Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M512694200 on March 8, 2006

J. Biol. Chem., Vol. 281, Issue 19, 13525-13532, May 12, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/19/13525    most recent
M512694200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hernandez-Pigeon, H.
Right arrow Articles by Laurent, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hernandez-Pigeon, H.
Right arrow Articles by Laurent, G.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Human Keratinocytes Acquire Cellular Cytotoxicity under UV-B Irradiation

IMPLICATION OF GRANZYME B AND PERFORIN*

Hélène Hernandez-Pigeon{ddagger}12, Christine Jean{ddagger}1, Alexandra Charruyer{ddagger}, Marie-José Haure§, Matthias Titeux{ddagger}, Laure Tonasso{ddagger}, Anne Quillet-Mary{ddagger}, Caroline Baudouin§, Marie Charveron§, and Guy Laurent{ddagger}

From the {ddagger}INSERM U563, CPTP, Bat B, Pavillon Lefebvre, Place du Dr. Baylac, Centre Hospitalier Universitaire Purpan, BP 3028, 31024 Toulouse cedex 3, France, §CERPER, Institut de Recherche Pierre Fabre, Laboratoire de Biologie Cellulaire Cutanée, Toulouse, France, and the Service d'Hématologie, Centre Hospitalier Universitaire Purpan, 31059 Toulouse, France

Received for publication, November 28, 2005 , and in revised form, February 22, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ultraviolet (UV) radiation from the sun is widely considered as a major cause of human skin photoaging and skin cancer. Granzyme B (GrB) and perforin (PFN) are two proteins contained in granules and implicated in one of the mechanisms by which cytotoxic lymphocytes and natural killer cells exert their cytotoxicity against virus-infected, alloreactive, or transformed cells. The distribution of GrB and PFN in the skin has received little attention. However, Berthou and co-workers (Berthou, C., Michel, L., Soulie, A., Jean-Louis, F., Flageul, B., Dubertret, L., Sigaux, F., Zhang, Y., and Sasportes, M. (1997) J. Immunol. 159, 5293-5300) described that, whereas freshly isolated epidermal cells did not express GrB or PFN, keratinocyte growth to confluence was associated with GrB and PFN mRNA and protein synthesis. In this work, we have investigated the possible role of UV-B on GrB and PFN expression in keratinocytes. We found that UV-B induces GrB and PFN expression in these cells through redox-, epidermal growth factor receptor-, and mitogen-activated protein kinase-dependent signaling. Furthermore, under UV irradiation, keratinocytes acquire a significant cytotoxicity, which is GrB and PFN dependent, toward a variety of cellular targets including transformed T-lymphocytes, melanocytes, and keratinocytes. This phenomenon may have important functional consequences in the regulation of skin inflammatory response and in the emergence of cancer skin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human skin, unlike all other organs, is continuously and directly exposed to environmental influences. Ultraviolet (UV) radiation from the sun is among the most ubiquitous damaging environmental factors from which human skin must protect itself. UV radiation in sunlight is divided into three regions depending on wavelength, short-wave UV-C (200-280 nm), mid-wave UV-B (280-320 nm), and long-wave UV-A (320-400 nm). UV-C has the highest energy and, hence, is the most biologically damaging region of UV radiation. However, UV-C in solar radiation is filtered out by the ozone layer of the atmosphere of the Earth, and therefore, its role in human pathogenesis is minimal. Both UV-B, and to a lesser extent, UV-A radiation are responsible as causative factors for various skin disorders including photoaging and skin cancer (1-3). UV radiation, in particular UV-B from sunlight is known to alter cellular function via DNA damage (4), generation of radical oxygen species (ROS)3 (5), and the resultant alterations in a large variety of signaling events (6, 7). UV-B is therefore one of the most important external stimuli that affects skin by inducing immunosuppression, cancer, premature skin aging, inflammation, and cell death (2, 3, 8, 9).

The granule secretory pathway is one of the mechanisms by which cytotoxic lymphocytes and natural killer (NK) cells exert their cytotoxicity against virus-infected, alloreactive, or transformed cells. The contact between effector cells and aberrant target cells induces exocytosis from the cytotoxic lymphocyte or the NK cells of granules that contain the potential cytolytic effector molecules (10). This mechanism depends on the actions of several constituents of the secreted granules that contain the pore-forming molecule perforin (PFN) together with a variety of granule-associated enzymes (such as granzymes and granulysin) (11). Among of them, granzyme B (GrB) is the main effector when dealing with target cell death (12). Although the precise molecular and cellular pathways through which PFN and GrB cooperate to bring about cell death are still highly controversial, experiments indicate an absolute dependence on PFN for all granule-mediated cell death (13). In vivo, this is illustrated by the fact that targeted mutation of the pfn gene in mice results in profound immunosuppression and altered skin and tumor allograft rejections (14). By contrast, deficiency of grB has a severely depressed ability to cause target cell lysis (12). Previous studies have described that GrB and PFN are not strictly distributed in lymphoid cells. Indeed, GrB and PFN are contained in other hematopoietic cells, such as normal and leukemic myeloid cells (15), as well as non-hematopoietic cells, such as chondrocytes of articular cartilage (16) and cells of the reproductive system, including spermatogenic cells (17), granulosa cells of human ovary (18), or placental trophoblasts (17).

The distribution of GrB and PFN in the skin has received little attention except in the context of immune skin disorders, such as in allergy, psoriasis, vitiligo, and lichen planus, or eventually in graft versus host disease related skin injury. These situations are often associated with dermal or epidermal infiltration by GrB- and PFN-producing activated T cells. However, Berthou and co-workers (19) described that, whereas freshly isolated epidermal cells did not express GrB or PFN, keratinocyte growth to confluence was associated to GrB and PFN mRNA and protein synthesis. In this study, the authors proposed that these proteins could be involved in epidermal homeostasis by preventing invasion by pathogens or migration of inflammatory cells (19). The role of GrB and PFN in keratinocytes has not deserved further attention. This is surprising based on the importance of PFN-dependent cytolysis not only in a large variety of bacterial and viral infections but also in tumor surveillance (for review, see Ref. 20). This is also surprising based on the role of GrB, which is not simply implied in cytolysis, but also exerts, as an extracellular enzyme, PFN-independent important functions in extracellular matrix remodeling as well as in cell adhesion and motility (21).

