Effects of the N-terminal cysteine mutation on prolactin expression and secretion in transfected cells

Molecular and Cellular Endocrinology

117 (1996) 59973

Effects of the N-terminal cysteine mutation on prolactin and secretion in transfected cells A. Morin*,

R. Picart,

expression

A. Tixier-Vidal

Coii+~ de France, Groupe de Neuroendocrinologie et Neurobiologie Cellulaires et Mokcubires,

URA 11 I.5 CNRS.

F-75231 Paris Cedez 05 France

Received 18 September 1995; accepted 15 November 1995

Abstract We developed an experimental cell model to look for motif(s) of rat PRL sequence encoding a sorting signal to secretory granules. An efficient expression vector (pCMV-rPRL) was used to transfect several eukaryotic cell lines in culture, i.e., one neuronal cell line (C6) and three glandular pituitary derived cell lines (AtT20, GC, GH3CDL). Despite the ubiquitous transcription of pCMV-rPRL, the synthesis and secretion of rPRL were detected primarily in GH3CDL cells that derived from lactotrophs, suggesting a cell-specific post-transcriptional control of rPRL expression. During transient expression, exogenous native PRL was transported through intracellular compartments of the secretory pathway and underwent regulated release. Abolition by mutagenesis (C4S) of the N-terminal disulfide bond increased by 50% the PRL secretion rate (medium to cell ratio) and multiplied by 5 the specific activity of medium PRL from pulse-labeled cells. These results support the hypothesis that N-terminal disulfide bond plays a role in the control of PRL intracellular transit and storage. Keywords: Prolactin; Expression; Secretion; Transfection;

Mutagenesis; Disulfide bridge

1. Introduction Prolactin (PRL) is a multifunctional polypeptide hormone secreted by specialized endocrine cells within the anterior pituitary gland. In these cells, as in other endocrine cells, PRL is synthesized, processed and transported through successive compartments of the endoplasmic reticulum (rough endoplasmic reticulum, Golgi subcompartments) and finally stored in dense core secretory granules before being released in response to specific extracellular signals (Tixier-Vidal and Picart, 1967; Farquhar et al., 1978, Tougard et al., 1980, 1982). Thus PRL follows a regulated secretory pathway (Kelly, 1985). However, previous works (Morin et al., 1984a,b) have suggested the existence of several intracellular routes for the release of the hormone (see review Tougard and Tixier-Vidal, 1994). As with other peptide hormones, PRL was shown to

* Corresponding author, Laboratoire d’Enzymologie, Centre National de la Recherche Scientifique, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex. France. Tel.: 33 I 69823498; Fax: 33 1 69823129.

be highly concentrated in secretory granules. Biochemical (Zanini et al., 1980) as well as immunocytochemical (Tougard et al., 1983, 1989) studies have shown that the secretory granules of lactotropes contain a large proportion of PRL (85%), in addition to other components of the secretory granule matrix, namely secretogranin I and II which belong to a family of acidic sulfated proteins: the granins (Huttner et al., 1991). PRL secretory granules are known to be formed in a specialized post-Golgi compartment - the trans-Golgi network (TGN) (Griffith and Simon, 1986) - by budding of immature secretory granules which give rise to mature secretory granules characterized by an electron dense homogenous content. The mechanisms by which PRL is concentrated and sorted in secretory granules are not yet resolved. Two hypotheses have been proposed to explain concentration and sorting events in regulated secretory cells: receptor mediated sorting versus aggregation mediated sorting. The prevalent view is the passive aggregation model which received experimental support from studies performed in various cell types including a rat

0303-7207/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved SSDl0303-7207(95)03730-N

60

splicing

region

poly

A+ signal

pCMV(T7/Neo)-rPRL

splicing

Fig.

I.

Main

modifications

clcmenta

of the rat PRL

indicated in Materials

under the transcriptional

cxpresGon

and methods.

control of cytomegalovirus

transcripts are I .9 kb in length and vector-derived whereas cellular rPRL

messenger RNA

are

I

vector.

pCMV-rPRL

bettor

IS originally

region

derived

from

It contains finally the rat PRL coding sequence (823.bp (CMV)

enhancer-promoter

rPRL-RNA

sequence. As calculated

pMAM-neo

cDNA

(Clontech)

with

the

cloned by Cooke et al. (1980))

from the restriction map. rPRL

are expected to be 2 kb (or more) after lengthening

by polyadenylation

primary process.

hb in length onI>.

pituitary mammosomatotrope cell line (GH4Cl). In these cells, granins were found to spontaneously aggregate in the TGN due to a decrease in pH and an increase in calcium concentration, in conjunction with their specific physical properties (Chanat and Huttner, 1991). Additional mechanisms such as homophilic and heterophilic protein interactions and binding to membrane components are likely to participate in the formation of mature secretory granules (Arvan and Castle, 1992; Tooze et al., 1993). As far as PRL is concerned. information is still lacking about the role of its structural features in intracellular sorting and transport. PRL belongs to a family including growth hormone (GH) and placental lactogen (PL), that exhibit common structural and biological features. When aligned to optimize the homology of the peptidic sequences, four cysteine residues located at positions 56, 172, 189 and 197 are found to be invariably conserved among all PRLs, GHs and PLs, whereas two cysteine residues located at positions 4 and 9 (C4 and C9) are conserved among the PRLs only. These different degrees of conservation suggest a relative importance of these cysteines in the biological functions of proteins (Nicoll et al., 1986), as well as in their subcellular targeting and destiny within the secretory pathway. In the present work, we developed a model of transfected cells to analyse by an approach of in vitro mutagenesis, the importance of the N-terminal disulfide bond, which is specific to PRL. in its intracellular transport within the lactotropes. For that purpose. we carried out the construction of a eukaryotic expression vector (pCMV-rPRL) and we investigated several mammalian cell lines in culture (C6. AtT20, GC, GH3CDL)

as host cells. for transfection experiments leading to an efficient expression of rat PRL. Only the GH3CDL cells. derived from the mammosomatotrope GH3B6 line and which secrete a very low level of endogenous PRL. allowed us to study the subcellular distribution and the regulation of the secretion of the vector-derived (exogenous) PRL. As expected, the exogenous native PRL displayed a similar behavior with the endogenous native rat PRL, suggesting that it was intracellularly transported through the same pathway and released by the same mechanisms. Using in vitro site-specific mutagenesis to modify the PRL coding sequence within pCMV-rPRL vector, in transfected GH3CDL cells, we have obtained experimental evidence for a role of the N-terminal cysteine-4 in the control of intracellular PRL transport and storage.

