epitope tag sequence (FLAG),
DYKDDDDK, upstream from the multiple cloning sites. The construct was
sequenced to confirm that annexin V cDNA was in the appropriate reading
frame and that there were no errors produced during the PCR
amplification. The (SP)-FLAG-annexin V construct was subcloned into
pUHG10-3, which has a coding sequence for the tetracycline
operator upstream from a minimal cytomegalovirus (CMV) promoter
(12). The final construct in pUHG 10-3 was used to
transfect PC-rTTA7 cells.
Establishment of stable cell lines expressing and secreting
(SP)-FLAG-annexin V.
PC-rTTA7 cells expressing rTTA were transfected with the
(SP)-FLAG-annexin V plasmid DNA by using Lipofectamine (GIBCO) as described below. Cells were trypsinized, resuspended, and counted. The
cells (2 × 105) were plated in 35-mm culture dishes
and grown overnight. To prepare the DNA-lipid mixture, 1 µl of
(SP)-FLAG-annexin V DNA (1 µg/µl), 1 µl of pTK-Hyg (plasmid DNA
with hygromycin resistance gene; Clontech), and 6 µl of Lipofectamine
reagent were mixed by vortexing with 200 µl of serum-free F-12
Coon's medium. The DNA-lipid mixture was incubated at room temperature
for 30 min, and 800 µl of serum-free medium were added and mixed by
vortexing. Cells were rinsed once with PBS. One milliliter of the
DNA-lipid transfection mixture was added to each 35-mm culture dish,
followed by incubation at 37°C for 5 h in 5% CO2.
The DNA-lipid transfection mixture was then aspirated, and the cells
were rinsed with PBS. Modified Coon's culture medium supplemented with
5% FBS, 1 mIU/ml TSH, 10 µg/ml insulin, 5 µg/ml transferrin, and
10 nM hydrocortisone, antibiotic/antimycotic reagent (GIBCO), and 0.15 mg/ml G418 (GIBCO) was added, and the cells were returned to 37°C
incubation in 5% CO2. After 48 h, the cells were
trypsinized and split into five 100-mm culture dishes with selection
medium containing 0.15 mg/ml hygromycin (Calbiochem) in addition to
G418. The selection medium was changed every 4 days until individual
colonies were 1 mm in diameter (~20 days). The colonies were grown to
confluence on six-well plates. One half of the cells of each colony
were stored in liquid nitrogen, and the corresponding half were used
for characterization.
Expression and secretion of (SP)-FLAG-annexin V in PC-rTTA7
cells.
The stable cell lines were selected and characterized by
immunofluorescence microscopy and immunoblot analysis. In all the experiments with doxycycline induction, cells were induced with 1 µg/ml doxycycline (Calbiochem). The stable cell lines were grown on
11 × 11-mm glass coverslips and were induced with doxycycline for
24 h (GIBCO). After fixation with 10% PBS-buffered formalin for
20 min and permeabilization with acetone (
20°C) for 7 min, the
coverslips were incubated with mouse anti-FLAG monoclonal antibody
(Sigma) for 2 h, followed by fluorescein-tagged goat anti-mouse
secondary antibodies for 1 h. Expression of the FLAG epitope was
visualized by using fluorescence microscopy with a Nikon Optiphot.
Images were recorded on Ektachrome 200 film (Eastman Kodak).
For immunoblot analysis, cells were grown in six-well plates and
induced with doxycycline for 5 days with a change of inducing medium on
days 2 and 4. Culture media from induced and
noninduced cells were collected and combined from all six wells and
then immediately applied to a column of phenyl-Sepharose previously treated with brain phospholipids (Sigma). The column was extensively washed with buffers containing NaCl concentrations of 0.5 and 1.0 M in
the presence of 1 mM CaCl2. FLAG-annexin V was eluted from
the column with 1 mM EGTA. Fractions (15 µl) were subjected to 12%
SDS-PAGE. The cells previously washed with PBS were lysed 1 h on
ice in 1 ml of buffer containing 1% Triton X-100, 100 mM NaCl, and 25 mM Tris (pH 6.8). The mixture was centrifuged at 5,000 rpm (12,000 g) for 5 min to remove cellular debris. The supernatant was
mixed with SDS-PAGE sample buffer and subjected to 12% SDS-PAGE. After
electrophoresis, the proteins were transferred to nitrocellulose
membranes, probed with either mouse anti-FLAG antibodies (Sigma) or
rabbit anti-annexin V antibodies (13), and detected by
horseradish peroxidase-conjugated secondary antibodies.
