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J. Biol. Chem., Vol. 275, Issue 41, 31946-31953, October 13, 2000

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Cell Type-dependent Differences in Thyroid Peroxidase Cell Surface Expression^*

Xiaoqing Zhang^§ and Peter Arvan^§¶

From the  Division of Endocrinology and ^§ Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461

Received for publication, April 26, 2000, and in revised form, July 8, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recently, it has been suggested that only ~2% of human thyroid peroxidase (hTPO₉₃₃) reaches the surface of stably transfected (Chinese hamster ovary) cells, most being degraded intracellularly, and this might be representative of thyroid peroxidase (TPO) behavior in thyrocytes (Fayadat, L., Siffroi-Fernandez, S., Lanet, J., and Franc, J.-L. (2000) J. Biol. Chem. 275, 15948-15954). In agreement, in stably transfected Madin-Darby canine kidney clones, nonpermeabilized cells exhibit wild-type hTPO₉₃₃ immunofluorescence (apically) on <10% of that found in permeabilized cells, where an endoplasmic reticulum pattern is observed. Further, a C-terminally truncated, membrane-anchorless hTPO₈₄₈ is also retained in the endoplasmic reticulum of stably transfected Madin-Darby canine kidney cells. However, by contrast, in Chinese hamster ovary cells after transient transfection, hTPO₉₃₃ immunofluorescence is detected equally well in nonpermeabilized and permeabilized cells, indicating that a large portion of hTPO₉₃₃ is present at the cell surface; furthermore, hTPO₈₄₈ is efficiently secreted. Further, using an antiserum not cross-reacting with rat TPO, we find by immunofluorescence that in stable clones of PC Cl3 (rat) thyrocytes, considerably more (~50%) of the cells exhibit hTPO₉₃₃ at the cell surface. However, cell surface biotinylation and endoglycosidase H digestion assays appear to under-represent the extent of hTPO₉₃₃ transport, presumably because protein folding limits both Golgi carbohydrate modification and accessibility of lysines in the extracellular domain. We conclude that cell type-specific factors may facilitate stable expression of TPO at the cell surface of thyrocytes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the past decade, cell type-dependent differences in membrane and secretory protein trafficking have been increasingly recognized. Certainly, the rate and efficiency of protein folding and export from the endoplasmic reticulum (ER)¹ varies between cell types (1). Further, in the distal secretory pathway, the expression of cDNAs encoding regulated secretory proteins has been found to result in storage in secretory granules in some regulated secretory cell types but not others (2-5). Additionally, cell surface polarity signals can be differentially interpreted by different polarized cell types (6-10). The identification of cell type-dependent differences in protein sorting and trafficking represents a crucial first step in elucidating the underlying molecular mechanisms that account for such differences (11).

Thyroid peroxidase (TPO) is a key enzyme responsible for iodination of thyroglobulin during the synthesis of thyroid hormones. Electron microscope autoradiography studies have established that iodination activity is localized primarily at the (extracellular aspect of the) apical surface of thyrocytes delimiting the thyroid follicle lumen; suggesting that under physiological circumstances, TPO is primarily an apical plasma membrane enzyme (12). TPO is neither the sole gene product responsible for the diaminobenzidine oxidation reaction (a technique that has been used to detect peroxidase activity cytochemically) nor the sole protein immunoreacting with thyroid autoimmune antisera (used to immunolocalize the "anti-microsomal antigen" (Ref. 13)). Nevertheless, TPO cytochemical activity in thyrocytes is associated with apical membrane vesicles and the outer surfaces of apical microvilli (14), while additional reaction product can be detected in the thyrocyte ER and Golgi complex. Moreover, using patients' antisera, the thyroid microsomal antigen is immunolocalized to the apical surface of thyrocytes (15), while additional immunoreaction is found in the cytoplasm (16, 17). Acute thyroid-stimulating hormone stimulation of the thyroid gland causes additional TPO enzymatic activity and immunoreactivity at the apical cell surface (18-23). These results all indicate that TPO travels via the secretory pathway to the plasma membrane.

Despite this, lingering questions persist about the trafficking of TPO to the cell surface in the thyroid and in various cell culture systems (24). In primary porcine thyrocytes after 7 days in culture, only ~30% of endogenously expressed TPO could be chemically modified at the cell surface with a non-permeant biotinylation reagent (25). Further, it has recently been reported that only ~2% of recombinant wild-type hTPO (full-length hTPO₉₃₃) reaches the surface of a stably transfected CHO cell clone, while most hTPO₉₃₃ is retained in the ER without intracellular transport (26, 27), and this fraction at the cell surface is only negligibly increased by enhanced heme availability (28). From this it has been argued that impaired intracellular traffic and massive degradation of recombinant hTPO₉₃₃ (29) might be representative of the situation found in thyrocytes.

