臺灣博碩士論文加值系統

退化性關節炎隨著人口的老化已經成為全世界所有健康照護系統的一大負擔。關節炎的生成是受到許多因素的影響，臨床上常把這些影響分類為外因性或內因性。然而在病生理機轉上，它的生成與軟骨組織的恆定調節有著密不可分的關係。肥胖長久以來都被認為是增加機械力負擔的危險因子，然而隨著脂肪激素的發現， 它在生物化學上對於退化性關節炎的生成的影響，開啟了人們對於關節炎病生理上新的視野。一個完整的治療方案必須透過對於力學性與生物性訊息相互作用在軟骨組織上的了解才能完成。
骨塑型蛋白(bone morphogenic protein, BMP)對於軟骨的生成可提供正向調控的作用，然而在健康的關節軟骨內卻很少發現它們的存在。有些研究發現在受關節炎破壞的軟骨中可以發現BMP的存在，但是仍不確定它們在這其中所扮演的角色。另外有研究在肥胖者的關節中發現極高濃度的瘦素(leptin)，而且和關節炎的產生有著密切相關。我們第一個研究旨在探討人類初代軟骨細胞在瘦素的刺激下是否會激發BMP的產生?而其中機轉又是如何? 本研究顯示在瘦素的刺激下，人類初代軟骨細胞的第二型環氧合酶(COX-2)和BMP-2會增加表現，而BMP-4,6 和 7則無。再者，該BMP-2 的誘增是藉由上調JAK2-ERK1/2促進Ser727的磷酸化以激活STAT3路徑。值得注意的是，組織蛋白去乙醯酶HDAC中的 HDAC-3和HDAC-4都有參與該BMP-2 的誘增，但是所經由的路徑不同。HDAC-3會和STAT形成複合體共同作用，而HDAC-4則是透過其他路徑來調控BMP-2 的誘增。另外，BMP-2的增加會促進軟骨細胞製造第二型膠原蛋白(typeII collagen)。本研究確認了人類初代軟骨細胞在瘦素的刺激發炎作用會藉由BMP-2的自分泌效應(autocrine effect)啟動修復作用，並明確描述其中的分子生物學機轉，對於將來發展新藥有重要參考價值。
有些研究曾提出低度剪應力在調控軟骨恆定中可以扮演修復的角色；而近來有流行病學研究顯示血液和血清中的阻抗素(resistin)濃度和患者的關節炎嚴重度有正相關。然而低度剪應力對於關節炎軟骨細胞在受到阻抗素刺激下所引發的分解代謝(catabolic effect)是否有調節作用則仍不清楚。因此我們的第二個研究旨在釐清低度剪應力是否可以調控人類軟骨細胞在阻抗素刺激下由COX-2所引發的分解代謝。在本實驗中使用人類初代軟骨細胞和SW1343惡性軟骨肉瘤細胞株進行研究。兩種低度應力(2 dyn/cm2)模式被施予在接受阻抗素刺激軟骨細胞:刺激前施予(pre-shear)和刺激後施予(post-shear)，藉以研究COX-2表現的不同；更進一步利用各種專一性的活化因子和沉默核醣核酸(siRNA)來研究細部調控機轉。我們發現軟骨細胞在阻抗素刺激下COX-2的表現會在不同低剪力施予模式下產生截然不同的反應。阻抗素刺激前施予低剪力會降低COX-2表現，而刺激後施予則會增強其表現。Pre-shear效應是透過抑制(NF)-kB-p65 subunit和cAMP結合蛋白來達成，post-shear效應則是只有透過活化(NF)-kB-p65 subunit表現來完成。而兩者最後都是透過AMPK路徑的激活再經sirtuin 1 (長壽蛋白-1)表現來調控COX-2生成。本研究釐清了低剪力應力對於調控阻抗素所引起的軟骨細胞發炎反應的詳細機轉，並或許可以指引其應用在產生修復作用。