MTT assay to evaluate cell proliferation.
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide] assay was used for assessing cell growth as described by King
et al. (18). PC-rTTA7 cells were seeded in 96-well plates in triplicate at 1,000 cells/well in medium with or without
doxycycline. Relative cell number was measured at 24, 48, and 72 h
by adding 25 µl of MTT (5 mg/ml in PBS) at each time point. After
incubation at 37°C for 2 h, 100 µl of extraction buffer (50%
dimethylformamide and 10% SDS, pH 4.7) was added and incubated for
24 h at 37°C. The absorbance at 570 nm was recorded using a
Microelisa Auto Reader (Dynatech).
45Ca2+ uptake and
release.
The procedures were modified from those reported previously (21,
28). Noninduced and doxycycline-induced cells were grown in
six-well plates and loaded with 2 µCi/ml of
45Ca2+ for 48 h. These cells
(106) were washed five times with Krebs-Ringer-HEPES (KRH)
buffer (125 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4,
1.2 mM MgSO4, 2 mM CaCl2, 6 mM glucose, and 25 mM HEPES, pH 7.4) and lysed in a 25 mM Tris buffer (pH 6.8) containing
1% Triton X-100, 100 mM NaCl, and 5 mM EGTA. For measuring
45Ca2+ release by thapsigargin, washed cells
were incubated for 20 min with 20 µM thapsigargin in KRH supplemented
with 3 mM EGTA. The supernatants were collected, and cells were lysed
as described above. The radioactivity associated with the samples was
determined using a scintillation counter (Beckman).
Mobilization of stored
45Ca2+ by IP3 in
saponin-permeabilized cells.
The procedures were modified from those reported by Lin et al.
(21) and Missiaen et al. (28). Noninduced and
doxycycline-induced cells (106) were grown for 5 days in
six-well plates. Cells were permeabilized with saponin (50 µg/ml, 1 ml/well; Sigma) for 5 min at room temperature in loading buffer (140 mM
KCl, 20 mM NaCl, 2 mM MgCl2, 2 mM ATP, and 30 mM
imidazole-HCl, pH 6.8) and then washed three times with PBS.
45Ca2+ (10 µCi/ml) was added to the cells in
loading buffer for 45 min at room temperature. After cells had been
rapidly washed five times with efflux buffer (120 mM KCl, 2 mM
MgCl2, 1 mM ATP, 1 mM EGTA, 5 mM NaN3, 2 µM
thapsigargin, and 30 mM imidazole-HCl, pH 6.8), they were treated with
20 µM IP3 (D-myo-inositol
1,4,5-trisphosphate sodium salt, 1 ml/well; Calbiochem) in efflux
buffer. Aliquots were collected every 2 min for 20 min. Another
six-well plate of cells was labeled with 10 µCi/ml
45Ca2+ for 15-60 min, and the cells were
lysed and collected as described above. Radioactivity associated with
the samples was determined using a scintillation counter (Beckman).
Assessment of Tg synthesis, processing, and secretion.