2. Materials and methods 2. I. Muterids

Restriction and DNA modifying enzymes were either from Gibco-BRL (Cergy-Pontoise, France) or from Biolabs (Beverly, MA, USA). pMAM-neo expression vector was obtained from Clontech Laboratories, Inc. (Palo Alto, CA, USA). The oligonucleotides were either from Eurogentec SA (Liege. Belgium) or from Appligene (Illkirch, France). The DNA sequencing kit and “P-rdATP were purchased from Amersham (Les Ulis, France). Culture media and sera were from Gibco-BRL (Cergy-Pontoise. France). Forskolin, TPA (phorbol 12.13-didecanoate), NP40 (Nonidet P40), DOC (deoxy-

61

A. Morin et al. / Molecular and Cellular Endocrino1og.v I I7 (1996) 59- 73

16 24 48 72 -

d

Fig. 2. Transcription of pCMV-rPRL vector pCMV-rPRL and taken 6, 16, 24, 48 or 72 somatotropes, GH3CDL lactotrope variant onto GeneScreen membrane and hybridized ( -) Negative control: 20 pg of total RNA vector-derived PRL transcripts.

in transfected cells. Northern blot analysis of total cellular RNA extracted from cells tr ‘an sfected with h after the electroporation, as indicated. For every cell line, C6 glial cells, AtT20 corticotropes, GC cells: 20 pg samples of glyoxalated RNA were submitted to agarose gel electrophoresis, transfered with “P-labeled rPRL-cDNA. ( + ) Positive control: 5 pg of total RNA from GH3B6 lactotropes. from cells transfected with pMAM-neo. Arrow indicates 1 kb rPRL-mRNA. Arrowhead indicates

cholic acid), SDS (sodium dodecyl sulfate) and DTT (dithiothreitol) were obtained from Sigma Chimie (St Quentin-Fallavier, France). Aprotinin, PMSF (phenylmethylsulfonyl fluoride), Leupeptin and pepstatin were purchased from Boehringer Mannheim (Meylan, France). Tran3’S-label was obtained from ICN Biomedicals, Inc. (Orsay, France). Protein A-Sepharose (CL4B) was from Pharmacia-LKB (St Quentin-Yvelines, France). Immunoreagents were purchased from Biosys (Compiegne, France). Transfer nitrocellulose membrane (BAS 83) was from Schleicher & Schuell (CERA-LABO, Ecquevilly, France). The RNase-free DNase RQl was from Promega Corporation (Madison, WI, USA). 2.2. Recombinant DNA construction and mutagenesis The 823-bp cDNA cloned by Cooke et al. (1980) including the entire coding sequence for rat PRL (rPRL), its signal peptide, and parts of the 5’- and 3’-untranslated of the mRNA, have been first subcloned into the multiple cloning site of pMAM-neo mammalian expression vector. The recombinant vector (pMAM-rPRL) was found not to have enough efficient expression so that it was modified as follows: the fragment including the RSV-LTR linked to the dexamethasone-inducible MMTV-LTR was removed and replaced by a CMV promoter and enhancer sequence linked to the bacteriophage T7 promoter, finally, to obtain the expression vector: pCMV-rPRL (Fig. 1).

In vitro site-specific mutagenesis was applied directly on pCMV-rPRL expression vector according to the ‘USE method’ described by Deng and Nickoloff (1992). Briefly, double-stranded vector was heat denatured and two mutagenic primers were then annealed. Thus, through primer-directed DNA synthesis by T7 DNA polymerase, the first ‘USE primer’ (S’-CGTCTTCAAGATATCCTTTGCCT-3’) eliminates a unique EcoRI site of pCMV-rPRL vector by modifying it into a EcoRV site, and the second ‘mutagenic primer’ (5’CGCCACCAGAAGAGACTGGCAGGG-3’) substitutes the cysteine-4 codon of rPRL coding sequence to a serine codon (C4S mutation). Transformant clones were screened for the EcoRI/EcoRV change of DNA vector and finally, the C4S mutation was confirmed by sequencing with the dideoxy chain termination method of Sanger et al. (1977) using a T7-promoter primer (5’-TAATACGACTCACTATAGGGAGA-3’) and ‘Multiwell microtitre plate DNA sequencing system with T7 DNA polymerase’ (Amersham). Both native pCMV-rPRL and mutated pCMVC4S vectors were prepared in large scale and purified by equilibrium centrifugation in cesium chloride-ethidium bromide continuous gradient (Sambrook et al., 1989). 2.3. Cell cultures and transfection The mouse glioma C6 cells were grown in Ham’s FlO medium containing 10% fetal calf serum, in 10% CO, in

air. The corticotrope AtT20 line derived from a mouse pituitary tumor was grown in I: I mixture of DMEM and Ham’s F12 media supplemented by 10% fetal calf serum, in IO”/0 CO? in air. The somatotrope GC line derived from a rat anterior pituitary tumor was grown in Ham’s F12 medium containing 15% heat-inactivated horse serum and 2.5% fetal calf serum, in 5% CO, in air. The rat pituitary GHSCDL cells, a stable variant of the mammosomatotrope GH3 line (Brunet et al., 1977). were maintained in Ham’s F12 medium supplemented with charcoal-dextran stripped sera (18% horse serum and 3% fetal calf serum), in 5% CO, in air. For transfection by electroporation, subconfluent cultures were harvested by mild trypsinization and washed twice with serum-free medium. Cell suspension (25 x lo6 cells in 0.8 ml serum-free medium) was mixed with supercoiled plasmid DNA (30 ,~g of either expression vector and 10 pg of pCMV-/?Gal used as an internal control for transfection efficiency) and exposed to a single 1800 PF and 250 V/4 mm discharge delivered by an ‘Electropore 2000’ apparatus (Eurogentec, Liege, Belgium). An equal volume (0.8 ml) of culture medium containing 2 x -concentration of serum was immediately added to the cell suspension. Then, transfected cells were transferred into tissue culture dishes and maintained under culture conditions appropriate to each cell line for the indicated duration. Despite the presence of a dominant selectable marker (NeoR) in the expression vector, attempts to clone GH3CDL cells transfected with pCMV-rPRL using the G418 selection method, led to a low level of rPRL secretion. Thus the analysis of rPRL expression and secretion were performed in transient expression experiments, using a similar batch of transfected cells for comparative studies (effects of secretagogues).

