- Open Access
14-3-3ζ loss leads to neonatal lethality by microRNA-126 downregulation-mediated developmental defects in lung vasculature
- Jun Yang†1, 3,
- Sonali Joshi†1,
- Qingfei Wang1,
- Ping Li1,
- Hai Wang1,
- Yan Xiong1,
- Yi Xiao1,
- Jinyang Wang1,
- Jan Parker-Thornburg2,
- Richard R. Behringer2, 3 and
- Dihua Yu1, 3, 4Email author
© The Author(s) 2017
- Received: 28 June 2017
- Accepted: 23 October 2017
- Published: 2 November 2017
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.
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.
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.
- Lung development
The mammalian 14-3-3 protein family consists of seven isoforms (β, τ, ε, ζ, η, γ and σ). They modulate multiple signaling pathways in diverse biological processes by binding to specific phospho-specific motifs on target proteins and thereby regulate their function, activity, stability, or localization [1, 2]. Due to their ability to interact with a plethora of target proteins, they are reported to play an important role in the regulation of cell cycle, DNA damage repair, cell proliferation, cell polarity, programmed cell death and cell metabolism, etc. [3–8]. In spite of exhibiting a high degree of homology, knockout mouse models of different 14-3-3 family members have revealed distinctive biological functions for the different isoforms [1, 9, 10], indicating that the various isoforms can exert unique functions which cannot be compensated by other members of the 14-3-3 family.
14-3-3ζ has been implicated in various human diseases such as Alzheimer’s disease [11, 12], progressive multifocal leukoencephalopathy , Huntington disease , Creutzfeldt–Jakob disease  and is reported to be upregulated in multiple cancer types, including lung [16, 17], breast [2, 18, 19], ovarian , head and neck  as well as lymphomas .
Previous studies on the developmental role of 14-3-3ζ have identified that 14-3-3ζ functions in neuronal development and in regulation of adipogenesis . On a SV129 background, 14-3-3ζ−/− mice were reported to exhibit behavioral and cognitive defects similar to those observed in schizophrenia, autism spectrum disorder and bipolar disorder . Additionally, these mice were found to manifest enhanced locomotor hyperactivity due to dysregulation of dopamine signaling . On a Balb/c background, deletion of 14-3-3ζ was found to result in hippocampal defects with a decrease in spatial memory but they did not exhibit enhanced locomotor activity as observed on a SV129 background . 14-3-3ζ−/− mice have also been reported to exhibit reduced deposition of visceral fat as loss of 14-3-3ζ promotes autophagy mediated degradation of CEBP/δ preventing induction of the master adipogenesis regulators PPARγ and C/EBPα . However, these previous studies did not reveal a role of 14-3-3ζ in lung development.
In this study, we generated a 14-3-3ζ hypomorphic mutant mouse (14-3-3ζ−/−) model for studying the 14-3-3ζ gene function in vivo. We found that targeted deletion of 14-3-3ζ on a C57Bl/6J genetic background led to neonatal lethality due to respiratory distress, which resulted from miR-126 downregulation-induced defects in lung vasculature. Our findings demonstrate a unique and essential role of 14-3-3ζ in normal development in a genetic background specific manner.
