(Received for publication, April 27, 1994; and in revised form, September 28, 1994)
From the
We have previously reported that two closely related protein
kinase C (PKC) isoforms, PKC and PKC
I, had divergent effects
on the growth and transformation of the same parental R6 rat embryo
fibroblast cell line (Housey, G. M., Johnson, M. D., Hsiao, W.-L. W.,
O'Brian, C. A., Murphey, J. P., Kirschmeier, P., and Weinstein,
I. B.(1988) Cell 52, 343-354; Borner, C., Filipuzzi, I.,
Weinstein, I. B., and Imber, R. (1991) Nature 353,
78-80). Whereas cells that overexpress PKC
I lost anchorage
dependence, grew to higher saturation densities, and generated small
tumors when injected into nude mice, none of these properties were seen
with cells that overexpress PKC
. In fact, the latter cells grew
even slower and to lower saturation densities as compared to control
cells. Here we investigate possible molecular mechanisms underlying the
reciprocal effects of PKC
and PKC
I. Overexpression of both
isoforms enhanced 12-O-tetradecanoyl phorbol-13
acetate-induced expression of the growth regulatory genes
c-jun, c-myc, and collagenase and enhanced feedback
inhibition of epidermal growth factor receptor binding and cellular
levels of diacylglycerol. However, the cells overexpressing PKC
I
differed from those overexpressing PKC
by displaying a decreased
requirement for growth factors and by the production of a mitogenic
factor. Thus, the basis for enhanced growth and transformation of cells
overexpressing PKC
I may be the establishment of an autocrine
growth factor loop. These findings may be relevant to the roles of
specific isoforms of PKC in carcinogenesis and tumor growth.
Protein kinase C (PKC) ()occupies a pivotal role in
signal tranduction pathways that influence numerous cellular functions,
including cell proliferation and
tumorigenesis(1, 2, 3) . PKC-mediated
signaling systems are initiated by the stimulation of cell-surface
receptors by their respective ligands. This triggers the breakdown of
phospholipids by phospholipase C (PLC) and D (PLD) enzymes to produce,
among other products, diacylglycerol (DAG)(4) . DAG binds to
and activates PKC. This activation is frequently accompanied by
increased association of PKC with cellular membranes and the subsequent
phosphorylation of various soluble and membrane-bound proteins
(reviewed in Refs. 1, 3). Once phosphorylated, PKC substrates may
provoke the activation of c-ras(6) ,
c-raf(7, 8) , MEK/MAP kinases(9) ,
and I
B/NF
B(10) . These latter proteins relay the
agonist-evoked signal to the nucleus where gene expression and cell
cycle processes are altered(11, 12) . Prominent
nuclear changes that occur as a result of PKC activation consist of
increased expression of immediate early response genes such as
c-myc, c-fos/c-jun (AP-1) and
others(13) . These genes encode transcription factors which
alter the expression of secondary response genes whose protein products
play major roles in proliferation and differentiation (reviewed in (14) ). In addition to its positive effects on signal
transduction and gene expression, PKC can also act as a cellular
guardian to prevent overstimulation of growth factor-elicited signaling
pathways(1, 2, 3) . This is achieved through
feedback inhibition of the activity of growth factor receptors, as well
as inhibition of phospholipid hydrolysis and Ca
transients.
In view of these complex and reciprocal functions of PKC, it is difficult to provide a simple description of the role of this enzyme in the multistage process of carcinogenesis. Nevertheless, it is now widely accepted that PKC constitutes the major cellular receptor for the potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and related compounds(15) . Presumably, the activation of PKC by these compounds enhances the promotion phase of carcinogenesis by altering gene expression and stimulating the growth of initiated cells that carry mutations in oncogenes and/or tumor suppressor genes. The resulting clonal expansion also increases the likelihood of additional genetic changes(16, 17) . Since TPA can enter cells and directly stimulate PKC, it mimics the action of DAG while bypassing the normal agonist-mediated control of this enzyme(18) . In addition, since it is much more potent than DAG and only slowly metabolized, TPA causes marked and prolonged activation of PKC(5) . Activation of PKC by TPA is also associated with membrane translocation and marked phenotypic effects, including alterations in morphology and stimulation or inhibition of cell growth, depending on the cell system(19, 20, 21, 22, 23, 24) . At later time points, phorbol esters usually provoke proteolytic down-regulation of PKC, thereby terminating the effects of PKC activation(1, 3, 25) . This down-regulation abrogates the positive as well as the above-mentioned negative feedback effects of PKC on signal transduction. It is, therefore, not entirely clear whether phorbol esters contribute to tumorigenesis via PKC activation, PKC down-regulation, or a combination of the two processes.
