Regulation of Interleukin-1{alpha} Expression by Integrins and Epidermal Growth Factor Receptor in Keratinocytes from a Mouse Model of Inflammatory Skin Disease*

Robin M. Hobbs and Fiona M. Watt {ddagger}

From the Keratinocyte Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

Received for publication, January 16, 2003 , and in revised form, March 21, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic mice expressing {beta}1 integrins in the suprabasal epidermal layers have sporadic skin hyperproliferation and inflammation correlated with activation of extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase and increased interleukin (IL)-1{alpha} production. We investigated the link between aberrant integrin expression, Erk activation, and expression of IL-1{alpha}. Transgenic keratinocytes had higher basal Erk activity and IL-1{alpha} levels than nontransgenic controls and were more sensitive to stimulation of Erk activity and IL-1{alpha} production by IL-1{alpha}, 12-O-tetradecanoylphorbol-13-acetate (TPA), epidermal growth factor (EGF), and serum. Inhibition of Erk in transgenic keratinocytes reduced basal IL-1{alpha} levels and the stimulation of IL-1{alpha} production by serum or phorbol ester, demonstrating that Erk could regulate IL-1{alpha} expression. TPA or IL-1{alpha} treatment resulted in rapid down-regulation of the EGF receptor in transgenic cells, indicative of transactivation. Inhibition of transactivation blocked basal and TPA or IL-1{alpha} induced Erk activation, but not I{kappa}B{alpha} degradation, and abolished increased IL-1{alpha} production in transgenic cells. In transgene-negative cells, constitutive activation of IL-1-dependent signaling by wild type or kinase-dead IRAK1 stimulated IL-1{alpha} production independent of Erk. We conclude that suprabasal integrin expression leads to Erk activation and increased IL-1{alpha} expression by potentiating activation of the EGF receptor. These results provide a mechanism by which aberrant integrin expression triggers epidermal hyperproliferation and inflammation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Extracellular matrix receptors of the {beta}1 integrin family play a role in normal epidermal proliferation, differentiation, and homeostasis (1). Integrin expression is normally confined to the basal layer of the epidermis. However, in benign hyperproliferative conditions, such as wound healing and psoriasis, integrins are also expressed in the suprabasal, differentiating cell layers.

To analyze the significance of suprabasal integrin expression, transgenic mice have been generated in which individual human integrin subunits are expressed under the control of the involucrin promoter (2, 3). Mice expressing a {beta}1 transgene (Inv{beta}1 mice) are crossed with mice expressing {alpha}2, {alpha}3, or {alpha}5 integrin transgenes (Inv{alpha}2, Inv{alpha}3, or Inv{alpha}5 animals) to generate mice with the corresponding suprabasal integrin heterodimers: {alpha}2{beta}1, {alpha}3{beta}1, or {alpha}5{beta}1. The mice have a sporadic hyperproliferative phenotype with skin inflammation that bears some resemblance to the human skin disease, psoriasis. The presence of the transgenic integrins increases the sensitivity of the skin to external stimuli: when phenotypically normal skin is treated with the phorbol ester TPA1 suprabasal proliferation is observed in transgenic (Inv{alpha}2{beta}1 and Inv{alpha}3{beta}1) but not wild type epidermis (4).

In affected lesions of integrin transgenic mice and human psoriasis, there is activation of Erk MAPK in suprabasal integrin-positive cells (5). Constitutive activation of Erk in cultured human keratinocytes recreates the hyperproliferation and per-turbed differentiation that are characteristic of psoriasis. In addition, keratinocytes from transgenic mice produce more interleukin-1{alpha} (IL-1{alpha}) than cells from wild type mice independent of integrin ligation (5).

Keratinocyte-derived IL-1 is known to be an important initiator of wound healing in response to skin injury (6, 7). Disruption of the epidermis causes the release of pre-formed pools of IL-1 (8, 9), which, through stimulation of endothelial and immune cells, can generate an inflammatory infiltrate at the site of injury (7, 10). Keratinocytes themselves are activated by the released IL-1; they become more migratory and express keratins associated with a hyperproliferative state (11, 12). Overexpression of IL-1{alpha} in murine epidermis produces spontaneous inflammatory lesions, illustrating the potency of IL-1 in driving this process (13). Keratinocytes also contain large stores of an intracellular variant of IL-1 receptor antagonist (icIL-1ra) (10, 14), which is released alongside IL-1 (9) and blocks access to the IL-1 receptor (IL-1R1) (15).

Elevated IL-1{alpha} production in transgenic mice that express suprabasal integrins is believed to be of central importance to the psoriatic phenotype. It contributes to keratinocyte hyperproliferation through its ability to activate Erk (5) in addition to triggering the inflammatory infiltrate in the skin. The aim of the present experiments was therefore to investigate the mechanism that links suprabasal integrin expression to IL-1{alpha} expression.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—U0126 was purchased from Promega (Madison, WI); CAPE was from BIOMOL Research Laboratories (Plymouth Meeting, PA); SB203580, LY294002, bisindolylmaleimide, AG1478, TPA, and cell culture grade heparin were from Sigma. Recombinant human IL-1{alpha}, porcine TGF-{beta}1, recombinant human IL-6 and ELISA kits for murine IL-1{alpha} were purchased from R&D Systems (Minneapolis, MN). EGF was from Peprotech (Rocky Hill, NJ). Neutralizing antibodies to mIL-1{alpha} (both goat polyclonal and rat monoclonal), mIL-6 (goat polyclonal), mGM-CSF (goat polyclonal), mTNF-{alpha} (goat polyclonal) and TGF-{beta}1,2,3 (mouse monoclonal) were also from R&D Systems. The mouse monoclonal antibody to human {beta}1 integrin (P5D2) has been used previously (5). Antibodies specific for phospho-Erk MAPK (both a rabbit polyclonal and E10 mouse monoclonal), total Erk1/2 (rabbit polyclonal), phospho-I{kappa}B{alpha} Ser32/36 (5A5 mouse monoclonal), and total I{kappa}B{alpha} (rabbit polyclonal) were from Cell Signaling Technology Inc. (Beverly, MA). The antibody to EGFR used for Western blotting (sheep polyclonal) and antibodies to phosphotyrosine (mouse monoclonal, clone 4G10) and Shc (rabbit polyclonal) were from Upstate Biotechnology (Lake Placid, NY). The antibody used to immunoprecipitate the EGFR was a rabbit polyclonal from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies used to detect mIL-1{alpha} and mIcIL-1ra by Western blot were goat polyclonals from R&D Systems. The antibody to actin (AC40 monoclonal) was from Sigma and to total Erk2 (monoclonal) and IRAK1 (rabbit polyclonal) from Santa Cruz Biotechnology. Anti-cornifin was a kind gift of A. Jetten (NIEHS, National Institutes of Health, Research Triangle Park, NC) (16). The rabbit antiserum to mouse involucrin antibody is described elsewhere (17). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit were from Amersham Biosciences (Bucks, UK) and anti-goat from Sigma. A protease inhibitor mixture for mammalian tissues and phosphatase inhibitor mixtures 1 and 2 were also from Sigma. BCA protein assay was purchased from Pierce.

