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J. Embryol. exp. Morph. 95,193-212(1986)
193
Printed in Great Britain © The Company of Biologists Limited 1986
Developmentally regulated expression of insulin-like
growth factors by differentiated murine
teratocarcinomas and extraembryonic mesoderm
J O H N K. H E A T H AND WAI- KANG SHI*
Department of Zoology, University of Oxford, South Parks Road,
Oxford OX1 3PS, UK
SUMMARY
The expression of plasma membrane receptors for insulin-like growth factors (IGFs) by PC13
embryonal carcinoma (EC) cells, and their immediate differentiated progeny PC13END was
examined by binding radiolabelled IGF-I to cell monolayers. Both cell types express highaffinity IGF receptors, but the apparent number of unoccupied receptor sites falls by about 60 %
upon differentiation. Crosslinking studies reveal that both type 1 and type 2 IGF receptors are
expressed by PC13EC cells.
PC13END-cell-conditioned medium contains developmentally regulated, separable activities,
one of which reacts directly with IGF-II, and the other with IGF for plasma membrane
receptors. The former activity represents a soluble secreted IGF-binding protein. The latter
activity is structurally and functionally similar to rat IGF-II.
Polyclonal antibodies raised against purified rat IGF-II specifically recognize multiple forms
of IGF in radiolabelled culture supernatants and material which closely resembles the soluble
IGF-binding protein. Immunoprecipitation of radiolabelled culture supernatants with anti-rat
IGF-II reveals that the differentiation of PC13EC cells is accompanied by the coexpression of
IGF-like molecules and the soluble binding protein, and that IGF-like molecules are expressed
by extraembryonic tissues of mesodermal origin in the early postimplantation mouse embryo.
These findings show that IGF-like molecules are expressed in early mammalian development
and may act in an autocrine fashion in vivo.
INTRODUCTION
The insulin-like growth factors (IGF) I and II are single-chain polypeptide
hormones, which have structural similarities to insulin (Rindernecht & Humbel,
1918a,b). IGFs exert a number of biological effects upon cells in culture including
the induction of cellular DNA synthesis and cell multiplication, and stimulation of
sugar and amino acid transport (reviewed by Rechler etal. 1981). The levels of
circulating IGF-I in plasma are regulated by growth hormone and IGF-I is
consequently implicated as a mediating agent in the effects of growth hormone on
skeletal growth during puberty (reviewed by Van Wyk et al. 1974). Serum IGF-II
concentrations are not regulated by growth hormone but are elevated in the foetus
* Present address: Shanghai Institute of Cell Biology, Academia Sinica, 320 Yo-Yang Rd,
Shanghai, China.
Key words: insulin, growth factors, teratocarcinoma, mouse embryo, extraembryonic meso­
derm, expression.
194
J. K. HEATH AND W.-K.
SHI
(D'Ercole & Underwood, 1980; Moses etal. 1981), leading to the suggestion that
IGF-II may regulate foetal growth in vivo (D'Ercole et al. 1980). There is, in fact,
evidence that IGF-II-like molecules are synthesized by multiple tissues in the
foetus, including liver (Rechler etal. 1979), fibroblasts (Adams etal. 1983), myo­
blasts (Hill etal. 1984), kidney, heart, lung, limb bud mesenchyme, intestine and
brain (D'Ercole etal. 1980). More recently Scott etal. (1985) have shown that
IGF-II transcripts are widespread in first trimester human foetal tissues. These
findings have consequently led to the proposition that IGF-II acts as a paracrine
growth factor in vivo where local secretion results in the proliferation of
neighbouring cell types (D'Ercole etal. 1980, Hill etal. 1984).
The biological effects of IGFs are exerted through association with specific
plasma membrane IGF receptors. Two distinct species of IGF receptor have
been demonstrated by crosslinking radiolabelled IGFs to plasma membranes. The
type 1 IGF receptor structurally resembles the insulin receptor, and comprises a
tetramer of two or chains (relative molecular mass Mx 130 000) and two ß chains (Mr
60000) linked by disulphide bonds (Masague & Czech, 1982). The type 1 IGF
receptor binds IGF-I and IGF-II with high affinity, and insulin with lower but
significant affinity. The type 2 IGF-receptor is a single-chain molecule of apparent
relative molecular mass 260000 (Masague et al. 1981) which binds both IGF-I and
IGF-II with high affinity, but has no significant affinity for insulin (Masague &
Czech, 1982).
That IGF-II-like molecules may be expressed and function, in embryonic stages
of mammalian development, arises from the finding that murine embryo-derived
embryonal carcinoma (EC) cells express receptors for, and respond to, exogenous
IGF-II in vitro (Nagarajan et al. 1982; Stern & Heath, 1983; Heath & Délier, 1983;
Heath & Rees, 1985). Here we report that the induction of PC13EC cell differ­
entiation in vitro by retinoic acid is accompanied by a drop in the number of
available plasma membrane IGF receptor sites and in the developmentally regu­
lated expression of molecules which, in structure and activity, resemble IGF-II.
The use of antibodies directed against rat IGF-II reveals that multiple forms of
IGF are expressed in vitro and that IGF-like molecules are synthesized in the
embryo by extraembryonic tissues of mesodermal derivation.
MATERIALS AND METHODS
Cells and cell culture
The origin and preparation of cell culture materials has been previously described (Heath &
Délier, 1983). The Buffalo rat liver (BRL) cell line was obtained from the cell bank, Sir William
Dunn School of Pathology, University of Oxford, and cultured as described (Heath & Délier,
1983). The maintenance and retinoic-acid-induced differentiation of PC13EC clone 1A4 cells
was as described by Heath & Délier (1983).