Generation and maintenance of 14-3-3ζ knockout mouse
The ES cell line RRR334, in which 14-3-3ζ was targeted according to the 5′RACE data from the Baygenomics database, was obtained from Mutant Mouse Regional Resource Center (MMRRC). RT-PCR was performed to confirm that the cell line inactivates 14-3-3ζ. The primer sequence for the exogenous was forward: 5′-TGCTGAGAAAAAGCAGCAGA and reverse: 5′-GACAGTATCGGCCTCAGGAAGATCG. The primer sequence for endogenous 14-3-3ζ control PCR was forward: 5′-TGCTGAGAAAAAGCAGCAGA and reverse: 5′-TTGTCATCACCAGCAGCAAC. The genetic engineered mouse facility at MD Anderson Cancer Center injected the cells into B6(Cg)-Tyrc − 2j/J (The Jackson Laboratory, Bar Harbour, ME, Stock #000058) blastocysts. Chimeras were mated with B6(Cg)-Tyrc − 2j/J mice to test germline transmission and obtain 14-3-3ζ knockout founder mice. The primer sequences for PCR genotyping were forward: 5′-CAACCATGTTGGGATAGAGG homologous to 14-3-3ζ intron 3, reverse: 5′-CCAAATAAGCCTTCCCTTCC homologous to intron 3 and 5′-AAGGGTCTTTGAGCACCAGA homologous to the gene trap vector. PCR resulted in 954-bp fragment from the wild-type allele and a 544-bp fragment from the mutant allele. Mice were backcrossed into C57Bl/6J and FVB/NJ congenic background as determined by genome scan using a panel of simple sequence length polymorphism (SSLP) (microsatellite) markers, or outbred to CD-1 genetic background respectively and maintained thereafter. The C57Bl/6J and FVB/NJ mouse breeders were purchased from the Jackson Laboratory (Bar Harbor, ME). The CD-1 breeders were purchased from Charles Rivers (Wilmington, MA). All animal work was performed under an IACUC-approved protocol. University of Texas MD Anderson Cancer Center is an AAALAC accredited institution.
The mouse endothelial cell line ARBEC was obtained from Dr. Fidler’s lab at MDACC. The endothelial cells were generated from H-2Kb-tsA58 mice immortalized with SV40 large T antigen. The ARBEC cells were cultured using DMEM supplemented with 10% FBS. The transfection was performed using Lipofectamine 2000 using standard protocol.
MCF7 cells were cultured using DMEM supplemented with 10% FBS. N-terminal 139aa of 14-3-3ζ was cloned into pcDNA3 and later transfected into MCF7 cell lines. Stable clones (∆C1 and ∆C12) were selected with G418 and high expressers were examined by western blot and maintained since. Mouse embryonic fibroblast (MEF) cells were obtained from E13.5 B6 embryos. The embryos were minced, cut and filtered. Cells were then centrifuged, resuspended in DMEM supplemented with 10% FBS and plated. Control pre-microRNA and pre-miR126 were ordered from Exiqon.
Tissue collection and histological analysis
The mouse embryos were collected by C-section at 18.5 dpc (days post coitum). The lung tissues were collected after euthanasia following an IACUC-approved protocol. Tissues were fixed in 10% neutral buffered formalin for 12–18 h. The samples were stored in 70% ethanol and then embedded in paraffin. Paraffin sections (5 µm) were stained with hematoxylin and eosin. Histological analysis were independently evaluated by at least two pathologists (Y.X., W.H. and Q.Z.). Immunohistochemistry (IHC) was performed as previously described . Antibodies used were Ki67 (DAKO, Carpentaria, CA M7249), CD34 (eBioscience, San Diego, CA 14-0341), 14-3-3ζ (C-16, Santa Cruz, Santa Cruz, CA sc-1019), SP-A (Santa Cruz sc-13977), and AQP5 (Calbiochem, Germany 178615). For IHC analysis and quantification, 10 fields were randomly chosen at 200 × magnification. The total number of cells and positive cells were counted, and the average percentage of positive cells was determined.
Tissues were collected from the mice. Protein extracts were prepared by homogenizing samples in tissue lysis PBSTDS buffer [10 mmol/L sodium phosphate (pH 7.3), 154 mmol/L NaCl, 5% sodium deoxycholate, 1% SDS] using a tissue grinder, followed by centrifugation to remove particulate matter and lipids. Immunoblotting was performed as previously described by Lu et al. . Antibodies used were anti-HA high affinity (clone 3F10, Roche 11867423001), 14-3-3ζ (C-16, Santa Cruz sc-1019), Erk (Cell Signaling 4695), phosphor-Erk (T202/Y204, Cell Signaling 4370), Mek1 (Cell Signaling 9126), phosphor-Mek1 (S221, Cell Signaling 2338), Ets1 (c-20, Santa Cruz sc-350), Ets2 (c-20, Santa Crux sc-351), Akt (Cell Signaling 9272), phosphor-Akt (S473, Cell Signaling 3787), β-actin (Sigma A5441), and tubulin (Sigma T5168). The antibodies were mainly used at a 1:1000 dilution.