Pulse-chase experiments were performed as described by Kim and Arvan
(15). Noninduced and doxycycline-induced cells were incubated in DMEM without methionine and cysteine (Sigma) for 20 min
and then labeled with 1 mCi [35S]methionine (NEN) per
well for 60 min. Cells were then incubated for 1-5 h with
"chase" medium containing 135 mg/l unlabeled methionine and
cysteine. At specified times (0, 1, 2, 3, 4 and 5 h), media were
collected and a mixture of protease inhibitors (10 mIU/ml aprotinin, 2 mM leupeptin, 10 mM pepstatin, 10 mM iodoacetamide, 5 mM EDTA and 2 mM
phenylmethylsulfonyl fluoride) was added. The cells were then treated
with 50 mM iodoacetamide in PBS for 5 min, followed by lysis on ice in
buffer containing 1% Triton X-100, 100 mM NaCl, 5 mM EDTA, 25 mM Tris
(pH 6.8), and the protease inhibitor mixture. The cell lysates were
centrifuged at 5,000 rpm (12,000 g) for 10 min and the media
at 5,000 rpm (12,000 g) for 5 min. The percentage of Tg
secretion at each time point was calculated by dividing the amount of
labeled Tg in the medium by the sum of labeled Tg in the medium plus
that in the cell lysate (medium radioactivity/total radioactivity). The
percentage of labeled Tg retained in the cells was calculated from the
Tg in the cell lysate at each time point divided by that in the cell lysate at time 0 (cell radioactivity at each time point/cell
radioactivity at time 0). Tg synthesis was evaluated at the
labeling time points of 15, 30, 45, 60, and 90 min. The samples were
subjected to 4% SDS-PAGE. After electrophoresis, the gels were dried
and then exposed in PhosphorImager cassettes overnight. Labeled Tg
was quantitated by using the PhosphorImager (Molecular Dynamics), and
data were analyzed with Microsoft Excel.
Immunoblot analysis of ER resident proteins.
Noninduced and induced cells were trypsinized, resuspended in PBS, and
counted. Cells (106) were pelleted by centrifuging at 5,000 rpm (12,000 g) for 5 min and then lysed as described above.
The samples were subjected to 10% SDS-PAGE, transferred to
nitrocellulose membrane, probed with antibodies against calreticulin,
GRP78/BiP, ERp72, protein disulfide isomerase (PDI), and GRP94, and
visualized by using peroxidase-conjugated secondary antibodies. These
antibodies have been previously characterized (17).
Statistical analysis.
The quantitative data were calculated as means ± SD. Student's
t-test was used to analyze the differences between groups.
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RESULTS |
Targeting (SP)-FLAG-annexin V to the secretory pathway in PC-rTTA7
thyroid epithelial cells.
To target annexin V to the secretory pathway, a hydrophobic signal
peptide (SP) from preprotrypsin (MSALLILALVGAAVA) was engineered to its
NH2 terminus. The epitope tag FLAG (DYKDDDDK) was
incorporated to distinguish the transgenic annexin V from endogenous
annexin V, which is an abundant intracellular protein in thyrocytes.
Because the signal peptide is rapidly cleaved following entry in the ER lumen, the transgene protein product is referred to as
(SP)-FLAG-annexin V. Transcription of the construct was driven by a
minimal CMV promoter, which in turn was regulated by a tetracycline
operator (Fig. 1, top).
Immunostaining with mouse anti-FLAG monoclonal antibody showed
that, in doxycycline-induced cells, (SP)-FLAG-annexin V is expressed
and localized to the ER/Golgi complex (Fig. 1, middle). ER
localization was confirmed by the colocalization of (SP)-FLAG-annexin V
with an ER-resident protein, BiP (data not shown). The distribution
pattern of (SP)-FLAG-annexin V is distinct from that of the endogenous
annexin V, which was not associated with ER/Golgi (Fig. 1,
bottom). There was no staining of (SP)-FLAG-annexin V in
cells not induced by doxycycline.

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Fig. 1.
Targeting of (SP)-FLAG-annexin V to the endoplasmic
reticulum (ER)/Golgi secretory pathway. Top: annexin V cDNA
was tagged with FLAG epitope (DYKDDDDK). A 17-amino acid secretory
signal peptide (SP: MSALLILALVGAAVA), derived from preprotrypsin, was
incorporated to the NH2 terminus for targeting to ER.
