SREBP2 promotes the viability, proliferation, and migration and inhibits apoptosis in TGF-β1-induced airway smooth muscle cells by regulating TLR2/NF-κB/NFATc1/ABCA1 regulatory network

Abstract

Asthma is a respiratory disease with complex pathogenesis. Sterol-responsive element-binding proteins 2 (SREBP2) was found to bind to promoter sequences of ABCA1 to suppress ABCA1 promoter activity. This study aimed to explore the expression level of SREBP2 and ATP-binding cassette transporter A1 (ABCA1), and their effects on the development of airway smooth muscle cells (ASMCs) in asthma. ASMCs were treated with different concentrations of TGF-β1 (0, 0.5, 1, 5 and 10 ng/mL). Short hairpin SREBP2 (shSREBP2), SREBP2, shABCA1 or ABCA1 were transfected into ASMCs. Cell viability, proliferation, apoptosis, migration, and the expression of SREBP2, ABCA1 and related pathway proteins were detected by MTT assay, Brdu staining, flow cytometer, Transwell assay, qRT-PCR, and Western blotting, respectively. The results showed that TGF-β1 increased the viability, proliferation, migration and inhibited apoptosis in ASMCs. Moreover, TGF-β1 also decreased the expression of ABCA1, cleaved caspase-3, cleaved PARP, E-cadherin, and increased the expression of vimentin, TLR2, p-p65 and NFATc1. SREBP2 knockdown alleviated these TGF-β1-induced changes. SREBP2 overexpression inhibited ABCA1 expression and apoptosis, and promoted cell migration and the expression of TLR2, p-p65, NFATc1 in ASMCs. ABCA1 overexpression alleviated these SREBP2-induced promoting and inhibition effects. In conclusion, SREBP2 activates TLR2/NF-κB/NFATc1 regulatory network and promotes TGF-β1-induced cell movement through inhibiting ABCA1 expression.

Keywords: ABCA1; NFATC1; SREBP2; TGF-Β1; TLR2.

Graphical abstract

Figure 1.

TGF-β1 increased SREBP2 level and decreased ABCA1 level in ASMCs. Different concentrations of TGF-β1 (0, 0.5, 1, 5 and 10 ng/mL) were used to treat ASMCs. (a) Cell viability, the relative SREBP2 and ABCA1 (b) mRNA level and (c) expression level was detected by MTT assay, qRT-PCR, and Western blotting. ^# p < 0.05, ^## p < 0.01; ^# compare with the 0 ng/mL TGF-β1 group.

Figure 2.

SREBP2 knockdown inhibited cell proliferation in ASMCs. shSREBP2 or shNC were transfected into ASMCs, or 5 ng/mL TGF-β1 were treated with ASMCs. (a) The relative SREBP2 and ABCA1 level, (b) cell viability, and (c) cell proliferation was detected by Western blotting, MTT assay and Brdu staining. ^## p < 0.01; ^# compare with the control group. ^@ p < 0.05, ^@@ p < 0.01; ^@ compare with the TGF-β1 + shNC group.

Figure 3.

SREBP2 knockdown promoted apoptosis in ASMCs. shSREBP2 or shNC were transfected into ASMCs, or 5 ng/mL TGF-β1 was treated with ASMCs. (a) Apoptosis and (b) apoptosis-related proteins, caspase-3 and cleaved PARP levels were detected by flow cytometer and Western blotting. ^# p < 0.05, ^## p < 0.01; ^# compare with the control group. ^@ p < 0.05, ^@@ p < 0.01; ^@ compare with the TGF-β1 + shNC group.

Figure 4.

SREBP2 knockdown inhibited cell migration in ASMCs. shSREBP2 or shNC was transfected into ASMCs, or 5 ng/mL TGF-β1 was treated with ASMCs. (a) Cell migration and (b) migration-related proteins, E-cad and vimentin levels were detected by Transwell assay and Western blotting. ^# p < 0.05, ^## p < 0.01; ^# compare with the control group. ^@ p < 0.05, ^@@ p < 0.01; ^@ compare with the TGF-β1 + shNC group.

Figure 5.

SREBP2 promoted cell viability, migration, and inhibited apoptosis in ASMCs. SREBP2 vector or empty vector were transfected into ASMCs, or 5 ng/mL TGF-β1 was treated with ASMCs. (a) Cell viability, (b) apoptosis and (c) cell migration were detected by MTT assay, flow cytometer and Transwell assay. ^# p < 0.05, ^## p < 0.01; ^# compare with the control group. ^@ p < 0.05, ^@@ p < 0.01; ^@ compare with the TGF-β1 + shNC group.

Figure 6.

SREBP2 activated the TLR2/NF-κB/NFATc1 pathway by inhibiting the expression of ABCA1. shSREBP2, SREBP2 vector or ABCA1 vector were transfected into ASMCs, or 5 ng/mL TGF-β1 was treated with ASMCs. (a) The level of ABCA1, TLR2, p-p65, p65 and NFATc1, and (b) SREBP2, ABCA1, TLR2, p-p65, p65 and NFATc1 were detected by Western blotting. ^## p < 0.01; ^# compare with the control group. ^@ p < 0.05, ^@@ p < 0.01; ^@ compare with the TGF-β1 + shNC or SREBP2 group.

Figure 7.

SREBP2 promoted TGF-β1-induced cell motility through regulating ABCA1 expression. shSREBP2, SREBP2 vector, shABCA1 or ABCA1 vector were transfected into ASMCs. (a and c) Cell viability, (b and d) apoptosis and (e and f) cell migration was detected by MTT assay, flow cytometer and Transwell assay. ^## p < 0.01; ^# compare with the control group. ^@@ p < 0.01; ^@ compare with the SREBP2 group or the shSREBP2 group.