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The ER membrane protein complex subunit Emc3 controls angiogenesis via the FZD4/WNT signaling axis

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Abstract

The endoplasmic reticulum (ER) membrane protein complex (EMC) regulates the synthesis and quality control of membrane proteins with multiple transmembrane domains. One of the membrane spanning subunits, EMC3, is a core member of the EMC complex that provides essential hydrophilic vestibule for substrate insertion. Here, we show that the EMC subunit Emc3 plays critical roles in the retinal vascular angiogenesis by regulating Norrin/Wnt signaling. Postnatal endothelial cell (EC)-specific deletion of Emc3 led to retarded retinal vascular development with a hyperpruned vascular network, the appearance of blunt-ended, aneurysm-like tip endothelial cells (ECs) with reduced numbers of filopodia and leakage of erythrocytes at the vascular front. Diminished tube formation and cell proliferation were also observed in EMC3 depleted human retinal endothelial cells (HRECs). We then discovered a critical role for EMC3 in expression of FZD4 receptor of β-catenin signaling using RNA sequencing, real-time quantitative PCR (RT-qPCR) and luciferase reporter assay. Moreover, augmentation of Wnt activity via lithium chloride (LiCl) treatment remarkably enhanced β-catenin signaling and cell proliferation of HRECs. Additionally, LiCl partially reversed the angiogenesis defects in Emc3-cKO mice. Our data reveal that Emc3 plays essential roles in angiogenesis through direct control of FZD4 expression and Norrin/β-catenin signaling.

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References

  • Anghel, S.A., McGilvray, P.T., Hegde, R.S., and Keenan, R.J. (2017). Identification of Oxa1 homologs operating in the eukaryotic endoplasmic reticulum. Cell Rep 21, 3708–3716.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bagchi, P., Inoue, T., and Tsai, B. (2016). EMC1-dependent stabilization drives membrane penetration of a partially destabilized non-enveloped virus. eLife 5, e21470.

    Article  PubMed  PubMed Central  Google Scholar 

  • Bai, L., You, Q., Feng, X., Kovach, A., and Li, H. (2020). Structure of the ER membrane complex, a transmembrane-domain insertase. Nature 584, 475–478.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cabet, S., Lesca, G., Labalme, A., Des Portes, V., Guibaud, L., Sanlaville, D., and Pons, L. (2020). Novel truncating and missense variants extending the spectrum of EMC1-related phenotypes, causing autism spectrum disorder, severe global development delay and visual impairment. Eur J Med Genet 63, 103897.

    Article  PubMed  Google Scholar 

  • Carmeliet, P., and Jain, R.K. (2000). Angiogenesis in cancer and other diseases. Nature 407, 249–257.

    Article  CAS  PubMed  Google Scholar 

  • Chitwood, P.J., and Hegde, R.S. (2019). The role of EMC during membrane protein biogenesis. Trends Cell Biol 29, 371–384.

    Article  CAS  PubMed  Google Scholar 

  • Chitwood, P.J., Juszkiewicz, S., Guna, A., Shao, S., and Hegde, R.S. (2018). EMC is required to initiate accurate membrane protein topogenesis. Cell 175, 1507–1519.e16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chung, A.S., and Ferrara, N. (2011). Developmental and pathological angiogenesis. Annu Rev Cell Dev Biol 27, 563–584.

    Article  CAS  PubMed  Google Scholar 

  • Coelho, J.P.L., Stahl, M., Bloemeke, N., Meighen-Berger, K., Alvira, C.P., Zhang, Z.R., Sieber, S.A., and Feige, M.J. (2019). A network of chaperones prevents and detects failures in membrane protein lipid bilayer integration. Nat Commun 10, 672.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fan, J., Ponferrada, V.G., Sato, T., Vemaraju, S., Fruttiger, M., Gerhardt, H., Ferrara, N., and Lang, R.A. (2014). Crim1 maintains retinal vascular stability during development by regulating endothelial cell Vegfa autocrine signaling. Development 141, 448–459.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Folkman, F. (1971). Tumor angiogenesis: therapeutic implications. N Engl J Med 285, 1182–1186.