In this study, we have investigated the possible role of UV-B on GrB and PFN expression in keratinocytes. We found that UV-B induces GrB and PFN expression in these cells through redox-, epidermal growth factor receptor (EGFR)-, and MAPK-dependent signaling. Furthermore, under UV irradiation, keratinocytes acquire a significant cytotoxicity that is GrB- and PFN-dependent toward a variety of cellular targets including transformed T-lymphocytes, melanocytes, and keratinocytes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Chemicals—Immortalized human HaCaT keratinocytes, provided by CERPER (Pierre-Fabre, France), were cultured in Dulbecco's modified Eagle's medium at 37 °C in 5% CO2. Culture medium was supplemented with 10% heat-inactivated fetal calf serum (FCS), glutamine (2 mM), streptomycin (100 µg/ml), and penicillin (200 units/ml) (all these reagents were purchased from Eurobio, les Ulis, France). Normal human primary keratinocytes (HK) were kindly provided by Prof. Hovnanian (Toulouse, France). HK were isolated from skin biopsies as previously described (22) and were expanded on a feeder layer of lethally irradiated 3T3-J2 mouse fibroblasts in keratinocyte growth medium, following the method described by Rheinwald and Green (23). K562 and Jurkat cell lines were obtained from the ATCC (Rockville, MD). The K562 cell line was stably transfected with pEGFP/PI-9 plasmid, kindly provided by Dr. P. I. Bird (Monash University, Victoria, Australia), using LipofectamineTM 2000 (Invitrogen) according to manufacturer's recommendations. Selection of transfected cells was done with 500 µg/ml geneticin. The melanoma cell line (KAL) was established by Cohen-Knafo et al. (24). K562, Jurkat, and KAL cells were grown in RPMI 1640 containing 10% FCS, glutamine (2 mM), streptomycin (100 µg/ml), and penicillin (200 units/ml).

The UV-B irradiation source was a fluorescent lamp that emitted an energy peak at 310 nm. The emitted dose was calculated using a UV-B radiometer photodetector. UV-B irradiation was performed in cells incubated in phosphate-buffered saline (PBS).

Drugs and Reagents—The rabbit polyclonal antibodies (Abs) against PFN and JNK (used at 1/500) were purchased from Santa Cruz Biotechnology. The rabbit polyclonal anti-MAPKAPK-2 and anti-phospho-MAPKAPK-2 Thr222 Abs (used at 1/1000) were purchased from Cell Signaling Technology (St. Quentin en Yvelines, France). The mouse monoclonal anti-GrB (clone GrB-7; used at 1/200), anti-beta-actin (used at 1/1000), anti-phospho-JNK (used at 1/1000), and anti-GFP (used at 1/500) Abs were purchased, respectively, from Euromedex (Souffelweyersheim, France), NeoMarkers (Interchim, Montluçon, France), Cell Signaling Technology, and Santa Cruz Biotechnology. Affinity purified secondary Abs were purchased from Jackson ImmunoResearch Laboratories Inc. (Beckman Coulter Co., Marseille, France).

3,4-Dichloroisocoumarin (DCIC) was purchased from ICN Biomedical (Aurora, OH) and dissolved in dimethyl sulfoxide. SB203580, SP600125, and PD98059 were purchased from VWR International (Fontenay sous Bois, France). Antisense oligonucleotides and controls directed against GrB have been designed and manufactured by Biognostik (Euromedex) (15). Other products were purchased from Sigma.

Western Blot Analysis—Cells were washed with cold PBS and lysed, and then scrapped in lysis buffer (30 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 10% glycerol, 1 mM Na3VO4, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 mM beta-glycerophosphate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) for 5 min on ice, followed by centrifugation at 13,000 x g for 5 min at 4 °C. For each lysate, 30 µg of total protein was boiled for 5 min at 95 °C in the presence of 5% beta-mercaptoethanol.

Proteins were separated in a 10% SDS-PAGE and transferred onto nitrocellulose membranes (Amersham Biosciences). Nonspecific binding to the membrane was blocked for 1 h at room temperature with 10% nonfat milk in PBS containing 0.1% Tween 20 (PBST). Membranes were incubated overnight at 4 °C with specific primary antibody diluted at an appropriate concentration in PBST containing 1% nonfat milk. Membranes were then washed three times at room temperature and bound immunoglobulins were detected with horseradish peroxidase-conjugated secondary Ab for 30 min at room temperature in 1% nonfat milk dissolved in PBST. Membranes were then washed with PBST and bound Abs were detected by the enhanced chemiluminescence system ECL kit (Amersham Biosciences).

Real-time Quantitative PCR—Total cellular RNA was extracted with TRIzol®. The cDNA was synthesized using random hexamers and oligo(dT) from 4 µg of mRNA and performed with the SuperScriptTM First Strand Synthesis System for RT-PCR (Invitrogen). Forward and reverse primers were designed following Guilloton et al. (25) for GrB and following Simon et al. (26) for PFN (Table 1). Real-time PCR was performed using an iCycler thermal cycler (Applied Biosystems 7000 Real-Time PCR System, Courtaboeuf, France) according to the manufacturer's instructions. Reactions were performed with 0.3 µM primers. Nucleotides, Taq DNA polymerase, and buffer were included in SYBR Green JumpStartTM Taq ReadyMixTM for quantitative PCR. cDNA amplification consisted of one cycle at 95 °C for 1 min and 30 s, followed by 40 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 1 min. The threshold cycle (CT) values were determined by iCycler software (ABI Prism 7000) and the quantification data were analyzed following the {Delta}{Delta}CT method using S14 as reference. We have checked that PCR efficiency (E) of the amplification was similar whatever the primers and we calculated the relative amount (RA): RA = (1 + E)-{Delta}{Delta}CT. Electrophoresis of PCR products on a 2% agarose gel was undertaken when no gene expression was observed in non-treated cells.


View this table:
[in this window]
[in a new window]
  		TABLE 1
Oligonucleotide primer sequences for real-time quantitative PCR

 
Confocal Laser Scanning Microscopy Analysis—Cells were fixed with 4% (w/v) paraformaldehyde (10 min at room temperature), washed twice with PBS, and permeabilized using 100% methanol (2 min at room temperature). Coverslips were washed with PBS, saturated with 20% FCS/PBS for 20 min at room temperature, and incubated with anti-GrB and/or anti-PFN Abs, both at a 1/20 dilution in 1% FCS/PBS, followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG and/or Cy3 goat anti-mouse IgG (Beckman Coulters) diluted at 1/100 in 1% FCS/PBS for 25 min at room temperature. After washing, the coverslips were sealed and examined with the confocal imaging system that was a Zeiss (Oberkochen, Germany) scanning assembly incorporating argon and helium/neon lasers coupled to a Zeiss Axiovert 100 fluorescence microscope.