-7.4. Anui~~sis of’ rPRL

2.4.2 Protein level Both intracellular and culture medium rPRL were measured either by radioimmunoassay (RIA) using an rPRL reference preparation (RP3 kindly provided by the NIADDK) as standard and “‘I-labeled rPRL as tracer, or by enzyme immunoassay (EIA) using the same rPRL standard and rPRL-RP3 covalently linked to acetylcholinesterase as tracer (Duhau et al.. 1991). The rabbit antiserum against purified rPRL (kindly provided by the NIADDK) used in both these assays (diluted to l/24000) was obtained by D. Grouselle in our laboratory and its specificity was previously tested (Morin et al.. 1994a). The assay sensitivity was 200 and 50 pg of rPRL by sample test, in RIA and EIA, respectively. The intracellular distribution of rPRL in transfected cells was analyzed in situ by immunofluorescence (IFL). and at the electron microscope level by immunocytochemistry (ICE), 20 or 40 h after the electroporation. The same rabbit antiserum against rPRL as for RIA and EIA was used for both procedures (diluted to I /300).

The transfected cells were fixed with formaldehyde, permeabilized with saponin and intracellular rPRL was detected by IFL using goat immunoglobulins (IgG) against rabbit IgG (GAR) labeled with rhodamine (TRITC) as previously described (Tougard et al., 1982). 2.4.4. Electron microscopr procedures The transfected cells were fixed in situ either for conventional ultrastructural study or for electron microscopic ICE using immunoperoxidase localization of intracellular rPRL according to Tougard et al. (1980, 1982).

espressiorl

The transient expression of rPRL was studied from 6 up to 72 h after the electroporation, at both the mRNA and the protein levels, in parallel with four eukaryotic cell lines (C6, AtT20, GC or GH3CDL) transfected by pMAM-neo (as control vector) or by pCMV-rPRL.

Total cellular RNA were prepared as described by Cathala et al. (1983) then further digested by RNasefree DNase RQl to eliminate plasmid DNA contamination, and quantified by ethidium bromide fluorescence. Total cellular RNA purified from transfected cells were analyzed by Northern blotting as previously described (Laverriere et al.. 1983) using rPRL-cDNA (3’P-rdATP labeled by nick-translation) as a probe to detect the specific transcripts (rPRL-RNA).

The regulation of rPRL secretion was further studied only in GH3CDL cells transfected in parallel with control vector (pMAM-neo) and with either expression vector (pCMV-rPRL or pCMV-C4S). 2.5.1. Ejkts oj’ secretugogues The culture medium was renewed 20 h after the electroporation. After 40 h, transfected cells were washed twice and preincubated for 30 min in Ham’s F12 serum-free medium. Then, cells were further incubated for 30 min in control medium (Ham’s F12) or in Ham’s F12 containing either depolarizing potassium (30 mM KC]), or phorbol ester (16 nM or 1.6 ,uM TPA), or Forskolin (1 or 10 ,uM Fk). At the end of incubations. the culture media were clarified by centrifugation to remove cell fragments and stored at - 30°C. Cell extracts were prepared using non-ionic

A. Morin cl al. i Molecular and Cellular Endocrinology 117 (1996) 59- 73

detergents (0.5% NP40 and 0.5% DOC), as previously described by Green and Shields (1984), and stored at - 20°C. Total cellular proteins contained in the cell extracts were quantified using Coomassie blue dye reagent (BioRad SA, Ivry-sur-Seine, France) according to Bradford (1976). 2.5.2. Metabolic labeling Twenty-eight hours after the electroporation, transfected cells were washed twice and preincubated for 10 min in methionine-free and cysteine-free Ham’s F12 medium. Then, cells were pulse-labeled for 20 min in the same medium containing 200 LlCi/ml of 35S-methionine and 35S-cysteine mixture (Tran3?j-label). Chase incubations were performed by washing the cells three times in Ham’s F12 medium supplemented with a large excess of unlabeled methionine (200 PM) and cysteine (250 PM), and then culturing for 40 min in the same medium containing or not secretagogues (30 mM KC1 and 1.6 p M TPA). At the end of the chase, media were clarified and cell extracts were performed as described above. 2.5.3. Immunoprecipitation Thb procedure for indirect immunoprecipitation was adapted from Ledbetter et al. (1985). Briefly, three batches of protein A-Sepharose beads were prepared according to the manufacturer, resuspended in IP buffer (PBS; containing 0.1% BSA, 0.5% NP40 and 0.5% DOG and incubated at 4°C for 1 h, with normal rabbit seru4 (NRS) diluted to l/20 (batch A), or NRS diluted to lh0 (batch B), or with rabbit antiserum against rPRL! (the same antiserum as for RIA, and EIA) diluted to l/40 (batch C). We determined the antiserum dilution to ensure a large excess of specific antibodies and so the completeness of immunoprecipitation in all samples. NRS-beads of batch A were used to pre-adsorb the samples (cell extract or medium) and thus reduce background. The pre-adsorbed samples were then incubated at 4°C for 2 h, with either batch B or batch C. Finally, Sepharose beads were washed extensively: once in IP buffer, three times in IP buffer without BSA containing 1 M NaCl and finally, four times in IP buffer without BSA. The immunoprecipitated proteins were eluted from the Sepharose beads by boiling for 3 min in electrophoresis buffer (Laemmli, 1970) containing 4% SDS and 100 mM DTT, and further reduced by incubating at 50°C for 1 h. The eluted proteins were then analyzed by electrophoresis and Western blotting (see below). The 35S-labeled proteins were quantified by counting in scintillation fluid (Aquasol, DuPont-NEN, Les Ulis, France). For each sample, the amount of immunoprecipitated 35S-rPRL was calculated by subtracting background (radioactivity associated with beads of batch B) from radioactivity associated with beads of batch C.

25.4.

