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Transfection with c-Ha rasEJ modulates alpha-actin and alpha 1B-adrenoceptor gene expression in vascular smooth
M S Lundberg1, D N Sadhu, W M Chilian
1Department of Veterinary Physiology, Texas A&M University, College Station 77843, USA.
Abstract:
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.
Insights
The c-Ha-rasEJ oncogene alters smooth muscle cell gene expression, reducing muscle-specific genes and increasing proliferation. This implicates c-Ha-ras in regulating vascular smooth muscle cell phenotype, potentially impacting atherosclerosis.
Area of Science:
- Vascular Biology
- Molecular Biology
- Oncology
Background:
- Atherosclerosis involves smooth muscle cell (SMC) gene down-regulation and increased proliferation.
- The role of oncogenes in SMC phenotypic changes during atherosclerosis requires further investigation.
Purpose of the Study:
- To investigate the impact of c-Ha-rasEJ oncogene transfection on alpha-smooth muscle (SM) actin and alpha 1B-adrenoceptor (ADR) gene expression in vascular SMCs.
- To determine how c-Ha-rasEJ affects SMC proliferation and gene expression under varying serum concentrations and mechanical stretch.
Main Methods:
- Vascular SMCs were transfected with the c-Ha-rasEJ oncogene or a vector control using lipofection.
- Cells were cultured in different serum concentrations (0.1% vs. 10%) and subjected to cyclical mechanical stretch.
- Gene expression of alpha-SM actin, beta/gamma-actin, and alpha 1B-ADR was analyzed using mRNA levels.
- DNA synthesis rates were measured by [1H]thymidine incorporation.
Main Results:
- c-Ha-rasEJ transfection enhanced SMC DNA synthesis and reduced alpha-SM actin and beta/gamma-actin mRNA levels.
- Low serum conditions differentially affected alpha-SM actin expression in ras-transfected vs. control cells.
- Alpha 1B-ADR mRNA levels were higher in ras-transfected cells, and stretch modulated ADR expression differently between cell types.
- Mechanical stretch inhibited DNA synthesis in both cell types but did not significantly alter actin mRNA expression patterns.
Conclusions:
- c-Ha-rasEJ oncogene transfection alters the expression of muscle-specific genes in vascular SMCs.
- The c-Ha-ras oncogene plays a role in regulating the phenotypic expression of vascular SMCs.
- These findings provide insights into the molecular mechanisms underlying SMC alterations in atherosclerosis.