The above scenario is further complicated by the fact that PKC
comprises a multigene family that encodes at least eleven distinct
isoforms(1, 2) . These isoforms can be divided into
three subgroups based on sequence homologies and cofactor requirements:
conventional PKC (,
I,
II,
) that are dependent on
Ca
for activity and membrane localization;
non-conventional PKCs (
,
,
/L,
) that are not
dependent on Ca
; and atypical PKCs (
,
,
) that are not dependent on Ca
and are not
stimulated by DAG or TPA (reviewed in Refs. 1, 2). The expression of
these isoforms varies between tissues and cell
types(1, 2, 26) . It seems likely, therefore,
that individual isoforms differ with respect to their roles in growth
control and other specific biologic effect, but possible redundancies
in their functions may also exist. It has been difficult to examine
this question since individual cells often express more than one
isoform of PKC and at the present time isoform specific inhibitors are
not available.
We have pursued a genetic approach to analyze the
question of isoform specificity by utilizing gene transfer methods to
develop derivatives of cell lines that stably overexpress high levels
of a particular PKC isoform and then examining various biochemical and
phenotypic properties of these derivatives. We previously reported that
overexpression of PKC and PKC
, two closely related
conventional PKC isoforms, had opposite effects on the growth of the
rat embryo fibroblast cell line R6(19, 22) .
In the
present study we investigate the mechanisms underlying these divergent
growth effects of PKCI and PKC
. Our results provide some of
the first direct evidence that, even when expressed in the same cell
type, two isoforms of PKC can be redundant with respect to certain
biologic effects but differ markedly with respect to other biologic
effects.
Doubling times and saturation densities of the various cell derivatives were determined exactly as described (19, 22) with the modification that the cells were grown in 1% instead of 10% CS.
For
immunocytochemical analysis, subconfluent R6 cell derivatives were
fixed with paraformaldehyde, permeabilized with methanol, stained with
M4 (PKC-specific) or M7 (PKC
- and PKC
-specific)
monoclonal antibodies and finally with fluorescein-conjugated second
antibody, as described previously(33) .
The relative abundance of RNA/lane was judged to be similar by comparing the ethidium bromide staining of the ribosomal bands. For further confirmation, the blots were hybridized with a probe for an endogenous housekeeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH). In all cases, the ethidium bromide staining reflected the results obtained by the GAPDH probe.
Conditioned medium (CM) was
collected from PKC-overexpressing cells as follows. Complete medium was
removed from subconfluent cultures, and the cultures were washed with
DMEM and incubated for 30 h in DMEM in the absence of serum. This CM
was collected, cleared of any cell debris by centrifugation at 5000
g for 90 min, and a 100-200-µl aliquot was
added to serum-starved control R6 cells to test for its capacity to
induce DNA synthesis.
Figure 5:
Growth properties of PKCI- and
PKC
-overproducing R6 cells in low serum. Cells were seeded and
treated as described under ``Experimental Procedures.''
Growth curves were determined in the absence (Ctrl) or
presence of 16 or 160 nM TPA (10 or 100 ng/ml) in 1% calf
serum. The doubling times (A) relate to the initial
exponential phase of cell growth, the saturation density (B)
to the number of cells/35-mm plate on day 28. The results are the
average ± S.E. of three independent
experiments.
