Mitogenic effects and nuclear localisation of procorticotrophin-releasing hormone expressed within stably transfected fibroblast cells (CHO-K1)

ELSEVIER

Molecular and Cellular Endocrinology

107 (1995) 17-27

Mitogenic effects and nuclear localisation of procorticotrophin-releasing hormone expressed within stably transfected fibroblast cells (CHO-Kl) Maria G. Castrob*,

P. Tomaseca, E. Morrisona, C.A. Murraya, P. Hodges, P. Blanninga, E. Lintonb, P.J. Lowryc, Pedro R. Lowensteina

af.aboratory of Cellular and Molecular Neurobiology, Department of Physiology, University of Wales College of Card& Museum Avenue, P.O. Box 902, Cardift; CFl ISS, Wales, lJK bDepartment of Obstetrics and Gynaecology, John Radcliffe Maternity Hospital, University of Oxford, Headington, Oxford, OX3 9DV* UK ‘Department of Biochemistry and Physiology, University of Reading, P.O. Box 228. Reading, RG2 2AJ. UK Received 28 July 1994; accepted

8 October

1994

Abstract To investigate the intracellular localisation and biological activity of procorticotrophin-releasing hormone (proCRH), we have established stably transfected CHO-Kl cells expressing the rat pre-proCRH cDNA. Using immunoblot analysis of cell lysates of transfected CHO-Kl cells, we detected a major CRH immunoreactive band with an apparent molecular weight of approximately 19 kDa. This 19 kDa band could account for full length proCRH molecule which has not undergone post-translational modifications. Metabolic labelling followed by immunoprecipitation, SDS-PAGE and autoradiography indicated that no endoproteolytic processing of proCRH takes place within the transfected CHO-KI cells. Immunofluorescence staining localises the CRH precursor to both the cytoplasm and to the nucleus in transfected CHO-Kl cells. This result was confirmed using subcellular fractionation techniques on radiolabelled CHO-Kl cells expressing immunoreactive CRH. A major CRH-immunoreactive band of 19 kDa was detected both in the microsomal and secreted fractions, indicating the presence of proCRH within the secretory pathway of these cells. This was also evident in the nuclear fraction, therefore confirming the nuclear localisation of proCRH. Analysis of DNA concentration, ceil number and DNA synthesis showed that stably transfected CHO-Kl cells expressing proCRH have a higher proliferation and DNA synthesis rate than wildtype CHO-Kl cells or CHO-Kl cells transfected with pEE14 alone. Our results therefore suggest a mitogenic role for the intact proCRH molecule within CHOKl cells. Furthermore, treatment of mouse corticotrophic tumour cells (AtT20/D16-16) with conditioned medium from transfected CHOKl cells expressing proCRH, stimulated both DNA synthesis and cell proliferation above basal levels. Our results constitute the first reported direct evidence of a mitogenic role for proCRH acting on a corticotrophic cell population. Keywords:

Corticotrophin-releasing

hormone; Intracellular trafficking; Secretory pathway; Nuclear translocation; Mitogenic activity

1. Introduction Most peptide hormones and neuropeptides are synthesized in the form of precursor molecules, i.e. prohormones or proneuropeptides. These are post-translationally processed to yield the biologically active peptides, by specific endoproteolytic cleavages, glycosylation, phosphorylation and amidation (Docherty and Steiner, 1982; Eipper and Mains, 1988; Castro et al., 1989). Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which is cleaved from a large * Corresponding 874094.

author,

Tel.: +44 0222

874561;

Fax: +44 0222

precursor molecule (pre-proCRH) by the action of specific endoproteases at pairs of dibasic amino acids (Thompson et al., 1987; Castro et al., 1991). As is the case with other hormone and neuropeptide precursors, intact proCRH is present in very low abundance within cells and is also highly unstable. For this reason the studies on its biological activity, biochemical and physicochemical characteristics and its biosynthetic pathway are poorly understood. The method of choice for large scale expression of proteins within mammalian cell lines has been dihydrofolate reductase coupled amplification (Kaufman and Sharp, 1982). Vectors containing hamster glutamine synthetase (GS) encoding sequences have recently been described

0303~7207/95/$09.50 0 1995Eisevier Science Ireland Ltd. All rights reserved SSDI

0303-7207(94)03416-Q

18

M.C. Custrol et ~11./ Moleculur

und Cellulur

that can be used as dominant selectable markers in cells already containing active GS genes yet can still be amplified to high copy number by selection with methionine sulphoximine (MSX) (Cockett et al., 1990). Whether the recombinant proteins produced by these methods resemble naturally occurring proteins with respect to posttranslational modifications is of crucial importance since the presence or absence of such modifications may have profound implications with respect to bioactivity as welt as endoproteolytic processing. This paper describes the use of GS vectors for efficient expression of recombinant proCRH in CHO-Kl cells, containing the selectable GS marker with a highly efficient transcription unit for the recombinant gene (proCRH). We provide evidence which indicates that proCRH produced in CHO-Kl cells is not endoproteolytitally processed (19 kDa band) within these cells. Immunofluorescence studies show that proCRH is targeted to the cytoplasm with a staining pattern compatible with the presence of the prohormone within the secretory pathway of CHO-Kl cells and is also localised within the nucleus of these cells. We also provide data indicating that the CRH precursor molecule stimulates cell proliferation in CHO-Kl cells and in mouse corticotrophic tumour cells (AtT20/D16- 16). This work constitutes the first reported evidence of a mitogenic role for the intact corticotrophin-releasing hormone precursor. 2. Materials and methods Chinese hamster ovary (CHO-Kl) cells were obtained from the European Collection of Animal Cell Cultures, Porton Down, UK; pEE14 plasmid was obtained from Dr. M Cockett (Cockett et al., 1990) (Celltech Limited, Slough, Berkshire, UK). This vector drives transcription of the cloned gene using the hCMV promoter/enhancer sequences and carries an SV40 poly(A) tail. In addition to this, it contains the glutamine synthetase (GS) minigene as a dominant selectable marker. This allows amplification of the cloned gene in transfected cells under selection with methionine sulphoximine (MSX), an inhibitor of the glutamine biosynthetic pathway. Relatively high copy numbers of the cloned gene can be obtained, resulting in the rapid achievement of high level expression without the requirement for a mutant cell line (Cockett et al., 1990). pSP65/rat-preproCRH cDNA was obtained from Dr. R. Thompson (Mental Health Research Institute, Michigan, USA). Restriction enzymes were obtained from Promega Limited (Chilworth Research Centre, Southampton, UK), cell culture reagents were obtained from Gibco BRL (Paisley, UK) and all other reagents were obtained from Sigma (Poole, Dorset, UK) unless otherwise stated. Rat pre-proCRH cDNA was excised from the plasmid pSP6YrCRH by EcoRI digestion and was cloned into the EcoRI site within pEE14.

