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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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.

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.

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).

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).

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.

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 282(6):C1313-C1321
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society




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