To understand the basis for the disparate growth
effects of PKCI and PKC
, we performed a series of biochemical
and molecular analyses on two PKC
I- and three
PKC
-overexpressing cell lines. We show here the results of
R6-PKC3
I (overexpressing PKC
I 50-fold) and R6-bPKC4
cells (overexpressing bovine PKC
40-fold) as representatives.
Similar results were obtained with R6 cell derivatives overexpressing
PKC
I or PKC
10-20-fold (data not shown).
We first
examined whether the growth differences can be explained by
differential activation/translocation or down-regulation of the
overexpressed or endogenously expressed PKC isoforms in response to the
phorbol esters TPA or PDBu. This is because these agents were
previously shown to activate PKC in vivo(18) and to
exaggerate the disparate effects of PKCI and PKC
on the
growth of R6 cells(19, 22) .
Immunocytochemical
analysis on intact R6-PKC3I and R6-bPKC4
cells revealed that
the overexpressed PKC
and PKC
I resided mainly in the
cytoplasm of cells when the cells were grown under standard conditions (Fig. 1). Upon treatment of the cells with phorbol esters, both
PKC
and PKC
I rapidly redistributed from the cytoplasm to the
plasma membrane. This event, called translocation, has been shown to be
associated with PKC activation(5) . There was also intensive
membrane ruffling (Fig. 1) presumably associated with
rearrangements of the underlying cytoskeleton. Since membrane ruffling
was exaggerated in both the PKC
- and PKC
I-overexpressing
cells, it is apparent that both of these isoforms can induce this
morphologic change. With prolonged treatment of cells with TPA or PDBu,
the translocation of PKC isoforms is usually associated with
``down-regulation'' of these enzymes due to
proteolysis(25) . Isoforms of PKC differ in their
susceptibility to down-regulation, and this is also a function of cell
type (reviewed in (1) ). Here we found that whereas
overexpressed PKC
I was almost completely depleted from
R6-PKC3
I cells, overexpressed PKC
is partially resistant to
down-regulation in R6-bPKC4
cells at 24 h after treatment with
phorbol esters (Fig. 1). This could be shown both by
immunocytochemical analysis on intact cells (Fig. 1) as well as
by Western blots on cytosolic and membrane fractions (Fig. 2A and (40) ). Appreciable amounts of
PKC
but not PKC
I were still immunodetectable in cells treated
with repetitive doses of TPA or PDBu for 28 days (data not shown).
Figure 1:
Immunocytochemical analysis of R6 cells
overproducing PKCI or PKC
. Immunofluorescence of PKC
I or
PKC
in intact R6-PKC3
I, R6-bPKC4
, or R6-C1 vector
control cells treated with or without 200 nM PDBu for the
indicated time periods. Monolayer cultures were treated with PDBu where
indicated, fixed with formaldehyde, permeabilized with methanol,
stained with the monoclonal antibody M4 (specific for PKC
) or M7
(specific for PKC
/
), and finally stained with a
fluorescein-conjugated secondary antibody, as described previously (33) . All photomicrographs were exposed and printed for the
same times to allow for quantitative
comparisons.
Figure 2:
Subcellular distribution and
down-regulation of ectopically overexpressed PKC and endogenously
expressed PKC
and PKC
in R6 cells treated with TPA. Protein
immunoblots were performed on cytosol (Cyt) and membrane (M) fractions of R6-PKC3
I or R6-bPKC4
cells treated
without (Ctrl) or with 100 ng/ml TPA for 30 min (30`)
or 24 h. Cyt and M samples from both cell lines were analyzed on the
same gel; autoradiographs are depicted separately for the sake of
clarity. Immunodetection was performed with monoclonal PKC
- or
polyclonal PKC
- or PKC
-specific antibodies as described under
``Experimental Procedures.'' A, overexpressed
PKC
in R6-bPKC4
cells. B, endogenous PKC
and
PKC
in R6-PKC3
I and R6-bPKC4
cells as indicated. The
apparent molecular masses in kilodaltons (kDa) of the PKC isoforms are
indicated: p81 for PKC
, p76 for PKC
, and p89 for
PKC
.