Cytotoxicity Assays—The CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI) was used to evaluate stress-induced cytotoxicity (15). This assay is a colorimetric alternative to the 51Cr-release cytotoxicity assay based on the measurement of lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released on cell lysis, in much the same way as 51Cr is released in radioactive assay and allows discrimination between effectors and targets LDH release (27). Variations on this technology have been reported for measuring natural cytotoxicity and have been demonstrated to be identical (within experimental error) to values determined in parallel 51Cr-release assays and was performed according to the manufacturer's recommendations. Briefly, effector cells (HaCaT: 4 x 106 cells/ml) were irradiated with 0.2 kJ/m2 UV-B, incubated for 16 h, trypsinized, washed, resuspended in RPMI 1640 supplemented with 5% FCS, incubated for 1 h, and mixed with target cells (K562, HaCaT, and HK cells: 1 x 105 cells/ml) in U-bottom 96-microwell plates (Nunc, Roskilde, Denmark) at various effector to target (E:T) ratios in triplicate. Microplates were spun for 3 min at 200 x g and incubated for 4 h at 37°C, 5% CO2. Supernatant (50 µl) was collected from each well and added to 50 µl of reconstituted Substrate Mix for 30 min in the dark at room temperature. Enzymatic reaction was stopped by adding Stop Solution (50 µl). Counting was realized by recording absorbance at 490 nm. Maximum release (TM) was determined by lysing target cells with 20 µl of Lysis Solution. Spontaneous release (TS) was determined by incubation of target cells in medium in the absence of effectors cells. Effectors spontaneous release (ES) was done with effector cells alone at the same E:T ratio. Results are expressed as a percentage of cytotoxicity, using the formula: % cytotoxicity = [(experimental - ES - TS)/(TM - TS)] x 100.

Blocking experiments were performed with DCIC (20 µmol/liter), a broadly reactive serine esterase inhibitor, which has been shown to neutralize GrB enzymatic activity (28, 29), MgCl2 (1.5 mM)/EGTA (1 mM), which blocks degranulation and prevents PFN polymerization (30), anti-CD95 (Fas/APO-1) blocking Ab (ZB4) (500 ng/ml), or antisense or sense phosphorothioate oligodeoxynucleotides (5 µmol/liter) directed against GrB.

Statistics—The Student's t test was performed to evaluate the statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
UV-B-induced GrB and PFN mRNA Expression in Keratinocytes—GrB and PFN mRNA expressions in the HaCaT cell line were analyzed by real-time quantitative PCR followed by a separation of PCR products in 2% agarose gel. We first examined whether UV-B irradiation was able to stimulate production of GrB and PFN in HaCaT cells. Cells were irradiated with UV-B at different doses. In untreated non-confluent HaCaT cells, GrB and PFN mRNA were undetectable as previously described (19) (Fig. 1, A and B). However, UV-B irradiation induced GrB and PFN mRNA in a dose- and time-dependent manner (Fig. 1, A and B). At the dose of 0.2 kJ/m2, cell loss was about 15% after 48 h (data not shown). Therefore, this dose was used for further experiments.

In HaCaT cells grown at confluence, GrB and PFN mRNA were detectable as previously described (19). However, in these conditions of culture, UV-B irradiation was still able to increase 3-4-fold GrB and PFN expression (Fig. 1C).

In primary normal human keratinocytes (HK) from different donors, UV-B also increased GrB and PFN expression (Fig. 1D). It is interesting to note that, contrary to HaCaT cells, untreated fresh human keratinocytes do express GrB but not PFN. S14, a ribosomal protein, was used as an internal control as previously described (31, 32). Indeed, S14 mRNA expression was found to be invariable, whereas we detected minor but significant changes in beta-actin in UV-irradiated HaCaT cells. These results demonstrate that treatment with UV-B irradiation resulted in a significant increase in grB and pfn gene expression in both HaCaT and fresh human keratinocytes.


Figure 1
View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 1.
Expression of GrB and PFN mRNA measured by real-time quantitative PCR. A, HaCaT cells were irradiated at different doses of UV-B and incubated for 16 h before mRNA extraction. B, HaCaT cells were irradiated at 0.2 kJ/m2 and incubated for different times before mRNA extraction. Electrophoresis of PCR products on a 2% agarose gel was undertaken because no gene expression was observed in non-treated cells. C, HaCaT cells at confluence were irradiated at 0.2 kJ/m2 and incubated for 16 h ({blacksquare}). The data were expressed as the relative amounts compared with values from non-treated cells (Figure 1). These data are the mean of three independent experiments. D, primary HK cells were irradiated at 0.2 kJ/m2 and incubated for 16 h before mRNA extraction ({blacksquare}). The data for GrB was expressed as the relative amounts compared with values from non-treated cells (Figure 1). The data for PFN was shown on a 2% agarose gel because no PFN expression was observed in non-treated cells. *, p < 0.05 and **, p < 0.01. These data are representative of three independent experiments.

 
UV-B-induced GrB and PFN Protein Expression in Keratinocytes—GrB and PFN protein expressions in the HaCaT cell line were analyzed by Western blot and confocal microscopy. Western blot analysis revealed that treatment with UV-B (0.2 kJ/m2) resulted in an increase in GrB and PFN protein expression 16 h after irradiation, as illustrated in Fig. 2A. Confocal analysis confirmed the lack of GrB or PFN in untreated cells (Fig. 2B). However, after irradiation, PFN was easily detected in the cytoplasm of HaCaT cells, whereas immunostaining with anti-GrB showed a more diffuse distribution with a predominant cytoplasmic distribution associated with a weak but detectable accumulation in the nucleus (Fig. 2B). Merging revealed that in the cytoplasm, PFN and GrB co-localized at 72.6% (Fig. 2B). This co-localization was also tested with Cy5 goat anti-mouse IgG and we obtained the same results (data not shown). These data suggested that these two lytic proteins accumulated into granules as they do in cytotoxic granules contained in activated immune effectors. These results demonstrate that in keratinocytes, UV-B irradiation induces the expression of GrB and PFN proteins that co-localize in cytoplasm.

Role of MAPK Pathway in the Induction of GrB and PFN by UV-B Irradiation—UV-B irradiation activates a large variety of signaling pathways, including c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinases (ERK), and p38 MAPK modules (33-35). To assess the involvement of MAPK activation in HaCaT cells, we examined whether different inhibitors suppressed GrB and/or PFN induction in response to UV-B irradiation.


Figure 2
View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 2.
Expression of GrB and PFN proteins in HaCaT cells. Cells were irradiated with UV-B (0.2 kJ/m2) and incubated for 16 h before protein extraction (A) or confocal analysis (B). Western blot and confocal analysis was performed using anti-GrB and anti-PFN Abs. beta-Actin was used as a loading control for Western blot (A). These data are representative of three independent experiments.