Cell Culture—Spontaneously immortalized murine keratinocyte lines derived from the skin of adult Inv{alpha}2{beta}1 and negative control mice (passages 25–45) were cultured in FAD medium (one part Ham's F-12 medium, three parts Dulbecco's modified Eagle's medium, and 0.18 mM adenine) supplemented with 10% FCS and HICE mixture (0.5 µg/ml hydrocortisone, 5 µg/ml insulin, 1010 M cholera toxin, and 10 ng/ml epidermal growth factor) (complete medium) in the absence of J2–3T3 feeder cells at 32 °C in an atmosphere of 5% CO2 (3, 5). Cells were split at 1:3 to 1:5 at each passage.

Primary murine keratinocytes were isolated from 3-day-old newborn mice generated from Inv{beta}1 and Inv{alpha}2 crosses (2, 4) and cultured in low calcium FAD + 10% FCS + HICE as described previously (18). To induce stratification of primary keratinocytes, cells were transferred to standard ("high calcium") complete medium overnight. For some experiments preconfluent cells were starved overnight in FAD + 0.5% FCS (without HICE mixture) before addition of cytokines, growth factors, or other stimuli.

IL-1{alpha} ELISAs—To determine intracellular levels of IL-1{alpha}, cells were washed in PBS and then lysed in 1.0% Nonidet P-40, 150 mM NaCl, and 50 mM Tris, pH 8.0, containing protease inhibitor mixture. Lysates were assayed for protein content using the BCA assay and diluted to 10 µg of total protein/ml in 5% bovine serum albumin in PBS. Samples were assayed for IL-1{alpha} levels by ELISA according to the instructions from the manufacturer. Data were analyzed for statistical significance using a paired Student's t test.

Protein Extraction, Immunoprecipitation, and Western Blotting—For Western blotting cells were washed twice in PBS and lysed in radioimmune precipitation assay buffer as described previously (5), except that protease and phosphatase (1 and 2) inhibitor mixtures were used in place of individual inhibitor components. Western blots were performed as previously described (5, 19), and antibodies were used according to instructions from the manufacturers. The methods for combined immunoprecipitation and Western blotting of EGFR (20) and Shc (21) have also been described previously. Detection of IL-1{alpha} and icIL-1ra on blots required an overnight incubation with the antibodies in 5% milk, 0.1% Tween 20 in Tris-buffered saline. Densitometry of bands produced by Western blotting was carried out using NIH Image version 1.58.

Cytokine Neutralization by Antibodies—Cells were washed twice in FAD + 0.5% FCS and then transferred for ~16 h to FAD + 0.5% FCS, containing the appropriate antibodies at the following concentrations: goat anti-IL-1{alpha}, 0.2 µg/ml; rat anti-IL-1{alpha}, 5 µg/ml; anti-IL-6, 0.4 µg/ml; anti-GM-CSF, 0.4 µg/ml; anti-TNF-{alpha},1 µg/ml; anti-TGF-{beta},1 µg/ml; and anti-human {beta}1 integrin, 0.1 mg/ml. Cells were washed in PBS and harvested in radioimmune precipitation assay buffer as described above. Western blots and IL-1{alpha} ELISAs were performed as described above.

IRAK1 Retroviral Infection—Wild type and kinase-dead (D340N) IRAK1 constructs in the pBK-RSV expression vector (Stratagene) were kindly provided by Dr. Keith Ray (Department of Cell Biology, Glaxo-SmithKline, Stevenage, UK) (22). Both IRAK1 constructs were removed from pBK-RSV by EcoRI/XbaI digestion and blunted with Klenow fragment plus dNTPs. The resulting purified cassettes were ligated into the retroviral vector pBabe puro that had been digested with BamHI and also blunted. The pBabe puro vector (23) was a modified version made by Dr. Sally Lowell with an internal ribosome entry site driving an enhanced green fluorescent protein construct following the multiple cloning site (pBabe puro IRES-EGFP).

To generate retrovirus, constructs were transfected via a calcium phosphate method into the Phoenix amphotropic packaging line obtained from ATCC with kind permission of Dr. G. Nolan (Stanford University School of Medicine, Stanford, CA) (24). Retrovirus-containing supernatant was incubated with the transgene-negative murine keratinocyte line in the presence of 2.5 µg/ml Polybrene. After 2–3 days, 2.5 µg/ml puromycin was added to select for infected cells. Flow cytometric analysis of EGFP expression in live cells showed that, after selection, ~40% of cells were expressing the retroviral vector.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular Levels of IL-1{alpha} Correlate with Erk MAPK Activity in Primary Transgenic Mouse Keratinocytes—We previously reported that immortalized keratinocytes derived from an Inv{alpha}2{beta}1 transgenic mouse express more IL-1{alpha} than keratinocytes from a transgene-negative control (5). We confirmed and extended the correlation between suprabasal integrin expression and IL-1{alpha} expression by examining primary keratinocytes from transgene-negative mice and from Inv{beta}1 and Inv{alpha}2{beta}1 animals. When grown as a monolayer in low calcium medium, transgene-positive keratinocytes contained more pro-IL-1{alpha} (Fig. 1) and secreted more IL-1{alpha} (data not shown) than transgene-negative cells. The increased IL-1{alpha} expression correlated with increased basal Erk activity, both in low serum medium (Fig. 1) and in complete medium (data not shown).



