14-3-3ζ loss leads to neonatal lethality by microRNA-126 downregulation-mediated developmental defects in lung vasculature

Abstract

Background: The 14-3-3 family of proteins have been reported to play an important role in development in various mouse models, but the context specific developmental functions of 14-3-3ζ remain to be determined. In this study, we identified a context specific developmental function of 14-3-3ζ.

Results: Targeted deletion of 14-3-3ζ in the C57Bl/6J murine genetic background led to neonatal lethality due to respiratory distress and could be rescued by out-breeding to the CD-1 or backcrossing to the FVB/NJ congenic background. Histological analysis of lung sections from 18.5 days post coitum embryos (dpc) showed that 14-3-3ζ-/- lung development is arrested at the pseudoglandular stage and exhibits vascular defects. The expression of miR-126, an endothelial-specific miRNA known to regulate lung vascular integrity was down-regulated in the lungs of the 14-3-3ζ-/- embryos in the C57Bl/6J background as compared to their wild-type counterparts. Loss of 14-3-3ζ in endothelial cells inhibited the angiogenic capability of the endothelial cells as determined by both trans-well migration assays and tube formation assays and these defects could be rescued by re-expressing miR-126. Mechanistically, loss of 14-3-3ζ led to reduced Erk1/2 phosphorylation resulting in attenuated binding of the transcription factor Ets2 on the miR-126 promoter which ultimately reduced expression of miR-126.

Conclusion: Our data demonstrates that miR-126 is an important angiogenesis regulator that functions downstream of 14-3-3ζ and downregulation of miR-126 plays a critical role in 14-3-3ζ-loss induced defects in lung vasculature in the C57Bl/6J genetic background.

Keywords: 14-3-3ζ; Angiogenesis; Lung development; miR-126.

Fig. 1

Generation and characterization of 14-3-3ζ knockout mice. a Schematic map showing that the gene trap vector integrated into intron 3 of the 14-3-3ζ gene (Ywhaz). Ywhaz is the HUGO Gene Nomenclature Committee—approved gene symbol for 14-3-3ζ. The lines represent the introns for the 14-3-3ζ gene and the rectangles signify the exons. The solid sections indicate the coding region for 14-3-3ζ protein. The gene trap vector pGT0Lxf was integrated ~ 3.3 kb downstream of exon 3. SA indicates the splice acceptor sequence of mouse En2 exon 2. β-Geo/pA indicates the fusion of β-galactosidase and neomycin transferase followed by SV40 polyadenylation signal. The arrows indicate the primers for genotyping. The scale bar represents 1 kb length of DNA sequence. b PCR genotyping for 14-3-3ζ knockout mice: +/+, +/-, and −/− indicate 14-3-3ζ wild-type, heterozygous, and homozygous mutant alleles, respectively. M indicates marker. Wild-type allele generated a 954-bp band, and the mutant allele generated a 544-bp PCR product. c Western blot confirming the loss of 14-3-3ζ expression in mouse embryonic fibroblast (MEF) cells. β-Actin served as loading control. Quantification of relative 14-3-3ζ expression level was shown below the western panel. d Immunohistochemical (IHC) staining of 14-3-3ζ expression in the neonatal lung tissue dissected from +/+ and −/− mice. Scale bar indicates 50 µm in length

Fig. 2

14-3-3ζ mediated neonatal lethality and associated defects in lung development. a 18.5 dpc embryos were dissected using caesarean section. Representative photos of 14-3-3ζ wild type (+/+) and homozygous mutant (−/−) are shown as indicated. b Lung tissues from the embryos were immersed in water to determine buoyancy. c Hematoxylin and eosin staining of the lungs dissected from the embryos. Sample size is indicated. d IHC staining for Ki-67 on the lungs dissected from +/+ and −/− embryos. e IHC staining for SP-A on the lungs dissected from +/+ and −/− embryos. f IHC staining for AQP5 on the lungs dissected from +/+ and −/− embryos. g IHC staining for CD34 on the lungs dissected from +/+ and −/− embryos. Length of the scale bar represents 50 µm in each panel. d–g Right: the quantification of the IHC stainings by immunoreactive score (IRS). ***, **, * Indicate p < 0.001, 0.01, and 0.05, respectively. n.s.: not significant

