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Department of Biology (K.Y., J.R.B., M.B.L., C.D.), University of Michigan, Natural Science Building, Ann Arbor, Michigan 48109; and Department of Medicine, University of North Carolina (D.R.C)., Chapel Hill, North Carolina 27599
Address all correspondence and requests for reprints to: Cunming Duan, Ph.D., Department of Biology, The University of Michigan, Natural Science Building, Ann Arbor, Michigan 48109-1048. E-mail: cduan{at}umich.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, -ßI, -
, -
, -
,
, and -
, were detectable by Western immunoblot analysis in these
cells. Further analysis indicated that the subcellular distribution of
several PKC isoforms is regulated by IGF-I. While IGF-I stimulated
membrane translocation of PKC-, -, and - and regulated the
cytosolic levels of PKC-ßI, it had no such effect on PKC- and
-. To examine whether PKC activation is required for the
IGF-I-regulated biological responses, phorbol myristate acetate (PMA)
and GF109203X were used to down-regulate or inhibit PKC activity. Both
PMA (1 µM) and GF109203X (20 µM) nearly
completely suppressed the total PKC activity after a 30-min incubation
(> 90%), and this inhibition lasted for at least 24 h.
Down-regulation or inhibition of PKC activity abolished the
IGF-I-induced DNA synthesis, migration and IGFBP-5 gene expression. In
contrast, the IGFBP-5 expression induced by forskolin was unaffected by
PKC down-regulation or inhibition, suggesting that PKC activation is
required for the IGF-regulated but not the cAMP-regulated events.
Because the actions of IGF-I on DNA synthesis and IGFBP-5 gene
expression in VSMCs have been shown to be mediated through the
phosphatidylinositol 3-kinase (PI3 kinase) signaling pathway in porcine
VSMCs, the potential role of PKC in IGF-I-induced activation of PI3
kinase and PKB/Akt were examined. Treatment with either PMA or
GF109203X did not significantly affect the effects of IGF-I on PI3
kinase activation or PKB/Akt phosphorylation. These results indicated
that PKC-ßI, -, -, and - may play an essential role(s) in
IGF-I regulation of VSMC migration, DNA synthesis and gene expression,
and that these PKC isoforms may either act independently of the PI3
kinase pathway or act further downstream of PKB/Akt in the IGF
signaling network. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While it is known that the IGF-I actions on VSMC growth, migration, differentiation, and apoptosis are mediated through the IGF-IR, it is not clear how one receptor can mediate these diverse, sometimes even mutually exclusive, cellular responses to the same ligand. Studies using model systems (i.e. various tumor or transformed cell lines) indicate that one of the earliest steps in IGF-IR signaling is the phosphorylation of a number of signaling molecules such as insulin receptor substrate(IRS)-1 or -2, Shc, Grb2, and Grb10 (14). These molecules then interact with SH2 domain-containing proteins, including the Grb2/SOS and phosphatidylinositol 3-kinase (PI3 kinase), resulting in activation of the ras-raf-MEK-MAPK (mitogen-activated protein kinase) and PI3 kinase signaling pathways, respectively. Activation of the MAPK pathway is critical for cell proliferation, whereas the PI3 kinase pathway is considered to be responsible for mediating the metabolic and antiapoptotic actions of IGF-I. It should be emphasized that intracellular signaling pathways induced by a specific receptor is highly cell-type specific. Diploid, normal VSMCs in culture, which are untransformed, may respond differently than 3T3 cells or other immortalized cell lines often used for signal transduction studies (15). Thus, observations of IGF actions made in immortalized cell lines may not be applicable to primary VSMCs. Indeed, previous studies using cultured bovine and human VSMCs indicated that IGF-I neither stimulated MAPK activity nor elevated intracellular cAMP levels in these cells (16, 17). Using primary cultures of porcine VSMCs, we have recently shown that while IGF-I activates both the PI3 kinase and MAPK signaling cascades, inhibition of PI3 kinase activation, but not that of MAPK, abolishes the IGF-I-stimulated IGFBP-5 gene expression (18). These results suggest that, unlike those observations made in tumor cells, activation of PI3 kinase rather than MAPK is required for IGF-I action in primary, diploid VSMCs. Despite this progress, many important questions still remain. For instance, are other signaling components/pathways involved in IGF signaling in VSMCs ? If so, what are their relationships with the PI3 kinase and MAPK signaling pathways? To understand how the IGF-IR can mediate the diverse actions of IGF-I in VSMCs, the intracellular signaling network initiated from the IGF-IR must be understood in greater detail.