Quantitative RT-PCR for microRNA quantification
RNA of the tissue samples was extracted using Trizol (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. The specific primers and probe for miR-126 real-time PCR were purchased from Applied Biosystems. Reverse transcription was performed using the Taqman MicroRNA Reverse Transcription kit (Applied Biosystems 4366569), and real-time PCR was performed using the Taqman Universal PCR Master Kit (Applied Biosystems 4324018) and iQ SyBR Green Supermix (Bio-Rad 170-8882). The primer sequences for PECAM1 were forward: 5′-CTGGTGCTCTATGCAAGCCT and reverse: 5′-AGTTGCTGCCCATTCATCAC. The primer sequences for 18S ribosomal RNA were forward: 5′-AACCCGTTGAACCCCATT and reverse: 5′-CCATCCAATCGGTAGTAGCG. Control pre-microRNA and pre-miR126 were ordered from Exiqon.
Transwell migration assay
Mouse endothelial cells (3 × 104) resuspended in 500 µL of serum-free medium were seeded in the top chamber of 24-well 8-µm pore transwell plates. 600 µL of medium containing 10% FBS was used as chemo-attractant in the bottom chamber. The plates were incubated at 37 °C for 4 h. The cells were fixed in 10% neutral buffered formalin for 1 h before staining with 0.5% crystal violet for 1 h. The plates were then flushed under tap water to remove excessive dye. The cells that did not migrate through the wells were wiped away by cotton tips. Images were taken with a Zeiss Discovery V20 microscope with the supplied software Axiom. The entire wells were quantified and three replicates were performed for statistical analysis.
Quantification of the migrated cells was performed using Adobe Photoshop and ImageJ software.
Tube formation assay
Matrigel (BD, Franklin Lakes, NJ 356231) was thawed at 4 °C overnight before assay, and then 500 µL of Matrigel was added into each well of the 24-well plate. The plates were incubated at 37 °C for 30 min to 1 h to form gel. Cells (3 × 105) resuspended in DMEM/F-12 medium containing 10% FBS were plated above the Matrigel and incubated for 6 h at 37 °C. The formation of the tube structure by the endothelial cells was imaged with an Olympus inverted fluorescence microscope and quantified using the ImageJ software. Three replicates were performed and five images were taken from each well for analysis.
Statistical differences were assessed with two-tailed Student’s t test or one-way ANOVA. A p value of < 0.05 was considered statistically significant.
Chromatin proteins were cross linked to chromatin with formaldehyde and sheared into 400–1000 bp fragments. Nucleoprotein complexes were immunoprecipitated using antibody to Ets-1, Ets-2, or control IgG antibody. The precipitated DNA fractions were analyzed by Quantitative-PCR for the presence of the miR-126 proximal regulatory region encompassing the EBS1 and EBS2 (region − 150 to + 100 bp). Input DNA was used as a positive control.
Generation of 14-3-3ζ knockout mice
Based on the integration site of the gene trap vector, the knockout mouse should express a truncated 14-3-3ζ protein containing 139 amino acids of the N-terminal part of 14-3-3ζ protein fused to the β-geo cassette. To test whether the N-terminal part of 14-3-3ζ protein is functional, a vector containing the HA-tagged N-terminal fragment of 14-3-3ζ with deletion of the C-terminal target protein binding groove (ΔC14-3-3ζ) [27, 28] was transfected into MCF7 breast cancer cells. The expression of the HA-tagged ∆C14-3-3ζ was detectable only in the presence of the proteosomal inhibitor MG132 (Additional file 1: Figure S2A) and it did not enhance cell proliferation or induce activation of the Ras/Raf/Erk and PI3K/Akt pathways (Additional file 1: Figure S2B, C) as would be expected with full length 14-3-3ζ . Thus the N-terminal fragment of 14-3-3ζ is non-functional and the 14-3-3ζ−/− mouse generated by the gene-trap approach is a strong hypomorphic model for studying the function of 14-3-3ζ in vivo.