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FIG. 1.
Correlation between phospho-Erk levels and intracellular IL-1{alpha} in keratinocytes. Inv{beta}1, Inv{alpha}2{beta}1, and transgene-negative (Neg) primary mouse keratinocytes (passages 3–4) were cultured overnight in low (— Ca2+) or high (+ Ca2+) calcium-containing medium either with 10%FCS + HICE mixture (complete medium) or 0.5% FCS alone (0.5% serum medium). Cells were lysed and analyzed by Western blotting for the proteins indicated. The loading control was actin.

 

When transgene-negative or -positive cells were induced to stratify and accumulate differentiated cells in high calcium medium, the levels of IL-1{alpha} and active Erk decreased in unison (Fig. 1). The decrease in Erk activity is consistent with the described role of Erk in maintaining proliferation and inhibiting differentiation of keratinocytes (5, 19, 25, 26), and the decline in IL-1{alpha} levels supports earlier observations that IL-1{alpha} expression decreases during keratinocyte differentiation both in vitro and in vivo (27, 28, 29). In contrast, icIL-1ra tended to accumulate along with differentiation markers such as the cornified envelope precursor cornifin (Fig. 1), also in agreement with earlier observations (28, 30).

These data confirm that transgenic keratinocytes express more IL-1{alpha} than controls and establish a correlation with increased basal Erk MAPK activity. They also suggest that icIL-1ra is not positively regulated by Erk.

Transgenic Keratinocytes Show Enhanced Induction of IL-1{alpha} and Erk Activity in Response to External Stimuli—The increased sensitivity of Inv{alpha}2{beta}1 and Inv{alpha}3{beta}1 mouse skin to TPA (4) led us to investigate whether the enhanced Erk activity and IL-1{alpha} production in transgenic keratinocytes extended to cells exposed to TPA and a variety of other stimuli. Negative and Inv{alpha}2{beta}1 cells that had been starved overnight were treated with IL-1{alpha}, TPA, EGF, TGF-{beta}, or IL-6. NF{kappa}B activity is known to regulate IL-1{alpha} expression in fibroblasts (31, 32), and degradation of I{kappa}B{alpha} was used to monitor NF{kappa}B pathway activation (33). The level of phospho-Erk1/2 was used to measure Erk activation (Fig. 2).



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FIG. 2.
Cytokine and phorbol ester stimulation of Inv{alpha}2{beta}1 and transgene-negative keratinocytes. Cells were starved overnight and then treated with various stimuli (IL-1{alpha}, 10 ng/ml; TPA, 25 ng/ml; EGF, 10 ng/ml; TGF-{beta}, 10 ng/ml; IL-6, 10 ng/ml) for either 20 min or 6 h before harvesting. Lysates were analyzed by Western blotting for the proteins indicated. Loading controls were total Erk1/2 and actin. Samples were compared with untreated controls (—).

 

IL-6 and TGF-{beta} did not activate the Erk or NF{kappa}B pathways to any great extent and did not induce IL-1{alpha} (Fig. 2). Instead, TGF-{beta} reduced IL-1{alpha} expression in Inv{alpha}2{beta}1 cells, probably reflecting its known inhibitory effect on NF{kappa}B signaling (34). In transgene-positive and -negative cells, IL-1{alpha} activated Erk and induced degradation of I{kappa}B{alpha}; TPA and EGF activated Erk but did not affect I{kappa}B{alpha} levels. All three stimuli efficiently induced pro IL-1{alpha}. icIL-1ra expression, on the other hand, was not responsive to any of the stimuli tested, agreeing with the suggestion that regulation of the IL-1 network in keratinocytes is principally dependent upon modulation of IL-1{alpha} and not icIL-1ra levels (35).

Transgene-positive cells showed increased sensitivity to IL-1{alpha}, TPA, and EGF, in terms of Erk activation, I{kappa}B{alpha} degradation, and pro-IL-1{alpha} expression (Fig. 2). In transgene-negative cells, the order of efficiency of Erk activation was IL-1{alpha} < TPA < EGF, whereas in Inv{alpha}2{beta}1 cells it was IL-1{alpha} < EGF < TPA.

The time course of Erk and NF{kappa}B activation was monitored following IL-1{alpha}, EGF, or TPA treatment of transgene-negative and Inv{alpha}2{beta}1 cells that had been starved overnight (Fig. 3). The kinetics of Erk activation triggered by IL-1{alpha} were largely similar in both cell types, except that activation was slightly more sustained in Inv{alpha}2{beta}1 cells (see 30-min time point in Fig. 3A). The kinetics of activation triggered by EGF were also similar in transgene-negative and Inv{alpha}2{beta}1 cells, although again the activation was extended in the Inv{alpha}2{beta}1 cells (see 30-min time point in Fig. 3A). Although transgene-positive cells had a lower basal level of I{kappa}B{alpha} (Figs. 2 and 3B), activation of the NF{kappa}B pathway in response to IL-1{alpha} was essentially identical in negative and Inv{alpha}2{beta}1 cells, as shown by similar time courses of I{kappa}B{alpha} Ser32/36 phosphorylation and subsequent degradation and resynthesis of total I{kappa}B{alpha} (Fig. 3B).



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FIG. 3.
Activation of Erk and NF{kappa}B pathways by IL-1{alpha}-inducing stimuli. Inv{alpha}2{beta}1 and transgene-negative keratinocytes were starved overnight and then treated with 10 ng/ml IL-1{alpha}, 10 ng/ml EGF, or 25 ng/ml TPA for the times indicated. Cells were harvested and analyzed by Western blotting using antibodies to the proteins shown.

 

In response to TPA, a massively sustained Erk activation was observed in Inv{alpha}2{beta}1 cells compared with negative cells (Fig. 3A). Significant Erk activity was still observable in Inv{alpha}2{beta}1 cells after6hofTPA treatment, whereas in transgene-negative cells Erk activation was down-regulated within 30 min after TPA treatment. The time course of I{kappa}B{alpha} phosphorylation and degradation in response to TPA was similar in both negative and Inv{alpha}2{beta}1 cells, indicating equal activation of the NF{kappa}B pathway (Fig. 3B).