We next examined the possibility that overexpression of exogenous
PKC or PKC
I isoforms might alter the sensitivity of
endogenous PKC isoforms to phorbol ester-induced translocation and
down-regulation. We previously showed that R6 cells normally express
four endogenous PKC isoforms, PKC
, PKC
, PKC
, and
PKC
(27) . Whereas PKC
and PKC
are present at low
abundance and are mainly confined to the cytosol, PKC
and PKC
are more abundant and are mainly membrane-bound(27) . This
distribution of the endogenous isoforms of PKC was unaltered in either
untreated R6-bPKC4
or R6-PKC3
I cells (Fig. 2B). By contrast, PKC
and PKC
exhibited
differential extents of down-regulation in R6-bPKC4
or
R6-PKC3
I cells following treatment with TPA. While they were
almost completely down-regulated in cells overexpressing PKC
, they
were highly resistant to TPA-induced down-regulation in cells
overexpressing PKC
I (Fig. 2B).
Taken together
these results indicate that the overexpressed PKC and PKC
I
isoforms undergo similar translocation when the respective cells are
treated with phorbol esters. In addition, their overexpression does not
perturb the subcellular distribution of endogenous PKC isoforms. By
contrast, in response to long term phorbol ester treatment, the
overexpressed isoforms are differentially down-regulated and they
appear to influence the down-regulation of endogenous PKC
and
PKC
isoforms by an unknown mechanism. In any case, neither
PKC
- nor PKC
I-overexpressing R6 cells are fully depleted of
PKC following prolonged phorbol ester treatment, thus enabling these
cells to mainain PKC-mediated responses for prolonged periods of time
(see below).
With the exception of c-fos, the basal levels
of all of these mRNAs were increased in both R6-bPKC4 and
R6-PKC3
I cells when compared to the control cells (Fig. 3).
Thus, overexpression of PKC
and PKC
I increases the levels of
expression of both immediate early and secondary response genes. This
could be mediated by the 10-20% of the overexpressed isoforms
that were tightly associated with the membrane fraction ( Fig. 2and (40) ) because these membrane-associated PKCs
might reflect activated molecules(41) . Treatment of the
control cells with 100 ng/ml TPA caused transient increases in the
levels of c-fos, c-myc, c-jun, and
collagenase mRNAs, with maximal induction at 30 min, and 2, 3, and 6 h,
respectively (Fig. 3). In TPA-treated R6-PKC3
I cells, the
kinetics of induction of these mRNAs were similar but the induced
levels of c-myc, c-jun, and collagenase mRNAs were
higher and more sustained than in control cells, presumably due to the
intense and prolonged activation of PKC
I in these cells (Fig. 3). A different result was obtained with c-fos mRNA in R6-PKC3
I cells; the level of induction obtained with
TPA was not increased, but it persisted for a longer period of time
than in the control cells (Fig. 3). Surprisingly, R6-bPKC4
cells displayed changes in the levels of all of these mRNAs, in
response to TPA, that were very similar to those seen in R6-PKC3
I
cells (Fig. 3). These results indicate that the reciprocal
effects of PKC
and PKC
I overexpression on growth and cell
transformation are not due to differences in the regulation of this set
of genes.
Figure 3:
Basal and TPA-induced mRNA levels of
immediate early and secondary genes in PKCI- and
PKC
-overexpressing R6 cells. RNA Northern blots showing the levels
of c-myc, c-fos, c-jun, and collagenase
transcripts in control R6-C1, R6-PKC3
I, and R6-bPKC4
cells
treated without (0) or with 100 ng/ml TPA for the indicated times.
Total RNA was isolated from subconfluent cell cultures and hybridized
to corresponding
P-labeled cDNA probes as described under
``Experimental Procedures.'' To assure equal RNA loading,
these samples were also hybridized to a GAPDH cDNA
probe.
Analysis of the control cell line R6-SVXc1 revealed a
high level of saturable binding of I-EGF to EGF
receptors. Following exposure to 100 ng/ml TPA for 30 min, this binding
was markedly reduced (Fig. 4A). This reduction was due
to a decreased affinity of the EGF receptor for its ligand and not a
decrease in the number of receptors (data not shown). By 24 h of TPA
treatment, EGF binding returned to the control level (Fig. 4A), presumably reflecting extensive
down-regulation of the endogenous isoforms of PKC(27) .
