Regulation of calprotectin expression by interleukin-1alpha and transforming growth factor-beta in human gingival keratinocytes.
OBJECTIVE
Calprotectin, a heterodimer of S100A8 and S100A9 with antimicrobial properties, is expressed in gingival keratinocytes and plays an important role in innate immunity. Because calprotectin expression is localized in the spinous cell layer of the gingival epithelium, we hypothesized that the expression of calprotectin in keratinocytes is related to the differentiation stage. The aim of the present study was to investigate the relationship between calprotectin expression and keratinocyte differentiation using some factors that regulated its differentiation.


METHODS
Normal human gingival keratinocytes were isolated from gingival tissues obtained at the extraction of wisdom teeth, and were cultured in serum-free keratinocyte medium supplemented with interleukin-1alpha or calcium, which promote keratinocyte differentiation, and transforming frowth factor-beta (TGF-beta) or retinoic acid, which suppress its differentiation. The expression of S100A8/A9 mRNA and the production of calprotectin in normal human gingival keratinocytes were examined by northern blotting and enzyme-linked immunosorbent assay, respectively. The expression of cytokeratin 14, involucrin and filaggrin (marker proteins of keratinocyte differentiation) was investigated by immunohistochemical staining, and the DNA-binding activity of CCAAT/enhancer binding protein alpha (C/EBPalpha), a transcription factor, was examined by electrophoretic mobility shift assay.


RESULTS
The expression of S100A8/A9 mRNA and the production of calprotectin were increased by interleukin-1alpha and calcium, but decreased by TGF-beta. RA inhibited the expression of S100A8/A9 and keratinocyte differentiation, which were induced by interleukin-1alpha. C/EBPalpha DNA-binding activity in normal human gingival keratinocytes was enhanced by interleukin-1alpha and calcium, but suppressed by TGF-beta.


CONCLUSIONS
The present study suggests that calprotectin expression is related to keratinocyte differentiation and that C/EBPalpha is a regulator of calprotectin expression in keratinocytes.
Hayashi
N
N
Department of Periodontology and Endodontology, Oral and Maxillofacial Dentistry, Division of Medico-Dental Dynamics and Reconstruction, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.
Kido
J
J
Kido
R
R
Wada
C
C
Kataoka
M
M
Shinohara
Y
Y
Nagata
T
T
D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000032:analysis; D002118:Calcium / Q000494:pharmacology; D040501:Calgranulin A / Q000032:analysis / Q000187:drug effects; D040502:Calgranulin B / Q000032:analysis / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D000091344:Filaggrin Proteins; D005786:Gene Expression Regulation / Q000187:drug effects; D005881:Gingiva / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D053582:Interleukin-1alpha / Q000494:pharmacology*; D053547:Keratin-14 / Q000032:analysis; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism; D039841:Leukocyte L1 Antigen Complex / Q000032:analysis / Q000187:drug effects*; D010750:Phosphoproteins / Q000032:analysis; D011498:Protein Precursors / Q000032:analysis; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014212:Tretinoin / Q000494:pharmacology
D022763:CCAAT-Enhancer-Binding Protein-alpha; D040501:Calgranulin A; D040502:Calgranulin B; D016895:Culture Media, Serum-Free; C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D053582:Interleukin-1alpha; D053547:Keratin-14; D039841:Leukocyte L1 Antigen Complex; D010750:Phosphoproteins; D011498:Protein Precursors; D016212:Transforming Growth Factor beta; D014212:Tretinoin; C032253:involucrin; D002118:Calcium