Fig. 3

miR-126 regulates angiogenesis downstream of 14-3-3ζ. a qRT-PCR analysis of miR-126 expression normalized to PECAM1 in the lungs from 14-3-3ζ wild type (+/+) and knockout (−/−) embryo lungs, ± SD (n = 3) *p < 0.05. b qRT-PCR analysis of miR-126 expression normalized to PECAM1 in the lungs from 14-3-3ζ wild type (+/+) mice (6 weeks) from the indicated genetic backgrounds. c Detection of 14-3-3ζ and β-actin by western blotting in mouse endothelial cells expressing either a scrambled shRNA or shRNA targeting 14-3-3ζ. d qRT-PCR analysis of miR-126 expression normalized to PECAM1 in mouse endothelial cells expressing either a scrambled shRNA or shRNA targeting 14-3-3ζ. ± SD (n = 3)

Fig. 4

14-3-3ζ and miR-126 regulate endothelial migration and tube formation. a Transwell migration assay of mouse endothelial cells with 14-3-3ζ knockdown and rescue by overexpression of exogenous miR-126. Cells stably expressing scramble or 14-3-3ζ shRNA were transfected with control or pre-miR-126. Representative result is shown in the top panel and the bottom panel shows the quantification. Scale bar indicates 50 μm in length. b Tube formation assay with exogenously expressed miR-126 in the 14-3-3ζ knockdown mouse endothelial cells. Representative results are shown in the top panel and the bottom panel shows the quantification. Scale bar indicates 50 μm in length. c Mouse endothelial cells stably transfected with either scrambled shRNA or shRNA targeting 14-3-3ζ were transfected with pre-miR-126. MiR-126 expression was analyzed by qRT-PCR and normalized to PECAM1, ± SD (n = 3). Three independent transwell experiments were performed and three random fields from the tube formation assay were analyzed for statistical analysis

Fig. 5

14-3-3ζ mediates miR-126 transcription by modulating Ets2 activity. a Detection of 14-3-3ζ, phosphor-Erk 1/2 (Thr202/Tyr204), total Erk1/2 and β-actin by western blotting in mouse endothelial cells expressing either a scrambled shRNA or shRNA targeting 14-3-3ζ. b, c ChIP analysis of Ets2 and Ets1 binding to the miR-126 promoter in mouse endothelial cells expressing either a scrambled shRNA or shRNA targeting 14-3-3ζ, ± SD (n = 3). d Analysis of phosho-Erk (Thr202/Tyr204) and Erk1/2 in mouse endothelial cells treated with either DMSO or the ERK1/2 inhibitor AZD6244 by western blotting. e qRT-PCR analysis of miR-126 expression relative to PECAM1 in mouse endothelial cells treated with either DMSO or AZD6244, ± SD (n = 3). f ChIP analysis of Ets2 binding to the miR-126 promoter in mouse endothelial cells treated with either DMSO or AZD6244. ± SD. g ChIP analysis of Ets1 binding to the miR-126 promoter in mouse endothelial cells treated with either DMSO or AZD6244. ± SD (n = 3)

Fig. 6

Schematic model of 14-3-3ζ mediated lung vascular integrity regulation and respiratory distress mediated neonatal lethality. On C57Bl/6J background, 14-3-3ζ is critical for the activation of multiple signaling pathways including the Raf/Mek/Erk pathway, facilitating the Ets2 mediated transcription of the pro-angiogenic miR-126, which promotes lung vascular integrity (left). In the absence of 14-3-3ζ, the Raf/Mek/Erk/Ets2 pathway, along with other signaling pathways, is inhibited and miR-126 expression is attenuated, resulting in defects in lung vascular integrity that leads to neonatal lethality (right)