Smad3 controls β-1,3-glucuronosyltransferase 1 expression in rat nucleus pulposus cells: implications of dysregulated expression in disc disease.
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.


CONCLUSIONS
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.
Thomas Jefferson University, Philadelphia, Pennsylvania, PA 19107, USA.
Qianghua
Q
Wu
Jianru
J
Wang
Renata
R
Skubutyte
Christopher K
CK
Kepler
Zonggui
Z
Huang
D Greg
DG
Anderson
Irving M
IM
Shapiro
Makarand V
MV
Risbud
D000818:Animals; D015870:Gene Expression / Q000187:drug effects; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007403:Intervertebral Disc / Q000187:drug effects / Q000378:metabolism*; D055959:Intervertebral Disc Degeneration / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000187:drug effects; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; C067806:glucuronyltransferase GlcAT-1; D014453:Glucuronosyltransferase