Osteoarthritis is one of the major burden of health care system worldwide and earning more attentions along with aging of population. Its development can be attributed to extrinsic or intrinsic factors but, in pathophysiology, all are closely related to the homeostasis of cartilage. Obesity has been taken as a mechanical risk factor for the progress of osteoarthritis. However, its biochemical influence on osteoarthritis has opened a new window since the discovery of adipokines. Treatment of osteoarthritis cannot be completed without understanding the crosstalk between mechanical and biological signaling.
Bone morphogenetic proteins (BMPs) play positive roles in cartilage development, but they can barely be detected in healthy articular cartilage. Recent evidence has indicated that BMPs could be detected in osteoarthritic and damaged cartilage and their precise roles have not been well defined. Extremely high amounts of leptin have been reported in obese individuals, which can be associated with osteoarthritis (OA) development. The aim of our first study was to investigate whether BMPs could be induced in human primary chondrocytes during leptin-stimulated OA development and the underlying mechanism. We found that expression of COX-2 as well as BMP-2 mRNA, but not BMP-4, BMP-6, or BMP-7 mRNA, could be increased in human primary chondrocytes under leptin stimulation. Moreover, this BMP-2 induction was mediated through transcription factor-signal transducer and activator of transcription (STAT) 3 activation via JAK2-ERK1/2-induced Ser727-phosphorylation. Of note, histone deacetylases (HDACs) 3 and 4 were both involved in modulating leptin-induced BMP-2 mRNA expression through different pathways: HDAC3, but not HDAC4, associated with STAT3 to form a complex. Our results further demonstrated that the role of BMP-2 induction under leptin stimulation is to increase collagen II expression. The findings in this study provide new insights into the regulatory mechanism of BMP-2 induction in leptin-stimulated chondrocytes and suggest that BMP-2 may play a reparative role in regulating leptininduced OA development.
Low shear stress has been proposed to play a reparative role in modulating cartilage homeostasis. Recently, epidemiological studies have found a positive correlation between the resistin level in serum and synovial fluid and osteoarthritis (OA) severity in patients. However, the effect of low shear stress on the catabolic stimulation of resistin in OA chondrocytes remains unclear. Hence, our second study was to investigate whether low shear stress could regulate resistin-induced catabolic cyclooxygenase (COX)-2 expression in human OA chondrocytes and the underlying mechanism. Human OA chondrocytes and SW1353 chondrosarcoma cells were used in this study. Two modes of low shear stress (2 dyn/cm2), pre-shear and post-shear, were applied to the chondrocytes. A specific activator and siRNAs were used to investigate the mechanism of low shear stress-regulated COX-2 expression of resistin induction. We found that human OA chondrocytes exposed to different modes of low shear stress elicit an opposite effect on resistin-induced COX-2 expression: pre-shear for a short duration attenuates the resistin effect by inhibiting the transcription factor nuclear factor (NF)-kB-p65 subunit and the cAMP response element binding protein; however, post-shear over a longer duration enhances the resistin effect by activating only the NFkB-p65 subunit. Moreover, our results demonstrated that the regulation of both shear modes in resistin-stimulated COX-2 expression occurs through increasing AMP-activated protein kinase activation and then sirtuin 1 expression. This study elucidates the detailed mechanism of low shear stress regulating the resistin-induced catabolic COX-2 expression and indicates a possible reparative role of moderate shear force in resistin-stimulated OA development.

Contents

摘要 i
Abstract iii
Contents v
List of Figures viii

Chapter 1
Molecular insights of osteoarthritis chondrocytes involving mechanical stresses, adipokines and bone morphogenic proteins 1
1.1 Osteoarthritis 1
1.2 Mechanical Stresses 2
1.3 Adipokines 3
1.4 Bone morphogenic proteins 5
Chapter 2
Upregulation of Bone Morphogenetic Protein-2 Synthesis and Consequent Collagen II Expression in Leptin-stimulated Human Chondrocytes 7
2.1 Introduction 7
2.2 Materials and methods 8
2.2.1 Materials 8
2.2.2 Cell culture 9
2.2.3 Quantitative real-time PCR 9
2.2.4 Western blot analysis 10
2.2.5 siRNA transfection 10
2.2.6 Immunoprecipitation 10
2.2.7 Chromatin immunoprecipitation (ChIP) assay 10
2.2.8 Statistical analysis 11
2.3 Results 12
2.3.1 Leptin induces BMP-2 expression in human primary chondrocytes 12
2.3.2 Leptin-induced BMP-2 mRNA expression is mediated by JAK2-ERK1/2 signaling in human primary chondrocytes 12
2.3.3 STAT3 regulates leptin-induced BMP-2 mRNA in human primary chondrocytes 12
2.3.4 HDAC3/4 mediate leptin-induced BMP-2 mRNA expression in human primary chondrocytes 13
2.3.5 STAT3-HDAC3 and HDAC4 are divergent pathways mediating leptininduced BMP-2 mRNA expression in human primary chondrocytes 13
2.3.6 BMP-2 induction in leptin-stimulated human primary chondrocytes increases collagen II expression 14
2.4 Discussion 15

