Transforming growth factor beta (TGF-beta) contributes to the progression of pulmonary fibrosis through up-regulation of alpha-smooth muscle actin (alpha-SMA) as lung myofibroblast differentiation. Bioactive sphingosine 1-phosphate (S1P) has been shown to mimic TGF-beta signals; however, the function of S1P in lung fibrotic process has not been well documented. We found, in a mouse model of bleomycin lung fibrosis, that SPHK1 and alpha-SMA were colocalized within lung fibrotic foci and that these expressions were significantly increased in primary cultured fibroblasts. Using human lung fibroblasts WI-38, we explored the rationale of sphingosine kinase (SPHK) with TGF-beta1 stimulation. SPHK inhibitors and small interference RNA (siRNA) targeted SPHK1 decreased alpha-SMA and fibronectin expression up-regulated by TGF-beta1. In the meantime, SPHK1 inhibition did not affect smad2 phosphorylation in response to TGF-beta1. Then we examined whether S1P receptors transactivation may affect TGF-beta signals. siRNA against S1P(2) and S1P(3), but not S1P(1), reduced alpha-SMA expression as well as Y-27632, Rho kinase inhibitor. We also detected activation of Rho GTPase upon stimulation of TGF-beta1 on the cell membrane where S1P(2) or S1P(3) was overexpressed. These data suggested that SPHK1 activation by TGF-beta1 leads to Rho-associated myofibroblasts differentiation mediated by transactivated S1P receptors in the lung fibrogenic process.

Sphingosine 1-phosphate (S1P) produced by sphingosine kinase (SPHK) is implicated in acute immunoresponses, however, mechanisms of SPHK/S1P signaling in the pathogenesis of bronchial asthma are poorly understood. In this study, we hypothesized that SPHK inhibition could ameliorate lung inflammation in ovalbumin (OVA)-challenged mouse lungs. Six- to eight-week-old C57BL/6J mice were sensitized and exposed to OVA for 3 consecutive days. Twenty-four hours later, mice lungs and bronchoalveolar lavage (BAL) fluid were analyzed. For an inhibitory effect, either of the two different SPHK inhibitors, N,N-dimethylsphingosine (DMS) or SPHK inhibitor [SK-I; 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole], was nebulized for 30 min before OVA inhalation. OVA inhalation caused S1P release into BAL fluid and high expression of SPHK1 around bronchial epithelial walls and inflammatory areas. DMS or SK-I inhalation resulted in a decrease in S1P amounts in BAL fluid to basal levels, accompanied by decreased eosinophil infiltration and peroxidase activity. The extent of inhibition caused by DMS inhalation was higher than that caused by SK-I. Like T helper 2 (Th2) cytokine release, OVA inhalation-induced increase in eotaxin expression was significantly suppressed by DMS pretreatment both at protein level in BAL fluid and at mRNA level in lung homogenates. Moreover, bronchial hyperresponsiveness to inhaled methacholine and goblet cell hyperplasia were improved by SPHK inhibitors. These data suggest that the inhibition of SPHK affected acute eosinophilic inflammation induced in antigen-challenged mouse model and that targeting SPHK may provide a novel therapeutic tool to treat bronchial asthma.

No abstract

In this study, we determined whether the proinflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin-1 beta contribute to the regulation of matrix metalloproteinase (MMP)-9 in human bronchial epithelial cells and whether the induction of MMP-9 is regulated by the transcription factor nuclear factor (NF)-kappa B. We demonstrated that TNF-alpha induced MMP-9 at both the protein and mRNA levels in human bronchial epithelial cells and that interleukin-1 beta did not. In contrast, induction of the tissue inhibitor of metalloproteinase-1 by TNF-alpha was less than that of interleukin-1 beta. Increased expression of MMP-9 and NF-kappa B activation induced by TNF-alpha were inhibited by pyrrolidine dithiocarbamate and N-acetyl-L-cysteine but were not inhibited by curcumin. These results suggest that TNF-alpha induces the expression of MMP-9 in human bronchial epithelial cells and that this induction is mediated via the NF-kappa B-mediated pathway.

Mechanical ventilation can paradoxically cause acute lung injury, which is termed ventilator-induced lung injury. Neutrophil recruitment and neutrophil elastase release play a central role in the pathogenesis of ventilator-induced lung injury including cell damage, extracellular matrix degradation and alveolar-capillary hyperpermeability. We therefore speculated that neutrophil elastase inhibition ameliorates ventilator-induced lung injury. Anesthetized C57/BL6 mice received mechanical ventilation with a high tidal volume (V(T); 20 ml/kg) for 4 h. The neutrophil elastase inhibitor (sivelestat, 100 mg/kg) or saline was given intraperitoneally (i.p.) 30 min before ventilation. Sivelestat completely inhibited both neutrophil elastase and myeloperoxidase activities that were increased by ventilation, and attenuated the histopathological degree of lung damage, neutrophil accumulation and lung water content, as well as the concentration of macrophage inflammatory protein (MIP)-2, interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha in bronchoalveolar lavage fluid and serum. Moreover, mechanical ventilation increased the phosphorylation of c-Jun NH2-terminal kinase (JNK) and the expression of early growth response gene-1 (Egr-1) mRNA, and these increases were also recovered by sivelestat. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining revealed apoptotic cells mainly in alveolar epithelial cells and their numbers corresponded to histological damage. These data suggested that sivelestat could protect against ventilator-induced lung injury by suppressing apoptotic responses through mechanical stress-induced cell signaling in addition to inhibiting neutrophil chemotaxis.