Transgene expression was under control of a minimal cytomegalovirus
promoter (mCMV) and a tetracycline operator (tetO). Rat thyroid PCrTTA7
cells constitutively expressing reverse tetracycline transactivator
(rTTA) were stably transfected with the transgene and induced with
doxycycline (Dox), as described in text. Transfected cells were stained
with anti-FLAG monoclonal antibody that only recognizes the
(SP)-FLAG-annexin V. Middle: (SP)-FLAG-annexin V is
localized in the ER and Golgi of the cells. Bottom:
anti-annexin V antibodies stain endogenous annexin V in nontransfected
cells.
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The secretion of (SP)-FLAG-annexin V was confirmed by immunoblot
analysis of the culture medium. Anti-FLAG antibodies demonstrated that
there is (SP)-FLAG-annexin V in both the cell lysate and the culture
medium of doxycycline-induced cells (Fig.
2). The fusion protein containing the
FLAG epitope migrated at a molecular mass of 36 kDa instead of 35 kDa
for native annexin V (Fig. 2). The samples were applied to a
phospholipid-treated phenyl-Sepharose resin that binds annexins in a
Ca2+-dependent manner (13). The protein was
then selectively eluted with EDTA. These procedures showed that
(SP)-FLAG-annexin V retains the property of Ca2+-dependent
phospholipid binding. There was no (SP)-FLAG-annexin V in either the
cell lysate or culture medium of control cells not induced by
doxycycline, confirming that the tetracycline-inducible system is not
"leaky" (Fig. 2). The 36-kDa (SP)-FLAG-annexin V appeared in the
culture medium and cell lysate of doxycycline-induced cells. The 35-kDa
endogenous annexin V was present only in the cell lysate (Fig. 2).
These data suggest that endogenous annexin V is not secreted from
thyroid cells, further confirming our earlier finding that, without a
signal peptide, annexin V is not secreted into extracellular spaces
(40).

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Fig. 2.
Characterization of (SP)-FLAG-annexin V secretion from
PCrTTA7 cells. Cells were transfected and induced with doxycycline as
described in text. Media were collected and applied to a
phospholipid-treated phenyl-Sepharose column to selectively bind and
concentrate annexin V. The purified annexin V was eluted with 1 mM
EDTA. Fractions from columns were subjected to SDS-PAGE and transferred
to nitrocellulose membrane. Both anti-FLAG (top) and
anti-annexin V antibodies (bottom) were employed to detect
FLAG-annexin V in the culture medium and in cell lysates of noninduced
control cells and doxycycline-induced cells expressing
(SP)-FLAG-annexin V in the ER. FLAG-annexin V (36 kDa) was not detected
in the medium and cell lysate of noninduced cells but was detected in
both the medium and cell lysate of induced cells. The noninduced cells
contained endogenous 35-kDa annexin V in the cell lysate but not in the
medium. fAV, FLAG-annexin V standard; AV, recombinant annexin V
standard.
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Ca2+ dynamics in the ER.
The ER is the major Ca2+ storage and release organelle in
cells. The specialized Ca2+-ATPase in the ER membrane
[sarco(endo)plasmic reticulum Ca2+-ATPase, or SERCA]
pumps Ca2+ into the ER, where the ions are sequestered and
buffered by low-affinity, high-capacity Ca2+-binding
proteins such as GRP94 and GRP78/BiP, calreticulin, calsequestrin, and
PDI (4, 20, 23, 25, 26, 35). This sequestered Ca2+ is thapsigargin sensitive and can be readily released
by IP3 (8, 20, 23). The Ca2+
concentration in the ER lumen is also important for the function of the
enzymes involved in protein processing and folding (22). To evaluate whether overexpression of (SP)-FLAG-annexin V, a
Ca2+-binding protein, in the ER would affect the
Ca2+ sequestration/release dynamics and the
Ca2+ storage capacity, 45Ca2+ was
loaded for 48 h to reach equilibrium (21).