In this study, we report that several protein kinase C (PKC) isoforms are expressed in cultured procine VSMCs and their subcellular distribution is regulated by IGF-I. Furthermore, down-regulation or inhibition of PKC inhibited IGF-I-stimulated VSMC DNA synthesis, migration, and gene expression. These data indicate that these PKC isoforms may be essential signaling intermediates for IGF-I actions in porcine VSMCs in addition to the previously identified PI3 kinase and MAPK.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
Porcine aortic smooth muscle cells (VSMC) were isolated from the
thoracic aorta of a 3-week-old piglet. The cells were grown in DMEM
supplemented with 4 mM glutamine, penicillin (100 U/ml),
and streptomycin (100 µg/ml) plus 10% FBS at 37 C in 10-cm dishes
(Falcon, Becton Dickinson and Co. Labware, Franklin Lakes,
NJ). Medium was changed every fourth day until cells became confluent.
For stimulation experiments, cells were kept in serum-free medium (SFM)
for 18–24 h and then treated with indicated growth factors for the
various times indicated. The protein concentration of the cleared
lysates was determined by the copper-bicinchoninic acid method with a
kit from Pierce Chemical Co. (Rockford, IL).
Cell fractionation
Preparation of cytosolic and membrane fractions was as described
previously (19) with slight modification. Briefly, cells were treated
with IGF-I or PMA for indicated periods of times after overnight
serum-free medium starvation. After treatments, cells were scraped in
buffer A (25 mM Tris-HCl, pH 7.5, containing 250
mM sucrose, 0.5 mM EDTA, 0.5 mM
EGTA, 20 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride (PMSF)), sheared by passages through a
26.5 gauge needle, and centrifuged at 100,000 x g for
1 h. The supernatant was designated as the cytosolic fraction. The
pellet was extracted with buffer B (20 mM Tris-HCl, pH 7.5,
containing 1% SDS, 150 mM NaCl, 1 mM EDTA, 1
mM EGTA,, 20 µg/ml leupeptin, 2 µg/ml aprotinin, and 1
mM PMSF), and centrifuged at 30,000 x g
for 20 min. The supernatant was saved as the membrane fraction. Protein
concentrations of the samples were determined by the
copper-bicinchoninic acid method using a kit from Pierce Chemical Co. (Rockford, IL).
Western immunoblot analysis
For anti-PKB/Akt blotting, cells were lysed with lysis buffer
(65 mM Tris, 0.2 M NaCl, 0.5% IGEPAL CA-630,
0.1% BSA, 5 mM EDTA, 1 mM PMSF, 5 µg/ml
leupeptin, 5 µg/ml aprotinin). Equal amounts of lysates were
separated by 12.5% SDS-PAGE and transferred to Immunobilon P membrane
(0.45 µm pore size, Millipore Corp., Bedford, MA). The
membranes were blocked in a Tris-buffered saline-Tween 20 (TBST)
containing 3% BSA (Fisher Scientific, Pittsburgh, PA) and
subsequently incubated with a 1:1000 dilution of either
phospho-specific PKB/Akt antibody or a control antibody overnight at 4
C. After washing with TBST, the blots were incubated with a horseradish
peroxidase-linked secondary antibody for 2 h. Again, the blots
were washed, and enhanced chemiluminescence was performed according to
the manufacturer’s instructions (Amersham Pharmacia Biotech). Densitometry was performed by scanning the films
(ScanJet IIcx, Hewlett-Packard Co.) and then analyzing the
images using Scion Image software (Frederick, MD). The protocol for
phospho-specific and control MAPK was the same except that the dilution
of the first antibodies were 1:1000 and 1:5000, respectively, and that
the incubation with the first antibodies was at room temperature for
2 h. For PKC, cell lysates or cytosolic and membrane fractions
were separated by 7.5% SDS-PAGE. The dilution of the antibodies
against PKC isoforms were 1:500 for PKC-, -ßI, -ßII, -
, -,
-
, -, -, and - and 1:2000 for PKC-.
PI3 kinase assay
Confluent cells in 10-cm plates (Falcon) were washed with
serum-free medium and kept in the SFM overnight before stimulation with
indicated concentrations of IGF-I for 10 min. After IGF-I stimulation,
cells were lysed with lysis buffer (1% IGEPAL CA-630, 137
mM NaCl, 1 mM MgCl2, 1
mM CaCl2, 150 µM vanadate, 1
µg/ml leupeptin, and 1 µg/ml aprotinin). The PI3 kinase assay was
performed as described previously (20) with some modifications.