 


Figure 3
View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 3.
Role of MAPK pathway in the induction of GrB and PFN by UV-B irradiation. A and B, HaCaT cells were pre-treated with SB203580 (1 µM), an inhibitor for p38 MAPK, and/or SP600125 (5 µM), a specific inhibitor for JNK, or PD98059 (25 µM), a specific inhibitor of ERK for 1 h. Cells were then irradiated with UV-B (0.2 kJ/m2) and incubated for 16 h before mRNA extraction (A) or confocal analysis (B). A, expression of GrB and PFN mRNA was measured by real-time quantitative PCR. Electrophoresis of PCR products on a 2% agarose gel was undertaken because no gene expression was observed in non-treated cells. B, confocal analysis was performed using anti-GrB and anti-PFN Abs. C and D, HaCaT cells were pre-treated with SB203580 (1 µM) or SP600125 (5 µM) for 1 h. Cells were or were not irradiated with UV-B (0.2 kJ/m2) and incubated for 1 h before protein extraction. Expression of phosphorylated MAP-KAPK-2 (C) and phospho-JNK (D) was analyzed by Western blot. These data are representative of three independent experiments.

 
As shown in Fig. 3, A and B, SB203580, a specific and potent chemical inhibitor for p38 MAPK, totally inhibited GrB mRNA (A) and protein (B) expression. In contrast, SP600125, a specific inhibitor for JNK, and PD98059, a specific inhibitor for ERK, did not significantly reduce GrB mRNA and protein induction. Consistent with these results, phosphorylation of MAPKAPK-2, a substrate of p38 MAPK (36), was also observed in HaCaT cells irradiated with UV-B, indicating that UV-B activates p38 MAPK (Fig. 3C). Then, grB gene stimulation was found to be specifically dependent from p38 MAPK module.

As shown in Fig. 3, A and B, SP600125, but not SB203580 or PD98059, inhibited pfn gene stimulation in irradiated HaCaT cells. Consistent with these results, in these cells, UV-B-induced SP600125-inhibitable JNK phosphorylation (Fig. 3D). pfn gene stimulation was then found to be specifically dependent from JNK module. Moreover, as expected, co-treatment with SB203580 and SP600125 resulted in total inhibition of both GrB and PFN expression (Fig. 3B). Altogether, these results show that UV-B irradiation activates GrB and PFN through p38 MAPK and JNK, respectively.

Role of Epithelial Growth Factor Receptor in the Induction of GrB and PFN by UV-B Irradiation—Previous studies have documented that UV-B-induced rapid activation of EGFR, and that this event is critical for most components of the UV signaling response, including ERK, phosphatidylinositol 3-kinase/Akt, p38 MAPK, and JNK pathways (34, 35, 37). Based on the presumed role of p38 MAPK and JNK activation in GrB and PFN induction (see above), we examined whether EGFR activation could play a role in regulating these two proteins. For this reason, we investigated the effect of AG1478, a specific inhibitor of EGFR, on UV-induced GrB and PFN stimulation by UV-B in HaCaT cells. We found that pre-treatment with AG1478 used in conditions that allowed abrogation of p38 MAPK and JNK phosphorylation (Fig. 4, A and B) resulted in total inhibition of UV-B-induced GrB and PFN gene stimulation (Fig. 4C). These results suggested that EGFR activation is critical for grB and pfn induction by UV-B in keratinocytes.

Role of Reactive Oxygen Species in the Induction of GrB and PFN by UV-B Irradiation—Previous studies have indicated that ROS are also important mediators for the UV response. For example, ROS production is involved in UV-induced EGFR activation (38-40) as well as in coupling between EGFR and MAPK signaling. Therefore, we examined whether ROS could be involved in GrB and PFN regulation upon UV exposure in HaCaT cells. As shown in Fig. 5, A and B, pre-treatment with N-acetylcysteine, a potent antioxidant agent, at a dose of 25 mM for 2 h, allowed abrogation of UV-induced p38 MAPK (Fig. 5A) and JNK (Fig. 5B) phosphorylation. Treatment with N-acetylcysteine resulted in total inhibition of GrB and PFN mRNA and protein expression after UV irradiation (Fig. 5, C and D). Conversely, treatment with hydrogen peroxide (H2O2) resulted in increased GrB and PFN mRNA (Fig. 5E), thus mimicking the effects of UV-B. Altogether, these results showed that upon UV-B stimulation, grB and pfn gene activation is controlled by a signaling cascade that involves ROS, EGFR, p38 MAPK, and JNK.

Cellular Cytotoxicity of Irradiated HaCaT Cells—In the immune system, the GrB/PFN system confers to activated T or NK cells potent cellular cytotoxicity against infected or transformed cells. Therefore, we hypothesized that UV-B-induced GrB/PFN stimulation should also confer to keratinocytes some ability to destroy cells through cytotoxic granules. Cellular toxicity was measured using K562 target cells, this leukemia cell line being the standard cellular model to investigate GrB/PFN-mediated cellular cytotoxicity of innate immune effectors. UV-B-irradiated or untreated HaCaT cells were co-cultured with the K562 cell target at an effector:target (E:T) ratio between 80:1 and 10:1. Untreated HaCaT cells displayed no cytotoxicity toward K562 cells (Fig. 6A). When irradiated at 0.2 kJ/m2 and then incubated for 16 h, HaCaT cells exhibited cytotoxicity toward K562 cells in a 4-h cytotoxic assay (Fig. 6A). Albeit low, compared with NK or T cells, cellular cytotoxicity was significant with a maximum of 48% at a ratio of 80:1. To confirm these results, we have also shown that irradiated HaCaT cells displayed no cytotoxicity toward K562 cells when HaCaT cells were pre-treated with both SB203580 and SP600125 (Fig. 6A) and, in consequence, when GrB and PFN proteins were not expressed. To assess the role of the GrB/PFN system in the acquisition by keratinocytes of cellular cytotoxicity, we used pharmacological agents currently used to inhibit either GrB intrinsic cytotoxicity properties (DCIC) (28, 29) or PFN function by limiting its polymerization (MgCl2/EGTA) (30). Therefore, irradiated HaCaT cells were preincubated with DCIC and MgCl2/EGTA, and allowed to react with K562 target cells (E:T of 80:1). As depicted in Fig. 6B, pretreatment with DCIC and MgCl2/EGTA resulted in the abrogation of irradiated HaCaT cell cytotoxicity, whereas the anti-CD95 (Fas/APO-1) blocking Ab (ZB4), used as control reagents, had no effect (Fig. 6B). The role of GrB in keratinocyte-irradiated cellular cytotoxicity was also investigated by transfecting the serine protease inhibitor PI-9 in K562 target cells. Indeed, PI-9 blocks specifically GrB/PFN-mediated cellular cytotoxicity. As a matter of fact this serpin was used to ascertain the role of GrB in immune effector-mediated cell lysis (41, 42). As shown in Fig. 6C, PI-9 overexpression resulted in an inhibition of HaCaT-irradiated cytotoxicity. Finally we used antisense oligonucleotides directed against GrB. Cells were then irradiated at 0.2 kJ/m2 and then allowed to react with K562 used as target cells at various E:T ratios for 4 h. In fact, GrB antisense, but not sense, oligonucleotides strongly diminished irradiated HaCaT cell cytotoxicity as depicted in Fig. 6D for an E:T ratio of 80:1. In parallel, exposure to antisense oligonucleotides resulted in an inhibition of GrB-induced UV-B expression, whereas exposition to control oligonucleotide did not affect significantly GrB expression. These results show that UV-B conferred to keratinocytes cellular cytotoxicity through the GrB/PFN system.


