Qiuhong Fang

Cigarette Smoke Stimulates MMP‐1 Production by Human Lung Fibroblasts Through the ERK1/2 Pathway

An imbalance between proteases and anti‐proteases is believed to play an important role in the pathogenesis of emphysema. In this study, we explored the hypothesis that cigarette smoke can alter tissue structure through an effect on the release of matrix metalloproteinase‐1 (MMP‐1) and type I tissue inhibitor of metalloproteinases (TIMP‐1). Cigarette smoke extract (CSE) significantly stimulated pro‐MMP‐1 production (determined by ELISA and immunoblots) and mRNA expression (by real‐time RT‐PCR) by human fetal lung fibroblasts (HFL‐1) in a concentration‐dependent manner (2.5–10%). High concentrations of CSE (10%) could potentially activate the latent form of MMP‐1 as the high molecular weight (52 kDa) form was converted into a low molecular weight (42 kDa) form consistent with active MMP‐1. TIMP‐1 production, however, was not significantly altered by the concentrations of CSE tested. After 30 min exposure, CSE significantly induced ERK1/2 phosphorylation, which then gradually decreased from 90 minutes to 3 hours. PD98059, a specific inhibitor of ERK‐MAPK, significantly blocked the CSE effect on ERK1/2 phosphorylation. Furthermore, PD98059 significantly inhibited the CSE effect on MMP‐1 production and mRNA expression by fibroblasts. These results suggest that cigarette smoke stimulates production and likely activates MMP‐1 through activating ERK1/2 signal transduction pathway. By inducing MMP‐1, cigarette smoke may result in excess tissue destruction and contribute to the development of emphysema.

Thrombin induces collagen gel contraction partially through PAR1 activation and PKC-e

The ability of fibroblasts to contract three-dimensional collagen gels has been used as an in vitro model of the tissue contraction which characterises both normal repair and fibrosis. Among its actions, thrombin can activate the protease-activated receptor (PAR)1 and, thereby, stimulate inflammation and repair. The current study evaluated whether thrombin could stimulate fibroblast-mediated collagen gel contraction by activating PAR1 and whether its downstream signalling depends on protein kinase C (PKC)-ϵ. Human foetal lung fibroblasts (HFL-1) were cultured in three-dimensional collagen gels and the area of the gels was measured by image analyser. Both thrombin and TFLLR, a selective PAR1 agonist, stimulated collagen gel contraction mediated by HFL-1. After RNA interference-mediated PAR1 knockdown in HFL-1, both thrombin and the PAR1 agonist-induced gel contraction were partially inhibited (by 22.4±2.2% and 17.6±5.6%, respectively). The gel contraction stimulated by thrombin was also reduced by a nonspecific PKC inhibitor and a calcium-independent PKC-ϵ inhibitor. Both thrombin and TFLLR significantly increased PKC-ϵ activity, and this effect was blocked by PAR1 knockdown. Thrombin stimulates collagen gel contraction at least partially through activation of protease-activated receptor 1 and protein kinase C-ϵ, and may contribute to tissue remodelling in inflammatory airway and lung diseases.

Effect of budesonide on fibroblast-mediated collagen gel contraction and degradation.

Background The balance between production and degradation of extracellular matrix is crucial in maintaining normal tissue structure. This study was designed to investigate the effect of budesonide on fibroblast-mediated tissue repair and remodeling. Methods Using human fetal lung fibroblasts in a three-dimensional collagen gel culture system, we investigated the effect of budesonide (1-1000 nM) on collagen gel contraction and degradation in the presence or absence of Inflammatory cytokines (interleukin-1β and tumor necrosis factor α; 5 ng/mL each) and, in order to activate latent proteases, serine protease trypsin 0.25 μg/mL. The effects of budesonide on metalloproteinase production and activation were also investigated. Results Inflammatory cytokines significantly inhibited collagen gel contraction mediated by lung fibroblasts. Budesonide counteracted the effect of cytokines in a concentration-dependent manner (to 50%, P< 0.01). Budesonide 100 nM almost completely inhibited the release and mRNA expression of metalloproteinase-1, metalloproteinase-3, and metalloproteinase-9 induced by the cytokines (P< 0.05). Exposure to the cytokines plus trypsin increased collagen degradation and conversion of the metalloproteinases to lower molecular weight forms corresponding to their active forms. Budesonide blocked both enhanced collagen degradation (P< 0.01) and suppressed trypsin-mediated conversion of cytokine-induced metalloproteinase-9 and metalloproteinase-3 to lower molecular weight forms. Similar effects were observed with dexamethasone 1 μM, suggesting a class effect. Conclusion These findings demonstrate that budesonide directly modulates contraction of collagen gels and can decrease collagen degradation under Inflammatory conditions. The mechanism of this effect is through suppressing gene expression, release, and activation of metalloproteinases. By modulating the release and activity of metalloproteinases, inhaled budesonide may be able to modify airway tissue repair and remodeling.

PGE 2 desensitizes β -agonist effect on human lung fibroblast-mediated collagen gel contraction through upregulating PDE4.

In the current study, we investigated the effect of a long-acting β-agonist (salmeterol) and a phosphodiesterase 4 (PDE4) inhibitor (cilomilast) on human lung fibroblast-mediated collagen gel contraction. Higher concentrations of salmeterol (10−7 and 10−6 M) inhibited fibroblast-mediated collagen gel contraction. No effect was observed with cilomilast alone (up to 10−5 M). In the presence of 10−8 M salmeterol, however, cilomilast could significantly inhibit fibroblast-mediated collagen gel contraction in a concentration-dependent manner (10−7 ~10−5 M). Blockade of endogenous PGE2 by indomethacin further potentiated the inhibitory effect of salmeterol on fibroblast-mediated collagen gel contraction, but it did not affect cilomilasts effect. Pretreatment with PGE2 abolished the inhibitory effect of salmeterol, but it potentiated the inhibitory effect of cilomilast on fibroblast-mediated collagen gel contraction. Finally, indomethacin slightly inhibited PDE4C expression, while PGE2 stimulated the expression of PDE4A and -4C in human lung fibroblasts. These findings suggest that long-acting β-agonist and PDE4 inhibitor have a synergistic effect in regulating fibroblast tissue repair functions and that PGE2 can modulate the effect of β-agonist and PDE4 inhibitor at least in part through the mechanism of regulating PDE4 expression.