(Received for publication, May 25, 1995; and in revised form, September 11, 1995)
From the
Protein kinase C (PKC) activation enhances neurite outgrowth in
several cell lines and primary neurons. The PKC isozymes that mediate
this response are unknown. One clue to their identity has come from
studies using PC12 cells treated with ethanol. In these cells, ethanol
increases levels of -PKC and
-PKC and markedly enhances nerve
growth factor (NGF)-induced neurite outgrowth and activation of
mitogen-activated protein (MAP) kinases by a PKC-dependent mechanism.
Since these findings suggest that
-PKC or
-PKC can promote
neural differentiation, we studied neurite outgrowth in stably
transfected PC12 cell lines that overexpress these isozymes.
Overexpression of
-PKC markedly increased NGF-induced neurite
outgrowth. This effect was blocked by down-regulating PKC or by
treating cells with the PKC inhibitor GF 109203X. In addition,
overexpression of
-PKC enhanced NGF-induced phosphorylation of MAP
kinases. In contrast, overexpression of
-PKC did not alter
responses to NGF. These results demonstrate that
-PKC promotes
NGF-induced neurite outgrowth by enhancing NGF signal transduction.
These findings suggest a role for
-PKC in neural differentiation
and plasticity.
Protein kinase C (PKC) ()is a multigene family of
phospholipid-dependent, serine-threonine kinases central to many signal
transduction pathways. The endogenous activators of PKC isozymes are
thought to be sn-1,2-diacylglycerols, free cis-unsaturated fatty acids, and polyphosphoinositides,
produced as lipid second messengers from turnover of membrane
phospholipids(1, 2, 3) . So far, 10 family
members encoded by 9 different mRNAs have been described and divided
into three structurally related groups: ``conventional''
cPKCs (
,
,
, and
) which
are regulated by calcium and diacylglycerols or tumor-promoting phorbol
esters; ``novel'' nPKCs which are sensitive to
diacylglycerols and phorbol esters but are calcium-independent (
,
,
, and
); and ``atypical'' aPKCs (
and
/
) which are insensitive to calcium, diacylglycerols, and
phorbol esters(2, 4) . In addition, two recently
cloned, highly related, phospholipid-dependent kinases, µ-PKC and
PKD, share significant homology to nPKCs in their regulatory domains
and appear to constitute a new PKC subgroup(5, 6) .
Studies with tumor-promoting phorbol esters that potently activate
most PKC isozymes have implicated PKC in the differentiation of several
cell types, including neurons(7) . In Xenopus embryos,
phorbol esters stimulate the induction of neural cells from
neuroectoderm (8) and induce neurite outgrowth from chick
sensory ganglia(9) , chick ciliary ganglion
neurons(10) , and several neuroblastoma cell lines (11, 12) . In the neural crest-derived cell line
PC12(13) , phorbol esters enhance NGF-induced MAP kinase
activation (14) and neurite
outgrowth(15, 16) . Recent studies with purified
isozymes, mutant cell lines, or transfected cells have implicated
-,
-,
-, and
-PKC isozymes in the differentiation
of non-neural cells(7, 17, 18, 19) .
In Xenopus embryos, overexpression of
- or
-PKC
enhances neural induction in dorsal and ventral ectoderm(8) .
However, little else is known about the role of specific PKC isozymes
in neural differentiation, particularly in the regulation of neurite
outgrowth.
Using PC12 cells to study mechanisms by which ethanol
alters neural growth, we found that ethanol increases neurite outgrowth
stimulated by NGF or basic fibroblast growth factor
(bFGF)(20) . A key event in signal transduction by growth
factors, including NGF and bFGF, is the activation of the closely
related serine-threonine mitogen-activated protein (MAP) kinases (also
known as extracellular signal-regulated kinases, or ERKs)
p44 (ERK1) and p42
(ERK2) by phosphorylation on tyrosine and threonine
residues(21, 22) . We recently found that ethanol
increases NGF- and bFGF-induced activation of ERK1 and
ERK2(14) . Enhancement of neurite outgrowth and MAP kinase
activation by ethanol is blocked by down-regulation of
-,
-, and
-PKC
isozymes(14, 16) . Ethanol also stimulates
PKC-mediated phosphorylation in PC12 cells (23) , and this is
associated with increased levels of mRNA (24) and protein (23) for
- and
-PKC. These results suggest that
ethanol enhances NGF-induced signal transduction and neurite formation
by increasing levels of
- and
-PKC.
