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The neurite-promoting effect of fibroblast growth factor on PC12 cells
Vol. 114, No. 3, 1983 August
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 1189-1193
12, 1983
THE NEURITE-PROMOTING Akifumi
Togari,
July
Dole
Baker,
GROWTH FACTOR ON PC12 CELLS
Geneva Dickens,
and Gordon
Guroff
Section on Growth Factors Institute of Child Health and Human Development National Institutes of Health Bethesda, MD 20205
National
Received
EFFECT OF FIBROBLAST
6, 1983
Treatment of PC12 cells with fibroblast growth factor(s) from P: rain or pituitary caused neurite outgrowth comparable to that produced nerve growth factor. The neurite outgrowth was preceded by a substantial in the activity of ornithine decarboxylase.
The PC12 clone model are
for
the
treated
study
with
electrically
pheochromocytoma
(l-5)
of the
actions
of nerve
growth
amounts
of the
nanomolar
excitable,
PC12 cells
will
addition,
the
and elaborate
synapse nerve
with
growth
factor
neurotransmitters. parallel
those
neurons.
seen when nerve
The mechanism
it
is
thought
some series
that
the
of second
actions
nuclear
(13-15).
phosphorylation
in the
various
epidermal
growth
other
acting
factor
and CAMP derivatives these
at the
effecters
(16), (19)
have
are the
acts
tumor
some effect
as a model It
functional; (6).
is
In
of proteins of closely
sympathetic
not
known in detail, and, These
by changes
perhaps
by
latter,
in cytoplasmic
has led
and
to an interest
has been shown that
(17), on these
same in some cases
I189
on normal
cells become
of a number
(10-12).
cells.
promoters
in culture
at the membrane,
may be mediated
on the
are
in PC12 cells
occur
nucleus
use of the PC12 cells
effecters
occur
changes both
dividing,
in the metabolism
which
premiere
When these
stop
cells
involved
acts
messengers,
they
induction
factor
these
factor.
The neurites
the
changes
factor
transcriptionally-based
The wide-spread
causes
growth
by which
factor
muscle
enzymes
Many of the
has become the
neurites.
appropriate
among them some of the
17-91,
but
of rat
either by rise
adenosine cells.
as those
analogues The actions
of nerve
(18), of
growth
0006-291X/83 $1.50 Copyright 0 1983 by Academic Press, Inc. AN rights of reproduction in arty form reserved.
Vol. 114, No. 3, 1983
factor.
BIOCHEMICAL
However,
characteristic
purified
none of these
action
We report
of cell
outgrowth This
that
from either types,
is
decarboxylase
and,
growth
those
produces
namely,
neurite
factor
to that
(ZO-22), with
effects
of nerve
growth
single
most
outgrowth. a partially
mitogenic
seen with
the
the
origin,
on the marker
actions, those
agents
of mesodermal
by an action
with
RESEARCH COMMUNICATIONS
or pituitary,
comparable
in both
seem to be additive
factor,
brain
primarily
preceded
other
growth
fibroblast
in PC12 cells
action
several
of nerve
here
material
number
AND BIOPHYSICAL
actions
produces
nerve
growth
enzyme,
on a
neurite
factor.
ornithine
of fibroblast
growth
factor
factor.
Methods PC12 cells were grown flasks at 37°C in 5% CO . supplemented with 7% fe z al and 100 ug/ml penicillin, by the method of Bocchini purchased from Collaborative
as described previously (13, 16) in 150 cm2 Costar The medium was Oulbecco's modified Eagle's medium bovine serum, 7% horse serum, 100 U/ml of of streptomycin. Nerve growth factor was prepared and Angeletti (23); fibroblast growth factors were Research and from Bethesda Research Laboratories.
Ornithine decarboxylase was assayed Ashman (24) as modified by Oka and Perry publications (26).
by the method of Pegg and Williams(25) and described in previous
Results When PC12 cells formed.
