Rac inhibition reverses the phenotype of fibrotic fibroblasts - PubMed (original) (raw)
Rac inhibition reverses the phenotype of fibrotic fibroblasts
Shi-wen Xu et al. PLoS One. 2009.
Abstract
Background: Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce alpha-smooth muscle actin (alpha-SMA), type I collagen and CCN2 (connective tissue growth factor, CTGF). The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies.
Methods and findings: Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc) patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766.
Conclusion: Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Rac activity is elevated in dermal fibroblasts cultured from scars of SSc patients.
Cells were loaded with or without GTPγS (positive control, to assess maximal Rac activation) or GDP (as a negative control). Rac-GTP was immunoprecipitated from cell lysates. The precipitated Rac-GTP was detected by immunoblot analysis using anti-Rac. In parallel, whole cell lysates were subjected to SDS/PAGE and Western blot analysis with an anti-rac antibody. Quantitative densitometry data is indicated on the right. Lanes are: GTPγS, GDP, cells from three normal individuals (N1, N2, N3) and three individuals with SSc (SScF1, SScF2, SScF3). Experiments were performed on cells derived from 6 normal individuals and 6 SSc patients. Quantitative densitometry data is indicated on the right (* = p<0.05 relative to wild-type control).
Figure 2. Rac inhibition suppresses the pro-fibrotic phenotype of lesional SSc fibroblasts. mRNA and protein analysis.
Fibroblasts were from normal individuals (NF) and individuals with SSc (SScF) were assessed in the presence or absence of the rac-specific inhibitor (NSC23766, 24 hour treatment 50 µM). (A) Real-time PCR analysis. Messenger RNA was harvested from cells and subjected to real-time PCR analysis to detect the mRNAs indicated. Fibroblasts from 6 individuals were analyzed. Data represent averages and standard deviation. As a control, 28S RNA was amplified. Average of three replicates from three separate individuals, adjusted for 28S RNA expression values, are shown (+/− SD * = p<0.05 significant inhibition by NSC23766 compared to untreated controls, Student's paired t test). (B) Western blot analysis. Proteins were harvested and subjected to Western blot analysis with antibodies directed against the proteins indicated. Densitometry is on the right (+/− SD, * = p<0.05 significant inhibition by NSC23766 compared to untreated controls, Student's paired t test) (C) Immunofluorescence analysis. Cells were fixed and stained with anti-vinculin antibody to detect focal adhesion and rhodamine phalloidin to detect actin and α-SMA.
Figure 3. Rac inhibition suppresses the pro-fibrotic phenotype of lesional SSc fibroblasts. Gel contraction analysis.
Rac inhibitor 50 µM NSC23766 (24 hour treatment) reduced the ability of lesional SSc fibroblasts to contract a collagen gel matrix: (A) FPCL analysis. The effect of loss of rac inhibition on the ability of fibroblasts to exert contractiles force in a fixed, tethered floating collagen gel lattice was investigated using a Culture Force Monitor. Forces generated by fibroblasts were measured over 24 hours; a representative trace is shown (N = 3). (B) Floating gel analysis. The effect of rac inhibition on ECM contraction over 24 hours generated by fibroblasts embedded in a floating collagen gel matrix was evaluated. Contraction was assessed photographically and by measuring gel weight iameter of contracted gels (n = 6; Average +/− standard deviation is indicated; * = p<0.05, statistically different from control untreated normal fibroblasts, ANOVA). Note that NSC23766 suppressed the enhanced ECM contraction by lesional SSc fibroblasts.
Figure 4. Rac inhibition reduces the enhanced migration of lesional SSc fibroblasts.
Fibroblasts were subjected to a scratch wound assay in the presence or absence of 50 µM NSC23766. Cells were cultured on fibronectin until reaching confluence. A uniform linear scrape wound was made across the cell layer. Three independent experiments were performed Gap size expressed as a percentage of the original wound is shown (average +/− standard deviation) (* = p<0.05 statistically different relative to untreated healthy controls).
Figure 5. Overexpression of rac in normal fibroblasts results in a profibrotic phenotype in a PI3kinase/Akt-dependent fashion: protein analysis.
(A) Transfection of constitutively active rac (ca rac), compared to empty expression vector (empty), increases profibrotic protein expression. Twenty four hours post-transfection, protein was harvested and subjected to Western blot analysis was conducted with antibodies detecting the proteins indicated. Average +/− standard deviation is shown (N = 3, * = p<0.05). (B) The effect of rac overexpression is reduced by PI3 kinase inhibition. Cells were transfected with empty expression vector or constitutively active rac (ca rac) in the presence or absence of 100 nM wortmannin, 10 µM Ly294002, 10 µM U0126 and 10 µM SP600125. Cells were then processed for Western blot analysis as described in (A).
Figure 6. Akt phosphorylation is elevated in dermal fibroblasts and is reduced by rac inhibition.
Whole cell lysates were subjected to SDS/PAGE and Western blot analysis with an anti-phospho Akt and anti-Akt antibodies in the absence (TOP PANEL) or presence or absence (BOTTOM PANEL) of 50 µM NSC23766. Quantitative densitometry data is indicated on the right. Experiments were performed on cells derived from 6 normal individuals and 6 SSc patients. Quantitative densitometry data is indicated on the right (* = p<0.05 relative to wild-type control).
Figure 7. Overexpression of rac in normal fibroblasts results in a profibrotic phenotype phenotype in a PI3kinase/Akt-dependent fashion: collagen gel contraction analysis.
(A) Transfection of constitutively active rac (ca rac), compared to empty expression vector (empty), increases ECM contraction by normal fibroblasts. Eighteen hours post-transfection, cells were subjected to the floating collagen gel model of ECM contraction in the presence or absence of 100 nM wortmannin, 10 µM Ly294002, 10 µM U0126 or 10 µM SP600125. Average +/− standard deviation is shown (N = 3, * = p<0.05). (B) Rac overexpression rescues the reduced ECM contraction in rac knockout fibroblasts (K/K) versus control fibroblasts (C/C). Cells were transfected with empty expression vector or constitutively active rac (ca rac) in the presence or absence of wortmannin or Ly294002. (*, p<0.05, Student's t test).
References
- Gabbiani G. The myofibroblast in wound healing and fibrocontractive diseases. J Pathol. 2003;200:500–3. - PubMed
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