細胞間質在骨代謝及骨腫瘤形成之探討

Studies of extracelluar matrix in bone metabolism and osteosarocoma formation

湯智昕(Chih-Hsin Tang)

指導教授 ： 符文美

共同指導教授 ： 楊榮森

國立臺灣大學/醫學院/藥理學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.00977

摘要

Fibronectin (Fn) 為造骨細胞 (osteoblast) 分泌的骨細胞間質，Fn在調控細胞黏著 (adhesion)、移行 (migration) 及造骨細胞的成熟中扮演了一個非常重要的角色。而Fn的纖維固化 (fibrillogenesis) 也參與骨骼礦化過程中，Bone morphogenetic protein-4 (BMP-4) 可從骨間質中抽取出來且有促進骨質形成的作用。在本論研究當中我們研究BMP-4對老鼠造骨細胞Fn間質纖維固化的調控作用。BMP-4可以增加Fn合成及細胞外的纖維固化，BMP-4也可以增加由外界直接加入soluble Fn的纖維固化。在之前的報告有指出α5 及β1 integrins與Fn fibrillogenesis有關，BMP-4也可以增加α5 及β1 integrins的合成，利用免疫螢光染色法也可看到促進α5 及β1 integrins的聚合。BMP-4可以增加造骨細胞黏著到Fn間質上，而蛇毒蛋白triflavin及Gly-Arg-Gly-Asp-Ser (GRGDS) 可以拮抗其增加的作用。BMP-4可以促進extracellular signal-regulated kinase (ERK) 及 focal adhesion kinase (FAK) 的磷酸化。BMP-4增加細胞Fn纖維固化及 β1 mRNA的表現會受到ERK kinase (MEK) 抑制劑PD 98059所拮抗。本結果顯示BMP-4可以增加造骨細胞Fn纖維聚合能力，此作用與增加Fn合成及α5 及β1 integrins聚合有關。

basic fibroblast growth factor (bFGF) 為一個重要調控骨合成的因子，bFGF可增加造骨細胞分泌Fn及外給soluble human Fn纖維固化能力，使用西方點墨法也發現bFGF增加Fn蛋白量，Protein kinase C (PKC) 抑制劑H7、GF109203X、Ro 318220 及Gő 6976都可拮抗bFGF所增加Fn合成。給予造骨細胞bFGF可促進PKCα、β、ε及δ轉移至細胞膜，然而使用不同的PKC antisense oligonucleotides (AS-ODN)，證明只有PKCα 及β參與bFGF增加Fn的作用中。使用高濃度TPA (1 uM) 處理24小時來down-regulate PKC則可抑制bFGF增加Fn的纖維固化。使用免疫螢光染色法發現bFGF促進 α5 integrins的聚合，利用流式細胞分析也觀察到bFGF增加 α5 及 β1 integrins在細胞上的表現，PKC抑制劑可拮抗bFGF促進 α5 及 β1 integrins的作用。經由事先埋在幼鼠脛骨內的針頭局部給予bFGF，可以增加脛骨內Fn蛋白合成，若同時給予PKC抑制劑則可拮抗此作用。長時間局部給予bFGF可以增加次級海綿骨的骨量，且PKC抑制劑可阻斷bFGF的作用。因此，bFGF可以經由PKC訊息傳遞路徑增加骨合成及Fn 纖維固化。