Figure 4:
Enhanced negative feedback regulation of
EGF binding and DAG production in PKCI- and
PKC
-overexpressing R6 cells.
I-EGF binding (A) and cellular levels of DAG (B) were determined
for subconfluent control R6-SVX, R6-PKC3
I, and R6-bPKC4
cells
following treatment without (Ctrl) or with 100 ng/ml (160
nM) TPA for 30 min (30`), 6 h and 48 h, as described
under ``Experimental Procedures.'' All values shown are means
of three independent assays ± SE.
In
untreated R6-bPKC4 cells, EGF binding was similar to that of the
control cells. Incubation of these cells with TPA for 30 min decreased
EGF binding by an extent that was similar to that seen with the control
cells (Fig. 4A). However, EGF binding did not return to
the control level in long term TPA-treated R6-bPKC4
cells (Fig. 4A). Since in both the control and the
PKC
-overexpressing cell lines the levels of EGF receptor protein
and mRNA were not affected by prolonged treatment with the TPA (data
not shown), the latter result is probably due to persistent
phosphorylation of the EGF receptor by PKC
in the overexpresser
cells. This interpretation is in agreement with the relative resistance
of the overexpressed PKC
to TPA-induced down-regulation ( Fig. 1and Fig. 2A) and may indeed contribute to
the growth inhibition mediated by TPA in PKC
-overexpressing cells.
Prolonged inhibition of EGF receptor binding was also seen in
TPA-treated R6-PKC3
I cells (Fig. 4A). In these
cells, even the basal level of EGF binding was slightly decreased (Fig. 4A). Presumably, these effects also reflect
prolonged phosphorylation of the EGF receptor by the overexpressed
PKC
I, although the persistent levels of PKC
and PKC
in
TPA-treated R6-PKC3
I cells (Fig. 2B) may also
contribute to these effects.
When we analyzed cellular levels of
DAG, we found that the untreated control cells had a relatively high
level of DAG. This level was markedly decreased in control cells
treated with TPA for 30 min (Fig. 4B). By 48 h,
however, it had returned to the original level, presumably reflecting
down-regulation of the endogenous isoforms of PKC(27) . The
cellular levels of DAG in untreated R6-bPKC4 and R6-PKC3
I
cells were much lower than that in the control cells and remained low
following treatment with TPA for either 30 min or 48 h (Fig. 4B). Thus, high levels of either PKC
or
PKC
I exert a negative effect on cellular levels of DAG, presumably
by inhibiting the activity of PI-specific PLC, although other
explanations have not been excluded.
Therefore, we carried out growth studies in the
presence of a limiting concentration of CS. When grown in 1% CS,
control R6-SVXc1 cells exhibited a doubling time of 23 h (Fig. 5A) and grew to a saturation density of about 3
10
(Fig. 5B). TPA did not
significantly influence these growth properties (Fig. 5).
R6-bPKC4
cells were more compromised with respect to growth rate
and saturation density when compared to the control cells, and the
presence of low or high doses of TPA led to a further growth inhibition (Fig. 5). On the other hand, the doubling time of R6-PKC3
I
cells was shorter than that of control or PKC
-overexpressing
cells, and the former cells reached a higher saturation density,
especially in the presence of TPA.
These results indicate that
PKC overexpression may increase the dependence of R6 cells on
growth factors, hence the growth inhibitory effect. By contrast,
R6-PKC3
I cells appear to have a decreased growth factor
requirement, especially in the presence of TPA. We have also found that
R6-PKC3
I cells survive for a longer period of time in serum-free
medium than control or R6-bPKC4
cells (data not shown).
