Long-term cyclosporin A exposure suppresses cathepsin-B and -L activity in gingival fibroblasts.
BACKGROUND
Gingival overgrowth is a common side-effect following administration of cyclosporin A. We reported previously that lysosomal protease cathepsin-L activity, but not cathepsin-B, was significantly suppressed by short-term cyclosporin A exposure in human gingival fibroblasts. Although this suppression may lead to decreased degradation of gingival connective tissue, a net increase in matrix proteins, and gingival overgrowth, the effects of cyclosporin A need to be more elucidated, considering the long-term use for patients following organ transplantation.


OBJECTIVE
The aim of the present study was to evaluate the effects of clinically relevant doses of cyclosporin A on cultured human gingival fibroblasts. We evaluated the effects of long-term cyclosporin A exposure on cell proliferation, mRNA expression of various proteases and both cathepsin-B and -L activity in human gingival fibroblasts.


METHODS
Human gingival fibroblasts were isolated from three donors' healthy gingiva and cultured from five to eight passages with or without 200 ng/ml of cyclosporin A. Proliferative activity of cyclosporin A-treated cells was examined using MTT assay. Total RNA and cellular proteins were collected for semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis and for measurement of the cathepsin-B and -L activity.


RESULTS
Long-term cyclosporin A exposure had no effects on cell proliferation. Accumulation of cathepsin-B, -H and -L mRNA was markedly suppressed by long-term cyclosporin A exposure, whereas accumulation of another lysosomal enzyme N-acetyl-beta-D-glucosaminidase mRNA, which is involved in remodeling of gingival epithelium, was not apparently impaired in cyclosporin A-treated cells. Accumulation of matrix metalloprotease-1 (MMP-1) and tissue inhibitor of matrix metalloprotease-1 (TIMP-1) mRNA, which are involved in remodeling of extracellular matrix, also was not impaired. In addition, we demonstrated that long-term cyclosporin A exposure significantly suppressed not only the activity of the active form of cathepsin-(B + L) compared to the activity in non-treated cells (p = 0.0458), but also the activity of the active form of cathepsin-B (p < 0.0001) in human gingival fibroblasts.


CONCLUSIONS
The decreased ability of protein degradation by not only cathepsin-L but also cathepsin-B is, at least, one of the several factors developing the cyclosporin A-induced gingival overgrowth.
Yamaguchi
Mayumi
M
Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
Naruishi
Koji
K
Yamada-Naruishi
Hisa
H
Omori
Kazuhiro
K
Nishimura
Fusanori
F
Takashiba
Shogo
S
D002401:Cathepsin B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology*; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D005881:Gingiva / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D015853:Cysteine Proteinase Inhibitors; D007166:Immunosuppressive Agents; D012333:RNA, Messenger; D016572:Cyclosporine; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L