The Relation Between Sox9, TGF-beta1, and Proteoglycan in Human Intervertebral Disc Cells.
OBJECTIVE
The aim of this study is to elucidate the effects of transforming growth factor-beta (TGF-beta)1 and L-ascorbic acid on proteoglycan synthesis, and the relationship between Sox9, proteoglycan, and TGF-beta1 in intervertebral disc cells.


METHODS
Human intervertebral disc tissue was sequentially digested to 0.2% pronase and 0.025% collagenase in DMEM/F-12 media and extracted cells were cultured in 37, 5% CO(2) incubator. When intervertebral disc cells were cultured with TGF-beta1 or L-ascorbic acid, the production level of sulfated glycosaminoglycan (sGAG) was estimated by dimethyl methyleneblue (DMMB) assay. The changes of Sox9 mRNA and protein levels via TGF-beta1 were detected by RT-PCR and Western blot analysis in each.


RESULTS
The amount of sGAG was increased with the lapse of time during incubation, and sGAG content of pellet cultured cells was much larger than monolayer culture. When primary cultured intervertebral disc cells in monolayer and pellet cultures were treated by TGF-beta1 20 ng, sGAG content of experimental group was increased significantly compared to control group in both cultures. L-Ascorbic acid of serial concentrations (50-300 ug/ml) increased sGAG content of mono layer cultured intervertebral disc cells significantly in statistics. The co-treatment of TGF-beta1 and L-ascorbic acid increased more sGAG production than respective treatment. After treating with TGF-beta1, Sox9 mRNA and protein expression rates were significantly increased in disc cells compared with the control group.


CONCLUSIONS
This study suggests that TGF-beta1 would increase sulfated glycosaminoglycan (sGAG) and other proteoglycans such as versican by elevating Sox9 mRNA and protein expressions in order.
Lee
Yong-Jik
YJ
Division of Clinical Research , Seoul Medical Center Research Institute, Seoul, Korea.
Kong
Min-Ho
MH
Song
Kwan-Young
KY
Lee
Kye-Heui
KH
Heo
Su-Hak
SH