Figure 6:
Effects of serum, single growth factors,
and the conditioned medium of PKCI-overexpressing cells on the DNA
synthesis of R6 cells. A, induction of DNA synthesis (measured
as [
H]thymidine incorporation) 15-18 h
following the addition of 10% serum (CS) or a single dose of EGF (5
ng/ml) or PDGF (10 ng/ml) to serum-starved control R6-C1,
R6-PKC3
I, or R6-bPKC4
cells. In the absence of any additions,
[
H]thymidine incorporation with R6-C1 cells was
less than 1% of the control value (plus 10% serum); with
PKC-overexpressing cells, the corresponding value was between 5 and
7.5%. B, induction of DNA synthesis following addition of CM
from R6-PKC3
I (CM
3), R6-PKC5
I (CM
5), 10T1/2-PKC4
I (CM
4), or
R6-bPKC4
(CM
) cells to serum-starved R6-SVXc1 vector
control cells. The additions were in the presence or absence of 5 ng/ml
EGF. [
H]Thymidine incorporation assays were done
in triplicates in 96-microwell plates (10
cells/well), and
the results are expressed as mean values/well. The error bars indicate the standard deviations.
R6-PKC3I cells might represent a
clone which fortuitously contains an activated oncogene that abrogates
the need for a second growth factor. This seems to be unlikely since we
found that other clones that overexpress PKC
I also displayed a
decreased requirement for growth factors. (
)Therefore, we
examined the possibility that the cells that overexpress PKC
I
might excrete a growth factor. Serum-free CM was collected from
cultures of R6-bPKC4
and R6-PKC3
I cells and tested on
quiescent serum-starved R6-SVXc1 cells for its capacity to stimulate
DNA synthesis. The CM from the PKC
I-overexpressing cells induced
DNA synthesis to a level that was 30% of that obtained with 10% CS, and
in combination with EGF the extent of DNA synthesis was about 70% of
that obtained with 10% CS (Fig. 6B). On the other hand,
the CM from the PKC
-overexpressing cell line when tested either
alone or in combination with EGF did not significantly stimulate DNA
synthesis in the serum-starved vector control cells (Fig. 6B). Thus, R6 cells that express high levels of
PKC
I generate and secrete a growth factor(s) (see also Refs. 39,
44, 45), but this is not the case for R6 cells that express high levels
of PKC
.
We wondered whether the amount of secreted growth
factor correlated with the extent of PKCI activation. We therefore
exposed R6-PKC3
I cells to various doses (10-300 nM)
of TPA for 0-24 h and then collected the respective conditioned
media. Surprisingly, none of these media were capable of inducing DNA
synthesis of quiescent R6-SVXc1 cells better than the medium from
untreated R6-PKC3
I cells (data not shown).
Finally, to ensure
that the production of the growth factor results from the
overexpression of PKCI and not just from a rare mutagenic event,
we collected the conditioned media of another R6 cell clone
overexpressing PKC
I 20-fold (R6-PKC5
I) as well as from a
mouse 10T1/2 fibroblast cell clone overexpressing PKC
I 11-fold
(10T1/2-PKC4
I). In both cases we detected a growth stimulatory
activity in the conditioned medium although this activity was somewhat
less than that of R6-PKC3
I cells (Fig. 6B).
The purpose of the present study was to explore molecular
mechanisms which might explain why, when stably overexpressed in a rat
embryo fibroblast cell line, PKCI and PKC
, two closely
related PKC isoforms, exert different effects on growth control. We
find that both isoforms, when overexpressed, provoke similar extents
and kinetics of immediate early gene induction and negative feedback
responses despite the fact that PKC
I stimulates and PKC
inhibits growth. However, R6 cells overexpressing PKC
I secrete a
growth factor which is not produced by R6 cells that overexpress
PKC
. This growth factor may act in an autocrine fashion to
stimulate autonomous growth and transformation of cells overexpressing
PKC
I.
The reciprocal effects of overexpression of PKC and
PKC
I on R6 cells is somewhat surprising because the two isoforms
share considerable homology and cofactor requirements, and in
subcellular systems they phosphorylate similar
substrates(1, 2, 3, 28, 40) .