Doxycycline-induced cells with (SP)-FLAG-annexin V in the ER stored
~30% more 45Ca2+ in 48 h than
noninduced control cells (n = 6, P < 0.01) (Fig. 3). Thapsigargin inhibits ER
membrane Ca2+ reuptake by SERCA. After
45Ca2+ had been loaded for 48 h to reach
equilibrium, cells were treated with 3 mM thapsigargin. Cells
expressing (SP)-FLAG-annexin V in the ER released more
45Ca2+ than those not expressing annexin V in
the ER (n = 6, P < 0.01) (Fig. 3).
Under either condition the 45Ca2+ concentration
retained after thapsigargin treatment was very similar.

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Fig. 3.
45Ca2+ storage capacity.
Noninduced control cells and doxycycline-induced cells expressing
(SP)-FLAG-annexin V in the ER were loaded with 2 µCi/ml of
45Ca2+ for 48 h, followed by 3 washes with
Krebs-Ringer-HEPES (KRH) buffer and PBS. Cells were lysed and then
mixed with scintillation solution, and radioactivity was determined
(filled bars, n = 6, P < 0.01). For
measurement of 45Ca2+ release induced by
thapsigargin, washed cells were treated with 20 µM thapsigargin for
20 min in KRH supplemented with 3 mM EGTA. Supernatants and cell lysate
were counted (open bars, n = 6, P < 0.01). Equal numbers of cells (106) were used for the
experiments.
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Cells permeabilized with saponin rapidly incorporated
45Ca2+ from the medium and reach equilibrium by
45 min. Similar to nonpermeabilized cells, at equilibrium
doxycycline-induced cells with (SP)-FLAG-annexin V in the ER
sequestered 25% more 45Ca2+ than noninduced
control cells (Fig. 4A)
(n = 6, P < 0.05). There is little
difference in the rate of 45Ca2+ uptake between
the two groups of cells. Permeabilized cells were loaded with
45Ca2+ for 45 min and then challenged with
IP3 to stimulate Ca2+ release. After treatment
with IP3, doxycycline-induced cells with (SP)-FLAG-annexin
V in the ER released more 45Ca2+ than
noninduced control cells (n = 6, P < 0.01; calculated by comparing areas under the curves) (Fig.
4B). At the peak response, the induced cells released
~30% more Ca2+ than noninduced control cells
(n = 6, P < 0.05). These results demonstrate that the Ca2+ sequestered by the ER is
releasable upon stimulation with IP3.

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Fig. 4.
45Ca2+-uptake and release by
saponin-permeabilized cells. Noninduced control cells and
doxycycline-induced cells expressing (SP)-FLAG-annexin V in the ER were
treated with 50 µg/ml saponin in Ca2+ loading buffer for
5 min at room temperature, followed by 3 washes with PBS. For measuring
45Ca2+ uptake, cells were incubated with 10 µCi 45Ca2+ from 10 to 60 min and then washed
and lysed at each specified time point (A). For measuring
1,4,5-trisphosphate (IP3)-induced release, cells were
incubated with 10 µCi 45Ca2+ for 45 min
followed by incubation with 20 µM IP3 in efflux buffer.
Buffer was collected at 2-min intervals for 20 min (B)
(n = 6; P < 0.01).
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Evaluation of cell proliferation.
To assess whether the targeting of annexin V to the ER would affect
cellular growth, the MTT assay was used to evaluate the respective cell
numbers at 24, 48, and 72 h after doxycycline induction. The
growth and viability of the cells are essentially same, because the
growth curves of noninduced control cells and doxycycline-induced cells
with (SP)-FLAG-annexin V in the ER were coincident (data not shown).
Synthesis, processing, and secretion of Tg.
Experiments were designed to investigate whether the presence of
annexin V in the secretory pathway would affect the synthesis, processing, and secretion of Tg. The rates of Tg synthesis in control
cells and cells with (SP)-FLAG-annexin V were evaluated by
[35S]methionine incorporation from 15 to 90 min. There is
no significant difference between incorporation in noninduced control
cells and doxycycline-induced cells (n = 3, P > 0.05) (Fig. 5).

