Smad3调控大鼠髓核细胞中β-1,3-葡糖醛酸基转移酶1的表达：表达失调在椎间盘疾病中的意义
Smad3 controls β-1,3-glucuronosyltransferase 1 expression in rat nucleus pulposus cells: implications of dysregulated expression in disc disease.
作者信息
Wu Qianghua, Wang Jianru, Skubutyte Renata, Kepler Christopher K, Huang Zonggui, Anderson D Greg, Shapiro Irving M, Risbud Makarand V
机构信息
Thomas Jefferson University, Philadelphia, Pennsylvania, PA 19107, USA.
出版信息
Arthritis Rheum. 2012 Oct;64(10):3324-33. doi: 10.1002/art.34570.
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.
目的
研究Smad3对髓核（NP）细胞中β-1,3-葡糖醛酸基转移酶1（GlcAT-1，糖胺聚糖（GAG）合成的重要调节因子）表达的调控作用。
方法
检测转化生长因子β（TGFβ）处理的大鼠NP细胞和纤维环（AF）细胞中GlcAT-1的表达。在大鼠NP细胞中检测Smad信号和Smad抑制对GlcAT-1的影响。分析Smad3基因敲除小鼠椎间盘和退变人NP组织中GlcAT-1的表达。
结果
TGFβ增加大鼠NP细胞中GlcAT-1的表达，但不增加大鼠AF细胞中GlcAT-1的表达。显性负性ALK-5（DN-ALK-5）明显抑制GlcAT-1启动子活性。与Smad3共转染可强烈诱导启动子活性，且不依赖TGFβ。生物信息学分析表明启动子中存在多个Smad结合位点；缺失分析表明-274至-123 bp之间的区域是Smad3应答所必需的。DN-Smad3、Smad 3小干扰RNA和Smad7强烈抑制基础及TGFβ诱导的启动子活性。Smad3诱导的启动子活性被DN-Smad3显著阻断；Smad7的作用非常小。用短发夹RNA Smad3慢病毒转导NP细胞导致GlcAT-1表达降低和GAG积累。与野生型小鼠相比，Smad3基因敲除小鼠椎间盘内GlcAT-1的表达明显降低。退变人NP组织标本分析显示GlcAT-1与TGFβ表达之间无正相关。此外，从退变人组织中分离的细胞在TGFβ处理后未显示GlcAT-1表达的诱导，提示反应改变。
结论
我们的研究结果表明，在健康的NP细胞中，TGFβ-Smad3轴作为GlcAT-1表达的调节因子。然而，椎间盘退变过程中对TGFβ的反应性改变可能会损害GAG合成。
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