Briefly, equal amounts of cell lysates were incubated with specific
IRS-I antibody overnight at a 1:500 dilution and the immune complexes
were precipitated by protein A-Sepharose. The pellets were resuspended
in 50 µl of PI3 kinase buffer (20 mM Tris, pH 7.5, 100
mM NaCl, 0.5 mM EGTA) and 10 µg of
phosphatidylinositol was added followed by 10 µCi of
[32P]ATP. After a 10-min reaction at room temperature,
lipids were extracted using 160 µl of MeOH:CHCl3 (1:1).
The extracts were spotted on 1% potassium oxalate-treated TLC plates
(Analtech, Newark, DE) and developed in
CHCl3:MeOH:NH4OH (75:58:17, vol/vol/vol). The
highest migrating spots on the TLC plate, representing
phosphatidylinositol phosphate, were quantitated by performing
densitometry as described above.
Protein kinase C assay
Subconfluent cells cultured in 12-well plates (Falcon) were
washed with 1x PBS for three times and total cellular extracts were
prepared by sonicating in an extraction buffer containing 25
mM Tris-HCl (pH 7.5), 250 mM sucrose, 0.5
mM EDTA, 0.5 mM EGTA, 20 µg/ml leupeptin, 2
µg/ml aprotinin, and 1 mM DTT (21). The extracts were
cleared by centrifuging (8,000 x g) for 10 min at 4 C
and PKC activity was determined immediately using the method of Yasuda
et al. (22) with some modification. Briefly, phosphorylation
of the PKC-specific substrate peptide, Ac-MBP (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14), was carried out
in 50 µl assay buffer containing 20 mM Tris-HCl (pH 7.5),
20 mM MgCl2, 1 mM
CaCl2, 20 µM ATP, 0.25 µCi
[-32P]ATP, 50 µM Ac-MBP (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14), 10
µM phorbol 12-myristate 13- acetate (PMA), 280 µg/ml
phosphatidyl serine, 0.3% Triton-X 100 plus 25 µl cellular extract
in the presence or the absence of the PKC pseudosubstrate inhibitor
peptide, PKC (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) (25 µM). The mixture was incubated
for 5 min at 30 C and a 25 µl aliquot was spotted onto a P-81
phosphocellulose membrane. The membrane was washed twice with 1%
H3PO4 followed by two more washes with water
and the membrane-bound radioactivity was counted by liquid
scintillation counter. Specific PKC activity was calculated by
subtracting the nonspecific activity in the presence of PKC (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
from the total activity and expressed as pmol 32P bound to
Ac-MBP per min per mg protein. PMA, a PKC regulator, and GF109203X, a
PKC specific inhibitor, were used to down-regulate total cellular PKC
activity. To determine concentrations and time required by PMA and
GF109203X to down-regulate PKC activity, cells were washed five times
with SFM and then incubated in SFM containing either PMA (0.1–1000
nM) or GF109203X (0.02–200 µM) for up to
24 h. At each time point, the activity was expressed as percentage
of the activity that has been treated with SFM only. To examine the
effects of PKC down-regulation on IGF-I actions, cells were starved for
18–24 h with SFM and incubated for 2 h in SFM with indicated
concentrations of either PMA or GF109203X before growth factor
treatment.
RNA isolation and Northern blot analysis
Total RNA was isolated from cell cultures using TriReagent
following the manufacturer’s instructions (Molecular Research Center, Inc., Cincinnati, OH) and was quantified by measuring UV
absorption at O.D. 260 nm. RNA samples were size-fractionated on
a 1.0% agarose gel, blotted and fixed onto a Hybond-N
membrane (Amersham Pharmacia Biotech,
Amersham, UK). They were hybridized with the
[32P] dCTP labeled IGFBP and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) complementary DNAs (cDNAs) (23). Densitometry was
performed as described above.
[3H]-thymidine incorporation assay
To determine the rate of DNA synthesis, cells were plated onto
96-well plates (Falcon) at 15,000 cells/well in DMEM supplemented with
10% FBS and incubated for 3–5 days without a medium change. After
washing three times with serum-free DMEM, the cultures were exposed to
DMEM that contained 1 µCi [3H]-thymidine (ICN Biochemicals, Inc., Inc., Costa Mesa, CA), and the stated
concentration of IGF-I and/or inhibitors in a final volume of 200 µl.