Endocrinology

107 (1995)

17-27

2.1. Cell culture Chinese hamster ovary (CHO-Kl) cells were grown in culture medium consisting of DMEM supplemented with 10% (v/v) horse serum, 5% (v/v) newborn calf serum, 2 mM glutamine, 100 U/ml penicillin and lOOpg/ml streptomycin sulphate, and split when they reached 7080% confluency. The stably transfected cell lines (CHOKl clone l/22 and CHO-Kl clone 2/l) were grown in the selection medium containing 50pM of methionine sulphoximine (MSX) (Cockett et al., 1990). Cells were maintained at 37°C in 6% CO, with high humidity. Transfection and selection of stably transfected CHO-Kl cells were carried out as described previously by Cockett et al. (1990). Immunoreactive corticotrophin-releasing hormone (IR-CRH) synthesized by the transfected cells was assayed using a specific radioimmunoassay (RIA) described previously (Castro et al., 1990a). Immunoblotting was carried out in wildtype and transfected CHO-Kl cell extracts as described previously (Castro et al., 1990b). 2.2. Metabolic labelling and immunoprecipitation Cells at 80% confluency in 25 cm2 flasks were washed twice with methionine/cysteine free DMEM (ICN-Flow, Irvine, UK) and starved for 30 min in the same medium. Cells were then labelled for increasing periods of time, IO-320 min (time course study) with 200&i of [35S]methionine/[35S]cysteine per ml of medium at 37°C. After metabolic labelling, the cells were washed twice in cold PBS, scraped from the dishes, spun down at 800 rev./min for 20 min at 4°C and the cell pellets lysed in 100~1 RIPA buffer (150 mM NaCl, 50 mM Tris, pH 8, 1% (v/v) NP-40, 0.5% (w/v) DOC, 0.1% (w/v) SDS, 1 mM PMSF) for 30 min at 4°C. Samples were clarified by centrifugation at 13 000 rev./min for 30 min, precleared with 50,u.l of SAC (Staphylococcus aureus protein A positive fixed cell suspension, Sigma Chemical Co., Poole, UK) for 40 min at 4°C on a rotating wheel and spun down at 13 000 rev./min for 10 min to pellet the SAC. Supernatants were then incubated with rabbit polyclonal antibody MP2 at 1:lO dilution overnight at 4°C. Cell supernatants were supplemented with 1 mM PMSF, precleared with SAC and incubated with MP2 antibody diluted 1: 10 as described above. Antibody-bound proteins were precipitated using SAC at a 1: 10 final dilution, precipitates were washed several times with RIPA buffer, boiled in 50-lOO@ of SDS PAGE sample buffer and analyzed by 0.1% (w/v) SDS, 13.5% (w/v) polyacrylamide gels. Proteins in gels were fixed by immersing them for 30 min in 35% (v/v) isopropanol, 10% (v/v) acetic acid. The fixed gels were then soaked in Amplify (Amersham UK, Aylesbury, UK) for 30 min, dried and exposed to X-ray films (Kodak). 2.3. Subcellular fractionation Approximately 30 x lo6 cells were used in this proce-

M.G. Castro1 et al. I Molecular and Cellular Endocrinology 107 (1995) 17-27

dure. Cells were grown to confluency in tissue culture flasks, scraped into PBS and pelleted by centrifugation at 1000 x g for 10 min. The resultant cell pellet was resuspended in 1 ml of homogenisation buffer (10 mM triethanolamine, pH 7.4; 0.25 M sucrose; 0.15 M sodium chloride; 1 mM magnesium chloride; 1 mM EDTA and 1 mM dithiothreitol) followed by 50 strokes in a dounce homogenizer. The volume of the cellular homogenate was made up to 15 ml with homogenisation buffer and spun at 1000 x g for 10 min to yield a crude nuclear pellet. The supernatant from this spin was centrifuged at 27 000 x g to yield a mitochondrial pellet, followed by a final spin at 120 000 x g resulting in microsomal and membrane fraction pellets. The crude nuclear pellet was further purified by two washes in 15 ml HS buffer (10 mM HEPES, pH 7.4; 0.25 M sucrose; 3 mM magnesium chloride) and three washes in 15 ml TESM buffer (10 mM Tris, pH 7.4; 2 mM magnesium chloride; 1 mM EDTA and 0.25 M sucrose before being resuspended in 40% w/w sucrose in 10 mM Tris (pH 7.4), 1 mM magnesium chloride and centrifuged against a cushion of 61.5% w/w sucrose for 2 h at 80 000 x g to isolate morphologically intact nuclei. These were recovered and washed twice in T’ESM buffer containing 2% (v/v) CHAPS followed by washes in TESM buffer containing 2% (v/v) Triton X-100. This purified nuclear pellet was resuspended in 0.5 ml RIPA buffer, as were the mitochondrial and microsomal pellets, and immunoreactive CRH was detected in the subcellular fractions by immunoprecipitation, followed by SDS PAGE and autoradiography. All manipulations were carried out on ice, all spins were carried out at 4°C and all buffers contained the following protease inhibitors; 1 mM PMSF, 1 pg/ml each of antipain, chymostatin and pepstatin A and 2 pg/ml of aprotinin. 2.4. Immunojluorescence Cells were washed once in phosphate-buffered saline (PBS, pH 7 4) then fixed in 4% (w/v) paraformaldehyde in PBS for 20 min. The method for immunofluorescence staining has been described in detail previously (Lowenstein et al., 1994). In nuclear co-localisation experiments, the mounting medium also contained the DNA specific dye 4’,6-diamino-2-phenylindole (DAPI) (Molecular Probes) at a final concentration of 1 pg/ml. In some experiments the cell nucleus (DNA and RNA) was visualised by incubating cells with propidium iodide at a final concentration of O.OS~g/ml in PBS at 37°C for 0.5 h. The Golgi apparatus in CHO-Kl cells was visualized using an antibody specific for a trans-Golgi network (TGN) protein, i.e. TGN38 (Reaves and Banting, 1992). Slides were observed and photographed using an Olympus Vanox AHBS microscope with the fluorescence attachment AHZRFL, using Kodak T-Max 3200 film.