Basal and Induced IL-1{alpha} Levels Are Sensitive to Inhibition of the Erk MAPK Pathway—To test the hypothesis that Erk could regulate IL-1{alpha} expression, Inv{alpha}2{beta}1 keratinocytes were treated for 16 h in complete medium with inhibitors of a variety of signaling molecules. IL-1{alpha} levels in cell lysates were determined by ELISA and compared with those present in lysates from untreated (DMSO) transgene-positive (Inv{alpha}2{beta}1) and -negative (Neg) cells (Fig. 4A). Inhibitors of p38 MAPK (SB203580), protein kinase C (Bisindolylmaleimide), and phosphatidylinositol 3-kinase (LY294002) had only minor effects on IL-1{alpha} levels. However, treatment with the MEK1/2 inhibitor U0126 or the reportedly potent and specific NF{kappa}B inhibitor CAPE (36, 37) reduced IL-1{alpha} expression to the levels found in control cells. These data indicate that increased activation of both the NF{kappa}B and Erk pathways could account for the increased IL-1{alpha} production in Inv{alpha}2{beta}1 cells.



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FIG. 4.
Dependence of IL-1{alpha} expression on Erk and NF{kappa}B signaling. A, Inv{alpha}2{beta}1 keratinocytes were incubated for 16 h in complete medium supplemented with the following inhibitors: U0126 (10 µM), SB203580 (10 µM), bisindolylmaleimide (5 µM), LY294002 (50 µM), or CAPE (25 µg/ml). Cells were harvested and assayed for intracellular IL-1{alpha} levels by ELISA. Controls were Inv{alpha}2{beta}1 and transgene-negative (Neg) keratinocytes treated with diluent alone (DMSO). B, Inv{alpha}2{beta}1 and transgene-negative (Neg) keratinocytes were starved overnight and then stimulated with 10% FCS, 25 ng/ml TPA, or 10 ng/ml IL-1{alpha}, with or without a 30-min preincubation with inhibitor (plain bars, Me2SO (DMSO) control; shaded bars, 10 µM U0126; speckled bars, 10 µM SB203580). Cells were assayed for intracellular IL-1{alpha} levels by ELISA, and values were expressed as -fold induction compared with unstimulated, Me2SO-treated controls (horizontal dashed line). Data averaged from three independent experiments with standard errors are shown in A and B. A, differences in IL-1{alpha} levels of U0126 and CAPE-treated Inv{alpha}2{beta}1 cells and Me2SO-treated controls are statistically significant (p < 0.05). B, inhibitor treatments that caused a significant (p < 0.05) decrease in IL-1{alpha} induction are indicated with an asterisk.

 

To investigate whether induced as well as basal IL-1{alpha} production required Erk activity, Inv{alpha}2{beta}1 and negative control keratinocytes that had been starved overnight were simulated with TPA, IL-1{alpha}, or 10% serum for 6 h and then IL-1{alpha} levels in lysates were assayed by ELISA (Fig. 4B). All three stimuli induced IL-1{alpha} in both negative and Inv{alpha}2{beta}1 cells, with TPA having the greatest effect (see also Fig. 2). However, the increase in IL-1{alpha} production was considerably greater in transgene-positive cells, particularly in response to TPA, which increased IL-1{alpha} levels ~16-fold in Inv{alpha}2{beta}1 cells compared with 5-fold in control cells.

Cells were incubated with inhibitors to either the Erk or p38 MAPK pathways prior to stimulation and IL-1{alpha} levels assayed as before (Fig. 4B). The p38 inhibitor was included because IL-1{alpha} activates p38 in keratinocytes (5). Inhibiting the Erk pathway significantly reduced induction of IL-1{alpha} in response to both serum and TPA, although not to IL-1{alpha} itself. p38 MAPK inhibition had little effect on induction of IL-1{alpha} except in the case of IL-1{alpha}-stimulated Inv{alpha}2{beta}1 cells.

Activity of the Epidermal Growth Factor Receptor Is Required for TPA-induced Erk Activation and IL-1{alpha} Synthesis—Activation of Erk by TPA has recently been shown to involve transactivation of the EGF receptor via MMP activation and shedding of membrane-bound HB-EGF (38). As Erk is involved in TPA-induced IL-1{alpha} production and Inv{alpha}2{beta}1 cells produce a sustained Erk activation in response to TPA, a role for the EGFR and its ligands was investigated. To manipulate the activity of the EGFR, we used the specific inhibitor AG1478 and to alter the activity of heparin-binding EGFR ligands, soluble heparin was added to the culture medium (39, 40).

Preincubation with AG1478 largely abolished TPA-induced Erk activation in both negative and Inv{alpha}2{beta}1 cells, whereas addition of heparin slightly inhibited the duration of Erk activation i.e. potentiated the down-regulation of Erk activity (Fig. 5, A and B). These results suggested that TPA-induced Erk activation was indeed dependent on EGFR transactivation and that shedding of heparin-binding EGFR ligands could have a role in this process.



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FIG. 5.
Modification of the response to TPA by inhibition of EGFR. A, Inv{alpha}2{beta}1 and transgene-negative keratinocytes were starved overnight and then stimulated for the times indicated with 25 ng/ml TPA alone or following 30-min pretreatment with 1 µM AG1478 or 500 µg/ml heparin. Lysates were analyzed by Western blot for the proteins shown. B, the average density of the phospho-Erk2 band for all time points and treatments for both cell types was determined and plotted. C, cells with or without AG1478 or heparin that had been simulated for 6 h with TPA were harvested and assayed by ELISA for intracellular levels of IL-1{alpha}. Results in C are expressed as -fold induction compared with untreated controls and are the average of two experiments with standard deviations. Asterisk (*) indicates inhibitor treatment that caused a significant (p < 0.05) decrease in IL-1{alpha} induction.

 

We also examined the effects of AG1478 and heparin on TPA-induced IL-1{alpha} synthesis (Fig. 5C). AG1478 reduced IL-1{alpha} induction in Inv{alpha}2{beta}1 cells from 18- to 12-fold, whereas heparin produced a small degree of inhibition that was not statistically significant. It is interesting to note that in Inv{alpha}2{beta}1 cells TPA-induced IL-1{alpha} synthesis was blocked to a similar extent with both the EGFR inhibitor AG1478 and the MEK1/2 inhibitor U0126 (Figs. 5C and 4B). This implies that EGFR transactivation is responsible for the Erk pathway activation that occurs in TPA-induced IL-1{alpha} synthesis.

