COX-2, PPAR-γ, TGF-β及各種細胞激素在甲狀腺眼外肌病變所扮演的角色

The role of COX-2, PPAR-γ, TGF-β and different kinds of cytokines in the pathogenesis of dysthyroid myopathy

鄭敏蟬(Man sim cheng)

指導教授 ： 廖述朗

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.00495

摘要

研究目的:
葛瑞夫氏眼部病變是自體免疫引起的發炎反應所造成的後果。近年來研究顯示，甲狀腺刺激素受體是眼球很重要的自體抗原。典型發炎反應會引起脂肪增生和眼外肌的纖維化肥厚，從而引起眼球外凸，眼瞼發炎，以及眼周圍組織的水腫。
抗體抗原反應引發一系列的連鎖機制，包含單核白血球及巨噬細胞釋放激素。以IL-1為例子，IL-1可以引發COX-2表現，促使產生PGH2。PGH2之後轉變成PGE2，然後引起發炎反應。另一方面，PGH2可被轉變成PGD2，再經由cyclopentane ring脫水和代謝形成15d-PGJ2， 其為PPAR-γ受體的一個天然刺激原。當PPAR-γ受體活化後，可以產生抗發炎反應，同時合併脂肪增生。葛瑞夫氏眼病變有一個很特殊的組織病理學變化，葡萄醣胺類會堆積在眼球的結締組織,主要是玻尿酸的成份。最近研究顯示，眼球纖維母細胞合成玻尿酸會受到TGF-β刺激而增加。TGF-β是個與細胞生長分化有關的細胞激素。但是TGF-β過量會造成過度的疤痕組織形成以及纖維化。通過PGE2，COX-2可誘導TGF-β後產生纖維化，並激活HAS而開始堆積聚醣胺。TGF-β可增加玻尿酸形成。接著，玻尿酸和TGF-β也可以刺激COX-2和前列腺素的生成。先前研究顯示IGF-1α, IGF-1R, IL-1β, IFN-γ, PPAR-γ, TSH-R, COX-2, HAS等等物質的基因表現在葛瑞夫氏眼病變組織中都是增加的。
另一方面，PPAR-γ可破壞TGF-β信號傳導路徑，並且中斷纖維化之前的反應。這些反應可能是葛瑞夫氏眼睛病變發病機制中的一部分。在製造前列腺素PGE2和玻尿酸，或那些調節糖胺聚醣生產和脂肪分化的的途徑中，協同或拮抗作用都會發生。
本研究主要是探討細胞激素如何影響體外培養之眼窩眼外肌母細胞釋放PPAR-γ，COX-2，TGF-β，Adiponectin，CXCL10和TSH-R。本研究亦評估PPAR -γ促進劑，抑制劑和COX-2抑制劑對於眼外肌母細胞中TGF-β，玻尿酸合成酶（HAS1，HAS2，HAS3）和產生玻尿酸的影響效果。
研究方法:
我們以培養之眼外肌母細胞經IFN-γ，IGF-1，IL-1β和TNF-α刺激後，以real time PCR分析PPAR -γ，COX-2，TGF-β，CXCL10，Adiponectin，TSH- R的訊息核醣核酸(mRNA)的基因表現量。並以酶聯免疫吸附試驗（ELISA）和免疫印跡分析(Western blot)來確定TGF-β，CXCL10，COX-2，TSH-R蛋白質表現量。
此外，我們將培養之眼外肌母細胞經TNF-α刺激後，同時加入PPAR-γ促進劑、抑制劑，COX-2抑制劑，以酶聯免疫吸附試驗（ELISA）和免疫印跡分析(Western blot) 來確定TGF-β，玻尿酸合成酶（HAS1，HAS2，HAS3）和產生玻尿酸的蛋白質表現量。經由上述實驗期望可以進一步釐清葛瑞夫氏眼部病變的分子機制。
結果:
葛瑞夫氏眼病變的病人組眼外肌母細胞尤其在IL-1β或TNF-α刺激24小時下，PPAR-γ，COX-2，TGF-β，CXCL10，Adiponectin，TSH-R的訊息核醣核酸的基因表現量皆有顯著增加。TGF-β和CXCL10的酶聯免疫吸附試驗及COX-2和TSH-R的西方點墨法檢測結果和RT-PCR結果吻合。對照組則全無顯著變化。受了細胞激素刺激之後，葛瑞夫氏眼病變眼外肌母細胞基因表現的確比對照組強。在本次體外實驗的葛瑞夫氏眼病變眼外肌母細胞，COX-2抑制劑(Celecoxib)和PPAR-γ促進劑(Pioglitazone)抑制TGF-β，HAS或玻尿酸蛋白質表現未達統計上顯著，但是，我們仍然發現經由COX-2抑制劑和PPAR促進劑，有抑制TGFβ，HAS和玻尿酸表現的趨勢。我們推測玻尿酸合成增加是透過HAS基因之誘發，而這是透過TGF-β刺激所致。Celecoxib和Pioglitazone作用都可以抑制因細胞激素誘發的HAS3表現，但對HAS1或HAS2表現則無影響。TGF-β透過某種未確定的信號傳導通路來刺激HAS3表現。細胞中的玻尿酸合成可在HAS3刺激下受到誘發，這可能和葛瑞夫氏眼病變的發病有極為重要的關聯。
結論:
這些數據佐證了:細胞激素可誘導葛瑞夫氏眼部病變眼外肌母細胞表現多種基因。我們也確認了眼窩眼外肌在葛瑞夫氏眼病變的致病機制中佔很重要的角色。葛瑞夫氏眼病變自體免疫過程中，纖維母細胞以外，眼外肌應該是另一個同等重要的目標。目前的數據支持我們的假設:在葛瑞夫氏眼部病變患者的眼外肌母細胞裡，依賴COX-2的前列腺素，可以透過TGF-β訊息作用，誘導被HAS激活的玻尿酸合成反應。我們觀察到COX-2抑制劑及PPAR-γ促進劑抑制了TGF-β，HAS3和玻尿酸蛋白質的製造。未來發展中，針對葛瑞夫氏眼部病變併牽制性肌肉病變和脂肪增生，需要更多的廣泛研究以開發治療藥物。

