Vol.
172,
October
No.
BIOCHEMICAL
2, 1990
30.
RESEARCH
BIOPHYSICAL
FACTOR-IYMANNOSE Mercedes Department
COMMUNICATIONS
775779
Pages
1990
DEVELOPMENTAL
Received
AND
REGULATION
OF INSULIN-LIKE
GROWTH
6-PHOSPHATE
RECEPTOR
IN THE RAT
Ballesteros,
Carolyn
D. Scott*and
mRNA Robert
of Endocrinology, Royal Prince Alfred Hospital, South Wales 2050, Australia
September
C. Baxter Camperdown,
New
5, 1990
This study examined levels of insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MGPR) mRNA in tissues of rats at different stages of growth. Northern blot analysis of total RNA from tissues of rats aged 2, 9, 21 and 42 days and from 21 day fetal rats was carried out using a cDNA probe to the IGFII/MGPR. Northern blots showed this probe hybridized to a single 9kb band in all tissues tested. Highest hybridization signals were detected in fetal and neonatal tissues with levels rapidly decreasing after birth. For all age groups tested the highest signal was obtained with heart followed by muscle, lung, and kidney, with liver and brain showing lower levels of message. These results indicate that IGF-II/MGPR mRNA is developmentally regulated, and suggest a role for the 0 1990Acddemlc Press.Inc. IGF-II/MGPR in fetal and neonatal growth.
The insulin-like growth factor-II/mannose binds the mitogenic polypeptide IGF-II transport receptor
B-phosphate receptor (IGF-II/MGPR) and is involved in the uptake and
of proteins bearing mannose 6-phosphate residues (1). The role of this in targeting
lysosomal enzymes containing
lysosomes has been well characterized interaction
with IGF-II
is unclear. IGF-II
mannose 6-phosphate
(2); however
the importance
to
of its
has been implicated in fetal rat growth
and development. High levels of this growth factor are present in serum of fetal and neonatal rats with levels 20-100 fold lower in adult rat serum (3). Furthermore developmentally
the
mRNA
regulated
for
this
growth
factor
has been shown
to be
with elevated levels detected in tissues of fetal and
newborn rats and levels almost negligible in tissues of older rats (4). Certain IGFII actions have been shown to specifically
occur via the IGF-II/MGPR
(5)
and
levels of this receptor have been shown to correlate with rates of cell growth (61
*To whom correspondence should be addressed. Abbreviations: Insulin-like growth factor II/ mannose 6-phosphate receptor (IGFII/MGPR), 3-[N-Morpholino] propanesulfonic acid (MOPS), Kilobases (kb), ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS). 0006-291X/90
775
$1.50
Copyright 0 1990 by Academic Press, ITIC. All rights of reproduction in any form reservrd.
Vol.
172,
No.
suggesting
2, 1990
BIOCHEMICAL
the receptor
has a role in growth.
be present in a soluble truncated levels of receptor were recently tissues than in adult rat tissues determine if the developmental mRNA
level. This
rats at different
MATERIALS
study
AND
BIOPHYSICAL
RESEARCH
The IGF-II/MGPR
COMMUNICATIONS
has been found to
form in serum of fetal and neonatal rats (7) and shown to be higher in fetal and neonatal rat (8). In view regulation
examined
of these findings it was of interest to of the IGF-IIMGPR occurs at the
levels of IGF-IIAIIGPR
message
in tissues
of
stages of development.