In this report, we have compared the surface expression of recombinant hTPO in three very different cell culture models. Our data provide strong evidence to suggest that delivery of hTPO to the cell surface varies widely depending upon the system in which it is studied. We find that clonal MDCK cells fail to efficiently export wild-type hTPO₉₃₃ to the plasma membrane, and fail to secrete a truncated hTPO₈₄₈ luminal domain. However, we find that both hTPO₉₃₃ and hTPO₈₄₈ are exported quite efficiently in transiently transfected CHO cells despite the fact that after clonal CHO cell selection and expansion, only little hTPO₉₃₃ is found at the cell surface (26). More importantly, in stably transfected clones of PC Cl3 (rat thyrocyte) cells, we also find a significantly higher fraction of hTPO₉₃₃ distributed on the plasma membrane. These results suggest that cell type-specific factors may allow for stable surface expression of hTPO in thyrocytes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- A rabbit antiserum (serum L-0666) against hTPO generously provided by Dr. A. Taurog (University of Texas Southwestern Medical Center, Dallas, TX) was initially prepared by Dr. Paul Banga (King's College, London, United Kingdom) against the whole purified molecule. The full-length hTPO₉₃₃ and C-terminally truncated hTPO₈₄₈ cDNAs were the kind gifts of Drs. R. Magnusson (Mt. Sinai Medical Center, New York, NY) and B. Rapoport, Cedars-Sinai Medical Center, Los Angeles, CA). Both cDNAs were subcloned into pCDNA3 (Invitrogen) in which hTPO expression was driven by the immediate early cytomegalovirus promoter. Sulfo-NHS-biotin (used at 0.6 mg/ml) was from Pierce. Avidin-agarose was from Roche Molecular Biochemicals. For secondary antibodies, goat anti-rabbit horseradish peroxidase conjugate and goat anti-rabbit Cy3 conjugate were from Jackson Immunochemicals. For immunoprecipitation, the secondary precipitant was Zysorbin (Zymed Laboratories Inc., South San Francisco, CA). The enhanced chemiluminescence substrate for blotting was from Amersham Pharmacia Biotech, and the ³⁵S-amino acid mixture (Expre³⁵S³⁵S) was from PerkinElmer Life Sciences. Recombinant endoglycosidase H was from New England Biolabs and was used according to the manufacturer's instructions.

Cell Culture-- MDCK strain II and CHO cells were grown in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. PC Cl3 cells were obtained from Dr. James Fagin (University of Cincinnati, Cincinnati, OH) and were grown in Coon's medium plus 5% calf serum, 10 microunits/ml thyroid-stimulating hormone, 1 µg/ml insulin, 5 µg/ml transferrin, and 10 nM hydrocortisone. Antibiotics (streptomycin-penicillin from Life Technologies, Inc.) were added to all cell culture media.

Transfection-- In 35-mm tissue culture dishes, 2× 10⁵ MDCK, CHO, or PC Cl3 cells were seeded in complete growth medium. The cells were incubated at 37 °C in a 5% CO₂ incubator until 50-80% confluent. For each transfection, 1-2 µg of plasmid DNA was diluted into 100 µl of Opti-MEM (Life Technologies, Inc.). Separately, 15 µl of LipofectAMINE was diluted to 100 µl in the serum-free medium. The two solutions were gently mixed and incubated for 30 min at room temperature, before diluting to 1 ml in serum-free medium. Each dish of cells was rinsed twice with the same medium before the LipofectAMINE/DNA mix was added. After overnight incubation with the cells, the medium was replaced with fresh complete growth medium. For transient transfection of CHO cells, assays were performed after 48 h. For stable transfection of MDCK and PC Cl3 cells, after 48 h, the cells were passaged to large Petri dishes and incubated in complete medium containing 0.8 mg/ml G418. Individual clones were isolated, expanded, and maintained in selection medium for 3-6 months prior to experiments. Of note in one instance (Fig. 6B), 16 h before the experiment, cell culture medium was modified to include 10 mM sodium butyrate to enhance expression from the cytomegalovirus promoter.

Immunofluorescence-- Cells grown on uncoated glass coverslips (Bellco) were washed three times in PBS+ (PBS containing calcium and magnesium (each at 1.0 mM)). The cells were then fixed with 3.7% formaldehyde for 15 min at room temperature, followed by a brief wash in PBS+ and quenching with 50 mM NH₄Cl in PBS for 5 min. After two additional washes in PBS+, the cells were incubating in a blocking solution containing 3% bovine serum albumin in PBS+. For permeabilization, the blocking and all subsequent incubation steps included 0.1% Triton X-100. Anti-TPO was diluted 1:2000 in PBS+ and was incubated with the cells overnight at 4 °C. The cells were washed five times with PBS+ and then incubated with goat anti-rabbit Cy3 conjugate, diluted 1:2000, for 60 min at room temperature. The cells were finally washed five further times in PBS+ before mounting and visualization with a Nikon Eclipse E400 microscope equipped with epifluorescence optics and a digital camera; images were captured as Adobe Photoshop files.