Figure 4
View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 4.
Role of EGFR in the induction of GrB and PFN by UV-B irradiation. HaCaT cells were or were not pre-treated with AG1478 (10 µM), an inhibitor for EGFR, for 1 h, irradiated with UV-B (0.2 kJ/m2), and incubated for 1 h before protein extraction. Expression of phosphorylated MAPKAPK-2 (A) and phospho-JNK (B) was analyzed by Western blot. C, HaCaT cells were pre-treated with AG1478 (10 µM) for 1 h. Cells were then irradiated with UV-B (0.2 kJ/m2) and incubated for 16 h before mRNA extraction. Expression of GrB and PFN mRNA were measured by real-time quantitative PCR. Electrophoresis of PCR products on a 2% agarose gel was undertaken because no gene expression was observed in non-treated cells. These data are representative of three independent experiments.

 


Figure 5
View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 5.
Role of reactive oxygen species in the induction of GrB and PFN by UV-B irradiation. HaCaT cells were or were not pre-treated with N-acetylcysteine (NAC) (25 mM), an inhibitor of oxidative stress, for 2 h, irradiated with UV-B (0.2 kJ/m2), and incubated for 1 h before protein extraction. Expression of phosphorylated MAP-KAPK-2 (A) and phospho-JNK (B) was analyzed by Western blot. C and D, HaCaT cells were pretreated with NAC (25 mM) for 2 h. Cells were then irradiated with UV-B (0.2 kJ/m2) and incubated for 16 h before mRNA extraction (C) or confocal analysis (D). D, confocal analysis was performed using anti-GrB and anti-PFN Abs. E, HaCaT cells were treated with H2O2 at different concentrations for 1 h in PBS. Cells were then incubated for 16 h before mRNA extraction. C-E, expression of GrB and PFN mRNA was measured by real-time quantitative PCR. Electrophoresis of PCR products on a 2% agarose gel was undertaken because no gene expression was observed in non-treated cells. These data are representative of three independent experiments.

 
Cellular Cytotoxicity of Irradiated HaCaT Cells to Skin Cells—Based on these results, we hypothesized that irradiated keratinocytes could destroy other cells and particularly other skin cells. To test this assumption, UV-B-irradiated HaCaT were co-cultured with HaCaT naive cells, HK cells, Jurkat T cells, or KAL melanoma cells (Fig. 7) at an E:T ratio of 80:1. Naive HaCaT displayed no cytotoxicity against all these cells. However, when irradiated at 0.2 kJ/m2 and then incubated for 16 h, HaCaT cells exhibited significant cytotoxicity toward all these skin cells in a 4-h cytotoxic assay. These results suggest that UV-B may activate a cell to cell destruction among the keratinocyte population through the GrB/PFN system.


Figure 6
View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 6.
Cellular cytotoxicity of irradiated HaCaT cells toward the K562 cell line. Irradiated HaCaT cells were cultured for 16 h. Cytotoxicity was measured by using a 4-h non-radioactive LDH-release assay at the indicated E:T ratio. A, irradiated ({blacktriangleup} or gray circle) or not ({diamondsuit} or Figure 6) HaCaT cells were pre-treated (gray symbols) or not (black symbols) with SB203580 (1 µM) and SP600125 (5 µM), before co-incubation with K562 cells. B, percentage of cytotoxicity of UV-B-irradiated HaCaT cells pre-treated or not with MgCl2/EGTA, DCIC, or ZB4 Ab, toward K562 cells at an E:T ratio of 80:1. C, untreated HaCaT ({diamondsuit} or Figure 6) and irradiated HaCaT ({blacktriangleup} or gray circle) cells were co-incubated with K562 (black symbols) or GFP-PI-9-transfected K562 (gray symbols) cells as targets. Expression of GFP-PI-9 proteins in transfected (2) or not (1) K562 cells. Western blot analysis was performed using anti-GFP Ab. beta-Actin was used as a loading control for Western blot. D, HaCaT cells were incubated with antisense GrB or sense (control) oligonucleotides for 6 h, irradiated ({blacksquare}) or not (Figure 6), and cultured for 16 h, and then co-incubated with K562 cells as targets. Cytotoxicity was measured using a 4-h non-radioactive LDH-release assay at an 80:1 E:T ratio. Western blot analysis of antisense or sense oligonucleotides on GrB expression after irradiation. S, sense; AS, antisense. These data are representative of three independent experiments. Results are the mean ± S.D. of three independent experiments performed in triplicate. **, p = 0.02.

 


Figure 7
View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 7.
Cellular cytotoxicity of irradiated HaCaT cells toward different skin cells. Irradiated HaCaT cells were cultured for 16 h. Cytotoxicity was measured using a 4-h non-radioactive LDH-release assay at 80:1 ratio. Untreated HaCaT (Figure 7) and irradiated HaCaT ({blacksquare}) cells were co-incubated with non-irradiated HaCaT (1), HK (2), Jurkat (3), or KAL (4) cells as targets. These data are the mean ± S.D. of three independent experiments performed in triplicate. **, p < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have demonstrate for the first time that UV-B irradiation induces grB and pfn gene and protein expression in keratinocytes. Our data supports a model, in which, in irradiated keratinocytes, oxidative stress and EGFR activation results in the activation of p38 MAPK and JNK pathways, which in turn mediates the up-regulation of functional GrB and PFN proteins. It is important to note that GrB and PFN induction by UV-B was observed not only in the transformed HaCaT cell line but also in human normal primary keratinocytes, and in both non-confluent and confluent cells, suggesting that these events were not dependent on the culture conditions. Moreover, although GrB and PFN accumulated in keratinocytes in a dose-dependent fashion, it is important to note that gene activation was detected for non-toxic doses, suggesting that this event does not correlate with cell death.

UV-B light is known to induce diverse DNA damage, including DNA adducts, DNA strand breaks, DNA cross-links, and DNA-protein cross-links. The fact that GrB and PFN up-regulation is part of the cellular response to DNA damage induced by UV irradiation was not totally unexpected. Indeed, in a previous study, we have described that ionizing radiation or anti-cancer agents may induce GrB and PFN in acute myeloid leukemia cells through a transcriptional mechanism, and that this confers to cells a potent cellular cytotoxicity capacity, which was abrogated by GrB and PFN inhibitors (15). More recently, we have also reported similar events in acute myeloid leukemia cells treated by inflammatory cytokines such as tumor necrosis factor-{alpha} (25). Together with the present study, these findings suggest that, at least in some non-lymphoid tissues, diverse conditions of stress may lead to GrB and PFN induction.

