IL-1β-stimulated β-catenin up-regulation promotes angiogenesis in human lung-derived mesenchymal stromal cells through a NF-κB-dependent microRNA-433 induction

Abstract

Considerable attentions have been focused on the treatment of lung injury using mesenchymal stem cells that can replenish damaged tissues including the blood vessels. In human lung-derived mesenchymal stem cells (hL-MSC), we investigated the potential role of an IL-1β-stimulated miR-433 pathway in angiogenesis in vitro. The expressions of miR-433 and its target genes were examined in cells treated with IL-1β. The angiogenic activity of hL-MSC was studied by cell migration and tube formation assays in which miR-433 levels were manipulated. The reporter assay and chromatin immunoprecipitation (ChIP) were also performed to analyze the underlying regulations. We found that the expression of miR-433 was enhanced in hL-MSC by IL-1β in a NF-κB dependent manner via a NF-κB binding site at its promoter region. The effects of IL-1β on promoting angiogenic activities in hL-MSC can be mimicked by the overexpression of miR-433 and were blocked by anti-miR-433. Mechanistically, our data suggested that miR-433 directly targets the 3'-UTR of Dickkopf Wnt signaling pathway inhibitor 1 (DKK1) mRNA and decreases its expression. Consistently, the expression of β-catenin, the major mediator of canonical Wnt pathway that is capable of inducing endothelial differentiation and angiogenesis, was upregulated by IL-1β through miR-433. Thus, increasing miR-433 expression by IL-1β in mesenchymal stem cells could stimulate their capacity of vascular remodeling for efficient repair processes, which may be utilized as a therapeutic target in patients suffering from severe lung injury.

Keywords: Wnt/β-catenin; angiogenesis; lung injury; mesenchymal stem cell; microRNA.

Figure 1. Identification of human lung-derived MSC

Cells were characterized by flow cytometry using FITC- or PE-conjugated antibodies against negative surface markers CD14, CD34, CD45 and positive surface markers CD73, CD 90, CD105.

Figure 2. IL-1β treatment upregulated miR-433 and down-regulated DKK1 expressions in hL-MSC

A. Levels of miR-433 in hL-MSC after IL-1β treatment, with PBS as control. B. mRNA levels of genes known to be inhibited by IL-1β after IL-1β treatment, with PBS as control. C. mRNA levels of genes in hL-MSC transfected with either miR-negative control (NC) or miR-433. Values were mean ± SD from three independent experiments. ** P < 0.01, * P < 0.05 vs PBS or miR-NC, respectively.

Figure 3. miR-433 was required for IL-1β-induced enhancement of angiogenesis in hL-MSC derived endothelial cells

A. and B. Wound healing (A) and tube formation (B) assays were performed in hL-MSC derived endothelial cells treated with PBS or IL-1β. C. and D. Wound healing (C) and tube formation (D) assays were performed in hL-MSC derived endothelial cells transfected with miR-NC or miR-433. E. and F. hL-MSC derived endothelial cells treated with PBS or IL-1β were also transfected with either miR-NC or anti-miR-433, followed by wound healing (E) and tube formation (F) assays to assess their angiogenic capacity. Values were mean ± SD from three independent experiments. ** P < 0.01, * P < 0.05, ns not significant vs respective control.

Figure 4. IL-1β treatment upregulated miR-433, which directly targeted the 3′-UTR on DKK1 mRNA in hL-MSC

A. Sequence of the putative miR-433 targeting site (capitalized) on the 3′-UTR of DKK1 mRNA. B. Wild type (-Wt) or mutated (-Mut) versions of putative targeting sequence from the 3′-UTR of DKK1 mRNA were fused after the downstream of a luciferase reporter (Luc) open reading frame. C. and D. Luciferase activities of Luc-Wt and Luc-Mut constructs were measured in hL-MSC after transfection with either miR-NC or miR-433 (C), or treatment with either PBS or IL-1β (D). E. DKK1 protein levels in hL-MSC after transfection with either miR-NC or miR-433. F. hL-MSC treated with PBS or IL-1β were also transfected with either miR-NC or miR-433 inhibitor (anti-miR-433), followed by Western blot analysis to examine DKK1 protein levels. Values were mean ± SD from three independent experiments. ** P < 0.01, * P < 0.05, ns not significant vs PBS or miR-NC, respectively.

Figure 5. NF-кB activation was required for IL-1β induced upregulation of miR-433 in hL-MSC

A. Levels of miR-433 in the presence of IL-1β were examined in hL-MSC after treatment with either NF-кB inhibitor TPCA-1, p38 inhibitor BIX02188 or JNK inhibitor SP600125, respectively. B. and C. Levels of NF-кB protein (B) and miR-433 (C) in hL-MSC following NF-кB siRNA knock-down were examined by Western blot and RT-PCR, respectively. D. Protein levels of DKK1 in hL-MSC in the presence of IL-1β were examined by Western blot, following treatment with the inhibitors used in (A) or NF-кB siRNA knock-down used in (B). Values were mean ± SD from three independent experiments. ** P < 0.01, ns not significant vs mock or siRNA NC, respectively.

Figure 6. NF-κB induced miR-433 expression by directly binding to its promoter region

A. Promoter region of human miR-433 contains two putative binding sites for NF-κB, which was then clone to the upstream of a luciferase reporter (Luc) open reading frame. B. Binding of NF-κB to the promoter of miR-433 in hL-MSC was examined by ChIP assay using control IgG or NF-κB antibody. C. Luciferase activities of AB-Luc, A-Luc and B-Luc constructs were measured in hL-MSC after treatment with either PBS or NF-κB. Values were mean ± SD from three independent experiments. ** P < 0.01, ns not significant vs control IgG or PBS, respectively.

Figure 7. β-catenin expression was upregulated by IL-1β induced miR-433, in a NF-κB dependent manner

A. Levels of β-catenin mRNA in hL-MSC transfected with either miR-NC or miR-433. B. hL-MSC treated with PBS or IL-1β were also transfected with either miR-NC or anti-miR-433, followed by RT-PCR analysis to examine β-catenin mRNA levels. C. Levels of β-catenin mRNA in hL-MSC treated with PBS, IL-1β or IL-1β + NF-кB inhibitor TPCA-1. Values were mean ± SD from three independent experiments. ** P < 0.01, * P < 0.05, ns not significant vs miR-NC or PBS, respectively. D. A schematic diagram illustrating the mechanism of IL-1β-stimulated β-catenin up-regulation, mediated by NF-κb-dependent miRNA-433 induction, to promote angiogenesis in hL-MSC.