Surface Biotinylation and SDS-PAGE-- In preparation for biotinylation, cells were grown either on standard tissue-culture treated plasticware (MDCK, CHO, PC Cl3) or on Transwell-Clear filters (for MDCK cells, Costar-Corning Corp.). The cells were washed three times with ice-cold PBS+ and then incubated for 15 min with 0.6 mg/ml sulfo-NHS biotin in PBS+ at 4 °C. In some experiments, two sequential 15-min incubations with sulfo-NHS biotin were performed; however, this had no effect on the extent of surface biotinylation of hTPO or the outcome of our experiments. After biotinylation, the cells were washed twice in ice-cold PBS+ and then quenched with 50 mM NH₄Cl in PBS for 5 min.

For a blotting approach, the biotinylated cells were lysed at 4 °C in precipitation buffer (1% Triton X-100, 0.1% SDS, 0.2% sodium deoxycholate, 100 mM NaCl, 10 mM Tris, pH 7.4) plus a mixture of protease inhibitors: aprotinin (2 µg/ml), leupeptin (100 µM), pepstatin (10 µM), EDTA (10 mM), and diisopropyl fluorophosphate (1 mM). The biotinylated samples were then precipitated with avidin-agarose. Unless otherwise indicated, all of the avidin-agarose precipitate was loaded into a single gel lane, while only a portion of the supernatant was analyzed in parallel (see figures). The samples were resolved by reducing 5% SDS-PAGE, electrotransferred to nitrocellulose, and then blotted with anti-TPO at a dilution of 1:5000.

For an immunoprecipitation approach, the cells were metabolically labeled with 300 µCi of ³⁵S-amino acids, and then chased in complete growth medium. At a given chase time, biotinylated cells were lysed as above and immunoprecipitated with anti-TPO. After washing, immunoprecipitates were incubated with 100 µl of boiling 1% SDS to denature the antibody. The samples were then diluted 10-fold in precipitation buffer lacking SDS and re-precipitated with avidin-agarose before analysis by reducing 5% SDS-PAGE. Gels were impregnated with 1 M sodium salicylate, dried, and exposed to x-ray film at 70 °C.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stable Expression of hTPO in Clonal MDCK Cells-- The cloned TPO cDNA predicts a type 1 membrane protein topology with an N-terminal signal peptide (involved in protein translocation across the ER membrane), followed by a large extracellular catalytic domain, a single transmembrane span, and a short cytosolic tail at the C terminus (30). The original reports of recombinant hTPO expression indicated the presence of a subpopulation of antibiotic-resistant CHO cells in which stably expressed, full-length wild-type hTPO₉₃₃ was clearly detected on the plasma membrane (31, 32) and a truncated hTPO₈₄₈ (mutated to place a stop codon that eliminates the transmembrane span and cytosolic tail, as in Fig. 1) was efficiently secreted into the medium (33-35); both of these recombinant hTPO constructs were proven to function as enzymatically active peroxidases. Notably, Rapoport and colleagues (36) went on to hypothesize that expression of TPO at the cell surface might create a selective growth disadvantage for cells in culture. In such a case, TPO-expressing cells might require mechanisms to suppress surface localization or would be outgrown during cell passage. We recently made similar observations concerning the instability in culture of surface expression of recombinant wild-type (enzymatically active) viral hemagglutinin-neuraminidase (37).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Schematic of hTPO cDNAs expressed in this paper. Sig, cleaved signal peptide; TM, transmembrane span; C, cytosolic tail domain. The arrow above identifies the location of residue 848 at the beginning of the transmembrane span; truncation at this position in the construct below has been reported to produce a secreted, bioactive hTPO (33). wt, wild type.

We transfected MDCK (strain II) cells with the wild-type hTPO₉₃₃ cDNA in the pCDNA3 vector, selected for G418 resistance, and expanded clones for screening by Western blot. After confirming hTPO expression, a pulse-chase protocol followed by immunoprecipitation with a rabbit polyclonal antibody showed that hTPO₉₃₃ in MDCK cells was relatively stable (see upper panel of Fig. 2). To initially estimate the fraction of hTPO₉₃₃ molecules transported through the secretory pathway to the plasma membrane, two biochemical assays were employed. First, we used digestion with endoglycosidase H of hTPO₉₃₃ immunoprecipitated from the whole cell lysate at different chase times after metabolic labeling. Endoglycosidase H digests high mannose oligosaccharides; failure of exportable glycoproteins to be delivered to the Golgi (or failure of side chain conversion to complex glycans within the Golgi) leads to persistent sensitivity to endoglycosidase H digestion. Indeed, as shown in the lower panel of Fig. 2, the fraction of total hTPO₉₃₃ acquiring resistance to digestion with endoglycosidase H in MDCK cells was undetectable. The same behavior was detected in all MDCK clones (data not shown).

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Stability and sensitivity to endoglycosidase H (Endo H) of recombinant hTPO933 in stably transfected MDCK cells. Confluent cells grown in six-well plates were pulse-labeled with 35S-amino acids for 30 min, and then individual wells of cells were lysed at each of the chase times indicated, immunoprecipitated with a rabbit polyclonal antiserum against hTPO, and analyzed by SDS-PAGE and fluorography. To be representative, two different clones (upper panel, clone 2a; lower panel, clone 3c) are shown in the figure. The same patterns shown here were observed in all MDCK clones tested.