The differences in their effects on growth do not appear to be due to
different extents of activation of the two isoforms in the R6
derivatives, since both isoforms underwent complete membrane
translocation in response to treatment with TPA. The two isoforms,
however, exhibit different sensitivities to down-regulation in response
to TPA and, in addition, appear to influence the down-regulation of the
endogenous PKC isoforms PKC
and PKC
. It is therefore possible
that the high transformation state of TPA-treated R6-PKC3
I cells
is not only due to the initial activation of overexpressed PKC
I
but also to the persistent presence and/or activation of endogenous
PKC
. Consistent with this idea is our recent finding that
overexpression of PKC
in R6 cells is associated with enhanced
cellular growth and transformation(23) . By contrast, PKC
appears to be an isoform which impedes with the growth of fibroblasts,
and the partial resistance of this PKC isoform to TPA-induced
down-regulation even enhances the growth inhibitory effect in
TPA-treated cells. To obtain a more concise picture of the role of PKC
isoforms in cell growth, we attempted to block TPA-induced
down-regulation of the overexpressed and endogenous PKC isoforms in
R6-PKC3
I and R6-bPKC4
cells by cellular treatment with
cell-permeable calpain inhibitors (E64-d and calpain inhibitor I and
II). Although down-regulation of the PKC isoforms could be protracted
by 3-6 h, no complete blockage was observed. In addition,
prolonged exposure of fibroblasts to single or repetitive doses of
calpain inhibitors turned out to be cytotoxic (data not shown).
Another reason for the differential growth effects of PKC and
PKC
I may be their association with distinct subcellular
structures, thereby having access to different substrates and signaling
pathways. There are reports that PKC
can be detected in the
nucleus (46) and in focal adhesion plaques (33) in
fibroblasts and that PKC
can be detected in the nucleus of liver (47) and leukemic cells(48) . However, neither the
nuclei, nor focal adhesion plaques were enriched in PKC
or
PKC
I in the R6 cell derivatives used in the present studies. (
)Differential association with other subcellular
structures, such as the Golgi, mitochondria, endo- or exocytotic
vesicles, or gap junctions, have, however, not been ruled out. We are
currently searching for isoform-specific cell substrates by comparing
phosphoprotein maps (40) between phorbol ester-treated
R6-bPKC4
and R6-PKC
I cells. It will also be of interest to
examine whether putative down-stream effectors of PKC such as
c-raf(7, 8) , MEK/MAP
kinases(9, 49) , ras-GAP (6, 50) , S6 kinases(51) , or glycogen
synthase kinase 3
(GSK-3) (52) are differentially
phosphorylated and/or activated in response to phorbol ester treatment
of R6-bPKC4
and R6-PKC3
I cells.
It is astonishing to find
that overexpression of both PKC and PKC
I has similar effects
on the induction of immediate early genes, given the fact that higher
expression of these gene products has been shown to be associated with
enhanced proliferation(11, 13, 14) . An
additional regulation of the transcriptional activity of these gene
products, however, often occurs by post-translational phosphorylation.
It will therefore be important to compare the phosphorylation state and
gel shift activity of c-jun, c-fos, and c-myc between untreated and TPA-treated R6-bPKC4
or R6-PKC3
I
cells. In addition, differences in the expression of other immediate
early gene products (11, 13, 14, 52) or proteins that
regulate phases of the cell cycle, such as the retinoblastoma gene
product(53) , p53(54) , cdc2-like kinases(55) ,
or specific cyclins (55) , remain to be explored.
PKC has
been shown to negatively influence growth by feedback inhibiting EGF
binding and DAG production through phosphorylation of the EGF receptor
and PLC,
respectively(1, 2, 3, 42, 43) .
In the present study, we found that TPA-induced inhibition of EGF
receptor binding and TPA-induced decreased cellular levels of DAG were
enhanced in the cells that overexpressed either PKC or PKC
.
