©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Secretion, Surface Localization, Turnover, and Steady State Expression of Protein Disulfide Isomerase in Rat Hepatocytes (*)

(Received for publication, March 22, 1995; and in revised form, June 22, 1995)

Kunihiko Terada (1) (2) Parthasarathi Manchikalapudi (1) Robert Noiva (3) Hugo O. Jauregui (4) Richard J. Stockert (1) Michael L. Schilsky (1)(§)

From the  (1)Department of Medicine and Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461, the (2)National Center for the Study of Wilson's Disease, Inc., New York, New York 10019, the (3)Department of Biochemistry and Molecular Biology, University of South Dakota School of Medicine, Vermillion, South Dakota 57069, and the (4)Department of Pathology, Rhode Island Hospital, Brown University, Providence, Rhode Island 02903

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Protein disulfide isomerase in isolated rat hepatocytes was present at a concentration of 7 µg/mg cell protein, representing a 2-fold enrichment compared to isolated hepatic non-parenchymal cells. Though localized mainly in microsomal fractions of hepatocytes, direct immunofluorescence and cell surface radioiodination followed by immunoprecipitation revealed the presence of M(r) 57,000 disulfide isomerase at the cell surface. Electrostatic interaction of the protein with the cell surface was suggested by susceptibility to carbonate washing. Metabolic radiolabeling and immunoprecipitation studies also indicated that some of the newly synthesized M(r) 57,000 disulfide isomerase was secreted. Treatment of cells with colchicine markedly reduced the recovery of disulfide isomerase from the media, indicating microtubular-directed secretion of the protein. Partial staphlococcal V8 proteolytic digestion of the secreted protein revealed a peptide pattern similar to that of the cellular protein. Immunoprecipitation with antibody specific to the -KDEL peptide retention sequence confirmed the presence of this sequence in the secreted protein. Studies of the turnover of disulfide isomerase revealed a half-life of approximately 96 h. Treatment of cells with tunicamycin or heat shock resulted in an increased recovery of newly synthesized disulfide isomerase from cell lysates but diminished recovery from the media. The secretion and cell surface distribution of disulfide isomerase in hepatocytes may be important for the pathogenesis of immune mediated liver injury.


INTRODUCTION

Protein disulfide isomerase (PDI) (^1)is a cellular protein which was first postulated to catalyze thiol-disulfide interchange reactions in vivo almost 30 years ago(1) . Since then a great deal has been learned about the structure and function of the protein (reviewed in Refs. 2, 3) and its potential substrate specificities(4) . PDI has also been identified as a subunit of prolyl hydroxylase (5) and of the microsomal triglyceride-transfer protein complex(6) . Recently it has been suggested that PDI also serves as a molecular chaperone(7, 8, 9) . PDI is reported to be present in all eukaryotic cells in proportion to the secretory capacity of the specific cell type(2) . It is particularly abundant in liver where it is reported to constitute 0.4% of the total protein(10) . Consistent with its proposed function, the main subcellular localization of PDI in liver and other cells examined is the endoplasmic reticulum (ER), as determined by its immunodetection in intact hepatocytes (11) and purification from liver microsomes(12) . The localization of PDI to the ER is presumed due to the presence of a carboxyl terminus -KDEL retention sequence(13) .

Two studies of the distribution of immunodetectable PDI in rat hepatocytes noted the presence of a small amount of cell surface and secretory vesicle reactivity along with abundant reactivity of the microsomes and nuclear membrane(11, 14) . In another study utilizing Chinese hamster ovary cells, disulfide reduction was found to be catalyzed by a surface-associated PDI(15) . The first evidence for the secretion of PDI came from studies by Yoshimori et al.(16) who demonstrated that while the majority of PDI in a rat pancreatic exocrine cell line, ARIP, was concentrated in the ER, there was also PDI present on the cell surface and PDI which was secreted in a form containing the -KDEL retention sequence. No studies to date have described the turnover or secretion of PDI in primary cells.

In the following studies we have utilized isolated primary rat hepatocytes to better define the subcellular localization and metabolism of PDI in liver. We present new evidence for the cell surface localization of PDI and its secretion and report on the distribution of newly synthesized PDI following treatment with tunicamycin or heat shock.


