轉化生長因子-beta家族之訊息傳遞對物理性與化學性刺激調節細胞功能之機制探討:TGF-β1與流體剪力對平滑肌細胞分化與癌細胞程序性死亡之影響

Physical and Chemical Regulation of Cell Fate by Transforming Growth Factor Superfamily Signaling: Roles of TGF-β1 and Fluid Shear Stress in Smooth Muscle Cell Differentiation and Cancer Cell Death Programming

連聖傑(Sheng-Chieh Lien)

指導教授 ： 裘正健 ； 張俊彥 ； 張大慈

國立清華大學/生命科學院/分子與細胞生物研究所/博士(2013年)

摘要

細胞經常性地受到生化與物理微環境的影響，生化反應藉由化學物質與其特定受體結合產生刺激而機械力則藉由調節細胞膜上的力學感應器進而傳導訊息進入胞內並調控細胞功能。因此，化學與物理性因子在細胞訊息傳遞、基因表現、細胞結構與功能的調節作用上扮演重要角色。
為了證實轉化生長因子β1 (transforming growth factor beta 1, TGF-1)在調控間質幹細胞細胞週期與進行平滑肌細胞分化之機制，我們檢測了C3H10T1/2間質細胞受TGF-β1所調控之細胞週期與平滑肌細胞型態轉變功能。此外，近來在平滑肌功能調節的研究中，過氧化物酶增生劑活化受體 (peroxisome proliferator activated receptor, PPAR)與其興奮劑受到相當大的關注，在本研究的結果中，發現了(1) TGF-β1誘導C3H10T1/2細胞分化為平滑肌細胞型態是透過PI3K/Akt/p70S6K 訊息傳遞路徑。 (2) PPAR-α興奮劑(即WY14,643與clofibrate)可抑制C3H10T1/2細胞受TGF-β1誘發生成之平滑肌細胞標記蛋白表現和SRF與DNA結合活性的能力，但 PPAR-δ/β興奮劑 (GW501516)或PPAR-γ興奮劑(troglitazone)則無此功能。 (3) WY14,643與clofibrate 可抑制TGF-β1所造成的Smad3/Akt/P70S6K訊息傳遞路徑活化。 (4) Smad3分子調節 TGF-β1造成之細胞週期停滯 。 (5) PPAR-α調控10T1/2細胞週期停滯於G0/G1時期，而此現象與 TGF-β受體無關。以上結果可推測PPAR-α 分子調控10T1/2 cells之細胞週期與其受TGF-β1誘導之平滑肌細胞型態轉變。
間隙流所造成的機械力可調控腫瘤細胞轉移與侵入以及抗癌藥物輸送等功能。為了釐清流體剪力調節腫瘤細胞生存之機制，我們將四種不同腫瘤細胞株施以不同剪力大小之層流或震盪流。實驗結果發現，當施以0.5到12 dynes/cm2之層流剪力時，將造成四種腫瘤細胞株的死亡。以Hep3B肝癌細胞作為模型進行研究時，發現此死亡現象僅在層流作用下可見，在震盪型剪力則不可見。兩種型態的流場對於正常的肝細胞則不會造成其死亡。在四種腫瘤細胞株中，層流剪力會增加細胞中凋亡標記annexin V-FITC之螢光染色以及cleaved caspase-8, -9, -3與PARP蛋白的表現。此外，藉由偵測酸性液泡微粒之生成、LC3B蛋白轉型以及p62/SQSTM1 降解等現象，發現層流剪力亦誘發Hep3B細胞之自噬反應。以小片段干擾型RNA進行短期轉殖，我們發現剪力造成Hep3B細胞所產生的凋亡與自噬反應是經由骨型態蛋白受體1B (BMPRIB)、Smad1/5與 p38 MAPK等分子所調控。
綜觀來說，我們的結果指出TGF-β 家族之訊息傳遞會調控間質細胞的平滑肌細胞型態轉變與機械性流體力所誘導的凋亡與自噬反應。 我們的發現也提供機械性微環境調節分子信息、基因表現、細胞存亡與功能之機制的新觀點。TGF-β 家族之訊息傳遞調控可能對建立改善心血管或癌症疾病的新方法有所助益。此外，機械流體力學與TGF-β 家族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。