These
were
neurites
72 hours,
the
(Fig.
and appeared
11,
maximal
growth as that
fibroblast give
were time
produced
by nerve
amounts
then
with did
Fibroblast decarboxylase produced the
in
factor
factor
per ml;
growth
of nerve
factor,
amounts
growth
were
factor
the
of nerve
brain
factor
factor,
for
and thin
formation
was
and pituitary
combination
growth
factor
was not of
seemed to
The addition T-cell
growth
as
of factor,
or
effect.
activity
was usually factor
and fibroblast 1190
to grow
The outgrowth
alone.
growth
neurites
long
Process
and the
without
The increase
growth
were
of nerve
growth
stimulated
amount
factor,
and continued
to be equivalent.
nerve
PC12 cells.
by an equivalent
combination
cones.
maximal
activity growth
24 hours
in growth
of platelet-derived
multiplication-stimulating
growth
The processes
growth appeared
more outgrowth
within
to terminate
factor
fibroblast
investigated.
factors
growth
comparable
with
evident
at 50 ng of fibroblast
fibroblast profuse
longest
treated
of ornithine not
as great
(Table growth
l), factor
as that and,
again,
seemed to
Vol. 114, No. 3, 1983
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Control
FGF
Fig.
1.
NGF+FGF
Neurite outgrowth in PC12 cells grown conditions (control) or in the presence rig/ml (NGF), fibroblast growth factor, growth factor, 10 rig/ml and fibroblast FGF).
be additive.
Neither
had a major
effect
T-cell
growth
on ornithine
factor
for
72 hours under standard of nerve growth factor, 10 50 rig/ml (FGF), or nerve growth factor, 25 rig/ml (NGF
nor
decarboxylase
platelet-derived
growth
t
factor
levels.
Discussion There here. which
One is it
second
acts, is
primarily
that
that
points
that
fibroblast
seems to act fibroblast
medulla, is
that
PC12, which
factor,
factor
cells the
the observations
a mitogen
as a differentiating
growth
from which the
can be made about
growth
on mesoderm-derived
adrenal point
are three
agent
has previously
and the embryonic
PC12 arises,
is
quite
has become a fundamental 1191
reported
for
most cells
upon
for
PC12 cells.
A
been shown to act derivation
of the
different. model
A third for
the
study
of
BIOCHEMICAL
Vol. 114, No. 3, 1983
AND BIOPHYSICAL
Table The
action
of
fibroblast
growth
ornithine
RESEARCH COMMUNICATIONS
1
factor
decarboxylase
and of
activity
nerve
in
Additions
growth
PC12
factor
Ornithine
decarboxylase
activity
pmol[14C021/pg
Expt.
Control Nerve
growth
on
cells
protein
1
Expt.
0.06
0.20
1.90
2.1
0.81 1.15
2:7
factor
(50 w/ml 1 Fibroblast
growth
factor
(25 w/ml 1 (50 w/ml) (100 rig/ml)
0.90
Nerve growth factor and fibroblast growth factor (50 rig/ml each)
Additions figures
nerve
were made at 0 time represent the average
growth
which
is
from It
is
the
actions
quite
sources
evidence
the
decarboxylase
can
a maximal
dose
growth
these
in
of nerve
the
It
cells.
effecters
also
adreneral
medulla
light
is act
be
that of
act
the
and the
through
to
shown
be
factor
is
the
many
not
factor,
factor,
resemble
additive,
The
to be quite a factor
and which
growth
separate and
each
their be
is
factor. other
pathways.
to
and,
Thus, actions
additive,
their on
even
when
used. of PC12 as a model
effecters
to know,
which
in this
counterparts
sympathetic
growth
quantitatively
relevance
normal
growth
do
later.
was thought
or no nerve
two factors
the
important on
factor
nerve
little
appear
ornithine
considered
from
here,
outgrowth
growth
5 hours
way to fibroblast
contain
presented
suggest
nerve
different
that
neurite
The data
and cells were collected of duplicate flasks.
which
which
interesting
on
6.6
in a similar
chemically
isolated
from
and for
factor,
responds
specific,
2
have
regard, of
the
system
been
shown
whether PC12
should to
these
cells,
be
act
on
several
namely,
the
neurons. References
1. 2. 3.
Greene, L.A. 2424-2428.
and Tischler,
Greene,
and
Dichter, 504.
L.A.
M.A.,
Rein,
Tischler,
A.S.
(1976)
Proc.
G. (1977) Nature 268, A.S., and Green3.A.
1192
Natl.
Acad.