19

2.5. Cell proliferation studies The effects of proCRH expression upon cell prolifera-

tion in transfected CHO-Kl cells were analysed using a variety of methods, the first of which was cell counts. Wildtype and transfected cells were grown to confluency before incubation in serum-free medium for 3 days (as described earlier, but with 0.2% (w/v) BSA in place of serum) to synchronise the cell population in Ga. Cells were harvested and plated out at a density of lo4 cells/well in serum-free medium in 24-well tissue culture plates and allowed to attach overnight. The medium was then replaced with fresh serum-free medium and the cells were incubated for 24, 48, 72 and 96 h. The cells were then harvested by trypsinisation and counted using a haemocytometer. DNA concentration was also used as an index of cell proliferation, and was measured using the Cell Titer 96 non-radioactive cell proliferation assay from Promega (Madison, WI, USA) according to the manufacturer’s instructions. The assay is based upon the cellular conversion of a tetrazolium salt into a formazan product that is easily detected by an ELISA plate reader. Briefly, a premixed dye solution was added to culture wells of a 96well plate containing the cells and the appropriate experimental treatment (serum-free or serum-containing medium). After a 4-h incubation, the solubilisation/stop solution was added to the culture wells to solubilise the formazan product, and the absorbance at 570 nm was recorded using an ELISA plate reader. The absorbance at 570 nm is directly proportional to the number of cells present in each well. Cells were plated at a density of 2500 cells/well in 100~ 1 of DMEM containing 10% FCS. After the cells reached confluency, they were serum starved for 3 days until a stable baseline of BrdU incorporation levels were obtained (results not shown). Following serum starvation, fresh medium was added to either CHOKl, CHO-Kl (pEE14) or CHO-Kl-2/l cells (either serum-free or serum-containing medium). After incubation for 48 h at 37°C the medium was aspirated, the cells washed twice in PBS and 100~1 of PBS added to each well. Dye solution (15 ~1) was added to each well and the plate incubated for 4 h. At the end of this time, 100~1 of the solubilisation/stop solution was added to each well and the wells incubated at 37°C for 1 h. The absorbance was recorded at 570 nm using a Bio-Rad ELISA plate reader. Thymidine incorporation assay was also used as an index of DNA synthesis in wildtype and stably transfected CHO-Kl cells (expressing pEE14 or pEE141rCRH cDNA). Cells were synchronized in Go by serum starvation for 3 days. To synchronize cells at Gl/S, serum was added to the Go-synchronized cells (final concentration 10% (v/v) calf serum) followed by addition of hydroxyurea (2 mM final concentration) 2 h later. Cells were plated at a density of 100 000 cells/well in 24-well plates

M.G.

20

Glstrol

et ui. I Molecular

und Cellultrr

and incubated with hydroxyurea for I6 h before use. Cells were washed twice with PBS and the medium was replaced with medium containing serum or serum free medium. Cells were incubated at 37°C 5% CO, for different periods of time from 2 h to 10 h. Thymidine incorporation assay was also used as an index of DNA synthesis in the corticotrophic tumour cell line, AtT20/Dl6-16. Cells were synchronised in Ga by serum starvation for 3 days. Cells were mechanically dispersed and 100 000 cells/well were plated onto 24-well plates which had been previously coated with poly-L-lysine. Cells were allowed to attach overnight, and the next day cells were treated with (i) medium containing 10% serum (+S), (ii) serum free medium, (iii) conditioned serum free medium from wildtype CHO-Kl cells, (iv) conditioned serum free medium from transfected CHO-Kl cells expressing proCRH (2/l), and (v) conditioned serum free medium from transfected CHO-Kl cells expressing proCRH (2/l) which had been immunoprecipitated using a CRH-specific antibody (Matthew) at a final dilution of 1: 100. The CRH-antibody complexes were separated by immunoprecipitation using SAC (final concentration 1: IO). Cells were incubated at 37°C 5% COT for different periods of time from 3 h to 12 h. [3H]Thymidine (0.5,~Ci) was added to the cultures 2 h before the end of the incubation period. The medium was carefully removed by aspiration, and the cells were fixed with 10% trichloroacetic acid (0.2 ml/well). Fixed cells were washed four times with 5% trichloroacetic acid and the acid-insoluble pellets were dissolved by adding 0.2 ml of 0.5 M NaOH/O.l% (w/v) SDS. The samples were neutralized by the addition of 200~ 1 of 0.5 M acetic acid and were transferred to a scintillation vial. Then 5 ml of scintillation liquid was added to each vial and radioactivity was measured in a /?counter.