AND
METHODS
Materials: Genescreen Plus nylon membrane and [“sP] dCTP were purchased from DuPont (Wilmington, DE). Multiprime random primer DNA labeling kit and Hyperfilm MP were obtained from Amersham Corp. (Bucks,UK). RNA molecular weight markers and Eco Rl restriction endonuclease were from Boehringer Mannheim (W. Germany). Geneclean DNA Purification Kit was from Bio 101 Inc. (La Jolla, CA).All other chemicals were of analytical grade. An IGF-II/MGPR cDNA probe (9) was a gift from Genentech Inc., South San Francisco, CA, kindly provided by Dr. A. Ullrich. Animals: Wistar-Furth rats aged 2, 9, 21 and 42 days and fetuses from pregnant rats at day 21 of gestation were euthanased under halothane anesthesia and used for tissue extractions. RNA Extraction: Total RNA was prepared from brain, liver, lung, kidney, muscle and heart for each age group using a single step acid guanidinium thiocyanate phenol-chloroform extraction protocol (10). Total RNA was estimated by absorbance readings at 260nm where 1 OD unit was assumed to be equal to 4Opg of RNA. Probe preparation and labeling: A 616 basepair insert cDNA to the IGF-IUMGPR was extracted from plasmid DNA by digestion with Eco Rl restriction enzyme. The digest was electrophoresed on 1.5% agarose gel and the cDNA insert was purified using Geneclean kit. The cDNA probe was labeled with [““PI dCTP using a Multiprime random primer DNA4 labeling kit: Slot blots: RNA was denatured by incubation at 50C for 1 h in 50% formamide and 6% formaldehyde then chilled on ice. Samples (2O>ug) were diluted and applied to Genescreen Plus nylon membranes usin, u a Bio-Rad (Richmond, CA) slot blotting apparatus. Northern blots: Rat tissue RNA samples (151.18) and RNA molecular weight markers (1Oug) were denatured by heating for 10min at 65G in 25~1 loading buffer consisting of 50% formamide, 6% formaldehyde, 1xMOPS buffer (0.2M MOPS/O.O05M sodium acetate/O.OOlM EDTA pH7.5) and 0.01% bromophenol blue. Samples were then chilled and 1~1 of ethidium bromide (lmg/ml) added. RNA was fractionated by electrophoresis on a 1% agarose gel containing 0.66M formaldehyde in Ix MOPS buffer. Gels were photographed under UV light and RNA was transferred to nylon membranes by capillary transfer in 10x SSC buffer (0.15M tri-sodium citrate/l .5M sodium chloride). Hybridization to IGF-IIjM6PR cDNA: Northern and slot blot hybridizations were carried out with a 132P] dCTP labeled cDNA probe to the IGF-II/MGPR at 42C for 16h in hybridization buffer (0.04M sodium phosphate, pH 7.7, 0.004M EDTA, 0.72M sodium chloride, 50% formamide, 1% SDS, 10% dextran sulfate, 0.5% skim milk powder and 0.5mg/ml sheared herring sperm DNA) (ll).For each tissue and age group studied, triplicate RNA preparations were hybridized twice. Autoradiography and Densitometry: Membranes were washed as recommended by the manufacturers, dried and set up for autoradiography using Hyperfilm MP for 72h at -70C. The density of the slot blot autoradiographs was quantitated using a Bio-Rad video densitometer model 620. 776
Vol.
172,
No.
RESULTS
BIOCHEMICAL
2, 1990
AND
Total RNA
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
DISCUSSION
was prepared
from liver,
lung, kidney,
muscle,
brain
and heart
rats at different stages of growth and subjected to Northern analysis using a dCTP labeled receptor cDNA. As shown in Figure 1 the cDNA probe hybridized a single RNA species of approximately 9kb in all tissues. An mRNA transcript this size has been reported differences however
(-1OkD)
decreasing of 18s
have been reported
we found
tissues. As shown hybridization
for the IGF-II/MGPR
no difference
in Fig. 2a for signal in fetal
after birth
and 28s
bands
Lung,
to almost
under
slot blot hybridizations,
kidney,
liver
levels after birth Developmental
and brain
whereas
rat tissues
for the receptor
from
followed
indicated
for this pattern results
lowest
levels
of message.
lung,
of which
that
variations
are represented large
and muscle levels decreased
various
and kidney,
in RNA
with
liver
between
in Figure
decreases steadily
has been reported
We found no difference
1.9 1.6
(13);
(Fig. 2b). This was quantitated
relatively
of the IGF-IIMGPR
by muscle,
size
levels by day 21. Detection
until
day 21.
showing
levels
the
of muscle
+ +
Fig. 1. Northern blot of total RNA (15ug) from tissues of day 9 rats. RNA was electrophoresed on 1% agarose gel containing 0.66M formaldehyde and transferred to nylon membranes as described in Materials and Methods. Membranes were hybridized overnight at 42C with a 132Pl dCTP labelled cDNA probe to the IGF-WMGPR, washed, and autoradiographed at -70C for 72h. Standards are in kilobases. 771
(8)
for the protein hybridization
and brain mRNA
3.
in receptor
in rat tissues
and we found the receptor mRNA levels paralleled those reported at different stages of development. Heart showed the strongest signal
to of
cells (12). Small
in different
undetectable
UV light
all showed
heart
regulation
PPI
muscle, Northern blots showed the strongest and neonatal tissues with levels of receptor
loaded onto gels could not account by scanning
for the receptor
in size of mRNA
and falling
RNA
in BRL-3A
from
Vol.
