Abstract
The goal of this investigation was to study the expression and regulation of beta1,3-Glucuronosyltransferase-I (GlcAT-I), a key enzyme regulating GAG synthesis in cells of the intervertebral disc. There was a robust expression of GlcAT-I in the nucleus pulposus in vivo. Treatment with the calcium ionophore ionomycin resulted in increased GlcAT-I expression, whereas GlcAT-I promoter constructs lacking TonE site or a mutant TonE were unresponsive to the ionophore. Experiments using TonEBP and DN-TonEBP constructs showed that TonEBP positively regulated GlcAT-I promoter activity. ChIP analysis confirmed binding of TonEBP to the promoter. We further validated the role of TonEBP in controlling GlcAT-I expression using mouse embryo fibroblasts from TonEBP null mice. GlcAT-I promoter activity in null cells was significantly lower than the wild type cells. In contrast to wild type cells, treatment with ionomycin failed to increase GlcAT-I promoter activity in null cells. We then investigated if calcineurin (Cn)-NFAT signaling played a regulatory role in GlcAT-I expression. Inhibition of Cn following ionomycin treatment did not block GlcAT-I and tauT, a TonEBP-responsive reporter activity. GlcAT-I promoter activity was suppressed by co-expression of Cn, NFAT2, NFAT3, and NFAT4. Moreover, following ionomycin treatment, fibroblasts from CnAalpha and CnAbeta null mice exhibited robust induction in GlcAT-I promoter activity compared with wild type cells. Results of these studies demonstrate that calcium regulates GlcAT-I expression in cells of the nucleus pulposus through a signaling network comprising both activator and suppressor molecules. The results suggest that by controlling both GAG and aggrecan synthesis, disc cells can autoregulate their osmotic environment and accommodate mechanical loading.
Highlights
The intervertebral disc is a specialized structure that permits rotation as well as flexure and extension of the human spine
We showed that nucleus pulposus cells responded to changes in osmotic pressure by up-regulating the transcription factor, TonEBP [4]
It is noteworthy that other workers have shown that galactose-1,3-glucuronysltransferase- 1 (GlcAT-I) activity was required for GAG chain synthesis [5]; there is the possibility that this enzyme serves as the rate-limiting step in GAG synthesis for chondrocytes and possibly other cell types [6, 7]
Summary
Plasmids and Reagents—Rabbit polyclonal TonEBP antibody was a kind gift from Dr H. Plasmids were kindly provided by Dr. Takashi Ito (Osaka University, Japan) (tauT (taurine transporter) wild type (WT) and mutant (MT). NFAT2 (CA-NFAT2) (catalog strated that GAG synthesis was inhibited by calcium blocking number 11102) with key serine residues changed to alanine to agents [12]. Calcium was shown to control GlcAT-I number 17870) developed by Dr Gerald Crabtree [18] were expression through Sp1 transcription factor [13]. After thoroughly washing the sections, the bound primary antibody was incubated with biotinylated universal secondary antibody, at a dilution of 1:20 (Vector Laboratories) for 10 min at room temperature. Sections were incubated with a streptavidin-peroxidase complex for 5 min and washed with PBS, and color was developed using 3Ј,3-
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