Lundberg M S, Sadhu D N, Chilian W M, Ramos K S
Department of Veterinary Physiology, Texas A&M University, College Station 77843, USA.
J Mol Cell Cardiol. 1997 Jun;29(6):1695-702. doi: 10.1006/jmcc.1997.0423.
Co-ordinate down-regulation of smooth muscle-specific genes and acquisition of unregulated proliferative characteristics have been proposed as hallmarks of the atherosclerotic process. In the present study, we have evaluated this reciprocal relationship by examining the impact of c-Ha-rasEJ oncogene transfection on alpha-smooth muscle (SM) actin and alpha 1B-adrenoceptor (ADR) gene expression in vascular (aortic) smooth muscle cells (SMCs), c-Ha-rasEJ transfection of SMCs by lipofection (LF-1) was associated with enhanced DNA synthetic rates relative to vector controls and a significant reduction in alpha-SM actin and beta/gamma-actin mRNAs. Incubation of ras- and neo-LF-1 SMCs in a restrictive serum concentration (0.1%) for 72 h inhibited DNA synthesis in both cell types, but differentially influenced the pattern of alpha-actin gene expression. While neo-LF-1 cells incubated in 0.1% exhibited increased alpha-SM actin mRNA levels relative to 10% serum, slight decreases in alpha-SM actin were observed in ras-LF-1 cells under the same conditions. Cyclical stretch of randomly cycling cells, seeded on a flexible elastin substrate at a rate of 100 cycles/min for 72 h, did not significantly influence the pattern of alpha-SM or beta/gamma-actin mRNA expression in neo-LF-1 or ras-LF-1 cells. Steady-state mRNA levels of alpha 1B-ADR were higher in ras-LF-1 SMCs relative to neo-LF-1 cells, and stretch increased alpha 1B-ADR mRNA levels in neo-LF-1, but not ras-LF-1 cells. Stretch inhibited [1H]thymidine incorporation into DNA in both neo- and ras-LF-1 cells relative to unstretched counterparts. These results demonstrate that c-Ha-rasEJ transfection is associated with alterations in the expression of genes associated with muscle-specific functions in vascular SMCs and implicate c-Ha-ras in the regulation of phenotypic expression in SMCs.
平滑肌特异性基因的协同下调和获得不受调控的增殖特性被认为是动脉粥样硬化过程的标志。在本研究中，我们通过检测c-Ha-rasEJ癌基因转染对血管（主动脉）平滑肌细胞（SMC）中α-平滑肌（SM）肌动蛋白和α1B-肾上腺素能受体（ADR）基因表达的影响，评估了这种相互关系。通过脂质转染（LF-1）将c-Ha-rasEJ转染到SMC中，相对于载体对照，DNA合成速率增强，α-SM肌动蛋白和β/γ-肌动蛋白mRNA显著减少。将ras-LF-1和neo-LF-1 SMC在限制性血清浓度（0.1%）中孵育72小时，抑制了两种细胞类型的DNA合成，但对α-肌动蛋白基因表达模式有不同影响。虽然在0.1%血清中孵育的neo-LF-1细胞相对于10%血清时α-SM肌动蛋白mRNA水平增加，但在相同条件下，ras-LF-1细胞中α-SM肌动蛋白略有下降。以100次循环/分钟的速率在柔性弹性蛋白底物上接种随机循环的细胞，进行72小时的周期性拉伸，对neo-LF-1或ras-LF-1细胞中α-SM或β/γ-肌动蛋白mRNA表达模式没有显著影响。相对于neo-LF-1细胞，ras-LF-1 SMC中α1B-ADR的稳态mRNA水平更高，拉伸增加了neo-LF-1细胞中α1B-ADR mRNA水平，但ras-LF-1细胞中没有增加。相对于未拉伸的细胞，拉伸抑制了neo-LF-1和ras-LF-1细胞中[1H]胸苷掺入DNA。这些结果表明，c-Ha-rasEJ转染与血管SMC中与肌肉特异性功能相关的基因表达改变有关，并暗示c-Ha-ras参与SMC表型表达的调节。