We then examined biotinylation of hTPO₉₃₃ at the cell surface. The method involves employing a membrane-impermeant reagent that can covalently tag (with a biotin moiety) the -amino groups of lysine residues that are extracytoplasmically disposed and freely accessible to solvent. Fig. 3A shows the results from MDCK cells grown to confluence on porous filters, surface-tagged with sulfo-NHS-biotin (either from the apical or basolateral side), washed and quenched, and finally lysed and precipitated with avidin-agarose to selectively sediment the biotinylated proteins. Both the precipitates and a fraction of the supernatants containing protein that had not been biotinylated were analyzed by SDS-PAGE and immunoblotting with anti-hTPO. A quantitative analysis suggested that surface hTPO in MDCK cells exhibited an apical:basolateral ratio of ~5:1 (e.g. see left panel of Fig. 3A). More significantly, however, the biotinylated surface molecules represented only ~5% of total hTPO detected (Fig. 3A). A very similar outcome was obtained when pulse-labeled hTPO₉₃₃ at the 6-h chase time was analyzed by sequential immunoprecipitation and avidin-agarose precipitation (Fig. 3C). When sufficient amounts of surface-biotinylated hTPO₉₃₃ were collected for digestion with endoglycosidase H (Fig. 3A, right), even this subfraction appeared sensitive to digestion, indicating that in MDCK cells, surface hTPO₉₃₃ molecules escape typical modification of their N-linked glycans upon passage through the Golgi complex, as has been suggested previously (38-40). No TPO biotinylation was detected in MDCK cells expressing truncated hTPO₈₄₈ (of which no portion is localized to the cell surface, see below); this control establishes that the biotinylated hTPO₉₃₃ indeed represents a subpopulation localized at the surface and not in the ER. Increased availability of heme, known to be required for recombinant TPO enzyme activity (28, 41-43), did not change either the small fraction of hTPO₉₃₃ at the cell surface or the endoglycosidase H sensitivity of the biotinylated surface molecules (Fig. 3B).

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Cell surface biotinylation of hTPO933 expressed in MDCK cells cultured on porous filters. MDCK cells (clone 3c) were grown for 3 days on Transwell-Clear filters. At this time transepithelial resistances of 100 ohms-cm2 were confirmed. The cells were then biotinylated either apically or basolaterally as described under "Experimental Procedures." A, apically (A) and basolaterally (B) biotinylated proteins bound to avidin-agarose were detected by immunoblotting with anti-hTPO, in comparison to one-tenth of the avidin-unbound supernatant. In control experiments, re-precipitation of the supernatant with additional avidin-agarose recovered no additional hTPO, while precipitation of non-biotinylated cell lysates with avidin-agarose also recovered no hTPO (data not shown). In the right panel of A, biotinylated hTPO933 from additional MDCK cells was mock-digested () or digested (+) with endoglycosidase H (Endo H). B, the same experimental protocol was repeated but this time including 30 µM hemin in the medium throughout the experiment. In this case, only one-twentieth of the avidin-unbound supernatant was analyzed on the gel. Note that increased heme availability did not appreciably increase the avidin-bound fraction of hTPO933, which was again sensitive to digestion with endoglycosidase H. C, the cells were pulse-labeled as in Fig. 2 and chased for 6 h before surface biotinylation from either apical (A) or basolateral (B) sides. Total labeled hTPO933 at the 6-h chase time from a parallel filter is shown for comparison.

We then examined the immunofluorescence localization of hTPO₉₃₃ in nonpermeabilized clonal MDCK cells. Interestingly, we found that among the population of cells derived from a clone, surface expression of hTPO₉₃₃ was limited to only a few (<10%) of the cells. When observed, surface hTPO₉₃₃ always showed an apical pattern of immunofluorescence, and this was especially clear in cells grown on porous filters (Fig. 4). However, in the vast majority of cells, significant hTPO₉₃₃ expression became apparent only after permeabilization (Fig. 5, upper panels). Further, the permeabilized MDCK cells showed strong perinuclear and reticular fluorescence throughout the cytoplasm, suggesting an ER localization.

View larger version (57K): [in this window] [in a new window]   Fig. 4.   Surface hTPO expression in MDCK cells (clone 3c) grown on Transwell-Clear filters. The cells were processed for immunofluorescence with a polyclonal anti-hTPO under nonpermeabilized conditions (upper panel) and counterstained with DAPI (middle panel) to identify nuclei. The lower panel is a merged view. Note that most nonpermeabilized cells exhibit no surface immunofluorescence.

View larger version (81K): [in this window] [in a new window]   Fig. 5.   Immunofluorescence distribution of full-length hTPO933 and truncated hTPO848 in MDCK cells. MDCK cells expressing hTPO933 (clone 3c, upper panels) or hTPO848 (clone HB3, lower panels), grown on glass coverslips, were processed for immunofluorescence with anti-hTPO under nonpermeabilized or permeabilized conditions as indicated.