The isozyme of
PKC is expressed predominantly in the nervous system with only trace
amounts detected in non-neuronal tissues (25) while
-PKC
is expressed widely in neuronal and non-neuronal tissues(26) .
Little is known about
-PKC in regulating neural function, but
recent evidence suggests a role for
-PKC in neural development. In
rat brain,
-PKC is particularly abundant in the hippocampus,
olfactory tubercle, and layers I and II of cerebral
cortex(27) . Within immunoreactive neurons, it is localized to
the Golgi apparatus and to axons and presynaptic nerve
terminals(27) . It is activated by growth factors that
stimulate neural differentiation and diacylglycerol formation such as
insulin, which activates
-PKC in cultured fetal chick brain
neurons(28) , and nerve growth factor (NGF), which activates
-PKC in PC12 cells(29) . In addition, in developing chick
brain,
-PKC is the major isozyme found in nondividing,
differentiating neurons(30) . However, despite this suggestive
evidence, no studies have yet directly demonstrated that
-PKC
regulates neural differentiation.
In the present study, we generated
PC12 cell lines that overexpress - or
-PKC to investigate the
role of these isozymes in promoting neurite formation. We found that
overexpression of
-PKC, but not of
-PKC, enhanced NGF-induced
neurite outgrowth and MAP kinase activation. The findings demonstrate
that
-PKC is a positive regulator of NGF signal transduction and
neurite growth.
Figure 1:
Immunofluorescence detection of -
and
-PKC in differentiating PC12 cells. PC12 cells were treated
without (A, B, D, and E) or with (C and F) 50 ng/ml NGF for 4 days prior to fixation
and staining with antibodies against
-PKC (A-C) or
-PKC (D-F). Immunoreactivity was inhibited when
antibodies were preincubated with their corresponding peptide antigens (B and E). Bar = 50
µm.
Figure 2:
Western analysis of -PKC and
-PKC immunoreactivity in transfected PC12 cells. PC12 cells were
transfected by electroporation with the parent vector pRc/RSV (C1, C2),
a vector containing the cDNA sequence for
-PKC (
1,
2) or
a vector containing the sequence for
-PKC (
1,
2). Stable
transfectants were selected in G418 and expanded. A, Western
blots showing increased
-PKC immunoreactivity in clones
1 and
2 and increased
-PKC immunoreactivity in
1 and
2 in
comparison with empty vector control clones C1 and C2. Immunoreactivity
was similar in control clones and parental PC12 cells. B, bar
graph showing percent increase in
- or
-PKC immunoreactivity
above controls C1 or C2 in
- and
-PKC-transfected clones.
Data are from 3-6 experiments. p < 0.04 for all
PKC-transfected clones compared to control (two-tailed t-test).
To determine whether the increase
in immunoreactivity reflected increased expression of an active PKC
isozyme, PKC activity was measured in Triton X-100-solubilized lysates
of cloned cell lines. To remove endogenous PKC inhibitors that prevent
assay of PKC activity (32) and to separate -PKC from
-PKC, the cell lysates were subjected to ion exchange
chromatography using a Mono Q column. Western analysis of fractions
eluted from the column revealed that
-PKC immunoreactivity was
present in fractions 16-27 with a peak in fraction 17 (Fig. 3A). In contrast,
-PKC was present only in
fractions 23-27 (Fig. 3A) with the strongest
immunoreactivity in fractions 25-27.
Figure 3:
Calcium-independent PKC activity in
transfected clones. PC12 clones were homogenized and solubilized in
Triton X-100, and proteins were separated after centrifugation by Mono
Q chromatography. A, immunoblots of column fractions from
2 cells stained with antibody to
-PKC or
-PKC as
indicated. B, Ca
-independent PKC activity in
Mono Q column fractions from
-PKC-transfected clones assayed in
parallel with fractions from C1 control cells. C,
Ca
-independent PKC activity in Mono Q column
fractions from
-PKC-transfected clones assayed in parallel with
fractions from parental PC12 or C1 control cells. Activity in fractions
15-20 which lack
-PKC immunoreactivity were similar in
-PKC transfected and control cell lines (data not
shown).
