Bond Mark, Sala-Newby Graciela B, Newby Andrew C
Bristol Heart Institute, University of Bristol, Bristol BS2 8HW, United Kingdom.
J Biol Chem. 2004 Sep 3;279(36):37304-10. doi: 10.1074/jbc.M404307200. Epub 2004 Jun 18.
Smooth muscle cell (SMC) proliferation is suppressed in intact blood vessels but stimulated in atherosclerosis, restenosis after angioplasty, and vein graft disease. The cyclin-dependent kinase inhibitors, including p27(Kip1), play important roles in maintaining SMC quiescence. Levels of p27(Kip1) are dependent on attachment to and the composition of the extracellular matrix (ECM). Here we sought to elucidate mechanisms underlying the ECM-dependent regulation of p27(Kip1) and hence, SMC proliferation. Serum stimulation decreased p27(Kip1) levels in isolated SMC but not in rat aorta. The effect was post-translational and mediated by proteasomal degradation. We studied the S-phase-associated kinase protein-2 (Skp-2), an F-box protein involved in ubiquitination and proteasome-mediated degradation. Skp-2 protein is strongly induced by serum from undetectable levels in isolated SMCs but remains undetectable in aorta; Skp-2 mRNA is also lower in aorta. Overexpression of wild-type Skp-2 in SMCs decreased p27(Kip1) levels, whereas dominant negative F-box deleted mutant (DeltaF-Skp-2) Skp-2 increased p27(Kip1) levels. Furthermore, hyperphosphorylation of retinoblastoma protein and SMC proliferation were also reciprocally affected by wild-type and dominant negative Skp-2. Skp-2 expression was absolutely dependent on cell attachment to the ECM and was inhibited by laminin and type-1 fibrillar collagen but increased by fibronectin. Expression of Skp-2 protein, but not mRNA, was associated with focal adhesion kinase (FAK) activity and inhibited by overexpression of FAK-related non-kinase and a dominant negative FAK(Y397F) mutant. Furthermore, the inhibition of Skp-2 expression by dominant negative FAK was reversed by the proteasome inhibitor MG-132. Taken together, these data demonstrate that the vascular ECM controls SMC proliferation via FAK-dependent regulation of Skp-2 protein stability.
在完整血管中，平滑肌细胞（SMC）增殖受到抑制，但在动脉粥样硬化、血管成形术后再狭窄及静脉移植物疾病中则受到刺激。细胞周期蛋白依赖性激酶抑制剂，包括p27（Kip1），在维持SMC静止状态中发挥重要作用。p27（Kip1）的水平取决于与细胞外基质（ECM）的附着及ECM的组成。在此，我们试图阐明ECM依赖性调节p27（Kip1）进而调节SMC增殖的潜在机制。血清刺激可降低分离的SMC中p27（Kip1）水平，但对大鼠主动脉无此作用。该效应是翻译后水平的，且由蛋白酶体降解介导。我们研究了S期相关激酶蛋白-2（Skp-2），一种参与泛素化和蛋白酶体介导降解的F盒蛋白。Skp-2蛋白在分离的SMC中由血清从不可检测水平强烈诱导产生，但在主动脉中仍不可检测；Skp-2 mRNA在主动脉中也较低。在SMC中过表达野生型Skp-2可降低p27（Kip1）水平，而显性负性F盒缺失突变体（DeltaF-Skp-2）Skp-2则增加p27（Kip1）水平。此外，视网膜母细胞瘤蛋白的过度磷酸化及SMC增殖也分别受到野生型和显性负性Skp-2的相互影响。Skp-2的表达绝对依赖于细胞与ECM的附着，并受到层粘连蛋白和I型纤维状胶原的抑制，但被纤连蛋白增加。Skp-2蛋白而非mRNA的表达与粘着斑激酶（FAK）活性相关，并被FAK相关非激酶及显性负性FAK（Y397F）突变体的过表达所抑制。此外，显性负性FAK对Skp-2表达的抑制被蛋白酶体抑制剂MG-132逆转。综上所述，这些数据表明血管ECM通过FAK依赖性调节Skp-2蛋白稳定性来控制SMC增殖。