Inhibition of the EGFR Blocks IL-1{alpha}-stimulated Erk Activation but Has No Effect on NF{kappa}B Activation—TNF-{alpha}, which activates similar signaling pathways to IL-1, transactivates the EGFR and other members of the ErbB receptor family (41, 42), and it has been reported that IL-1{beta}-induced Erk activation and subsequent MMP-1 expression in human keratinocytes requires EGFR activity (43). Given these observations, we investigated whether inhibition of the EGFR had any effect on induction of IL-1{alpha} or on activation of IL-1{alpha}-dependent signaling in our system.

Inv{alpha}2{beta}1 cells that had been starved overnight were stimulated with IL-1{alpha} with or without a preincubation with AG1478 or heparin. Neither heparin or AG1478 had any effect on the degradation of I{kappa}B{alpha} in response to IL-1, suggesting that activation of the NF{kappa}B pathway was unaffected (Fig. 6A). AG1478 almost completely blocked activation of Erk by IL-1, whereas heparin had no effect on activation (Fig. 6A). In addition AG1478 significantly inhibited the induction of IL-1{alpha} by IL-1 treatment (Fig. 6B). Heparin also inhibited IL-1{alpha} induction, but to a lesser extent than AG1478.



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FIG. 6.
Role of EGFR in IL-1-induced signaling and IL-1{alpha} synthesis. A, Inv{alpha}2{beta}1 cells that had been starved overnight were stimulated for the times indicated with 10 ng/ml IL-1{alpha} alone or following pretreatment for 30 min with 1 µM AG1478 or 500 µg/ml heparin. Samples were analyzed by Western blot for the proteins shown. B, starved cells pretreated with AG1478 or heparin plus untreated controls were stimulated for 6 h with 10 ng/ml IL-1{alpha}, harvested, and assayed by ELISA for intracellular levels of IL-1{alpha}. The average of two runs with standard deviation is shown. Results are expressed as -fold induction compared with untreated control cells. Heparin and AG1478 inhibited IL-1{alpha} induction in transgene-negative and Inv{alpha}2{beta}1 cells significantly (p < 0.05). C, cells were starved overnight in 0.5% serum medium and then left untreated (—) or stimulated with TPA (25 ng/ml) or IL-1{alpha} (10 ng/ml) for the indicated times. AG1478 inhibitor was added to a final concentration of 1 µM for 30 min. Upper panel, immunoprecipitation (IP) of the EGFR followed by Western blotting with an antibody to phosphotyrosine or, as a loading control, total EGFR. Lower panel, lysates were immunoprecipitated (IP) with antibodies to the EGFR or Shc and then Western blotted (WB) with antibodies to EGFR, phosphotyrosine, or total Shc. Arrows indicate ShcA band; the lower band is ShcB.

 

We conclude that transactivation of the EGFR is required for activation of Erk but not NF{kappa}B in response to IL-1. In Inv{alpha}2{beta}1 cells inhibition of the EGFR was more effective in blocking induction of IL-1 than inhibiting MEK1/2 (Figs. 6B and 4B). This implies that, in the induction of IL-1{alpha}, the EGFR activates downstream pathways in addition to Erk.

Comparison of EGFR Activation in Control and Transgenic Keratinocytes—Previous studies have demonstrated EGFR phosphorylation in response to integrin ligation and co-immunoprecipitation of EGFR and ligated integrins, indicative of formation of a macromolecular complex (21, 44). In keratinocytes the suprabasal integrins are unligated and we could not co-immunoprecipitate them with the EGFR (data not shown). This does not exclude the possibility that the EGFR and integrins form a complex, as it could reflect the low levels of EGFR in keratinocytes and the fact that in the absence of ligands the integrins are not clustered. To gain more insight into potential mechanisms by which the EGFR contributed to the differential responsiveness of control and Inv{alpha}2{beta}1 cells to TPA and IL-1{alpha}, we examined the levels of total and phosphorylated EGFR and phosphorylation of the adaptor protein, Shc. Ligation of EGFR (45) or {beta}1 integrins (21, 46) results in rapid EGFR phosphorylation and binding of Shc to phosphorylated tyrosine residues on the receptor. Shc is involved in the activation of Erk downstream of both EGFR (45) and integrins (47).

Addition of TPA for 5 min led to a small increase in EGFR phosphorylation in Inv{alpha}2{beta}1 and transgene-negative cells, which was slightly more pronounced in the transgene-positive cells (Fig. 6C, upper panel). However, 5 min of IL-1{alpha} treatment did not affect EGFR phosphorylation appreciably in either cell population (Fig. 6C, upper panel). Shc phosphorylation was reduced in Inv{alpha}2{beta}1 and transgene-negative cells by treatment with AG1478, confirming its dependence on EGFR activity (Fig. 6C, lower panel). TPA, but not IL-1{alpha}, increased Shc phosphorylation in Inv{alpha}2{beta}1 cells (Fig. 6C, lower panel). Neither treatment altered Shc phosphorylation in control cells (Fig. 6C, lower panel).

Down-regulation of phosphorylated EGFR occurs through endocytosis and subsequent degradation (45, 48). The most striking effect of TPA and IL-1{alpha} was on total EGFR levels, measured at 15 min, a time point when there is appreciable endocytosis of activated EGFR (45, 48) (Fig. 6C, lower panel). Treatment of Inv{alpha}2{beta}1 and transgene-negative cells with AG1478 led to much higher levels of receptor than in untreated cells, confirming that EGFR activation was linked with down-regulation. In transgene-negative cells treatment with TPA or IL-1{alpha} had no effect on EGFR levels. However, TPA and IL-1{alpha} caused a marked reduction in EGFR in Inv{alpha}2{beta}1 cells (Fig. 6C, lower panel).

These results provide evidence for increased activity of the EGFR in Inv{alpha}2{beta}1 cells compared with control cells in the absence of exogenous EGF and integrin ligands. Differential sensitivity to TPA was observed at the level of EGFR phosphorylation, Shc phosphorylation, and EGFR down-regulation, whereas sensitivity to IL-1{alpha} was seen at the level of EGFR down-regulation only. The greater effects of TPA correlate with the more efficient Erk activation by TPA than IL-1{alpha} in Inv{alpha}2{beta}1 cells (Fig. 2).

