- 學位論文
PPARγ配體、第一型血基質氧化酵素與去組合蛋白在調控骨代謝與發炎之探討
Studies of PPARγ ligands, heme oxygenase-1, and disintegrin in bone metabolism and inflammation
摘要
核內受體，過氧化體增殖劑活化受體γ(peroxisome proliferators-activated receptor gamma, PPARγ)被視為是一種抑制骨合成的因子，因為PPARγ會增加脂肪細胞的形成以及直接抑制造骨細胞，而引發老年性骨質疏鬆症。我們發現到餵食大鼠PPARγ配體(活化劑) rosiglitazone會降低大鼠脛骨之骨密度以及血清中鹼性磷酸酶(ALP)的表現，但是並不影響與骨代謝相關的血鈣以及破骨細胞指標CTx。我們進一步使用兩種PPARγ活化劑，包括ciglitazone以及體內可自行合成的15d-PGJ2，來驗證PPARγ活化劑抑制骨合成的機轉。我們的研究指出在初級造骨細胞中，PPARγ活化劑會抑制造骨細胞的鈣化能力以及鹼性磷酸酶(ALP)的活性。我們接著利用反轉錄酶連鎖反應中(RT-PCR)，發現ciglitazone和15d-PGJ2會抑制造骨細胞表現骨形成蛋白第二型(BMP-2)以及骨鈣素(OCN)。更進一步地，PPARγ活化劑會抑制轉錄因子NF-κB所調控的兩個造骨相關蛋白：環氧化酶二型(COX-2)和誘導型一氧化氮合成酶(iNOS)的表現。利用治療性超音波來刺激造骨細胞可誘導造骨細胞表現COX-2以及iNOS和一氧化氮，然而在PPARγ活化劑的存在下會中止治療性超音波的作用。在動物實驗中，我們利用局部注射的方法在年輕大鼠的脛骨遠端注射PPARγ活化劑，而PPARγ活化劑會降低次級海綿骨的骨容積。這些結果證實，PPARγ活化劑會直接抑制造骨細胞的分化以及會抑制造骨細胞所表現的多種骨合成促進因子。本研究可以用來解釋年長者以及服用thiazolidinediones (TZD)類藥物的糖尿病患易得到骨質疏鬆症的原因。 第一型血基質氧化酵素(heme oxygenase-1, HO-1)能夠將血基質(heme)代謝成一氧化氮、膽綠素(biliverdin)以及鐵離子，HO-1在血管疾病、器官移植以及發炎反應中扮演重要角色。在骨組織中，有報告指出過度表現HO-1會抑制破骨細胞的形成。然而HO-1對於造骨細胞的作用仍是未知。本研究利用攜帶人類HO-1基因的腺病毒以及HO-1的誘導劑hemin來探討HO-1在初級造骨細胞中的作用。我們發現過度表現HO-1會抑制造骨細胞的鈣化能力以及鹼性磷酸酶(ALP)的活性，並且會抑制多種造骨細胞分化指標如鹼性磷酸酶、骨鈣素(OCN)以及轉譯因子RUNX2。我們也發現到HO-1的三個下游：一氧化碳、膽色素(bilirubin)以及鐵離子均參與HO-1對於造骨細胞分化的抑制作用。在造骨細胞中，HO-1可以被雙氧水(H2O2)、內毒素(LPS)以及發炎性細胞介素(cytokine)包含IL-1β以及TNF-α所誘導。在動物實驗中我們也在鏈菌素(streptozotocin, STZ)所誘發的糖尿病小鼠中，在造骨細胞上觀察到HO-1的表現。我們進一步發現到15d-PGJ2在造骨細胞中會經由PI3K-Akt以及MAPK訊息路徑來大量表現HO-1。利用HO-1抑制劑ZnPP IX可以拮抗hemin以及15d-PGJ2對造骨細胞所表現骨鈣素的抑制。我們的研究證實誘導HO-1表現會抑制造骨細胞的分化及成熟且可能參與氧化壓力以及發炎所造成的骨流失。 組合蛋白(integrins)是一群異型二聚體的(heterodimeric)穿膜受體，可以調控細胞與細胞間以及細胞與基質間的交互作用。αvβ3組合蛋白是玻璃體結合蛋白(vitronectin)以及骨橋蛋白(osteopontin)的受體，αvβ3大量表現於破骨細胞並且參與破骨細胞的貼附、活化以及破骨細胞在骨表面上的轉移。在細胞實驗以及動物實驗中，利用αvβ3的拮抗劑例如:含有精氨酸(Arg)-甘氨酸(Gly)-天門冬安酸(Asp)片段的蛋白藥物、小分子抑制劑以及抑制性抗體均被報導到可以抑制破骨細胞所造成的骨代謝。在本研究中，我們將蛇毒所萃取出來的數種組合蛋白(rhodostomin derivatives)結合人類血清蛋白以增加其生物體內穩定性，這些組合蛋白(HSA-ARLDDL、derivative P、derivative X)對於αvβ3組合蛋白擁有高度的專一性，我們進一步利用這些組合蛋白來研究其在破骨細胞分化以及卵巢切除所造成的骨質疏鬆中所扮演的角色。在破骨細胞分化中，rhodostomin的衍生物顯著地抑制了破骨細胞的形成而且在奈米莫爾濃度的範圍內即可達到半數抑制濃度(IC50)。在破骨細胞分化的後期給予HSA-ARLDDL依舊可以抑制破骨細胞的形成。在卵巢切除小鼠的動物實驗中，每週給予一次HSA-ARLDDL以及derivative P可以顯著地抑制血清中破骨細胞活性指標(CTx)的增加以及脛骨和股骨中海綿骨的流失。而在卵巢切除大鼠中，rhodostomin衍生物亦抑制了血清中破骨細胞活性指標(CTx)的表現以及減緩了髖骨骨密度的流失。本研究指出這些rhodostomin的衍生物對於αvβ3組合蛋白有高度專一性且能在動物體內長時間作用，這些衍生物可以發展為臨床上抑制骨代謝，並治療停經後骨質疏鬆症的藥物。 金屬蛋白酶(matrix metalloproteinase, MMP)中的膠原蛋白酶三型(collagenase-3, MMP-13)在病理情況下的軟骨退化扮演重要的角色。在類風濕性關節炎(RA)以及骨關節炎(OA)的關節組織中可以看到MMP-13的大量表現。而且在這些病症中，滑囊纖維母細胞(synovial fibroblast)會分泌MMP-13以及其他的發炎性細胞介素引起進一步的病症。PPARγ訊息主要調控脂肪形成以及醣類代謝。越來越多證據指出PPARγ在發炎性關節炎中扮演了重要的角色，主要是經由調控B細胞的反應以及在受刺激的滑囊纖維母細胞以及軟骨細胞中抑制MMP-13的產生。然而文獻指出：內生性的PPARγ配體，15d-PGJ2在抑制發炎性關節炎中比其他化學合成的PPARγ活化劑擁有更佳藥效，且其藥效有部份不經由PPARγ的訊息機轉(PPARγ-independent)。然而詳細機轉仍是未知。在本研究中，我們發現15d-PGJ2在人類滑囊纖維母細胞中顯著抑制內毒素(LPS)或TNF-α所誘發的MMP-13的表現。我們進一步地發現到，在滑囊纖維母細胞中，轉錄因子NF-κB在TNF-α誘導MMP-13的表現中扮演重要角色，15d-PGJ2會經由直接抑制IKK的活性來顯著地抑制NF-κB的入核與活化，而這個步驟是不經由PPARγ訊息的。本研究結果指出在發炎的關節中提升15d-PGJ2的含量在臨床上可發展為避免軟骨退化的治療方法。
並列摘要
Peroxisome proliferators-activated receptor gamma (PPARγ), a ligand-activated transcription factor, is considered as an anti-osteoblastic factor associated with adiposity and the elderly osteoporosis due to a defect in osteoblastogenesis. We have found that oral administration of PPARγ activator rosiglitazone decreased tibia BMD and serum ALP but left serum calcium and osteoclast marker c-terminal telopeptide unaffected. In addition, we examined the inhibitory mechanisms of PPARγ on the bone formation by using PPARγ activators ciglitazone and 15-deoxy-Δ12,14-prostaglandin-J2 (15d-PGJ2). Our data indicated that PPARγ ligands inhibited both mineralized bone nodules and alkaline phosphatase (ALP) activities in cultured primary osteoblasts. Reverse transcription polymerase chain reaction (RT-PCR) showed that the expression of bone morphogenetic protein-2 (BMP-2) and osteocalcin (OCN) was inhibited by ciglitizone and 15d-PGJ2. Furthermore, PPARγ ligands inhibited NFκB associated downstream COX-2 and iNOS osteogenic signaling. The ultrasound (US)-induced elevation of COX-2 and iNOS expression and nitric oxide (NO) production were attenuated in the presence of PPARγ ligands. Furthermore, local administration of PPARγ ligands into the metaphysis of rat tibia decreased the bone volume in secondary spongiosa. These results suggest that the activation of PPARγ inhibits osteoblastic differentiation and the expression of several anabolic mediators involved in bone formation. These data may reflect osteoporosis and less bone formation in the aging people and patients treated with thiazolidinediones. Heme-oxygenase-1 (HO-1), an important enzyme involved in vascular disease, transplantation, and inflammation, catalyzes the degradation of heme into carbon monoxide and biliverdin. It has been reported that overexpression of HO-1 inhibits osteoclastogenesis. However, the effect of HO-1 on osteoblast differentiation is still not clear. We here used adenoviral vector expressing recombinant human HO-1 and HO-1 inducer hemin to study the effects of HO-1 in primary cultured osteoblasts. The results showed that induction of HO-1 inhibited the maturation of osteoblasts including mineralized bone nodule formation, alkaline phosphatase activity and decreased mRNA expression of several differentiation markers such as alkaline phosphatase, osteocalcin, and RUNX2. Furthermore, downstream products of HO-1, bilirubin, carbon monoxide and iron, are involved in the inhibitory action of HO-1. HO-1 can be induced by H2O2, lipopolysaccharide (LPS) and inflammatory cytokines such as TNF-α and IL-1β in osteoblasts and also in STZ-induced diabetic mice. In addition, endogenous PPARγ ligand, 15d-PGJ2 markedly increased both mRNA and protein levels of HO-1 in osteoblasts via PI3K-Akt and MAPK pathways. Blockade of HO activity by ZnPP IX antagonized the inhibitory action on osteocalcin expression by hemin and 15d-PGJ2. Our results indicate that upregulation of HO-1 inhibits the maturation of osteoblasts and HO-1 may be involved in oxidative- or inflammation-induced bone loss. Integrins are heterodimeric cell surface receptors which mediate cell-cell and cell-matrix interaction. The vitronectin and osteopontin receptors, αvβ3 integrin, is highly expressed and implicated in the adhesion, activation and migration of osteoclasts on the bone surface as well as osteoclast polarization. αvβ3 integrin plays an important role in osteoclast differentiation and resorption. In addition, Arg-Gly-Asp (RGD)-containing peptides, small molecular inhibitors and blocking antibodies to αvβ3 integrin have been shown to inhibit bone resorption in vitro and in vivo. In this study, we examined the effects disintegrins, derivatives of rhodostomin conjugated with human serum albumin (HSA-ARLDDL, derivative P and derivative X) and highly selective to αvβ3, on RANKL-induced osteoclastogenesis and ovariectomy (OVX)-induced osteoporosis. In RANKL-induced osteoclastogenesis, derivatives of rhodostomin significantly inhibited osteoclast formation and IC50 was at nM range. Post-treatment of HSA-ARLDDL also inhibits osteoclast formation. Furthermore, weekly administration of HSA-ARLDDL and derivative P significantly inhibited the increase of serum bone resorption marker and decrease of cancellous bone loss in tibia and femur induced in OVX mice. Rhodostomin derivatives also decreased bone resorption markers and ameliorated the reduction of total hip BMD in OVX rats. These results suggest that the highly selective and long-duration of αvβ3 integrin antagonists, rhodostomin derivatives, could be developed for effective drugs for the treatment of postmenopausal osteoporosis. Matrix metalloproteinase (MMP) of collagenase-3 (MMP-13) plays an important role in the degradation of cartilage in pathologic conditions. MMP-13 is found to be elevated in joint tissues in both rheumatoid arthritis (RA) and osteoarthritis (OA) patients. In addition, inflammation-stimulated synovial fibroblasts are able to release MMP-13 and other cytokines related to these syndromes. PPARγ signaling controls adipogenesis and glucose metabolism. A growing evidence indicated that PPARγ plays a critical role in inflammatory arthritis by regulating B cell response and inhibiting MMP-13 production in stimulated synovial fibroblasts and chondrocytes and natural PPARγ ligand, 15d-PGJ2 is reported to be more potent than other synthetic PPARγ compounds in the inhibition of inflammatory arthritis. 15d-PGJ2 may exert its effects via PPARγ-dependent and also PPARγ-independent pathways. In this study, it was found that 15d-PGJ2 markedly inhibited LPS- and TNF-α-induced MMP-13 production in human synovial fibroblasts. In addition, activation of nuclear factor κB (NF-κB) is strongly associated with MMP-13 induction by TNF-α and 15d-PGJ2 markedly attenuated the translocation of NF-κB by direct inhibition of the activation of IKK via a PPARγ-independent manner. These results indicate that elevation of 15d-PGJ2 levels in the inflammatory joint disease may have therapeutic potential in the prevention of cartilage degradation.