Preparation of conditioned media
PC13END-cell-conditioned medium was prepared from PC13END cells generated by RA
exposure of PC13EC cells in 175 cm2 culture flasks. Following 5 days' exposure to differ­
entiation-inducing conditions PC13END cell monolayers (approximately 107 cells per flask)
Expression of insulin-like growth
195
were washed extensively with basal medium and then cultured for 2 days in 100 ml ECM serumfree supplemented medium (Heath & Délier, 1983) per flask at 37 °C in an atmosphere of 5 %
C0 2 in air. Conditioned medium was decanted, centrifuged at 9000g for 30min and
concentrated approximately 150-fold in a stirred cell ultrafiltration device (Amicon, UK)
equipped with YM2 membranes.
BRL-cell-conditioned medium was prepared from cells cultured in 1030 cm2 glass roller
culture vessels in 200 ml DME: F12 (50:50 vol: vol) supplemented with 5 % calf serum (Gibco) at
a density of approximately 106 cells per vessel. The cells were cultured with continuous rotation
at 37°C for 3 days in an atmosphere of 5% C0 2 in air. The medium was changed to 200 ml
DME:F12 (50:50 vol: vol) supplemented with iron-conjugated transferrin at 5jUgml-1 and
cultured as above. The medium was removed after 24 h and replaced with 200 ml of fresh
DME:F12: transferrin, the first serum-free medium collection was discarded and the medium
was subsequently collected and replaced every 2-3 days with fresh serum-free medium as above.
Media collections could be made for up to 14 days before overt cell degeneration became
evident. Conditioned medium (approximately 101 per batch) was centrifuged at 9000g for
30 min to remove cells and debris and concentrated 50-fold in a hollow fibre filtration apparatus
(DC2 Amicon, UK, HIP2-43 cartridges).
IGF radioreceptor assay and crosslinking
IGF-I (Amersham, UK), IGF-II, and experimental fractions were iodinated by the iodogen
method. Freeze-dried peptides were dissolved in 10/xl 0 1 % TFA for 20 min at room tempera­
ture and diluted to 100 /A by addition of 0-2M-phosphate buffer pH7-8. The dissolved peptides
were added to a vial precoated with lOjUg iodogen (Pierce, UK) and the reaction initiated by
addition of 0-5-1 mCi 125I (Amersham, UK). The mixture was incubated for 20 min at room
temperature and then terminated by addition of KI and tyrosine to a final concentration of
0-1 M. Following a further 5 min incubation the mixture was diluted to 2 ml with 0-1 % TFA and
applied to a Sep-pak ODS cartridge (Waters, UK). The cartridge was washed with 10 ml 0-1 %
TFA in water, and iodinated peptides eluted with two 1 ml portions of 50 % acetonitrile/0-1 %
TFA in water. Specific activities thus obtained ranged from 46 000-104 000 ets min -1 ng - 1 .
IGF radioreceptor assays were performed on cell monolayers essentially as described by
Heath et al. (1981). PC13EC and PC13END cells were plated into gelatin-coated 3-5 cm
diameter dishes at a density of 105 cells/dish (EC) or 2x 105 cells/dish (END) in 4 ml F12: DME
supplemented with 5% foetal calf serum. After 24h culture the medium was changed to 4ml
ECM serum-free supplemented media (Heath & Délier, 1983) and the cell monolayers used for
binding assays 24 h later. PYS-2 cells were plated at a density of 5xl0 4 cells per well in 24-well
cluster plates in F12:DME 5% calf serum and used for radioligand binding assays 48 h later.
PC13 membranes were prepared by homogenization of PC13EC cells in lOmM-tris, lmMEDTA, 10% sucrose, pH7-2, followed by centrifugation at 3000g for 30 min at 4°C to remove
nuclei. A crude membrane fraction was prepared by centrifugation of the supernatant at
100000g for 1 h, the membrane pellet was resuspended in PBS and rehomogenized. Membranes
were stored in liquid nitrogen before use. For crosslinking studies, membranes were incubated
at 250 ng ml - 1 in binding buffer (Earles balanced salt solution, 20mM-Hepes, 0-5% bovine
serum albumin pH7-2) with 125I-IGF-I to a final concentration of InM, with or without l|Ug
unlabelled IGF-I. Following 2 h incubation at 4°C the membranes were pelleted by
centrifugation at 15000g for 10 min, resuspended in PBS and bound IGF-crosslinked by addition
of disuccinimidyl suberate (Pierce, UK, freshly dissolved in dimethyl sulphoxide) to a final
concentration of 0-25 mM or 0-5 IHM. Following 15 min incubation on ice the membranes were
repelleted as above and dissolved in 50/il SDS sample buffer with or without 50 mMdithiothreitol, separated by SDS-PAGE (5 % acrylamide resolving gels, Laemmli, 1970), dried,
and exposed to Fuji-RX X-ray film at -70°C in the presence of intensifying screens.
Purification ofIGF-II and separation methods
Rat IGF-II was purified by a modification of the methods described by Marquardt et al.
(1981). Concentrated BRL-conditioned media were dialysed at 4°C for 48 h against 0-1 M-acetic
