|
Vascular smooth muscle cells (VSMCs) undergo phenotypic changes in response to environmental cues. Normally, VSMCs have a contractile phenotype physiologically. An increased synthetic phenotype, characterized by higher growth and migration, has been implicated in restenosis and atherosclerosis. The precise mechanism of redifferentiation is still not completely understood. Our laboratory recently has demonstrated that reactive oxygen species (ROS), through NADPH oxidase 4 (NOX4), play a critical role in the differentiation of VSMC (2).
Actin is an ATPase that cycle between monomeric (G-actin) and polymerized (F-actin) states. In addition to its classical role as a cytoskeletal component, actin has a long history of proposed connections with nuclear events that occur in the cell. Numerous studies have suggested potential roles for actin controlling its own expression both transcriptionally and at the level of mRNA translation and stability (6). Autoregulation of actin transcription occurs via a mechanism in which G-actin binds to and controls the activity of MAL/MKL1, a coactivator of the serum response factor (SRF) transcription factor (6). SRF, a MADS-box protein, controls a large number of growth factor-inducible and muscle-specific genes through the mutually exclusive association of different SRF cofactors, and the expression of many of these genes is thus influenced by G-actin level (6). Alterations in actin dynamics are required for RhoA-mediated SRF activation, which is inhibited upon treatment of cells with the G-actin binding drug latrunculin or C2 toxin (6). The RhoA-actin pathway controls a subset of SRF target genes, including the immediate-early genes β-actin, vinculin, srf, smooth muscle-specific 22 and SM α-actin genes (6). Cytochalasins are fungal metabolites that have the ability to bind to actin filaments and block polymerization and the elongation of actin. As a result of the inhibition of actin polymerization, cytochalasins can change cellular morphology and inhibit cellular processes such as cell division (4). Cytochalasins are known to bind to the barbed, fast growing plus ends of microfilaments, which then blocks both the assembly and disassembly of individual actin monomers from the bound end (2). Due to its effects on actin polymerization, cytochalasins are good tools to help researchers understand the importance of actin in various biological processes (2).
p38 mitogen-activated kinase (p38) has been linked to the differentiation process in fibroblasts and cardiomyocytes. Moreover, several studies from our labs and others have demonstrated that p38 is ROS dependent. We hypothesize that ROS generation via NOX4, as well as p38 activation, regulates the redifferentiation of VSMC induced by changes in G-actin.
Cell Culture
After treatment, human VSMCs were cultured in 10% fetal bovine serum (FBS) growth media. Prior to treatment, the cells were serum-starved for 24 h. All cells were harvested at passages 4 to 7 and used at 70% to 80% confluence for differentiation marker expression, inhibition and time course experiments.
Western Blotting
After treatment, human VSMCs were washed three times with cold PBS. Then, HVSMC were lysated using Hunters buffer and sonicated for 10 seconds. The protein concentrations were determined by the method of Bradford with BSA as a standard. Fractions were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked and incubated with specific corresponding primary antibodies. Proteins were detected by ECL. Band intensity was quantified by densitometry of immunoblots using Image J.
Measurement of p38 activity
p38 activity was measurement by the determination of phospho Heat Shock Protein 27 (HSP27), which is one the its substrated, by Western Blot using a specific antibody against phospho HSP27.
ROS generation via NOX4, as well as p38 activation, regulates the redifferentiation of VSMC induced by changes in G-actin
CD induces SMα Actin in HVSMC in a dose response manner
Figure 1. CD induces SMα Actin. HAVSMC were treated with different doses (1,2,5 and 10 μM) of CD for 24 hours. SMα Actin was measured by Western Blot using an antibody specific against SMα Actin and Tubulin (as a loading control).
CD induces p38 activity
Figure 2. CD induces p38 activation and kinase activity. HAVSMC were treated with 5µM of CD for 1,5,10,30 and 60 minutes. The p38 activation level was measured by Western blot, using antibodies against phospho p38 (A) or phospho HSP27 (B).
p38 and ROS production are critical in the SMα Actin expression induced by
Figure 3. ROS production and p38 activation are essentials in the SMα Actin expression by CD. HAVSMC were preincubated with NADPH oxidase inhibitor (DPI, 10μM), p38 inhibitor (SB-203580, 10 μM), RhoA kinase or ROCK (Y-27632, 10 μM) or TGFβ receptor inhibitor (SB-431542, 10 μM) for 90 minutes. Then, HVSMC were treated with CD (5 μM) during 24 hours. SMα Actin was measured by Western Blot using an antibody specific against SMα Actin and Tubulin (as a loading control).
Summary
CD induces SMα Actin in HVSMC
CD increases p38 activation and kinase activity
p38 MAPK is important in the expression of the differentiation marker, SMα actin, in VSMCs
ROS production is necessary for CD induction of SMα actin in human VSMCs
ROS production and p38 activity are necessary in the expression of Smooth Muscle α Actin induced by changes in the G-actin pool
Future Directions
Identify the NADPH oxidase implicated in this process
Determine whether p38 activation by CD is ROS dependent
Study the transcription factors implicated in this process as well as its sensitivity for ROS and p38
David I. Brown and Mitsuhisa Koga.
This material is based upon work supported by the Howard Hughes Medical Institute under Grant No.52005873 and by the Graduate Division of Biological and Biomedical Sciences, Emory University
1. Clempus RE, Sorescu D, Dikalova AE, Pounkova L, Jo P, Sorescu GP, Schmidt HH, Lassegue B and Griendling KK. Nox4 is required for maintenance of the differentiated vascular smooth muscle cell phenotype. Arterioscler Thromb Vasc Biol 27(1), 2007.
2. Cooper, John A. (1987). Effects of Cytochalasin and Phalloidin on Actin. J. Cell Biol. 105, 1473-1478.
3. Griendling, K. K., D. Sorescu, et al. (2000). NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res 86(5): 494-501.
4. Haidle, Andrew and Myers, Andrew (2004). An enantioselective, modular, and general route to the cytochalasins: Synthesis of L-696,474 and cytochalasin B. Proceedings of the National Academy of Sciences of the United States of America 101, 12048-12053.
5. Hilenski, L. L., R. E. Clempus, et al. (2004). Distinct subcellular localizations of Nox1 and Nox4 in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 24(4): 677-83.
6. Posern G, Sotiropoulos A and Treisman R. Mutant actins reveal a role for unpolymerised actin in control of transcription by Serum Response Factor. Mol Biol Cell 2002; 13: 4167-4178
|