After approximately 48 h, cells were washed twice with 1 x
PBS and twice with cold 5% trichloroacetic acid for 10 min at 4 C and
then solubilized in 200 µl 0.1 M NaOH and 1% SDS at room
temperature. The solubilized DNA was harvested for liquid scintillation
counting. The results are expressed as the percent change from the
controls.
Cell migration assay
VSMC were grown to confluence on six-well plates (Falcon). The
confluent monolayers were wounded with a single razed blade as
described previously (24). The plate was rinsed once and incubated in
DMEM plus 0.2% FBS with indicated concentrations of IGF-I and/or PMA
for 2 days at 37 C. After the incubation, the number of cells migrating
across the wound area was determined. Each data point represents a mean
of seven to ten 1-mm regions that were selected immediately after
wounding.
Statistical analysis
One-way ANOVA followed by Fisher PLSD test was used to compare
differences between control and test groups. Values are means ±
SE.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ßI, ßII, and ), novel (n) PKC (, , , ) and
atypical (a) PKC (, , 
) (25). These PKC isoforms are subject
to differential tissue distribution and may have distinct functions.
For instance, PKC- is growth inhibitory, whereas PKC- exerts
transforming activity when overexpressed (26, 27). To elucidate the
possible role(s) of PKC in IGF-I signaling, we first examined the
specific PKC isoforms expressed in porcine VSMCs by Western immunoblot
analysis. The results are summarized in Table 1
. Seven PKC isoforms, namely PKC-,
-ßI, -, -, -, , and -, were detectable in porcine
VSMCs. No specific signal was detected for PKC-ßII, - and - in
these cells.
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and 2A,
the two detectable cPKC isoforms, PKC- and -ßI, were predominantly
found in the cytosolic fraction in the serum-starved, resting cells. As
expected, PMA dose dependently stimulated membrane translocation of
PKC-. At the concentration of 1 µM PMA, the majority
of PKC- (
90%) was detected in the membrane fraction (Fig. 2A). A
notable effect of PMA on PKC-ßI levels was also detected. The
cytosolic levels of PKC-ßI decreased to undetectable level at the
high concentration (1 µM), while low concentration of PMA
(0.1 nM) increased the levels of cytosolic PKC-ßI (Fig. 1A). Of the two cPKC isoforms, only PKC-ßI was regulated by IGF-I
(Fig. 1B). This regulation appeared to be transient. IGF-I (100 ng/ml)
significantly increased the cytosolic levels of PKC-ßI at 30 min
(P < 0.05, n = 3) (Fig. 1B). No significant
change was observed at earlier or later time points. IGF-I did not
affect the subcellular distribution of PKC- throughout the
experiment (Fig. 2B).
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and - (Fig. 1B
). PKC- was
present predominantly in the membrane fraction in serum-starved,
resting cells (70%). When treated with 1 µM PMA, the
cytosolic levels of PKC- decreased to 38% of the control levels
(P < 0.05, n = 5) and its membrane levels showed
a consistent increase, although this increase was not statistically
significant (Fig. 1A). IGF-I (100 ng/ml) treatment decreased the
cytosolic PKC- by 33% at 10 min (P < 0.05, n
= 4) and 22% at 120 min (P < 0.05, n = 4) and
caused a consistent but statistically insignificant increase in the
membrane fraction at these same time points (Fig. 1B). No significant
change was detected at 2, 5, or 30 min after IGF-I treatment. PKC-
was most abundant in the cytosol fraction (> 90%) in serum-starved,
resting cells. In two out of the three experiments performed, the
cytosolic levels of PKC- were reduced by 30% with PMA (1
nM or 1 µM), and the membrane levels were
increased by 5.5-fold (1 nM) and 2.4-fold (1
µM), respectively (Fig. 1A). In all three experiments,
IGF-I treatment resulted in a significant increase in the membrane
PKC- levels at 10 and 30 min (P < 0.05) (Fig. 1B).
As predicted, the subcellular distribution of PKC- and PKC-, two
atypical PKCs expressed in porcine VSMCs, was unchanged by PMA (Figs. 1A and 2A). IGF-I had no effect on the distribution of PKC- (Fig. 2B). In contrast, IGF-I treatment caused a 2-fold increase in the
membrane fraction levels of PKC- at 30 min. These results indicate
that IGF-I stimulation altered the subcellular distribution or
concentration of several PKC isoforms including PKC-ßI, -, -,
and -, in primary cultured porcine VSMCs.