196
J. K. H E A T H AND W . - K .
SHI
acid (201, two changes) in 'Spectrapor' dialysis bags (nominal cut-off 3000 daltons, Spectrum
Medical Industries, USA). Acid-insoluble material was removed by centrifugation (100000g
for 30min). The acid-soluble portion was freeze-dried, reconstituted in lM-acetic acid (1ml
for every 400ml of the original concentrated medium), and subjected to gel filtration at 4°C
on a 2-5 cm diameter x 90 cm length column of Biogel P-60 (200-400 mesh, BioRad, UK)
equilibrated in 1 M-acetic acid. The column was eluted at a flow rate of 10 ml h - 1 collecting 10 ml
fractions. 1ml samples from column fractions were freeze-dried and reconstituted in 1ml
phosphate-buffered saline (PBS) for the determination of growth-promoting activity and
protein concentration (method of Read & Northcote, 1982). IGF activity was assayed by
incorporation of trichloracetic-acid-insoluble [3H]thymidine into quiescent NRK 49F fibroblasts
as described (Marquardt et al. 1981) or by inhibition of 125I-IGF-binding to PYS cells. Three
zones of mitogenic (or IGF competing) activity were routinely observed. The latest eluting
fractions containing mitogenic activity (corresponding to molecular mass about 7000 daltons)
were pooled, freeze-dried, reconstituted in 0-01 M-trifluoroacetic acid (TFA, solvent A,
sequencer grade, Rathburn Chemicals, UK) and subjected to reverse-phase high-pressure liquid
chromatography (rpHPLC) on an Altex model 344 gradient liquid chromatography apparatus
with a C3 30nm pore size support (Ultrapore RSPC, Beekman, UK). Absorbed material was
eluted by application of a gradient of acetonitrile (HPLC grade S, Rathburn, UK), 0-01 M-TFA
(solvent B) at a flow rate of 1 ml min -1 . Sample elution was monitored with a Beekman (UK)
model 160 fixed wavelength u.v. detector equipped with 214 nm filters. A single peak of
mitogenic activity eluted at a nominal concentration of 28% solvent B. Peak activities were
concentrated by solvent evaporation and subjected to size exclusion chromatography on a TSKSW2000 (7 mm X 60 cm) column in 10% acetonitrile/0-1% TFA, in water at a flow rate of
0-5 ml min -1 . Two peaks of activity were obtained with Mr = 10000 and 7000.
Concentrated PC13END-cell-conditioned medium was adjusted to 1 M-acetic acid, centrifuged for 30 min at 100 000 g to remove acid-insoluble material and fractionated by Biogel P-60
chromatography and rpHPLC as above.
IGF-binding protein identification by chemical crosslinking
Freeze-dried fractions were reconstituted in phosphate-buffered saline. Crosslinking
reactions took place in 200 jul phosphate-buffered saline/1 % (by weight) bovine serum albumin
at a final sample protein concentration of 50jugml-1. The reaction was initiated by addition of
125
I-rat IGF-I to a final concentration of 0-5 nM in the presence or absence of 500 ng unlabelled
IGF-I, and incubated for 2 h at room temperature. Crosslinking was achieved by addition of
DSS to a final concentration of 0-2 mM followed by incubation at 4°C for 15 min. The crosslinking reaction was terminated by addition of double-strength SDS sample buffer and boiling
for 5 min followed by electrophoresis in 12-5 % Polyacrylamide gels containing SDS.
Preparation of antisera
IGF-II peptide (Mr = 7000 prepared as above) was coupled to keyhole limpet haemocyanin
(KLH) before immunization. 5mg KLH (Calbiochem-Behring) was dissolved in 450/il 0-1 MNaHC0 3 (pH9-0) and reacted with 50fû of 2-5% glutaraldehyde (BDH, UK) at room
temperature with continuous agitation. Free glutaraldehyde was removed after 2 h incubation
by desalting on a 0-9 cm diameterxllcm column of Biogel P-2 (200-400 mesh, Bio-Rad)
equilibrated in 0-lM-NaHCO3 (pH9-0). The glutaraldehyde-activated KLH was mixed with
250 jug of IGF-II peptide and incubated overnight at 4°C with constant agitation. The con­
jugation mixture (2 ml) was dialysed for 24 h at 4°C against 51 of 0-1 M-acetic acid in a 3000 Mr
cut-off 'Spectrapor' dialysis bag, divided into four 500 fi\ portions and freeze-dried. No attempt
was made to quantify the extent of conjugation. The freeze-dried portions were reconstituted in
200 jul of PBS and emulsified with Freunds adjuvant. Male New Zealand white rabbits were
injected subcutaneously on days 0 (complete adjuvant), 14, 28 and 38 (incomplete adjuvant).
Blood was taken on day 44 and at intervals thereafter. IgG fractions were prepared from
immune serum by 40 % ammonium sulphate precipitation, followed by dialysis of the precipitate
against 20mM-sodium phosphate (pH7-0) and chromatography on a 2-5 cm diameterx 10 cm
column DEAE-trisacryl (LKB, UK) equilibrated in the same buffer. Unabsorbed fractions
containing immune IgG were dialysed against PBS containing 0-01 % sodium azide and
Expression of insulin-like growth
197
concentrated by ultrafiltration to a final concentration of 2mgml~1. Antibody species directed
against KLH were removed by passing the immune IgG fractions through a 5 ml column of
KLH-Affigel (Bio-Rad, approx. 5mg KLH ml - 1 Affigel-10 prepared according to the manu­
facturers' recommendations) equilibrated in phosphate-buffered saline (PBS) and collecting the
unabsorbed material. The specificity of KLH-absorbed anti-IGF-II antisera was tested by
titration in a solid-phase ELISA assay against a variety of target peptides, including: porcine
insulin; desoctapeptide insulin; glucagon; epidermal growth factor; nerve growth factor;
platelet-derived growth factor; ovine growth hormone; bovine prolactin; human placental
lactogen; and four different preparations of rat IGF-II, two prepared in this laboratory, a
commercial preparation (rat multiplication-stimulating activity, Collaborative Research) and a
preparation of rat IGF-II generously provided by Dr M. Czech (Dept of Biochemistry,
University of Massachussets Health Centre, Worcester, MA, USA). Significant reactivity was
only observed with the preparations of IGF-II. Significant binding of the immune IgG
preparations to IGF-II peptides was observed at IgG concentrations above 20ngml -1 .
Embryos
Embryonic tissues were obtained from natural matings of C3HXC57/B16 ¥x mice. Embryos
were staged and dissected as described by Shi & Heath (1984).
