Transforming growth factor-beta promotes pro-fibrotic behavior by serosal fibroblasts via PKC and ERK1/2 mitogen activated protein kinase cell signaling - PubMed (original) (raw)

Transforming growth factor-beta promotes pro-fibrotic behavior by serosal fibroblasts via PKC and ERK1/2 mitogen activated protein kinase cell signaling

Jurgen J W Mulsow et al. Ann Surg. 2005 Dec.

Abstract

Objective: To assess the role of fibroblasts, transforming growth factor (TGF)-beta, and cell signal pathways in promoting fibrosis in Crohn's disease (CD).

Summary background data: Intestinal strictures are a major source of morbidity in CD. Fibroblasts found at sites of stricture promote fibrogenesis. The mechanisms underlying this pro-fibrotic behavior remain elusive.

Methods: Fibroblasts were isolated from strictured and macroscopically normal serosa in patients with CD and from normal serosa in patients with colorectal cancer. Whole cell connective tissue growth factor (CTGF) and fibronectin expression were determined by Western blot analysis. Fibroblast type I collagen expression was evaluated by real-time PCR, while fibroblast contractile activity was measured using fibroblast populated collagen lattices. Cells were stimulated with TGF-beta1 and inhibitors of the protein kinase C (PKC) and ERK 1/2 mitogen activated protein (MAP) kinase cell signaling pathways.

Results: Stricture fibroblasts displayed enhanced constitutive expression of fibronectin. TGF-beta promoted fibroblast CTGF, fibronectin, and type I collagen expression and enhanced fibroblast contractile activity. Inhibition of PKC reduced basal collagen expression and contractile activity in Crohn's fibroblasts and attenuated the effect of TGF-beta on fibroblast CTGF, fibronectin, and collagen I expression as well as fibroblast contractility. ERK 1/2 inhibition had a similar effect on TGF-beta-induced CTGF and fibronectin expression.

Conclusions: TGF-beta is a critical pro-fibrotic growth factor in CD, and its effects are mediated via PKC and ERK 1/2 MAP kinase cell signaling. These pathways may represent novel therapeutic targets for patients with CD characterized by recurrent intestinal stricture formation.

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Figures

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FIGURE 1. TGF-β cell signaling via Smad proteins and ERK 1/2 MAP kinase pathways. The ERK 1/2 pathway is activated by phosphorylation in a PKC-dependent or -independent manner and signals to the nucleus where gene transcription is regulated. Go6850 is a broad spectrum PKC inhibitor, PD98059 inhibits MEK 1, while Uo126 inhibits both MEK 1 and 2 subtypes.

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FIGURE 2. CTGF and fibronectin expression by intestinal fibroblasts. Basal and TGF-β1 (1 ng/mL for 24 hours) induced CTGF and fibronectin expression were assessed in control and Crohn's fibroblasts by Western blot analysis of whole cell protein extracts. Equal protein loading was confirmed by staining for Coommaisse blue and β-actin. Western blots were scanned and assessed for protein densitometry (B, C). A, Representative Western blots from 2 patients with Crohn's disease comparing CTGF and fibronectin expression by fibroblasts cultured from strictured and nonstrictured intestine. Fibronectin protein band densitometry is shown (B) and is expressed relative to unstimulated fibronectin levels in nonstricture fibroblasts (n = 5 patients with Crohn's disease). Data are mean ± SEM. *P < 0.05 versus unstimulated nonstricture fibroblasts (analysis of variance [ANOVA]). C, Relative induction of fibronectin by TGF-β1 in control (n = 3) and nonstricture (n = 5) fibroblasts. Data are mean ± SEM. *P < 0.05 versus unstimulated cells (Student t test).

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FIGURE 3. Collagen I expression and contraction by intestinal fibroblasts following stimulation with TGF-β1. (A) Relative induction of collagen type I assessed by real-time PCR following stimulation with TGF-β1 (1ng/mL) for 24 hours in nonstricture (n = 7) and stricture (n = 4) fibroblasts. Data are mean + SEM. *P < 0.05 relative to unstimulated fibroblasts (Student t test). (B) TGF-β1 stimulated collagen lattice contraction by nonstricture fibroblasts (n = 3). The area of contracting lattices is shown after 5 days of culture in the presence of TGF-β1 and is expressed as a percentage of the starting FPCL size. Medium containing 1% FCS acted as a control. Data are mean ± SEM. *P < 0.05 relative to 1% FCS control (analysis of variance [ANOVA]).

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FIGURE 4. Phosphorylation of ERK 1/2 MAP kinase in nonstricture and stricture fibroblasts following stimulation with TGF-β1 for 30 minutes. Protein band densitometry for phosphorylated ERK 1/2 is shown graphically below representative Western blots and is adjusted for total ERK 1/2 expression.

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FIGURE 5. Effect of PKC and ERK 1/2 inhibition on the TGF-β1 induction of CTGF (A, n = 10), and fibronectin (B, n = 7) in control and nonstricture fibroblasts. Cells were pretreated with Go6850 (Go, 10 μmol/L), Uo126 (Uo, 50 μmol/L), and PD98059 (PD, 50 μmol/L) for 45 minutes prior to the addition of TGF-β1 (1 ng/mL) for 24 hours. Fibroblast CTGF and fibronectin expression were determined by Western blot analysis. Protein band densitometry is shown graphically beneath representative Western blots. Data are mean ± SEM. *P < 0.05 versus TGF-β1 alone (analysis of variance [ANOVA]).

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FIGURE 6. Effect of PKC and ERK 1/2 inhibition on collagen expression by nonstricture fibroblasts cultured from patients with Crohn's disease. The effect of Go6850 (Go, 10 μmol/L) and PD98059 (PD, 50 μmol/L) on basal (A, n = 4) and TGF-β1 (1 ng/mL) (B, n = 5) induced collagen type I expression after 24 hours was assessed using real-time PCR. Median, interquartile range, and range are indicated by horizontal bar, rectangular box, and error bars, respectively. *P < 0.05 versus TGF-β alone (Mann-Whitney U test).

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FIGURE 7. The effect of Go6850 and PD98059 on basal (A, n = 6), and TGF-β1 (B, n = 3) induced contractile activity by nonstricture fibroblasts. Fibroblast populated collagen lattices were treated with TGF-β1 (1 ng/mL), Go6850 (10 μmol/L), and PD98059 (50 μmol/L). Data are shown following 3 days contraction and are plotted as the percentage inhibition of contraction, relative to unstimulated (A) or TGF-β1 (B) induced contraction. Data are mean ± SEM. *P < 0.05 versus unstimulated (A) or TGF-β1 (B) alone (analysis of variance [ANOVA]).

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