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E.L. Humphrey a, J.H.H. Williams a,b, M.W.J. Davie a, M.J. Marshall a, 姓名 : 萬映汝

Received 15 June 2005; revised 12 September 2005; accepted 7 October 2005 Effects of dissociated glucocorticoids on OPG and RANKL in osteoblastic cells. E.L. Humphrey a, J.H.H. Williams a,b, M.W.J. Davie a, M.J. Marshall a, 姓名 : 萬映汝 系級 : 生物科技學系四年級

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E.L. Humphrey a, J.H.H. Williams a,b, M.W.J. Davie a, M.J. Marshall a, 姓名 : 萬映汝

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  1. Received 15 June 2005; revised 12 September 2005; accepted 7 October 2005Effects of dissociated glucocorticoids on OPG and RANKL in osteoblastic cells E.L. Humphrey a, J.H.H. Williams a,b, M.W.J. Davie a, M.J. Marshall a, 姓名:萬映汝 系級:生物科技學系四年級 學號:91390496

  2. Abbreviation • GIOP- glucocorticoid-induced osteoporosis • GR- glucocorticoid receptor • hFOB- human fetal osteoblast • nGRE- negative glucocorticoid response element • OPG- osteoprotegerin促骨生成蛋白受體為蝕骨細胞分化抑制因子 • RANKL- receptor activator of nuclear factor kappa B ligand促骨生成蛋白結合子為促進蝕骨細胞生成的主因子

  3. Introduction • Glucocorticoid treatment often leads to unwanted side effects including weight gain, skin thinning, osteopenia, eye defects, depression, diabetes, adrenal insufficiency and peptic ulcers，但長期使用Glucocorticoid治療會導致GIOP. • Adult bone remodeling is carried out by osteoblasts and osteoclasts, which are controlled by various hormones, cytokines and growth factors.

  4. 骨骼新陳代謝動態之平衡造骨速率與蝕骨速率相當骨骼新陳代謝動態之平衡造骨速率與蝕骨速率相當

  5. 骨骼新陳代謝正常狀況

  6. hormonal和cytokine會調控造骨細胞產生RANKL的可溶性cytokine，會跟蝕骨細胞上的RANK receptor 鍵結或是跟它的precursor,刺激骨骼的再吸收 • 造骨細胞會產生OPG,跟RANKL binding阻止與RANK receptor 鍵結來抑制蝕骨細胞的生成 • GR與nGRE結合會抑制基因轉錄及物理性阻斷轉錄機制運行，或阻斷一正向調控的轉錄因子去跟它的response element結合使基因轉錄被抑制cis-repression

  7. Actived GR可抑制一持續性的活化，或抑制具誘導性的轉錄因子，利用protein-protein的交互作用不需與DNA binding來防止activating基因轉錄trans-repression • Ligand與GR鍵結造成receptor構型改變transrepress基因轉錄，但有較少甚至無transactivation活性dissociated glucocorticoid (symmetrical glucocorticoid則會造成GR的transactivation及transrepression) • 結果顯示dissociated glucocorticoid 及deflazacort較symmetrical glucocorticoid prednisolone 對RANKL的活化作用低，且在某些情況下較無法抑制OPG製造

  8. 實驗方法 細胞培養 MG63cell(European collection of cell cultures) 培養在α-minimal essential media (αMEM) at 37°C +10%胎牛血清 +100 IU/ml苯甲基青黴素 + 100μg/ml鏈黴素 +2 mM L-glutamine 在含有5 %carbon dioxide 潮濕的環境下培養

  9. 人類胚胎造骨細胞hFOB cells培養在DMEM/HAMs F12 medium +10%胎牛血清 +100 IU/ml苯甲基青黴素 + 100μg/ml鏈黴素 +2 mM L-glutamine +0.3mg/ml geneticin 在潮濕環境33.5°C下培養7天-細胞增生 39.5°C2天-細胞分化

  10. Both cell types were seeded in 96-well plates at 2.8 × 10−3 cells/cm2for OPG protein determination • 24-well plates for total RNA extraction • after 48h and stored at −20°C for OPG measurements • 加入Tetrazolium (MTS 190 μg/ml)及phenazine ethosulphate (PES 30 μM)溶液到毎個well作用30min

  11. OPG ELISA • 用goat anti-human OPG capture antibody • Captured OPG was detected with biotinylated anti-OPG antibody • 用10% sulphuric acid停止反應 • 用Dynatech Microtitre plate reader • 用Sigma plot software

  12. RNA extraction and first strand cDNA synthesis • Qiagen RNeasy kit Real-time PCR • 利用the ABI Assays on Demand primers and Taqman universal PCR master mix

  13. 在MG63 cell中dexamethasone抑制效果最佳

  14. 在hFOB cell中dexamethasone抑制效果最佳

  15. The potency of each GR ligand is described by itsIC50, and its effectiveness is described by the maximuminhibition. 達最大抑制OPG產量 一半所需的化合物濃度 6倍 10倍 12倍

  16. 在MG63cell中加入10μM 的anti-glucocorticoid RU486 OPG濃度皆提高

  17. control為anti-glucocorticoid control RU43044( in MG63 cell )

  18. control為anti-glucocorticoid control RU43044(in hFOB cell )

  19. control為inactive racemer AL438-F2( in MG63 cell )

  20. control為inactive racemer AL438-F2( in hFOB cell )

  21. 三個RU compond的抑制能力及最大抑制效果相近，但較prednisolone低 AL438-F1及ZK216348抑制效能力低近50 %

  22. Effect of Glucocorticoids on OPG and RANKL expression

  23. hFOB cell內OPG與RANKL訊息的相對調控造成的message比例 • Prednisolone因為對OPG抑制效果佳 • 所以相對比值就較高

  24. Discussion • Glucocorticoid會刺激蝕骨細胞分化，藉由直接增強RANKL訊號，間接抑制INF-β的產生，並刺激造骨細胞產生RANKL抑制產生OPG，以利蝕骨細胞的生成及骨骼的再吸收能力上升 • Deflazacort相較於prednisolone對造骨細胞OPG抑制能力低10倍，但在高濃度下兩者效果相當，且 Deflazacort對RANKL的刺激能力低於prednisolone (都是由類固醇的GR ligand；deflazacort被視為”bone sparing”Glucocorticoid) • dissociated glucocorticoid屬非類固醇的GR ligand，仍具有抗炎的活性但呈現較少的transactivation的活性

  25. dissociated glucocorticoid AL438對MG63cell的OPG產生及骨鈣蛋白質(視為是骨骼形成及造骨功能的marker， prednisolone會抑制它的產生)產生的抑制較prednisolone弱 • 所有活化的dissociated glucocorticoid對造骨細胞OPG產生的抑制能力都較prednisolone低；所有的高濃度的RU compounds對OPG的抑制效果和prednisolone相當 • AL438-F1和ZK21648在hFOB、MG63 cell中對OPG抑制效果明顯低於prednisolone • 近來發現GR會藉由招集不同co-regulator對基因產生不同的影響，因為GR的ligand binding domain有一個高度保留且含有glutamate及lysine residues的序列charge clamp

  26. charge clamp可與GR的co-regulator鍵結，當被GR ligand binding改變結構使基因轉錄被抑制 • AL438不與co-activator PGC-1反應，但prednisolone會(PGC-1牽涉到glucocorticoid對肝臟glucose產量的調控)，因此相較於prednisolone可導致較少的血糖過高問題 • 利用GR ligand這種co-regulator去改變GR的結構使不會刺激 RANKL的表現及抑制OPG的產生，因此不會刺激骨骼的再吸收，降低骨質疏鬆的風險

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