關鍵字

葛瑞夫氏眼部病變 ； 眼窩自體抗原 ； 細胞激素 ； 眼窩脂肪增生 ； 玻尿酸堆積

並列摘要

Purpose:
Graves’ophthalmopathy (GO) is an autoimmune inflammatory disorder.Recent evidence implicates the TSH receptor (TSH-R) as an important orbital autoantigen in GO. The characteristic clinical manifestations of GO, including proptosis, chemosis, periorbital edema, and extraocular muscle dysfunction, result from the intraorbital inflammatory process and enlargement of the adipose tissue and muscles within the orbit.
After antibody binding with antigen, a cascade of inflammation starts, including monocytes and macrophages releasing cytokines, eg. IL-1β. IL-1β can induce COX-2, which can produce prostaglandin H2. After the conversion of prostaglandin H2 into prostaglandin E2, inflammation is induced. On the other hand, prostaglandin H2 can be converted to prostaglandin D2. Prostaglandin J2 is formed after dehydration of cyclopentane ring and is further metabolized to yield 15d-PGJ2, a natural ligand of PPAR-γ. After the binding of 15d-PGJ2, PPAR-γ will be activated and induce anti-inflammatory activity, combined with adipogenesis.
A distinctive histopathological feature associated with GO is the accumulation of the glycosaminoglycan, mainly hyaluronan, in orbital connective tissues. In recent study, hyaluronan synthesis in orbital fibroblasts has been shown to be up-regulated by TGF-β. Overproduction of TGF-β will result in excessive scar formation and fibrosis. Through action of PGE2, COX-2 induces TGF-β and produces fibrosis, and activates HAS, starting accumulation of glycosaminoglycan. TGF-β can increase the formation of hyaluronan. In turn, hyaluronan and TGF-β can stimulate the production of COX-2 and prostaglandin. Previous study found that mRNA expression of IGF-1α, IGF-1R, IL-1β, IFN-γ, PPAR-γ, TSH-R, COX-2, HAS in GO orbital fat tissues by real time PCR were up regulated as compared to normal orbital tissues.
PPAR-γ disrupts TGF-β signal transduction and blocks profibrotic responses. Some of these responses may take part in the pathogenesis of GO. Synergistic and antagonistic interactions can occur between the pathways producing prostaglandin (PGE2) and hyaluronan or those regulating glycosaminoglycan production and adipogenesis.
In this study, we will investigate the effect of different cytokines on the expression of PPAR-γ, COX-2, TGF-β, Adiponectin, CXCL10, TSH-R mRNA and production of protein by myoblasts cultured from the extraocular muscles. We will also try to evaluate the effect of PPAR-γ agonist, antagonist and COX-2 inhibitor on the expression of TGF-β, hyaluronan synthase (HAS1, HAS2, HAS3) and hyaluronan.
Methods:
To determine whether cytokines induce these genes expression, we treated orbital myoblasts with or without IFN-γ, IGF-1, IL-1β , TNF-α. Data were analyzed by real time PCR for PPAR-γ, COX-2, TGF-β, CXCL10, Adiponectin, TSH-R mRNA. ELISA and Western blot were used to confirm the TGF-β, CXCL10, COX-2, TSH-R protein expression in cytokines treated orbital myoblasts.
We cultured orbital extraocular muscles and treated them with TNF-α in the presence of the PPAR-γ agonist, antagonist and COX-2 inhibitor. ELISA and Western blot were used to determine their effect on TGF-β, hyaluronan synthase (HAS1, HAS2, HAS3) and hyaluronan protein synthesis.