Table

1

lmmunoreactive culture

(IR) CRH stored intracellularly

medium

from

stably transfected

incubated in selective medium containing SOpM 72h.96hand

120h

Time

lmmunoreactive

and released into the

CHO-KI

CRH (pg/,,g)

(clone MSX

2/l)

Ratio stored/secreted

(h) Secreted

Stored 72.0 + 10.0

8.5 + I.1

48

I I .4 f 2.4

80.0 f 6.2

7.2+

72

IS.1 k 2.9

89.2 2 9.0

5.8 + 0.6

96

18.5 f 1.4

109.7 f 8.0

5.9 * 0.2

120

16.5 + 2.4

24

IR-CRH

cells

for 24 h. 48 h,

8.5 ? I .O

92.7 + I I .8

5.6 + 0.2

was measured using a specific radioimmunoassay

scribed in Section 2 (pg IR-CRH/pg

I.1

(RIA)

de-

of total cellular proteins). The

results are expressed as the mean + the standard error of the mean (x 2 SEM) for n = 4. Each sample was assayed in duplicate and at two different dilutions to ensure that the CRH concentration would fall within the linear range of the RIA standard curve.

Endndocrinology

107 (1995)

17-27

3. Results 3. I. Establishment of stably transfected CHO-Kl cells CHO-Kl cells were transfected using the expression vector pEEl4 (Cockett et al., 1990). CHO-Kl cell transfection, using the calcium phosphate co-precipitation method, resulted in the isolation of 57 MSX resistant clones. These were screened for CRH production using a specific radioimmunoassay for CRH (l-41), which also cross-reacts with intact proCRH. All of the isolated clones secreted detectable levels of CRH, ranging from 1.2 to 17.8 nglml per 24 h. Two of the highest producing clones (clone l/22 and clone 2/l) were chosen for further characterisation. Approximately 80% of the cells in the transfected CHO-Kl cells were positive for CRH expression as assessed by immunofluorescence. 3.2. Synthesis and post-translational processing proCRH in transfected CHO-Kl

of

cells

We also used a specific radioimmunoassay to assess the relative storage capacities of the transfected cell lines. The results (Table 1) show that transfected CHO-Kl cells store and release IR-CRH. Interestingly, the majority of this IR-CRH is present intracellularly (ranging from 5.6 to 8.5 times more IR-CRH being stored than secreted), although CHO-Kl cells do not possess a regulated secretory pathway and, therefore, would not be expected to store proCRH intracellularly. Cell extracts from stably transfected CHO-Kl cells were examined for the presence of IR-CRH using SDSPAGE followed by Western blotting (Fig. 1). A major immunoreactive band of approximately 19 kDa was detected using six different CRH-specific antibodies: MP2, B3, Charlie, Matthew, Hannah and Horns. No immunoreactive bands corresponding to smaller CRH immunoreactive peptides were observed using this technique. The other higher molecular bands detected in CHO-Kl cells expressing proCRH are also present in wildtype CHO-Kl cells (Fig. 1, lanes 1) and in CHO-Kl cells stably transfected with pEE14 (results not shown). We therefore conclude that these bands are cross-reactive substances present in wildtype CHO-Kl cells. Transfected cells were metabolically labelled using [ 3SS]methionine/[35S]cysteine to determine the kinetics of appearance of radiolabelled IR-CRH in both cell extracts and culture medium, and to investigate endoproteolytic processing of the precursor. This method gives a higher degree of sensitivity than Western blotting. IR-CRH was detected using immunoprecipitation with CRH-specific antibodies, followed by separation of the immunoprecipitate using SDS-PAGE and visualisation of immunoreactive radiolabelled CRH by exposure to X-ray film. Stably transfected CHO-KI cells were metabolically labelled for up to 320 min (Fig. 2). IR-CRH was detected in cell extracts after IO min labelling (Fig. 2, lane 2) and first appeared in culture supernatant after 40 min labelling (Fig.

M.C. Castro1 et al. I Molecular and Cellular Endocrinology 107 (1995) 17-27

MP2

B3

Charlie

Matthew

Hannah

21

Horns

Fig. 1. SDS-PAGE of cellular extracts of CHO-Kl cells stably transfected with the recombinant plasmid pEE14/rat pre-proCRH cDNA followed by immunoblotting with CRH-specific antibodies. Lane 1 in each panel am cellular extracts from wildtype CHO-Kl cells, lane 2 in each panel are cellular extracts from CHO-Kl cells stably transfected with the rat pre-proCRH cDNA. Molecular weight markers are indicated to the left and the anti-CRH (l41) antibodies used am indicated below each lane. Primary antibodies were used at a dilution of 1:lOO in 10% w/v skimmed milk in PBS. Major ProCRH immunoreactive band is indicated with an arrow to the right hand side.

2, lane 5). The amount of IR-CRH detectable in cell extracts remained relatively constant throughout the course of the experiment, whereas IR-CRH accumulated to high levels in the culture supernatant (Fig. 2, lanes 7, 9 and 11). The major immunoreactive product of labelling in transfected CHO-Kl cells was a band of approximately 19 kDa. No IR-CRH was detected in wildtype CHO-Kl culture medium or cell extract after 320 min labelling (Fig. 2, lanes 15 and 16, respectively) or in transfected CHO-Kl (l/22) cells labelled for 320 min and immunoprecipitated using normal rabbit serum (Fig. 2, lanes 13 and 14, culture medium and cell extract, respectively). CRH (l-41) or other smaller molecular weight IR-CRH products derived from the CRH precursor molecule were not detected in cellular extracts or culture medium from stably transfected CHO-Kl cells. 3.3. Intracellular localisation of immunoreactive CRH within stably transfected CHO-Kl cells To address the intracellular localisation of IR-CRH within transfected CHO-Kl cells, we analysed the microsomal, purified nuclei and mitochrondrial subcellular fractions (Fig. 3, lanes 4, 5 and 6, respectively) using metabolic labelling of the transfected cells prior to fractionation. The fractions obtained were analysed using immunoprecipitation followed by SDS-PAGE and exposure to X-ray film. The experiments revealed an immunoreactive band of approximately 19 kDa in total cell extract, microsomal and purified nuclear fractions from stably transfected CHO-Kl cells (Fig. 3, lanes 3, 4 and 5, respectively), confirming that no processing of proCRH was occurring in these cells and that intact proCRH was

