PKC Activation

A Divergence Point in the Signaling of Insulin and

IGF-1Induced Proliferation of Skin Keratinocytes
Shlomzion Shen, Addy Alt, Efrat Wertheimer, Marina Gartsbein, Toshio Kuroki, Motoi Ohba,
Liora Braiman, Sanford R. Sampson, and Tamar Tennenbaum

Insulin and insulin-like growth factor-1 (IGF-1) are

members of the family of the insulin family of growth
factors, which activate similar cellular downstream
pathways. In this study, we analyzed the effects of
insulin and IGF-1 on the proliferation of murine skin
keratinocytes in an attempt to determine whether
these hormones trigger the same signaling pathways.
Increasing doses of insulin and IGF-1 promote keratinocyte proliferation in an additive manner. We identified downstream pathways specifically involved in
insulin signaling that are known to play a role in skin
physiology; these include activation of the Na+/K+ pump
and protein kinase C (PKC). Insulin, but not IGF-1,
stimulated Na+/K+ pump activity. Furthermore, ouabain,
a specific Na+/K+ pump inhibitor, abolished the proliferative effect of insulin but not that of IGF-1. Insulin
and IGF-1 also differentially regulated PKC activation.
Insulin, but not IGF-1, specifically activated and
translocated the PKC isoform to the membrane fraction. There was no effect on PKC isoforms , , , and ,
which are expressed in skin. PKC overexpression
increased keratinocyte proliferation and Na+/K+ pump
activity to a degree similar to that induced by insulin
but had no affect on IGF-1induced proliferation. Furthermore, a dominant negative form of PKC abolished
the effects of insulin on both proliferation and Na+/K+
pump activity but did not abrogate induction of keratinocyte proliferation induced by other growth factors. These data indicate that though insulin or IGF-1
stimulation induce keratinocyte proliferation, only
insulin action is specifically mediated via PKC and
involves activation of the Na+/K+ pump. Diabetes
50:255264, 2001

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, RamatAviv, 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@mail.biu.ac.il.
Received for publication 21 March 2000 and accepted in revised form
23 October 2000.
DNPKC, dominant-negative PKC; DTT, dithiothreitol; EcGF, endothelial cell growth factor; EGF, epidermal growth factor; IGFR, IGF-1 receptor; IR, insulin receptor; KGF, keratinocyte growth factor; MEM, minimum
essential medium; PBS, phosphate-buffered saline; PDGF, platelet-derived
growth factor; PI3K, phosphatidylinositol 3 kinase, PKC, protein kinase C;
PMSF, phenylmethylsulfonyl fluoride; RIPA, radioimmunoprecipitation
assay; WTPKC; wild-type PKC
DIABETES, VOL. 50, FEBRUARY 2001

nsulin and IGF-1 are members of the insulin family of

growth factors and exert their mitogenic and metabolic
effects in different tissues via distinct receptors (14).
Both of these growth factors are implicated in cellular
growth and differentiation and are essential components of
the growth medium of cells in vitro (59). However, although
the mitogenic effects of IGF-1 are well documented, insulininduced proliferation has been mainly attributed to its transactivation of the IGF-1 receptor (IGFR) (2,8).
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 (24). 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 (PI3K) 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
(1416). 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).
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INSULIN ACTIVATION OF PKC IN PROLIFERATION