Results:
After treating patients with Graves' ophthalmopathy group myoblasts especially in IL-1β or TNF-α for 24 hours, gene expression of the mRNA of PPAR-γ, COX-2, TGF-β, CXCL10, Adiponectin, TSH-R show significantly increase. ELISA results of TGF-β, CXCL10 and Western blot results of COX-2, TSH-R were compatable with RT-PCR. There was no significant gene expression in normal group. All myoblasts gene expressions show differences between the disease and control groups. In this in vitro experiment of cytokine treated GO myoblasts, there is a trend that the TGF-β and hyaluronan gene expression was under inhibition with COX-2 inhibitor (Celecoxib) or PPAR-γ agonist (Pioglitazone), though not reach significant level. We hypothesis that TGF-β activate hyaluronan synthesis via HAS signals. Both Celecoxib and Pioglitazone treatment inhibited cytokine induced HAS3 expression, but did not afftect HAS1 and HAS2 expression. The signaling pathways by which TGF-β regulates HAS3 are still poorly understood. HAS3 can induce hyaluronan production which may be important pathogenesis to Graves' ophthalmopathy.
Conclusion:
To our knowledge, this is the first in vitro experiment demonstrates PPAR-γ, COX-2, TGF-β, CXCL10, Adiponectin, TSH-R production stimulated by cytokines in GO extraocular muscles. These results suggest that the extraocular muscles indeed play an important role in GO pathogenesis besides orbital fibroblasts. Taking together, the present data did partly support our working hypothesis that COX-2 dependent PGs induce TGF-β signaling, stimulate hyaluronan production via HAS activation in GO myoblasts. In our study, we observed COX2 inhibitor and PPAR-γ agonist suppress TGF-β, HAS and hyaluronan protein production. In the future, more studies will be helpful to get useful information in the treatment of Graves’ophthalmopathy with restrictive myopathy and adipogenesis.

並列關鍵字

Graves’ophthalmopathy ； orbital autoantigen ； cytokines ； orbital adipogenesis ； hyaluronan deposition

參考文獻

Alevizaki M, Mantzou E, Cimponeriu A, Saltiki K, Philippou G, Wiersinga W. The Pro 12 Ala PPAR gamma gene polymorphism: possible modifier of the activity and severity of thyroid-associated orbitopathy (TAO). Clin Endocrinol (Oxf). 2009 Mar;70(3):464-8. Epub 2008 Jul 7.

Allahabadia A, Heward JM, Nithiyananthan R, Gibson SM, Reuser TT, Dodson PM, Franklyn JA, Gough SC. MHC class II region, CTLA4 gene, and ophthalmopathy in patients with Graves' disease. Lancet. 2001 Sep 22;358(9286):984-5.

Aniszewski JP, Valyasevi RW, Bahn RS.Relationship between disease duration and predominant orbital T cell subset in Graves' ophthalmopathy.J Clin Endocrinol Metab. 2000 Feb;85(2):776-80.

Antonelli A, Rotondi M, Ferrari SM, Fallahi P, Romagnani P, Franceschini SS, Serio M, Ferrannini E. Interferon-gamma-inducible alpha-chemokine CXCL10 involvement in Graves' ophthalmopathy: modulation by peroxisome proliferator-activated receptor-gamma agonists. J Clin Endocrinol Metab. 2006 Feb;91(2):614-20.

Arnold R, and Konig W. Peroxisome-proliferator-activated receptor-gamma agonists inhibit the release of proinflammatory cytokines from RSV-infected epithelial cells. Virology. 2006;346:427-39.