It has been clearly shown that CHO cells extensively secrete a truncated hTPO₈₄₈ (see Fig. 1) expressed from a dicistronic mRNA that includes dihydrofolate reductase for amplified recombinant gene expression (36). In our case, we introduced hTPO₈₄₈ into MDCK cells using the pCDNA3 vector. However, after isolating G418-resistant colonies and then screening the medium bathing confluent cells (collected for 24-36 h), no positive clones could be detected. We therefore went on to repeat the examination but now included analysis of the cell lysates. Fig. 6A shows 10 such clones with varying degrees of cellular hTPO₈₄₈ expression. In no case was any hTPO detected in the medium by immunoblotting after a 1-day collection. This was true also in one of our stronger expressors, clone HB3, even after treatment with 10 mM sodium butyrate to boost expression further (Fig. 6B). Similarly, the truncated protein could not be detected by immunoprecipitation of the medium after pulse-chase, although intracellular degradation comparable to that seen with the full-length hTPO constructed was noted (Fig. 6C). When examined by immunofluorescence, no hTPO₈₄₈ immunofluorescence could be observed under nonpermeabilized conditions, whereas in permeabilized cells, an ER localization pattern was again detected (Fig. 5, lower panels).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Lack of secretion of truncated hTPO848 from different MDCK cell clones. A, for each of 10 different confluent G418-resistant clones of MDCK cells transfected with the hTPO848 cDNA, growth medium was collected for 1 day. The media were trichloroacetic acid-precipitated, cells lysed, and both were analyzed by immunoblotting with anti-hTPO. B, clone HB3, one of our highest expressors, was grown overnight in the presence or absence of butyrate to further increase hTPO848 expression, but this did not lead to detectable secretion. C, after pulse labeling with 35S-amino acids, clone HB3 was chased for 0.5 or 6 h and the cell lysates (C) and medium (M) analyzed by immunoprecipitation with anti-hTPO, SDS-PAGE, and fluorography. Some decrease in intracellular hTPO848 is apparent over this time, but no hTPO848 is detected in the medium.

Transient Expression of hTPO in CHO Cells-- The foregoing experiments seemed quite consistent with recent reports indicating that only 2% of recombinant hTPO₉₃₃ can be detected at the surface of a stably transfected clone of CHO cells (26-29). However, these findings seem at odds both with the original studies of recombinant hTPO expression in other stably transfected CHO cells (31, 33), as well as a recent suggestion of hTPO₉₃₃ surface expression upon transient transfection of CHO cells (42). We therefore decided to reinvestigate expression of hTPO in transiently transfected CHO cells.

We began by examining the fate of the truncated hTPO₈₄₈ construct. Importantly, nontransfected CHO cells exhibit essentially no background fluorescence when probed with anti-hTPO (see below). Using LipofectAMINE, we found the transfection efficiency using a green fluorescent protein cDNA in these cells to be ~10%, and immunofluorescence detection of hTPO₈₄₈ (observed only after permeabilization) was also positive in only ~10% of the cells (data not shown). We then examined a 30-h collection of conditioned medium; from clonal MDCK cells there was no secreted hTPO₈₄₈, while transiently transfected CHO cells secreted the majority of the protein (Fig. 7A). We confirmed this result upon pulse-chase, where nearly equal concentrations of hTPO₈₄₈ were found in the CHO cells and medium at 8 h of chase, still more was recovered in the medium at 24 h of chase (Fig. 7B), and the secreted hTPO₈₄₈ appeared resistant to digestion with endoglycosidase H (Fig. 7B, bottom panel). These kinetics of hTPO₈₄₈ secretion agree with those reported previously (33). As a control, a negligible fraction of full-length (membrane-bound) hTPO₉₃₃ was found in the chase medium bathing CHO cells at the 24-h chase time (Fig. 7B, right panel). We therefore proceeded to examine hTPO₉₃₃ expression by immunofluorescence.

View larger version (44K): [in this window] [in a new window]   Fig. 7.   Truncated hTPO848 is efficiently secreted from transiently transfected CHO cells. A, beginning at 48 h after transfection, conditioned medium was collected for 30 h from transiently transfected CHO cells or, as a control, from stably transfected MDCK cells (clone HB3). The cells were lysed and media trichloroacetic acid-precipitated, before analysis by SDS-PAGE and immunoblotting. In the first lane (Con), MDCK cells expressing full-length hTPO933 were analyzed as positive control. wt, wild type; trunc., truncated. B, after pulse labeling with 35S-amino acids, CHO cells transiently transfected to express truncated hTPO848 (left part of figure) or full-length hTPO933 (right part of figure) were chased for 8 or 24 h and the cell lysates (C) and media (M) were analyzed by immunoprecipitation (I.P.) with anti-hTPO followed by SDS-PAGE and fluorography. Endo H, endoglycosidase. Bottom panel, endoglycosidase H digest of cellular and secreted hTPO848 at the 8-h chase time.