Autocrine EGFR Activation and IL-1 Signaling Regulate Basal Levels of IL-1{alpha}Keratinocytes produce and respond to a wide range of cytokines (49), including EGFR ligands (50, 51) and pro-inflammatory cytokines such as TNF-{alpha} and IL-1 (6). In addition, IL-1{alpha} is known to stimulate its own expression in keratinocytes (30, 52). The possibility that Inv{alpha}2{beta}1 cells had higher basal IL-1{alpha} levels compared with negative cells as a result of increased production and responsiveness to one or more cytokines/growth factors was investigated.

Inv{alpha}2{beta}1 cells were maintained in low serum medium without added growth factors. Addition of a panel of neutralizing antibodies to the medium for 16 h and subsequent monitoring of basal IL-1{alpha} levels and activation states of the Erk and NF{kappa}B pathways were then performed (Fig. 7). Neutralization of IL-6, GM-CSF, TNF-{alpha}, or TGF-{beta} had little effect on levels of IL-1{alpha}. However, addition of neutralizing antibodies to IL-1{alpha} significantly inhibited endogenous IL-1{alpha} levels. This effect was robust because both a goat polyclonal and rat monoclonal antibody to IL-1{alpha} were inhibitory. Also, incubation with a blocking antibody to the IL-1R1, to prevent IL-1-dependent signaling (53, 54) significantly inhibited endogenous IL-1{alpha} levels (data not shown). Addition of the EGFR inhibitor AG1478 significantly decreased endogenous IL-1{alpha} in the cells (Fig. 7A), consistent with its effects on induction of IL-1{alpha} by TPA and IL-1{alpha} (Figs. 5C and 6B). Adhesion blocking antibodies to transgenic (i.e. human) {beta}1 integrins did not affect IL-1{alpha} levels, confirming that elevated IL-1{alpha} production is independent of integrin ligation (5).



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FIG. 7.
Effects of neutralizing antibodies on IL-1{alpha} levels and basal Erk/NF{kappa}B pathway activation. Inv{alpha}2{beta}1 cells (plus a transgene-negative control, Neg) were incubated for 16–17 h in medium containing 0.5% FCS alone or supplemented with various neutralizing antibodies or AG1478. Cells were harvested and assayed for intracellular levels of IL-1{alpha} by ELISA (A) or analyzed by Western blot for phospho-Erk1/2 and I{kappa}B{alpha} with total Erk1/2 as a loading control (B). Results in A are expressed as a percentage of IL-1{alpha} levels in the untreated Inv{alpha}2{beta}1 cells and are the average of three independent experiments with standard errors. {alpha}IL-1{alpha} gt and {alpha}IL-1{alpha} rt are goat polyclonal and rat monoclonal neutralizing antibodies to IL-1{alpha}, respectively. {alpha} h{beta}1 integrin is an adhesion blocking antibody specific for the transgenic human {beta}1 integrin (P5D2). The reduction in IL-1{alpha} levels in response to the anti-IL-1{alpha} antibodies and AG1478 is statistically significant (p < 0.05).

 

The higher basal level of Erk activity in Inv{alpha}2{beta}1 cells than transgene-negative cells was abolished by treatment with AG1478 (Fig. 7B). In contrast, the inhibitory effect of neutralizing IL-1{alpha} antibodies on IL-1{alpha} levels (Fig. 7A) was not accompanied by a reduction in basal active Erk levels, which remained higher than in transgene-negative cells (Fig. 7B). This indicates that the increased Erk activity in transgene-positive cells is not attributable solely to autocrine production of IL-1{alpha} and that autocrine IL-1{alpha} production can operate via an Erk-independent mechanism.

Constitutive Activation of IL-1-dependent Signaling Pathways in Transgene-negative Cells Generates a Massive Accumulation of IL-1{alpha} without Enhanced Erk Activity—Although the increased production of IL-1{alpha} in transgenic keratinocytes is attributable to activation of the EGFR, transgene-negative cells and AG1478-treated transgene-positive cells still express detectable amounts of IL-1{alpha} (see Figs. 1 and 7A). To test whether increased IL-1{alpha} expression in the absence of aberrant integrin expression resulted in Erk activation, we overexpressed the key upstream regulator of IL-1-dependent signaling, the Ser/Thr kinase IL-1 receptor-associated kinase 1 (IRAK1), in keratinocytes with the aim of constitutively activating and/or potentiating IL-1 signaling pathways (55, 56, 57). IRAK1 plays a vital role in transducing IL-1-dependent signals, acting just downstream of the IL-1R (58, 59, 60, 61). Overexpression of IRAK1 can constitutively activate and potentiate IL-1-induced NF{kappa}B activation, an effect not dependent on its kinase activity (22, 55, 56, 57, 60). In addition, both wild type (wt) and kinase-dead (kd) IRAK1 overexpression potentiates IL-1-induced Jun N-terminal kinase MAPK activity (56, 57). Overexpression of IRAK1 bypasses the negative regulators icIL-1ra and the decoy receptor IL-1R2, which are expressed by keratinocytes (15, 30, 62, 63, 64).

The IRAK1 constructs could be specifically detected by an anti-human IRAK1 antibody, which has no appreciable cross-reactivity with the endogenous murine IRAK homologue (Fig. 8A). Upon stimulation with IL-1 for 7 h, the level of wt IRAK1 decreased slightly, consistent with the degradation that is suggested to occur upon IRAK1 activation and phosphorylation (65). Multiple IRAK1 forms of increased apparent molecular weight were detected in cells expressing kd IRAK, presumably representing multiply phosphorylated or possibly ubiquitinated forms (22, 60, 65). The reason why kd IRAK1 accumulated in these forms but wt IRAK1 did not is unknown but may be because of a decreased rate of proteolysis of the phosphorylated/ubiquitinated kd IRAK1 compared with wt IRAK1.