Metabolic labelling and immunoprecipitation
Embryonic tissues were metabolically labelled with [35S]methionine in conditions described
previously (Shi & Heath, 1984). PC13EC, differentiated PC13EC cells (derived by retinoic acid
treatment) and BRL cells were plated at a density of 106 cells in a 25 cm2 tissue culture flask in
DME: F12 (50:50) supplemented with FCS (5 % by volume). The cells were incubated overnight
at 37°C in 5 % C0 2 in air. The cell monolayers were then washed once with PBS and the medium
changed to 5 ml methionine-free minimal essential medium (Gibco, UK) supplemented with
FCS (0-5% by volume) and 50//Ci ml"1 [35S]methionine (specific activity 2000 mCi mmol"1,
Amersham, UK), followed by culture for 16-18h in the conditions described above. Culture
supernatants were centrifugea! at 15000g for 15 min and divided into 1ml or 0-5 ml samples
which were either used for immunoprecipitation immediately or stored frozen at -70°C for up
to 2 weeks before use. No differences were noted between fresh or frozen material.
The procedures for immunoprecipitation and electrophoretic analysis of secreted products in
labelled culture media were as described (Shi & Heath, 1984). Immune IgG was used at a
concentration of 200 ^g ml"1.
Immunoblotting
Samples resolved by discontinuous electrophoresis in Polyacrylamide gels containing sodium
lauryl sulphate (buffer system of Laemmli, 1970) were electrophoretically transferred (Towbin
et al. 1979) to nitrocellulose paper (Sartorius, UK, type SM) and reacted with immune IgG and
alkaline-phosphatase-conjugated sheep anti-rabbit IgG (Serotec, UK, diluted 1/1000) as
described by Blake et al. (1984).
Peptide mapping
Acid-extracted BRL-conditioned medium was subjected to SDS-PAGE (resolving gel 15 %
acrylamide) in multiple sample slots (approx. 250 jug of protein per slot). Paired sample lanes
were excised from the gel after the bromophenol dye front had migrated to approx. 1 cm from
the end of the gel, sliced into 3 mm sections, and then cut in halves representing each sample
lane. Each section from one of the paired lanes was stored at -70°C in SDS sample buffer and
the sections from the other lane were equilibrated with SDS sample buffer and electrophoresed
on a second 15 % SDS-PAGE gel followed by transfer to nitrocellulose paper and reaction with
rabbit anti-IGF-II IgG to locate sections containing immunoreactive material. The corres­
ponding sections from the paired sample lane were then subjected to partial proteolysis with
198
J. K. H E A T H AND W . - K .
SHI
Staphylococcus protease V8 (Worthington, UK, lug/slot) and electrophoresis according to
Cleveland et al. (1977). Immunoreactive peptides were detected by immunoblotting as above.
RESULTS
(A) Expression of IGF receptors by murine teratocarcinomas
The expression of IGF receptors by teratocarcinoma cells was examined by
testing the ability of 125I-IGF-I to bind specifically to PC13EC cells and their
differentiated derivatives. Significant specific saturable binding of IGF-I to EC
cells and their differentiated derivatives was observed. Replotting the data
according to the method of Scatchard (1949) permitted an estimation of IGF
receptor number and affinity (Fig. 1); PC13EC cells express approximately 80000
sites (Kd 0-98 nM) per cell. These findings confirm the earlier reports of IGF
receptor expression by F9EC (Nagarajan et al. 1982) and PC13EC (Stern & Heath,
1983) employing IGF-II as the radiolabelled ligand. Upon RA-induced differ­
entiation the number of sites fell by about 60 % (to 20 000 sites per cell) whilst the
apparent affinity of IGF-I for PC13END cell receptor sites remained similar
(ATd = 0-83nM). Since the number of contaminating residual EC cells found in
PC13END cell preparations under our differentiation-inducing conditions is very
low (less than 0-01 %, Heath & Délier, 1983) it is probable that this represents a
genuine decline in the apparent number of unoccupied IGF receptor sites avail­
able after RA-induced differentiation.
0-04
0-03
3
/F
0-02-
0-01
10
20
30
40
IGF-1 bound (fmoles)
Fig. 1. Binding of 125 MGF-I to PC13EC ( • ) and PC13END ( ■ ) cells. 4 x l 0 5 cells
were incubated with varying concentrations of 125 I-IGF-I (0-01-2-5nM). Nonspecific
binding was determined in the presence of 1 ^g IGF-I. The data were plotted according
to the method of Scatchard (1949).
Expression of insulin-like growth
a
Mrx
1CT3
240 ►
215^
«1
88K
Fig. 2. Crosslinking 125I-IGF-I to PC13EC cell membranes. 125I-IGF-I (2nM) was
bound to EC cell membranes in the presence (lane a) or absence (lanes b, c, d, e) of
1 jug unlabelled IGF-I. Bound IGF-I was crosslinked with either 0-25 mM (lanes a, b, c)
or 0-5 mM (lanes d, e) disuccinimidyl suberate followed by SDS-PAGE (5% acrylamide in resolving gel) under reducing (lanes a, c, e,) or nonreducing (lanes b, d)
conditions. MT, relative molecular mass of marker proteins. 1 indicates position of a
chain of type 1 receptor and 2 the position of the type 2 receptor.
IGF-I and IGF-II have been found to react with two different receptor species
on a variety of cultured cell types when analysed by crosslinking labelled ligand to
membrane receptor sites (Masague & Czech, 1982). The identity of IGF receptors
expressed by PC13EC cells was determined by allowing radiolabelled IGF-I to
react with PC13EC cell membranes followed by crosslinking with disuccinimidyl
suberate (DSS, Pilch & Czech, 1980) and electrophoresis in Polyacrylamide gels
containing SDS (SDS-PAGE). Electrophoresis under nonreducing conditions
revealed a number of specifically labelled molecular species ranging from Mr =
280000 to Mr = 330000 (Fig. 2). Electrophoresis under reducing conditions
yielded two molecular species; one of Mr = 260000 which corresponds to the
reported size of the type 2 IGF receptor, and a second of MT = 130000 corres­
ponding to the or chain of the type I (insulin-like receptor). In common with other
studies (e.g. Masague & Czech, 1982) the ß chain of the type 1 receptor, which
does not interact directly with the ligand, would not be detected under the
200
J. K. H E A T H AND W . - K .
BSA OV
T
V
SHI
LY
DNP
T
V
0-2-1
;
♦
A
i
1
♦
<001
5
10
15
20
25
30
Fraction number
Fig. 3. Fractionation of PC13END-cell-conditioned media on Biogel P-60 in 1Macetic acid. 41 of PC13END-cell-conditioned media was concentrated by ultrafil­
tration, solubilized in 1 M-acetic acid and subjected to gel filtration on Biogel P-60.