Ca-independent PKC activity in the
- and
-PKC-transfected clones was compared with activity in wild-type
PC12 cells or with activity in the empty vector control clone C1 (Fig. 3, B and C). In fractions containing the
highest
-PKC immunoreactivity, Ca
-independent
PKC activity was increased by 2.2-fold in clone
1 (fractions
16-21) and by 1.4-fold in clone
2 (fractions 14-18)
compared to activity in corresponding fractions from C1 cells. In
fractions 23-27, which contained
-PKC immunoreactivity,
Ca
-independent PKC activity was increased by 1.6-fold
in clone
1 and by 2.6-fold in clone
2 compared to control
cells. Thus,
1 and
2 cells contained elevated
Ca
-independent PKC activity in fractions with the
greatest
-PKC immunoreactivity and
1 and
2 cells showed
increased PKC activity only in fractions with
-PKC
immunoreactivity.
Figure 4:
Morphology of wild-type and
PKC-transfected PC12 cells after exposure to NGF. Cells were cultured
for 4 days on glass coverslips treated with poly-L-ornithine
and laminin in the presence of 50 ng/ml NGF. Parental PC12 cells (PC) and clones transfected with the empty pRc/RSV vector (C1), -PKC (
1 and
2), or
-PKC (
1 and
2) were visualized by
transmitted light interference contrast microscopy. Bar = 25 µm.
The time course and concentration
dependence of NGF-induced neurite outgrowth was examined in clones that
overexpressed -PKC. Differentiation occurred more rapidly in
clones
1 and
2 than in control cells (Fig. 5A). In addition, as shown in Fig. 5B, the percentage of cells with neurites in
cultures treated for 4 days with a maximally effective concentration of
NGF (100 ng/ml) was significantly greater in
1 (56 ± 2%)
and
2 (64 ± 1%) cells than in C1 (45 ± 2%) or
parental PC12 (47 ± 2%) cells (ANOVA, Scheffe F-test). In
contrast, the EC
for NGF-induced neurite outgrowth was
similar in PC12 (125 ± 28 pM), C1 control (124 ±
44 pM),
1 (191 ± 63), and
2 (182 ± 11
pM) cells (p = 0.54, n = 3).
Figure 5:
Concentration dependence and time course
of NGF-induced neurite outgrowth in -PKC-transfected cells. Cells
were cultured on plastic tissue culture dishes treated with
poly-L-ornithine and laminin in 0.08-100 ng/ml NGF for 4
days (A) or 50 ng/ml NGF for 0-10 days (B).
Data shown are mean ± S.D. values from representative
experiments repeated twice with similar results. Means ± S.E.
for EC
values and maximal responses in cells treated with
NGF for 4 days are given in the text.
Figure 6:
Inhibition and down-regulation of PKC
reduce neurite outgrowth in PKC-transfected clones. Clones were
cultured on poly-L-ornithine-treated tissue culture dishes for
4 days in the presence of 50 ng/ml NGF, with (striped bars) or
without (black bars) 1 µM GF 109203X. In
addition, some cells were treated with 100 nM PMA for 24 h
before NGF was added to the cultures in the continued presence of PMA
for 4 days (gray bars). Data are from 3-6 experiments.
*, significantly different compared with 1 cells treated with NGF
alone;**, significantly different compared with
2 cells treated
with NGF alone (ANOVA, Scheffe F-test).
To test this
hypothesis, we measured NGF-stimulated tyrosine phosphorylation of ERK1
and ERK2 in clones that overexpress - or
-PKC. In cells
transfected with the empty vector pRc/RSV, NGF stimulated
phosphorylation of 44- and 42-kDa proteins (Fig. 7), which
correspond to ERK1 and ERK2(36) . Phosphorylation was maximal
after 5 min and then persisted at a lower level for at least 1 h. This
pattern of phosphorylation is identical with that observed in the
parent PC12 cell line(14) . In the
-PKC-transfected clone
2, ERK1 and ERK2 phosphorylation was maximal after 5 min of NGF
treatment and remained elevated near maximal levels for at least 60 min (Fig. 7, A and B). This pattern of increased
ERK phosphorylation resembles that seen in ethanol-treated PC12
cells(14) . A similar persistent increase in phosphorylation
was observed in
1 cells (Fig. 7C). In the
-PKC-transfected clones
1 and
2, the time course (data
not shown) and extent (Fig. 7C) of NGF-stimulated
phosphorylation of ERK1 and ERK2 was similar to phosphorylation in
control cells. Thus,
-PKC overexpression increased NGF-stimulated
ERK phosphorylation while overexpression of
-PKC did not.