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FIG. 8.
Transduction of transgene-negative keratinocytes with IRAK1 constructs. The transgene-negative keratinocyte line was infected with empty retroviral vector (ev) or wild type (wt) or kinase-dead (kd) IRAK1. For each IRAK1 construct, cells from two independent infections (-1, -2) were compared. Cells were starved overnight and then stimulated for either 7 h (A and B) or 15 min (C) with 10 ng/ml IL-1{alpha}. Cell lysates were analyzed by Western blot for the proteins indicated, including IRAK (A), keratinocyte differentiation markers (B), and components of downstream IL-1 signaling pathways (C). Actin and total Erk2 loading controls are included.

 

Overexpression of wt and kd IRAK1 had no effect on icIL-1ra levels or expression of keratinocyte differentiation markers such as involucrin and cornifin in the presence or absence of exogenous IL-1{alpha} (Fig. 8, A and B). Overexpression of wild type or kinase-dead IRAK1 led transgene-negative cells to produce large amounts of IL-1{alpha} in the absence of an exogenous IL-1{alpha} stimulus (Fig. 8A). However, in the absence of exogenous IL-1{alpha}, expression of IRAK had no effect on Erk phosphorylation (Fig. 8C).

When transgene-negative cells were treated with IL-1{alpha}, Erk activation was observed (Fig. 8C). Overexpression of the kinase-dead version of IRAK1 reduced the activation of Erk in response to IL-1, whereas the wild type version did not (Fig. 8C). The kinase-dead construct also reduced degradation of I{kappa}B{alpha} in response to IL-1 (Fig. 8C).

We conclude that, although exogenous IL-1 can activate Erk in transgene-negative cells, Erk activation is not obligatorily coupled to autocrine IL-1 production.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic mice expressing {beta}1 integrins in the suprabasal, differentiated layers of the epidermis develop a sporadic hyperproliferative and inflammatory skin disease (2), which correlates with activation of Erk MAPK and elevated production of IL-1{alpha} in keratinocytes (5). Given the importance of IL-1 in skin inflammation and the role of Erk activation in epidermal hyperproliferation (5, 26), we investigated the connection between increased IL-1{alpha} production and Erk activity in cultured keratinocytes from the transgenic mice. Our data form the basis of the model presented in Fig. 9.



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FIG. 9.
Interaction between integrins, EGFR, and IL-1R1 in the regulation of IL-1{alpha} synthesis: proposed model for increased IL-1{alpha} expression in Inv{alpha}2{beta}1 keratinocytes. Model depicts the EGFR as a key regulator of Erk activation and pro-inflammatory IL-1{alpha} synthesis via trans/autocrine activation. Suprabasal integrins are shown potentiating phorbol ester (TPA)-induced EGFR transactivation and subsequent Erk activation, whereas the IL-1R1 depends on EGFR activity for Erk activation. Autocrine IL-1 signaling and IRAK1 action also contribute significantly to synthesis of IL-1{alpha}.

 

Transgenic cells had elevated IL-1{alpha} levels and a higher basal Erk activity than controls (Fig. 1). IL-1{alpha} levels in Inv{alpha}2{beta}1 cells were reduced to those in transgene-negative cells when incubated with the MEK1 inhibitor U0126 (Fig. 4A). An inhibitor of NF{kappa}B, CAPE, also effectively reduced IL-1{alpha} levels in the cells (Fig. 4A), showing that NF{kappa}B activity, as well as Erk, was important in regulating IL-1{alpha} synthesis. Our results agree with the well studied role of NF{kappa}B activity in regulating IL-1{alpha} expression in fibroblasts (31, 32). Induction of IL-1{alpha} in response to serum and phorbol ester (TPA) was more sensitive to U0126 than induction by IL-1 itself (Fig. 4B). It thus appears that both Erk and NF{kappa}B contribute to IL-1{alpha} synthesis in keratinocytes, with the dominant pathway depending on the nature of the external stimulus. Erk is known to inhibit NF{kappa}B-dependent transcriptional responses (34, 66); therefore, inhibiting Erk can be expected to enhance the NF{kappa}B-dependent pathway of IL-1{alpha} induction.

Inv{alpha}2{beta}1 cells were most sensitive to IL-1{alpha} induction by the phorbol ester TPA (Fig. 4B). TPA produced a corresponding massively potentiated Erk activation without affecting NF{kappa}B pathway activation (Fig. 3). When Inv{alpha}2{beta}1 or Inv{alpha}3{beta}1 mice are treated with TPA, transgene-positive, suprabasal epidermal cells are induced to enter S phase of the cell cycle, but this does not occur in control epidermis (4). The in vivo hypersensitivity of the skin to TPA may reflect enhanced Erk activation and subsequent IL-1 production.

Transactivation of the EGFR is required for efficient activation of Erk in TPA-treated epidermal cells (38), and inhibition of EGFR kinase activity with AG1478 largely blocked the sustained Erk activation in Inv{alpha}2{beta}1 cells treated with TPA (Fig. 5). TPA treatment stimulated EGFR and Shc phosphorylation independent of exogenous EGF or integrin ligands and also stimulated down-regulation of the EGFR in Inv{alpha}2{beta}1 but not control cells (Fig. 6C). The conclusion from these experiments, that suprabasal integrins potentiate EGFR transactivation (Fig. 9), is attractive for a number of reasons. Like {beta}1 integrins, the EGFR is localized to the basal layer of normal epidermis and its expression extends into the suprabasal layers when the epidermis is hyperproliferative (67, 68, 69). Activation of the EGFR has been demonstrated in response to {beta}1 integrin ligation by extracellular matrix (21, 44, 46), and integrin {alpha}2{beta}1-dependent EGFR activation has been observed at cell-cell contact sites (70). Transactivation of the EGFR in response to TPA can occur through MMP-mediated cleavage of membrane-associated pro-HB-EGF (38) and the {alpha}3{beta}1 integrin interacts with pro-HB-EGF via the tetraspanin CD9 (71). TPA is also known to induce expression of TGF-{alpha} in keratinocytes (72, 73). In addition, {beta}1 integrins form a complex with protein kinase C{alpha} and TPA stimulates integrin endocytosis (74). Thus, the potentiation of TPA-induced Erk activation by suprabasal {beta}1 integrins could occur through direct interaction between the integrin and EGFR, via TPA-mediated release of EGFR ligands, or via an effect of TPA on integrin and EGFR trafficking (Fig. 9).