14-3-3ζ−/− mice are neonatal lethal with defective lung development
Genotype of pups generated by mating heterozygous breeders under different genetic background
Hematoxylin and eosin (H&E) staining of the lung sections of the 18.5 dpc embryos showed that the lungs of the 14-3-3ζ−/− mice lacked saccular structures and exhibited increased mesenchymal compartments and thickened saccular septae (Fig. 2c). Amid that 14-3-3ζ expression level was significantly downregulated (Fig. 1d), no significant difference in pneumocyte proliferation was detected by Ki-67 immunohistochemical (IHC) staining (Fig. 2d). Pneumocyte differentiation was not altered by loss of 14-3-3ζ as evident by IHC staining of type II pneumocyte marker, pulmonary surfactant-associated protein A (SP-A), and type I pneumocyte marker, aquaporin isoform 5 (AQP5) (Fig. 2e, f). Loss of 14-3-3ζ resulted in fragmented blood vessels that were surrounded by leukocyte aggregates and also decreased endothelial cells as analyzed by CD34 staining (Fig. 2g). Thus defects in lung vasculature result in the respiratory distress observed in the 14-3-3ζ−/− mice.
Loss of 14-3-3ζ impedes angiogenesis via downregulation of miR-126
14-3-3 isoforms exhibit unique non-overlapping biological functions. 14-3-3σ stabilizes p53, thus suppresses tumor growth [38–40] and is frequently lost in cancer [41–43]. 14-3-3σ mediates keratinocyte proliferation and differentiation in vivo . 14-3-3ε can enhance TGF-β signaling , bind to poly A polymerase and regulate its cellular localization . Targeted disruption of 14-3-3ε in mice results in embryonic lethality from hippocampal and cortical defects while 14-3-3ε heterozygous mice exhibited neuro-developmental defects that mimic the Miller–Dieker syndrome . 14-3-3γ knockout mice do not display a discernible phenotype  but it can affect PDGF signaling in smooth muscle cells . 14-3-3τ regulates E2F stability, is required for autophagy  and targeted deletion of 14-3-3τ in mice leads to embryonic lethality from gross developmental defects . We uncovered an unexpected role for 14-3-3ζ in mediating vascular integrity during lung development. Thus loss of 14-3-3ζ in mice leads to distinct physiological defects and pathological effects in various genetic backgrounds that cannot be compensated by other 14-3-3 isoforms in vivo. Together, these studies indicated that specific 14-3-3 isoforms, including 14-3-3ζ, have distinctive biological functions in normal development and in diseases.
Loss of 14-3-3ζ leads to neonatal lethality with defects in lung development on the C57Bl/6J genetic background, indicating that 14-3-3ζ plays an indispensable role in normal development. Mechanistic insights came from our finding that loss of 14-3-3ζ on the C57Bl/6J background phenocopies miR-126 knockout mice. MiR-126 is an endothelial cell-specific microRNA, and miR-126 loss resulted in neonatal lethality and lung deflation due to vascular disintegration and inhibition of angiogenesis . Additionally, zebrafish studies have suggested a role of miR-126 in facilitating the lung vasculature integrity by reducing the expression of negative regulators of the VEGF pathway, such as SPRED-1 (Sprouty related protein 1) and the PI3K regulatory subunit 2 . Several miR-126 targets have been identified that potentially account for the importance of miR-126 expression in endothelial cells. MiR-126 has also been shown to enhance endothelial cell proliferation by targeting the 3′UTR of the Notch1 inhibitor delta-like 1 homolog (Dlk1) . MiR-126 was also found to suppress the expression of the endothelial adhesion molecule vascular cell adhesion molecule 1 (VCAM1) and thereby plays a role in regulating vascular inflammation . Other miR-126 targets that play a role in angiogenesis include VEGF , epidermal growth factor like domain 7 (EGFL7) , etc. The lungs of our 14-3-3ζ−/− mice had a significant miR-126 down-regulation, consistent with the defective lung development phenotype. Furthermore, reintroducing miR-126 to 14-3-3ζ knockdown endothelial cells rescued endothelial cell functions. Together, these data indicate that miR-126 down-regulation plays a critical role in the defective lung vasculature-mediated respiratory failure and neonatal lethality in 14-3-3ζ knockout mice, which may also have relevance for clinical syndromes of neonatal respiratory distress . Notably, 14-3-3ζ binds to many target proteins and regulates a wide variety of biological processes, thus miR-126 down-regulation in 14-3-3ζ−/− mice is likely one of the mechanisms underlying the developmental defects. A more comprehensive and in-depth future study will define other molecular mechanisms at play.
