Proteomic analysis in cyclosporin A-induced overgrowth of human gingival fibroblasts.
Cyclosporin A (CsA) has been used as an immunosuppressive drug to prevent organ transplant rejection and to treat autoimmune diseases. CsA has a proliferative effect on human gingival fibroblasts (HGF) in vitro. However, the molecular mechanisms underlying CsA-induced proliferation in HGF remain to be elucidated. This study was aimed to investigate the CsA responsive proteins in HGF using systematic proteomic approach. Cell viability was determined by MTT assay and reactive oxygen species (ROS) was measured by fluorescent spectrometer. Proteins profiled by two-dimensional gel electrophoresis (2-DE) were identified by matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) and electrospray ionization quadrupole time-of-flight mass spectrometry (EIQ-TOF MS). To confirm the expression changes of proteins by proteomics analysis, Western blot was performed using specific antibody. CsA increased the cell viability of HGF in a dose- and time-dependent manner. Significantly, seventeen proteins were overexpressed in the CsA-treated HGF, whereas three proteins were found to be expressed less than the untreated cells. The identified proteins were mainly related with cell proliferation, metabolism, and oxidation. The overexpression of peroxiredoxin 1 (Prx 1) confirmed by Western blotting and reduction of cytosolic reactive oxygen species (ROS) levels in the CsA-treated HGF demonstrated that Prx 1 may play a crucial role in the HGF proliferation induced by CsA. Upregulation of Galectin 3 in CsA-treated HGF indicated that it is related to CsA-induced proliferation. These proteomic analysis data will provide an efficient approach in understanding the mechanisms of HGF proliferation by CsA.
Jung
Ji Yeon
JY
Department of Oral Physiology, Dental Science Research Institute, 2nd Stage of Brain Korea 21 for School of Dentistry, Chonnam National University School of Dentistry, Gwangju, Republic of Korea.
Kang
Gi Chang
GC
Jeong
Yeon Jin
YJ
Kim
Sun Hun
SH
Kwak
Yong Geun
YG
Kim
Won Jae
WJ
D015153:Blotting, Western; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016572:Cyclosporine / Q000009:adverse effects* / Q000494:pharmacology; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D037502:Galectin 3 / Q000096:biosynthesis; D005881:Gingiva / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D019214:Gingival Overgrowth / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007166:Immunosuppressive Agents / Q000009:adverse effects* / Q000494:pharmacology; D054464:Peroxiredoxins / Q000096:biosynthesis; D020543:Proteome / Q000096:biosynthesis*; D040901:Proteomics; D017382:Reactive Oxygen Species / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015854:Up-Regulation
D037502:Galectin 3; D007166:Immunosuppressive Agents; D020543:Proteome; D017382:Reactive Oxygen Species; D016572:Cyclosporine; C515940:PRDX1 protein, human; D054464:Peroxiredoxins