Down-regulation and inhibition of PKC abolish IGF-I-dependent VSMC
proliferation, migration and IGFBP-5 gene expression
To determine the possible role(s) of PKC in IGF-I signaling, PMA
was used to down-regulate PKC activity in these cells. Because PMA can
either activate or down-regulate PKC depending on concentrations and
cellular context, the concentration- and time-dependent effects of PMA
on total cellular PKC activity in cultured porcine VSMCs were
determined first. The total cellular PKC activity of fed cells was
158.5 ± 20.9 pmol 32P/min/mg (n = 12).
Serum-starvation of the cells for 5 min significantly decreased the
activity to 73.2 ± 33.9 32P/min/mg (n = 3)
(P < 0.05). Further starvation up to 24 h did not
cause an additional decrease. As shown in Fig. 3, treatment of VSMCs with low
concentrations of PMA (0.1 and 1 nM) resulted in a
transient increase in PKC activity. At 5 and 10 min after incubation,
PMA at 0.1 nM caused 184 ± 61% and 146 ± 39%
increases, respectively. The activity returned to the basal level after
3 h. Similarly, up-regulation by PMA (1 nM) was
greatest at 5 and 10 min (206 ± 45% and 129 ± 20%,
respectively) and returned to the initial basal level after 30 min. 10
nM of PMA resulted a transient up-regulation in two out of
three experiments. After 30-min incubation, it led to a modest
down-regulation of PKC activity (69% inhibition). In contrast,
treatment of cells with 1 µM PMA for 5 min decreased the
activity to 34 ± 7% (n = 3) of the control. After 30 min, 1
µM PMA resulted in a 97% inhibition and this inhibition
was sustained for 24 h (Fig. 3). These results indicate that,
within the time range tested, PMA activates total PKC activity at
concentrations below 10 nM but down-regulates PKC activity
at concentrations higher than 1 µM.
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, GF109203X caused a dose-dependent decrease in total PKC activity in
porcine VSMCs. The estimated IC50 value was 3.6
µM in porcine VSMCs, which is comparable with the
reported IC50 values for this inhibitor (8.4 nM
to 5.8 µM) (28, 29). At 20 µM, it resulted
in a 90 ± 1% inhibition (n = 2). The inhibitory effect of
GF109203X occurred within 5 min and lasted for at least 24 h (Fig. 4B).
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, IGF-I stimulated
3H-thymidine incorporation in a dose-dependent manner. At
10 ng/ml, IGF-I induced a maximum increase of 236% (P
< 0.01). When PMA (1.6 µM), which nearly completely
suppressed PKC activity in this system, was added, the IGF-I-stimulated
increase in 3H-thymidine incorporation was negated.
Similarly, inhibition of PKC activity by 10 µM GF109203X
abolished IGF-I-stimulated DNA synthesis (Fig. 5). PMA (1.6
µM) or GF109203X (10 µM) alone resulted in
a 37% and 49% decreases in thymidine incorporation, respectively. In
contrast, PMA at 16 nM, resulted in a moderate increase and
this effect was additive with IGF-I in stimulating
3H-thymidine incorporation (Fig. 5). When 10
µM of GF109203X, which inhibits binding of ATP to PKC,
was added together with 16 nM PMA in the presence and
absence of IGF-I, it inhibited both the PMA (16 nM) and
IGF-I-induced increases. This result indicates that the increased DNA
synthesis with PMA (16 nM) is due to its transient
up-regulation of PKC.
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, IGF-I (100 ng/ml) induced a 3.6-fold
increase in cell migration (P < 0.01). In comparison,
PDGF (20 ng/ml), the strongest known chemoattractant for VSMCs, caused
a 6.7-fold increase while no increase was observed with 500 ng/ml of
angiotensin II. Incubation of the cells with 1 µM PMA
alone did not significantly alter the basal migration levels, but it
completely abolished the IGF-I-induced increase (Fig. 6).
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, treatment of porcine VSMCs with
IGF-I (100 ng/ml) resulted in a 123-fold increase in the steady-state
levels of IGFBP-5 messenger RNA (mRNA) (n = 3). There was no
significant change in the levels of IGFBP-2 mRNA or GAPDH mRNA.
Down-regulation of PKC activity with 1 µM PMA abolished
this IGF-I-induced increase in IGFBP-5 expression. To ascertain that
the change to IGF-I was not due to any toxic effect of PMA, we examined
its effect on the forskolin-induced IGFBP-5 gene expression. Our
previous results indicated that forskolin treatment increases IGFBP-5
gene expression through a cAMP-mediated mechanism (11). As shown in
Fig. 7, forskolin (10 µM) treatment resulted in a 86-fold
increase in IGFBP-5 mRNA abundance, and the effect was additive with
IGF-I-induced IGFBP-5 gene expression, suggesting that they may act
through different mechanisms. When PMA (1 µM) was added
with forskolin, it did not significantly affect its induction of
IGFBP-5 gene expression. Likewise, the response to IGF-I plus
forskolin was reduced by PMA to the same level as forskolin alone (Fig. 7) indicating that only IGF-I induced expression was inhibited by the
treatment.