Previously, we reported that 14-3-3ζ regulates transcription of miR-221 via the Erk target c-fos and the Jnk target c-Jun . In this study, we found that 14-3-3ζ regulates miR-126 via the Erk/Ets2 pathway. These suggest an important role for 14-3-3ζ in controlling miRNA transcription via its regulation of the Raf/Mek/Erk pathway. Ets1 and Ets2 have been shown to regulate the transcription of multiple miRNAs such as miR-196b , miR-155  which also play an important role in mediating endothelial cell function [65, 66]. Additionally Ets2 regulates the transcription of c-Myc  which can regulate miRNA processing via regulating drosha transcription . Thus, it is plausible that 14-3-3ζ may regulate miRNA transcription and miRNA processing, ultimately miRNA expression and activity, which merits future in-depth studies. Additional tissue/organ defects due to 14-3-3ζ loss and other mechanisms may also contribute to the neonatal lethality observed on the C57Bl/6J genetic background.
The lethality phenotype from 14-3-3ζ loss was most severe on a C57Bl/6J genetic background and could be rescued by outbreeding to CD-1 and backcrossing to FVB/NJ, suggesting that the genetic background plays an important role in this phenotype. The molecular mechanisms of this dramatic phenotypic difference in different genetic backgrounds remains to be fully understood. Since 14-3-3ζ can bind to many different target proteins to regulate various biological processes, it is possible that one or more such target proteins important for lung development are coded by gene(s) hypomorphic on the C57Bl/6J genetic background. Interestingly, we have observed that miR-126 expression in the lungs of C57Bl/6J mice is significantly lower as compared to that in the FVB/NJ and CD-1 mice, suggesting that downregulation of miR-126 from 14-3-3ζ-loss may lead to more severe biological consequences on the C57Bl/6J mice than in the FVB/NJ and CD-1 mice. Identifying the genes differentially expressed on the C57Bl/6J mouse strain that contributed to 14-3-3ζ-mediated lethality may have important clinical implications. For example, the genes that contributed to 14-3-3ζ-mediated lethality on the C57Bl/6J mouse strain may predict patients’ therapeutic response versus adverse side effects when targeting the 14-3-3ζ and downstream pathways which can guide the optimization of personalized therapy in the future.
14-3-3ζ plays important functions in mouse lung development. Loss of 14-3-3ζ resulted in neonatal lethality in C57Bl/6 genetic background due to respiratory distress. 14-3-3ζ hypomorphic lungs had dysregulated angiogenesis mediated by inhibition of Erk-Ets2-miR-126 signaling pathway.
Conception and design: JY, SJ, QW, DY. Acquisition of data: JY, SJ, QW, PL, HW, YX, JW. Analysis and interpretation of data: JY, SJ, QW, YX, DY. Drafting or revising the article: JY, SJ, JP, RB, DY. Contributed essential reagents: JP. All authors read and approved the final manuscript.
The authors thanks the following MDACC core facilities: Genetically engineered mouse facility for pro-nuclei injections, Laboratory Animal Genetic Services at Science Park for analysis of SSLP, the Histology Core Lab for tissue preparations, the shRNA and ORFome for shRNA constructs.
The authors declare that they have no competing interests.
Availability of data and materials
The data analyzed in this study were available at Mouse Genome Informatics site http://www.informatics.jax.org/.
Consent for publication
Ethics approval and consent to participate
All animal work was performed under an IACUC-approved protocol. University of Texas MD Anderson Cancer Center is an AAALAC accredited institution.
Grant support: This work was supported partially by National Institutes of Health Cancer Center Support Grant 5 P30-CA016672-39 (MDACC), DOD Postdoctoral Fellowship W81XWH-13-1-0022 (SJ), PO1-CA099031 project 4 (DY), RO1-CA112567-06 (DY), R01CA184836 (DY), R01CA208213 (DY), and China Medical University research fund (DY). D. Yu is the Hubert L. and Olive Stringer Distinguished Chair in Basic Science at MDACC.
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