Mechanism of Proteoglycan Synthesis by Bovine and Human Link N

Abstract

Introduction Link N peptide is the N-terminal region of link protein which stabilizes the interaction between aggrecan and hyaluronan in proteoglycan aggregates. In vivo, during tissue turnover, link protein is subject to proteolytic degradation by stromelysin and gelatinase A and B generating Link N peptide. It has been shown that human Link N (DHLSDNYTLDHDRAIH) can act as a growth factor and stimulate the synthesis of proteoglycans and collagen by IVD cells in vitro and improve disc height and proteoglycan levels in vivo in a rabbit model of IVD degeneration.1-3 To date, there have been no reports on the effect of bovine Link N (DHHSDNYTVDHDRVIH) on disc cells. The specific aim of this study is to compare the effects of bovine Link N (BLN) and human Link N (HLN) on bovine IVD cells to determine whether substitution of residues, as occurs in the BLN sequence, can alter Link N function. Materials and Methods Coccygeal IVDs from healthy 20 to 24 months old steers were obtained from a local abattoir at 2 to 3 hours after slaughter. The IVDs were separated from their adjacent vertebral bodies, and the NP and AF cells were isolated from the nucleus pulposus (NP) and annulus fibrosus (AF) regions by sequential digestion with 0.2% pronase followed by 0.125% collagenase digestion as previously described.4 After isolation, the NP and AF cells were either immediately embedded in 1.2% alginate beads (350,000 cell/bead) for proteoglycan synthesis or were plated in six well plates for protein extraction. Proteoglycan synthesis After 7 days of stabilization in complete DMEM high glucose medium, the alginate beads were placed in 24 well plates at a density of 9 bead/well and were incubated for 18 days in media supplemented with 1 µg/mL of either HLN or BLN (CanPeptide, Montreal). Beads cultured in media alone for the same period of time were used as the control (CTL). The culture media with or without Link N was changed every three days for 18 days and the sulfated glycosaminoglycan (GAG, predominantly aggrecan) content of the media was analyzed using the 1,9-dimethylmethylene blue (DMMB) dye-binding assay. Canonical SMAD-Mediated Signaling AF and NP cells were expanded in culture medium (Dulbecco's Modified Eagle Medium high glucose supplemented with 10% fetal bovine serum) into six well plates (7.5 × 105 cells/well) until reaching 80 to 90% confluence. The cells were preincubated overnight in serum-free medium, then were incubated in 1μg/mL HLN or BLN for different time points up to 6 hours. Cells incubated in medium alone were use as the control (CTL). After 6 hours of incubation, AF and NP cells were then lysed and Western blots were used to measure protein expression using specific antibodies directed against P-Smad ?, P-Smad-2 (Cell Signaling Technology, Danvers, MA). The phosphorylation of Smad ? and Smad-2 were normalized to the corresponding Smad 1 and Smad 2 total proteins. All experiments were performed in triplicate and were repeated with three independent cultures. The effect of treatment and culture period as well as the significance of differences among the treatment groups (CTL, BLN, and HLN) at each time point was assessed by repeated measures one-way ANOVA followed by Tukey Multiple Comparison Test with a significance level of p < 0.05 Results Most of the GAG synthesis was detected in the culture media and there was minimal retention (approximately 1%) in the alginate beads. Both NP and AF cells incubated with Link N (BLN or HLN) had an increased rate of GAG release into the culture media with time. This increase was significantly higher for AF cells incubated with BLN or HLN compared with the AF control cells ( p < 0.001) after 9 days of incubation. On the contrary, the NP cells had a significant and consistent increase only for HLN. Western blot results revealed that HLN activates tSMAD ? in bovine AF cells within 5 minutes, while the activation with BLN occurred within 10 minutes, achieving maximum activation at 30 minutes (Fig. 1). For both Link N supplementations, SMAD ? levels in AF cells decreased to below the control levels after 6 hours. In NP cells, BLN and HLN supplementation significantly stimulated SMAD ? after 30 minutes and continued to increase with time. However, for both IVD cells, HLN appeared to be more effective at SMAD ? activation than BLN. In AF cells, incubation in either HLN or BLN seemed to induce a slightly increased SMAD 2 activation up to 3 hours. In contrast, no SMAD 2 activation was detected in NP cells incubated with Link N. Conclusion BLN is also capable of stimulating GAG release in vitro in bovine IVD cells by activation of SMAD ?. The rapid activation within 10 minutes of SMAD ? by BLN in AF cells may explain our finding that AF cells respond better than NP cells to BLN supplementation in promoting proteoglycan synthesis. Therefore, in principle, BLN supplementation could also be an option for treating disc degeneration. However, HLN at the concentration of 1 μg/mL is more effective at stimulating proteoglycan synthesis and can directly activate SMAD ? signaling (within 5 minutes) in the AF, which is the main source of proteoglycan synthesis with age and degeneration. Although, both peptides have features needed for any agent designed to stimulate disc repair, HLN supplementation could be a better option for treating disc degeneration during its early stages, while the AF is still intact. Disclosure of Interest None declared References Mwale F, Demers CN, Petit A, et al. A synthetic peptide of link protein stimulates the biosynthesis of collagens II, IX and proteoglycan by cells of the intervertebral disc. J Cell Biochem 2003;88(6):1202–1213 Mwale F, Masuda K, Pichika R, et al. The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration. Arthritis Res Ther 2011;13(4):R120 Petit A, Yao G, Rowas SA, et al. Effect of synthetic link N peptide on the expression of type I and type II collagens in human intervertebral disc cells. Tissue Eng Part A 2011;17(7-8):899–904 Mwale F, Ciobanu I, Giannitsios D, Roughley P, Steffen T, Antoniou J. Effect of oxygen levels on proteoglycan synthesis by intervertebral disc cells. Spine 2011;36(2):E131-E138