Roxithromycin suppresses involucrin expression by modulation of activator protein-1 and nuclear factor-kappaB activities of keratinocytes.
BACKGROUND
Roxithromycin (RXM), a new 14-member macrolide antibiotic, is effective for chronic airway diseases such as diffuse panbronchiolitis and bronchial asthma. Recent study disclosed that RXM inhibits nuclear factor-kappaB (NF-kappaB)-mediated inflammation. Involucrin is one of the precursor proteins of the cornified cell envelope (CE) and is markedly increased in inflammatory skin diseases such as psoriasis. However, its molecular mechanism of action remains unknown.


OBJECTIVE
To determine the effect of RXM on involucrin expression of keratinocytes.


METHODS
We constructed chloramphenicol acetyltransferase (CAT)-involucrin promoter expression vector and CAT assay was performed. Furthermore, western blot and RT-PCR were performed to examine the expression of involucrin in RXM-treated cultured human keratinocytes.


RESULTS
The increased involucrin expression by 12-O-tetradecanoylphorbol acetate (TPA) was suppressed by 10(-6) M RXM and the maximal inhibitory effect was observed at 48 h. RXM suppressed increased CAT activity by TPA and the effect was not inhibited by H-7 or cafferic acid phenethyl ester (CAPE). Deletion of T1 region (-119 to -113) of involucrin promoter completely abolished TPA-dependent stimulatory and RXM-dependent inhibitory promoter activity. Gel shift assay showed that c-Jun (but not p65) selectively binds to the T1 region. The assay of activator protein-1 (AP-1) and NF-kappaB activities revealed that RXM decreased both transcriptional activities. Co-transfection of c-jun and c-fos expression vectors, or p65 and p50 expression vectors, rescued decreased CAT activity by RXM, respectively.


CONCLUSIONS
Our study demonstrated for the first time that involucrin expression of keratinocytes is suppressed by RXM through direct inhibition of AP-1 and indirect inhibition of NF-kappaB.
Takahashi
Hidetoshi
H
Department of Dermatology, Asahikawa Medical College, 2-1-1-1 Midorigaokahigashi, Asahikawa 078-8510, Japan. ht@asahikawa-med.ac.jp
Hashimoto
Yoshio
Y
Ishida-Yamamoto
Akemi
A
Iizuka
Hajime
H
D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D015603:Keratinocytes / Q000187:drug effects / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics*; D015575:Roxithromycin / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014162:Transfection
D016328:NF-kappa B; D011498:Protein Precursors; D018808:Transcription Factor AP-1; D015575:Roxithromycin; C032253:involucrin; D015500:Chloramphenicol O-Acetyltransferase