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, IGF-I treatment resulted in an approximately 10-fold increase in PI3
kinase activity. This activation was dose dependent and sustained for
up to 6 h (data not shown). Pretreatment with two PI3 kinase
inhibitors, wortmannin (20 µM) and LY294002 (10
µM), inhibited this activation. Preincubation with either
PMA (0.1 nM and 1 µM) or 20 µM
GF109203X did not significantly change the IGF-I-induced increase in
PI3 kinase activity (Fig. 8). The effect of IGF-I on the
phosphorylation status of PKB/Akt, a downstream target of PI3 kinase,
was also examined using phospho-specific and control PKB/Akt
antibodies. As shown in Fig. 9A, neither
PMA nor GF109203X had any significant effect on IGF-I-stimulated
PKB/Akt phosphorylation. PMA or GF109203X alone did not change the
basal level, either. These data suggest that down-regulation and
inhibition of total PKC activity do not affect the IGF-I-induced
activation of PI3 kinase and PKB/Akt.
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). This increase was additive
to the IGF-I-induced MAPK phophorylation. In contrast to PMA, treatment
of porcine VSMCs with GF109203X (20 µM) resulted in a
modest decrease in IGF-I-induced MAPK phosphorylation (Fig. 9B).
GF109203X treatment alone resulted in a decline in basal MAPK
phosphorylation. These opposite effects of PMA (1 µM) and
GF109203X (20 µM) suggest that these drugs have different
effects on MAPK activation, either independent of PKC or through their
differential regulation of c, nPKC and aPKC. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, -ßI, -,
-, -, -, and - are expressed in cultured procine VSMCs. Of
these PKC isoforms, the subcellular distribution or concentration of
PKC-ßI, -, -, and - is regulated by IGF-I. Furthermore,
up-regulation of PKC activity enhanced the IGF-I-stimulated DNA
synthesis, whereas down-regulation or inhibition of PKC inhibited
IGF-I-stimulated VSMC DNA synthesis, migration, and IGFBP-5 gene
expression. These data indicate that PKC may be involved in mediating
the biological responses of VSMCs to IGF-I in primary cultured procine
VSMCs. Our data also indicate that inhibition or down-regulation of PKC
does not affect IGF-I-induced PI3 kinase and PKB/Akt activation,
suggesting that these PKC isoforms either act independently of the PI3
kinase pathway or act further downstream of PKB/Akt in the IGF
signaling network.
Five PKC isoforms, including PKC-, -ß, -, - and -, have
been previously reported in rodent and human VSMCs (26, 30, 31). In the
present study, we have identified PKC- and - in cultured primary
porcine VSMCs in addition to previously reported PKC-, -ß, -
and -. Contrary to a previous study in rat VSMCs (31), we failed to
detect any specific signal for PKC-. This may be due to differences
that exist among mammalian species. The possible effect(s) of IGF-I in
regulating these PKC isoforms has not been well studied in this cell
type. To date, there is only one published observation showing that
IGF-I stimulation causes membrane translocation of PKC-, but not
that of PKC-, -ß, and - in rat VSMCs (31). In good agreement
with this report (31), we found that IGF-I stimulation caused transient
but significant decrease in cytosolic PKC- levels in porcine VSMCs.
This decrease in cytosolic PKC- was associated with a consistent but
statistically insignificant increase in its membrane presence,
suggesting the subcellular distribution of PKC- is also regulated by
IGF-I in the porcine system. Furthermore, the present study
demonstrates that IGF-I also stimulated the membrane translocation of
PKC- and - and regulated the cytosolic levels of PKC-ßI in this
cell type. These effects of IGF-I are specific because IGF-I did not
affect subcellular distribution of PKC-, -, whereas PMA induced
the membrane translocation of PKC- in cultured porcine VSMCs.
Therefore, not only PKC- but PKC-ßI, - and - are also
regulated/activated by IGF-I and may be involved in IGF-I signaling in
VSMCs.