the molecular form being translocated to the nucleus. To eliminate the possibility that the nuclear IR-proCRH was derived from membranous contamination from organelles within the secretory pathway, the nuclear fraction from radiolabelled cells was extensively washed using detergents (2% CHAPS and 2% Triton X-100) and the purified nuclear pellet was analysed by immunoprecipitation using CRH-specific antibodies followed by SDS-PAGE and autoradiography. This treatment did not abolish IRproCRH in the nuclear fraction. (Fig. 3, lane 5). No CRHimmunoreactive bands were detected in total cell extract from wildtype CHO-Kl cells or stably transfected CHOKl cells with the plasmid pEE14 (Fig. 3, lanes 1 and 2, respectively). The distribution of IR-CRH in stably transfected CHOKl cells was also examined using immunofluorescence microscopy (Fig. 4). Cytoplasmic IR-CRH staining was predominantly perinuclear (Fig. 4A-Q and co-localised with both fluorescently labelled WGA (Fig. 4E) and an antibody against a membrane protein specifically located in the trans-Golgi network, TGN38 (Fig. 4F). IR-CRH did not co-localise with WGA labelling of endosomes (results not shown). IR-CRH staining was also seen in the nuclei of transfected CHO-Kl cells (Fig. 4A,B). Nuclear IRCRH co-localised with the DNA-specific dye DAPI (Fig. 4D), confirming its nuclear location. 3.4. The effects of proCRH expression in stably transfected CHO-Kl cells upon cell proliferation The growth properties of transfected CHO-Kl cells were compared to those of wildtype CHO-Kl cells and of CHO-Kl cells transfected with pEE14 by a variety of

M.G. Custrol et (11.I Molecular nnd Cellular Endocrinology 107 (1995) 17-27

22

ma

1

16

2

15

3

14

4

13

5

12

6

11

7

6

lo 9 *".,'h

431,

29+

Fig. 2. Metabolic labelling of CHO-Kl cells stably transfected with the recombinant plasmid pEEl4lrat pm-proCRH cDNA. (A) Immunoprecipitated culture medium and cell extracts, respectively, from transfected CHO-Kl cells. Lanes I, 2, after 10 min labelling; lanes 3, 4, after 20 min labelling; lanes 5.6. after 40 min labelling; lanes 7. 8 after 80 min labelling. (B) Immunoprecipitated culture medium and cell extracts, respectively, from transfected CHO-Kl cells. Lanes 9, 10, after 160 min labeling; lanes 1 I, 12, after 320 min labelling; lanes 13, 14, culture medium and cell extract from transfected CHO-K 1 cells immunoprecipitated with normal rabbit serum after 320 min labelling; lanes 15, 16, culture medium and cell extract from wildtype CHO-Kl cells after 320 min labelling. All samples were immunoprccipitated using the anti-proCRH antibody 3B3 at a 1150 final dilution. Protein molecular weight markers am indicated to the left of the figure. The major proCRH immunoreactive band is indicated with an arrow to the right of the figure.

methods. Cell counts of wildtype cells showed arrested growth after 3 days of serum starvation (Table 2), as did cells transfected with pEE14. Transfected cells, however, were still capable of dividing for up to 72 h (Table 2) in serum-free medium. Similar results were obtained with two different clones of stably transfected CHO-Kl cells expressing proCRH (not shown). The total cellular DNA content of transfected cells expressing proCRH was significantly higher than that of wildtype CHO-Kl cells both in the presence and in the absence of serum after 48 h incubation when utilizing the Promega cell proliferation assay, which measures DNA concentration (Table 3). The DNA content of transfected CHO-Kl cells compared to wildtype cells in the presence of serum is 1.6 times higher (P < O.OOl), while in the absence of serum it is 2.9 times higher (P < 0.001). This is due to the fact that serum contains uncharacterized factors which stimulate DNA synthesis, while in serum-free medium, proCRH is the only putative mitogenic factor

which is not present in wildtype CHO-Kl cells or in CHO-Kl cells stably transfected with pEE14. Similar results have also been obtained with CHO-Kl cells transfected with pEE14. Since transfected CHO-Kl cells would be constantly releasing proCRH into the culture medium (which could act as a mitogenic factor), we decided to synchronize the cell cultures at the GUS interface by exposure to hydroxyurea (Han et al., 1993). After removal of the drug, S-phase progression was monitored by incorporation of [3H]thymidine. Results from these studies (Table 4) shown for wildtype CHO-Kl cells, CHO-Kl cells transfected with pEE14 and transfected CHO-Kl cells expressing proCRH (clone 2/l), demonstrate that the transfected cells expressing proCRH have higher incorporation of r3H]thymidine over a time course ranging from 2 h to 10 h after the removal of the hydroxyurea block and incubation in serum-free medium. The maximal stimulation in the incorporation of [3H]thymidine is seen 10 h after the removal of the hydroxyurea block with an

M.G. Castro1 et al. I Molecular and Cellular Endocrinology 107 (1995) 17-27

1

2

3

4

5

6

43-l--+

M-7+

5.54

Fig. 3. Metabolic labelling followed by subcellular fractionation immunoprecipitation with the CRH-specific antibody 3B3 at a final dilution of 1150, followed by SDS-PAGE and autobradiography of tiansfected CHO-Kl cells. Lane 1, total wildtype CHO-Kl cell extract; lane 2, total cell extract from CHO-Kl cells stably transfected with pEE14; lane 3, total cell extract from CHO-Kl cells stably transfected with the plasmid pEEl4/rat pre-proCRH (CHO-Kl clone 2/l); lane 4, microsomal fraction from CHO-Kl cells clone 2/l; lane 5, nuclear fraction from CHO-Kl cells clone 2/l; lane 6, mitochrondial fraction from CHO-Kl cells clone 2/l. Protein molecular weight markers are indicated to the left of the figure.