Prostaglandin E (PGE) 是另一個重要調控骨合成的因子，使用免疫螢光染色法及enzyme-linked immunosorbent assay (ELISA) 發現PGE2增加Fn纖維固化，西方點墨法觀察到PGE2增加Fn蛋白量。藥物抑制劑或利用AS-ODN基因抑制法發現EP1接受器參與PGE2增加Fn合成作用中，其他EP接受器的AS-ODN並無此效果。在訊息傳遞方面，鈣離子螯合劑BAPTA-AM、phosphatidylinositol-phospholipase C (PI-PLC) 抑制劑 U73122 或 Src抑制劑 PP2都可拮抗PGE2增加Fn的作用。使用PKC抑制劑GF109203X也可阻斷PGE2的作用。利用PKCα、β、ε及δAS-ODN發現只有PKCα參與PGE2增加Fn合成。使用流式細胞儀及RT-PCR分析，PGE2 及 17-phenyl trinor PGE2 (EP1/EP3 作用劑) 都可增加α5 或β1 integrins的表現。轉殖pGL2F1900-Luc後給予PGE2 或 17-phenyl trinor PGE2可以增加Fn promoter activity，共同轉殖PKCα或c-Src的dominant negative mutants可拮抗PGE2增加Fn promoter activity。局部給予PGE2或17-phenyl trinor PGE2在幼鼠的脛骨可增加次級海綿骨內Fn及α5β1 integrin 免疫染色含量及骨量。因此PGE2增加Fn合成及促進骨合成是經由EP1/ PLC/PKCαc-Src 訊息傳遞路徑。
一氧化碳 (NO) 為一種自由基，在骨合成中也扮演重要角色。YC-1可加強NO對soluble guanylate cyclase (sGC) 的活化，在本論文我們研究YC-1對於骨合成的作用。使用免疫螢光染色法發現YC-1增加培養骨細胞Fn纖維固化，流式細胞分析也發現YC-1促進造骨細胞的α5, α2, β1 integrin表現。給予YC-1二星期會增加 alkaline phosphatase (ALP) 活性，NOS抑制劑L-NAME、sGC抑制劑 ODQ及cyclic GMP-dependent protein kinases (PKG) 抑制劑KT 5823都可拮抗YC-1的作用。處理造骨細胞YC-1二星期後會促進骨小結 (bone nodule) 及collagen合成。破骨前驅細胞加入分化劑RNAKL (50 ng/ml)及MCS-F (20 ng/ml) 分化2星期以形成破骨細胞，YC-1可抑制破骨細胞的分化。使用osteoclast activity assay substrate (OAAS) plate分析破骨細胞吸收能力，YC-1隨著濃度抑制破骨細胞再吸收的作用。經由22G的針頭局部給予YC-1在幼鼠脛骨內，可以增加次級海綿骨的骨量，而L-NAME、ODQ及KT 5823都可拮抗YC-1的作用。每天腹腔注射YC-1 (1 mg/kg/day) 可以防止由卵巢切除所引起的骨質流失。因此，YC-1應可發展為臨床上促進骨合成、治療骨質疏鬆症的藥物。

細胞間質的交互作用在細胞的黏著、移行、增殖 (proliferation) 及分化 (differentiation) 扮演了重要的角色。癌細胞表現多種不同的黏著因子，這與癌症轉移 (metastasis) 有密不可分的關係。使用ROS 17/2.8骨癌細胞與初級造骨細胞比較二者黏著因子的差異，發現二者在α5 integrin或Fn 纖維固化上有不同的分佈。使用免疫螢光染色法及流式細胞分析發現，正常造骨細胞比ROS 17/2.8有較高的α5、β1、α2 integrins及FAK。Crovidisin為snake venom metalloproteinase (SVMP)，從Crotalus viridis 的蛇毒中所分離出來，具有collagen-binding activity及matrix metalloproteinase (MMP) activity双重活性。Crovidisin可選擇性造成ROS 17/2.8骨癌細胞的脫落 (detachment) 但對正常造骨細胞没有影響。在另一方面triflavin為RGD-dependent disintegrin從Trimersurus flavoviridis的蛇毒中所分離出來，都不會造成ROS 17/2.8骨癌細胞及正常造骨細胞的脫落。ROS 17/2.8處理crovidisin 在造成細胞脫落的24小時的時間點上，並不會引起粒線體膜電位的改變，但處理36小時則造成粒腺體膜電位下降。這結果顯示正常造骨細胞與ROS 17/2.8骨癌細胞表現不同的integrin及細胞間質的環境，造成ROS 17/2.8對crovidisin較敏感。而間質的分解則是crovidisin引起ROS 17/2.8脫落的主要原因。

腫瘤細胞轉移至骨頭的過程包括癌細胞的移行、侵入 (invasion) 及黏著至骨頭。乳癌及前列腺癌是最常轉移至骨頭的二種癌細胞。從蛇毒分離出來的RGD-containing disintegrin (e.g. rhodostomin) 可以抑制癌細胞黏著。Rhodostomin可抑制乳癌及前列腺癌細胞黏著至由造骨細胞所分泌未礦化或礦化的間質上，這抑制黏著的作用並不會影響癌細胞的存活率。另外，rhodostomin也可抑制乳癌及前列腺癌細胞的移行及侵入作用。分析disintegrin所能結合至癌細胞上的integrin，只有7E3 monoclonal antibody (MoAb) (已證實可結合至細胞的αvβ3 integrin上) 可抑制rhodostomin結合至癌細胞，其他α2、α3、α5及β1 integrins MoAb並無此作用。乳癌細胞MDA-MB-231局部注射至裸鼠的脛骨內，經過28天後發現腫瘤生長及骨破壞的現象，同時注射trigramin則可以抑制腫瘤生長及其骨破壞的面積。Disintegrin為強力癌細胞黏著、移行、侵入的抑制劑，且抑制乳癌細胞在骨頭上的生長。因此，disintegrin可發展為治療腫瘤轉移至骨頭的臨床藥物。

細胞間質與其接受器在細胞黏著、移行、增殖及分化中扮演了重要的角色，在本論文中我們發現成長因子 (BMP、bFGF、PGE2及NO)都可促進Fn合成進而增加骨生成，因此這些成長因子都是發展治療骨質疏鬆症的標的。在另一方面，蛇毒蛋白成份disintgrin具有integrin-dependent抑制癌細胞黏著、移行及侵入到骨骼的作用，應是一個良好抑制癌症轉移至骨頭的藥物。

關鍵字

造骨細胞 ； 破骨細胞 ； 骨合成 ； 骨質疏鬆症 ； 細胞間質

並列摘要

The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin (Fn), which regulates the adhesion, differentiation, and function of osteoblasts. Fn fibrillogenesis is involved in the process of bone mineralization. Bone morphogenetic proteins (BMPs) can be isolated from organic bone matrix and are able to initiate de novo cartilage and bone formation. In this study, the effect of BMP-4 on Fn fibrillogenesis in cultured rat osteoblasts was examined. BMP-4 enhanced Fn synthesis and extracellular Fn assembly in primary cultured osteoblasts. In addition, the extracellular assembly of Fn from exogenously applied soluble human Fn was also increased by BMP-4. It has been reported thatα5β1 integrin is related to Fn fibrillogenesis. The synthesis of bothα5 andβ1 integrins was up-regulated by BMP-4. Immunocytochemistry showed that the clustering ofα5 andβ1 integrins was also increased by BMP-4. BMP-4 increased fibril formation of Fn and the adhesion of osteoblasts onto Fn matrix, which was inhibited by disintegrin triflavin and Gly-Arg-Gly-Asp-Ser (GRGDS) peptide. Phosphorylation of extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK) was increased by BMP-4. Enhancement of extracellular Fn fibrillogenesis and the mRNA expression ofβ1 integrin by BMP-4 were inhibited by ERK kinase (MEK) inhibitor PD98059. These results suggest that the enhancement of extracellular Fn fibrillogenesis by BMP-4 in cultured osteoblasts is related to the increase of the synthesis of Fn and clustering ofα5 andβ1 integrins. ERK is involved in the signaling pathway of BMP-4 in regulating Fn fibrillogenesis in osteoblasts.