349-351. (1977)
Sci. Nature
U.S.A. 268,
73, 501-
Vol. 114, No. 3, 1983
4. 5. 6. k 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 2:
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Greene, L.A. and Rein, G. (1977 Brain Res. 129, 247-263. Greene, L.A. and Rein, G. (1977 1 Brain Res.m, 521-528. Schubert, D., Heinemann, S., and Kidokoro, Y-977) Proc. Natl. Acad. Sci. U.S.A. 74, 2579-2583. Edgar, D.H. and Thoenen, H. (1978) Brain Res. 154, 186-190. Rieger, F., Shelanski, M.L., and Greene, L.A. Tf480) Dev. Biol. 76, 238253. H. (1978) FEBS Lett. 92, 313-316. Hatanaka, H., Otten, U., and Thoenen, Bradshaw, R.A. (1978) Annu. Rev. Biochem.47, 191-216. Vinores, S. and Guroff, G. (1980) Annu. Rev. Biophys. Bioeng.2, 223-257. Yankner, B.A. and Shooter, E.M. (1982) Annu. Rev. Biochem. 51, 845-868. Yu, M.W., Tolson, N.W., and Guroff, G. (1980) J. Biol. Chem. 255, 1048110492. Halegoua, S. and Patrick, J. (1980) Cell 22, 571-581. End, D., Hanson, M., Hashimoto, S., and Guroff, G. (1982) J. Biol. Chem. 257, 9223-9225. Huff, K., End, D., and Guroff, G. (1981) J. Cell Biol. 88, 189-198. End, D., Tolson, N., Yu, M., and Guroff, G. (1982) J. Cn. Physiol. 111, 140-148. Guroff, G., Dickens, G., End, D., and Londos, C. (1981) J. Neurochem.37, 1431-1439. Gunning, P.W., Landreth, G.E., Bothwell, M.A., and Shooter, E.M. (1981) J. Cell Biol. 89, 240-245. Gospodarowicz, D. and Moran, J.S. (1976) Annu. Rev. Biochem. 45, 531-558. Gospodarowicz, D. (1974) J. Biol. Chem. 250, 2515-2520. Gospodarowicz, D., Bialecki, H., and Grexurg, G. (1978) J. Biol. Chem. 253, 3636-3743. Bocchini, V. and Angeletti, P.U. (1969) Proc. Natl. Acad. Sci. U.S.A.3, 787-794. Pegg, A.E. and Williams-Ashman, H.G. (1968) Biochem. J.108, 533-539. Oka, T. and Perry, J.W. (1976) J. Biol. Chem. 251, 1738-1744. Guroff, G., Dickens, G., and End, D. (1981) J.-?&rochem. 37, 342-349.
1193
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 1189-1193
12, 1983
THE NEURITE-PROMOTING Akifumi
Togari,
July
Dole
Baker,
GROWTH FACTOR ON PC12 CELLS
Geneva Dickens,
and Gordon
Guroff
Section on Growth Factors Institute of Child Health and Human Development National Institutes of Health Bethesda, MD 20205
National
Received
EFFECT OF FIBROBLAST
6, 1983
Treatment of PC12 cells with fibroblast growth factor(s) from P: rain or pituitary caused neurite outgrowth comparable to that produced nerve growth factor. The neurite outgrowth was preceded by a substantial in the activity of ornithine decarboxylase.
The PC12 clone model are
for
the
treated
study
with
electrically
pheochromocytoma
(l-5)
of the
actions
of nerve
growth
amounts
of the
nanomolar
excitable,
PC12 cells
will
addition,
the
and elaborate
synapse nerve
with
growth
factor
neurotransmitters. parallel
those
neurons.
seen when nerve
The mechanism
it
is
thought
some series
that
the
of second
actions
nuclear
(13-15).
phosphorylation
in the
various
epidermal
growth
other
acting
factor
and CAMP derivatives these
at the
effecters
(16), (19)
have
are the
acts
tumor
some effect
as a model It
functional; (6).
is
In
of proteins of closely
sympathetic
not
known in detail, and, These
by changes
perhaps
by
latter,
in cytoplasmic
has led
and
to an interest
has been shown that
(17), on these
same in some cases
I189
on normal
cells become
of a number
(10-12).
cells.
promoters
in culture
at the membrane,
may be mediated
on the
are
in PC12 cells
occur
nucleus
use of the PC12 cells
effecters
occur
changes both
dividing,
in the metabolism
which
premiere
When these
stop
cells
involved
acts
messengers,
they
induction
factor
these
factor.
The neurites
the
changes
factor
transcriptionally-based
The wide-spread
causes
growth
by which
factor
muscle
enzymes
Many of the
has become the
neurites.
appropriate
among them some of the
17-91,
but
of rat
either by rise
adenosine cells.
as those
analogues The actions
of nerve
(18), of
growth
0006-291X/83 $1.50 Copyright 0 1983 by Academic Press, Inc. AN rights of reproduction in arty form reserved.