63

Electrophoresis and Western blotting

The immunoprecipitated proteins were separated by electrophoresis on 13% polyacrylamide slab gels using the discontinuous buffer system of Laemmli (1970). The proteins were then transferred onto nitfocellulose membranes using a semi-dry electroblotting apparatus (Multiphor II Novablot, Pharmacia) as described by the manufacturer. The transfer-membranes were blocked in TBST (10 mM Tris-HCl pH 7.6, 150 mM NaCl and 0.2% Tween 20) containing 5% non-fat powdered milk for 30-60 min at room temperature (RT). They were subsequently probed with the same antiserum against rPRL as for IP, diluted to l/500 in TBST containing 0.5% milk, for 90 min at RT and washed extensively. Then, they were inchbated with anti-rabbit IgG alkaline phosphatase conjugate for 60 min in the same buffer. After extensive washes, the chromogenic reaction was developed using NBT (nitro blue tetrazolium) and BCIP (5-bromo-4-chloro-3-indolylphosphate) substrates according to the manufacturer’s procedure (Promega Corp., Madison, WI, USA). The 35S-labeled proteins transferred onto the nitrocellulose membranes were detected by autoradiography using X-Omat AR X-ray films (Kodak, Rochester, NY, USA).

3. Results 3.1. Choice of the host cells

Mouse glioma C6 cells and both mouse AtT20 and rat GC pituitary cells were selected as host cells for transfection with pCMV-rPRL vector because they do not express rat PRL gene. In addition, AtT20 and GC cells have been shown to possess a well-characterized regulated secretory pathway and AtT20 cells have been used extensively in transfection experiments to study intracellular trafficking and processing of several secretory proteins such as insulin (Quinn et al., 1991; Ferber et al., 1991), POMC (Noel et al., 1991), and ELH (Jung et al., 1993). GH3CDL cells were selected because they expressed a very low level of rPRL mRNA and produce minute amount of rPRL (Laverriere et al., 1986). 3.2. Transcription of pCMV-rPRL

vector in

transfected cells

From the respective positions of CMV promoter and SV40 polyadenylation signal within pCMV-rPRL expression vector (Fig. l), the size of exogenous PRL transcripts in transfected cells was anticipated to be larger ( - 1.9 kb) than the size of native rPRL-mRNA ( w 1 kb) in lactotrophs. The Northern blot analysis of total cellular RNA (Fig. 2) showed the appearance of PRL-specific RNA of the expected size in C6, AtT20

16H

Incubation time

24H

30H

48H

72H

48H

72H

Incubation time

24H

Incubation time

30H

Incubation time

Fig. 3. Induction of rPRL secretion in transfected cells. Kinetic analysis of rPRL secretion after transfection with pCMV-rPRL of C6. AtT30, CC and GH3CDL cells. The amount of rPRL released in the culture medium was measured by RIA. 6. 16, 24, 30. 48 or 73 h after the electroporation. in the same dishes as used for RNA cell extraction (see Fig. 2). The results are expressed in ng of rPRL per culture dish and are the mean of triplicate determinations i SD. After transfection with pMAM-neo. no rPRL secretion was detected in C6. AtT20 or CC cells, whereas GH3CDL cells secreted low levels of endogenous rPRL. When GH3CDL cells were transfected with pCMV-rPRL. they secreted higher levels of rPRL and the exogenous secretion of vector-derived rPRL (induced) was calculated by subtraction.

and GC cells after cell transfection with pCMV-rPRL (but not with pMAM-neo used as a ‘mock’ vector without rPRL sequence). The concentration of exogenous PRL transcripts was roughly unchanged from 6 up to 72 h in GC cells, whereas it decreased in C6 and AtT20 cells. In GH3CDL cells, the endogenous (1 kb) rPRL-mRNA was observed in cells transfected with either vector, whereas additional 1.9-kb PRL-RNA was only present after the transfection with pCMV-rPRL. As in the other cell types, the exogenous PRL transcripts were detected from 6 h in GH3CDL cells. Its concentration was maximum at 16 h, then slightly

decreased 72 h.

at 24 h and remained

roughly

constant

up to

3.3. Induction of’ sPRL secretion in trunsfected cells In C6. AtT20 and GC cell lines transfected with pMAM-neo, no rPRL could be detected (RIA or EIA) in both cell extracts and culture media. The induction of PRL secretion was closely related to the appearance of exogenous PRL transcripts after electroporation with pCMV-rPRL. Nevertheless, the amounts of PRL released in the culture media greatly differed depending

A. Morin et al. 1 Molecular and Cellular Endocrinology II 7 (1996) 59-73

65

Fig. 4. Immunolocahzation of rPRL within transfected GH3CDL cells. The intracellular rPRL was detected in situ by immunofluorescence 40 h after the electroporation: GH3CDL transfected with pMAM-neo (A) or with pCMV-rPRL (B). Cells were fixed with paraformaldehyde, permeabilized with saponine and immunostained using rabbit antiserum against rPRL and goat anti-rabbit IgG labeled with rhodamine (see Materials and methods). PRL immunoreactivity was predominantly localized in a juxta-nuclear Golgi-like region (arrowhead) in both transfected cells, but was found in a larger number of cells in (A) than in (B). One can notice a punctate distribution of PRL immunoreactivity in the peripheral cytoplasm of cells (arrow) in (B); (magnification: x 125).

on cell lines (Fig. 3). The non-pituitary C6 cells displayed the lowest level of PRL secretion ( - 10 ng/mg cell protein/48 h). The two non-lactotrope pituitary lines, i.e., AtT20 and GC cells, showed rather moderate PRL secretion ( - 40 ng/mg cell protein/48 h). As expected, GH3CDL cells transfected with pMAM-neo (Fig. 3, black bars) displayed a very low level of PRL secretion ( - 20 ng/mg cell protein/48 h) which was comparable to that of non-transfected GHXDL cells (Brunet et al., 1977). This was the endogenous PRL secretion. In contrast, GH3CDL cells transfected with pCMV-rPRL secreted about ten-fold larger amounts of PRL ( - 200 ng/mg cell protein/48 h) that corresponded to both endogenous and exogenous PRL secretion. Thus, the vector-derived PRL secretion was further calculated by subtraction (Fig. 3, grey bars). Taken together, these results suggest the existence of a cell-specific control of rPRL gene expression, most probably at a post-transcriptional level, considering that all cell types were transfected with the same expres-

sion vector (pCMV-rPRL). In addition, cells were transfected at equal efficiency since cotransfection with pCMV-PGal vector indicated a same level of the reporter gene expression (data not shown). 3.4. Immunolocalization of intracellular rPRL The in situ detection of intracellular PRL by immunofluorescence (Fig. 4) pointed out the large increase of the percentage of immunoreactive cells, 20-40 h after the electroporation of GH3CDL cells with pCMVrPRL (up to 90-95%), whereas there was no change after the transfection with pMAM-neo (5-10%). After transfection with either vector, PRL immunoreactivity was mainly concentrated in a juxta-nuclear Golgi-like position (Fig. 4A and B) within all the immunoreactive cells. In addition, a punctate PRL immunoreactivity was observed in the cytoplasm of some pCMV-rPRL transfected cells (Fig. 4B). Using the same procedure, intracellular PRL was not detectable in the other host cells tested: i.e., C6, AtT20 and GC cells (not shown).

