(Received for publication, December 6, 1995; and in revised form, February 15, 1996)
From the
Most cytokines stimulate the p21pathway,
leading to MAP kinase activation. One exception is interleukin-4
(IL-4), which has been shown not to activate this pathway in
hematopoietic cells. However, IL-4 acts on a broad range of cells,
including keratinocytes, in which it induces IL-6 production. We report
here that IL-4 stimulation of human keratinocytic cell lines or primary
cultures activates MAP kinase. In these cells, IL-4 stimulation induces
the tyrosine phosphorylation of p42/44 MAP kinase as well as its
catalytic activity. We also observed an increased phosphorylation of
p46
, an SH2-containing protein involved in the
Ras pathway, as a result of IL-4 stimulation in human keratinocytic
cell lines but not in T lymphocytes.
IL-4 ()is a cytokine produced by cells of
hematopoietic origin, which acts on a broad range of cells of both
hematopoietic and non-hematopoietic origin(1, 2) .
Among the latter, epidermal keratinocytes, which represent the major
cell type of the skin, are able to produce IL-6 in response to IL-4
stimulation(3) . Even though the IL-4 receptor (IL-4R) lacks a
tyrosine kinase domain, IL-4 stimulation results in an increased
tyrosine phosphorylation of a variety of intracellular substrates,
including a 170-kDa protein, designated as 4PS in hematopoietic
cells(4, 5, 6) . 4PS is both structurally and
functionally similar to IRS-1, the most prominent substrate
phosphorylated in response to insulin or insulin-like growth factor-1
stimulation in cells of connective tissue
origin(7, 8) . The amino acid sequence of 4PS (now
called IRS-2) has recently been reported, and its alignment with that
of IRS-1 revealed highly conserved stretches, which encompass the
domains of interaction with SH2-containing proteins p85 and
Grb2(8) . IL-4 induces the phosphorylation of 4PS/IRS-1 but
fails to activate the p21
pathway in
hematopoietic cells. Indeed, neither phosphorylation of Shc nor the
conversion of Ras to its active GTP-bound conformation has been
observed, correlating with a lack of Raf-1 or MAP kinase
activation(9, 10) . However, IL-4 does activate the
phosphatidylinositol 3-kinase whose p85 subunit is known to bind to 4PS
in an IL-4-inducible manner (4, 11) .
IL-4 down-regulates IL-6 production in human and murine monocytes, in contrast to what is described for keratinocytes(3, 12) . Thus, IL-4 regulates the IL-6 gene in opposite ways depending on the origin of its target cells (hematopoietic or not). We therefore investigated the possibility that alternate signal transduction pathways, which could account for these differences, may be triggered in various cell types. We indeed observed an increased tyrosine phosphorylation of MAP kinase, which is accompanied by an enhanced catalytic activity of the enzyme following IL-4 stimulation. We also report here that in keratinocytes, but not in T cell blasts, Shc is in fact phosphorylated, suggesting its possible implication in MAP kinase activation in keratinocytes in response to IL-4.
Peripheral
blood lymphocytes were obtained from lymphocyte concentrates of healthy
blood donors by Ficoll concentration. Cells (10/ml) were
prestimulated with 1 µg/ml purified phytohemagglutinin (Wellcome,
France) for 5 days. The cells were further expanded with IL-2 (7.5
ng/ml) for 5-7 days before use. These cells are IL-2-dependent
for growth and were rendered quiescent by IL-2 deprivation for 24 h
before stimulation with IL-4 (50 ng/ml).
Figure 1:
MAP
kinase activity after IL-4 stimulation. A, phosphorylation of
exogenous substrate. Cell lines were stimulated for increasing periods
of time and lysed. MAP kinase activity was determined by incubating the
lysate of SVK14 cells () or A431 cells (
) with MAP kinase
substrate peptide and [
-
P]ATP. The average
of six independent experiments is shown, and MAP kinase activity is
represented as -fold activation of the level observed in unstimulated
cells. B, in-gel kinase assay. Lysates from untreated or
IL-4-stimulated A431 or SVK14 cells were used to determine MAP kinase
activity in an in-gel kinase assay of renatured proteins in PAGE in the
presence of MBP as substrate. Inset,
P
incorporated into the p42/p44 bands. The graph indicates
quantification of radioactivity incorporated in the two
bands.
Figure 2:
Kinetics of MAP kinase activation in PHK
after EGF and IL-4 stimulation. PHK were stimulated with EGF (10 ng/ml,
) or with IL-4 (50 ng/ml,
) for increasing periods of time
and lysed. MAP kinase activity was determined as in Fig. 1A. The average of three independent experiments
is shown.
