Knockdown of asporin affects transforming growth factor-β1-induced matrix synthesis in human intervertebral annulus cells.
Jiang X1, Wu CA2, Wang Y2, Shi KJ2, Jiang XZ1, Zheng S1, Tian W1.
1Department of Orthopaedics, Beijing Jishuitan Hospital, 4 Clinical Medical College of Peking University, Beijing, China.2Department of Molecular Orthopaedics, Beijing Institute of Traumatology and Orthopaedics, Beijing, China.
Asporin is associated with osteoarthritis and lumbar disk degeneration. Previous studies in chondrocytes showed that asporin can bind to transforming growth factor-β1 (TGF-β1) and downregulate matrix biosynthesis. However, this has not been studied in intervertebral disk (IVD) cells. This study aimed to inspect the expression of asporin under TGF-β1 stimulation and its effect on TGF-β1-induced matrix biosynthesis in human intervertebral annulus cells.
Human intervertebral annulus cells were obtained from the pathological region of IVD in eight patients. After primary culture and redifferentiation in alginate beads, cells were reseeded and treated with different concentrations (5 ng/mL, 10 ng/mL, and 15 ng/mL) of TGF-β1 for up to 24 hours. Total RNA extracted from the cells and those with asporin knockdown were subjected to real-time polymerase chain reaction analysis to examine the expression of asporin and extracellular matrix genes.
TGF-β1 stimulation induces asporin transcription significantly in a dose- and time-dependent manner. Knockdown of endogenous asporin leads to the upregulated expression of collagen II alpha 1 and aggrecan.
Our results have verified a functional feedback loop between TGF-β1 and asporin in human intervertebral annulus cells indicating that TGF-β1-induced annulus matrix biosynthesis can be significantly upregulated by knockdown of asporin. Therefore, asporin could be a potential new therapeutic target and inhibition of asporin could be adopted to enhance the anabolic effect of TGF-β1 in human intervertebral annulus cells in degenerative IVD diseases.
TGF-β1; aggrecan; asporin; collagen II alpha 1; human intervertebral annulus cells