66

A.

btori?lof 01.i hioiecuiur mci Cellular Endocrinology I1 7 (1996) 59- 7.1

At the electron microscope level, the immunodetection of PRL indicated that in pCMV-rPRL transfected

cells, intracellular PRL was distributed in the same subcellular compartments as in parent GH3B6 cells, that is Golgi stacks and a few small secretory granules and immunoreactive vesicles throughout the cytoplasm and near the plasma membrane (Fig. 5). 3.5. Western blot analysis of rPRL expressed in transfected GH3CDL cells Western blot analysis of immunoprecipitated material (Fig. 6) from culture medium and cell extracts of GH3CDL cells, 28 h after transfection with either expression vector (pMAM-neo, pCMV-rPRL), revealed the presence of only one specific band which co-migrates with the NIH rat PRL standard (23 kDa) and some weaker unspecific bands which correspond to serum components since they were observed with both normal serum (ns) and A-PRL specific antiserum (sa) (see Fig. 6). Moreover, the intensity of the specific immunoreactive band in each sample correlated with the concentration of rPRL as determined by EIA (see below). Thus, vector-derived PRL (both intracellular and secreted proteins) was well recognized by the rPRL-specific antiserum and migrated on PAGE as endogenous 23-kDa rPRL. The autoradiography of immunoblot membranes revealed one main labeled protein in chase medium that again corresponded to immunoreactive 23-kDa rPRL (Fig. 6). The density of this band correlated with the amount of radioactivity immunoprecipitated in each sample (see below). In cell extracts, the major part of the radioactivity also fitted with 23-kDa 35%PRL (Fig. 6). Scattered bands were also revealed by autoradiography in both control (ns) and specific (sa) immunoprecipitates. This unspecific radioactivity was subtracted from the total to calculate the radioactivity incorporated into ‘%-PRL. 3.6. Regulation of rPRL release by transfected GH3CDL

Fig. 5. Immunoelectron microscopic localization of rPRL within transfected GH3CDL cells. lmmunoperoxidase detection of intracellular rPRL at the electron microscope level, 40 h after the electroporation: GH3CDL cells transfected with pCMV-rPRL were fixed with paraformaldehyde, permeabilized with saponine and then, immunocytochemically stained using rabbit antiserum against rPRL and sheep anti-rabbit IgG conjugated with peroxidase. The immunoreactive material is distributed among membrane-bound intracellular compartments, that is the Golgi cisternae (G) and vesicles (arrow), as well as in small secretory granules (arrowhead) which are located in the Golgi zone and beneath the plasma membrane: (magnification: x 24 000).

cells

The regulation of PRL release was further studied in GH3CDL cells through transient expression experiments, 40 h after the electroporation. Various factors were tested in parallel which stimulates the short-term (30 min) release of PRL via distinct intracellular mechanisms: depolarizing potassium (30 mM KCI) that involves calcium ion influx (Wang et al., 1992) Forskolin ( 1- 10 ,uM) that increases the intracellular cyclic AMP (Thompson et al., 1992) and phorbol ester TPA ( 16 nM or 1.6 PM) that activates the protein kinase C pathway (Laverriere et al., 1988). All of these factors stimulated the short-term release of both exogenous PRL and endogenous PRL (Fig. 7). However, the percentage of stimulation by every factor was 2-4times larger for endogenous PRL than for exogenous PRL.

A. Morin er al. / Molecular and Cellular Endocrinology II 7 (1996) 59- 73

67

Fig. 6. Western blot analysis of rPRL expressed in transfected GH3CDL cells. Culture medium and cell extracts from GH3CDL transfected with pMAM-neo (MAM), pCMV-rPRL (PRL) or pCMV-C4S (C4S) were immunoprecipitated using specific antiserum (sa) against rat PRL, or normal serum (ns) as control. The immunoprecipitated material was resolved on PAGE under dissociating (SDS) and reducing (DTT) conditions (see Materials and methods). After the transfer onto membrane, the same PRL-specific antiserum and IgG alkaline phosphatase conjugate were used to visualize secreted (medium) and intracellular (cell) immunoreactive proteins. The transfer membrane was then submitted to direct autoradiography to detect %-proteins immunoprecipitated from medium or cells. Arrowhead indicates the position of native 23-kDa rPRL.

These findings, together with the former immunolocalization of PRL within tranfected cells, indicate that PRL expressed from pCMV-rPRL presents important similarities with endogenous PRL: first, it is localized in the same intracellular compartments of the secretory pathway and, second, it is released by the same regulatory factors. 3.7. Release of newly synthesized rPRL In basal conditions (Table l), both endogenous and vector-induced 35S-PRL (i.e., newly synthesized PRL) were released in chase medium together with unlabeled PRL. Besides, the secretagogues stimulated the short-term release of both newly synthesized and total PRL (Table 1) whether their origin was endogenous (pMAM-neo) or exogenous (pCMVrPRL). We calculated the PRL specific activity (S.A.) as the ratio: dpm of 3SS-PRL/ng of total PRL. As shown by Table 1, the S.A. of secreted exogenous PRL was larger than that of secreted endogenous PRL, whatever conditions of chase. These data suggest a ‘faster’ intracellular transit of the newly synthesized exogenous PRL in transfected GH3CDL cells. However, in control conditions the secretion rates of both endogenous and exogenous PRL, as estimated by the ratio of released PRL to PRL cell content, were similar indicating that, in this experiment, exogenous PRL was stored in the same proportions as endogenous PRL (Table 1).