Figure 7:
NGF-stimulated MAP kinase phosphorylation
in PKC-transfected PC12 cells. Clones were cultured for 24 h on
poly-L-ornithine-treated dishes and then treated with 50 ng/ml
NGF. ERK1 and ERK2 tyrosine phosphorylation was detected by Western
analysis of cell lysates using anti-phosphotyrosine antibody. A, time course of ERK1 and ERK2 tyrosine phosphorylation in
clones C1 and 2. B, quantitation of ERK1 and ERK2
tyrosine phosphorylation by scanning densitometry in
2 cells
(
) and C1 cells (
). C, tyrosine phosphorylation of
ERK1 and ERK2 in control cells and PKC-transfected clones after 20 min
of treatment with 50 ng/ml NGF. Data are expressed as a percentage of
maximal phosphotyrosine immunoreactivity in the parental PC12 cell line (PC) measured after 5 min of NGF treatment and are from 4
experiments. *, significantly different compared with phosphotyrosine
immunoreactivity present in PC12 or C1 cells after 20 min of NGF
treatment (ANOVA, Scheffe F-test).
Our findings provide the first evidence that -PKC
regulates NGF signaling and neurite outgrowth. Previously, we
demonstrated that chronic exposure to ethanol enhances NGF responses in
PC12 cells, and this enhancement is abolished by down-regulation of
-,
-, and
-PKC(16) . Since we also
found that ethanol increases PKC activity and levels of
- and
-PKC(23) , we hypothesized that one of these two PKC
isozymes mediates ethanol's effects on NGF-induced neurite
outgrowth. In the present study, we found that
-PKC was abundant
in growth cones and neurites of differentiating PC12 cells, while
-PKC was not. The localization of
-PKC in growth cones and
neurites is consistent with a role in regulating neurite
outgrowth(37) . Moreover, overexpression of
-PKC enhanced
NGF-induced activation of MAP kinases and NGF-induced neurite
outgrowth, whereas overexpression of
-PKC did not. Thus, our
results clearly identify
-PKC as a positive modulator of NGF
signal transduction and NGF-induced neurite outgrowth.
Overexpression of -PKC in 3T3 fibroblasts stimulates cell
division and induces anchorage-independent growth(38) . In
contrast, in PC12 cells, overexpression of
-PKC facilitated
neuronal differentiation without affecting the growth rate. Similar
results have been observed with expression of oncogenic Ras mutants
that stimulate cell division and transformation in fibroblasts, but
induce neuronal differentiation of PC12 cells(39) . These
parallel observations suggest that
-PKC modulates signaling
pathways regulated by Ras. This is supported by our finding that
overexpression of
-PKC increased NGF-induced phosphorylation of
MAP kinases, a process known to be Ras-dependent in PC12
cells(40) . The mechanisms responsible for translating these
events into signals for cell division or differentiation appear
cell-specific and are currently unknown, but are likely to involve
signaling pathways downstream of Ras and
-PKC.
We do not know
the mechanism by which -PKC enhances NGF-induced activation of MAP
kinases. Ras is active when GTP is bound and is inactivated by the
GTPase activating protein Ras-GAP which promotes the formation of
inactive Ras-GDP(41) . In lymphocytes, activation of PKC
inhibits Ras-GAP and increases the formation of Ras-GTP(42) .
Thus,
-PKC could enhance MAP kinase activation by decreasing
Ras-GAP activity. Alternatively,
-PKC may promote activation of
kinases downstream of Ras that lead to activation of MAP kinases. MAP
kinases are activated by phosphorylation on tyrosine and threonine
residues by MAPK (or ERK) kinases (MAPKKs or MEKs). In PC12 cells, NGF
stimulation activates MEK1 (MAPKK-1)(43, 44) . Recent
evidence in PC12 cells indicates that the Ras-regulated kinase B-Raf is
activated by NGF and in turn activates MEK1 by
phosphorylation(45) . Recombinant
-,
-, and
-PKC
expressed in insect cells (46) and
-PKC in fibroblasts (47) activate Raf-1, a kinase related to B-Raf, and studies in
fibroblasts indicate that
-PKC activates Raf-1 by direct
phosphorylation(47) . Further studies are needed to determine
whether
-PKC phosphorylates and promotes activation of B-Raf in
PC12 cells.
