The in vitro effects of dehydroepiandrosterone on human osteoarthritic chondrocytes.
OBJECTIVE
To investigate the in vitro effects of dehydroepiandrosterone (DHEA) on human osteoarthritic chondrocytes.


METHODS
Chondrocytes isolated from human osteoarthritic knee cartilage were three-dimensionally cultured in alginate beads, except for cell proliferation experiment. Cells were treated with DHEA in the presence or absence of IL-1beta. The effects on chondrocytes were analyzed using a 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay (for chondrocyte proliferation), a dimethylmethylene blue (DMB) assay (for glycosaminoglycan (GAG) synthesis), and an indole assay (for DNA amount). Gene expressions of type I and II collagen, metalloproteinase-1 and -3 (MMP-1 and -3), and tissue inhibitor of metalloproteinase-1 (TIMP-1) as well as the IL-1beta-induced gene expressions of MMP-1 and -3 were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The protein synthesis of MMP-1 and -3 and TIMP-1 was determined by Western blotting.


RESULTS
The treatment of chondrocytes with DHEA did not affect chondrocyte proliferation or GAG synthesis up to 100 micro M of concentration. The gene expression of type II collagen increased in a dose-dependent manner, while that of type I decreased. DHEA suppressed the expression of MMP-1 significantly at concentrations exceeding 50 micro M. The gene expression of MMP-3 was also suppressed, but this was without statistical significance. The expression of TIMP-1 was significantly increased by DHEA at concentrations exceeding 10 micro M. The effects of DHEA on the gene expressions of MMP-1 and -3 were more prominent in the presence of IL-1beta, in which DHEA suppressed not only MMP-1, but also MMP-3 at the lower concentrations, 10 and 50 micro M, respectively. Western blotting results were in agreement with RT-PCR, which indicates that DHEA acts at the gene transcription level.


CONCLUSIONS
Our study demonstrates that DHEA has no toxic effect on chondrocytes up to 100 micro M of concentration and has an ability to modulate the imbalance between MMPs and TIMP-1 during OA at the transcription level, which suggest that it has a protective role against articular cartilage loss.
Jo
H
H
Department of Orthopedic Surgery, Seoul National University Hospital, 28 Yongon-dong, Chongno-gu, 110-744, Seoul, South Korea.
Park
J S
JS
Kim
E M
EM
Jung
M Y
MY
Lee
S H
SH
Seong
S C
SC
Park
S C
SC
Kim
H J
HJ
Lee
M C
MC
D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D015153:Blotting, Western / Q000379:methods; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000276:immunology; D003094:Collagen / Q000032:analysis; D003687:Dehydroepiandrosterone / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D006025:Glycosaminoglycans / Q000096:biosynthesis; D006801:Humans; D007375:Interleukin-1 / Q000276:immunology; D020781:Matrix Metalloproteinase 1 / Q000032:analysis; D019278:Matrix Metalloproteinase 3 / Q000032:analysis; D020370:Osteoarthritis, Knee / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D014176:Protein Biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000032:analysis
D000276:Adjuvants, Immunologic; D006025:Glycosaminoglycans; D007375:Interleukin-1; D019715:Tissue Inhibitor of Metalloproteinase-1; D003687:Dehydroepiandrosterone; D003094:Collagen; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1