title: Activation of TonEBP by calcium controls {beta}1,3-glucuronosyltransferase-I expression, a key regulator of glycosaminoglycan synthesis in cells of the intervertebral disc.
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.
authors.affiliation: Department of Orthopedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Hiyama
authors.forename: Sachin
authors.initials: S
authors.lastname: Gajghate
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sakai
authors.forename: Joji
authors.initials: J
authors.lastname: Mochida
authors.forename: Irving M
authors.initials: IM
authors.lastname: Shapiro
authors.forename: Makarand V
authors.initials: MV
authors.lastname: Risbud
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism*; D007403:Intervertebral Disc / Q000378:metabolism*; D007476:Ionophores / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors / Q000378:metabolism*; D011817:Rabbits; D051381:Rats
chemical_list: D006025:Glycosaminoglycans; D007476:Ionophores; D050778:NFATC Transcription Factors; C111364:galactosylgalactoylxylosylprotein 3-beta-glucuronosyltransferase; D014453:Glucuronosyltransferase; D002118:Calcium