This study shows also for the first time that upon UV-B irradiation, keratinocytes acquired cytotoxic potential against a variety of cellular skin targets, including keratinocytes, T-lymphocytes, and melanoma cells. It should be noted that cell lysis was measured using a non-radioactive cytotoxicity assay, a simpler alternative than the conventional Cr51 release, now widely used (15, 25, 43, 44). It should be pointed out that the killing capacity of irradiated keratinocytes was lower than that usually observed for immune effectors. For example, comparative experiments revealed that at the E:T ratio of 80:1, K562 cell lysis was 80, 70, and 48% for interleukin 2-activated peripheral blood lymphocytes, NK cells, and irradiated HaCaT cells, respectively (data not shown). However, because of the keratinocytes density, one can speculate that, in vivo, the acquisition of cellular cytotoxicity could have profound consequences if irradiated keratinocytes are directed against minor cell populations, such as epidermal lymphocytes or melanocytes.

The fact that in irradiated keratinocytes, grB and pfn gene activation correlates with the acquisition of cellular cytotoxicity raised the possibility that the GrB/PFN system contributed to UV-B-induced cellular cytotoxicity. Several lines of evidence support this hypothesis. First, the GrB/PFN system is very efficient at inducing cell lysis as illustrated by its function in immune effectors such as activated T or NK cells. Second, the role of the GrB/PFN system in the acquisition of cellular cytotoxicity upon stress has been previously documented in other cellular models (15). Third, the present study shows that both PI-9 expression as well as GrB and PFN inhibitors abolished the lytic function of irradiated keratinocytes. Moreover, we have observed that irradiated HaCaT displayed no lytic ability when separated from target cells by nylon membrane (data not shown). The fact that cell-cell contact is required for cellular cytotoxicity precludes the role of soluble mediators, including those that have been found to be produced after UV exposure, such as tumor necrosis factor-{alpha}, transforming growth factor beta, interferon, and Fas-ligand (45, 46). In addition, irradiated keratinocytes were efficient against K562 cells that are known to be highly resistant to tumor necrosis factor-{alpha} (47) and Fas agonist (48). This observation renders unlikely the contribution of tumor necrosis factor-{alpha} or Fas-ligand in both their soluble and membrane-bound forms. However, one can speculate that in our model, cell-cell contact requires receptor/ligand interaction, such as described for NK cell lysis (49). This point is currently being investigated in our laboratory.

Altogether, these results support the fact that upon UV-B irradiation, not only keratinocytes produced GrB and PFN proteins, but also that the lytic function of these proteins was preserved. The fact that these two lytic proteins co-localize in the cytoplasm of keratinocytes as revealed by confocal microscopy raises the possibility that they are contained in cytotoxic granules like in activated T or NK cells.

The mechanism by which UV-B up-regulates GrB and PFN has also been examined. UV elicited a number of interconnected signaling pathways, known as the UV response, which includes ROS-dependent activation of phosphatidylinositol 3-kinase/Akt, p38 MAPK, JNK, ERK, and EGFR (6). By using chemical inhibitors, we describe that GrB induction is dependent on p38 MAPK, whereas for PFN, JNK is the most critical. However, ERK or phosphatidylinositol 3-kinase pathways play no role in this regulation (data not shown). Altogether these results demonstrate that p38 and JNK govern distinctly GrB and PFN expression after UV irradiation. This observation is rather intriguing and suggests that these two pathways are coordinated and likely under common regulators, among them ROS and EGFR play a critical role. Based on previous studies, which have indicated that the pfn promoter contains the c-fos region (50, 51), the role of JNK in PFN regulation can be anticipated. However, the link between p38 MAPK activation and grB gene activation could be more complex. Indeed, although p38 MAPK activates cAMP-response element-binding protein (52) and AP1, two putative regulators of the grB gene, other studies also indicate that the p38 MAPK pathway may also influence mRNA stability through a MAPKAPK2-dependent, AU-rich element-targeted mechanism (53). Thus, it is possible that p38 MAPK acts directly through a cAMP-response element-binding protein- or AP1-mediated transcriptional mechanism, and indirectly at a posttranscriptional level by stabilizing GrB mRNA.

The significance and the functional consequences of GrB/PFN accumulation on the epidermis homeostasis remain uncertain. Because of the relatively low efficiency of cell lysis capacity toward themselves, it is unlikely that this serves at eliminating DNA-damaged keratinocytes in the context of a cell to cell killing interaction. In contrast, we can speculate that the acquisition of cellular toxicity by keratinocytes results in the partial destruction of skin-resident immune cells (epidermal T-cell) or in the limitation of cell recruitment in the context of UV-induced acute inflammation. Therefore, it is possible that the GrB/PFN system may be involved in the regulation of the skin inflammatory process to maintain epidermal cell defense after UV stress. In our work, we have also shown that irradiated keratinocytes results in the destruction of melanoma cells. In vivo, melanocytes are in contact with keratinocytes. We can then hypothesize that DNA damaged melanocytes can be killed by irradiated keratinocytes. The acquisition of this cytotoxicity could then also prevent the emergence of melanoma cells.

Alternatively, it is possible that the consequence of GrB or PFN accumulation in irradiated keratinocytes implies other properties of these two proteins, totally distinct from their lytic function. For example, GrB has been shown to cleave proteoglycan (54) as well as major components of the extracellular matrix such as vitronectin, fibronectin, and laminin (21). Whether or not GrB accumulation in the epidermis may contribute to the alteration of skin extracellular composition after chronic UV exposure should be investigated.

To conclude, we propose a model in which after UV-B irradiation, both PFN and GrB accumulated under their active forms in cytoplasmic granules of keratinocytes and that conferred to these cells potent cellular cytotoxicity. Although the significance of this intriguing observation remains uncertain, this phenomenon may have important functional consequences in the regulation of skin inflammatory response and in the emergence of cancer skin.


   FOOTNOTES
 
* 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

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed. Tel.: 33-5-62-74-45-61; Fax: 33-5-62-74-45-58; E-mail: helene.hernandez{at}toulouse.inserm.fr.