10 ml fractions were collected, and 200/4 samples from each fraction were freezedried and tested for the ability to compete with 125 I-IGF-I binding to PYS cells.
100 000 ets min - 1 1 2 5 I-IGF-I were used for each fraction. Inhibition of IGF-I binding is
presented as a percentage of the inhibition of binding in the presence of 1 fig unlabeled
IGF-I. Fractions corresponding to inhibitory zones A and B are marked with a bar.
The column was calibrated with bovine serum albumin (BSA), ovalbumin (OV),
lysozyme (LY) and DNP-lysine.
conditions employed. PC13EC cells therefore express both type 1 and type 2 IGF
receptors.
(B) Expression oflGF-like molecules by PC13END cells
We considered the possibility that the apparent fall in the number of IGF
receptor sites expressed by PC13EC cells upon RA-induced differentiation was
due to the developmentally regulated endogenous expression, by PC13END cells,
of molecules which inhibited the binding of exogenous IGF-I either by reaction
with IGF-I itself, or with the corresponding plasma membrane receptors. Serumfree ECM media conditioned by PC13END cells were concentrated by ultra­
filtration, solubilized in 1 M-acetic acid and subjected to gel filtration on Biogel
P-60. Individual fractions were tested for the ability to inhibit the binding of IGF-I
to PYS-2 parietal yolk sac cells. Two zones of inhibitory activity were observed;
zone A eluting near the ovalbumin size marker and zone B eluting around and
after the lysozyme size marker (Fig. 3). No IGF-I-binding inhibition activity was
recovered by similar analysis of either PC13EC cell conditioned or unconditioned
ECM media (not shown).
Expression of insulin-like growth
201
a b e d
Mrx
10
-3
-
*m ■
#
67 ► «» a p
**.
46 ►
21^
Fig. 4. Identification of IGF-binding protein in zone A fractions by crosslinking. 200 fA
samples of Zone A fractions were pooled, freeze-dried and reconstituted in 200/il
PBS/1% BSA and incubated with 0-5 nM 125I-IGF-I for 2h at room temperature.
Following crosslinking with DSS and quenching with SDS sample buffer the samples
were separated by SDS-PAGE (12 % acrylamide). Mt, relative mobility of marker
proteins, (a) 125I-IGF-I alone, (b) 125I-IGF-I+500ng unlabelled IGF-I, (c) zone A
fraction omitted, (d) crosslinking reagent omitted.
The existence of two similar classes of IGF-inhibitory activity has been reported
in the analysis of media conditioned by other IGF-secreting cells (Marquardt et al.
1981; Anderson et al. 1984) and tissues (Rechler etal. 1979). The high molecular
weight material has been ascribed to the presence of a specific soluble IGF-binding
protein ( Knauer et al. 1981; Knauer & Smith, 1982). We examined the possibility
that zone A contained binding protein activity by testing the ability of zone A
fractions to bind IGF-I directly. Zone A fractions were pooled, freeze-dried,
reconstituted in PBS/BSA, incubated with 125I-IGF-I in the presence or absence
of excess unlabelled IGF-I and then subjected to crosslinking with DSS, followed
by SDS-PAGE. A single species of approximate M r = 40000 was specifically
labelled in the presence of 125I-IGF-I; this species was not labelled in the presence
of 500-fold excess unlabelled IGF-I or in the absence of zone A material or
crosslinking reagent (Fig. 4). We conclude that the zone A contains a species
which binds directly to IGF-I of approximate Mr = 35 000 (derived by subtracting
the molecular weight of IGF-I from 40000 and assuming that one molecule of
IGF-I binds one binding protein molecule.
Zone B fractions were pooled and further fractionated by reverse-phase highpressure liquid chromatography in a solvent of 0-1 % TFA in water, eluting with a
202
J. K. HEATH AND W.-K.
SHI
linear gradient of acetonitrile/0-1 % TFA (Fig. 5). Individual fractions were tested
for the ability to inhibit IGF-I binding to PYS-2 cells and stimulate DNA synthesis
in quiescent NRK rat fibroblasts by [3H]thymidine incorporation. A major peak
of IGF-binding inhibition, coincident with a peak of DNA-synthesis-promoting
activity and a u.v. absorption peak, was observed which eluted at a similar nominal
60-i
50-
0-01
optical units
214 nm
40-
< 30-
20-
**H»»MV»»*
(YflxPaoocPchono 6oQ
Fig. 5. Separation of pooled zone B fractions by reverse-phase high-pressure liquid
chromatography. Elution conditions are described in Materials and Methods. 1ml
fractions were collected and 50 [A samples tested for induction of DNA synthesis
in quiescent NRK fibroblasts by [3H]thymidine incorporation ( ♦ ) or inhibition of
125
MGF-I binding to PYS-2 cells (O).
Expression of insulin-like growth
203
acetonitrile concentration to authentic rat IGF-II (26% acetonitrile). A second
minor peak with mitogenic activity and IGF-inhibitory activity eluted about 8 min
later. This finding suggested that the IGF-I-binding inhibition activity present
following reverse phase chromatography separation was due to the presence of an
activity which, in structure and function, resembles IGF-II. Samples of the peak
fractions from reverse-phase separations were freeze-dried, radiolabelled with
125
I, and tested for their ability to bind to PYS-2 cells. Specific binding of
PC13END-cell-derived IGF-like material was observed (Table 1), which could be
inhibited by unlabelled END-cell-derived IGF, IGF-I, IGF-II, but not EGF, or
embryonal carcinoma derived growth factor (ECDGF).
