Expression and regulation of the osteoarthritis-associated protein asporin

Asporin (ASPN) is a small leucine-rich proteoglycan that is involved in pathological processes of osteoarthritis. Previously, we showed that asporin can inhibit transforming growth factor-beta1 (TGF-beta1)-mediated expression of cartilage matrix genes and chondrogenesis in vitro (Kizawa, H., Kou, I., Iida, A., Sudo, A., Miyamoto, Y., Fukuda, A., Mabuchi, A., Kotani, A., Kawakami, A., Yamamoto, S., Uchida, A., Nakamura, K., Notoya, K., Nakamura, Y., and Ikegawa, S. (2005) Nat. Genet. 37, 138-144). However, details about regulation of asporin itself are not yet known. Here, we examined ASPN expression in skeletal tissue and potential regulation of ASPN by TGF-beta. In situ hybridization revealed the presence of ASPN mRNA in the perichondrium/periosteum of long bones, but its absence in articular cartilage and growth plates. Immunohistochemical analysis also showed ASPN protein expression predominantly in the perichondrium/periosteum. TGF-beta1 induced endogenous ASPN mRNA expression over time in vitro, and this induction was suppressed by the TGF-beta type I receptor kinase inhibitor SB431542. Inhibition of Smad3 significantly reduced TGF-beta1-induced ASPN expression, whereas overexpression of Smad3 augmented the induction. Characterization of the human ASPN promoter region revealed a region from -126 to -82 that is sufficient for full promoter activity; however, TGF-beta1 failed to increase activity through the ASPN promoter. Our findings indicate that TGF-beta1 induces ASPN through Smad3 but that this induction is indirect.     

Mechanisms for asporin function and regulation in articular cartilage

Osteoarthritis (OA), the most prevalent form of skeletal disease, represents a leading cause of disability following middle age. OA is characterized by the loss of articular cartilage; however, the details of its etiology and pathogenesis remain unclear. Recently, we demonstrated a genetic association between the cartilage extracellular matrix protein asporin and OA (Kizawa, H., Kou, I., Iida, A., Sudo, A., Miyamoto, Y., Fukuda, A., Mabuchi, A., Kotani, A., Kawakami, A., Yamamoto, S., Uchida, A., Nakamura, K., Notoya, K., Nakamura, Y., and Ikegawa, S. (2005) Nat. Genet. 37, 138-144). Furthermore, we showed that asporin binds to transforming growth factor-beta (TGF-beta), a key cytokine in OA pathogenesis, and inhibits TGF-beta-induced chondrogenesis. To date, functional data for asporin have come primarily from mouse cell culture models of developing cartilage rather than from human articular cartilage cells, in which OA occurs. Here, we describe mechanisms for asporin function and regulation in human articular cartilage. Asporin blocks chondrogenesis and inhibits TGF-beta1-induced expression of matrix genes and the resulting chondrocyte phenotypes. Small interfering RNA-mediated knockdown of asporin increases the expression of cartilage marker genes and TGF-beta1; in turn, TGF-beta1 stimulates asporin expression in articular cartilage cells, suggesting that asporin and TGF-beta1 form a regulatory feedback loop. Asporin inhibits TGF-beta/Smad signaling upstream of TGF-beta type I receptor activation in vivo by co-localizing with TGF-beta1 on the cell surface and blocking its interaction with the TGF-beta type II receptor. Our results provide a basis for elucidating the role of asporin in the molecular pathogenesis of OA.     

UDP-glucose dehydrogenase modulates proteoglycan synthesis in articular chondrocytes: its possible involvement and regulation in osteoarthritis

Introduction

The objective of this study was to investigate the possible role of UDP-glucose dehydrogenase (UGDH) in osteoarthritis (OA) and uncover whether, furthermore how interleukin-1beta (IL-1β) affects UGDH gene expression.

Methods

UGDH specific siRNAs were applied to determine the role of UGDH in proteoglycan (PG) synthesis in human articular chondrocytes. Protein levels of UGDH and Sp1 in human and rat OA cartilage were detected. Then, human primary chondrocytes were treated with IL-1β to find out whether and how IL-1β could regulate the gene expression of UGDH and its trans-regulators, that is Sp1, Sp3 and c-Krox. Finally, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and stress-activated protein kinase/c-Jun N-terminal kinase (SAP/JNK) inhibitor SP600125 were used to pick out the pathway that mediated the IL-1β-modulated PGs synthesis and gene expression of UGDH, Sp1, Sp3 and c-Krox.