basic fibroblast growth factor (bFGF) is an important factor regulating osteogenesis. We here found that bFGF enhanced extracellular assembly from either endogenously released or exogenously applied soluble Fn in primary cultured osteoblasts. bFGF increased protein levels of Fn using Western blotting analysis. Protein kinase C (PKC) inhibitors such as H7, GF109203X, Ro 318220 or Gő 6976 antagonized the increase of Fn protein by bFGF. Treatment of osteoblasts with bFGF increased membrane translocation of various isoforms of PKC, including α, β, ε, and δ. However, treatment with antisense of various PKC isoforms demonstrated that α and β isozymes play important roles in the enhancement action of bFGF on Fn assembly. Down-regulation of PKC by prolonged treatment with 1 μM TPA for 24 h inhibited the potentiating action of bFGF. Immunocytochemistry showed that bFGF treatment increased the clustering of α5 integrins. Flow cytometry analysis demonstrated that bFGF increased cell surface expression of α5 and β1 integrins and PKC inhibitors antagonized the increase by bFGF. Local administration of bFGF into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the protein levels of Fn in the area of trabecular spongiosa, which was inhibited by coadministration of PKC inhibitors. Furthermore, local injection of bFGF increased the bone volume of secondary spongiosa in tibia, which was significantly antagonized by PKC inhibitors. These results suggest that bFGF increased bone formation and Fn fibrillogenesis both in vitro and in vivo via PKC-dependent pathway.

Prostaglandin E (PGE) is an other important factor regulating osteogenesis. Here we found that PGE2 enhanced extracellular Fn assembly in rat primary osteoblasts, as shown by immunofluorescence staining and enzyme-linked immunosorbent assay (ELISA). PGE2 also increased the protein levels of Fn using Western blotting analysis. Using pharmacological inhibitors or activators or genetic inhibition by EP receptor antisense oligonucleotides (AS-ODN) revealed that EP1 receptor but not other PGE receptors is involved in PGE2-mediated up-regulation of Fn. At the mechanistic level, Ca2+ chelater (BAPTA-AM), phosphatidylinositol-phospholipase C (PI-PLC) inhibitor (U73122) or Src inhibitor (PP2) attenuated the PGE2-induced Fn expression. PKC inhibitor (GF109203X) also inhibited the potentiating action of PGE2. Furthermore, treatment with AS-ODN of various PKC isoforms including α, β, ε, and δ demonstrated that α isozyme plays important role in the enhancement action of PGE2 on Fn assembly.Flow cytometry and RT-PCR showed that PGE2, 17-phenyl trinor PGE2 (EP1/EP3 agonist) increased the surface expression and mRNA level of α5 or β1 integrins. Fn promoter activity was enhanced by PGE2 and 17-phenyl trinor PGE2 in cells transfected with pGL2F1900-Luc. Cotransfection with dominant negative mutants of PKCαor c-Src inhibited the potentiating action of PGE2 on Fn promoter activity. Local administration of PGE2 or 17-phenyl trinor PGE2 into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the Fn andα5β1 integrin immuostaining and bone volume of secondary spongiosa in tibia. Taken together, our results provided evidence that PGE2 increased Fn and promotes bone formation in rat osteoblasts via the EP1/ PLC/PKCα/c-Src signaling pathway.

Nitric oxide (NO) is a free radical which has important effects on bone cell function. We have investigated the effect of YC-1, a drug known to modulate the response of soluble guanylyl cyclase (sGC) to NO, on the bone formation. Fn is involved in early stages of bone formation, using immunocytochemistry and flow cytometric analysis demonstrated that YC-1 increased Fn fibrillogenesis and cell surface expression of α5, α2, β1 integrin. Treatment of osteoblasts with YC-1 for 2 weeks increased the activity of alkaline phosphatase (ALP) and NO synthase (NOS) inhibitor L-NAME, sGC inhibitor ODQ, cyclic GMP-dependent protein kinases (PKG) inhibitor KT 5823 antagonized the potentiating action of YC-1. Incubation of YC-1 for 2 weeks increased the formation of bone nodule and collagen synthesis in primary cultured osteoblast. Treatment with YC-1 inhibited RNAKL (50 ng/ml) and MCS-F (20 ng/ml)-induced differentiation of osteoclasts derived from bone marrow stromal cells. In addition, assay of osteoclast activity on substrate (OAAS) plate, YC-1 significantly inhibited the resorption activity of osteoclasts in a concentration-dependent manner. Daily injection of YC-1 (0.1 nmole/day), via local intraosseous route into the metaphysis of the tibia in young male rats for 7 days increased metaphyseal trabecular bone. The potentiation of bone growth by YC-1 was greatly reduced by L-NAME, ODQ and KT 5823. NO-cGMP-PKG pathway is thus involved in the enhancement of bone growth by YC-1. Daily intraperitoneal injection of YC-1 (1 mg/kg/day) could prevent bone loss caused by ovarietomy in adult rats. YC-1 is thus a good drug candidate to enhance bone growth and for the treatment of osteoporosis.

The interaction of extracellular matrix with cells plays a key role in the regulation of cell adhesion, migration, proliferation as well as differentiation. Transformed cells express a different profile of adhesion molecules, which may mediate metastasis under specific matrix microenvironment. We here found that ROS 17/2.8 osteosarcoma cells and osteoblasts have different expression of α5 integrin, executing different Fn fibrillogenesis. As compared with ROS 17/2.8 cells, osteoblasts have higher expression of Fn, collagen, α5, β1, α2 integrins and FAK as examined by immunostaining and flow cytometry. Crovidisin, a PIII snake venom metalloproteinase (SVMP) purified from venom of Crotalus viridis, exhibits collagen-binding activity and matrix metalloproteinase activity. Crovidisin selectively caused the detachment of ROS 17/2.8 osteosarcoma cells but not of primary cultured osteoblasts. On the other hand, triflavin, an RGD-dependent disintegrin purified from venom of Trimeresurus flavoviridis, did not cause the detachment of both osteoblasts and ROS 17/2.8 cells. Although ROS 17/2.8 cells detached from substratum after crovidisin treatment for 24 h, the loss of mitochondrial membrane potential was not observed unless a prolonged treatment for longer than 36 h. These results suggest that cultured primary rat osteoblasts and ROS 17/2.8 osteosarcoma cells possess different expression of integrins and matrix environment, and ROS 17/2.8 is much more susceptible to be detached by crovidisin. The matrix degradation by crovidisin may be responsible for the preferential detachment of ROS 17/2.8 osteosarcoma cells.

The metastasis of tumor cells to bone involves migration, invasion and adhesion to bone. Breast and prostate cancer cells have predilection for spreading to bone. Snake venom-derived RGD-containing disintegrins (e.g. rhodostomin) have been demonstrated to inhibit cell adhesion. Here, we found that rhodostomin inhibited the adhesion of breast and prostate carcinoma cells to both unmineralized and mineralized bone extracellular matrices in a dose-dependent manner, without affecting the viability of tumor cells. In addition, rhodostomin also inhibited the migration and invasion of breast and prostate carcinoma cells. It specifically inhibited the binding of monoclonal antibody (MoAb) 7E3, which recognizes integrin αvβ3, to tumor cells, but not those of other MoAbs against other integrin subunits such as α2, α3, α5 and β1. As breast cancer cells MDA-MB-231 were locally injected into tibia in nude mice, histological examination of the tibia of control group revealed that most of the cancellous bone had been replaced by the breast cancer cells after 28 days’ inoculation. In contrast, co-administration of trigramin with cancer cells markedly inhibited tumor growth and bone destruction. Taken together, disintegrins strongly inhibit the adhesion, migration, invasion of tumor cells and also tumor growth of human breast cancer cells in bone as well. Therefore, disintegrins may be developed as alternate therapy for bone metastasis of cancer cells.

The extracellular matrix and its receptor plays a key role in the regulation of cell adhesion, migration, proliferation as well as differentiation. Our results provided evidence that growth factors (BMP, bFGF, PGE2 and NO) increased Fn expression and promotes bone formation. Therefore, growth factors may be a good target to develop for the treatment of osteoporosis. In the other hand, disintegrin inhibit integrin-dependent cell adhesion, migration and invasion. Disintegrin is a good candidate to therapy for bone metastasis.

並列關鍵字

osteoblast ； osteoclast ； bone formation ； osteoporosis ； extracelluar matrix

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