We conclude that PC13END-cell-conditioned medium contains a developmentally regulated IGF-like activity based on its ability to interact specifically with IGF
receptors and stimulate DNA synthesis in quiescent NRK fibroblasts.
(C) Immunoprecipitation of IGF-like molecules
Although the presence of IGF-competing activity in conditioned medium
provides circumstantial evidence for IGF synthesis, it would be advantageous to
have independent confirmation of IGF-II expression by metabolic labelling of
PC13END-cell-derived IGF.
We accordingly raised polyclonal antibodies to BRL-cell-derived rat IGF-II,
conjugated to KLH carrier protein. Immune IgGs from two rabbits were analysed
in detail with essentially similar results. BRL cells were metabolically labelled with
[35S] methionine and immunoprecipitated with rabbit anti-IGF-II IgG. Three
radiolabelled species (Fig. 6A) of apparent MT = 35000, 18000 and 16000 were
observed. In some experiments a fourth species of apparent Mr = 14000 (doublet)
was also observed. Immunoprecipitation of all these species was inhibited in the
presence of IGF-II. Similar species (including the Mr = 14000 form in this
instance) were also observed by immunoblotting acid-extracted BRL-conditioned
media (Fig. 6B) with our rabbit anti-IGF-II demonstrating that these forms were
recognized directly by the antibody and were not the result of coprecipitation.
Immunoprecipitation of [35S]methionine-labelled PC13EC and PC13END cell
Table 1. Binding of125I-PC13END-cell-derived IGF to PYS-2 cells
Cts min -1 10" 5 cells
Additive
125
I-END-IGF
125
I-END-IGF
125
I-END-IGF
125
I-END-IGF
125
I-END-IGF
125
—
1 ng IGF-II
ljug IGF-I
l^gEND-IGF
1/zgEGF
I-END-IGF 50 ng ECDGF
(±S.E.M.)
1914 ± 217
723 ± 60
790 ± 117
818 ± 195
2111 ± 411
1916 ± 101
1-8 jug of peak fractions125from reverse-phase HPLC separation1 of PC13END-cell-conditioned
media
was labelled with I to a specific activity of 69JuCiJug~ . Binding of 200 000 ets min-1
125
I-END-IGF to PYS-2 cells was determined in the presence of additives as indicated.
204
J. K. H E A T H AND W.-K.
a
b
c
Wmm
MrX
3
^m
M rX
3
■
10"
SHI
10"
35^
^35
<«18
*16
16^ *~*
A
B
Fig. 6. (A). Immunoprecipitation of IGF-II from [35S]methionine-labelled BRL cell
culture supernatant. Precipitates were subjected to electrophoresis with resolving gels
containing 12-5% acrylamide. Mr, relative mobility of marker proteins, a, total
secreted proteins; b, immunoprecipitation with rabbit anti-rat IGF-II IGg; c, immuno­
precipitation with rabbit anti-rat IGF-II IGg; c, immunoprecipitation with rabbit antirat IGF-II in the presence of 5 fig unlabelled rat IGF-II. (B) Immunoblot of protein
species recognized by rabbit anti-rat IGF-II IGg. MT, relative molecular mass of
marker proteins.
culture supernatant resulted in the specific precipitation of the MT = 35 000 and
MT = 14000 doublet and minor bands of 18000 and 16000 species from PC13END
but not PC13EC cells (Fig. 7). We conclude that our rabbit anti-IGF-II IgG
recognizes molecules which are expressed by BRL cells and PC13END cells but
not PC13EC cells and whose expression is developmentally regulated during
teratocarcinoma differentiation.
The 18000, 16000 and 14000 forms observed in these experiments probably
represent precursor forms of an IGF-II prohormone since our antibodies were
raised against size exclusion chromatography purified Mr = 7000 rat IGF-II.
Sequence analysis of rat IGF-II cDNAs (Bell et al. 1984; Dull et al. 1984; Whitfield
Expression of insulin-like growth
3 4
5 6 7 8
■m.
-f^
14*
Fig. 7. Immunoprecipitation of [35S]methionine-labelled PC13EC and PC13END cell
culture supernatant by rabbit anti-rat-IGF-II IGg. Precipitates were subjected to elec­
trophoresis with resolving gels containing 12-5% acrylamide. MT, relative molec­
ular mass of marker proteins. Lanes 1, 5, 14C-labelled molecular weight standards
(lysozyme, MT= 14300; carbonic anhydrase, Mr = 30000; ovalbumin, Mr = 46000;
bovine serum albumin, Mr = 69000; Phosphorylase b, MT = 92500; and myosin, MT =
200000). Lanes 2-4, PC13EC cells; lanes 6-8, PC13END cells. Lanes 2, 5, total
secreted proteins. Lanes 3, 7, immunoprecipitation by rabbit anti-rat-IGF-II IGg.
Lanes 4, 8, immunoprecipitation by control (rabbit anti-KLH) IGg.
etal 1984; Soares et al. 1985) have revealed that sequences encoding the 7000
dalton form of IGF-II are embedded in a longer open reading frame with the
capacity to code for a 156 amino acid precursor whose predicted amino sequence
contains a number of paired basic amino acid residues forming potential proteo­
lytic processing sites. The expression of these proposed IGF-II prohormone forms
by BRL cells has been established by Acquaviva et al (1983) and Yang et al (1985)
who showed that immunoprecipitation of either radiolabelled BRL cell extracts,
or in vitro translation reactions primed with BRL mRNA, with antibodies directed
against IGF-II yielded a variety of secreted IGF species of approximate relative
molecular masses 19000, 15000 (doublet), 10000, 8000 and 7000 derived from
an Mr = 20000 primary intracellular IGF-II translation product. It is important
to note that in our studies radiolabelling with [35S] methionine would only be
expected to detect proforms of IGF-II since thefirstmethionine residue occurs at
amino acid 117 in the predicted rat IGF-II amino acid sequence (assuming
sequence conservation between rat and mouse IGF genes).
