Neuroleukin Promotes Proliferation And Matrix Synthesis Of Articular Chondrocytes During In Vitro Culture

Due to the low proliferative and migrative capacities of chondrocytes, cartilage repair remains one of the most challenging clinical problems. Therapeutic strategies for cartilage repair are far from optimal due to dedifferentiation of chondrocytes during in vitro expansion. Neuroleukin(NLK) is a multifunctional protein that stimulates cell growth and migration, together with its receptorautocrine motility factor receptor(AMFR). We investigated expression of NLK and AMFR during cartilage development and in cultured articular chondrocytes in vitro, and found the pair associates with chondrocyte proliferation and differentiation. As we investigated the implication of NLK in chondrocyte proliferation in vitro we discussed its potential to act as an exogenous growth factor during ACI. Furthermore, we optimized concentration of NLK for in vitro chondrocyte cultures with microfluidic devices and we report a concentration of 12.85 ng/ml as an optimal condition to promote chondrocyte proliferation for NLK. While applied to isolated articular chondrocytes, NLK promotes cell secretion of type II collagen, a marker of proliferating chondrocytes. Further work demonstrates that NLK up regulates p AKT and p Smad2/3, but down regulates p Smad1/5. These findings suggest that NLK can promote cell proliferation, type II collagen synthesis during in vitro chondrocyte propagation, and thus can serve as an exogenous factor for ACI. NLK is therefore a candidate factor that can be applied in treatment of cartilage defects.Experiment 1: Expression of Neuroleukin and its receptor AMFR/gp78 in articular chondrocytesObjective: The objective of this part was to find out whether Neuroleukin and its receptor AMFR/gp78 exist in articular chondrocyte as well as variations of expression between different ages.Method: We took primary chondrocytes from 4 weeks old rats, after primary culture we used P1 chondrocytes as mentioned before. Western blot was made for detection of Neuroleukin and AMFR/gp78 in chondrocytes, murine melanoma cell line(B16-F1), human fibrosarcoma cell line(HT1080), human umbilical vein endothelial cells(HUVEC) and murine fibroblast cell line(NIH3T3) were used as positive control. Immunohistochemical and immunofluorescent analysis was performed for localization of AMFR/gp78 in articular chondrocytes.Result: Based on results in western blotting we found a physiologically expression of Neuroleukin in articular chondrocytes, but higher expression was noticed in tumor cell lines which consistent with previous studies. Moreover, a secretion of Neuroleukin in chondrocytes was also found by western blotting. By analyze of rats form 1-4weeks during development period of articular cartilage, the expression of NLK and AMFR/gp78 was found to be increased, however, after the animal matured, form 4-12 months we noticed a decreased expression of NLK and AMFR/gp78.Conclusion: Neuroleukin is a physiological protein exist in and also secreted by articular chondrocytes, expression level of NLK and AMFR/gp78 elevated during cartilage development and bone formation will suppressed after mature.Experiment 2:Research on proliferation effect of Neuroleukin in articular chondrocytes and related signaling pathwaysObjective: The objective of this part was to evaluate the proliferative effect of Neuroleukin on articular chondrocytes and to investigate the related signaling pathways.Method: P1 chondrocytes were used from 4 weeks rats as mentioned before. In this part of our research, we develop a novel integrated microfluidic perfusion system to generate multiple concentrations of Neuroleukin by a concentration gradient generator CGG. By using this device we evaluate the proliferative effect of NLK on chondrocytes, furthermore,we detected an optimal concentration during a 6 days culture on the microfluidic device. In the end of culture we recorded the morphology of chondrocytes with or without NLK stimulation. Moreover, phosphorylation of Akt / and ERK was also tested as well as cell cycle distribution.Result: After the 6 days culture under condition of different concentrations of NLK, our result showed that NLK can promote proliferation of articular condrocytes by an optimal concentration of 12.85ng/ml, cell cycle analysis showed a increased proportion of S phase and G2 M phase cells after NLK stimulation. NLK also upregulated phosphorylate level of Akt and ERK to affect downstream Cyclins to perform proliferation effect.Conclusion: Neuroleukin promotes proliferation of chondrocytes at an optimal concentration of 12.85ng/ml.Experiment 3: Neuroleukin stimulates synthesis of type II collagen in articular chondrocytesObjective:The objective of this part was to find out whether Neuroleukin affect synthesis of type II collagen in articular chondrocytes.Method: We took primary chondrocytes from 4 weeks old rats, after primary culture we used P1 chondrocytes as mentioned before. In this part we performed realtime PCR to test m RNA expression of type II collagen and GAGs after NLK stimulation, which are specific matrix of articular chondrocytes. For collagen II synthesis detection during P0-P1 chondrocytes, western blot and immunofluorescent analysis was also performed. Furthermore, we investigated phosphorylation level of Smad2/3, Smad1/5/8 under condition of 12.85ng/ml and 25ng/ml NLK by western blotting.Result:Based on results in realtime PCR we found m RNA expression of type II collagen was evaluated by 48 hours Neuroleukin stimulation in articular chondrocytes, proportions of positive stain of collagen II cells in P0-P2 was found increased in NLK groups compare to control. Results from western blot showed that NLK stimulates phosphorylation of Smad2/3 but suppress p-Smad1/5/8 expression, which was also confirmed by immunofluorescent analysis.Conclusion: Neuroleukin stimulates synthesis of type II collagen in articular chondrocytes during in vitro culture.