172,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Fig. 2. Northern blot (a) and agarose gel (b) of RNA from muscle of rats at different stages of growth. Total RNA (15ug) was run under denaturing conditions, stained with ethidium bromide and photographed under LJV light (panel b) to check RNA loading. RNA was then transferred to nylon membranes and these were hybridized and autoradiographed as described in Figure 1 legend (panel a). Standards are shown in kilobases.
and lung although levels of receptor in lung had been reported to be higher (8). This may be due to different developmental
pattern
HEART
processing or turnover
of the receptor
MUSCLE
of the IGF-II/MGPR.
mRNA also correlates
LUNG
KIDNEY
LIVER
with
The
the IGF-II
BRAIN
TISSUE Fig. 3. Graph of densitometer scans from slot blot hybridizations. Total RNA (2Opg) from tissues of rats at different stages of growth was prepared in triplicate and slot blotted onto nylon membranes. Blots were hybridized as described in Materials and Methods and then scanned on a densitometer. Values are expressed as percentage of maximum optical density (fetal rat heart RNA = 100%) and are means f SEM for duplicate hybridizations of triplicate RNA preparations.
Vol.
BIOCHEMICAL
172, No. 2, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
message in most rat tissues (4). These findings suggest a role for the receptor in some aspect of growth. It is still not understood how the binding of the two ligands, IGF-II mannose 6-phosphate, to the IGF-IIfMGPR processes. Several
might result in initiation
studies suggest a direct
mitogenic
and
of growth
role for IGF-II.
For
example, a receptor antibody which apparently binds at the IGF-II site is able to stimulate DNA synthesis in Balb/c 3T3 cells, acting via a calcium gating system (14). IGF-II,
acting via the IGF-II/MGPR,
in human erythroleukemia
also appears to have a mitogenic action
cells, resulting in cell proliferation
(15). Alternatively,
it has been proposed that because of its role in targeting lysosomal enzymes, the IGF-IINGPR may be involved in tissue remodeling (8,ll). The high levels of receptor in rapidly-growing
tissues (for example during fetal growth) could thus
be explained since an increase in proteases and their transport
would be needed
for tissue remodeling. This is supported by studies using models of rapid cellular growth such as hepatoma cell lines and liver regeneration
where levels of IGF-
II/MGPR have been shown to be increased (6,16). In conclusion, we found levels of IGF-IL&tGPR mRNA were higher in fetal than in postnatal
rat tissues, with a developmental
pattern
paralleling
that of the
receptor protein and IGF-II mRNA. These data suggest that this receptor has a role in growth and development of rats. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
MacDonald, R.G., Pfeffer, S.R., Coussens, L., Tepper, M.A., Brocklebank, C.M., Mole, J.K., Chen, E., Czech, M.P. and Ullrich, A. (1988) Science 239, 11341137. von Figura, K.V. and Hasilik, A. (1986) Ann. Rev. Biochem. 55,167-193. Moses, A.C., Nissley, S.P., Short, P.A., Rechler, M.M., White, R.M., Knight, A.B., and Higa, O.Z. (1980) Proc. Natl. Acad. Sci. USA 77, 3649-3653. Brown, A.L., Graham, D.E., Nissley, S.P., Hill, J., Strain, A.J. and Rechler, M.M. (1986) J. Biol. Chem. 261,13144-13150. Hari, J., Pierce, S.B., Morgan, D.O., Sara, V., Smith, M.C. and Roth, R.A. (1987) EMBO J. 6,3367-3371. Scott, C.D. and Baxter, R.C. (1987) J.Cell Physiol. 133, 532-538. Kiess, W., Greenstein, L.A., White, R.M., Lee, L., Rechler, M.M. and Nissley, P.S. (1987) Proc. Natl. Acad. Sci. USA 84, 7720-7724. Sklar, M.M., Kiess, W., Thomas, C.L. and Nissley, P.S. (1989) J. Biol. Chem. 264,16733-l 6738. Laurys, G., Barton, D.E., Ullrich, A. and Francke, U. (1988) Genomics 3, 224229. Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162,156-159. Scott, C.D., Ballesteros, M. and Baxter, R.C. (1990) Endocrinology, in press. Morgan, D.O., Edman, J.C., Standring, D.N., Fried, V.A., Smith, M.C., Roth, R.A. and Ruttler, W.J. (1987) Nature 329, 301-307. Taylor, J.E., Scott, C.D. and Baxter, R.C. (1987) J. Endocrinol. 115, 35-41. Kojima, I., Nishimoto, I., Iiri, T., Ogata, E. and Rosenfeld, R. (1988) Biochem. Biophys. Res. Commun. 154, 9-19. Tally, M., Li, C. H. and Hall, K. (1987) Biochem. Biophys. Res. Commun. 148, 811816. Scott, C.D. and Baxter, R.C. (1990) Endocrinology 126, 2543-2549. 779