206
J. K. HEATH AND W.-K.
SHI
The prominent MT = 35 000 species observed here is not readily accommodated
within the predicted amino acid sequence of pro-IGF-II. The immunoblot analysis
(Fig. 6B) shows, however, that the 35000 species is directly recognized by anti­
bodies present in our rabbit anti-IGF-II IgG preparations. One-dimensional
peptide mapping of the 35 K and 19 K species present in BRL-conditioned media
did not reveal the presence of shared immunoreactive peptides, suggesting that the
35 K species was not directly related to the other forms (Fig. 8). The 35 K species
observed in our experments is, however, similar to the 33 K species identified in
immunoprecipitates of BRL-conditioned media by Yang et al. (1985) as the soluble
IGF-binding protein. A 35 K IGF-binding protein has also been identified by
crosslinking studies in liver perfusates by Schwander et al. (1984) and D'Ercole &
Wilkins (1984). We have confirmed the identification of the immunoreactive 35 K
species as the IGF-binding protein by its purification from BRL conditioned
medium (J. K. Heath & B. J. Smith, in preparation). Af-terminal amino acid
sequence analysis of the purified protein does not reveal any sequence homology
with either pro-IGF-II or the predicted translation products of the long open
reading frame described in the 5' extension of the pro-IGF-II cDNA clone
described by Dull etal. (1985). It is probable therefore that the IGF-binding
protein is encoded by a separate gene from IGF-II.
a
+
b
e
-
+ -
d
+
10"3
35 ►
19^
14^
w em WH*
4 | fÊk
é
Fig. 8. Peptide mapping of species recognized by rabbit anti-rat IGF-II IGg. Gel slices
containing MT = 35 000 species (a), MT = 18000 and 16000 species (b, c) or Mr = 14000
species (d) were digested with V8 protease, subjected to SDS-PAGE, followed by
transfer to nitrocellulose paper and reaction with rabbit anti-rat-IGF-II IGg. Immuno­
reactive peptides were revealed by reaction with alkaline-phosphatase-conjugated
sheep anti-rabbit IGg.
Expression of insulin-like growth
207
It is not clear why immunization with purifed IGF-II should yield antibodies
which react with the binding protein. Although Yang et ai (1985) ascribed the
presence of antibodies directed against the binding protein to immunization with
impure preparations of IGF, we consider this explanation unlikely in this case
since the preparations of IGF-II used for immunization were subjected to highresolution SEC as the final stage of purification. It may be significant, however,
that immunoprecipitation of the Mr = 35 000 species is inhibited by IGF-II (Fig. 6)
suggesting that the antibodies recognize sites which interact with IGF-II.
On the basis of these considerations we concluded that our antibodies recog­
nized prohormone forms of IGF and the soluble IGF-binding protein.
(D) Expression oflGF-like molecules by extraembryonic tissues of the mouse embryo
The availability of specific antibodies that recognize murine IGF-II and the IGFbinding protein allowed us to analyse the expression of IGF-like molecules by
tissues of the early postimplantation mouse embryo, which would be difficult to
study by direct methods. Extraembryonic tissues of the 9-5 days post coitum
(é.p.c.) embryo were explanted in culture and metabolically radiolabelled with
[35S]methionine. Radiolabelled culture supernatants were immunoprecipitated
with rabbit anti-IGF-II. Specific coexpression of the four IGF-related protein
species was observed in amnion and extraembryonic yolk sac mesoderm (Fig. 9).
However, no evidence was found for expression of IGF-like molecules or binding
protein by either parietal endoderm or visceral endoderm (Fig. 9). The results
suggest that IGF-like molecules and their cognate binding proteins are specifically
expressed in vitro by early postimplantation tissues of extraembryonic mesodermal
origin.
DISCUSSION
Here we report that PC13EC cells, and their retinoic-acid-induced differen­
tiated progeny PC13END express specific high-affinity receptors for insulin-like
growth factors. These receptors probably mediate the effects of physiological
concentrations of IGFs on EC cell viability, multiplication and differentiation
(Heath & Délier, 1983; Heath & Rees, 1985). Crosslinking studies show that
PC13EC cells express both type 1 and type 2 IGF receptors. Since type 1 IGF
receptors, unlike type 2, can bind insulin (Masague & Czech, 1982) and PC13EC
cells do not express detectable insulin receptors (Heath etal. 1981), the reported
(Heath & Délier, 1983) effects of relatively high concentrations of insulin and
insulin analogues on EC cell survival may be mediated through these type 1
receptors.
The apparent number of IGF receptors falls by about 60 % upon RA-induced
differentiation, and this is accompanied by the expression of both IGF-like
molecules and soluble IGF-binding proteins; PC13END cells are also responsive
to exogenous IGF-II. By analogy with similar findings in other cellular systems it is
208
J. K. H E A T H AND W.-K.
ve
Mrx
10-3
mes
SHI
pe
am
"i r z — - — ~ i r
1 2 3 4 5 6 7 8 910 11 12 13
IE?"'