關鍵字

凋亡 ； 自噬反應 ； 機械性流體力 ； 過氧化物酶增生劑活化受體 ； 平滑肌細胞型態轉變 ； 轉化生長因子β1

並列摘要

Cells are constantly influenced by their biochemical and physical microenvironments. Chemical ligands binding to their specific receptors under biochemical stimulation and mechanical forces regulating membrane mechanosensors, e.g., integrins, can transduce information into the cell and modulate cell functions. Thus, the modulation of cell signaling, gene expression, structure and function by both chemical and physical factors plays an important role in health and disease.
To demonstrate the mechanisms by which chemical stimuli of transforming growth factor-β1 (TGF-β1) regulating the cell cycle and the differentiation of mesenchymal cells into smooth muscle cells (SMCs), we examined the role of TGF-β1-mediated cell cycle control and SMC phenotypic modulation of C3H10T1/2 (10T1/2) mesenchymal cells. Furthermore, peroxisome proliferator-activated receptors (PPARs) and their agonists have recently gained more attention in the study of the SMC function. In this study, the results showed the following: (1) the PI3K/Akt/p70S6K signaling cascade is involved in TGF-β1-induced differentiation of 10T1/2 cells into cells with a SMC phenotype. (2) PPAR-α agonists (i.e., WY14,643 and clofibrate), but not a PPAR-δ/β agonist (GW501516) or PPAR-γ agonist (troglitazone), inhibit TGF-β1-induced SMC markers and the DNA binding activity of serum response factor (SRF) in 10T1/2 cells. (3) WY14,643 and clofibrate inhibit the TGF-β1 activation of the Smad3/Akt/P70S6K signaling cascade. (4) TGF-β1-induced cell cycle arrest at the G0/G1 phases is mediated by Smad3 in 10T1/2 cells. (5) The PPAR-α-mediated 10T1/2 cell cycle arrest at the G0/G1 phases is TGF-β receptor independent. These results suggest that PPAR-α mediates cell cycle control and TGF-β1-induced SMC phenotypic changes in 10T1/2 cells.
Mechanical forces, including interstitial fluid flow in and surrounding tissues, can modulate metastasis and invasion of tumor cells, and anticancer drug delivery. To elucidate the shear stress-regulated mechanism on tumor cell survival, four tumor cell lines (i.e., Hep3B, MG63, SCC25 and A549) were exposed to laminar or oscillatory flow at different magnitude of shear stress. We found that laminar shear stress (LSS) ranging from 0.5 to 12 dynes/cm2 induces death of these four tumor cell lines. Only laminar (0.5 dynes/cm2) but not oscillatory shear stress (0.5±4 dynes/cm2) could significantly induce the level of Hep3B hepatocarcinoma cell death. Both shear patterns had no effect on normal hepatocyte Chang liver cell line. LSS increased the percentage of cells positive for annexin V-FITC staining in all four cell lines up to 72 h after flow exposure, with cleaved caspase-8, -9, and -3, and PARP up-regulated by shear stress. In addition, LSS also induced Hep3B cell autophagy, as detected by acidic vesicular organelle formation, LC3B transformation, and p62/SQSTM1 degradation. By transiently transfecting small interfering RNA, we found that the shear-induced apoptosis and autophagy are mediated by bone morphogenetic protein receptor type 1B (BMPRIB), BMPR-specific Smad1 and Smad5, and p38 MAPK in Hep3B cells.
In summary, our results indicate that TGF-βsuperfamily signaling may mediate the smooth muscle phenotypic change of mesenchymal cells and the mechanical flow force-induced tumor cell apoptosis and autophagy. Our findings also provide new insights into the mechanisms by which the mechanical microenvironment modulates molecular signaling, gene expression, cell survival, and functions in tumor cells. The modulation of TGF-β-superfamily signaling may be useful in establishing new approaches to the treatment of a variety of cardiovascular or tumor disorders. Moreover, the communication between mechanical-flow forces and BMP signaling may contribute to new research directions for treating tumor patients, and further detailed investigations are needed.

並列關鍵字

Apoptosis ； Autophagy ； Mechanical Flow Force ； PPAR ； Smooth Muscle Cell Phenotypic Change ； TGF-β1

參考文獻

[62] S.F. Chang, C.A. Chang, D.Y. Lee, P.L. Lee, Y.M. Yeh, C.R. Yeh, C.K. Cheng, S. Chien, J.J. Chiu, Tumor cell cycle arrest induced by shear stress: Roles of integrins and Smad, Proceedings of the National Academy of Sciences of the United States of America, 105 (2008) 3927-3932.

[1] J. Massague, S.W. Blain, R.S. Lo, TGFbeta signaling in growth control, cancer, and heritable disorders, Cell, 103 (2000) 295-309.

[3] B. Schmierer, C.S. Hill, TGFbeta-SMAD signal transduction: molecular specificity and functional flexibility, Nature reviews. Molecular cell biology, 8 (2007) 970-982.

[4] K.H. Wrighton, X. Lin, X.H. Feng, Phospho-control of TGF-beta superfamily signaling, Cell research, 19 (2009) 8-20.

[5] X. Chen, L. Xu, Mechanism and regulation of nucleocytoplasmic trafficking of smad, Cell & bioscience, 1 (2011) 40.