3.8. EfSect of C4S mutation on rPRL immunoprecipitation and immunodetection PRL expressed in GH3CDL cells transfected with pCMV-C4S was analyzed by Western blotting as described above. Both immunoblots and autoradiographs (Fig. 6) clearly demonstrated that mutated C4S-PRL is immunoprecipitated and immunodetected by the rabbit antiserum against native rPRL. As expected, after SDS denaturation and DTT reduction, mutated PRL migrated on PAGE exactly as native rPRL (23 kDa), in cell extracts as well as in extracellular medium. 3.9. Eflect of C4S mutation on rPRL secretion In control conditions of culture, GH3CDL cells transfected with pCMV-C4S displayed a level of PRL secretion (50-60 ng/mg cell protein/48 h) much lower than GH3CDL cells transfected in parallel with pCMVrPRL (150- 180 ng/mg cell protein/48 h). Similar results were obtained in five independent experiments. However, the secretion rate of exogenous PRL, as estimated by the ratio of PRL released during a 30 min-incubation to PRL cell content, was 50% higher for mutated PRL than for native rPRL (Table 2), indicating a decreased storage capacity of cells transfected with mutated C4S-PRL sequence. The lower expression of mutated C4S-PRL is not likely to be related to a lower transcription efficiency, since pCMV-rPRL and pCMVC4S vectors contain identical DNA sequences with the unique exception of

C4S mutation. Moreover, expression of cotransfected pCMV-fiGal, as well as Northern blot analysis, did not reveal any significant difference in the transfection efficiency of the three vectors by electroporation. Thus, these data rather suggest that the mutation of rPRL coding sequence disturbed one post-transcriptional event-such as rPRL-RNA translation, or more likely, one post-translational step of protein intracellular processing and transport. In an attempt to further specify the disturbed of short-term PRL release step, the regulation was studied concurrently for mutated PRL and for native rPRL using the three secretagogues as described. Table 2 shows that all three secretagogues increased significantly the short-term release of mutated C4S-PRL. However, these increases were much lower in cells transfected with the mutated PRL sequence.

Pulse-chase experiments found for both endogenous

n pIM A H

revealed that, as previously and exogenous native PRL,

M

-n e o

pCMV-rPRL

0 . KCI: 30mM . Forskoline: bM 10pM

__ __

__ _-

++ -_

__ ++

-_ __

___

-__

-_ __

__ __

__ __

.TPA: 16nM 1.6pM

the newly synthesized (“?%labeled) C4S-PRL was released in chase medium together with unlabeled PRL. In these experiments, as in the previous ones (see Table 2). total mutated PRL was released and stored at lower levels than total native exogenous PRL. whereas its secretion rate was increased by 60% (Table 3), indicating a lower storage capacity of pCMV-C4S transfected cells. On the contrary, mutated ‘“S-PRL was released in much larger amounts than native “S-PRL (Fig. 8). These results were exemplified by the five-fold increase of C4S-PRL S.A. in the chase medium as compared to that of native rPRL (Table 3). Thus, the medium-to-cell S.A. ratio (R) was three-fold increased for pCM-C4S transfected cells (Table 3) whereas R was unchanged for pCMV-rPRL transfected cells as compared to pMAMneo transfected cells (see Tables l-3). This indicates a faster turnover of mutated PRL expressed within transfected GH3CDL cells under basal conditions. and strongly suggests the involvement of cysteine-4 residue in molecular interactions (most probably by disulfide bridging) at one step of the PRL secretory process (see Discussion).

++ __

The in situ detection of rPRL within GH3CDL cells by immunofluorescence showed no obvious modification of the subcellular distribution of the mutated hormone after transfection with pCMV-C4S. However. the decreased percentage of immunoreactive cells (3 1%. estimated from 500 cells) was in correlation with the intermediate rate of C4S-PRL secretion, as compared to cells transfected with pMAM-neo (5%) or with pCMV-rPRL (90%). At the electron microscope level, the intracellular localization of mutated C4S-PRL was not drastically different from that of native rPRL expressed in GH3CDL cells transfected with pCMV-rPRL although the immunoreactivity was less intense in the Golgi stacks (Fig. 9).

__ ++

Fig. 7. Regulation of the short-term rPRL release by transfected GH3CDL cells. Forty hours after the electroporation. GH3CDL cells transfected with pMAM-neo or with pCMV-rPRL were incubated for 30 min in the absence or in the presence of KCI (30 mM). or forskolin (Fk: I IO /rM) or phorbol ester (TPA: I6 nM 1.6 /tM). The amount of PRL released in the culture medium was measured by RIA and expressed in ng of rPRL per mg of cell protein. The results are the mean of triplicate determinations 2 SD. For endogenous PRL. the stimulated releases represent 703 (KCI), 866 (I ,uM Fk). 58 I ( IO /IM Fk). 464 (I6 nM TPA) and 844’!6#(I .6 /rM TPA) of release in control conditions. For exogenous PRL, the stimulated releases represent. respectively: 206. 136. 221. 120 and 317% of release in control conditions.

4. Discussion In order to get a better understanding of the molecular mechanisms which direct the sorting and intracellular transport of rPRL, we have used a transfection strategy. We have first developed a model of host cells which express and secrete native exogenous PRL. Then, using site-specific mutagenesis, we have obtained experimental evidence for a role of the N-terminal cysteine as a sorting signal involved in rPRL intracellular transport and storage.

A. Morin

Table 1 PRL turnover Incubation

in transfected

GH3CDL

er ul.

I Molecular

und Cellular

Endocrinolog!:

117 (1996)

69

59.- 73

cells

condition

medium PRL intracellular PRL medium-to-cell PRL ratio S.A. of medium rPRL S.A. of intracellular rPRL medium-to-cell S.A. ratio (R)

Endogenous

PRL (pMAM-neo”)

Exogenous

PRL (pCMV-rPRL”)

Control

K+TPA

Control

K+TPA

0.52 4.33 0.12 728 291

0.97 + 0.09 1.74*0.10 0.56 966 + 95 460 + 26

1.79kO.13 14.00 f 0.84 0.13 3305 * 198 1336k92

2.96 k 0.24 9.34 * 0.29 0.32 5238 + 361 645 + 56

2.10

2.47

8.10

+ 0.04 * 0.21 f 56 f 14

2.50

GH3CDL cells were transfected with pMAM-neo or pCMV-rPRL and submitted to pulse-chase experiments , 28 h after the electroporation. The ceils were pulsed for 20 min with ‘Wabeled methionine and cysteine, washed and then chased for 40 min in the absence (control) or presence of 30 mM KCI plus 1.6 PM TPA. Upper panel: total medium and intracellular PRL was measured by RIA. The amount of vector-derived PRL (exogenous) was calculated by subtracting, from total PRL in pCMV-rPRL transfected cells, the mean value of endogenous PRL as determined from pMAM-neo transfected cells in the same conditions. The results are expressed in ng of rPRL per mg of cell proteins. Then, the medium-to-cell ratio was calculated as an indicator of the PRL secretion rate, for each group of transfected cells, under either control or stimulated (K + TPA) conditions. Lower panel: the specific activity (S.A.) of intracellular PRL and of PRL secreted during chase incubation was calculated and results are expressed in dpm per ng of rPRL. Then. the ratio (R) of medium-to-cell PRL S.A. was calculated as an indicator of the PRL turnover rate, in each group of transfected cells. The results are the mean of triplicate determinations i S.D. ,‘Transfection vector.