In this case, ~10% of the cells were brightly immunofluorescent when probed with anti-hTPO under nonpermeabilized conditions (Fig. 8, left panels). Incidentally noted was the tendency of hTPO₉₃₃-expressing CHO cells to assume a slightly more elongated shape than surrounding nontransfected cells; the significance of this observation is unclear. Individual nonpermeabilized hTPO₉₃₃-positive CHO cells also exhibited immunofluorescence that tended to be brighter near the cell edges (where the cells are more flat) than over the cell midportion, which often had a darker central region (Fig. 8, left panels). Upon permeabilization, the fraction of hTPO₉₃₃-positive cells did not change and the immunofluorescence intensity was not increased over that of nonpermeabilized cells (Fig. 8, right panels). This result was repeated in several independent experiments. CHO cells are notably poor for high resolution analysis of intracellular organelle staining patterns by conventional immunofluorescence; thus, one cannot readily quantify the extent to which ER and/or Golgi staining can be superimposed upon the cell surface staining that is already apparent in the nonpermeabilized cells. Further, the biotinylation assay did not seem particularly advantageous in this case where the vast majority of cells are not expressing the hTPO₉₃₃ protein. However, the immunofluorescence data make clear that surface expression of hTPO₉₃₃ in transiently transfected CHO cells is much higher than in clonal MDCK cells (see above) or in selected clones of CHO cells (26-29). Since both our study and those reported previously (26-29) used the identical source of hTPO₉₃₃ cDNA for expression in the identical (CHO) cell type, the clear implication of these results is that upon selection, expansion, and maintenance of clones, some (and perhaps even most) clones are eventually propagated whose surface hTPO expression is no longer representative of the initial behavior of hTPO₉₃₃ in CHO cells.

View larger version (102K): [in this window] [in a new window]   Fig. 8.   Survey of the population of transiently transfected CHO cells indicates that full-length hTPO933 is strongly expressed at the cell surface. The cells at 48 h after transfection were processed for immunofluorescence with anti-hTPO under nonpermeabilized or permeabilized conditions. As indicated, the upper left panel demonstrates the degree of background fluorescence from a population of nontransfected CHO cells. Note that, unlike in MDCK cells (see Fig. 5), the frequency of positively expressing cells is equal under nonpermeabilized and permeabilized conditions.

Stable Expression of hTPO in the PC Cl3 (Rat Thyrocyte) Cell Line-- Because of the remarkably inefficient delivery of hTPO to the surface of stable clones of CHO and MDCK cells, we decided to examine the surface expression of hTPO₉₃₃ in a cell line derived from a thyrocyte lineage. PC Cl3 cells are a rat thyrocyte line similar in origin to FRTL5 cells that contain endogenous rTPO (20, 44) and have served as a suitable expression system for hTPO (45). When probed by Western blotting, there was no detectable hTPO cross-reactivity in untransfected PC Cl3 cells (Fig. 9A, lane 9). Eight independent G418-resistant clones of transfected PC Cl3 cells were selected and screened by immunoblotting for expression of the hTPO₉₃₃ cDNA (Fig. 9A, lanes 1-8). Three clones, C5 (lane 1), C4 (lane 6), and B6 (lane 7) were found to be positive and were studied further.

View larger version (34K): [in this window] [in a new window]   Fig. 9.   Western blot screening, cell surface biotinylation, and endoglycosidase digestion of PC Cl3 cells stably transfected with the cDNA encoding hTPO933. A, in the Western blot, lanes 1, 6, and 7 were positive for hTPO933 expression and were designated with the clone names shown above. Lane 9 shows untransfected PC Cl3 cells. Lane 10 shows MDCK cells expressing hTPO933 as a positive control. B, clone B6 cells were biotinylated, precipitated with avidin-agarose, and analyzed as in Fig. 3A, except that the biotinylated fraction was divided in three equal portions and digested with with PNGase F (F), endoglycosidase H (H), or mock-digested. Two different concentrations of the avidin-unbound fraction (one-fiftieth and one-twentieth) were loaded side-by-side. From these data, we estimate that 15% of hTPO molecules can be detected at the surface by this method, and this is probably an underestimate (see text). Moreover, the population of surface-biotinylated TPO is largely sensitive to digestion with endoglycosidase H, in agreement with published reports (39).

When hTPO₉₃₃ surface expression was examined by immunofluorescence in nonpermeabilized clonal cells, it was again surprising that not all cells in the population showed the same pattern. Specifically, about half the nonpermeabilized cells were bright while the other half exhibited no surface fluorescence (Fig. 10). Importantly, this pattern was observed in each of the three expressing clones. The surface hTPO appeared to be distributed in a dense punctate pattern (Fig. 11). Thus, although the frequency of surface-positive hTPO₉₃₃ expression in stable clones of PC Cl3 thyrocytes (~50%) was not as high as in transiently transfected cells (where it appeared to approach 100%, see Fig. 8), it was clearly much higher than in stably transfected MDCK cells (<10%, see Figs. 4 and 5). Moreover, the absence of surface immunofluorescence from the other portion of PC Cl3 cells was not due to a lack of hTPO₉₃₃ protein expression, as every cell in the population in these clones was positive for hTPO after permeabilization (Fig. 11, right panels), while immunofluorescence could not be detected in untransfected, permeabilized PC Cl3 cells (data not shown). Permeabilization primarily increased the fraction of positive cells rather than the immunofluorescence intensity of the cells. Nevertheless, in the permeabilized cells, an ER localization pattern was also suggested in addition to the surface fluorescence (Fig. 11). Together, these data clearly indicate a markedly increased distribution of recombinant hTPO₉₃₃ to the cell surface in thyrocytes versus other stably transfected cells that have been studied to date.