330 k 14.8% (P < 0.001) when compared to CHO-Kl cells or CHO-Kl cells transfected with plasmid pEE14 alone. Similar results were obtained with increase

of

wildtype

23

the second transfected CHO-Kl clonal cell line analysed l/22 (results not shown). These results suggest that proCRH expression in transfected CHO-Kl cells plays a role in stimulating DNA synthesis and cell proliferation in these cells. The effects of proCRH on cell proliferation and DNA synthesis were also studied using AtT20/D16-16 cells (Table 5). Total cellular DNA content in AtT20/D16-16 cells was stimulated by both serum-containing medium and serum-free conditioned media from transfected CHOKl cells expressing proCRH, when compared to basal DNA content (serum-free medium). No stimulation was observed after treatment with serum-free conditioned medium from wildtype CHO-Kl cells. In order to demonstrate that the increase in DNA content in AtT2OiD1616 cells was due to proCRH, serum-free conditioned media from transfected CHO-Kl cells expressing proCRH was immunmoprecipitated using a CRH-specific antibody (Matthew at a final dilution of 1: 100) which cross-reacts with the precursor (Table 5). The antibody-proCRH complexes were separated using SAC, and AtT2O/D16- 16 cells were exposed to the supernatant. No stimulation above basal DNA levels was observed, therefore demonstrating that the stimulation in DNA content seen was due to proCRH present in the serum-free conditioned medium for stably transfected CHO-Kl cells. ProCRH secreted from stably transfected CHO-Kl cells in serum-free medium also stimulated DNA synthesis as assessed by the incorporation of [3H]thymidine in AtT20/D16-16 cells (Table 6). This effect was abolished if cells were treated with immunoprecipitated serum-free medium from stably transfected CHO-Kl expressing proCRH, once again indicating the specificity of the effect seen. Maximum stimulating effects on DNA synthesis were seen with medium containing serum, due to the presence of various growth factors. No significant differ Table 3

Table 2 Comparison of cell proliferation properties of wildtype CHO-Kl cells, CHO-Kl (pEEl4) cells and CHO-Kl (2/l) cells, which express proCRH Treatment (h)

24 48 72 96

CHO-K 1 cells (cells/dish) DMEM (-S)

CHO-Kl (pEBl4) cells (cells/dish) DMEM (-1)

Transfected CHO-Kl(2.U) cells (ceils/dish) DMEM (-S)

14062 f 2790 16250 * 1005 14300 f 1895 1625Oi2338

13500 * 1800 15350 f 2350 15800*1985 1635Ok2280

17500 i 2338 26785 i 1944 37000*3100 36428 i 3524

Synchronized cells were plated at a density of lo4 cells/dish and incubated during 24 h, 48 h, 72 h and 96 h with DMEM without serum (-S). Cells were then released from each well by treatment with trypsin and were dispersed for counting in a haemocytometer. Means and SEM of each treatment group (n = 4) are presented.

Total cellular DNA content of wildtype CHO-Kl cells; CHO-Kl (pEEl4) cells and stably transfected CHO-KI (2/l) cells expressing proCRH Cell type

OD 570 nm

CHO-Kl (+S) CHO-Kl (pEE14) (+S) CHO-KI (2/l) (+S) CHO-Kl (-S) CHO-Kl (pEE14) (-S) CHO-Kl(2/1) (-S)

0.207 +-0.012 0.201 i 0.008 0.340 + 0.0122 0.021 i 0.002 0.023 + 0.003 0.061 f 0.005*

Cells were grown to confluency in 96-well plates, and serum starved for 48 h. The serum iiee medium was then replaced with either DMEM containing 10% FCS (+S) or DMEM serum free media (-S). After 48 h, the cells wem washed, and the cell proliferation assay was performed according to the supplier’s specifications. Results am expressed as the mean f SEM, for n = 8. *P
Fig. 4. Immunocytochemical localisation of CRH in CHO-Kl cells stably transfected with the recombinant plasmid pEEl4/rat pre-proCRH cDNA. (A),(B) Transfected CHO-Kl cell stained with the rabbit anti-CRH (l-41) antibody, 3B3. Note the strong perinuclear and reticular cytoplasmic staining. Note also the strong IR-CRH staining of structures within the nucleus of the cell. (C) Transfected CHO-Kl cell stained with the sheep anti-CRH (l-41) antibody, 116. Note the perinuclear staining pattern. (D) Cells shown in (A) stained with the DNA-specific dye DAPI. Compare with the CRH nuclear immunoreactivity in (A). (E) Histochernical staining of the Golgi apparatus in the cells shown in (B) using fluorescently labelled wheat germ agglutinin. The staining pattern co-localises with the cytoplasmic but not the nuclear CRH immunoreactivity observed in (B). (F) Immunofluorescent staining of the Golgi apparatus of the cell shown in (C), using a rabbit anti-TGN38 antibody. Note the co-localisation with the perinuclear staining in (C). (G),(H),(I) phase contrast images of the cells described above.

N P

M.G. Castro1 et al. I Molecular and Cellular Endocrinology 107 (1995) 17-27

25

Table 4 Incorporation of [3H] thymidine in GUS synchronized transfected with pEEl4lrCRH cDNA (clone 2/l) Treatment

wildtype CHO-Kl

cells, CHO-Kl

cells stably transfected

with pEEl4,

and CHO-Kl

cells stably

Time

CHO-Kl CHO-Kl

(-S) (pEE14) (-S) CHO-Kl (2/l) (pEEWrCRH)

(-S)

2h

4h

6h

10 h

9697 + 1050 9540 + 540 16618 + 150

9220 f 1200 9350 * 1050 20611 f 1500

9364+850 905Ok680 15138 k950

17655 f 3500 1554Oi2050 58400 f 4350

Cells were grown to confluency and synchronized in GUS as described in Section 2. The synchronized cells were detached by incubation with trypsin/EDTA, and seeded onto poly-t_-lysine coated 24-well plates. S-phase progression was assessed by measuring the incorporation of [3H]thymidine in serum free medium. At 2, 4, 6 and 10 h the cells were processed for TCA-insoluble radioactivity as described in Section 2. The results ate shown as mean f standard error of the mean for quadmplicate

determinations.

ences from basal DNA synthesis were seen when AtT20/D16-16 cells were treated with serum free conditioned medium from wildtype CHO-Kl cells (Table 6) or CHO-Kl cells transfected with pEE14 (not shown). 4. Discussion

The expression of secretory proteins in heterologous cell types is now widely accepted as a useful method for the study of processing and trafficking of these proteins and as an abundant source of material for studies of biological activity. The cell line used in this paper is one of the most commonly used for this type of work and was chosen because these cells were not expected to endoproteolytically cleave the CRH precursor molecule and would thus provide a good source of intact proCRH for biological activity studies.