4.1. Cell-specific expression of rPRL

We screened several mammalian cell lines in culture to express native rat PRL. In all the cell types, the induction of specific rat PRL transcripts required an expression vector containing the rat PRL coding sequence under the control of a strong enhancer-promoter (CMV) and an efficient transfection method (electroporation). All the host cells (C6, AtT20, GC and GH3CDL) were transfected with the same efficiency as seen by cotransfection with pCMV-/3Gal vector used as an internal control. However, the level of expression of vector-derived PRL (exogenous protein) within transfected cells was much higher in GH3CDL cells, which derived from the mammosomatotrope GH3 line, than in the other three host cell lines. Thus, the data suggest a strict cell-specific control of rat PRL biosynthesis and secretion, most probably at a posttranscriptional level. Such a strict cell-specific control of PRL secretion is to our knowledge reported here for the first time. This cannot be related to the cell-specific transcription factor Pit-l, since it is expressed at similar level in GH3CDL and GH3B6 cells (Ngo et al., 1994) as well as in GC cells (Billis et al., 1992). Interestingly, in contrast to PRL, GH was secreted by several transfected cell lines such as HeLa cells (Prager and Melmed, 1988) or mouse L-cells (Chen et al., 1992). A possible explanation for such a cell-specific control of rPRL secretion might be a role of the small amount of endogenous PRL in the coaggregation of vector-derived PRL.

In transfected GH3CDL cells, exogenous native PRL exhibited the same subcellular distribution as previously found for the parent cell line (GH3B6) which secrete large amounts of rPRL and contain a few small secretory granules (Tougard et al., 1982). The release of exogenous PRL was triggered by the same intracellular mechanisms in transfected GH3CDL cells, in control GH3CDL cells and in the parent GH3B6 cells (Laverriere et al., 1986). The higher specific activity of medium PRL displayed by pCMV-rPRL transfected cells (see Tables 1 and 3), together with the lower amplitude of their response to secretagogues (see Fig. 7, legend), suggest that a small part of the neosynthesized exogenous PRL might be diverted to an unregulated, constitutive-like route. In spite of these slight differences, which may be inherent to transfection artefacts, the data suggest that exogenous native PRL is, for a large part, intracellularly transported through the same pathway and released by the same mechanisms as endogenous PRL. Therefore, this experimental cell model was further used to look for sorting signal(s) included in the structure of the rPRL protein. 4.2. Importunce of the N-terminal cysteine-4 for rPRL secretion According to the current concept of aggregation-mediated sorting for regulated secretory proteins (see reviews: Arvan and Castle, 1992; Tooze et al., 1993), we have made the hypothesis that rPRL condensation within secretory granules could arise from disulfide

70

.4. Morin et al. I) Moleculur

Table 2 Effect of C4S mutation Transfection

on the secretion

rate of exogenous

Exogenous

vector

pCMV-rPRL

117 (1996) 59

7.1

PRL

PRL

Incubation

condition

Control

K’

Fk

TPA

1.29&0.17

2.16)

0.26

I .98 * 0.24

3.44 _+ 0.39

intracellular PRL medium-to-cell ratio

6.45 * 0.97 0.20

3.54 * 0.66 0.61

4.88 + 0.78 0.41

4.48 + 0.76 0.77

medium PRL intracellular PRL medium to cell ratio

0.66*U.l0 2.20 * 0.54 0.30

0.85 * 0.13 1.94~0.51 0.44

0.75 * (l.II I .64 i 0.52 0.46

I.25 + 0.18 2.06 + 0.54 0.61

medium

pCMVC4S

uttd Cellular Endocrinology

PRL

GH3CDL cells were transfected with pCMV-rPRL (upper panel). or pCMV-C4S (lower panel). Twenty-eight hours after the electroporation, cells were incubated for 30 min in the absence (Control). or in the presence of secretagogue: 30 mM KCI (K + ). 2 AIM forskolin (Fk) or I.6 /cM phorbol ester (TPA). The amount of vector-derived PRL was calculated by subtracting the mean value of endogenous PRL, as determined from triplicate dishes in parallel (pMAM-neo transfected cells). The results are expressed in ng of exogenous rPRL per mg of cell proteins as mean value of triplicate dishes + SD. Then, the medium-to-cell ratio was calculated as an indicator of the secretion rate for either

native.

or mutated.

exogenous

PRL.

by transfected

cells under

bond exchanges that involve PRL conserved cysteines. So we tested the possible role of the N-terminal C4/C9 disulfide bond that is specific to PRL. Many intracellular targeting signals are known to be present at the N-terminus of the targeted proteins. Disruption of disulfide bridges in the N-terminal portion of proopiomelanocortin (POMC) was shown to greatly perturb the processing and release of this regulated polyprotein (Roy et al., 1991). Moreover, the sorting of chromogranin B (CgB) to the secretory granules was shown to be dependent upon the integrity of its single N-terminal disulfide bond as revealed by DTT treatment (Chanat et al., 1993).

Table 3 Effect of C4S mutation Transfection

on the PRL turnover

vector

control

or stimulated

conditions.

