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Inhibitory effect of 14-3-3η on chondrogenesis of ATDC5 cell

초록/요약

Chondrogenesis in growth plate is regulated by the complex interaction of molecular signals. This complex is often dysregulated during chronic inflammatory disease, thus resulting in impaired growth plate chondrogenesis. It is known that chondrogenesis was inhibited by proinflammatory cytokine, markedly elevated in joint inflammatory disease. A recent study reported overexpression of 14-3-3η, one of the 14-3-3 families, in synovial fluid from patients with joint inflammation, often associated with growth failure. Therefore, we investigated the role of 14-3-3η in chondrogenesis using ATDC5, a clonal murine chondrogenic cell line, proper to study about ossification at growth plates. At first, we investigated that chondrogenic differentiation markers were expressed and cartilage nodule was formed in ATDC5 cell line, the endochondral model. We found that 14-3-3η was overexpressed in inhibition of chondrogenesis by proinflammatory cytokines. Furthermore, chondrogenesis was inhibited by overexpression of 14-3-3η without proinflammatory cytokines. Expression of Sox9, an essential transcription factor for chondrogenesis, was decreased by overexpression of 14-3-3η. And also, the formation of cartilage nodule was decreased in 14-3-3η-overexpressed ATDC5 cells. However, chondrogenesis of ATDC5 cells, knockdown the 14-3-3η using specific siRNA of 14-3-3η were stimulated,and formation of cartilage nodule was more increased. And overexpression of 14-3-3η inhibited G1 phase arrest that is important to chondrogenesis. Also, expression of p21cip was decreased in 14-3-3η overexpression cells. In this study indicated that 14-3-3η inhibits the chondrogenesis and, 14-3-3η is expected as a key regulator in impairment of growth from children with joint inflammatory disease.

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CONTENTS
CONTENTS -------------------------------------------------- i
LIST OF TABLES ----------------------------------------- iii
LIST OF FIGURES --------------------------------------- iv
ABSTRACT ------------------------------------------------- v
1. INTRODUCTION --------------------------------------- 1
2. MATERIALS AND METHODS ---------------------- 4
2.1. Materials ------------------------------------------------------------------- 4
2.2. Cell culture ---------------------------------------------------------------- 4
2.3. Reverse transcription PCR & Quantitative real-time PCR ----- 5
2.4. Western blot analysis --------------------------------------------- 6
2.5. Immunoprecitation analysis------------------------------------- 7
2.6. Flow cytometry ---------------------------------------------------- 7
2.7. Alcian blue staining ----------------------------------------------- 8
2.8. siRNA Experiments ----------------------------------------------- 8
3. RESULTS ------------------------------------------------ 10
3.1. Demonstration of the ATDC5 cells as a chondrogenic model -- 10
3.2. Formation of cartilage nodules in the presence of insulin ------ 13
3.3. 14-3-3η expression is increased by TNF-α in chondrogenesis. - 15
3.4. The chondrogenesis is inhibited by 14-3-3η overexpression in ATDC5. ------------------------------------------------------------------------- 17
3.5. The silencing of the 14-3-3η promotes chondrogenic differentiation in ATDC5. -------------------------------------------------- 20
3.6. Increased 14-3-3η inhibits cell cycle arrest of G1 phase induced by Insulin. --------------------------------------------------------------------- 23
3.7. The 14-3-3η interacts with Sox9 in differentiation of ATDC5 cells. ----------------------------------------------------------------------------- 27
DISCUSSION ---------------------------------------------- 29
REFERENCES -------------------------------------------- 32
ABSTRACT IN KORAEN ------------------------------ 39

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