3 The abbreviations used are: ROS, radical oxygen species; DCIC, 3,4-dichloroisocoumarin; E:T, effectors to target; GrB, granzyme B; LDH, lactate dehydrogenase; HK, normal human primary keratinocytes; PBS, phosphate-buffered saline; PFN, perforin; JNK, c-Jun N-terminal kinase; NK, natural killer; EGFR, epidermal growth factor receptor; MAPK, mitogen-activated protein kinase; FCS, fetal calf serum; Ab, antibody; ERK, extracellular signal-regulated kinase; MAPKAPK, mitogen-activated protein kinase-activated protein kinase. Back


   ACKNOWLEDGMENTS
 
Dr. P. I. Bird is gratefully acknowledged for the gift of pEGFP/PI-9 plasmid. We thank also Prof. A. Hovnanian and Dr. T. al Saati (INSERM U563, Toulouse, France) for the gift of primary normal human keratinocytes and the KAL melanoma cell line, respectively. We also thank C. Silvestri for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. de Gruijl, F. R., Sterenborg, H. J., Forbes, P. D., Davies, R. E., Cole, C., Kelfkens, G., van Weelden, H., Slaper, H., and van der Leun, J. C. (1993) Cancer Res. 53, 53-60[Abstract/Free Full Text]
  2. Goihman-Yahr, M. (1996) Clin. Dermatol. 14, 153-160[CrossRef][Medline] [Order article via Infotrieve]
  3. Young, A. R. (1990) Semin. Dermatol. 9, 25-31[Medline] [Order article via Infotrieve]
  4. Ananthaswamy, H. N., and Pierceall, W. E. (1992) Prog. Clin. Biol. Res. 376, 61-84[Medline] [Order article via Infotrieve]
  5. Sander, C. S., Chang, H., Hamm, F., Elsner, P., and Thiele, J. J. (2004) Int. J. Dermatol. 43, 326-335[CrossRef][Medline] [Order article via Infotrieve]
  6. Bowden, G. T. (2004) Nat. Rev. Cancer 4, 23-35[CrossRef][Medline] [Order article via Infotrieve]
  7. Katiyar, S. K., Afaq, F., Azizuddin, K., and Mukhtar, H. (2001) Toxicol. Appl. Pharmacol. 176, 110-117[CrossRef][Medline] [Order article via Infotrieve]
  8. Hart, P. H., Grimbaldeston, M. A., and Finlay-Jones, J. J. (2001) Clin. Exp. Pharmacol. Physiol. 28, 1-8[CrossRef][Medline] [Order article via Infotrieve]
  9. Schwarz, A., Bhardwaj, R., Aragane, Y., Mahnke, K., Riemann, H., Metze, D., Luger, T. A., and Schwarz, T. (1995) J. Investig. Dermatol. 104, 922-927[CrossRef][Medline] [Order article via Infotrieve]
  10. Barry, M., and Bleackley, R. C. (2002) Nat. Rev. Immunol. 2, 401-409[Medline] [Order article via Infotrieve]
  11. Trapani, J. A., and Smyth, M. J. (2002) Nat. Rev. Immunol. 2, 735-747[CrossRef][Medline] [Order article via Infotrieve]
  12. Heusel, J. W., Wesselschmidt, R. L., Shresta, S., Russell, J. H., and Ley, T. J. (1994) Cell 76, 977-987[CrossRef][Medline] [Order article via Infotrieve]
  13. Shi, L., Kam, C. M., Powers, J. C., Aebersold, R., and Greenberg, A. H. (1992) J. Exp. Med. 176, 1521-1529[Abstract/Free Full Text]
  14. Kagi, D., Ledermann, B., Burki, K., Seiler, P., Odermatt, B., Olsen, K. J., Podack, E. R., Zinkernagel, R. M., and Hengartner, H. (1994) Nature 369, 31-37[CrossRef][Medline] [Order article via Infotrieve]
  15. Bruno, A. P., Lautier, D., d'Orgeix, A. T., Laurent, G., and Quillet-Mary, A. (2000) Blood 96, 1914-1920[Abstract/Free Full Text]
  16. Horiuchi, K., Saito, S., Sasaki, R., Tomatsu, T., and Toyama, Y. (2003) J. Rheumatol. 30, 1799-1810[Abstract/Free Full Text]
  17. Hirst, C. E., Buzza, M. S., Sutton, V. R., Trapani, J. A., Loveland, K. L., and Bird, P. I. (2001) Mol. Hum. Reprod. 7, 1133-1142[Abstract/Free Full Text]
  18. Sasson, R., and Amsterdam, A. (2003) Biochem. Pharmacol. 66, 1393-1401[CrossRef][Medline] [Order article via Infotrieve]
  19. Berthou, C., Michel, L., Soulie, A., Jean-Louis, F., Flageul, B., Dubertret, L., Sigaux, F., Zhang, Y., and Sasportes, M. (1997) J. Immunol. 159, 5293-5300[Abstract]
  20. van den Broek, M. F., and Hengartner, H. (2000) Exp. Physiol. 85, 681-685[Abstract]
  21. Buzza, M. S., Zamurs, L., Sun, J., Bird, C. H., Smith, A. I., Trapani, J. A., Froelich, C. J., Nice, E. C., and Bird, P. I. (2005) J. Biol. Chem. 280, 23549-23558[Abstract/Free Full Text]
  22. Watt, F. M., Boukamp, P., Hornung, J., and Fusenig, N. E. (1987) Arch. Dermatol. Res. 279, 335-340[CrossRef][Medline] [Order article via Infotrieve]
  23. Rheinwald, J. G., and Green, H. (1975) Cell 6, 331-343[CrossRef][Medline] [Order article via Infotrieve]
  24. Cohen-Knafo, E., al Saati, T., Aziza, J., Ralfkiaer, E., Selves, J., Gorguet, B., and Delsol, G. (1995) J. Clin. Pathol. 48, 826-831[Abstract/Free Full Text]
  25. Guilloton, F., de Thonel, A., Jean, C., Demur, C., Mansat-De Mas, V., Laurent, G., and Quillet-Mary, A. (2005) Leukemia 19, 2206-2214[CrossRef][Medline] [Order article via Infotrieve]
  26. Simon, T., Opelz, G., Wiesel, M., Ott, R. C., and Susal, C. (2003) Am. J. Transplant. 3, 1121-1127[CrossRef][Medline] [Order article via Infotrieve]
  27. Brander, C., Wyss-Coray, T., Mauri, D., Bettens, F., and Pichler, W. J. (1993) Eur. J. Immunol. 23, 3217-3223[Medline] [Order article via Infotrieve]
  28. Hudig, D., Allison, N. J., Pickett, T. M., Winkler, U., Kam, C. M., and Powers, J. C. (1991) J. Immunol. 147, 1360-1368[Abstract]
  29. Odake, S., Kam, C. M., Narasimhan, L., Poe, M., Blake, J. T., Krahenbuhl, O., Tschopp, J., and Powers, J. C. (1991) Biochemistry 30, 2217-2227[CrossRef][Medline] [Order article via Infotrieve]