EXPERIMENTAL PROCEDURES

Materials

The following materials were used: Ultralink immobilized Protein A/G Sepharose (Pierce); Bio-Gel P-10 (Bio-Rad); Fast flow protein G-Sepharose (Pharmacia Biotech Inc.); goat anti-rabbit IgG-horseradish peroxidase linked (Cappel Laboratories, West Chester, PA); chemiluminescence reagent (RENAISSANCE, DuPont NEN); Immobilon-P polyvinyldifluoride membrane (Millipore, Bedford, MA); Amplify, S-Methionine/Cysteine Promix, [P]ATP (Amersham Corp.); colchicine (Sigma); tunicamycin (Genzyme, Boston); Texas Red (Molecular Probes Inc., Eugene, OR); Bio-Max film (Kodak); custom labeling media (Life Technologies, Inc.); sulfosuccinimidyl-3-(4-hydroxyphenyl)propionate (Sulfo-SHPP, Pierce). Fluorescent microscopy and photomicroscopy were performed using a Nikon Diaphot fluorescent microscope (Nikon Inc., Melville, NY). Modified Chee's Medium (MCM) and Vitrogen-coated tissue culture plates were prepared as described(17) .

Affinity-purified anti-KDEL antibody (16) was kindly provided from Drs. Yoshimori and Yamamoto (Kansai Medical University, Japan), and purified diferric transferrin was the kind gift of P. Aisen, Albert Einstein College of Medicine.

Methods

Isolation and Culture of Primary Hepatocytes

Cells were isolated from 250-300-g Wistar rats by collagenase perfusion of the portal vein of the liver(18) . Once the total population of isolated liver cells was obtained, parenchymal cells were separated from nonparenchymal cells by centrifugation at 50 g for 5 min. The pelleted cells, >95% hepatocytes, were subjected to three washings with Leffert's buffer with added CaCl(2) to 2.5 mM(18) and repeated centrifugations. Cell viability was geq90% in all hepatocyte preparations as determined by trypan blue exclusion. All cultured cells were maintained at 37 °C in 5% CO(2) atmosphere. Isolated cells (4 10^6/dish) were seeded onto 60-mm Vitrogen-coated tissue culture plates in MCM supplemented with 10% fetal bovine serum and incubated at 37 °C for 2 h. The medium and non-adherent cells were aspirated and replaced with serum-free MCM or with serum-free MCM supplemented with 10 µg/ml tunicamycin. Heat shock of cells was performed by incubation of adherent cells at 43 °C for 1 h followed by an overnight incubation at 39 °C. Cultured cells were maintained overnight prior to use.

Preparation of Antisera to PDI

PDI was purified from rat liver by the method of Lambert and Freedman(19) . Ten µg of PDI in Hunter's TiterMax adjuvant was inoculated into a New Zealand White rabbit. This was repeated 2 weeks after the first inoculation, and sera obtained at intervals afterward were tested for reactivity to PDI by Western blot analysis. An IgG fraction was purified from the PDI antisera by affinity chromatography with Protein G-Sepharose according to the manufacturer's instruction.

Preparation of Subcellular Fractions

The subcellular fractions were prepared from isolated primary rat hepatocytes by the following procedure performed at 4 °C. 6 10^7 cells were homogenized in 4 ml of homogenization buffer (250 mM sucrose, 5 mM Tris acetate, 1 µM EDTA, pH 8.0) using a Potter-Elvehjem homogenizer with seven strokes of a Teflon pestle. The homogenate was centrifuged for 30 min at 10,000 g, and the resulting supernatant was recentrifuged for 1 h at 100,000 g. The supernatant (cytosol) was removed, and the pellet (microsome fraction) was resuspended in homogenization buffer using a Potter-Elvehjem homogenizer with a Teflon pestle.

Western Blot Analysis

Proteins resolved by SDS-PAGE were transferred to Immobilon polyvinyldifluoride membrane using an LKB semi-dry blotter with 150 mA applied for 2 h. The blot was next incubated with Tris-buffered saline (TBS), 10 mM Tris, 0.15 M NaCl, pH 7.5, containing 10% non-fat dry milk, and 0.5% Tween-20 for 1 h, and then washed twice with TBS containing 0.5% Tween-20 (TBS-T). The blot was incubated in TBS-T containing 2% non-fat dry milk containing primary antibodies diluted as follows: rabbit anti-rat PDI IgG (1 µg/ml), rabbit ASGR antisera (1:500). The blot was then washed four times with TBS-T then incubated in 20 ml of TBS-T containing 1% non-fat dry milk and 4 µl of horseradish peroxidase-linked goat anti-rabbit IgG. After washing five times with TBS-T, the blot was incubated with a chemiluminescence substrate according to the manufacturer's instructions and exposed to x-ray film for detection of bound primary antibody.

