From the Faculty of Life Sciences (S.S. A.A., M.G., L.B., S.R.S., T.T.), Gonda-Goldschmeid Center, Bar-Ilan University, Ramat-Gan; Department of Pathology (E.W.), Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel Aviv, Israel; and the Institute of Molecular Oncology and Department of Microbiology (T.K., M.O.), Showa University, Tokyo, Japan.
Address correspondence and reprint requests to Dr. Tamar Tennenbaum, Faculty of Life Sciences, Bar Ilan University, Ramat-Gan, 52900, Israel. E-mail: tennet{at}mail.biu.ac.il .
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ABSTRACT |
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INTRODUCTION |
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Despite the extensive evidence showing remarkable homology between insulin and IGF-1 receptors and similarities in their signaling pathways, these two hormones are known to have distinct physiological functions. The insulin receptor (IR) and the IGFR differentially affect cell growth, apoptosis, differentiation, and transformation (2,3,4). However, to date, efforts to identify the selective downstream effectors of these two closely related receptors indicate more similarities than differences. When activated, both receptors use IRS-1, IRS-2, and Shc as immediate downstream adapter molecules leading to the activation of the Ras, Raf, extracellular signal-regulated kinase, and the phosphatidylinositol 3 kinase (P13K) pathways (3,10). This indicates that points of divergence in signaling are likely to be downstream of these pathways.
In the present study, we have focused on the signaling pathways of insulin and IGF-1 in skin keratinocyte proliferation. Keratinocytes are the major cellular component of the epidermis, the stratified squamous epithelia forming the outermost layer of skin. Keratinocytes lie on the basement membrane and are organized into distinct cell layers, which differ morphologically and biochemically (11,12). Cellular proliferation is restricted to the basal layer. Upon division, keratinocytes give rise to either replacement progenitor cells or to cells that are committed to undergo terminal differentiation. The latter cells leave the basal layer and gradually migrate upward, simultaneously progressing along the differentiation pathway and reaching the outer surface of the epidermis in the form of fully mature corneocytes (13).
Several endogenous substances regulate proliferation and growth of keratinocytes. Among these regulators are insulin and IGF-1 (9). Indeed, skin keratinocytes express IR and IGFR (14,15,16). Furthermore, it was shown that human keratinocytes are dependent on insulin for their growth (9) and IGF-1 is mitogenic to both mouse and human keratinocytes (5,6).
Of the various downstream elements of the insulin and IGF-1 signaling pathways, we have focused on two major downstream elements, the Na+/K+ pump and the protein kinase C (PKC) family of serine threonine protein kinases. Both of these protein families are known to be involved in the insulin and IGF-1 signaling pathway and are implicated in cellular proliferation processes (1).
The Na+/K+ pump, known to be regulated by insulin, is
an intrinsic plasma membrane enzyme, which hydrolyzes ATP to maintain
transmembrane gradients of Na+ and K+ in mammalian cells
(17). The enzyme consists of
two catalytic subunits and two regulatory ß subunits. At present,
as many as four
subunits (
1,
2,
3, and
4) and three ß subunits
(ß1, ß2, and ß3) have been
identified in mammalian cells. The multiple isoforms are known to be
differentially expressed and regulated in different tissues. Regulation of
Na+/K+ pump activity by insulin has been suggested to
occur by increasing the number of pump sites in the membrane or by increasing
the activity of existing pump units in the membrane
(18,19).
PKCs are a family of serine-threonine kinases, which play key functions in
cellular signal transduction
(20,21).
Three categories of PKC have been described depending on their mechanisms of
activation: conventional PKC (, ß, and
), nonconventional
PKC (
,
, and
) and atypical PKC (
,
, and
). In skin PKC isoforms
,
,
,
, and
have
been detected
(22,23).
However, their role in mediating the nonmetabolic effects of insulin in
keratinocytes has not been studied.
In our studies we have used a model system of murine keratinocytes in
culture. Cells are maintained in the proliferative state with a high growth
rate by culturing murine keratinocytes in medium containing low
Ca2+ concentrations (0.05 mmol/l)
(24). In the present study, we
identified a unique divergence point between insulin and IGF-1 mitogenic
signaling pathways. Insulin-induced proliferation was found to involve
specific activation of PKC and stimulation of the
Na+/K+ pump, whereas IGF-1induced proliferation
did not.
