Effect of phenytoin on collagen accumulation by human gingival fibroblasts exposed to TNF-alpha in vitro.
OBJECTIVE
Tumor necrosis factor (TNF)-alpha is associated with chronic gingival inflammation and reported to induce gingival overgrowth (GO), while phenytoin (PHT) is also known to be a causative agent of GO. We examined the synergistic effect of PHT and TNF-alpha on collagen metabolism in human gingival fibroblasts (HGFs).


METHODS
HGFs were cultured with TNF-alpha and PHT. Quantitative real-time RT-PCR was employed to determine the mRNA levels for collagen, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and integrin subunits. Cellular collagen endocytosis was determined using a flow-cytometry.


RESULTS
The proliferation of HGFs was not affected by TNF-alpha or PHT individually, whereas both synergistically increased collagen accumulation in HGFs. Further, collagen mRNA expression was not increased by TNF-alpha or PHT, although together they markedly prevented cellular collagen endocytosis, associated with the suppression of alpha2beta1-integrin mRNA expression. The mRNA expression of MMP-1 and-2 was suppressed by PHT, while TIMP-1 mRNA expression was enhanced by both TNF-alpha and PHT.


CONCLUSIONS
Our results suggest that TNF-alpha and PHT together cause impaired collagen metabolism by suppression of enzymatic degradation with MMPs/TIMP-1 and integrin-mediated endocytosis. These synergistic effects may also be involved in TNF-alpha- and PHT-induced collagen accumulation, leading to GO.
Department of Oral Frontier Biology, Osaka University Graduate School of Dentistry, Suita-Osaka, Japan.
T
T
Kato
N
N
Okahashi
T
T
Ohno
H
H
Inaba
S
S
Kawai
A
A
Amano
D000927:Anticonvulsants / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000187:drug effects* / Q000378:metabolism; D004357:Drug Synergism; D004705:Endocytosis / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects*; D005881:Gingiva / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D038982:Integrin alpha2beta1 / Q000187:drug effects; D016023:Integrins / Q000187:drug effects; D020781:Matrix Metalloproteinase 1 / Q000187:drug effects; D020778:Matrix Metalloproteinase 2 / Q000187:drug effects; D010672:Phenytoin / Q000494:pharmacology*; D021122:Protein Subunits / Q000187:drug effects; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000927:Anticonvulsants; D038982:Integrin alpha2beta1; D016023:Integrins; D021122:Protein Subunits; D019715:Tissue Inhibitor of Metalloproteinase-1; D014409:Tumor Necrosis Factor-alpha; D010672:Phenytoin; D003094:Collagen; D020778:Matrix Metalloproteinase 2; D020781:Matrix Metalloproteinase 1