View larger version (65K): [in this window] [in a new window]   Fig. 10.   Heterogeneity of cell surface expression of hTPO933 in stably transfected PC Cl3 cells. The upper panel shows an island of transfected PC Cl3 cells (clone B6) by phase-contrast microscopy. In the middle panel, the same cells were processed for immunofluorescence (I.F.) with anti-hTPO under nonpermeabilized conditions. The lower panel is a merged view. In multiple such samplings, approximately half the cells are strongly positive for cell surface immunofluorescence under nonpermeabilized conditions.

View larger version (116K): [in this window] [in a new window]   Fig. 11.   Survey of hTPO933 expression in PC Cl3 cells. The cells were processed for immunofluorescence with anti-hTPO under nonpermeabilized or permeabilized conditions. Upper panels, clone C4. Lower panels, clone C5. Note that both of the left-sided fields each show one continuous island of cells. The discontinuities of immunofluorescence in these islands represent cells with little or no surface expression of hTPO933; in multiple such samplings, approximately half the cells in these clones are strongly positive for cell surface immunofluorescence under nonpermeabilized conditions. However, in the right-sided fields, all PC Cl3 cells are positive for hTPO933 expression; discontinuities of fluorescence reflect only the extent to which the cells are subconfluent.

Finally, we examined surface biotinylation of hTPO933 in the stably transfected PC Cl3 cells. As quantitated from the data in Fig. 9B, only ~15% of the hTPO₉₃₃ could be surface-tagged in this experiment. In several other experiments (data not shown), the surface biotinylated fraction did not even achieve this level (i.e. <10%) despite the much higher surface expression that was apparent by immunofluorescence in these cells (Fig. 11) versus other cells (Fig. 5). This suggested the possibility that biotinylation might not efficiently tag all the surface hTPO₉₃₃ molecules. Further, when the surface-tagged subpopulation of hTPO₉₃₃ from PC Cl3 thyrocytes were examined by endoglycosidase digestion (Fig. 9B), all of the molecules were digested by PNGase F (indicating that they all had been N-glycosylated, lane 1) but most of the surface biotinylated molecules (~75%) were also digested with endoglycosidase H (indicating lack of conversion to complex carbohydrates upon passage through the Golgi complex). Thus, although both cell surface biotinylation and endoglycosidase H resistance assays have been validated in many cases, the ability of these assays to serve as valid reporters cannot be assumed for all surface glycoproteins (see "Discussion"). Indeed, the data suggest that both asssays under-represent the extent of hTPO₉₃₃ delivery to the cell surface, presumably because protein folding limits both Golgi carbohydrate modifications and the accessibility of lysines in the extracellular domain.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TPO has been a protein of special interest to thyroid biologists for the past 20 years. At this point, the evidence is overwhelming that this type 1 membrane protein is delivered to the apical plasma membrane of thyrocytes via the secretory pathway, and a an abnormal subcellular distribution of mutant hTPO may be associated with congenital hypothyroid goiter (42, 46). However, the fraction of cellular TPO that normally resides on the plasma membrane (a fraction that is clearly regulated by cell type-specific factors, including thyroid-stimulating hormone stimulation (see Refs. 18-24)) has been an area of confusion. Several technical limitations have compromised the analysis of hTPO trafficking. First, TPO may be modified by Golgi processing enzymes in some systems (Ref. 26 and Fig. 7B), not in others (38-40), and only partially modified in still other systems (Fig. 9B). Indeed, after TPO folding, it is not clear that the glycans themselves are needed for enzymatic activity or immunoreactivity, although it is difficult to deglycosylate the native protein which suggests that the glycans are relatively inaccessible in the tertiary structure (38, 47-49). Second, most antisera used to examine the intracellular distribution of thyroidal TPO (e.g. by immunofluorescence) are derived from patients who may have antibodies that simultaneously recognize other thyroid antigens.

