J Korean Neurosurg Soc.  2008 Mar;43(3):149-154. 10.3340/jkns.2008.43.3.149.

The Relation Between Sox9, TGF-beta1, and Proteoglycan in Human Intervertebral Disc Cells

• Lee YJ
• Kong MH
• Song KY
• Lee KH
• Heo SH

• KMID: 2067322
• DOI: http://doi.org/10.3340/jkns.2008.43.3.149

Abstract

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 37degrees C, 5% CO2 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.
CONCLUSION
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.

Keyword

Intervertebral disc; Proteoglycan; TGF-beta1; Sox9

MeSH Terms

Ascorbic Acid
Blotting, Western
Cells, Cultured
Collagenases
Glycosaminoglycans
Humans
Incubators
Intervertebral Disc
Pronase
Proteoglycans
RNA, Messenger
Transforming Growth Factor beta1
Versicans
Ascorbic Acid
Collagenases
Glycosaminoglycans
Pronase
Proteoglycans
RNA, Messenger
Transforming Growth Factor beta1
Versicans