Metabolic Labeling and Immunoprecipitation

Cells were washed with 20 mM sodium phosphate, 0.15 M NaCl, pH 7.4 (PBS) and incubated in cysteine- and methionine-free labeling medium supplemented with 10% dialyzed fetal bovine serum with a mixture of [S]cysteine and [S]methionine for 4 h at 37 °C. The labeling medium was removed and replaced with MCM for the chase period. For treatment of cells with colchicine, cells were incubated in MCM containing 40 µg/ml of colchicine for 30 min prior to labeling and chase periods in medium containing the same concentration of this drug. The medium was removed after the chase period and phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 1 µM. The cells were washed twice with ice-cold PBS and harvested by scraping. After centrifugation at 2000 revolutions/min for 5 min, the cells were suspended in 1.5 ml ice-cold PBS and recentrifuged. This PBS wash and centrifugation was repeated twice. The resulting cell pellets were dissolved in 1 ml of lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% bovine serum albumin (BSA), 1% Nonidet P-40, 1 mM EDTA, 2 µM PMSF) and kept on ice for 30 min. The cell lysates and medium were centrifuged at 15,000 g for 20 min to remove debris, and the resulting supernatants were precleared by incubation with 100 µl of 20% of non-fat dry milk, 10 µl of non-immune rabbit serum, and 20 µl of a 50% suspension of Protein A/G-Sepharose for 30 min at 4 °C. After centrifugation to remove the Protein A/G-Sepharose, the precleared cell lysates and medium were incubated with either 10 µg of anti-rat PDI antibody or 10 µl of anti-albumin antisera. The immune complexes were recovered by the addition of 20 µl of a 50% suspension of Protein A/G-Sepharose and incubation at 4 °C with rotation for 2 h. The suspension was then centrifuged at 15,000 g for 3 min, the supernatant discarded, and the pellet washed three times with RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS) and once with PBS. The immunoprecipitates were recovered in SDS-PAGE sample buffer containing 0.1 M dithiothreitol and 2% SDS.

For sequential immunoprecipitation, the first immunoprecipitates were eluted in 20 µl of PBS containing 2% SDS and 1% beta-mercaptoethanol by heating to 90 °C for 10 min, diluted to 1 ml in PBS, and incubated with 0.4 µg of anti-KDEL antibody for 18 h at 4 °C. The final immunoprecipitate was recovered as described above.

All immunoprecipitates were subjected to SDS-PAGE under reducing conditions and prepared for fluorography by fixation in 10% acetic acid and 50% methanol followed by incubation in Amplify or for autoradiography by fixation in 10% acetic acid and 50% methanol. Gels were then dried under vacuum and exposed to Bio-Max film at -70 °C.

Peptide Mapping

The radiolabeled PDI which was secreted into the medium after a 4-h chase period was recovered by immunoprecipitation as described above and subjected to a 7.5% SDS-PAGE together with 10 µg of PDI purified from rat liver microsomes. The PDI band visualized by Coomassie Blue staining was cut out, subjected to partial digestion with 50 ng of Staphylococcus aureus protease, and analyzed by 15% SDS-PAGE as described(20) . Peptide bands were visualized by silver staining and by fluorography.

Preparation of Texas Red Antibody and Transferrin Conjugates

Texas Red was dissolved in dimethyl formamide to a final concentration of 10 mg/ml and added sequentially in two 50-µl aliquots to 1 mg of anti-PDI antibody prepared as described above, or to 1 mg of diferric transferrin, dissolved in 1 ml of 0.1 M sodium bicarbonate, pH 9.0. Hydroxylamine, 100 µl of a 1.5 M solution, was added to terminate the reaction. The suspension was centrifuged at 15,000 g for 5 min and the supernatant subjected to gel filtration chromatography to remove unbound Texas Red utilizing Bio-Gel P-10 and a mobile phase of distilled deionized water.