Therefore, these negative feedback inhibitory effects cannot explain
why overexpression of these two isoforms exert reciprocal effects on
the growth of these cells. It has recently been recognized that DAG may
also be generated by a PLD-mediated hydrolysis of phosphatidylcholine
(reviewed in (56) ). In addition, Pai et al.(57) observed enhanced activation of PLD, and therefore
DAG production, by TPA and other agonists in R6 cells overexpressing
PKC
I (the presently described R6-PKC3
I). At first glance,
these results may contradict our present finding that DAG levels are
decreased in TPA-treated PKC
I-overexpressing cells. In the
previous study, the induction of DAG production by the PLD-mediated
pathway is, however, transient and takes place at very early time
points following PKC activation (5-15 min). Longer treatments of
R6-PKC3
I cells with phorbol ester (>30 min) also led to a
diminuation of DAG levels below control levels(57) , as
reported in the present study.
The most striking difference we
observed between R6-bPKC4 and R6-PKC3
I cells was that the
latter cells exhibited a decreased requirement for growth factors, most
likely due to the secretion of an autocrine acting growth factor(s)
into their medium. Studies in progress suggest that the autocrine
growth factor is a novel protein with a molecular mass of about 68
kDa.
Its production appears to be constitutive in at least
four different PKC
I-overexpressing fibroblast cell lines, two R6
fibroblast derivatives (R6-PKC3
I and R6-PKC5
I) (Fig. 6B), one 10T1/2 derivative (10T1/2-PKC4
I) (Fig. 6B), and one NIH/3T3 derivative (NIH-PKC6
I)
(data not shown). All these cell clones exhibit transformed phenotypes,
albeit to different degrees (Refs. 19, 30, and data not shown). Neither
various growth conditions (high/low serum, growth at post-confluence)
nor cellular treatments with defined growth factors or phorbol esters
resulted in an enhanced secretion of the autocrine growth factor into
the conditioned medium of these cells. A possible explanation for this
finding may be that the growth factor gene promoter becomes
demethylated (and thereby activated) as soon as the introduced
PKC
I has started to function within a cell. Alternatively, the
growth factor may be constitutively produced by normal fibroblasts but
reside at an intracellular site in an inactive, non-secreted form. A
small amount of active PKC
I would then trigger maturation and/or
secretion of the growth factor (for example through a PKC-evoked
signaling pathway). To resolve these issues definitely, we are awaiting
the molecular cloning and further characterization of the novel growth
factor.
The notion that the growth factor secreted from
PKCI-overexpressing cells contributes to the transformed phenotype
of these cells is further bolstered by the recent observation that
highly transformed R6 cells overexpressing PKC
also produce an
autocrine-acting growth factor. (
)Although the two factors
may not be the same molecular entity, this latter finding indicates
that the establishment of an autocrine growth factor loop may be a
general mechanism by which certain PKC isoforms transform cells. By
contrast, R6 cells overexpressing PKC
are obviously incapable of
producing a mitogenic factor. Apart from three different clones of R6
cells overexpressing PKC
to various extents (R6-bPKC4
,
R6-bPKC3
, and R6-bPKC7
), we have also examined Balb/c and
NIH3T3 fibroblasts overexpressing large amounts of either bovine or
mouse PKC
(22) (data not shown). In addition, we have
treated all the above cell lines with TPA for various time periods to
try to stimulate growth factor production in a PKC-dependent manner. In
no instances was a growth stimulatory activity detected in the
conditioned medium of PKC
-overexpressing cells.
It should be
stressed that the growth inhibition of R6-PKC4 cells is not solely
due to the inability of these cells to produce an autocrine growth
factor. We infer this because the conditioned medium of R6-PKC3
I
cells could neither stimulate the DNA synthesis nor overcome the
TPA-induced growth inhibition of R6-PKC4
cells (data not shown).
This finding suggests that PKC
is linked to a intracellular
signaling pathway which negatively regulates the cell cycle. We are
currently in progress to identify the molecular players of such a
signaling pathway.
Taken together, the present study indicates that two rather similar isoforms of PKC can produce both redundant effects (similar changes in morphology, expression of early response genes, and regulation of negative feedback circuits) and also opposite effects (growth inhibition or growth stimulation, respectively) when stably expressed at high levels in the same cell type. They may, therefore, play different roles in the process of carcinogenesis and the growth of established tumors.