So we substituted the N-terminal cysteine at position 4 (C4S mutation) of rat PRL by in vitro site-specific mutagenesis of pCMV-rPRL expression vector. Cysteine-to-serine change was selected to minimize the structural disturbances introduced in the mutated rPRL. As expected, the disruption of the N-terminal disulfide bond by this unique mutation did not modify the overall structure of rPRL molecules, since C4S-PRL was immunodetected in situ and immunoprecipitated by an antiserum against native rat PRL. In SDS-gel electrophoresis and after reduction with DTT, the mutated PRL behaved as native PRL (Fig. 6). However, C4S-PRL was produced at a lower level than native

rate Endogenous

PRL

Exogenous

PRL

__

medium PRL intracellular PRL medium-to-cell ratio S.A. of medium PRL S.A. of intracellular PRL medium-to-cell S.A. ratio (R)

pMAM-neo

pCMV-rPRL

pCMVC4S

0.22 * 0.02 4.14 + 0.25 0.05 814 * 80 376 i 36 2.16

1.03 6.78 0.15 2752 I’96 2.12

0.51 k 0.06 2.15 kO.38 0.24 14266 f 618 2209 f 155 6.46

*0.12 ir d.66 f 287 + I34

GH3CDL cells were transfected with pMAM-neo, pCMV-rPRL or pCMVC4S. Twenty-eight hours after the electroporation. the cells were pulsed for 20 min with “S-labeled methionine and cysteine, washed and then chased for 40 min. The results are expressed as in Table I: endogenous and vector-derived exogenous PRL (upper panel). and PRL specific activity (lower panel). The results are the mean of triplicate determinations + SD. One can notice that in these experiments, the basal level of endogenous PRL secreted by untransfected or pMAM-neo transfected cells (2-3 ngimg cell protein/48 h) was lower than that of previous experiments (see Table I), most probably in relation to culture medium variations (serum constituents) during cell line maintenance.

A. Morin et al. I Molecular and Cellular Endocrinology 117 (1996) 59-73

medium

cell

medium

cell

medium

cell

Fig. 8. Effect of C4S mutation on the release of newly synthesized %-PRL. GH3CDL were transfected with pMAM-neo, pCMV-rPRL or pCMV-C4S, and submitted to pulse-chase experiments: 28 h after the electroporation, transfected cells were pulsed for 20 min with %-labeled methionine and cysteine, washed and then chased for 40 min. The amount of ‘%-PRL released in the chase medium (medium) or contained within the cells (cell) was quantified by specific immunoprecipitation and expressed in dpm per mg of cell proteins. The results are the mean of triplicate determinations f S.D.

rPRL in spite of the fact that both native pCMV-rPRL and mutated pCMVC4S vectors were introduced and transcribed in transfected GH3CDL cells with the same efficiency (see Results). Two main important differences were found between the secretion of exogenous, mutated and native PRL, respectively. The secretion rate (medium-to-cell ratio) was increased by 50% for C4S-PRL (see Tables 2 and

11

Fig. 9. Immunoelectron microscopic localization of C4S-PRL within transfected GH3CDL cells. GH3CDL cells were transfected with pCMV-C4S and 40 h after the electroporation, intracellular PRL was visualized in situ by pre-embedding immunoperoxidase method at the electron microscope level. As compared to Fig. 5, PRL immunoreactivity is distributed in same compartments: Golgi cistemae (G) and a few small secretory granules (arrowhead); (magnification: x 24 000).

3) and the specific activity of medium C4S-PRL was increased 5 times (Table 3) as compared to exogenous native PRL. This result is reminiscent of the effect of reducing agent (DTT) that increased the release of newly synthesized CgB during 60 min chase (Chanat et al., 1993). Thus, the removal of the N-terminal disulfide

bond caused a faster rPRL turnover in basal conditions. In accordance with the concept of an aggregation mediated sorting, this suggests that part of C4S-PRL was excluded from the aggregation process and was then rapidly released from immature secretory granules or vesicles, via a constitutive-like secretion pathway (Kuliawat and Arvan, 1992). Obviously, in addition to the C4 N-terminal cysteine, other features of rPRL might participate in its intracellular sorting and condensation into secretory granules. Substitution of the central disulfide bond (C56/C172) which is involved in the stability of globular rPRL structure would induce the early degradation of the molecules within the rough ER (Helenius et al., 1992). The GH secretion by cultured cells was indeed drastically reduced when the homologous central bond of the bovine hormone was destroyed (Chen et al., 1992). The role of the C189; Cl97 disulfide bond of rat PRL is unlikely since bovine GH secretion was not affected when the homologous C-terminal bond was abolished (Chen et al., 1992). Besides, the 22K PRL variant (PRL I ~~173) was shown to be stored in secretory granules and released through the regulated pathway (Anthony et al., 1993). In contrast, interaction with divalent cations, as shown for Zn’+ induced hGH dimerization (Cunningham et al.. 1991) possibly participate in PRL oligomerization since Zn’+ and Mg2+ were found to play a role in the condensation and packaging of PRL within secretory granules (Greenan et al.. 1990). However. so far the mechanisms of these interactions are unknown. The role of the N-terminal disulfide bridge, that we have shown here by mutagenesis and pulse-chase experiments, is also supported by data from the literature using other approaches. In bovine PRL, the N-terminal bridge is the more sensitive one to reducing conditions (Doneen et al., 1979) and it makes a small loop that favors disulfide exchange reactions. Thus, an intragranular protein disulfide isomerase might catalyse the cross-linking of PRL molecules by interchain disulfide bridges. Such a mechanism was previously described for aggregation of pancreatic enzymes in the intracisternal granules (Tooze et al., 1989). Moreover, the morphology of PRL secretory granules in situ (Greenan et al.. 1990) and the release of rat PRL from isolated secretory granules (Lorenson and Jacobs, 1982) as well as from PRL cells (Mena et al., 1986) were found to be sensitive to thiol exposure. Cysteamine caused reduction of PRL monomers followed by PRL aggregation in the rat pituitary gland (Scammell et al., 1984) and the intragranular glutathione could serve as the reducing agent in vivo (Greenan et al., 1990). Together these data are consistent with a role of the highly conserved C4-C9 bond in the sorting and packaging of rat PRL into secretory granules. They cannot, however, exclude a role of the free cysteine C9 in the partial storage of mutated C4S-PRL.

Acknowledgements We would like to thank Dr C. Tougard for useful discussions and suggestions regarding the present study. We are grateful to other members of the lab for their constructive comments (J.N. Laverriere) and technical assistance (J. Catelon and A. Barret). We acknowledge E. Etienne for photographic illustrations. We are indebted to Dr J.A. Martial and A. Belayew (Universite de Liege, Belgium) for valuable help and advice on molecular biology strategy. This work was supported by grants from the CNRS (URA 1115) and from the INSERM ( # 894014).

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