Knockdown of asporin affects transforming growth factor-ß1-induced matrix synthesis in human intervertebral annulus cells.

BACKGROUND/OBJECTIVE: Asporin is associated with osteoarthritis and lumbar disk degeneration. Previous studies in chondrocytes showed that asporin can bind to transforming growth factor-ß1 (TGF-ß1) 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-ß1 stimulation and its effect on TGF-ß1-induced matrix biosynthesis in human intervertebral annulus cells. METHODS: 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-ß1 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. RESULTS: TGF-ß1 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. CONCLUSION: Our results have verified a functional feedback loop between TGF-ß1 and asporin in human intervertebral annulus cells indicating that TGF-ß1-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-ß1 in human intervertebral annulus cells in degenerative IVD diseases.

Thy28 partially prevents apoptosis induction following engagement of membrane immunoglobulin in WEHI-231 B lymphoma cells.

Thy28 protein is conserved among plants, bacteria, and mammalian cells. Nuclear Thy28 protein is substantially expressed in testis, liver, and immune cells such as lymphocytes. Lymphocyte apoptosis plays a crucial role in homeostasis and formation of a diverse lymphocyte repertoire. In this study, we examined whether Thy28 affects induction of apoptosis in WEHI-231 B lymphoma cells following engagement of membrane immunoglobulin (mIg). Once they were established, the Thy28-overexpressing WEHI-231 cells showed similar expression levels of IgM and class I major histocompatibility complex (MHC) molecule compared with controls. The Thy28-overexpressing cells were considerably resistant to loss of mitochondrial membrane potential (ΔΨm), caspase-3 activation, and increase in annexin-positive cells upon mIg engagement. These changes were concomitant with an increase in G1 phase associated with upregulation of p27(Kip1). The anti-IgM-induced sustained activation of c-Jun N-terminal kinase (JNK), which was associated with late-phase hydrogen peroxide (H(2)O(2)) production, was partially reduced in the Thy28-expressing cells relative to controls. Taken together, the data suggest that in WEHI-231 B lymphoma cells, Thy28 regulates mIg-mediated apoptotic events through the JNK-H(2)O(2) activation pathway, concomitant with an accumulation of cells in G1 phase associated with upregulation of p27(Kip1) in WEHI-231 B lymphoma cells.