Table 5 Effects on total cellular DNA content after treating AtT20iD16-16 cells with conditioned media from wildtype CHO-Kl cells and stably transfected CHO-Kl cells (clone 2/l) expressing r/pm-proCRH OD 570 nm

Cell type AtT20/D16-16 AtT2O/Dl6-16 AtT20/D16-16 AtT20/Dl6-16 AtT20/Dl6-16

(-S) (Kl) (2/l) (2/l; Ipp) (+S)

0.061 0.062 0.101 0.060 0.111

+ 0.004 k 0.003 * 0.012* i 0.005 *0.014*

AtT20/D16-16 cells were grown to confluency on poly-lysine coated %-well plates and serum starved for 48-72 h. The serum free medium was then replaced with either DMEM containing 10% FCS (+S); DMEM serum free medium (-S); conditioned DMEM medium without serum from wildtype CHO-Kl cells (Kl); conditioned DMEM medium without serum from stably transfected CHO-Kl cells, clone 2/l (WI), or conditioned DMEM medium without serum from stably transfected CHO-Kl cells, clone 2/l, which had been immunoptecipitated with anti-CRH antibody (Matthew) used at a final distribution of 1: 100 (2/l; Ipp). After 36 h, the cells were washed with PBS and the cell proliferation assay was performed, according to the supplier’s specifications (see Section 2). Results are expressed as the mean + SEM for n = 8. n.s., not sign&ant; *P < 0.05 versus AtT20/D16-16 (-S)).

The present study shows no evidence for endoproteolytic processing of proCRH expressed in CHO-Kl cells. We have detected no low molecular weight IR-CRH peptides in our transfected CHO-Kl ceI1 lines. Given the high levels of CRH expression in these cell lines any processing intermediates should have been readily detectable by immunoprecipitation and SDS-PAGEVautoradiography. The reasons for the lack of endoproteolytic processing of proCRH in transfected CHO-Kl cells are at present unclear, since these cells do possess furin-like enzymes within their secretory pathway (Hatsuzawa et al., 1992), and the CRH precursor molecule does possess furin-like cleavage sites (a.a. 89-93: Arg-Gly-Ser-Arg and a.a. 141144: Arg-Glu-Arg-Arg) within its primary amino acid sequence (Thompson et al., 1987). This finding suggests that tertiary conformation and/or cellular specific factors also play an important role in determining the cleavage products originated from one precursor molecule. A lack of a regulated secretory pathway is the major difference between CHO cells and cells which normally express CRH (e.g. hypothalamic neurons). Specialised secretory cells, such as those of the endocrine and nervous systems, possess two biochemically and morphologically distinct secretory pathways, the constitutive and regulated secretory pathways. Peptides secreted by the regulated secretory pathway are stored in large, densecore granules, and their release is stimulated by secretagogues. In contrast, all cells possess a constitutive secretory pathway. In cells which do not possess a regulated secretory pathway, dense-core vesicles are absent and proteins are exported continuously, with no storage. Transfected CHO-Kl cells expressing proCRH store more proCRH than they release into the culture medium (Table 1). These results could be explained by the fact that proCRH in these cells is also localized within the cell nucleus where it binds to DNA (Morrison et al., 1993), therefore preventing it from being secreted. The results from the metabolic labelling experiments show that newly synthesized proCRH is mainly secreted into the culture medium where with time, it accumulates to high levels. In contrast, the concentration of proCRH in

26

M.G. Cus~ml et (11.I Molecular and Cellular Endocrinology 107 (1995) 17-27

Table 6 Effects of proCRH secreted by stably transfected

CHO-KI

Treatment

cells (clone 2/l) upon incorporation

by AtT20/D16-I6

cells

Time 7h

3h Medium containing serum (+S) CHO-KI (2/l) conditioned medium (-S) CHO-KI conditioned medium (-S) Medium without serum (-S) CHO-KI (2/l) conditioned medium (-S), immunoprecipitated

of [3H]thymidine

396.5 196.8 85.4 78.8 80.2

r so.2* f 22.1* i S.S”-“i 6.2 -c 3.1”.‘.

431.1 341.7 168.8 158.1 170.1

12h +42.9* + 14.2* + I I.I”.“. T 9.8 + 10.1”.“.

