Open AccessSmad3 controls β‐1,3‐glucuronosyltransferase 1 expression in rat nucleus pulposus cells: Implications of dysregulated expression in disc disease
Author(s) -
Wu Qianghua,
Wang Jianru,
Skubutyte Renata,
Kepler Christopher K.,
Huang Zonggui,
Anderson D. Greg,
Shapiro Irving M.,
Risbud Makarand V.
Publication year - 2012
Publication title -
arthritis & rheumatism
Language(s) - English
Resource type - Journals
eISSN - 1529-0131
pISSN - 0004-3591
DOI - 10.1002/art.34570
Subject(s) - smad , transforming growth factor , chemistry , small hairpin rna , microbiology and biotechnology , downregulation and upregulation , glycosaminoglycan , small interfering rna , transfection , biology , rna , gene , biochemistry
Objective To study the regulation of expression of β‐1,3‐glucuronosyltransferase 1 (GlcAT‐1), an important regulator of glycosaminoglycan (GAG) synthesis, by Smad3 in nucleus pulposus (NP) cells. Methods GlcAT‐1 expression was examined in rat NP and anulus fibrosus (AF) cells treated with transforming growth factor β (TGFβ). The effects of Smad signaling and Smad suppression on GlcAT‐1 were examined in rat NP cells. GlcAT‐1 expression was analyzed in the discs of Smad3‐null mice and in degenerated human NP tissue. Results TGFβ increased the expression of GlcAT‐1 in rat NP but not rat AF cells. Suppression of GlcAT‐1 promoter activity was evident with dominant‐negative ALK‐5 (DN‐ALK‐5). Cotransfection with Smad3 strongly induced promoter activity independent of TGFβ. Bioinformatics analysis indicated the presence of several Smad binding sites in the promoter; deletion analysis showed that the region between −274 and −123 bp was required for Smad3 response. DN‐Smad3, Smad 3 small interfering RNA, and Smad7 strongly suppressed basal as well as TGFβ‐induced promoter activity. Induction of promoter activity by Smad3 was significantly blocked by DN‐Smad3; Smad7 had a very small effect. Lentiviral transduction of NP cells with short hairpin RNA Smad3 resulted in a decrease in GlcAT‐1 expression and accumulation of GAG. Compared to wild‐type mice, significantly lower expression of GlcAT‐1 was seen in the discs of Smad3‐null mice. Analysis of degenerated human NP tissue specimens showed no positive correlation between GlcAT‐1 and TGFβ expression. Moreover, isolated cells from degenerated human tissue showed a lack of induction of GlcAT‐1 expression following TGFβ treatment, suggesting an altered response. Conclusion Our findings demonstrate that in healthy NP cells, the TGFβ–Smad3 axis serves as a regulator of GlcAT‐1 expression. However, an altered responsiveness to TGFβ during disc degeneration may compromise GAG synthesis.