Chapter 3
Low shear stress attenuates COX-2 expression induced by resistin in human osteoarthritic chondrocytes 25

3.1 Introduction 25
3.2 Materials and Methods 27
3.2.1 Materials 27
3.2.2 Cell culture 27
3.2.3 Shear stress experiment 27
3.2.4 Quantitative real-time PCR 28
3.2.5 Western blot analysis 28
3.2.6 Reporter gene construct, siRNA, transfection, and luciferase assays 28
3.2.7 NF-kB and CREB activation ELISA assays 29
3.2.8 Statistical analysis 29
3.3 Results 30
3.3.1 Pre- and post-shear (2 dyn/cm2) effects on human OA chondrocytes result in differential regulations on resistin-induced COX-2 expression 30
3.3.2 Pre- and post-shear effects on human SW1353 chondrocytes result in differential regulations in resistin-increased COX-2 promoter activity 30
3.3.3 AMPK/SIRT1 signaling regulates the attenuated effect of pre-shear stress on resistin-induced COX-2 expression in human OA chondrocytes 31
3.3.4 AMPK/SIRT1 signaling regulates NF-kB and CREB activity under the pre-shear-inhibited resistin effect on human OA chondrocytes 32
3.3.5 AMPK/SIRT1 signaling regulates the enhancement of post-shear on resistin-induced COX-2 expression in human OA chondrocytes 32
3.3.6 AMPK signaling regulates NF-kB, but not CREB, activity under post-shear-enhanced resistin-induced COX-2 expression of human OA chondrocytes 32
3.4 Discussion 34
References 45

List of figures
Chapter 2
Figure 2-1 Leptin induces BMP-2 expression in human primary chondrocytes 18
Figure 2-2 Leptin-induced BMP-2 mRNA expression via JAK2-ERK 1/2 19
Figure 2-3 STAT3 regulates leptin-induced BMP-2 mRNA expression 20
Figure 2-4 HDAC3/4 mediates leptin-induced BMP-2 mRNA expression 21
Figure 2-5 STAT3-HDAC3 & HDAC4: divergent pathways for leptin-induced BMP-2 22
Figure 2-6 Leptin-induced BMP-2 increases collagen II expression 23
Figure 2-7 Signaling pathways: Leptin-induced BMP-2 increases collagen II 24

Chapter 3
Figure 3.1 Experimental setup for pre-shear and post-shear modes 37
Figure 3.2 Attenuation / enhancement of COX-2 expression modulated by shear stress
on human OA chondrocytes 38
Figure 3.3 Role of NF-kB & CREB regulating COX-2 response to shear stress on hum
-an SW1353 chondrocytes 39
Figure 3.4 AMPK/SIRT1 signaling for pre-shear attenuation of COX-2 on human OA chondrocytes 40
Figure 3.5 AMPK/SIRT1 signaling via NF-kB & CREB for pre-shear attenuation of
COX-2 on human OA chondrocytes 41
Figure 3.6 AMPK/SIRT1 signaling for post-shear enhancement of COX-2 on human
OA chondrocytes 42
Figure 3.7 AMPK signaling acts through NF-kB but not CREB for post-shear enhance
-ment of COX-2 43
Figure 3.8 Signaling pathway: effects of low shear stress on resistin-induced COX-2
expression 44

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(for Chapter 3)

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