215.8 + 172.6+ 96.7 f 102.1 k 98.4 k

26.5* 11.4* 5.F~ 4.3 3.8”,“,

AtT20/Dl6-16 cells were grown to confluency, the cells were arrested in GO by serum starvation during 3 days. After this time. the cells were mechanically dispersed and 100 000 cells/well were plated onto 24-well plates which had been previously coated with poly-L-lysine. Cells were allowed to attach overnight, and the next day they were treated with medium containing serum (+S); conditioned serum free medium (-S) from CHO-KI cells; conditioned serum free medium (-S) from transfected CHO-K I cells (clone 2.1); serum free medium (-S); and conditioned serum free medium (-S) from transfected CHO-Kl cells (clone 2/l) which had been immunoprecipitated using a CRH-specific antibody (Matthew) at a final dilution of l:lOO. [3H]Thymidine was added to each well 2 h prior the end of the incubation period (OS&i/ml). Cells were then washed twice with PBS, and DNA and proteins were precipitated with 10% TCA. After incubation for 30 min at 4°C. the pellet was resuspended with 0.5 M NaOH containing 0.1% SDS and then neutralized with an equal volume of 0.5 M acetic acid. Incorporated 13H]thymidine was determined by scintillation counting. Results arc expressed as dpm x 10-3/105 cells/well for n = 4. Not significantly different from treatment with medium without serum (basal), n.s.; *P < 0.001 when compared to treatment with medium without serum (basal).

the cell extract remains fairly constant during the same time course. These results are in agreement with work carried out in AtT20 cells and in rat intermediate pituitary melanotropes by Noel and Mains (1991). These authors demonstrate that both cell types showed preferential basal secretion of newly synthesized and not fully mature older peptides. Conversely, these cells showed preferential stimulated secretion of older peptides. Immunofluorescent localisation of IR-CRH in transfected CHO-Kl cells shows that the CRH precursor is present within the cytoplasm; it also shows that IR-CRH is present in a novel subcellular location, the nucleus of these cells (Morrison et al., 1993). This novel subcellular location was confirmed using subcellular fractionation techniques; a CRH-IR band of 19 kDa was detected within the purified nuclear fraction of transfected CHOKl cells. We have previously demonstrated nuclear IRCRH in the nucleus of human T-lymphocytes (Ekman et al., 1993). This would suggest a possible physiological role for nuclear CRH and it also supports the dual intracellular localisation of a secretory protein (CRH) in cells which produce this neuropeptide endogenously. This dual intracellular localisation has also been shown for the int-2 oncoprotein (Acland et al., 1990), PDGFrelated antigens (Yeh et al., 1994). ProCRH could be translocated to the cell nucleus through interactions with other cytoplasmic proteins destined to the nucleus which contain a nuclear localisation signal (NLS) for nuclear import. It has been demonstrated that proteins which lack an NLS can ‘piggy back’ into the nucleus in this way (Moreland et al., 1987; Booker et al., 1989). Another possibility which could explain nuclear translocation of proCRH is through the usage of alternative initiation cordons as has been described for fibroblast growth factor (Bouche et al., 1987), the product of the int-2 gene

(Acland et al., 1990) and the rat prostatic protein, probasin (Spence et al., 1989). Interestingly, we have presented evidence in this paper which suggests that expression of proCRH in transfected CHO-Kl cells has an effect upon the cell proliferation properties of these cells. It is tempting to speculate that proCRH could play an intracrine (O’Malley, 1989) stimulatory role on cell proliferation in transfected CHOKl cells. We have also shown that proCRH expressed in CHO-Kl cells can play a role in stimulating DNA synthesis and cell proliferation in anterior pituitary corticotrophic tumour cells (AtT20/D16-16). A possible mitogenie role for CRH (l-4 1) has been well documented, at the anterior pituitary level (McNicol et al., 1988; Gertz et al., 1987; Asa et al., 1992) where it exerts its effects upon corticotrophic cells which possess specific CRH receptors (Chan et al., 1993). A mitogenic role for CRH (1-41) has also been reported for rat P-lymphocytes (McGillis et al., 1989) although its action is not mediated through the same CRH receptor which is present in anterior pituitary corticotrophs. Our results using AtT20/D16-16 cells also suggest a role for the intact proCRH molecule in mediating cell proliferation at the corticotrophin-cell level. In stably transfected CHO-Kl cells it seems likely that such a mitogenic role could be mediated via intracellular proCRH, as has been suggested for other hormones and growth factors (Radulesu and Wendtner, 1993). Acknowledgements This work was supported by project grants from the MRC (UK) to MGC and PJL, and from the BBSRC (SERC) to MGC and PRL. MGC and PRL would also like to acknowledge the support received from The Wellcome Trust, The Welsh Scheme for the Development of

M.G. Custrol et al. I Molecular and Cellular Endocrinology 107 (1995) 17-27

Health and Social Research, The Sir Halley Stewart Trust, The Royal Society and the Department of Physiology, UWCC. PRL is a Research Fellow from the Lister Institute of Preventive Medicine. CAM wishes to acknowledge the BBSRC (SERC) for a studentship. We would like to express our appreciation to Dr. Y.A. Corredoira, Mr. S. Turner, Mr. H. Bines and Mr. J. Robertson for excellent technical and administrative assistance, and Mr. R. Jones for expert photographic work. We would like to express our appreciation to Professor D. WynfordThomas, Professor S. Lightman, Dr. A. White, Dr. D. Carter, Dr. G. Gillies and Dr. G. Banting for their careful review of our manuscript, and for many very useful suggestions. We thank Dr. M. Cockett (Celltech Ltd., Slough, UK) for his generous gift of the expression vector pEE14 and his advice to establish stably transfected CHO-Kl cells, Dr. R. Thompson (Mental Health Research Institute, University of Michigan, MI, USA) for the rat preproCRH cDNA clone and Mrs. C. Hayes and M. Treloar for their enthusiastic and skilful secretarial assistance. We also wish to acknowledge Professor V. Crunelli for his support and encouragement. References Acland, P.. Dixon, M., Peters, G. and Dickson, C. (1990) Nature. 343, 662-665. Booker, R.N., Alfar, C.E., Hyams, J.S. and Beech, D. (1989) Cell 58, 485497. Bouche, G., Gas, N., Prats, H., Baldin, V., Tauber, J.P., Teissie, J. and Almeric, F. (1987) Proc. Natl. Acad. Sci. USA 84,67706774. Castro, M.G., Birch. N.P. and Lob, Y.P. (1989) I. Neurochem. 52, 1619-1628. Castro, M.G., Spruce, B.A., Sawa, D. and Lowry, P.J. (1990a) Int. J. Biochem. 22, 1341-1349. Castro, M.G., Brooke, J., Bullman, A., Hannah, M., Glynn, B.P. and

27

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