Activation of the EGFR via autocrine production of heparin binding EGFR ligands such as HB-EGF and amphiregulin plays an important role in keratinocyte proliferation and wound healing responses (50, 51, 75, 76). Addition of soluble heparin to the culture medium of cells efficiently blocks amphiregulin activity (39, 75, 77). Heparin inhibited the sustained Erk activation induced by TPA but did not affect the initial peak of Erk activity (Fig. 5, A and B) and had only modest effects on IL-1{alpha} synthesis. Although heparin-binding EGFR ligands do appear to be involved in the response of transgenic keratinocytes to TPA, inhibition of EGFR kinase activity with AG1478 was significantly more efficient than heparin in per-turbing both Erk activation and IL-1{alpha} synthesis. This implies that heparin-unresponsive EGFR ligands, such as TGF-{alpha}, are also important. However, interpretation of the effects of heparin is complicated by the fact that heparin can inhibit amphiregulin activity but potentiate the effects of HB-EGF (39, 40, 78). Thus one reason why addition of heparin has a much weaker effect on IL-1{alpha} synthesis than AG1478 could be that heparin inhibits one pathway of EGFR transactivation but stimulates another.

Our experiments revealed a role for EGFR transactivation not only in the response of transgenic keratinocytes to TPA, but also to IL-1{alpha}. Heparin partially inhibited IL-1{alpha} induction by IL-1, and inhibition of EGFR kinase activity significantly reduced IL-1{alpha} induction and almost completely blocked Erk activation by IL-1 (Fig. 6). Furthermore, IL-1{alpha}, like TPA, caused down-regulation of the EGFR (Fig. 6C). The finding of a role for EGFR activity in mediating Erk activation by IL-1 in keratinocytes and subsequent IL-1{alpha} induction is novel. It fits well with the observations that IL-1{beta} activates the EGFR in CaCo-2 cells, possibly through increased sphingosine production (79), and that IL-1{beta}-induced Erk activation and MMP-1 production in human keratinocytes requires EGFR activity (43). The requirement of a novel EGFR-dependent pathway for IL-1 to activate Erk explains how alteration in the proliferative status of keratinocytes would specifically modulate responses to IL-1. Inhibition of the EGFR in Inv{alpha}2{beta}1 cells was more effective than inhibiting MEK1/2 in blocking induction of IL-1 (Figs. 4B and 6B). This suggests that the EGFR is responsible for the activation of additional downstream pathways in the induction of IL-1. One candidate pathway would involve phosphatidylinositol 3-kinase, which can regulate NF{kappa}B-dependent gene expression without affecting I{kappa}B{alpha} degradation (80).

Neutralizing antibodies to IL-1{alpha} reduced the intracellular levels of IL-1{alpha} in Inv{alpha}2{beta}1 keratinocytes by more than 50% (Fig. 7A), demonstrating a significant contribution of autocrine IL-1{alpha} signaling to the regulation of IL-1{alpha} expression in keratinocytes. This effect appeared to be Erk- and NF{kappa}B-independent, as levels of basal active Erk were not altered and I{kappa}B{alpha} was not increasingly stabilized (Fig. 7B). However, whether steady state I{kappa}B levels are a sensitive enough indicator of basal, as opposed to IL-1-induced (Fig. 3), NF{kappa}B activation remains to be shown. The p38 MAPK pathway may have a role in IL-1-induced IL-1{alpha} expression, as SB203580 could partially inhibit the induction in Inv{alpha}2{beta}1 cells (Fig. 4B). However, basal levels of active, phospho-p38 MAPK could not be reliably detected in the cells (data not shown).

We found that overexpression of wt and kd IRAK1, even in the absence of exogenous IL-1, induced a massive accumulation of IL-1{alpha} without affecting expression of icIL-1ra (Fig. 8), confirming the importance of autocrine IL-1 signaling in regulating IL-1{alpha} levels. We found two significant differences in the actions of wt and kd IRAK1; activation of Erk by IL-1 was inhibited by expression of kd but not wt IRAK1, and there was accumulation of hyperphosphorylated/ubiquitinated kd but not wt IRAK1 (Fig. 8). In the past, the kinase activity of IRAK has not been regarded as important for its ability to signal to downstream pathways in response to IL-1 (55, 57, 60). However, a number of recent reports do demonstrate kinase-dependent functions (81, 82, 83, 84). The kinase that phosphorylates kd IRAK1 in mouse keratinocytes is unknown, but possibilities include endogenous IRAK1 and a recently identified IRAK family member, IRAK4, which is critical in IL-1- and lipopolysaccharide-dependent responses (85) and acts upstream of IRAK1 (86).

In summary, our investigation of the mechanisms responsible for increased IL-1{alpha} expression in keratinocytes expressing suprabasal integrins has uncovered critical roles for EGFR-dependent Erk activation and autocrine IL-1 signaling in regulating IL-1{alpha} expression. Fig. 9 depicts a summary of the various contributions of these pathways.


    FOOTNOTES
 
* This work was supported by Cancer Research UK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 44-20-7269-3528; Fax: 44-20-7269-3078; E-mail: fiona.watt{at}cancer.org.uk.

1 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; IL, interleukin; icIL-1ra, intracellular IL-1 receptor antagonist; IL-1R, interleukin-1 receptor; CAPE, caffeic acid phenethyl ester; TGF, transforming growth factor; EGF, epidermal growth factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; TNF-{alpha}, tumor necrosis factor {alpha}; I{kappa}B, inhibitor of NF{kappa}B; IRAK, interleukin-1 receptor-associated kinase; FCS, fetal calf serum; PBS, phosphate-buffered saline; IRES, internal ribosome entry site; EGFP, enhanced green fluorescent protein; NF{kappa}B, nuclear factor {kappa}B; MEK, mitogen-activated protein kinase kinase; MMP, matrix metalloproteinase; HB-EGF, heparin-binding epidermal growth factor-like growth factor; EGFR, epidermal growth factor receptor; wt, wild type; kd, kinase-dead. Back


    ACKNOWLEDGMENTS
 
We are grateful to Keith Ray for supplying the IRAK1 constructs and to Anton Jetten for the anti-cornifin antibody. We thank David Owens for providing transgenic mice and Douglas Campbell for helpful discussions.



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 ABSTRACT
 INTRODUCTION
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 RESULTS
 DISCUSSION
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