Results

UGDH specific siRNAs markedly inhibited UGDH mRNA and protein expression, and thus led to an obvious suppression of PGs synthesis in human articular chondrocytes. UGDH protein level in human and rat OA cartilage were much lower than the corresponding controls and negatively correlated to the degree of OA. Decrease in Sp1 protein level was also observed in human and rat OA cartilage respectively. Meanwhile, IL-1β suppressed UGDH gene expression in human articular chondrocytes in the late phase, which also modulated gene expression of Sp1, Sp3 and c-Krox and increased both Sp3/Sp1 and c-Krox/Sp1 ratio. Moreover, the inhibition of SAP/JNK and p38 MAPK pathways both resulted in an obvious attenuation of the IL-1β-induced suppression on the UGDH gene expression.

Conclusions

UGDH is essential in the PGs synthesis of articular chondrocytes, while the suppressed expression of UGDH might probably be involved in advanced OA, partly due to the modulation of p38 MAPK and SAP/JNK pathways and its trans-regulators by IL-1β.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0484-2) contains supplementary material, which is available to authorized users.     

IL-1beta, but not BMP-7 leads to a dramatic change in the gene expression pattern of human adult articular chondrocytes--portraying the gene expression pattern in two donors

Anabolic and catabolic cytokines and growth factors such as BMP-7 and IL-1beta play a central role in controlling the balance between degradation and repair of normal and (osteo)arthritic articular cartilage matrix. In this report, we investigated the response of articular chondrocytes to these factors IL-1beta and BMP-7 in terms of changes in gene expression levels. Large scale analysis was performed on primary human adult articular chondrocytes isolated from two human, independent donors cultured in alginate beads (non-stimulated and stimulated with IL-1beta and BMP-7 for 48 h) using Affymetrix gene chips (oligo-arrays). Biostatistical and bioinformatic evaluation of gene expression pattern was performed using the Resolver software (Rosetta). Part of the results were confirmed using real-time PCR. IL-1beta modulated significantly 909 out of 3459 genes detectable, whereas BMP-7 influenced only 36 out of 3440. BMP-7 induced mainly anabolic activation of chondrocytes including classical target genes such as collagen type II and aggrecan, while IL-1beta, both, significantly modulated the gene expression levels of numerous genes; namely, IL-1beta down-regulated the expression of anabolic genes and induced catabolic genes and mediators. Our data indicate that BMP-7 has only a limited effect on differentiated cells, whereas IL-1beta causes a dramatic change in gene expression pattern, i.e. induced or repressed much more genes. This presumably reflects the fact that BMP-7 signaling is effected via one pathway only (i.e. Smad-pathway) whereas IL-1beta is able to signal via a broad variety of intracellular signaling cascades involving the JNK, p38, NFkB and Erk pathways and even influencing BMP signaling.     

The protective effects of grape seed procyanidin B2 against asporin mediates glycated low‐density lipoprotein induced‐cardiomyocyte apoptosis and fibrosis

The progression of diabetic cardiomyopathy is related to cardiomyocyte dysfunction and apoptosis. Our previous studies showed that asporin (ASPN) was significantly increased in the myocardium of db/db mice through proteomics, and grape seed procyanidin B2 (GSPB2) significantly inhibited the expression of ASPN in the heart of db/db mice. We report here that ASPN played a critical role in glycated low‐density lipoproteins (gly‐LDL) induced‐cardiomyocyte apoptosis. We found that gly‐LDL upregulated ASPN expression. ASPN increased H9C2 cardiomyocyte apoptosis with down‐regulation of Bcl‐2, upregulation of transforming growth factor‐β1, Bax, collagen III, fibronectin, and phosphorylation of smad2 and smad3. However, GSPB2 treatment reversed ASPN‐induced impairments in H9C2 cardiomyocytes. These results provide evidence for the cardioprotective action of GSPB2 against ASPN injury, and thus suggest a new target for fighting against diabetic cardiomyopathy.