Several mechanisms may underlie this IGF-I-regulated translocation of
PKC-ßI, -, -, and -. A recent study using Swiss 3T3 mouse
fibroblasts indicated that diacylglycerol (DAG) production stimulated
by IGF-I via activating phosphatidyl inositol-phospholipase C (PI-PLC)
may be the driving force for PKC translocation, which in turn leads to
cell proliferation (32). In VSMCs, IGF-I has been shown to regulate DAG
production and cytosolic Ca2+ influx (33). DAG is an
allosteric PKC activator produced endogenously which binds to the C1
domain of cPKCs and nPKCs. The domain contains a Cys-rich motif that
forms the DAG/phorbol ester binding site and serves as hydrophobic
anchor to recruit PKC to membrane. Membrane binding is followed by
enzyme activation (25). It is possible that the 3 c- and nPKCs
(i.e. PKC-ßI, -, and -) may be regulated by IGF-I
through this mechanism in VSMCs.
While the activity of both Ca2+-sensitive cPKC (, ßI,
ßII, and ) and Ca2+-insensitive nPKC (, , ,
) are regulated by their DAG/phorbol ester binding sites, aPKCs
including PKC-, -, - are not (25). Another mechanism that
IGF-I may use to regulate PKC is the activation of PI3 kinase. Several
reports indicate that the products of PI3 kinase including
phosphatidylinositol 3,4,5-trisphosphate are capable of activating
PKC- and - (34, 35, 36). In agreement with these observations, a
recent study in human HL-60 promyeloid cells indicated that PKC- is
a downstream target of PI 3-kinase that is activated during
IGF-I-promoted macrophage differentiation (37). Intriguingly,
IGF-I-induced PI3 kinase activation has been shown to be critical in
maintaining a differentiated phenotype in cultured chick Gizzard SMCs
(38). As we have shown in this and a previous study (18), IGF-I
stimulation strongly activates PI3 kinase in porcine VSMCs. In
addition, PKC- is translocated to the membrane fraction in IGF-I
treated cells following the activation of PI3 kinase. Experiments are
currently underway to determine whether specific inhibition of
IGF-I-induced PI3 kinase activation will abrogate the IGF-I-induced
PKC- translocation.
Activation of PKC by IGF-I has been previously documented in
fibroblasts, chondrocytes, myocytes, astrocytes, and VSMCs (30, 39, 40, 41, 42). However, the biological consequences of the activation is not
well understood. In rat astrocytes, inhibition of PKC activity
attenuates but does not abolish the IGF-I-stimulated DNA synthesis
(42). A recent study using rat VSMCs demonstrated that inhibition of
PKC by a specific inhibitor, RO31–8220, reduced IGF-I-stimulated
migration, whereas long-term incubation with PMA (100 nM),
which presumably down-regulated PKC, had little effect (43). In
contrast, it was reported that IGF-I-induced proliferation of neonatal
bovine VSMCs was unaffected by another PKC inhibitor,
dihydrosphingosine (44). It should be noted that, in these previous
studies, it was not determined whether the PKC activity was actually
effectively inhibited by these drugs. PMA is a member of phorbol esters
that are structurally related to DAG. PMA can both up- and
down-regulate PKC activity depending on the dose, time, and cellular
context. In the present study, we examined the dose- and time-dependent
regulation of PKC activity by PMA in cultured VSMCs for the first time.
Our data indicate that PMA can either up-regulate or down-regulate PKC
activity with the threshold being 10 nM. PKC activation
by the low concentrations of PMA is most apparent at 5 min and is
followed by a time-dependent loss of activity. This time course data
are consistent with a previous observation made in GH3
cells indicating that the maximum membrane translocation of PKC induced
by PMA happened at 15 min, followed by a time-dependent degradation of
the membrane-associated enzyme (45). PMA at a higher concentration (1
µM) causes a nearly complete suppression of PKC activity
(97%), and this effect is long lasting. In addition to PMA,
GF109203X, a drug that has been shown to inhibit all PKC isoforms (28, 29), was also used to inhibit PKC activity. While PMA down regulates
PKC by increasing its turnover through stimulation of membrane
association, GF109203X inhibits PKC by competing for the ATP binding
site (28, 45). We determined that the IC50 value of
GF109203X is 3.6 µM in cultured procine VSMCs. At the
concentration of 20 µM, it led to a more than 90%
inhibition. When the total PKC activity is suppressed by either high
concentrations of PMA or inhibited by GF109203X, the IGF-I-stimulated
VSMC migration and DNA synthesis were completely negated. In addition,
down-regulation of PKC activity by 1 µM PMA completely
abolished the IGF-I-induced IGFBP-5 expression. This effect was
specific because no change was detected in the expression of IGFBP-2
and GAPDH. The effect of PMA or GF109203X is probably not due to any
