Study of the role of connective tissue growth factor (CTGF) in human osteoarthritis

結締組織生長因子（connective tissue growth factor; CTGF）; 也稱為CCN2是一種發炎介質，而且已被證實在受傷損壞區域或是發炎相關疾病中會大量的表現。在退化性關節炎（Osteoarthritis; OA）的疾病中偵測到結締組織生長因子會大量表現的情形。然而，在退化性關節炎滑液囊纖維細胞（OASFs）中結締組織生長因子和介白素-6（IL-6）之間的相關性大多是未知的。在本篇研究中，我們發現CTGF的表現量在退化性關節炎滑液囊纖維細胞明顯高於正常關節炎滑液囊纖維細胞。同時也發現到CTGF能刺激退化性關節炎滑液囊纖維細胞產生介白素-6表現。除此之外，我們也發現CTGF會透過整合素avb5 (avb5 integrin)，ASK1，p38/JNK，AP-1/NF-kB的訊息傳遞增加IL-6生成。當我們前處理此路徑的抑制劑後，CTGF所增加的IL-6生成會明顯被抑制。 此外，在退化性關節炎的發病機制中常常可以看到單核球細胞的移動和浸潤現象產生在滑液囊發炎區域，促進發炎反應並且加劇退化性關節炎的病症。在發炎反應產生時，單核球趨化蛋白-1（MCP-1/CCL2）是關鍵的趨化因子，其能夠幫助調節單核球細胞的移動和浸潤至發炎區域。我們進一步發現MCP-1的表現量在退化性關節炎滑液囊纖維細胞明顯高於正常關節炎滑液囊纖維細胞。我們同時也發現到CTGF能刺激退化性關節炎滑液囊纖維細胞增加MCP-1表現。除此之外，我們也發現CTGF會刺激退化性關節炎滑液囊纖維細胞透過整合素avb5 (avb5 integrin)，FAK, MEK, ERK, and NF-kB/AP-1的訊息傳遞增加MCP-1生成。當我們前處理此路徑的抑制劑後，CTGF所增加的MCP-1生成會明顯被抑制。最後，我們也發現抑制CTGF所增加的MCP-1表現後，單核球細胞移行能力也會被抑制。綜合以上的研究結果，在初代培養退化性關節炎滑液囊纖維細胞實驗模式，利用結締組織生長因子的作用所引發的發炎反應，探討對於退化性關節炎滑液囊纖維細胞訊號傳遞機制的了解，期望可以帶來更多的降低發炎反應的治療新策略。Connective tissue growth factor (CTGF; also known as CCN2) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. CTGF is abundantly expressed in osteoarthritis (OA). Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of CTGF, and expression was higher than in normal SFs. Our results suggest that CTGF increased IL-6 production in OASFs via the avb5 integrin, ASK1, p38/JNK, and AP-1/NF-kB signaling pathways. Reagents that inhibit specific components of this pathway diminished the ability of CTGF to effect IL-6 expression. Moreover, migration and infiltration of mononuclear cells to inflammatory sites are also playing important role during OA pathogenesis. Monocyte chemoattractant protein-1 (MCP-1/CCL2) is the key chemokine that regulates migration and infiltration of monocytes. Our results showed that MCP-1 was highly expressed in OASFs as compared to normal SFs. Directly apply OASFs with CTGF increased MCP-1 expression by concentration and time-dependent manner. Our results indicated that CTGF enhances the migration of monocyte cells by increasing MCP-1 expression through the avb5 integrin, FAK, MEK, ERK, and NF-kB/AP-1 signal transduction pathway. On the other hand, In vitro chemotaxis assay showed that OA synovial fluid and supernatants from CTGF treated OASFs increased migration of monocyte. In addition, CTGF-mediated migration was inhibited by the FAK and MEK inhibitor. Taken together, these results provide insights into the mechanisms underlying CTGF-enhanced synovial inflammatory reponses in OA and many inspire new targeted therapeutic approaches.Contents 中文摘要 i Abstract ii Figure Contents v Table Contents vii Abbreviations viii CHAPTER 1 Background and significance 1 SECTION 1: General introduction 1 1.1. The Pathogenesis of osteoarthritis 1 1.2. Risk factors of osteoarthritis 1 1.2.1. Age: 1 1.2.2. Sex: 2 1.2.3. Joint injury: 2 1.2.4. Obesity: 2 1.2.5. Genetic factors: 3 1.3. Inflammation and osteoarthritis 3 1.4. Synovial fibroblast: key effector cells of osteoarthritis 4 1.5. The role of interleukin-6 (IL-6) in osteoarthritis synovial inflammation 5 1.6. The role of monocyte chemoattractant protein-1 (MCP-1) in osteoarthritis synovial inflammation 6 1.7. The role of Connective tissue growth factor (CTGF) in osteoarthritis 8 SECTION 2: Specific aims 23 CHAPTER 2 Materials and methods 24 CHAPTER 3 CTGF increases IL-6 expression in human synovial fibroblasts through integrin-dependent signaling pathway 30 SECTION 1: Results 31 SECTION 2: Discussion 34 SECTION 3: Figures and legends 37 CHAPTER 4 CTGF induces monocyte chemoattractant protein-1 expression to enhance monocyte migration in human synovial fibroblasts 48 SECTION 1: Results 49 SECTION 2: Discussion 54 SECTION 3: Figures and legends 57 CHAPTER 5 Conclusion and perspectives 73 References 76 Publication and honors 92 Figure Contents Appended Figure 1. Radiographs of osteoarthritis(OA) in hip, knee, lumbar, spine and hand. 11 Appended Figure 2. Theoretical model for the relationships of aging, oxidative stress, and the development of OA. 14 Appended Figure 3. Involvement of the synovium in OA pathophysiology. 15 Appended Figure 4. Chemokine receptors and disease. 18 Appended Figure 5. Factors that stimulate the MCP-1 transcription and some of the pathologies associated with MCP-1 overexpression. 19 Appended Figure 6. CTGF modular structure. 21 Appended Figure 7. Biological and pathological properties of CTGF. 22 Figure 3-1. CTGF increases IL-6 expression in human synovial fibroblasts through avb5 integrin. 38 Figure 3-2. ASK1 is involved in CTGF-induced IL-6 production. 39 Figure 3-3. JNK and p38 are involved in CTGF-mediated IL-6 production in synovial fibroblasts. 41 Figure 3-4. AP-1 and NF-kB are involved in the potentiation of IL-6 production by CTGF. 43 Figure 3-5. ASK1, p38, and JNK pathways are involved in CTGF-induced AP-1 and NF-kB activation. 44 Figure 3-6. Schematic presentation of the signaling pathways involved in CTGF-induced IL-6 expression of synovial fibroblasts. 46 Figure 3-S1. Curcumin and Tanshione IIA have no effect on NF-kB activation and ROS production in OASFs 47 Figure 4-1. CTGF increases MCP-1 expression in human synovial fibroblasts through avb5 integrin. 58 Figure 4-2. FAK is involved in CTGF-induced MCP-1 production. 60 Figure 4-3. MEK and ERK but not p38 and JNK are involved in CTGF-mediated MCP-1 production in synovial fibroblasts. 62 Figure 4-4. AP-1 and NF-kB are involved in the potentiation of MCP-1 production by CTGF. 64 Figure 4-5. FAK, MEK, and ERK pathways are involved in CTGF-induced AP-1 and NF-kB activation. 66 Figure 4-6. Conditioned media from the synovial fibroblasts-treated with CTGF promote monocyte migration. 68 Figure 4-7. Schematic presentation of the signaling pathways involved in CTGF-induced MCP-1 expression of synovial fibroblasts. 70 Figure 4-S1. The OASFs viability did not effect after antibodies or inhibitors treatment. 71 Figure 4-S2. The basal MCP-1 expression after antibodies or inhibitors treatment. 72 Summary: Schematic presentation of the signaling pathways involved in CTGF-induced IL-6 and MCP-1 expression of synovial fibroblasts. 75 Table Contents Appended Table 1. Classification of osteoarthritis 12 Appended Table 2. The Kellgren and Lawrence Scale 13 Appended Table 3. Evidence of the role of synovitis in OA. 16 Appended Table 4. Cytokines involved in the pathophysiology of OA 17 Appended Table 5. Summarizes the chemokine receptors and their known ligands. 2