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Fig. 5.
Rate of thyroglobulin (Tg) synthesis in cells with or
without ER (SP)-FLAG-annexin V expression. Cells were starved with
medium lacking methionine and cysteine for 20 min, followed by 1 mCi
[35S]methionine labeling for various times (15, 30, 45, 60, and 90 min). At each time point, cells were washed 3 times with
PBS, treated with 50 mM iodoacetamide, and lysed in lysis buffer with
protease inhibitors. Samples were subjected to 4% SDS-PAGE. Gels were
dried, exposed to X-ray film, and quantitated with a
PhosphorImager. Data were analyzed using Microsoft Excel. There is
no difference in labeled Tg between noninduced control cells and
doxycycline-induced cells expressing (SP)-FLAG-annexin V in the ER at
each time point (n = 3, P > 0.05).
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Tg is a glycoprotein synthesized and secreted by PC-rTTA7
cells. It is the major secretory protein in thyroid epithelial
cells and serves as a matrix for the synthesis and iodination of
thyroid hormones (9). The nascent protein contains a
single peptide of 2,750 amino acids with a molecular mass of 300 kDa.
Tg is folded and extensively modified while passing through the
secretory pathway, gaining 10% of its molecular mass from
glycosylation. It is secreted to the thyroid colloid compartment as a
660-kDa homodimer (24, 39). Therefore, Tg makes an ideal
candidate for studying the integrity of the secretory pathway. The
intracellular and secreted Tg from control cells has the same molecular
mass as that from (SP)-FLAG-annexin V-containing cells, as was
demonstrated by 4% SDS-PAGE (Fig. 6).
This finding indicates that the final protein product is processed
identically in both groups. Immunoblot analysis of Tg demonstrated that
there is only one band corresponding to intact Tg from both noninduced
and doxycycline-induced cells (Fig. 6). In addition, there are no
changes of the levels or molecular properties of five ER chaperones
(calreticulin, BiP, ERp72, PDI, and GRP94) (Fig.
7).

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Fig. 6.
Evaluation of Tg processing in cells with or without ER
(SP)-FLAG-annexin V. Cell lysates (A) and media
(B) from [35S]methionine-labeled cells were
subjected to 4-12% gradient SDS-PAGE. Gels were then dried and
exposed to X-ray film, followed by PhosphorImager analysis.
Left lanes: noninduced control cells. Right
lanes: doxycycline-induced cells expressing (SP)-FLAG-annexin V.
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Fig. 7.
Immunoblot analysis of Tg and ER chaperones. Noninduced
control cells and doxycycline-induced cells expressing
(SP)-FLAG-annexin V in the ER were trypsinized and counted. Cells
(106) were subjected to SDS-PAGE. Proteins were then
transferred to nitrocellulose membrane and probed with corresponding
antibodies, followed by peroxidase-conjugated secondary antibodies and
color reaction. CRTL, control; PDI, protein disulfide isomerase.
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Pulse-chase experiments confirmed that, at each chase time point, the
percentage of labeled Tg secreted from cells expressing (SP)-FLAG-annexin V in the ER is not significantly different from control cells (Fig. 8) (n = 8, P > 0.05). Collectively, these results
demonstrate that targeting annexin V to the ER lumen did not alter
thyroglobulin synthesis, processing, or secretion.

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Fig. 8.
Pulse-chase experiments for assessing Tg processing and
secretion. Noninduced control cells and doxycycline-induced cells
expressing (SP)-FLAG-annexin V in the ER were starved in medium lacking
methionine and cysteine for 20 min and then pulsed with 1 mCi
[35S]methionine for 60 min. After having been washed 3 times with PBS, cells were incubated with chase medium containing
methionine and cysteine. Media and cells were collected at 0, 1, 2, 3, 4, and 5 h, respectively. Cells were first treated with 50 mM
iodoacetamide for 10 min and then lysed in SDS buffer. Samples of both
cell lysate (A) and medium (B) were subjected to
4% SDS-PAGE. Gels were dried, exposed to X-ray film, and quantitated
by PhosphorImager analysis. Data were analyzed using Microsoft Excel
(n = 8, P > 0.05). HR,
hours.