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  subunits. At present, as many
as four  subunits (1, 2, 3, and 4) and three  subunits (1,
2, and 3) 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 (, , 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.
METHODS
Materials. Tissue culture media and serum were purchased from Biological
Industries (Beit HaEmek, Israel). Enhanced chemiluminescence was performed with a kit purchased from Bio-Rad (Israel). Polyclonal antibodies to
Na+/K+ pump isoforms and monoclonal antip-tyr antibody were purchased
from Upstate Biotechnology (Lake Placid, NY). Polyclonal and monoclonal
antibodies to PKC isoforms were purchased from Santa Cruz (California,
USA) and Transduction laboratories (Lexington, KY). Horseradish peroxidaseanti-rabbit and anti-mouse IgG were obtained from Bio-Rad (Israel).
Leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), dithiothreitol
(DTT), Na-orthovanadate, and pepstatin were purchased from Sigma Chemicals (St. Louis, MO). Insulin (recombinant human insulin [humulinR]) was purchased from Eli Lilly France SA (Fergersheim, France). IGF-1 was a gift from
Cytolab (Israel).
Isolation and culture of murine keratinocytes. Primary keratinocytes
were isolated from newborn BALB/C mice as described (25). Keratinocytes
were cultured in Eagles 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 57 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 g/ml aprotinin, 10 g/ml leupeptin,
2 g/ml pepstatin, 1 mmol/l PMSF, 10 mmol/l EDTA, 200 mol/l NaVO4, and
10 mmol/l NaF. After homogenization and four freeze/thaw cycles, lysates
were spun down at 4C 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 l PBS containing 1% Triton
X-100 with protease and phosphatase inhibitors, incubated for 30 min at 4C,
and spun down in a microcentrifuge at maximal speed at 4C. The supernatant
256

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 g/ml leupeptin, 10 g/ml aprotinin,
0.1 mmol/l PMSF, 1 mmol/l DTT, 200 mol/l orthovanadate; and 2 g/ml pepstatin). The preparation was centrifuged in a microcentrifuge at maximal
speed for 20 min at 4C. The supernatant was used for immunoprecipitation.
Immunoprecipitation. The lysate was precleared by mixing 0.3 ml of cell
lysate with 25 l of Protein A/G Sepharose (Santa Cruz, CA), and the suspension was rotated continuously for 30 min at 4C. The preparation was then centrifuged at maximal speed at 4C for 10 min, and 30 l 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 4C. The suspension was then centrifuged at maximal
speed for 10 min at 4C, and the pellet was washed with RIPA buffer. The suspension was again centrifuged at 15,000g (4C for 10 min) and washed four
times in TBST. Sample buffer (0.5M Tris HCl, pH 6.8, 10% SDS, 10% glycerol,
4% 2 -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 manufacturers 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 Ci/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 Ci 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 4C 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 104 mol/l
ouabain from the uptake determined in the absence of the inhibitor.

RESULTS

Effects of insulin and IGF-1 on keratinocyte proliferation.

Initially we wanted to characterize the mitogenic effects of
both insulin and IGF-1 on skin keratinocytes. The ability of
the hormones to induce keratinocyte proliferation was evaluated by measuring thymidine incorporation. As shown in
Fig. 1A, both insulin and IGF-1 stimulated thymidine incorporation in a dose-dependent manner with maximal induction
achieved at 107 and 108 mol/l, respectively. At each conDIABETES, VOL. 50, FEBRUARY 2001

S. SHEN AND ASSOCIATES

FIG. 1. Insulin and IGF-1 have an additive effect on keratinocyte proliferation. Primary keratinocytes were isolated and plated as
described in RESEARCH DESIGN AND METHODS. Proliferating keratinocytes
were maintained for 5 days in low Ca2+ medium (0.05 mmol/l) until they
reached 80% confluency. A: 5-day keratinocyte cultures were stimulated for 24 h with insulin or IGF-1 at the designated concentrations.
B: In parallel, keratinocytes were stimulated with 107mol/l insulin
(Ins) and increasing doses of IGF-1 (IGF). At each concentration, the
right column ( ) represents proliferation observed when both hormones were added together. The left bar demonstrates the separate
effect of 107 mol/l insulin ( ) and increasing concentrations of IGF-1
(). Thymidine incorporation was measured as described in RESEARCH
DESIGN AND METHODS. The results shown are representative of six
experiments. Each bar represents the mean SE of three determinations expressed as percent above control unstimulated keratinocytes.

