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Review
. 2015 Jan:57:55-63.
doi: 10.1016/j.jbior.2014.09.008. Epub 2014 Oct 13.

Sphingolipids in pulmonary fibrosis

Long Shuang Huang  1 , Viswanathan Natarajan  2
Affiliations
Review

Sphingolipids in pulmonary fibrosis

Long Shuang Huang et al. Adv Biol Regul. 2015 Jan.

Abstract

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by alveolar epithelial cell injury, accumulation of fibroblasts/myofibroblasts and deposition of extracellular matrix proteins. Levels of sphingosine-1-phosphate (S1P), a naturally occurring bioactive lipid, are elevated in bronchoalveolar fluids and lung tissues from IPF patients and animal models of pulmonary fibrosis. However, the in vivo contribution of S1P, regulated by its synthesis catalyzed by Sphingosine kinases (SphKs) 1 & 2 and catabolism by S1P phosphatases and S1P lyase (S1PL), in the pathogenesis of pulmonary fibrosis is not well defined. Microarray analysis of blood mononuclear cells from patients with IPF and SphK1-, SphK2- or S1PL-knockout mice and SphK inhibitor were used to assess the role of S1P in fibrogenesis. The expression of SphK1 negatively correlated with lung function and survival of patients with IPF. Further, the expressions of SphK1 and S1PL were increased in lung tissues from patients with IPF and bleomycin-challenged mice. Genetic knockdown of SphK1, but not SphK2, ameliorated bleomycin-induced pulmonary fibrosis in mice while deletion of S1PL (SGPL1(+/-)) in mice potentiated fibrosis post-bleomycin challenge. TGF-β increased the expression of SphK1 and S1PL in human lung fibroblasts and knockdown of SphK1 or treatment with SphK inhibitor attenuated S1P generation and TGF-β mediated signal transduction. Over-expression of S1PL attenuated bleomycin-induced TGF-β secretion and S1P mediated differentiation of human lung fibroblasts through regulation of autophagy. Administration of SphK1 inhibitor 8 days post-bleomycin challenge reduced bleomycin-induced mortality and pulmonary fibrosis. Our results suggest that SphK1 and S1PL play critical roles in the pathology of lung fibrosis and may be novel therapeutic targets.

Keywords: Autophagy; Pulmonary fibrosis; S1P lyase; Sphingosine kinase 1; Sphingosine-1-phosphate; TGF-β.

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Conflict of interest statement

Conflict of Interest

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
S1P and dihydro-S1P levels in lung tissues from mice post bleomycin challenge. C18-S1P (A) or C18-DHS1P (B) level in mouse lungs from C57BL/6J mice after bleomycin (2 U/kg, i.t.) treatment (0, 3, 7, 14, and 21 days). *P< 0.05, n= 4/group. S1P and dihydro-S1P levels were measured by LC/MS/MS and normalized to total lipid phosphorus in the samples.
Fig. 2
Fig. 2
Expression of SphK1, SphK2, fibronectin and α-smooth muscle actin in lung tissues from mice challenged with bleomycin. (A) Representative Western blot, and (B) quantification of the expression of SphK1, SphK2, FN and α-SMA in mouse lung tissue after bleomycin challenge (2 U/kg, i.t.) (0, 3, 7, and 14 days). Intensity of each band was quantified and normalized to GAPDH. Data are expressed as means ± SEM. *P< 0.05, n =8/group.
Fig. 3
Fig. 3
Expression of SphK1 and SphK2 in lung tissues from radiation challenged mice. Mice were administered a single dose of thoracic radiation (20 Gy) and lung tissues were collected at various time points as shown. (A) Representative Western blot, and quantification of the expression of SphK1 (B) and SphK2 (C) in lung tissues from mice 0, 4, 6, 8, 10, 14, 17 week post irradiation. Intensity of each band was quantified and normalized to GAPDH. Data are expressed as means ± SEM. *P< 0.05, n =4/group.
Fig. 4
Fig. 4
Bleomycin induces TGF-β and SphK1 expression in human lung fibroblasts. (A) mRNA levels of TGF-β and SphK1 in human lung fibroblast challenged with bleomycin (0–3 mU/ml, 48 h). *P< 0.05, n =4/group. (B) Representative Western blot, and (C) quantification of the expression of SphK1 in human lung fibroblast after bleomycin challenge.
Fig. 5
Fig. 5
SphK1 deficiency attenuates TGF-β induced expression of fibronectin and α-smooth muscle actin and activation of Akt and MAPKs in mouse lung fibroblasts. Primary mouse lung fibroblasts were isolated from wild type (WT) and SphK1 knockout (SphK1−/−) mice (male, 8–10 weeks). Following over-night starvation, cells were treated with vehicle (PBS) or TGF-β (5ng/ml) for 48 h. (A) Representative Western blot, and (B) quantification of the expression of fibronectin (Fn) and α- smooth muscle actin (α-SMA) in mouse lung fibroblasts after PBS or TGF-β challenge (5 ng/ml, 48 h). *P< 0.05, n =4/group. (C) Representative Western blot, and (D) quantification of the activation of Akt, JNK1 and p38 MAPK in mouse lung fibroblasts after PBS or TGF-β challenge. *P< 0.05, n =4/group.
Fig. 6
Fig. 6
S1P Lyase expression in lung tissues from bleomycin challenged mice. (A) Representative Western blot, and (B) quantification of the expression of S1PL in mouse lung tissues from mice after bleomycin (2 U/kg, i.t.) challenge (14 days). Intensity of each band was quantified and normalized to GAPDH. Data are expressed as means ± SEM. *P< 0.05, n =5/group.
Fig. 7
Fig. 7
Over-expression of S1P Lyase attenuates bleomycin-induced expression of TGF-β in human lung fibroblasts. Representative western blot (A) and quantification of TGF-β (B) in human lung fibroblasts after bleomycin challenge (3 mU/ml, 48 h). *P<0.05, n =4/group.
Fig. 8
Fig. 8
S1P Lyase over-expression enhances LC3 expression in human lung fibroblasts. Human lung fibroblasts were transfected with LC3-GFP plasmid (3 µg/ml/35-mm dish) and S1P lyase (S1PL) adenovirus (20 MOI/35-mm dish) for 48 h. (A), is a representative immunofluorescence micrograph and (B) Western blot of human lung fibroblasts transfected with LC3-GFP and S1PL adenovirus for 48 h followed by TGF-β challenge (5 ng/ml) for 48 h. *P< 0.05, n =4/group.
Fig. 9
Fig. 9
Schematic diagram illustrating the effects of TGF-β and S1P and transforming on fibrotic gene expression, differentiation, and activation in human lung fibroblasts. TGF-β binding to TGF-β receptor 1 and 2 induces activation, translocation and binding of Smad2/3 to Smad binding elements (SBE) in the promoter of target genes, and subsequent induction of the expression of fibrotic genes (collagen, fibronectin (FN) and α-smooth muscle actin (α-SMA) and S1P metabolizing enzymes (SphK1 and S1PL). In fibroblasts, SphK1 stimulates S1P generation through phosphorylation of sphingosine (Sph), and S1PL degrades S1P to ethanolamine phosphate and trans-2-hexadecenal. S1P generated intracellularly is transported outside the cell through the S1P transporter, Spns2. The intracellularly transported S1P binds to G-protein coupled S1P receptors (S1P1-5) and stimulates the activation of PI3K/Akt/MAPK to regulate the development of pulmonary fibrosis.
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