Gene expression profiling defines the role of ATP-exposed keratinocytes in skin inflammation.
BACKGROUND
Various environmental stimuli, e.g., mechanical stress, osmolarity change, oxidative stress, and microbial products trigger ATP release from cells. It is well known that ATP regulates cell growth, differentiation, terminal differentiation, and cell-to-cell communication in keratinocytes. Moreover, extracellular ATP stimulates the expression and release of IL-6 and modulates the production several chemokines by keratinocytes.


OBJECTIVE
To investigate the role of ATP-stimulated keratinocytes in skin inflammation and immune response.


METHODS
We identified genes whose expression is augmented in ATP-stimulated human keratinocytes by DNA microarray. These microarray data were validated by quantitative real-time RT-PCR. Furthermore, we confirmed the observed mRNA change at protein level by ELISA and Western blotting.


RESULTS
The statistical analysis of the microarray data revealed that, besides IL-6, the expression of several novel genes such as IL-20, CXCL1-3, and ATF3 was significantly augmented in ATP-stimulated keratinocytes. These data was validated by quantitative real-time RT-PCR. We also confirmed the augmented production of IL-6, IL-20, CXCL1 by ELISA and that of ATF3 by Western blotting. Since both IL-6 and IL-20 that can stimulate STAT3 were produced by the ATP-stimulated keratinocytes, we examined their phosphorylation of STAT3. The study demonstrated biphasic activation of STAT3 after ATP stimulation, which was composed of a first peak at 1-2 h and a second peak at 12-24 h. The latter peak was significantly suppressed by anti-IL-6 antibody.


CONCLUSIONS
These studies characterized (1) STAT3 activation, (2) chemotaxis for neutrophils via CXCL1-3, and (3) ATF3 activation as possible roles of ATP-stimulated keratinocytes in skin inflammation and immune response.
Ohara
Hiroshi
H
Department of Clinical Pharmacy, Tohoku University Graduate School of Pharmaceutical Sciences, 1-1 Seiryo-machi Aoba-ku, Sendai 980-8574, Japan.
Saito
Rumiko
R
Hirakawa
Satoshi
S
Shimada
Miki
M
Mano
Nariyasu
N
Okuyama
Ryuhei
R
Aiba
Setsuya
S
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D020869:Gene Expression Profiling; D006801:Humans; D007249:Inflammation; D015850:Interleukin-6 / Q000378:metabolism; D007378:Interleukins / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050796:STAT3 Transcription Factor / Q000378:metabolism; D012867:Skin / Q000166:cytology*
D015850:Interleukin-6; D007378:Interleukins; D012333:RNA, Messenger; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D000255:Adenosine Triphosphate; C422042:interleukin 20