Some of these problems may be circumvented by examining the expression of recombinant hTPO in heterologous cell types. However, there are additional technical limitations that must be considered. Notably, cell surface biotinylation requires that the protein tertiary structure allows lysines in the extracellular domain to be freely accessible to the reactive N-hydroxysuccinimidyl group (50). It cannot automatically be assumed that all membrane proteins under standard conditions will exhibit equally such reactivity (51), especially proteins like hTPO that have a complex tertiary structure (52). Indeed, in pilot studies in our laboratory (data not shown), we found that when hTPO₉₃₃-expressing cells were lysed before biotinylation, the efficiency of hTPO₉₃₃ recovery by precipitation with avidin-agarose varied between experiments in a manner that appeared to depend upon detergent conditions, suggesting that the native conformation of the hTPO₉₃₃ protein may limit its biotinylation efficiency. As a consequence, we have been careful to independently examine each of our cell culture systems by immunofluorescence with anti-hTPO under nonpermeabilized and permeabilized conditions. From a combination of these analyses, we now report that there are major cell type-dependent differences in the handling of the same hTPO construct in different stably transfected mammalian cell types. Further, these differences appear to follow as a direct consequence of heterogeneous cell surface expression that occurs even within a clonal population in which all cells are positively expressing hTPO as measured under permeabilized conditions (Figs. 4, 5, and 11).

First, in MDCK cells, we conclude that the hTPO₉₃₃ that can be biotinylated at the cell surface shows a predominantly apically polarized distribution; however, most hTPO₉₃₃ cannot be biotinylated and does not acquire endoglycosidase H resistance (Fig. 3). Instead, cell surface hTPO₉₃₃ in MDCK cell clones comes from a small fraction of cells that exhibit a relatively high level of surface expression (Fig. 4) while the remaining hTPO₉₃₃-positive cells express little or none at the cell surface (Fig. 5).

Second, in CHO cells, we conclude that the observations recently reported from one stable clone (26-29) are not representative of the very high hTPO trafficking to the cell surface that is initially obtained from these cells (Ref. 42 and Fig. 8). The data indicate that the problem is not a defect of protein expression or an intrinsic defect in the secretory pathway to accommodate hTPO. Rather, the data suggest that upon growth and expansion of hTPO₉₃₃-positive clones, an evolution in the cell population takes place such that many (but perhaps not all (see Refs. 31 and 32)) clones can be isolated in which hTPO fails to be efficiently delivered to the cell surface (26-29). This would be consistent with a tendency toward morphological changes in the cells (Fig. 8) and a growth disadvantage for cells in the population that exhibit high cell surface hTPO delivery. Indeed, nearly 10 years ago, McLachlan and Rapoport (36) postulated that there might be a growth disadvantage for certain cell cultures that externalize recombinant hTPO. We believe that the present observations support such an interpretation. Certainly, these data raise questions about (a) whether an individual clone can be considered representative of a population of transfected cells, and more importantly, (b) whether results from such clones can be used to reflect physiological mechanisms of hTPO regulation, such as targeting for intracellular degradation (26-29).

Finally, from our observations in clonal PC Cl3 cells, we conclude that the (rat) thyroid lineage may be more resistant to loss of cell surface hTPO₉₃₃ expression over time than are MDCK or CHO cells. Thus, thyrocytes may be to some extent growth-adapted to surface TPO expression. Nevertheless, even with continuous antibiotic selection pressure, and persistent hTPO expression in the entire population of permeabilized cells (Fig. 11), only about half the cells in our clones exhibited detectable surface staining at a moment in time in the steady state (Fig. 10). In this context, it is interesting to note a recent report that by immunofluorescence in thyrocytes after 18 days in primary culture, 5% of TPO is detected at the plasma membrane (24). Although such prolonged primary thyrocyte culture has not been extensively characterized, almost certainly these cells have an extremely low mitotic index. More study is needed to determine (a) whether the same fraction of surface hTPO₉₃₃ can be maintained in a clonal population (such as the PC Cl3 thyrocytes we have described), even after extensive passaging (e.g. for a year or more); (b) whether hTPO surface expression in thyrocytes might appear at different stages of the cell cycle, in which case, conceivably, all cells in the PC Cl3 line (e.g. Figs. 10 and 11) might exhibit surface expression at different times; (c) whether endogenous rTPO has a similar distribution in PC Cl3 cells (no good antibody to rTPO currently exists); and (d) whether surface expression of bioactive hTPO is selectively inhibitory to any particular stage of the cell cycle or whether it induces cell death.

Taken together, these results highlight previously unappreciated complexity in the intracellular transport of hTPO in selected cell culture systems, and suggest that cell type-specific factors may stabilize plasma membrane expression of hTPO in thyrocytes.

	ACKNOWLEDGEMENTS

We thank Dr. A. Taurog for helpful discussions and for transferring aliquots of anti-hTPO prepared by Dr. Paul Banga. We thank members of the Arvan laboratory for providing important teaching and advice in cell surface biotinylation, transfection, and immunoblotting. We thank members of the laboratory of Dr. A. Chang (Albert Einstein College of Medicine, Bronx, NY) for help with immunofluorescence microscopy.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant DK 40344 (to P. A.).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.

^¶ To whom correspondence should be addressed: Div. of Endocrinology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-8685; Fax: 718-430-8557; E-mail: arvan@aecom.yu.edu.

Published, JBC Papers in Press, August 2, 2000, DOI 10.1074/jbc.M003559200

	ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; MDCK, Madin-Darby canine kidney; PBS, phosphate-buffered saline; TPO, thyroid peroxidase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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