  30. Ostergaard, H. L., Kane, K. P., Mescher, M. F., and Clark, W. R. (1987) Nature 330, 71-72[CrossRef][Medline] [Order article via Infotrieve]
  31. Leonard, M., Brice, M., Engel, J. D., and Papayannopoulou, T. (1993) Blood 82, 1071-1079[Abstract/Free Full Text]
  32. Tchernitchko, D., Bourgeois, M., Martin, M. E., and Beaumont, C. (2002) Biochem. J. 363, 449-455[CrossRef][Medline] [Order article via Infotrieve]
  33. Devary, Y., Gottlieb, R. A., Lau, L. F., and Karin, M. (1991) Mol. Cell. Biol. 11, 2804-2811[Abstract/Free Full Text]
  34. El-Abaseri, T. B., Fuhrman, J., Trempus, C., Shendrik, I., Tennant, R. W., and Hansen, L. A. (2005) Cancer Res. 65, 3958-3965[Abstract/Free Full Text]
  35. Kitagawa, D., Tanemura, S., Ohata, S., Shimizu, N., Seo, J., Nishitai, G., Watanabe, T., Nakagawa, K., Kishimoto, H., Wada, T., Tezuka, T., Yamamoto, T., Nishina, H., and Katada, T. (2002) J. Biol. Chem. 277, 366-371[Abstract/Free Full Text]
  36. Cuenda, A., Rouse, J., Doza, Y. N., Meier, R., Cohen, P., Gallagher, T. F., Young, P. R., and Lee, J. C. (1995) FEBS Lett. 364, 229-233[CrossRef][Medline] [Order article via Infotrieve]
  37. Rosette, C., and Karin, M. (1996) Science 274, 1194-1197[Abstract/Free Full Text]
  38. Ashida, M., Bito, T., Budiyanto, A., Ichihashi, M., and Ueda, M. (2003) Exp. Dermatol. 12, 445-452[CrossRef][Medline] [Order article via Infotrieve]
  39. Chen, R., Lim, J. H., Jono, H., Gu, X. X., Kim, Y. S., Basbaum, C. B., Murphy, T. F., and Li, J. D. (2004) Biochem. Biophys. Res. Commun. 324, 1087-1094[CrossRef][Medline] [Order article via Infotrieve]
  40. Goldkorn, T., Balaban, N., Matsukuma, K., Chea, V., Gould, R., Last, J., Chan, C., and Chavez, C. (1998) Am. J. Respir. Cell Mol. Biol. 19, 786-798[Abstract/Free Full Text]
  41. Bird, C. H., Sutton, V. R., Sun, J., Hirst, C. E., Novak, A., Kumar, S., Trapani, J. A., and Bird, P. I. (1998) Mol. Cell. Biol. 18, 6387-6398[Abstract/Free Full Text]
  42. Sun, J., Bird, C. H., Sutton, V., McDonald, L., Coughlin, P. B., De Jong, T. A., Trapani, J. A., and Bird, P. I. (1996) J. Biol. Chem. 271, 27802-27809[Abstract/Free Full Text]
  43. Andre, N. D., Barbosa, D. S., Munhoz, E., Estevao, D., Cecchini, R., and Watanabe, M. A. (2004) J. Clin. Lab. Anal. 18, 27-30[CrossRef][Medline] [Order article via Infotrieve]
  44. Decker, T., and Lohmann-Matthes, M. L. (1988) J. Immunol. Methods 115, 61-69[CrossRef][Medline] [Order article via Infotrieve]
  45. Leverkus, M., Yaar, M., Eller, M. S., Tang, E. H., and Gilchrest, B. A. (1998) J. Investig. Dermatol. 110, 353-357[CrossRef][Medline] [Order article via Infotrieve]
  46. Leverkus, M., Yaar, M., and Gilchrest, B. A. (1997) Exp. Cell. Res. 232, 255-262[CrossRef][Medline] [Order article via Infotrieve]
  47. Munker, R., DiPersio, J., and Koeffler, H. P. (1987) Blood 70, 1730-1734[Abstract/Free Full Text]
  48. Robertson, M. J., Manley, T. J., Pichert, G., Cameron, C., Cochran, K. J., Levine, H., and Ritz, J. (1995) Leuk. Lymphoma 17, 51-61[Medline] [Order article via Infotrieve]
  49. Raulet, D. H. (2003) Nat. Rev. Immunol. 3, 781-790[CrossRef][Medline] [Order article via Infotrieve]
  50. Lichtenheld, M. G., and Podack, E. R. (1989) J. Immunol. 143, 4267-4274[Abstract]
  51. Youn, B. S., Liu, C. C., Kim, K. K., Young, J. D., Kwon, M. H., and Kwon, B. S. (1991) J. Exp. Med. 173, 813-822[Abstract/Free Full Text]
  52. Butler, M. P., Hanly, J. A., and Moynagh, P. N. (2005) J. Biol. Chem. 280, 27759-27768[Abstract/Free Full Text]
  53. Winzen, R., Kracht, M., Ritter, B., Wilhelm, A., Chen, C. Y., Shyu, A. B., Muller, M., Gaestel, M., Resch, K., and Holtmann, H. (1999) EMBO J. 18, 4969-4980[CrossRef][Medline] [Order article via Infotrieve]
  54. Ronday, H. K., van der Laan, W. H., Tak, P. P., de Roos, J. A., Bank, R. A., TeKoppele, J. M., Froelich, C. J., Hack, C. E., Hogendoorn, P. C., Breedveld, F. C., and Verheijen, J. H. (2001) Rheumatology (Oxford) 40, 55-61[Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Cancer Res.Home page
C. Jean, A. Blanc, N. Prade-Houdellier, L. Ysebaert, H. Hernandez-Pigeon, T. Al Saati, M.-J. Haure, A.-M.-L. Coluccia, M. Charveron, E. Delabesse, et al.
Epidermal Growth Factor Receptor/{beta}-Catenin/T-Cell Factor 4/Matrix Metalloproteinase 1: A New Pathway for Regulating Keratinocyte Invasiveness after UVA Irradiation
Cancer Res., April 15, 2009; 69(8): 3291 - 3299.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Hernandez-Pigeon, C. Jean, A. Charruyer, M.-J. Haure, C. Baudouin, M. Charveron, A. Quillet-Mary, and G. Laurent
UVA Induces Granzyme B in Human Keratinocytes through MIF: IMPLICATION IN EXTRACELLULAR MATRIX REMODELING
J. Biol. Chem., March 16, 2007; 282(11): 8157 - 8164.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/19/13525    most recent
M512694200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hernandez-Pigeon, H.
Right arrow Articles by Laurent, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hernandez-Pigeon, H.
Right arrow Articles by Laurent, G.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement