骨型態發生蛋白的訊息途徑對細胞功能之探討：BMP-4和流體剪力對骨母細胞分化作用及腫瘤細胞生長作用之影響

Abstract

骨型態發生蛋白（bone morphogenetic proteins, BMPs）是屬於轉型蛋白（transforming growth factor-beta, TGF-β）中的一種生長因子，因其具有在體內誘導異位（ectopic site）骨頭或軟骨生成作用的能力而被發現與命名，但近幾年研究發現，骨型態發生蛋白在腫瘤細胞上也具有重要的影響性。骨型態發生蛋白-4為骨格生成作用中最重要的生長因子之一，但經由其他研究發現，骨型態發生蛋白-4也對其他種類細胞的細胞功能具有調控作用，包含正常細胞和腫瘤細胞的生長或細胞凋亡。機械微環境（mechanical microenvironment）在組織的發展、維持、功能或致病上相當重要，近期研究發現，機械流體力學（mechanical flow forces）對腫瘤細胞的生長具有影響性，也會調控Smad訊息途徑來影響細胞功能。此外，科學家在哺乳動物的細胞功能研究上發現，影響細胞週期的運行結果可調控或決定細胞功能的作用。總和以上的論述，我們假設骨型態發生蛋白的訊息途徑可調控細胞週期的運行進而影響骨型態發生蛋白-4所調節骨母細胞（osteoblasts）增生（proliferation）及分化作用（differentiation）和機械流體力學調節腫瘤細胞生長作用。本論文的研究目的為（1）觀察骨型態發生蛋白-4是否會調控細胞週期的運行進而調節骨母細胞的分化作用；（2）研究骨型態發生蛋白-4是否藉由調節細胞週期調控因子（cell cycle regulators）的表現來影響此發生過程；（3）證實細胞膜上的integrins是否與骨型態發生蛋白的細胞膜上受體（receptors）交互作用，因而促進及加強骨型態發生蛋白-4的影響；（4）觀察機械流體力學是否會調控細胞週期的運行進而調節腫瘤細胞的生長作用；（5）研究機械流體力學是否藉由骨型態發生蛋白的訊息途徑來調節細胞週期調控因子的表現進而影響此發生過程；（6）觀察機械流體力學所刺激骨型態發生蛋白的訊息途徑是否是經由刺激細胞自體分泌（autocrine）骨型態發生蛋白所引起；（7）證實細胞膜上科學家認定的機械接受器（mechanosensors）-integrins是否為腫瘤細胞接受機械力的受體，因而影響細胞內部骨型態發生蛋白的訊息途徑。 研究發現，骨型態發生蛋白-4的刺激會遏止兩種不同的骨母細胞的細胞週期，並令其停止在G0/G1時期，此作用主要是藉由增加細胞週期調控因子p21CIP1 和p27KIP1的蛋白表現所引起，並因此造成細胞進行分化作用。研究中利用小干擾RNA （small interfering RNA, siRNA）技術發現此作用主要是受到骨型態發生蛋白的訊息途徑：骨型態發生蛋白的膜上第一型受體A和Smad5所調控。此外，骨型態發生蛋白-4也會刺激ERK基酶的短暫磷酸化，研究發現當ERK基酶的活性受到抑制時會抑制骨型態發生蛋白的訊息蛋白Smad1/5的活化，且抑制細胞膜上integrin β3的表現時，會同時造成骨型態發生蛋白-4刺激ERK和Smad1/5的活化被抑制。此結果提出骨型態發生蛋白-4所造成的反應及訊息途徑會受到integrin β3-ERK所調控。 在機械流體力學對腫瘤細胞生長調控的研究發現，當給予機械流體力學所產生的剪力刺激四種腫瘤細胞24和48小時後，會遏止細胞週期的運行，並使其停止在G2/M時期，此作用主要是藉由增加細胞週期調控因子cyclin B1和p21CIP1的蛋白表現及降低cyclins A, D1, and E, Cdk-1, -2, -4, and -6, and p27KIP的蛋白表現以及Cdk1的活性所引起。研究中利用抗體（antibodies）和小干擾RNA技術發現此作用主要是透過膜上integrins αvβ3 和β1經由骨型態發生蛋白的訊息途徑：骨型態發生蛋白的膜上第一型受體A和Smad1/5所調控。此外，研究也發現剪力會透過骨型態發生蛋白的訊息途徑抑制腫瘤細胞的分化作用，包含降低轉錄因子Runx2與DNA結合的活性和分化蛋白骨鈣素（osteocalcin）和鹼性磷酸酶（alkaline phosphatase, ALP）的基因和蛋白表現。 本論文的研究結果證實骨型態發生蛋白的訊息途徑可藉由調控細胞週期的運行而調節骨型態發生蛋白-4所誘導的骨母細胞分化作用和機械流體力學所誘導的腫瘤細胞生長抑制作用。也證實機械微環境具有調控腫瘤細胞的分子機制進而影響細胞功能的作用。我們提出對骨型態發生蛋白的訊息途徑活性的調控作用可能對治療骨骼或腫瘤相關疾病提供新的方法，另外，機械流體力學與骨型態發生蛋白的訊息途徑之間的相互關係對於治療腫瘤病人可能也可提供新的研究方向。

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-β superfamily, were originally identified by their unique ability to induce ectopic bone and cartilage formation in vivo. In recent studies, BMP signaling has been suggested to play an important role not only in bone cells but also in tumor cells. BMP-4 is one of the most potent inducers of bone formation and is also demonstrated to be a potent growth factor for modulating other cells’ functions, including normal and tumor cells’ growth or apoptosis. The importance of the mechanical microenvironment in tissue development, maintenance, function and pathogenesis has been well established for several decades. Recent studies demonstrated that mechanical flow forces may affect the growth of tumor cells. Moreover, it is also suggested that mechanical flow forces may mediate the Smad signaling pathway. In mammals, the regulation of cell cycle distribution can mediate cell functions. We hypothesize that BMP signaling may regulate the cell cycle distribution, thereby mediating osteoblast proliferation and differentiation under BMP-4 treatment and mediating tumor cell growth under mechanical flow forces. The aims of this study were: (1) to observe if BMP-4 regulates the cell cycle distribution in order to induce differentiation in osteoblasts; (2) to investigate if BMP-4 signaling mediates the expression of cell cycle regulators and hence modulates this process; (3) to demonstrate if membrane integrins interact with BMP-4 receptors for optimization of the BMP-4 effects; (4) to observe if mechanical flow forces regulate the cell cycle distribution in order to affect tumor cell growth; (5) to investigate if mechanical flow forces mediate the expression of cell cycle regulators through BMP signaling and hence modulate this process; (6) to observe if mechanical flow force-stimulated BMP signaling comes from BMP autocrine effects; and (7) to demonstrate if the mechanosensor (i.e., integrin) response to mechanical flow forces induces intracellular BMP signaling. In two osteoblast-like cell lines, BMP-4 stimulation induced G0/G1 arrest. BMP-4 induced the increased expression of p21CIP1 and p27KIP1 and hence cell differentiation, but had no effect on the expression of cyclins A, B1, D1, and E, and Cdk-2, -4, and -6. Using specific small interfering RNA (siRNA), we found that the BMP-4-induced G0/G1 arrest and increased expressions of p21CIP1 and p27KIP1 were mediated by BMP receptor type IA-specific Smad5. In addition, BMP-4 induced transient phosphorylation of extracellular signal-regulated kinase (ERK); transfection with ERK-specific siRNA inhibited BMP-4-induced Smad1/5 activation. Moreover, transfection with specific siRNA for β3, but not αv and β1, integrins inhibited the BMP-4-induced ERK and Smad1/5 phosphorylation, suggesting that the BMP-4-induced responses are mediated by the β3 integrin through ERK. In four tumor cell lines, incubation under static conditions for 24 or 48 h led to G0/G1 arrest; in contrast, shear stress (12 dynes/cm2) induced G2/M arrest. Shear stress induced increased expression of cyclin B1 and p21CIP1 and decreased expression of cyclins A, D1, and E, and cyclin-dependent protein kinases (Cdk)-1, -2, -4, and -6, and p27KIP1, as well as a decrease in Cdk1 activity. Using specific antibodies and siRNA, we found that the shear-induced G2/M arrest and corresponding changes in G2/M regulatory protein expression and activity were mediated by αvβ3 and β1 integrins through bone morphogenetic protein receptor type IA-specific Smad1 and Smad5. Shear stress also down-regulated runt-related transcription factor 2 (Runx2) binding activity and osteocalcin and alkaline phosphatase expression in tumor cells; these responses were mediated by αvβ3 and β1 integrins through Smad5. Our findings indicate that BMP signaling may mediate the cell cycle distribution to regulate BMP-4-induced osteoblast differentiation and the mechanical flow forces-induced tumor cell growth arrest. Our findings also provide new insights into the mechanisms by which the mechanical microenvironment modulates molecular signaling, gene expression, the cell cycle, and functions in tumor cells. Modulation of the activities of BMP signaling may be useful in establishing new approaches to the treatment of a variety of bone or tumor disorders. Moreover, the communications between mechanical flow forces and BMP signaling may contribute new research directions for treating tumor patients, and further detailed investigations are needed.