Immunodetection of Cell Surface PDI

Freshly isolated hepatocytes were washed with PBS and resuspended in MCM and incubated at 37 °C for 2 h in siliconized 1.5-ml tubes. The cells (0.5 10^5/tube) were centrifuged briefly, washed with ice-cold PBS, and then incubated with rotation at 4 °C in PBS containing 10 µg anti-rat PDI IgG-Texas Red conjugate and BSA (2 mg/ml) for 1 h. As a control for the specificity of the conjugate for PDI, 200 µg of purified PDI was added along with the anti-rat PDI to another tube. Diferric transferrin-Texas Red conjugate, 10 µg in PBS with 2 mg/ml BSA, was also added to cells for detection of a known surface receptor. The cell suspension was then centrifuged and the cells washed twice with PBS by resuspension and centrifugation. Aliquots of the suspension were added to a fluorescent mounting medium containing 22 mg/ml of N-propyl gallate in 50% glycerol and PBS. The presence of surface-bound anti-PDI or transferrin was detected by fluorescent microscopic visualization at 590 nm.

Cell Surface Labeling with I

Freshly iodinated sulfo-SHPP was prepared as follows. One mCi of Na I was added to 15 µl of a freshly prepared solution of sulfo-SHPP in dimethyl sulfoxide (200 µg/ml). To this was added sequentially 10 µl of chloramine-T (5 mg/ml in 0.5 M sodium phosphate, pH 7.4) and 10 µl of sodium metabisulfite (12 mg/ml in 0.5 M sodium phosphate, pH 7.4).

Two 60-mm dishes of cultured primary hepatocytes were washed twice with PBS at 4 °C and then incubated on ice. One dish was incubated for 5 min with 0.1 M sodium carbonate, pH 9, and washed twice with ice-cold PBS. One ml of PBS at 4 °C was added to each dish followed by 3 µg of freshly iodinated sulfo-SHPP, prepared as above, and incubated for 30 min on ice. The labeling medium was removed, and the cells were washed with PBS at 4 °C and harvested by scraping in ice-cold PBS containing 1 mg/ml of both potassium iodide and lysine. Cell pellets obtained by centrifugation were utilized for immunoprecipitation of PDI as described above.


RESULTS

Detection of PDI in the Subcellular Fractions

Western blot analysis of the total population of isolated liver cells, that were obtained prior to the 50 g centrifugations and washings, utilizing anti-PDI antibody reveals reactivity with a single band of M(r) 57,000 corresponding to PDI (Fig. 1, lane 1). No reactivity with preimmune sera was detected (data not shown). Densitometric scanning indicated a 1.9-fold enrichment of PDI in isolated hepatocytes compared to that present in the total population of isolated liver cells and 2.0-fold compared to isolated non-parenchymal cells (Fig. 1). Also ASGR, a known hepatocellular marker, was enriched 1.5- and 4-fold in isolated hepatocytes compared to that of the total population of isolated liver cells and non-parenchymal cells, respectively (Fig. 1).


Figure 1: Comparison of PDI content in isolated hepatocytes and hepatic non-parenchymal cells by Western blot analysis. Shown are the chemiluminescent blots of equivalent amounts of protein (20 µg) from total isolated liver cells (lanes 1 and 4), isolated primary hepatocytes (lanes 2 and 5), and isolated hepatic non-parenchymal cells (lanes 3 and 6) subjected to 10% SDS-PAGE, transfer to Immobilon polyvinyldifluoride membranes, and probing with anti-rat PDI IgG (lanes 1-3) or with anti-rat ASGR antisera (lanes 4-6) as described under ``Experimental Procedures.'' Molecular mass markers shown are kDa 10.



Western blot analysis of equal amounts of protein from isolated subcellular fractions from rat primary hepatocytes for PDI indicated an enrichment of PDI in the microsomal fraction compared to the whole cell homogenate (Fig. 2). Only a very small amount of PDI was detected in the cytosol (Fig. 2), its presence there likely due to disruption of a small fraction of the microsomes during their isolation.


Figure 2: Detection of PDI in subcellular fractions of primary rat hepatocytes by Western blot analysis. Shown is the chemiluminescent blot of equivalent amounts of protein samples (20 µg) obtained from primary rat hepatocyte homogenates (lane 1), and microsomal fractions (lane 2), cytosol (lane 3), and from normal rat serum (lane 4) subjected to 10% SDS-PAGE, transfer to Immobilon polyvinyldifluoride membranes, and probing with anti-rat PDI IgG as described under ``Experimental Procedures.'' Purified rat liver PDI (0.2 µg) is shown for comparison (lane 5). Molecular mass markers shown are kDa 10.



To quantitate the degree of microsomal enrichment, serially diluted protein samples from the microsomal fraction and cell homogenates were subjected to slot-blot analysis and compared with known amounts of serially diluted purified PDI standard. PDI concentrations in the microsomal fraction and in the homogenate of primary hepatocytes were estimated by densitometric scanning to be 10.5 and 7 µg/mg of protein, respectively.

Synthesis and Secretion of PDI by Primary Rat Hepatocytes

To examine the cellular metabolism of PDI, primary hepatocytes were metabolically radiolabeled, and PDI was recovered by immunoprecipitation from cell lysates and media. There was no change in the cellular levels of monomeric (57 kDa) or its presumed homodimer seen at 90-110 kDa (3) over the chase period for up to 4 h (Fig. 3A). PDI appeared in the media at 1 h following steady state labeling of cells, and increased in amount over the 4-h chase period (Fig. 3B). Radiolabeled PDI was also recovered by immunoprecipitation from cells incubated with the 2 h chase medium (equivalent to that used for Fig. 3, lane 6), indicating that the secreted protein may reassociate with the cell surface.


Figure 3: PDI synthesis and secretion by primary rat hepatocytes. Primary rat hepatocytes were radiolabeled with [S]cysteine and methionine for 4 h and chased for various periods as described. PDI (monomer at 57 kDa, presumed homodimer 90-110 kDa) recovered by immunoprecipitation from cell lysates (panel A) and media (panel B) was resolved by 10% SDS-PAGE and visualized by fluorography as described under ``Experimental Procedures.'' Chase periods were as follows: 0 min (lane 1); 15 min (lane 2); 30 min (lane 3); 60 min (lane 4); 120 min (lane 5); 240 min (lane 6). Radiolabeled PDI was also recovered by immunoprecipitation from a cell lysate incubated for 2 h with the 2-h chase medium containing radiolabeled secreted proteins (panel A, lane 7). Panel C, fluorogram showing the effect of addition of non-radiolabeled cell lysate on the recovery of radiolabeled secreted PDI. PDI recovered by immunoprecipitation from media of radiolabeled cells after a 4-h chase period following the addition of non-radiolabeled cell lysate obtained from one 60-mm culture dish was markedly reduced (lane 1) compared to PDI recovered from the media alone (lane 2). PDI is indicated by the arrow. Molecular mass markers shown are kDa 10.



Since the amount of radiolabeled PDI recovered from the media was much greater than that of cell lysates, we assumed that the specific activity (radioactivity/total protein) of intracellular PDI was much lower. To confirm this, non-radiolabeled cell lysates were added to the media containing radiolabeled secreted proteins following a 4-h chase period and then PDI was recovered by immunoprecipitation. As a result of the addition of the cell lysate, recovery of radiolabeled PDI from the media was reduced to 8.7% of the radiolabeled PDI recovered from media alone (Fig. 3C). This indicated that the reduced recovery of radiolabeled PDI from cell lysates compared to media was due to competition with unlabeled intracellular PDI.

To confirm that PDI recovered from the media was secreted and did not result from cell rupture, cells were treated with colchicine prior to and during labeling and chase periods. Results of densitometric analysis of fluorography of PDI immunoprecipitates revealed that the cellular content of newly synthesized PDI increased by almost 50% following colchicine treatment (control 0.031 ± 0, colchicine 0.047 ± 0.007, n = 2), while the amount of PDI in the media was reduced by 50% (control 0.124 ± 0.020, colchicine 0.057 ± 0.010, n = 2).

Estimation of Turnover Rates for PDI in Primary Rat Hepatocytes

The observation that the cellular levels of radiolabeled PDI over a 4-h chase period were relatively stable suggested that PDI was likely to have a relatively long half-life. Chase periods were therefore extended up to 72 h to estimate the turnover rate of intracellular PDI. While a slight increase in the intracellular level of radiolabeled PDI was observed at 24 h, levels of radiolabeled intracellular PDI gradually declined over the next 48 h to 60% of the value at the start of the chase period (Fig. 4). This allowed us to estimate the half-life of cellular PDI at approximately 96 h.


Figure 4: Turnover rates for PDI in primary rat hepatocytes. A, primary rat hepatocytes were radiolabeled with [S]cysteine and methionine for 4 h. PDI was recovered by immunoprecipitation from cell lysates at 0 h (lanes 1 and 5), 24 h (lanes 2 and 6), 48 h (lanes 3 and 7), and 72 h (lanes 4 and 8) after chase periods. The immunoprecipitates were analyzed by 10% SDS-PAGE and fluorography as described under ``Experimental Procedures.'' PDI is indicated by an arrow. Molecular mass markers shown are kDa 10. B, graphic representation of the amount of recoverable PDI from cell lysates shown in A. The relative amounts of PDI, determined by scanning laser densitometry, are shown as arbitrary units for each time point. The mean and standard deviation of the results of the two experiments and the first-order linear regression are shown.



Comparison of Cellular and Secreted PDI

To eliminate the possibility that newly synthesized PDI was modified during its transport through the secretory compartments, or that the secreted protein lacked the KDEL retention signal, peptide mapping of radiolabeled secreted PDI was compared to that of the protein purified from tissue. No difference was noted for peptides from cellular and secreted PDI (Fig. 5).


Figure 5: Peptide mapping of cellular and secreted PDI. The radiolabeled secreted PDI and purified rat liver PDI were subjected to 7.5% SDS-PAGE, and the bands corresponding to PDI were cut from the gel. These purified bands were subjected to partial digestion with S. aureus protease, and the resulting peptides resolved by 15% SDS-PAGE as described under ``Experimental Procedures.'' The peptides from purified rat liver PDI, visualized by silver staining (lane 1), and the visualized peptides from the secreted PDI, detected by fluorography, were similar (lanes 2 and 3). Shown are the same fluorogram exposed for 7 days (lane 2) and 17 days (lane 3). Molecular mass markers shown are kDa 10.



Since peptide mapping would not be sufficient to reveal the presence or absence of the KDEL sequence within PDI, sequential immunoprecipitation of secreted PDI using anti-PDI antibody and then an anti-KDEL antibody was performed. The secreted PDI was recovered with the anti-KDEL antibody following the immunoprecipitation with anti-PDI antibody, indicating the presence of the KDEL sequence in the secreted protein (Fig. 6, lane 2). The failure to recover albumin using the anti-KDEL antibody is consistent with the lack of the retention signal in this protein (Fig. 6, lane 4).


Figure 6: Immunodetection of KDEL sequence in secreted PDI. Primary rat hepatocytes (4 10^6/dish) were metabolically labeled for 4 h and the medium replaced for the 4-h chase period. The medium from each dish was subjected to immunoprecipitation, SDS-PAGE, and subsequent fluorographic analysis as described under ``Experimental Procedures.'' Shown are fluorographs of immunoprecipitates recovered with either anti-rat PDI IgG (lane 1) followed by the affinity purified anti-KDEL antibody (lane 2), or anti-albumin antisera (lane 3) followed by the affinity purified anti-KDEL antibody (lane 4). The M(r) of PDI and albumin standards are indicated. Molecular mass markers shown are kDa 10.



Effect of Heat Shock and Tunicamycin Treatment on the Distribution of Newly Synthesized PDI

Treatment of cells with either heat shock or incubation with tunicamycin resulted in an increased recovery of PDI from cell lysates and reduced secretion of the protein into media (Fig. 7).


Figure 7: Effect of tunicamycin and heat shock treatment on the distribution of PDI. Shown are the graphic representation of densitometric scans (in arbitrary units, means of duplicate experiments) of fluorographs of PDI recovered by immunoprecipitation from cell lysates (lanes 1-3) and from media (lanes 4-6). Lanes 2 and 5 show results of PDI recovered from cells subjected to tunicamycin treatment; lanes 3 and 6 PDI recovered from cells subject to heat shock as described under ``Experimental Procedures.'' The inset shows the fluorographic results from one experiment.



Detection of PDI on the Cell Surface of Primary Rat Hepatocytes

The presence of PDI on the cell surface of primary rat hepatocytes was confirmed by recovery of radiolabeled PDI by immunoprecipitation following radioiodination of cell surface protein (Fig. 8, lane 5). A corresponding band was observed in the total cell lysate (Fig. 8, lane 2). Carbonate washing of the cells prior to radioiodination resulted in minimal labeling of this band in the total lysate (Fig. 8, lane 1) and its reduced recovery by immunoprecipitation with antibody to PDI (Fig. 8, lane 4).


Figure 8: Biochemical identification of cell surface PDI. Primary rat hepatocytes were surface labeled with iodinated sulfo-SHPP following treatment with 0.1 M sodium carbonate, pH 9 (lanes 1 and 4) or with PBS alone (lanes 2 and 5). Cell lysates were either analyzed directly by SDS-PAGE and autoradiography (lanes 1 and 2) or subjected to immunoprecipitation with anti-rat PDI IgG followed by autoradiography as described under ``Experimental Procedures'' (lanes 4 and 5). Purified radioiodinated rat PDI (lane 3) is shown for comparison. PDI is indicated by the arrow. Molecular mass markers shown are kDa 10.



Both anti-PDI and transferrin Texas Red conjugates were visualized as present on the cell surface by immunofluorescent microscopy (Fig. 9, D and F), indicating the presence of PDI on the cell surface. The addition of unlabeled PDI to the suspension markedly reduced the detection of bound antibody (Fig. 9H), indicating the specificity of the binding of the Texas Red antibody conjugate for PDI. The failure to completely abolish the cell surface fluorescence by the addition of unlabeled PDI could be due to some association of the added PDI with the cell surface as seen in Fig. 3A, lane 7. No background autofluorescence was observed at this wavelength (Fig. 9B).


Figure 9: Immunofluorescent detection of cell surface PDI. Isolated primary rat hepatocytes were incubated at 4 °C without additional antibody (A and B), with Texas Red-labeled diferric transferrin (C and D), with Texas Red-labeled anti-PDI IgG (E and F), and with purified rat PDI and Texas Red-labeled anti-PDI IgG (G and H). Cells were visualized at 200 by light microscopy (A, C, E, and G) or by fluorescence microscopy at 590 nm (B, D, F, and H). All fluorescent photomicrographs were obtained with constant exposure times of 2 min.




DISCUSSION

In this study we provide evidence for the hepatocellular secretion of newly synthesized PDI, its surface membrane localization on rat hepatocytes, and information regarding PDI turnover in primary cultures of rat hepatocytes.

We confirmed that the majority of the detectable PDI in primary isolated rat hepatocytes was clearly localized to microsomes (Fig. 2), as previously reported. Quantitation of PDI by slot-blot and Western analyses indicated that PDI was present in primary hepatocytes at a concentration of 0.7% of cell protein (7 µg/mg), almost twice the 0.4% of the total liver protein reported by Freedman et al.(10) . This increased content of PDI in isolated hepatocytes compared to whole liver preparations is likely due to the exclusion of non-parenchymal cells which have significantly less PDI contents (Fig. 1).

Secretion of PDI from primary rat hepatocytes was detected by immunoprecipitation of the protein from the medium with PDI-specific antibody after metabolic labeling of cultured cells to steady state. The effect of colchicine treatment to reduce the recovery of PDI in the medium while increasing its recovery in cell lysates indicates microtubular directed secretion from cells. The identity of the secreted protein was similar to that of the cellular protein as determined by peptide mapping (Fig. 5). The presence of the carboxyl terminus ER retention signal was verified by immunoprecipitation with KDEL-specific antibody (Fig. 6), indicating that the recovery of secreted PDI was not due to the loss the peptide retention sequence. The amount of recoverable secreted PDI increased over time, reaching a steady state at approximately 4-6 h (Fig. 3B). The estimated t for secretion was between 1-2 h (Fig. 3B).

The recovery of the newly synthesized PDI from cell lysates by immunoprecipitation was relatively poor compared to that recovered from media. This raised the possibility that either the majority of newly synthesized PDI was secreted, or that there was competition for recovery of the labeled protein by the abundant quantities of unlabeled hepatocellular PDI. The latter possibility was suggested by experiments in which unlabeled cell lysates added to radiolabeled secreted proteins prior to immunoprecipitation with PDI-specific antibody significantly reduced the amount of PDI recovered (Fig. 3C) and is consistent with the calculated long half-life (t 96 h) in this study and the previously reported half-life of greater than 60 h reported by Berg et al.(21) for PDI from chicken tendon fibroblasts.