nonspecific toxic effect of these drugs, because 1) they did not
abolish the mitogenic activity of FBS in these cells (data not shown);
2) PMA did not affect c-AMP activated IGFBP-5 gene expression; and 3)
PMA treatment did not change the basal levels of cell migration. These
data, together with the observation that the subcellular distribution
of PKC-ßI, -, -, and - are regulated by IGF-I strongly
support the proposition that these PKC isoforms may be essential
signaling intermediates for IGF-I in VSMCs. Because our results show
that down-regulation of PKC by PMA completely inhibits IGF-I-stimulated
DNA synthesis, migration, and gene expression, we believe that either
PKC-ßI, -, - alone or in combination rather than the atypical
PKC- is involved in these actions of IGF-I in porcine VSMCs. Further
studies are needed to determine the specific role(s) for each of these
PKC isoforms by overexpression or antisense inhibition approach in
porcine VSMCs. The physiological consequence of the IGF-I-induced
PKC- is yet to be determined in VSMCs. As discussed earlier, PKC-
is a downstream target of PI 3-kinase and is involved in the action of
IGF-I on cell differentiation in other cell types. Because
IGF-I-induced PI3 kinase activation has been implicated in maintaining
a differentiated phenotype in cultured chick Gizzard SMCs (38), it
would be interesting to determine whether a similar mechanism operates
in VSMCs.
Recently, we have shown that specific inhibition of IGF-I-induced PI3 kinase activation, but not MAPK activation, abolished the IGF-I-stimulated DNA synthesis and IGFBP-5 gene expression in porcine VSMCs, indicating that the activation of PI3 kinase but not MAPK is essential for IGF-I actions in these cells (18). In the present study, we further explored possible "cross-talk" between the IGF-I-induced PKC activation and other pathways of the IGF signaling network. The results indicate that down-regulation or inhibition of PKC has no significant effect on the IGF-I-induced PI3 kinase activation. Similarly, no significant effect was found on the IGF-I-induced PKB/Akt phosphorylation. These data indicate that PKC is probably not involved in the IGF-I-induced PI3 kinase activation, suggesting that these PKC isoform(s) act either further downstream of PKB/Akt or independently from the PI3 kinase pathway. In contrast to its inability to affect PI3 kinase, both low and high concentrations of PMA induced the phosphorylation of MAPK in porcine VSMCs. This is not surprising because PMA has been shown to cause MAPK activation in a number of cell types (19, 46). A recent study using monkey kidney indicates that activation of PKC can stimulate ras (47). The activated ras, in turn, stimulates the small G protein-coupled, raf, MEK, and ultimately MAPK. Therefore, IGF-I and PMA may have acted via separate mechanisms to produce an additive effect on MAPK phosphorylation. Unlike PMA, inhibition of PKC by GF109203X reduced the basal and IGF-I-stimulated phosphorylation of MAPK. It is of interest that high concentrations of PMA abolished IGF-I-stimulated DNA synthesis and IGFBP-5 gene expression while activating MAPK in porcine VSMCs. This study further supports our conclusion that MAPK activation is not required for IGF-I-stimulated IGFBP-5 gene expression in porcine VSMC (18).
In summary, the results of this study has shown that several PKC
isoenzymes, including PKC-ßI, -, -, and - are regulated by
IGF-I, and that PKC activity is required for IGF-I-induced migration,
DNA synthesis and gene expression in porcine VSMCs. Therefore, these
PKC isoforms may be essential signaling intermediates for IGF-I acting
either independently from the PI3 kinase pathway or downstream of
PKB/Akt in the IGF signaling network in VSMCs. Further studies will
focus on the specific role(s) of these PKC isoforms in IGF-I signaling
and how they work together with other signaling intermediates to
specify the cellular responses to the plieotropic IGF-I. By identifying
the specific role(s) of each of these PKC isoform, insight may be
gained of how IGF-I regulates VSMC proliferation, migration, and
apoptosis and how it contributes to VSMC hyperplasia associated with
the development of atherosclerotic lesions.
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Acknowledgments |
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Footnotes |
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Received November 3, 1998.
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), 1 nM
(
), 10 nM (
) or 1 µM PMA (
) for 5,
10, and 30 min and 1, 3, 6, and 24 h. The specific PKC activity in
whole cell lysates was measured as described in Materials and
Methods. The results are expressed as percent change from the
controls. Values are the means ± SE of three
experiments.
) or presence of 16 nM PMA (
