成肌蛋白FHOD1分子羧基端新的Rac1结合域的确定及其在血管平滑肌细胞表型转变中的作用

Phenotype change of vascular smooth muscle cells (VSMC) from contractile to synthetic is a key step in the pathogenesis of atherosclerosis and post-stent re-stenosis of the coronary artery lumen due to the neointima formation. Restraining VSMC within the contractile phenotype as fully as possible is a novel approach for future prevention and treatment of atherosclerosis. Two famous signaling pathways, nitric oxide/ANP-sGC-cGMP-PKG, and RhoA-ROCK-actin-myocardin/MRTF-serum response factor, have been reported to be deeply involved in the process of keeping VSMC within the contractile phenotype by regulating actin stress fiber (SF) formation as well as a series of VSMC-specific protein expression, like SM-MHC, SM-alpha-actin, calponin, smoothelin, and SM22-alpha. In the downstream of both pathways, a key regulator of SF formation is formin homology domain-containing protein 1 (FHOD1), a member of formin in human. ..We have previously reported that FHOD1 is a substrate of PKG in VSMC. PKG phosphorylates FHOD1 1131S within the C-terminal auto-regulatory domain (DAD) and FHOD1 binds specially to Rac1, but not RhoA and Cdc42, in a GTP-independent way (Wang-YP, et al. J Biol Chem. 2004; 279:24420-6). However, the physiological significance of these two processes is still unclear. Four years later, on the basis of our report, a Japanese group reported that another two adjacent residues, 1137S and 1141T in the DAD, can be phosphorylated by ROCK. Only getting all three residues phosphorylated could almost fully activate FHOD1 (Takeya-R, et al. EMBO J. 2008; 27:618–28)...Our pilot studies include: 1) we discovered a novel Rac1 binding site in FHOD1 C-terminus, which is also GDP/GTP-independent; 2) P32 autoradiography phosphorylation assay of a series of FHOD1 phosphoresistent or phosphomimetic mutants by pure PKG, ROCK1, ROCK2 demonstrated that PKG only phosphorylates 1131S and that ROCK2, but not ROCK1, phosphorylates 1137S and 1141T; 3) As FHOD1 C-terminus gets more sites phosphorylated, it could recruit more Rac1; 4) FHOD1 is down-regulated in the neointima formed in the injured rat carotid artery. ..Next, we plan to: 1) ascertain both in vitro and in vivo that the FHOD1 C-terminus binding is specific to Rac1; 2) test whether Rac1-FHOD1 C-terminus interaction could regulate PKG/ROCK2 phosphorylation as well as FHOD1 activation; and 3) study whether Rac1-FHOD1 C-terminus interaction could regulate VSMC’s phenotype. Our study aims to investigate whether Rac1-FHOD1 C-terminus interaction is a new regulator of FHOD1 activation and a drug-discovery target for effectively restraining VSMC in the contractile phenotype.

血管平滑肌细胞（VSMC）表型转变是动脉粥样硬化及支架安置后再狭窄病理发生中的关键步骤。如何维持其收缩表型是防治粥样硬化的新思路。蛋白激酶G（PKG）及ROCK激酶是增加肌纤维合成从而阻止其表型转变的重要通路，二者下游有肌纤维合成的关键分子FHOD1。我们前期研究解明PKG能磷酸化FHOD1-1131S及FHOD1氨基端能特异结合Rac1，但二者的生理意义不明。在此基础上日本人报告了边上新位点1137S及1141T，三点都被ROCK磷酸化后能激活FHOD1。我们预实验发现FHOD1羧基端还有一个Rac1新结合域；突变体放射自显影揭示PKG只磷酸化1131S，ROCK2只磷酸化另两位点；随着磷酸化增多羧基端结合Rac1能力提高；大鼠颈动脉新生内膜中FHOD1表达下调；接下来要在细胞内证明Rac1新结合域；Rac1如何调节两激酶及其对VSMC表型转变的作用，从而提供维持收缩表型的新分子靶点。

近年我国心脑血管疾病的发病率和死亡率呈迅速上升趋势，而高血压又是造成心脑血管疾病的元凶，在世界范围内影响约30%的成年人口。血管平滑肌细胞（VSMC）肥大、增殖迁移及分泌细胞外基质蛋白能力的提高在血管中膜增厚变硬的过程中发挥重要角色。FHOD1作为一种促肌丝形成蛋白，极有可能促进VSMC肥大、迁移。且目前已有研究表明，FHOD1可促进肿瘤细胞增殖。因此，阐明FHOD1如何影响VSMC形态及生理功能对高血压血管病变有着极为重要的研究意义。.我们取得的主要结果有：.（1）高血压大鼠动脉组织中FHOD1的磷酸化水平及表达水平均增高。血管紧张素Ⅱ(Angll)刺激体外培养的大鼠VSMC，发现FHOD1的磷酸化水平及表达水平亦增高。.（2）为进一步确定高血压环境中FHOD1磷酸化水平及表达水平的变化对VSMC表型的影响，我们构建了6个FHOD1相关的腺病毒。通过转染VSMC，观察FHOD1磷酸化水平及表达水平对VSMC肥大、增殖、迁移及分泌能力的影响。结果发现具有持久活性的FHOD1-ΔN（421-1164）、FHOD1-3D（1-1131D-1137D-1141D-1164）可以显著提高VSMC内肌丝合成、促进VSMC肥大标志物aSMA的表达，促进细胞肥大、迁移。而不具有持久活性的FHOD1-Fulllength（1-1131S-1137S-1141T-1164）、FHOD1-2D（1-1131A-1137D-1141D-1164）、FHOD1-1D（1-1131A-1137A-1141D-1164）、FHOD1-3A（1-1131A-1137A-1141A-1164）则不影响细胞内肌丝合成、VSMC肥大标志物aSMA的表达，亦不影响细胞迁移，但可以轻度促进细胞面积增大。此外，我们发现无论是否具有持久活性，FHOD1过表达均可以促进VSMC增殖以及细胞外基质蛋白的表达。该结果提示我们，FHOD1发挥其作用不仅仅依赖其活性，无活性的FHOD1也可以发挥作用。.（3）为进一步探究FHOD1下游分子机制、以及具有活性的FHOD1和无活性的FHOD1作用机制的差别，我们将转染了FHOD1腺病毒的VSMC收样，进行蛋白质表达谱分析，质谱分析结果还需1月余返回。