Abstract
Introduction
Asporin, a member of the small leucine rich proteins, is an extracellular matrix protein with elevated expression level in cartilage tissues with OA and degenerative disc disease. Aspartate(D) repeat polymorphism (from 7-21) can be found in human and D14 (14 aspartate repeat) has been identified as the risk factor for both OA and DDD, while D13 (13 aspartate repeat) is a common allele. It has been shown that asporin negatively regulates Tgf-β signaling in vitro by ATDC5 cell culture as well as contributes to collagen fibrillogensis. However, the in vivo role of asporin in development and pathogenesis in cartilage tissues is still unclear. Thus, in vivo study using mice as a model was performed in this study.
Materials and Methods
In order to study the correlation of asporin and Tgf-β signaling, immunostaining of asporin and P-Smad2/3 was performed on mice disc samples induced with injuries by looping or puncture. To further understand the cause and consequence relationship between asporin and Tgf-β/Smad signaling and the impact of overexpressing asporin in the disc, transgenic mice overexpressed with human asporin D13 and D14 were generated with a cartilage specific promoter Col11a2 using LacZ as reporter. Histological and molecular analysis were performed to study the effect of overexpressing asporin in the cartilage tissues.
Results
Immunohistochemical results showed that asporin and P-Smad2/3 are colocalized in the disc and articular cartilages. Moreover, asporin and P-Smad2/3 also present in the degenerative models including natural degeneration, disc puncture and tail looping. These results suggested the possible correlation between asporin and Tgf-β/Smad signaling as well as the contribution of asporin and Tgf-β/Smad in the pathologenesis of disc degeneration.
Interestingly, in transgenic mice overexpressing asporin D13 and D14, overexpression of asporin induced glycosaminoglycans production in the transgenic nucleus pulposus, small cell clusters, and sparsely distributed cells with morphological differences comparing to clumps of vacuolated mouse nucleus pulposus cells in wildtype, indicated that overexpressing of asporin enhances differentiation or maturation process of notochordal-like cells in mouse nucleus pulposus toward the nucleus pulposus cells (NPCs) that is more indicative of human nucleus pulposus.
In situ hybridization showed that the differentiated cell showed increased Col2a1 and Agc expression, which indicated characteristics of NPCs. Marker analysis further showed that characteristics of chondrocyte-like cells (CLCs) with the molecular signature of Sox9+T- and CK18- cells exhibited in the nucleus pulposus of D13 transgenic mice. However, fibrosis and hypertrophic differentiation, which are commonly observed in human disc degeneration, were absent in the D13 transgenic. Intriguingly, phosphorylation of Smad2/3 was found to be up-regulated in the transgene-expressing cells, suggesting that overexpression of Asporin promotes Tgf-β signaling.
Conclusion
It is proposed that Asporin, as a risk factor, enhances cellular differentiation of cell types that can lead to degenerative progression possibly though promoting Tgf-β signaling. Further study of Tgf-β related pathway are need to be investigated in order to gain more insight in the involvement of Tgf-β in causing disc degeneration.
None declared