We found that the initial peak phase of NGF-stimulated
MAP kinase phosphorylation was not altered by overexpression of
-PKC. Instead,
-PKC overexpression prevented the subsequent
decline in MAP kinase phosphorylation. This suggests that
-PKC may
negatively regulate mechanisms that deactivate MAP kinases. At least
two phosphatases have been identified that dephosphorylate MAP
kinases(48) . In PC12 cells, NGF stimulates expression of MKP1,
a dual specificity threonine-tyrosine phosphatase that dephosphorylates
and deactivates MAP kinases. Moreover, protein phosphatase 2A (PP2A)
can dephosphorylate the activating phosphoserine in MEK1 and
phosphothreonines in ERK1 and ERK2 in vitro. In addition, ERK1
has recently been shown to retrophosphorylate MEK and reduce MEK
activity, thereby reducing ERK activation through negative feedback
control(49) . Thus, inhibition of phosphatases or MEK1
retrophosphorylation may be additional mechanisms by which
-PKC
sustains MAP kinase activation.
Other PKC isozymes besides -PKC
might promote neurite formation. NGF has recently been reported to
stimulate sustained translocation of
-PKC to the particulate
fraction of PC12 cells, suggesting a role for this isozyme in
NGF-induced neurite outgrowth(50) . However, we found that
overexpression of
-PKC did not increase NGF-induced neurite
outgrowth, suggesting that such a role for
-PKC is unlikely. In
neurons, the growth cone-associated protein GAP-43 (neuromodulin, B-50)
is regulated by PKC-mediated phosphorylation (51) and is a
particularly good substrate for
-PKC(52) .
GAP-43 appears to be important for neurite outgrowth since antisense
oligonucleotides against GAP-43 reduce neurite formation in PC12 cells (53) and suppress growth cone development and neurite branching
in chick dorsal root ganglion cells(54) . NGF treatment also
increases levels of
-PKC, which accumulates as
neurites elongate(55) . Although these findings are suggestive,
overexpression studies or studies with isozyme-selective inhibitors
will be needed to determine whether
-PKC contributes
to NGF-induced differentiation.
Although -PKC is activated by
NGF(29) , it does not appear to be required for NGF-induced
differentiation of PC12 cells since down-regulation of multiple PKC
isozymes, including
-PKC, does not inhibit NGF-induced neurite
outgrowth(16, 56) . Our findings indicate instead that
-PKC plays a modulatory role in neurite outgrowth. This may
provide a mechanism whereby neurotransmitters that stimulate
diacylglycerol formation and activate PKC could promote neurite
outgrowth. Such a mechanism could contribute to activity-dependent
remodeling of synaptic connections during normal development of the
nervous system(57) .
Our findings also suggest that
excessive activation of -PKC contributes to abnormal neurite
outgrowth observed in certain disease states. Excessive consumption of
ethanol can damage the nervous system by interfering with growth and
remodeling of neurites. Several reports indicate that ethanol enhances
the growth of dendrites and axons in certain brain
regions(58, 59, 60, 61, 62) .
Our studies using PC12 cells suggest that
-PKC could mediate this
process, since ethanol increases levels of
-PKC(23) , and
NGF-induced neural differentiation is similarly enhanced in
ethanol-treated cells (20) and in clones that overexpress
-PKC. Particularly striking changes in growth occur in the
hippocampus of rats exposed to ethanol in utero, where axons
of dentate granule cells (mossy fibers) grow excessively and invade the
stratum oriens of CA3(59) . Since
-PKC is expressed in
mossy fibers and their terminals(27) , increased expression of
-PKC induced by ethanol may mediate this overgrowth. In addition,
abnormal mossy fiber projections are found in the supragranular layer
of the hippocampal dentate gyrus in humans with epilepsy and in animals
following chemical or repetitive electrical stimuli that induce
epilepsy(63) . It is possible that increased activation of
-PKC during excessive neuronal activity promotes mossy fiber
sprouting associated with the kindling of epileptic foci. Further
investigations into the role of
-PKC in neural differentiation and
in these pathological states awaits the development of isozyme-specific
inhibitors that can be used in animal models of development, epilepsy,
and the fetal alcohol syndrome.