轉化生長因子beta調控平滑肌細胞中第二型富含半胱胺酸蛋白質表現之分子機轉

Transforming Growth Factor beta Induces Cysteine-rich Protein 2 Expression in Vascular Smooth Muscle Cells

鄔孟陵(Wu, Meng Ling)

指導教授 ： 陳令儀 ； 林秀芳

國立清華大學/生命科學院/分子醫學研究所/博士(2015年)

https://doi.org/10.6843/NTHU.2015.00447

摘要

動脈管壁中的平滑肌細胞，除了調控血壓此一重要的生理功能外，血管平滑肌細胞對於阻塞型血管疾病(例如：動脈硬化)的形成，也扮演很重要的角色。第二型富含半胱胺酸蛋白質主要表現在動脈平滑肌細胞中，其功能為限制血管平滑肌細胞移行，以保護血管免於病理性的血管內皮層增生。轉化生長因子β(Transforming Growth Factor β)為一多功能生長因子，動脈硬化形成時，轉化生長因子β透過其下游的許多訊息傳遞路徑參與其中，我們先前的研究發現，轉化生長因子β可增加第二型富含半胱胺酸蛋白質的表現，但其中調控的訊息傳遞路徑，仍尚待釐清。本篇研究旨在探討轉化生長因子β調控第二型富含半胱胺酸蛋白質表現之分子機制。以轉化生長因子β刺激血管平滑肌細胞後，除了會增加傳統的訊息傳遞分子Smad2及Smad3的磷酸化外，也會增加ATF2的磷酸化蛋白表現。利用RNA干擾(small interfering RNA)技術減少Smad2/3及ATF2的表現後，皆能有效地減少轉化生長因子β所增加的第二型富含半胱胺酸蛋白質表現，顯示Smad2/3及ATF2皆參與了轉化生長因子β對第二型富含半胱胺酸蛋白質的調控。不論利用第一型轉化生長因子β接受器激酶的抑制劑(SB431542)，抑制接受器上的激酶活性;或是利用RNA干擾技術，減少第一型轉化生長因子β接受器的表現，皆可有效降低轉化生長因子β所引起的Smad2/3磷酸化，但並不影響ATF2的磷酸化，由此可知，只有Smad2/3的磷酸化與第一型轉化生長因子β接受器有關，但ATF2的磷酸化與第一型轉化生長因子β接受器無關。利用Src家族酪氨酸激酶抑制劑(SU6656)，可降低轉化生長因子β所引起的RhoA和ATF2的活化，但不影響Smad2的活化。阻斷RhoA下游主要訊息分子－ROCK的活性，亦能降低轉化生長因子β所引起的ATF2磷酸化及第二型富含半胱胺酸蛋白質表現，但Smad2的磷酸化則不受影響。我們更進一步地發現，JNK激酶抑制劑(SP600125)也能抑制轉化生長因子β所引起的ATF2磷酸化及第二型富含半胱胺酸蛋白質表現，不過轉化生長因子β所引起的JNK激酶活化，也會受到ROCK抑制劑的阻斷。這些結果顯示，轉化生長因子β透過其第二型接受器，活化下游的Src家族酪氨酸激酶/RhoA/ROCK/JNK此一訊息傳遞路徑，調控ATF2的磷酸化。經由啟動子活性分析可知，轉化生長因子β透過啟動子上，CRE和SBE這兩個相鄰的結合位置，來增加第二型富含半胱胺酸蛋白質表現。上述的結果顯示，轉化生長因子β利用兩條不同的訊息傳遞路徑，分別作用在啟動子上CRE和SBE兩個結合位置，協同調控第二型富含半胱胺酸蛋白質表現，此一轉化生長因子β對於平滑肌細胞基因的特殊調控方式，是首度被報導。

關鍵字

轉化生長因子 ； 第二型富含半胱胺酸蛋白

並列摘要

Vascular smooth muscle cells (VSMCs) of the arterial wall play a critical role in the development of occlusive vascular diseases. Cysteine-rich protein 2 (CRP2) is a VSMC-expressed LIM-only protein, which functionally limits VSMC migration and protects against pathological vascular remodeling. The multifunctional cytokine TGFβ has been implicated to play a role in the pathogenesis of atherosclerosis through numerous downstream signaling pathways. We showed previously that TGFβ upregulates CRP2 expression; however, the detailed signaling mechanisms remain unclear. TGFβ treatment of VSMCs activated both Smad2/3 and ATF2 phosphorylation. Individually knocking down Smad2/3 or ATF2 pathways with siRNA impaired the TGFβ induction of CRP2, indicating that both contribute to CRP2 expression. Inhibiting TβRI kinase activity by SB431542 or TβRI knockdown abolished Smad2/3 phosphorylation but did not alter ATF2 phosphorylation, indicating while Smad2/3 phosphorylation was TβRI-dependent ATF2 phosphorylation was independent of TβRI. Inhibiting Src kinase activity by SU6656 suppressed TGFβ-induced RhoA and ATF2 activation but not Smad2 phosphorylation. Blocking ROCK activity, the major downstream target of RhoA, abolished ATF2 phosphorylation and CRP2 induction but not Smad2 phosphorylation. Furthermore, JNK inhibition with SP600125 reduced TGFβ-induced ATF2 (but not Smad2) phosphorylation and CRP2 protein expression while ROCK inhibition blocked JNK activation. These results indicate that downstream of TβRII, Src family kinase-RhoA-ROCK-JNK signaling pathway mediates TβRI-independent ATF2 activation. Promoter analysis revealed that the TGFβ induction of CRP2 was mediated through the CRE and SBE promoter elements that were located in close proximity. Our results demonstrate that two signaling pathways downstream of TGFβ converge on the CRE and SBE sites of the Csrp2 promoter to cooperatively control CRP2 induction in VSMCs, which represents a previously unrecognized mechanism of VSMC gene induction by TGFβ.

並列關鍵字

TGFbeta ； CRP2

參考文獻

Adam, P.J., Regan, C.P., Hautmann, M.B., and Owens, G.K. (2000). Positive- and negative-acting Kruppel-like transcription factors bind a transforming growth factor β control element required for expression of the smooth muscle cell differentiation marker SM22α in vivo. J. Biol. Chem. 275, 37798-37806.

Arber, S., Halder, G., and Caroni, P. (1994). Muscle LIM protein, a novel essential regulator of myogenesis, promotes myogenic differentiation. Cell 79, 221-231.

Arber, S., Hunter, J.J., Ross, J., Jr., Hongo, M., Sansig, G., Borg, J., Perriard, J.C., Chien, K.R., and Caroni, P. (1997). MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure. Cell 88, 393-403.

Bhoumik, A., Takahashi, S., Breitweiser, W., Shiloh, Y., Jones, N., and Ronai, Z. (2005). ATM-dependent phosphorylation of ATF2 is required for the DNA damage response. Mol. Cell 18, 577-587.

Bandyopadhyay, B., Han, A., Dai, J., Fan, J., Li, Y., Chen, M., Woodley, D.T., and Li, W. (2011). TβRI/Alk5-independent TβRII signaling to ERK1/2 in human skin cells according to distinct levels of TβRII expression. J. Cell Sci. 124, 19-24.