During development cartilage originates as condensations of primordial mesenchymal cells which go through a strict program whereby these cells differentiate into chondrocytes, undergo hypertrophy, and are subsequently replaced by osteoblasts. A zebrafish mutant termed gonzo has been isolated which displays a phenotype similar to human chondrodysplasias. Recently it was shown that this phenotype is due to a mutation in the zebrafish equivalent of a mammalian serine protease termed SKI-1 or Site-1 protease (S-1P). This enzyme is a member of the prohormone convertase family of subtilisin-like proteases which are known to play a critical role in controlling the processing of sterol regulatory element binding proteins and in the Unfolded Protein Response (UPR). The goal of this research project is to explore the expression and the function of S-1P in chondrocyte differentiation in vitro. / In this study, the chondrogenic mouse cell line ATDC5 which recapitulates the steps of cartilage development when cultured in the presence of insulin was used. Result showed that 10 mug/mL of insulin is the optimum concentration to be used to induce chondrogenesis in ATDC5 cells. This was shown by Alcian Blue staining and glycosaminoglycan quantification, where cells induced by 10 mug/mL of insulin produced highest amount of glycosaminoglycan. Using RT-PCR, it was shown that S-1P is constitutively expressed in ATDC5 cells both induced and non-induced with insulin. S-1P was also shown to be expressed in ATDC5 cells induced by ascorbic acid and a combination of ascorbic acid and insulin. Using Real Time-PCR, the expression levels of S-1P in the presence of insulin, ascorbic acid and the combination of both differentiation inducers was determined. The expression levels of type I collagen (early-phase chondrocyte differentiation expressing gene), type II collagen and aggrecan (mature chondrocyte matrix genes) and type X collagen (hypertrophic chondrocyte matrix gene) were also analyzed. / The effect of the inhibition of S-1P on the expression levels of cartilage related components was studied by inhibiting S-1P using an R134E prosegment mutant (pro-SKI-1), which has been shown a potent cellular inhibitor of S-1P. The DNA of the pro SKI-1 variant was subcloned into pIRES-EGFP (provided by Dr. Nabil Seidah) and transfected into ATDC 5 cells. Stably transfected cells were selected and the expression of S-1P and other cartilage components were quantitatively evaluated by studying the transfected cells using Real Time-PCR. Functional inhibition of S-1P was confirmed using an artificial substrate reporter construct adapted for specific cleavage by this protease. / Overall the results showed that inhibition of S-1P activity had drastic effects in chondrogenesis---the expression of cartilage components (Aggrecan, Collagen I, Collagen II and Collagen X) were significantly lowered. Inhibition of S-1P seemed to prevent the chondrogenesis process in ATDC 5 cells.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.101140 |
| Date | January 2006 |
| Creators | Ho, Yuen Yee, 1979- |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Division of Surgical Research.) |
| Rights | © Yuen Yee Ho, 2006 |
| Relation | alephsysno: 002587426, proquestno: AAIMR32719, Theses scanned by UMI/ProQuest. |
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