centration, the maximal stimulation by IGF-1 was greater

than that by insulin. Interestingly, when both hormones were
given together, their mitogenic effects were additive at all concentrations tested (Fig. 1B). These results suggest that
insulin and IGF-1 regulate keratinocyte proliferation through
distinct pathways.
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+
DIABETES, VOL. 50, FEBRUARY 2001

FIG. 2. Insulin but not IGF-1 induces Na+/K+ pump activity. Primary
keratinocytes were cultured as described in Fig. 1. For the pump
activity assay, 5-day-old keratinocytes were stimulated with 107 mol/l
insulin (Ins) or 108 mol/l IGF-1 (IGF) for the times indicated. A: Na+/K+
pump activity was evaluated by 86Rb uptake after 30 min stimulation
as described in RESEARCH DESIGN AND METHODS. Each bar represents the
mean SE of three determinations in three experiments performed on
separate cultures. Values are expressed as percent of control unstimulated cells from the same culture in each experiment. B: Na+/K+ pump
isoform expression was analyzed by Western blotting. Cell extracts
were prepared from control (Cont) keratinocytes and from cells stimulated with 107 mol/l insulin (Ins) or 108 mol/l IGF-1 (IGF) for the
times indicated. Whole-cell extracts (20 g protein) were subjected to
SDS-PAGE and transfer. Blots were probed with specific polyclonal
antibodies to each isoform. The blots shown are representative of
three different experiments.

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.
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, 1, and 2 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 1 or 2 subunits (results not
shown). Consistent with the lack of effect of IGF-1 on Na+/K+
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INSULIN ACTIVATION OF PKC IN PROLIFERATION

FIG. 3. Ouabain specifically blocks insulin-induced, but not IGF-1

induced, keratinocyte proliferation. Primary keratinocytes were cultured as in Fig. 1. After 5 days, keratinocyte cultures were either
untreated (Cont) or stimulated for 24 h with 107 mol/l insulin (Ins)
or 108mol/l IGF-1 (IGF) in the presence or the absence of ouabain
(104 mol/l). Thymidine incorporation was measured as described in
RESEARCH DESIGN AND METHODS. Each bar represents the mean SE of
three determinations in three separate experiments performed on
separate cultures. Values are expressed as percent of control unstimulated cells in the absence of ouabain from the same culture in each
experiment.

pump activity, this hormone did not affect protein expression

of either the  (Fig. 2B) or  (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 (104 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.
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 (3335). 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
258

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 or 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.
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 insulininduced 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.
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 insulininduced 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 insulininduced proliferation is independent of PI3K but is specifically
linked to PKC activation.
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.
DIABETES, VOL. 50, FEBRUARY 2001

S. SHEN AND ASSOCIATES

FIG. 4. Insulin, but not IGF-1 specifically, induces translocation of PKC in proliferating keratinocytes. Primary keratinocytes were isolated and
plated as described in RESEARCH DESIGN AND METHODS. Proliferating keratinocytes were maintained for 5 days in low Ca2+ medium (0.05 mmol/l)
until they reached 80% confluency. A: Cells were stimulated with 107 mol/l insulin (Ins) or 108 mol/l IGF-1 (IGF) for 5 min. Cells were lysed,
as described, and 20 g of membrane or cytosol extracts of stimulated and control unstimulated cells were subjected to SDS-PAGE and transfer. Blots were probed with specific polyclonal antibodies to each PKC isoform. B: Cells were stimulated with 107 mol/l insulin (Ins) or
108 mol/l IGF-1 (IGF) for the times indicated. Cells were lysed, as described, and 20 g of membrane or cytosol extracts of stimulated and control unstimulated (Cont) cells were subjected to SDS-PAGE and transfer. Blots were probed with PKC antibody. C: Cells were stimulated for
30 min with 107 mol/l insulin (Ins) in the presence or absence of ouabain. Cells were lysed, as described, and 20 g of membrane or cytosol extracts
of stimulated and control unstimulated (Cont) cells were subjected to SDS-PAGE and transfer. Blots were probed with specific polyclonal antibodies to PKC isoform. D: Cells were stimulated with 107 mol/l insulin (Ins) or with 10 ng/ml EGF for 10 min or maintained in 1 mmol/l Ca2+ for
18 h. After treatment, PKC or p-tyr immunoprecipitates were subjected to SDS-PAGE and transfer. Blots were probed with monoclonal antip-tyr
antibody (4G10, UBI) or anti-PKC and reblotted with anti-PKC. The data presented are representative of three separate experiments.

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 insulinmediated 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.
DIABETES, VOL. 50, FEBRUARY 2001

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 iso259

INSULIN ACTIVATION OF PKC IN PROLIFERATION

DISCUSSION

Insulin and IGF-1 exert their mitogenic and metabolic effects

in different tissues via distinct receptors (4,8). Both insulin and
IGF-1 are essential for the growth and maintenance of several
cell types including keratinocytes in culture and are essential
components of the growth medium of these cells (9). However, skin is not considered to be a classic insulin responsive
tissue, because glucose transport is not induced in response
to acute insulin stimulation. Therefore, the effects of insulin
in skin were mostly attributed to its ability to activate the
closely related IGFR (1). We have previously shown that in
keratinocytes, insulin and IGF-1 can both stimulate receptors
and activate similar downstream effectors (37). However,

FIG. 5. Insulin but not IGF-1 induces PKC activity. To determine PKC
activity, 5-day keratinocyte cultures were stimulated with 107 mol/l
insulin (Ins) or 108M IGF-1 (IGF) for the designated times (1, 15, or
30 min). PKC was immunoprecipitated from membrane ( ) and
cytosol ( ) fractions using specific anti-PKC antibody. PKC
immunoprecipitates were analyzed for PKC activity using an in vitro
kinase assay as described in RESEARCH DESIGN AND METHODS. Each bar
represents the mean SE of three determinations in three separate
experiments. Values are expressed as picomoles of ATP per dish per
minute.

forms, 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).

FIG. 6. Rottlerin specifically blocks insulin-induced proliferation. Primary keratinocytes were cultured as in Fig. 1. After 5 days, keratinocyte cultures were either untreated or stimulated for 24 h with
107 mol/l insulin (Ins) in the presence or the absence of rottlerin (Rot)
(5 mol/l) or wortmanin (Wort) (108 mol/l). Thymidine incorporation
was measured as described in RESEARCH DESIGN AND METHODS. Each bar
represents the mean SE of three determinations in three separate
experiments performed on separate cultures. Values are expressed as
percent of control unstimulated cells in the absence of inhibitors
from the same culture in each experiment.
260

FIG. 7. Overexpression of recombinant PKC adenovirus constructs.

Keratinocyte cultures were infected using recombinant adenovirus
constructs containing WTPKC, WTPKC, or a dominant DNPKC.
A: After infection, cells were cultured 24 h, harvested, and 20 g of
protein extracts were analyzed by Western blotting using specific
anti-PKC and anti-PKC antibodies. The blots presented are representative of five separate experiments. The relative increase in
expression of PKCs in overexpressing keratinocytes was analyzed by
densitometry. B: 24 h after infection, cells were harvested and PKC
and PKC immunoprecipitates were evaluated by in vitro kinase assay
as described in RESEARCH DESIGN AND METHODS.
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S. SHEN AND ASSOCIATES

FIG. 9. Inhibition of PKC activity specifically abrogates insulininduced keratinocyte proliferation. Primary keratinocytes were cultured as described in Fig.1. Noninfected cells or keratinocytes
infected with DNPKC were stimulated for 24 h with the following
growth factor concentrations: 107 mol/l insulin (Ins), 108 mol/l IGF-1
(IGF), 10 ng/ml EGF, 10 ng/ml PDGF, 1ng/ml KGF, or 5ng/ml EcGF.
Thymidine incorporation was measured as described in RESEARCH
DESIGN AND METHODS. Each bar represents the mean SE of three
determinations in three experiments done on separate cultures. Values are expressed as percent of control unstimulated cells from the
same culture in each experiment.

FIG. 8. Effects of PKC overexpression on insulin or IGF-1induced proliferation. Primary keratinocytes were cultured as described in Fig.1.
A: Mock-infected cells or keratinocytes infected with WTPKC or
WTPKC were either untreated (Cont) or were treated for 24 h with
107 mol/l insulin (Ins) or 108 mol/l IGF-1 (IGF). B: Noninfected ( )
cells overexpressing WTPKC ( ) or DNPKC ( ) were treated for
24 h with 107 mol/l insulin (Ins), 108 mol/l IGF-1 (IGF), or both
(Ins+IGF). Thymidine incorporation was measured as described in
RESEARCH DESIGN AND METHODS. Each bar represents the mean SE of
three determinations in three experiments done on separate cultures.
Values are expressed as percent of control unstimulated cells from the
same culture in each experiment.

the current study demonstrates that whereas both growth factors induce keratinocyte proliferation in a dose-dependent
manner, each hormone exert its effects through distinct signaling pathways. Our initial indication for differential regulation of keratinocyte proliferation by insulin and IGF-1 was
confirmed by our finding that these hormones had additive
effects on keratinocyte proliferation when given together, at
maximal proliferation-inducing concentrations for each hormone (Fig. 1). To identify the divergence point in insulinand IGF-1signaling pathway in regulation of keratinocyte
proliferation, we investigated elements known to both regulate keratinocyte proliferation and to act as downstream
effectors of insulin signaling. These studies revealed that
insulin but not IGF-1 signaling is mediated by PKC and
involves the stimulation of the Na+/K+ pump.
In this study, we determined that the Na+/K+ pump actively
participates in transmitting insulin but not IGF-1 signals, leadDIABETES, VOL. 50, FEBRUARY 2001

ing 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 (18,3841). 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 (19,4648). However, the functional significance
of the PKC-mediated changes in the phosphorylation state of
the Na+/K+ pump has not been conclusively demonstrated.
261

INSULIN ACTIVATION OF PKC IN PROLIFERATION

FIG. 10. Effects of overexpressed WTPKC and DNPKC on basal and

insulin-stimulated Na+/K+ pump. Primary keratinocytes were cultured
as in Fig. 1. Mock-infected keratinocytes ( ), keratinocytes infected
with WTPKC ( ), or DNPKC ( ) were either untreated or stimulated for 15 min with 107 mol/l insulin (Ins) 24 h after infection.
Na+/K+ pump activity was evaluated by 86Rb uptake as described in
RESEARCH DESIGN AND METHODS. Each bar represents the mean SE of
three determinations in three separate experiments. Values are
expressed as percent of control unstimulated cells from the same culture in each experiment. B: Mock-infected keratinocytes or keratinocytes infected with WTPKC or DNPKC were either untreated
or stimulated for 15 min with 107 mol/l insulin (Ins) 24 h after infection. Whole-cell extracts (20 g protein) were subjected to SDS-PAGE
and transfer. Blots were probed with specific polyclonal antibodies to
each isoform. The blots shown are representative of three different
experiments.

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.
262

Induction of Na+/K+ pump activity and isoform expression has

been linked to cell proliferation in different cell systems
(4951). However, this is the first report linking the insulininduced 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 (5254). 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 (59,6366).
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 (59,6367), 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 (3335,69). 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
DIABETES, VOL. 50, FEBRUARY 2001

S. SHEN AND ASSOCIATES

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.
ACKNOWLEDGMENTS

This study was supported by a Focus Giving Grant from Johnson & Johnson and in part by the Sorrell Foundation and
grants from the Israel Science Foundation founded by the
Israel Academy of Sciences and Humanities, and by the Chief
Scientists Office of the Israel Ministry of Health. E.W. is a recipient a Career Development Award from the Juvenile Diabetes
Foundation International. S.R.S. is the incumbent of the Louis
Fisher Chair in Cellular Pathology, Bar Ilan University, Israel.
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