Vol. 114, No. 3, 1983
factor.
BIOCHEMICAL
However,
characteristic
purified
none of these
action
We report
of cell
outgrowth This
that
from either types,
is
decarboxylase
and,
growth
those
produces
namely,
neurite
factor
to that
(ZO-22), with
effects
of nerve
growth
single
most
outgrowth. a partially
mitogenic
seen with
the
the
origin,
on the marker
actions, those
agents
of mesodermal
by an action
with
RESEARCH COMMUNICATIONS
or pituitary,
comparable
in both
seem to be additive
factor,
brain
primarily
preceded
other
growth
fibroblast
in PC12 cells
action
several
of nerve
here
material
number
AND BIOPHYSICAL
actions
produces
nerve
growth
enzyme,
on a
neurite
factor.
ornithine
of fibroblast
growth
factor
factor.
Methods PC12 cells were grown flasks at 37°C in 5% CO . supplemented with 7% fe z al and 100 ug/ml penicillin, by the method of Bocchini purchased from Collaborative
as described previously (13, 16) in 150 cm2 Costar The medium was Oulbecco's modified Eagle's medium bovine serum, 7% horse serum, 100 U/ml of of streptomycin. Nerve growth factor was prepared and Angeletti (23); fibroblast growth factors were Research and from Bethesda Research Laboratories.
Ornithine decarboxylase was assayed Ashman (24) as modified by Oka and Perry publications (26).
by the method of Pegg and Williams(25) and described in previous
Results When PC12 cells formed.
These
were
neurites
72 hours,
the
(Fig.
and appeared
11,
maximal
growth as that
fibroblast give
were time
produced
by nerve
amounts
then
with did
Fibroblast decarboxylase produced the
in
factor
factor
per ml;
growth
of nerve
factor,
amounts
growth
were
factor
the
of nerve
brain
factor
factor,
for
and thin
formation
was
and pituitary
combination
growth
factor
was not of
seemed to
The addition T-cell
growth
as
of factor,
or
effect.
activity
was usually factor
and fibroblast 1190
to grow
The outgrowth
alone.
growth
neurites
long
Process
and the
without
The increase
growth
were
of nerve
growth
stimulated
amount
factor,
and continued
to be equivalent.
nerve
PC12 cells.
by an equivalent
combination
cones.
maximal
activity growth
24 hours
in growth
of platelet-derived
multiplication-stimulating
growth
The processes
growth appeared
more outgrowth
within
to terminate
factor
fibroblast
investigated.
factors
growth
comparable
with
evident
at 50 ng of fibroblast
fibroblast profuse
longest
treated
of ornithine not
as great
(Table growth
l), factor
as that and,
again,
seemed to
Vol. 114, No. 3, 1983
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Control
FGF
Fig.
1.
NGF+FGF
Neurite outgrowth in PC12 cells grown conditions (control) or in the presence rig/ml (NGF), fibroblast growth factor, growth factor, 10 rig/ml and fibroblast FGF).
be additive.
Neither
had a major
effect
T-cell
growth
on ornithine
factor
for
72 hours under standard of nerve growth factor, 10 50 rig/ml (FGF), or nerve growth factor, 25 rig/ml (NGF
nor
decarboxylase
platelet-derived
growth
t
factor
levels.
Discussion There here. which
One is it
second
acts, is
primarily
that
that
points
that
fibroblast
seems to act fibroblast
medulla, is
that
PC12, which
factor,
factor
cells the
the observations
a mitogen
as a differentiating
growth
from which the
can be made about
growth
on mesoderm-derived
adrenal point
are three
agent
has previously
and the embryonic
PC12 arises,
is
quite
has become a fundamental 1191
reported
for
most cells
upon
for
PC12 cells.
A
been shown to act derivation
of the
different. model
A third for
the
study
of
BIOCHEMICAL
Vol. 114, No. 3, 1983
AND BIOPHYSICAL
Table The
action
of
fibroblast
growth
ornithine
RESEARCH COMMUNICATIONS
1
factor
decarboxylase
and of
activity
nerve
in
Additions
growth
PC12
factor
Ornithine
decarboxylase
activity
pmol[14C021/pg
Expt.
Control Nerve
growth
on
cells
protein
1
Expt.
