Phenytoin activates SMAD3 phosphorylation and periostin expression in drug-induced gingival enlargement
Shawna S. Kim1, Georgia Nikoloudaki1, Mark Darling2, Michael J. Rieder3 and Douglas W. Hamilton1,4
1Department of Anatomy and Cell Biology, 2Department of Pathology, 3Department of Physiology and Pharmacology and 4Division of Oral Biology, Schulich School of Medicine and Dentistry, The University of Western Ontario, Ontario, Canada
Offprint requests to: Dr. Douglas W. Hamilton, Division of Oral Biology, Schulich School of Medicine and Dentistry, Dental Sciences Building, The University of Western Ontario, London, Ontario, Canada, N6A 5C1. e-mail: dhamil2@uwo.ca
Summary. Drug-induced gingival enlargement (DIGE) is a fibrotic condition associated with systemic administration of the anti-epileptic drug, phenytoin. We have previously demonstrated that periostin, which is transforming growth factor-beta (TGF-β) inducible gene, is upregulated in various fibrotic conditions including gingival enlargement associated with nifedipine. The objective of this study was to assess periostin expression in phenytoin-induced gingival enlargement (PIGE) tissues and to investigate the mechanisms underlying periostin expression. Human PIGE tissues were assessed using Masson's trichrome, with cell infiltration and changes in extracellular matrix composition characterized through labeling with antibodies to periostin, phospho-SMAD 3, TGF-β, as well as the macrophage markers CD68 and RM3/1. Using human gingival fibroblasts (HGFs) in vitro, we examined the pathways through which phenytoin acts on fibroblasts. In PIGE tissues, which demonstrate altered collagen organization and increased inflammatory cell infiltration, periostin protein was increased compared with healthy tissues. p-SMAD2/3, the transcription factor associated with canonical TGF-β signaling, is localized to the nuclei in both gingival fibroblasts and oral epithelial cells in PIGE tissues, but not in healthy tissue. In vitro culture of HGFs with 15 and 30 ?g/ml of phenytoin increased periostin protein levels, which correlated with p-SMAD3 phosphorylation. Inhibition of canonical TGF-β signaling with SB431542 significantly reduced phenytoin induction of SMAD3 phosphorylation and periostin expression in HGFs. Analysis of PIGE tissues showed a subset of CD68 stained macrophages were TGF-β positive and that RM1/3 regenerative macrophages were present in the tissues. Our results demonstrate that phenytoin up-regulates periostin in HGFs in a TGF-β-dependent mannerGingiva, Fibrosis, Matricellular protein, Transforming growth factor signaling, Macrophages. Histol Histopathol 33, 1287-1298 (2018)
Key words: Gingiva, Fibrosis, Matricellular protein, Transforming growth factor signaling, Macrophages
DOI: 10.14670/HH-18-015