Stimulation of MAP kinase
activity was also analyzed using an in-gel kinase assay, with
immobilized MBP as substrate. Following IL-4 stimulation, two bands of M 42,000-44,000 appeared to be
phosphorylated reflecting the catalytic activity of kinases of the
corresponding M
(Fig. 1B, inset). Quantification of
P incorporated in the
two bands indicated that
2-fold activation of MAP kinase activity
was observed in SVK14 cells at 5 min, whereas a 1.7-fold enhancement
was observed in A431 cells at 20 min (Fig. 1B). MAP
kinase was therefore activated in the two keratinocytic cell lines.
Since in SVK14 cells, the time course and intensity of the response
were similar to those obtained with other growth factors, these cells
were chosen for further investigations.
Figure 3: MAP kinase tyrosine phosphorylation after IL-4 stimulation. Cells were treated for various times with IL-4 (50 ng/ml) and lysed. Cellular extracts were either immunoprecipitated with anti-Tyr(P) (P-Tyr, A) or anti-MAP kinase (MAP K, B) antibody and then immunoblotted with anti-Tyr(P) antibody (A, upper part, and B) or with anti-MAP kinase antibody (A, lower part).
Anti-MAP kinase immunoprecipitation followed by an anti-Tyr(P) immunoblotting in cellular extracts from SVK14 cells (Fig. 3B) also showed that MAP kinase is tyrosine-phosphorylated in keratinocytes but not in T cell blasts (not shown). These results suggested that specific signaling pathways might occur in keratinocytes.
Figure 4: Shc phosphorylation following IL-4 stimulation. Cells were either left untreated or stimulated for the indicated time with IL-4. Extracts (500 µg) were incubated with immobilized Grb2 protein, and complexes were further analyzed by SDS-PAGE and immunoblotted with anti-Shc or with anti-Tyr(P) (P-Tyr) antibodies (Ab).
Our results suggest that Shc phosphorylation may be the critical step leading to MAP kinase activation by IL-4 in keratinocytes. Indeed, using insulin receptor-transfected hematopoietic 32D cells, which are defective in 4PS/IRS-1 and cannot therefore use the IRS-1/Grb2 pathway, it has been shown that stimulation by insulin led to concomitant activation of MAP kinase and phosphorylation of Shc(24) . In addition, it has been shown that insulin or IL-4 induced similar levels of IRS-1 phosphorylation in an IL-4R-transfected, insulin-responsive, L6 myoblast cell line. Yet only insulin induces tyrosine phosphorylation of Shc and its association with Grb2, correlating with MAP kinase activation, and it has been suggested that this could explain the lack of MAP kinase activation by IL-4(25) . In addition Shc phosphorylation is induced by many cytokines, which activate the Ras/MAP kinase pathway (26, 27, 28) , suggesting that Shc could play a critical role in linking cytokine receptors to ras activation. The data presented support the hypothesis that in keratinocytes the phosphorylation of Shc may be the link between IL-4R and MAP kinase. It has been reported also that insulin can significantly stimulate phosphatidylinositol 3-kinase and MAP kinase activities, although to a lesser extent in IRS-1-deficient mice, reinforcing the idea that MAP kinase stimulation may be driven by IRS-1-independent signaling pathways(29) . These pathways could be used by IL-4R to activate the MAP kinase cascade.
Novel
phosphotyrosine binding (PTB) domains have been described(30) .
Insulin-stimulated phosphorylation of IRS-1 and Shc is mediated by
interaction of these two proteins via their PTB domain (31, 32) with a NPXY domain in the
juxtamembrane region of insulin receptor subunit. Although a
similar NPXY motif is also present in IL-4R and is required
for IRS-1 phosphorylation(33) , no tyrosine phosphorylation of
Shc after IL-4R stimulation has been reported(34) , suggesting
that this sequence is not sufficient to interact efficiently with Shc.
It has been suggested that amino acids surrounding the NPXY
motif may be critical in the regulation of interactions between
proteins that possess PTB motif(35) . To bypass the lack of
interaction, Shc can also be phosphorylated on tyrosine by certain
members of the Src tyrosine kinase family(36) . The
distribution of members of this family is different between lymphocytes
and keratinocytes, and implication of different Src kinases could
explain the difference of Shc phosphorylation observed between these
two cell types. Further experiments are currently in progress to
identify which members of the Src kinase family are involved in this
phosphorylation of Shc.