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METHODS |
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Isolation and culture of murine keratinocytes. Primary keratinocytes were isolated from newborn BALB/C mice as described (25). Keratinocytes were cultured in Eagle's minimal essential medium containing 8% Chelex- (Chelex-100, Bio-Rad) treated fetal calf serum. To maintain a proliferative basal cell phenotype, the final Ca2+ concentration was adjusted to 0.05 mmol/l. Experiments were performed 5-7 days after plating.
Preparation of cell extracts and Western blot analysis. For crude membrane fractions, whole-cell lysates were prepared by scraping cells into phosphate-buffered saline (PBS) containing 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml pepstatin, 1 mmol/l PMSF, 10 mmol/l EDTA, 200 µmol/l NaVO4, and 10 mmol/l NaF. After homogenization and four freeze/thaw cycles, lysates were spun down at 4°C for 20 min in a microcentrifuge at maximal speed. The supernatant containing the soluble cytosol protein fraction was transferred to another tube. The pellet was resuspended in 250 µl PBS containing 1% Triton X-100 with protease and phosphatase inhibitors, incubated for 30 min at 4°C, and spun down in a microcentrifuge at maximal speed at 4°C. The supernatant contains the membrane fraction. Protein concentrations were measured using a modified Lowry assay (Protein Assay Kit; Bio-Rad). Western blot analysis of cellular protein fractions was carried out as described (26).
Preparation of cell lysates for immunoprecipitation. Culture dishes containing keratinocytes were washed with Ca2+/Mg2+free PBS. Cells were mechanically detached in radioimmunoprecipitation assay (RIPA) buffer (50 mmol/l Tris HCl, pH 7.4, 150 mmol/l NaCl, 1 mmol/l EDTA, 10 mmol/l NaF, 1% Triton X-100, 0.1% SDS, and 1% Na deoxycholate) containing a cocktail of protease and phosphatase inhibitors (20 µg/ml leupeptin, 10 µg/ml aprotinin, 0.1 mmol/l PMSF, 1 mmol/l DTT, 200 µmol/l orthovanadate; and 2 µg/ml pepstatin). The preparation was centrifuged in a microcentrifuge at maximal speed for 20 min at 4°C. The supernatant was used for immunoprecipitation.
Immunoprecipitation. The lysate was precleared by mixing 0.3 ml of cell lysate with 25 µl of Protein A/G Sepharose (Santa Cruz, CA), and the suspension was rotated continuously for 30 min at 4°C. The preparation was then centrifuged at maximal speed at 4°C for 10 min, and 30 µl of A/G Sepharose was added to the supernatant along with specific polyclonal or monoclonal antibodies to the individual PKC isoforms (dilution 1:100). The samples were rotated overnight at 4°C. The suspension was then centrifuged at maximal speed for 10 min at 4°C, and the pellet was washed with RIPA buffer. The suspension was again centrifuged at 15,000g (4°C for 10 min) and washed four times in TBST. Sample buffer (0.5M Tris HCl, pH 6.8, 10% SDS, 10% glycerol, 4% 2 ß-mercaptoethanol, and 0.05% bromophenol blue) was added and the samples were boiled for 5 min and then subjected to SDS-PAGE.
Adenovirus constructs. The recombinant adenovirus vectors were
constructed as described (27).
The dominant negative mutant of mouse PKC was generated by the
substitution of the lysine residue at the ATP-binding site with alanine
(28). The mutant delta cDNA
was cut from SRD expression vector with EcoR I and ligated into the pAxCA1w
cosmid cassette to construct the Ax vector. The dominant negative activity of
this gene was demonstrated by the abrogation of its autophosphorylation
activity (29).
Transduction of keratinocytes with PKC isoform genes. The culture
medium was aspirated and keratinocyte cultures were infected with the viral
supernatant (29) containing
PKC recombinant adenoviruses for 1 h. The cultures were then washed
twice with low Ca2+-containing minimum essential medium (MEM) and
refed. Cells were transferred 10-h postinfection to serum-free low
Ca2+-containing MEM for 24 h. Keratinocytes from control and
insulin-treated cultures were used for proliferation assays, 86Rb
uptake, or extracted and fractionated into cytosol and membrane fractions for
immunoprecipitation and Western blotting.