Fig. 9. Immunoprecipitation of [35S]methionine-labelled material from cultured 9-5
d.p.c. embryonic tissues. Precipitates were subjected to electrophoresis with resolving
gels containing 15% acrylamide; MT, relative molecular mass of marker proteins, ve,
visceral endoderm; mes, extraembryonic mesoderm; pe, parietal endoderm; am,
amnion. Lane 1,14C-labelled molecular weight markers (as in legend to Fig. 7). Lanes
2, 5, 8, 11, total secreted proteins. Lanes 3, 6, 9, 12, immunoprecipitation with rabbit
anti-mouse transferrin control IGg. Lanes 4, 7, 10, 13, immunoprecipitation with
rabbit anti-rat IGF-II.
reasonable to suppose that these two phenomena are linked, in that the endo­
genous production of IGF-hke molecules results in the occupation of a fraction of
PC13END cell IGF receptors, resulting in an apparent decrease in the number of
sites available to bind exogenous IGF. This type of phenomenon has often been
linked with malignant cell behaviour (reviewed by Burk, 1980). It is of interest,
therefore, that PC13END cells are nontumourigenic in vivo and exhibit a finite
proliferative lifespan in vitro (reviewed by Heath, 1983). 'Autocrine' cell prolif­
eration may therefore be a feature of certain normal embryonic cell types as well
as malignant cells. The autocrine synthesis of IGF-like molecules by PC13END
cells may also provide an explanation for the observed dependency of PC13END
cell multiplication on cell density (Heath & Rees, 1985) in the absence of exo­
genous growth factors. Since insulin has been reported to increase the affinity of
IGF for the type 2 IGF receptor, at least in the case of adipocytes (Oppenheimer
et al. 1983), the mitogenic effects of low concentrations of insulin on PC13END
Expression of insulin-like growth
209
cells (Heath etal. 1981) may occur indirectly as a result of increased insulininduced occupancy of PC13END cell IGF receptors by endogenous IGF.
The expression of IGF receptors by PC13EC cells, and the synthesis of IGF-like
molecules by their immediate differentiated progeny points to the existence of a
feedback relationship whereby PC13END cells secrete factors which maintain
proliferation of their undifferentiated parents (reviewed by Heath & Rees, 1985).
Experimental support for this notion comes from the work of Isacke & Délier
(1983) who showed that PC13END cell feeders could substitute for exogenous
serum-derived factors to support PC13EC cell multiplication in vitro. (An opposite
feedforward growth dependency also occurs in this system through the mitogenic
action of PC13 embryonal carcinoma derived growth factor on PC13END cells
(Heath & Isacke, 1984).) Together these considerations suggest that a sophis­
ticated interactive network of growth and differentiation control pathways exists in
murine teratocarcinomas in which IGF-like molecules play a key role, acting
simultaneously as 'autocrine' and paracrine growth factors.
Further characterization of secreted forms of IGF-like molecules by immunoprecipitation of metabolically radiolabelled molecules from culture supernatants
with antibodies directed against rat IGF-II revealed multiple molecular species
consistent with the proposed production of IGF by proteolytic processing of a
longer precursor. The sizes of the secreted pro-IGF forms in the present study are
generally in accord with those observed for IGF-II secreted by BRL cells by Yang
et al. (1985) and in our own study. We have also observed, however, an immunoreactive putative MT = 14000 form, which is particularly prominent in immunoprecipitates of embryonic tissues. This may simply reflect quantitative, or
qualitative, differences between IGF processing in different tissues or technical
differences between the two studies. However, it is important to note firstly that
cDNA sequence analysis of the human IGF-I gene (Jansen et al. 1983) suggests
that it is also initially synthesized as a longer prohormone, and secondly,that the
possible existence of additional related IGF-like genes cannot as yet be elim­
inated. These considerations do not allow us at present definitively to identify
the species, recognized by antibodies directed against rat IGF-II, and secreted
by PC13END cells and embryonic tissues as IGF-II, IGF-I or other IGF-like
molecules. Furthermore, the existence of differences in 5' noncoding sequences
from separate IGF-II cDNA clones (Soares et al. 1985), combined with the
complexity of human IGF-II transcript sizes observed in normal tissues (Soares
etal. 1985; Scott etal. 1985) suggest that post-transcriptional processing of the
IGF-II transcript may occur prior to translation. It may be possible, therefore, to
generate multiple molecular forms of IGF-II by a variety of different mechanisms.
The expression of IGF and IGF receptors by embryo-derived teratocarcinomas
leads to the supposition that IGF is expressed in the early postimplantation
mouse embryo. Immunoprecipitation of radiolabelled culture supernatants from
9-5 d.p.c. extraembryonic tissues has shown the amnion and extraembryonic yolk
sac mesoderm, both mesodermal derivatives of the primitive ectoderm, to be the
principal extraembryonic sites of IGF and IGF-binding-protein synthesis. This
210
J. K. H E A T H AND W . - K .
SHI
expression of I G F by the extraembryonic membranes may account for the pres­
ence of relatively high levels of both IGF and IGF-binding-protein activity in
foetal amniotic fluid (Bala etal. 1978; Chochinov etal. 1978) and underlines the
proposed function of these tissues in supporting embryonic growth in utero (Heath
& Shi, 1984). It is, furthermore, important to note that extraembryonic
mesodermal cells, like PC13END cells, not only secrete IGF-like molecules but
are responsive to exogenous IGF-II in vitro (Heath & Rees, 1985). Together these
findings demonstrate that, as is the case for PC13END cells, the multiplication of
extraembryonic mesodermal cells is, at least in part, controlled by an 'autocrine'
mechanism, involving IGF-like molecules.
The widespread expression of IGF-II in later development by foetal organs
(Scott etal. 1985), many of which are initially derived from two component
epithelial/mesenchymal structures analogous to the yolk sac, may mean that, as in
the yolk sac, IGF-II is expressed by the mesodermal or mesenchymal cells in these
organs and that the type of interactive growth control system we have described in
murine teratocarcinomas may be recapitulated in later development.
Finally, our observations show that: (i) specific growth factors are expressed
during the early embryonic stages of mammalian development, (ii) the multipli­
cation of cells in the mammalian embryo may be regulated by both autocrine
and paracrine action of these growth factors, and (iii) IGFs may have an impor­
tant regulatory function in embryonic as well as later foetal and postnatal
development.
We thank Judy Morton, Hilary Oakley and John Eady for their assistance with this project,
and Michael Czech, Mathew Rechler and Chris Graham for helpful suggestions and discussion.
This work was funded by grants from the Cancer Research Campaign, the Royal Society and the
Medical Research Council. JH acknowledges the support of an MRC Senior Fellowship and
W-KS the support of the Chinese Academy of Sciences, the Henry Lester Fund and the British
Universities China Committee.
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