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Regulation of the mesangial cell myofibroblast phenotype by actin polymerization
Keyur Patel1, Pamela Harding, Lisa B Haney
1Department of Pathology and Anatomy, Eastern Virginia Medical School, Norfolk 23501, USA.
Abstract:
Mesangial cells in diverse glomerular diseases become myofibroblast-like, characterized by activation of smooth muscle alpha-actin (alpha-SMA) expression. In cultured mesangial cells, serum-deprivation markedly increases alpha-SMA expression, cell size, and stress fiber formation. Since stress fibers are assembled from actin monomers, we investigated the hypothesis that alterations in stress fiber formation regulate alpha-SMA expression and hypertrophy. Human mesangial cells were treated with agents that disrupt or stabilize actin stress fibers. Depolymerization of actin stress fibers in serum-deprived cells with actin-depolymerizing agents, cytochalasin B (CytB) and latrunculin B (LatB), or with inhibitors of Rho-kinase, Y-27632 and HA-1077 decreased alpha-SMA mRNA as judged by Northern blot analysis. Western blot analysis showed that CytB also reduced alpha-SMA protein levels. In serum-fed cells, agents that stabilized actin stress fibers, jasplakinolide (Jas) and phalloidin, increased alpha-SMA mRNA and protein. Treatment of human or rat mesangial cells with CytB, LatB, or Y-27632 decreased alpha-SMA promoter activity. In contrast, Jas increased promoter activity 5.6-fold in rat mesangial cells. The presence of an RNA polymerase inhibitor blocked degradation of alpha-SMA mRNA in cells treated with CytB suggesting that destabilization of this message is dependent on a newly transcribed or rapidly degraded factor. Inhibition of actin polymerization by CytB, LatB, Y-27623, and HA-1077 inhibited incorporation of (3)[H]-leucine into newly synthesized protein. Additionally, CytB and LatB decreased cell volume as determined by flow cytometry. Collectively, these results indicate that the state of polymerization of the actin cytoskeleton regulates alpha-SMA expression, hypertrophy, and myofibroblast differentiation in mesangial cells.
Insights
The actin cytoskeleton
Area of Science:
- Cell Biology
- Nephrology
- Biochemistry
Background:
- Glomerular diseases involve mesangial cells transforming into myofibroblast-like cells, marked by increased smooth muscle alpha-actin (alpha-SMA) expression.
- Serum deprivation in cultured mesangial cells significantly elevates alpha-SMA expression, cell size, and stress fiber formation.
- Stress fibers are constructed from actin monomers, prompting investigation into their role in regulating alpha-SMA expression and hypertrophy.
Purpose of the Study:
- To investigate the hypothesis that alterations in actin stress fiber formation regulate alpha-SMA expression and mesangial cell hypertrophy.
- To explore the impact of actin cytoskeleton polymerization state on alpha-SMA expression, cell size, and myofibroblast differentiation.
Main Methods:
- Human and rat mesangial cells were treated with agents that either disrupt (cytochalasin B, latrunculin B, Y-27632, HA-1077) or stabilize (jasplakinolide, phalloidin) actin stress fibers.
- Alpha-SMA mRNA and protein levels were assessed using Northern and Western blot analyses.
- Alpha-SMA promoter activity was measured, and cell volume was determined by flow cytometry. Protein synthesis was evaluated using (3)[H]-leucine incorporation.
Main Results:
- Disrupting actin stress fibers with cytochalasin B, latrunculin B, or Rho-kinase inhibitors decreased alpha-SMA mRNA and promoter activity, and cytochalasin B reduced alpha-SMA protein.
- Stabilizing actin stress fibers with jasplakinolide or phalloidin increased alpha-SMA mRNA and protein, and jasplakinolide significantly enhanced promoter activity.
- Inhibition of actin polymerization reduced protein synthesis and cell volume, while destabilization of alpha-SMA mRNA was dependent on a newly transcribed or rapidly degraded factor.
Conclusions:
- The state of actin cytoskeleton polymerization critically regulates alpha-SMA expression in mesangial cells.
- Actin stress fiber dynamics influence mesangial cell hypertrophy and the transition to a myofibroblast-like phenotype.
- Targeting actin polymerization pathways may offer therapeutic strategies for glomerular diseases characterized by mesangial cell activation.