PKC activity. Specific PKC activity was determined in freshly
prepared immunoprecipitates from keratinocyte cultures after appropriate
treatments. These lysates were prepared in RIPA buffer without NaF. Activity
was measured with the use of the SignaTECT PKC assay system (Promega, Madison,
WI) according to the manufacturer's instructions. PKC pseudosubstrate
was used as the substrate in these studies.
Cell proliferation. Cell proliferation was measured by [3H]thymidine incorporation in 24-well plates. Cells were pulsed with [3H]thymidine (1 µCi/ml) overnight. After incubation, cells were washed five times with PBS and 5% thrichloracetic acid was added to each well for 30 min. The solution was removed and cells were solubilized in 1% Triton X-100. The labeled thymidine incorporated into cells was counted in a 3H-window of a Tricarb liquid scintillation counter.
Na+/K+ pump activity. Na+/K+ pump activity was determined by the measurements of ouabain-sensitive uptake of 86Rb by whole cells in 1 ml of K+-free PBS containing 2 mmol/l RbCl and 2.5 µCi of 86Rb (30). Rb uptake was terminated after 15 min by aspiration of the medium, after which the cells were rinsed rapidly four times in cold 4°C K+-free PBS and solubilized in 1% Triton X-100. The cells from the dish were added to 3 ml H2O in a scintillation vial. Samples were counted in a 3H-window of a Tricarb liquid scintillation counter. Rb-uptake specifically related to Na+/K+ pump activity was determined by subtraction of the counts per minute accumulated in the presence of 10-4 mol/l ouabain from the uptake determined in the absence of the inhibitor.
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RESULTS |
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Effects of insulin and IGF-1 on regulation of the Na+/K+ pump. We next attempted to identify the possible downstream elements that could serve as a divergence point in mediating insulin- and IGF-1induced proliferation. We initially examined the effects of insulin and IGF-1 on Na+/K+ pump activity. The Na+/K+ pump is an established regulator of proliferation and differentiation of keratinocytes and is known to be regulated by insulin. Figure 2A demonstrates the effects of insulin and IGF-1 on Na+/K+ pump activity as measured by ouabain-sensitive 86Rb uptake. As seen, insulin but not IGF-1 significantly increased pump activity.
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Next, we examined the effects of insulin and IGF-1 on
Na+/K+ pump protein isoform expression
(Fig. 2B). Skin
keratinocytes express the 1,
2,
3, ß1, and ß2 isoforms of
the Na+/K+ pump. After insulin stimulation, expression
of the
2 and
3 but not the
1 isoforms was increased as early as 30 min after
stimulation (Fig. 2B).
The elevated expression was maintained for up to 24 h
(Fig. 2B). No change
was observed in the protein expression of the ß1 or
ß2 subunits (results not shown). Consistent with the lack of
effect of IGF-1 on Na+/K+ pump activity, this hormone
did not affect protein expression of either the
(Fig. 2B) or ß
(data not shown) subunits. Interestingly, in contrast to the differential
effects of insulin and IGF-1 on the Na+/K+ pump, both
factors similarly activated other immediate downstream elements of the
insulin- and IGF-1signaling pathway. These included the phosphorylation
and activation of IRS1, IRS2, MAPK, and PI3K (results not shown). Because the
Na+/K+ pump activity plays a role in skin proliferation,
we next wanted to determine whether the distinct regulation of
Na+/K+ pump activity by insulin is associated with
keratinocyte proliferation. Thus, we studied the effects of insulin and IGF-1
on keratinocyte proliferation in cells that were pretreated with ouabain, a
specific inhibitor of the Na+/K+ pump. As shown in
Fig. 3, ouabain
(10-4 mol/l) completely blocked insulin-induced thymidine
incorporation. In contrast, the proliferative effects of IGF-1 were
essentially unaffected by ouabain. Moreover, the addition of ouabain with both
insulin and IGF-1 reduced the increase in thymidine incorporation to the level
induced by IGF-1 alone. Thus, the ability of ouabain to block only the
insulin-associated component of proliferation further suggests that insulin
and IGF-1 use different signaling pathways to induce their respective
proliferative effects.
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Effects of insulin and IGF-1 on PKC isoform translocation and
activity. PKC is another major signaling pathway, which mediates
keratinocyte proliferation and differentiation
(28,31,32)
and was shown in other tissues to be regulated by insulin signaling
(33,34,35).
In skin, PKC isoforms ,
,
,
, and
are
expressed (36). Because the
activation of PKC isoforms is associated with their translocation to membrane
fractions, we first examined the effects of insulin and IGF-1 on translocation
of the various PKC isoforms from cytosol to the membrane. As seen in
Fig. 4B, as early as 1
min after stimulation, insulin specifically induced translocation of
PKC
from the cytosol to the membrane fractions. Membrane expression of
PKC
was maintained for several hours after insulin stimulation. In
contrast, IGF-1 reduced PKC
expression in the membrane and increased
its relative level of expression in the cytosol fraction. No change in
distribution of the other PKC isoforms was seen after stimulation by either
insulin of IGF-1 (Fig.
4A). Interestingly, whereas stimulation with epidermal
growth factor (EGF) and high calcium concentrations induced tyrosine
phosphorylation of PKC
, neither insulin nor IGF-1 induced tyrosine
phosphorylation of the PKC
isoform
(Fig. 4D). To
determine if the differential regulation of PKC
could be mediated by
the Na+/K+ pump, we further analyzed the effects of
ouabain on the expression and translocation of PKC
. As seen in
Fig.4C, ouabain, the
Na+/K+ pump inhibitor, did not affect PKC
distribution or expression in nonstimulated cells. Furthermore, ouabain did
not interfere with insulin-induced translocation of PKC
.
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To determine whether the translocation of PKC is sufficient for its
activation, we next measured kinase activity of PKC immunoprecipitates from
the cytoplasmic and membrane fractions of insulin- and IGF-1treated
keratinocytes. As shown in Fig.
5, insulin but not IGF-1 increased activity of PKC
in the
membrane fraction. No elevation in PKC
activity was observed in the
cytoplasmic fraction. The insulin-induced activation was specific for
PKC
and no activation of PKCs
,
,
, or
was
observed for up to 30 min after insulin stimulation (not shown). Altogether,
these results suggest selective PKC
activation specifically by insulin
but not by IGF-1 stimulation.
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To specifically link insulin-induced PKC activation to
insulin-induced keratinocyte proliferation we used rottlerin, a specific
inhibitor of PKC
, and studied its effects on insulin-induced
proliferation. As seen in Fig.
6, rottlerin inhibited keratinocyte proliferation induced by
insulin. In contrast, wortmanin, a PI3K inhibitor, did not have any effect on
insulin induced proliferation. These results suggest that insulin-induced
proliferation is independent of PI3K but is specifically linked to PKC
activation.
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To directly study the association between insulin-induced PKC
activation and insulin-induced keratinocyte proliferation, we used recombinant
PKC adenovirus constructs to overexpress both wild-type PKC
(WTPKC
) as well as a kinase-inactive dominant-negative PKC
(DNPKC
), which abrogates the endogenous PKC
activity. Both
constructs, as well as a PKC
construct, were efficiently expressed in
keratinocytes (Fig.
7A). Furthermore, overexpressing PKC
and
PKC
induced an increase in isoform-specific PKC activity several fold
above control levels (Fig.
7B). Next, we followed the effects of overexpressing
WTPKC
and DNPKC
on insulin-induced keratinocyte proliferation.
As can be seen in Fig.
8A, overexpression of WTPKC
without insulin
treatment, but not overexpression of PKC
, increased thymidine
incorporation. The increase was similar to the increase induced by insulin in
control cells. Moreover, insulin could not further increase the upregulated
proliferation of the WTPKC
overexpressing cells. In contrast,
stimulation by IGF-1 increased thymidine incorporation in a similar manner in
both noninfected cells and in cells overexpressing WTPKC
and PKC
(Fig. 8A). These
results indicate that insulin, but not IGF-1, mediates proliferation of
keratinocytes through a pathway involving PKC
.
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The direct involvement of PKC in insulin-induced proliferation was further proven by abrogating PKC
activity. As seen in Fig. 8B, basal thymidine incorporation in cells overexpressing the DNPKC
was slightly, but significantly, lower than that in noninfected cells. However, overexpression of DNPKC
completely eliminated insulin-induced proliferation but did not affect IGF-1induced proliferation. Moreover, the additive effects of insulin and IGF-1 were reduced to that of IGF-1 alone.
Finally, the specificity of PKC activation to the insulin-mediated
pathway was analyzed by investigating the effects of DNPKC
mutant on
the mitogenic response to a variety of growth factors including the following:
IGF-1, EGF, keratinocyte growth factor (KGF), endothelial cell growth factor
(EcGF), and platelet-derived growth factor (PDGF). As seen in
Fig. 9, the overexpression of
DNPKC
selectively eliminated the proliferative effects induced by
insulin but did not block those of any of the other growth factors tested.
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Effects of overexpressed WTPKC and DNPKC
on
insulin-induced 86Rb uptake. Our results so far demonstrate
that insulin-induced proliferation is selectively mediated by activation of
PKC
and is associated with stimulation of Na+/K+
pump activity. To demonstrate that these effects are causally related, we
examined effects of WTPKC
or DNPKC
on insulin-induced
Na+/K+ pump activity. As can be seen in
Fig. 10A,
overexpression of WTPKC
increased resting pump activity to a level
similar to that induced by insulin. Insulin did not cause a further increase
in pump activity in the cells overexpressing WTPKC
. Furthermore,
DNPKC
significantly reduced resting pump activity and blocked the
insulin-induced stimulation of the pump to a level lower than basal pump
activity in control unstimulated cells. Finally, we examined the effects of
WTPKC
and DNPKC
on the expression of
Na+/K+ pump isoforms
(Fig. 10B).
Interestingly, whereas insulin induced the expression of
2
and
3 isoforms, overexpression of PKC
increased
expression of
2 similarly to insulin stimulation, and
insulin could not further increase
2 expression. In
contrast, no change in
3 isoform expression was observed and
WTPKC
did not interfere with insulin-induced expression of
3. Furthermore, abrogating PKC
activation by
overexpressing DNPKC
completely inhibited insulin-induced
2 expression but had no effect on insulin-induced expression
of the
3 isoform (Fig.
10B).
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DISCUSSION |
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In this study, we determined that the Na+/K+ pump
actively participates in transmitting insulin but not IGF-1 signals, leading
to keratinocyte proliferation. Insulin-induced Na+/K+
pump activation was associated with selective increases in expression of the
2 and
3 Na+/K+ pump
subunit isoforms. The significant role of the Na+/K+
pump in the insulin-signaling pathway was also confirmed pharmacologically by
treatment of the cells with ouabain, a selective inhibitor of the
Na+/K+ pump. Pretreatment of keratinocytes with ouabain
completely blocked insulin-induced proliferation of keratinocytes but did not
affect proliferation induced by IGF-1. Furthermore, in studies in which
additive effects of insulin and IGF-1 were examined, ouabain inhibited only
the insulin component and reduced proliferation to a level induced by
stimulation with IGF-1 alone. These findings demonstrate the involvement of
the Na+/K+ pump in mitogenic effects of insulin and
further strengthen the idea that insulin and IGF-1 act via separate signaling
pathways to induce keratinocyte proliferation.
Na+/K+ pump activity has been demonstrated to be
regulated by a variety of hormones in different tissues
(17). After pump activation,
the Na+/K+ gradient provides the force for active
transport of amino acids, phosphate, and glucose. Several studies have
suggested the involvement of the Na+/K+ pump in
regulation of cellular proliferation in variety of cell types
(18,38,39,40,41).
However, whereas the activation of the Na+/K+ pump was
known to be an important target of insulin action
(42,43),
this is the first study that directly implicates specific regulation of the
Na+/K+ pump in insulin-induced keratinocyte
proliferation. The modulation of Na+/K+ pump activity is
thought to be regulated by direct phosphorylation and dephosphorylation of
Na+/K+ pump isoforms by protein kinases and protein
phosphatases
(44,45).
Specifically, PKC phosphorylation of the subunits of the
Na+/K+ pump was shown to affect the activation state of
the Na+/K+ pump in vitro and in vivo
(19,46,47,48).
However, the functional significance of the PKC-mediated changes in the
phosphorylation state of the Na+/K+ pump has not been
conclusively demonstrated. Furthermore, as the majority of the studies used
nonspecific pharmacological activators and inhibitors of PKC, a specific PKC
isoform or a distinct function for PKC could not be identified. In this study,
we directly linked hormonal stimulation of the Na+/K+
pump to specific activation of PKC
leading to the induction of cellular
proliferation. Activation of the Na+/K+ pump by
overexpression of PKC
and the fact that insulin could not further
increase this effect indicate that a common pathway is involved. Moreover, the
blockade of insulin-induced Na+/K+ pump activity by
overexpression of a DNPKC
mutant and the ability of ouabain, a specific
pump inhibitor, to abolish the effects of insulin on proliferation without
abrogating insulin-induced activation of PKC
places the
Na+/K+ pump downstream of insulin-mediated PKC
activation. However, whereas insulin stimulationinduced expression of
both
2 and
3 isoforms, insulin-induced
PKC
activation was only associated with changes in
2
expression. Induction of Na+/K+ pump activity and
isoform expression has been linked to cell proliferation in different cell
systems
(49,50,51).
However, this is the first report linking the insulin-induced proliferation
with PKC
- mediated induction of the Na+/K+ pump
in keratinocytes. These results are in accordance with the existence of an ion
gradient in skin in vivo and with the well-documented effects of
Ca2+, K+, and Na+ ions on keratinocyte
proliferation and differentiation
(52,53,54).
These observations are consistent with a role for both insulin and the
Na+/K+ pump in keratinocyte proliferation and may
explain the significance of insulin as an essential component of growth medium
of cultured keratinocytes.
Several isoforms of PKC, including ,
, and
, have been
shown to regulate growth and differentiation of skin keratinocytes
(28,31,55).
Our results provide further evidence for the role of PKC
in
keratinocyte proliferation. PKC
is a unique isoform among the PKC
family of proteins involved specifically in growth and maturation of various
cell types (56). This isoform
was shown to participate in apoptosis
(57,58)
differentiation
(59,60)
and cell-cycle retardation or arrest
(61,62).
However, PKC
was also shown to be specifically regulated by stimulation
of several growth factors including EGF, PDGF, and neurotransmitters, as well
as by the mitogenic signal by v-src and the oncogenic form of c-Ha-ras
(59,63,64,65,66).
Changes in PKC
regulation are usually associated with its translocation
to membranal fractions, tyrosine phosphorylation of the enzyme, and activation
or deactivation of its intrinsic kinase activity
(60,64).
In several of these studies, PKC
tyrosine phosphorylation was
associated with inhibition of PKC
activity or degradation of the enzyme
(64,65,67).
In the current study, we found that insulin-induced PKC
activity was
not associated with induction of tyrosine phosphorylation. Rather, PKC
activation was associated with translocation of the enzyme and stable
expression of PKC
in the membrane fraction for several hours. Because
the phosphorylation level of PKC
is thought to regulate its activity,
enzyme stability, and/or substrate specificity
(59,63,64,65,66,67),
the functional significance of the unphosphorylated state of PKC
in
this study could be related to its effect on keratinocyte growth. In contrast
to the effects of insulin, IGF-1 translocated PKC
from the membrane to
the cytosol but had no appreciable effect on PKC activity. The importance of
this effect to the mitogenic action of IGF-1 is currently unclear. However,
because mitogenic stimulation by EGF, KGF, PDGF, EcGF, or IGF-1 was not
abrogated by the dominant negative mutant of PKC
, insulin appears to be
the primary activator of this PKC isoform in the regulation of keratinocyte
proliferation.
The link between PKC and insulin signaling has also been established
in several other systems. For example, we have recently shown that in muscle
cultures, PKC
mediates insulin-induced glucose transport
(33,34).
Similarly, in cells overexpressing the IR, insulin stimulation was shown to be
associated with activation of PKC
(68,69).
Furthermore, the insulin stimulation was found to be specifically associated
with activation of PKC
(33,34,35,69).
In addition, we have shown in this study that whereas insulin-induced
proliferation of kertinocytes is mediated by PKC
, this pathway was
independent of PI3K, an important mediator of both insulin and IGF-1. Similar
to the findings in this study, in a previous report we have found that in
another model system of muscle myotubes, insulin-induced PKC
activation
was independent of PI3K activity
(33,34).
However, whereas in these studies insulin-mediated PKC
activation has
been linked to the metabolic effects of insulin, this is the first report
linking PKC
to insulin-mediated cell proliferation. In conclusion, this
study shows for the first time that PKC
, a multifunctional serine
kinase, serves as a divergence point in transmitting insulin but not IGF-1
mitogenic signals. Future studies will be aimed at elucidating the role of
insulin-induced PKC
-mediated proliferation and its effects on the
transmission of mitogenic signals by a variety of growth factors in skin
keratinocytes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication March 21, 2000 and accepted in revised form October 23, 2000
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REFERENCES |
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