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DISCUSSION |
Annexin V has been extensively studied as an anticoagulant and
anti-inflammatory protein with therapeutic potential (1-3, 5, 6, 11, 27, 29-31, 36, 37, 41). The circulating half-life
of annexin V injected into the circulatory system, however, is <15 min
(32). Synthesis and secretion of annexin V from
gene-transfected cells may prove to be valuable in the treatment of
thrombotic and inflammatory conditions in vivo. Our study has
demonstrated that targeting of annexin V to the secretory pathway has
little effect on cellular function.
The ER is a major organelle that is involved in the coordination of
intracellular Ca2+ homeostasis (7). The
expression of (SP)-FLAG-annexin V in the ER lumen moderately increased
Ca2+ storage and uptake by the ER as predicted by the
Ca2+ binding capacity of the protein (34). The
Ca2+ retained in the ER by (SP)-FLAG-annexin V is released
upon ligand (IP3) stimulation. The increase in ER luminal
Ca2+ does not alter cellular functions such as growth and
differentiation, as evaluated with several criteria.
Intraluminal Ca2+ also plays a critical role in the
processing of newly synthesized proteins destined for secretion. Like
most proteins, improperly modified Tg in the early secretory pathway is
prone to proteolysis and often results in the presence of Tg fragments
of various sizes in the cell lysates (16). In parallel, the levels of ER chaperones increase in response to the accumulation of
improperly processed Tg in the thyrocytes (16).
Intraluminal (SP)-FLAG-annexin V does not alter the synthesis and
processing of Tg. In addition, there are no detectable changes in the
levels of other resident ER Ca2+-binding proteins.
The balance of hemostasis is fragile. Under normal conditions
coagulation is tonically inhibited; clot-forming cascades are initiated
by trauma or by agents that disrupt the hemostatic equilibrium. Considerable attention has been focused on the development of annexin V
as a potent anticoagulant, which would be a potential therapeutic agent
for hypercoagulable conditions such as antiphospholipid syndrome
(1, 6, 11, 30, 31, 36, 37, 41). Patients, mainly young
women of child-bearing age, develop autoantibodies to anionic
phospholipids. These antibodies, in vitro, bind to negatively charged
phospholipids (cardiolipin, phosphatidylserine, phosphatidylinositol, and phosphatidic acid). Binding of the
antibodies to anionic phospholipid-containing membrane surfaces
prolongs several clinical tests of coagulation, including activated
partial thromboplastin time (aPTT), kaolin clotting time (KCT), diluted Russell viper venom time (dRVVT), and Textarin time (TT) (10, 14,
38). Annexin V may also exert anti-inflammatory properties by
depleting anionic phospholipid substrates of the enzyme phospholipase A2 and may consequently inhibit the production of
prostaglandin E2 and the febrile response induced by
cytokines (2, 3, 5, 27, 29). (SP)-FLAG-annexin V levels,
regulated by a ligand-inducible promoter, would compete with
anti-phospholipid antibodies and prevent activation of coagulation and
inflammatory processes. Secretion of a genetically engineered annexin V
from mammalian cells is feasible to introduce the protein into the circulation of whole animals. This approach would allow the direct analysis of the anticoagulant and anti-inflammatory activity of annexin
V in vivo and also exploration of the possibility of treating hypercoagulable conditions and inflammatory diseases with the circulating transgenic annexin V.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants
DK-46433 (J. R. Dedman and M. A. Kaetzel) and HL-60861
(J. R. Dedman).
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
J. R. Dedman, Dept. of Molecular and Cellular Physiology,
Univ. of Cincinnati, College of Medicine, Cincinnati, OH 45267-0576 (E-mail: john.dedman{at}uc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 27, 2002;10.1152/ajpcell.00553.2001
Received 18 November 2001; accepted in final form 9 January 2002.
 |
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