Although the vast majority of hepatocellular PDI remains cell associated and is concentrated in microsomes, the secretion of PDI by hepatocytes raises the question of how the ER retention signal is dealt with by cells. Two possibilities have been suggested: first, the presence of ER receptors specific for -KDEL-containing proteins, and second, a retrieval mechanism which may recognize -KDEL and possibly the neighboring peptide region as well. In the first an overflow of -KDEL-containing proteins should theoretically saturate their putative receptors. This possibility is not supported by a study in which the overexpression of PDI or ERp72, both -KDEL-containing proteins, led to their secretion but not to the secretion of other -KDEL proteins(22) . Alternatively, there may be sorting of the luminal proteins for recycling to the ER from a salvage compartment, with some escape from this pathway being a small but normal infidelity to the system. This is supported by the report that several -KDEL-containing proteins were secreted under normal conditions by ARIP cells (16) and in this study by primary rat hepatocytes (Fig. 6).

Earlier studies by Dorner et al.(22) indicated that steady state PDI mRNA levels were slightly increased in cells subjected to tunicamycin treatment, while the levels of mRNA for other ER proteins, glucose-regulated proteins 78 and 94 and ERp72, were markedly increased, suggesting a cellular stress response. We subjected primary rat hepatocytes to tunicamycin treatment and found a reduction in newly synthesized PDI that was secreted, and recovery from cell lysates was increased (Fig. 7). To determine whether this was indeed just a response to a generalized cellular stress as suggested by the studies of Dorner et al.(22) , we also subjected primary rat hepatocytes to heat shock treatment. Similar results were observed for heat shock treated cells (Fig. 7), suggesting that cellular stress responses result in an increased cellular retention of PDI. Some possible explanations for this outcome include the increased biosynthesis of retention molecules, an increased affinity of the retention mechanism for PDI, or increased association of PDI with other cellular proteins in the ER. Further experimentation is needed to resolve these or other possible explanations for these findings.

Surface localization of PDI was detected by both immunofluorescent labeling (Fig. 9) and by immunoprecipitation following cell surface labeling with radioiodine (Fig. 8). PDI on the cell surface appears to interact with the surface membrane electrostatically since the majority was removed by carbonate washing (Fig. 8) in contrast to the transmembrane localization proposed by Honscha et al.(14) . This loose association of PDI with membranes has been previously described for rat liver microsomes(12) . Though the source of the surface membrane PDI in rat hepatocytes was not directly determined, data from this study indicating recovery of newly synthesized PDI from unlabeled cells incubated with the radiolabeled secreted protein (Fig. 3A, lane 7) suggests that at least a fraction of the secreted protein is likely to reassociate with the surface membrane. We did not examine the disulfide isomerase activity of the surface protein; however the study by Mandel et al.(15) indicating disulfide isomerase on the surface of Chinese hamster ovary cells suggests the rat surface PDI should also be biologically active.

At present we can only speculate the presence of PDI on the cell surface of hepatocytes that may be important for certain immune mediated pathologic processes affecting the liver. Recent studies of two distinctly different modes of hepatic injury, halothane anesthesia-induced hepatitis in man (23) and hepatic copper toxicosis in the LEC rat(24) , demonstrate the presence of autoantibodies to PDI. Cell surface localization of this protein would be required for these autoantibodies to play a significant role in the pathogenesis of any autoimmune injury. Though the cell surface localization had been suggested for PDI in rat liver by previous immunolocalization studies (11, 14) , this methodology did not exclude the possibility of reaction of PDI antisera to common epitopes on other proteins or peptides. The data presented in this study confirm the cell surface localization of intact PDI, suggesting the hypothesis that its presence there plays a role in immune mediated liver injury.


FOOTNOTES

*
This research was supported in part by National Institutes of Health Grants CA61259 (to M. L. S.), DK32972 (to R. J. S.), by NIH Grant DK41296 to the Marion Bessin Liver Research Center, and by the National Center for the Study of Wilson's Disease, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Ullmann 517, Bronx, NY 10461. Tel.: 718-430-2091; Fax: 718-918-0857.

(^1)
The abbreviations used are: PDI, protein disulfide isomerase; ER, endoplasmic reticulum; MCM, modified Chee's medium; TBS, Tris-buffered saline; TBS-T, Tris-buffered saline with 0.5% Tween-20; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; ASGR, asialoglycoprotein receptor.


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