0.06
0.20
1.90
2.1
0.81 1.15
2:7
factor
(50 w/ml 1 Fibroblast
growth
factor
(25 w/ml 1 (50 w/ml) (100 rig/ml)
0.90
Nerve growth factor and fibroblast growth factor (50 rig/ml each)
Additions figures
nerve
were made at 0 time represent the average
growth
which
is
from It
is
the
actions
quite
sources
evidence
the
decarboxylase
can
a maximal
dose
growth
these
in
of nerve
the
It
cells.
effecters
also
adreneral
medulla
light
is act
be
that of
act
the
and the
through
to
shown
be
factor
is
the
many
not
factor,
factor,
resemble
additive,
The
to be quite a factor
and which
growth
separate and
each
their be
is
factor. other
pathways.
to
and,
Thus, actions
additive,
their on
even
when
used. of PC12 as a model
effecters
to know,
which
in this
counterparts
sympathetic
growth
quantitatively
relevance
normal
growth
do
later.
was thought
or no nerve
two factors
the
important on
factor
nerve
little
appear
ornithine
considered
from
here,
outgrowth
growth
5 hours
way to fibroblast
contain
presented
suggest
nerve
different
that
neurite
The data
and cells were collected of duplicate flasks.
which
which
interesting
on
6.6
in a similar
chemically
isolated
from
and for
factor,
responds
specific,
2
have
regard, of
the
system
been
shown
whether PC12
should to
these
cells,
be
act
on
several
namely,
the
neurons. References
1. 2. 3.
Greene, L.A. 2424-2428.
and Tischler,
Greene,
and
Dichter, 504.
L.A.
M.A.,
Rein,
Tischler,
A.S.
(1976)
Proc.
G. (1977) Nature 268, A.S., and Green3.A.
1192
Natl.
Acad.
349-351. (1977)
Sci. Nature
U.S.A. 268,
73, 501-
Vol. 114, No. 3, 1983
4. 5. 6. k 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 2:
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Greene, L.A. and Rein, G. (1977 Brain Res. 129, 247-263. Greene, L.A. and Rein, G. (1977 1 Brain Res.m, 521-528. Schubert, D., Heinemann, S., and Kidokoro, Y-977) Proc. Natl. Acad. Sci. U.S.A. 74, 2579-2583. Edgar, D.H. and Thoenen, H. (1978) Brain Res. 154, 186-190. Rieger, F., Shelanski, M.L., and Greene, L.A. Tf480) Dev. Biol. 76, 238253. H. (1978) FEBS Lett. 92, 313-316. Hatanaka, H., Otten, U., and Thoenen, Bradshaw, R.A. (1978) Annu. Rev. Biochem.47, 191-216. Vinores, S. and Guroff, G. (1980) Annu. Rev. Biophys. Bioeng.2, 223-257. Yankner, B.A. and Shooter, E.M. (1982) Annu. Rev. Biochem. 51, 845-868. Yu, M.W., Tolson, N.W., and Guroff, G. (1980) J. Biol. Chem. 255, 1048110492. Halegoua, S. and Patrick, J. (1980) Cell 22, 571-581. End, D., Hanson, M., Hashimoto, S., and Guroff, G. (1982) J. Biol. Chem. 257, 9223-9225. Huff, K., End, D., and Guroff, G. (1981) J. Cell Biol. 88, 189-198. End, D., Tolson, N., Yu, M., and Guroff, G. (1982) J. Cn. Physiol. 111, 140-148. Guroff, G., Dickens, G., End, D., and Londos, C. (1981) J. Neurochem.37, 1431-1439. Gunning, P.W., Landreth, G.E., Bothwell, M.A., and Shooter, E.M. (1981) J. Cell Biol. 89, 240-245. Gospodarowicz, D. and Moran, J.S. (1976) Annu. Rev. Biochem. 45, 531-558. Gospodarowicz, D. (1974) J. Biol. Chem. 250, 2515-2520. Gospodarowicz, D., Bialecki, H., and Grexurg, G. (1978) J. Biol. Chem. 253, 3636-3743. Bocchini, V. and Angeletti, P.U. (1969) Proc. Natl. Acad. Sci. U.S.A.3, 787-794. Pegg, A.E. and Williams-Ashman, H.G. (1968) Biochem. J.108, 533-539. Oka, T. and Perry, J.W. (1976) J. Biol. Chem. 251, 1738-1744. Guroff, G., Dickens, G., and End, D. (1981) J.-?&rochem. 37, 342-349.
1193