    Article  CAS  PubMed  Google Scholar 

  • Geetha, T.S., Lingappa, L., Jain, A.R., Govindan, H., Mandloi, N., Murugan, S., Gupta, R., and Vedam, R. (2018). A novel splice variant in EMC1 is associated with cerebellar atrophy, visual impairment, psychomotor retardation with epilepsy. Mol Genet Genomic Med 6, 282–287.

    Article  CAS  PubMed  Google Scholar 

  • Guna, A., Volkmar, N., Christianson, J.C., and Hegde, R.S. (2018). The ER membrane protein complex is a transmembrane domain insertase. Science 359, 470–473.

    Article  CAS  PubMed  Google Scholar 

  • Harel, T., Yesil, G., Bayram, Y., Coban-Akdemir, Z., Charng, W.L., Karaca, E., Al Asmari, A., Eldomery, M.K., Hunter, J.V., Jhangiani, S.N., et al. (2016). Monoallelic and biallelic variants in EMC1 identified in individuals with global developmental delay, hypotonia, scoliosis, and cerebellar atrophy. Am J Hum Genet 98, 562–570.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huang, L., Zhang, H., Cheng, C.Y., Wen, F., Tam, P.O.S., Zhao, P., Chen, H., Li, Z., Chen, L., Tai, Z., et al. (2016). A missense variant in FGD6 confers increased risk of polypoidal choroidal vasculopathy. Nat Genet 48, 640–647.

    Article  CAS  PubMed  Google Scholar 

  • Jonikas, M.C., Collins, S.R., Denic, V., Oh, E., Quan, E.M., Schmid, V., Weibezahn, J., Schwappach, B., Walter, P., Weissman, J.S., et al. (2009). Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum. Science 323, 1693–1697.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Junge, H.J., Yang, S., Burton, J.B., Paes, K., Shu, X., French, D.M., Costa, M., Rice, D.S., and Ye, W. (2009). TSPAN12 regulates retinal vascular development by promoting Norrin- but not Wnt-induced FZD4/β-catenin signaling. Cell 139, 299–311.

    Article  CAS  PubMed  Google Scholar 

  • Lahiri, S., Chao, J.T., Tavassoli, S., Wong, A.K.O., Choudhary, V., Young, B.P., Loewen, C.J.R., and Prinz, W.A. (2014). A conserved endoplasmic reticulum membrane protein complex (EMC) facilitates phospholipid transfer from the ER to mitochondria. PLoS Biol 12, e1001969.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Lai, M.B., Zhang, C., Shi, J., Johnson, V., Khandan, L., McVey, J., Klymkowsky, M.W., Chen, Z., and Junge, H.J. (2017). TSPAN12 is a Norrin co-receptor that amplifies Frizzled4 ligand selectivity and signaling. Cell Rep 19, 2809–2822.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li, V.S.W., Ng, S.S., Boersema, P.J., Low, T.Y., Karthaus, W.R., Gerlach, J. P., Mohammed, S., Heck, A.J.R., Maurice, M.M., Mahmoudi, T., et al. (2012). Wnt signaling through inhibition of β-catenin degradation in an intact Axin1 complex. Cell 149, 1245–1256.

    Article  CAS  PubMed  Google Scholar 

  • Lin, D.L., Inoue, T., Chen, Y.J., Chang, A., Tsai, B., and Tai, A.W. (2019). The ER membrane protein complex promotes biogenesis of dengue and Zika virus non-structural multi-pass transmembrane proteins to support infection. Cell Rep 27, 1666–1674.e4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Louie, R.J., Guo, J., Rodgers, J.W., White, R., Shah, N.A., Pagant, S., Kim, P., Livstone, M., Dolinski, K., McKinney, B.A., et al. (2012). A yeast phenomic model for the gene interaction network modulating CFTR-AF508 protein biogenesis. Genome Med 4, 103.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marquez, J., Criscione, J., Charney, R.M., Prasad, M.S., Hwang, W.Y., Mis, E.K., García-Castro, M.I., and Khokha, M.K. (2020). Disrupted ER membrane protein complex-mediated topogenesis drives congenital neural crest defects. J Clin Invest 130, 813–826.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mi, H., Muruganujan, A., Ebert, D., Huang, X., and Thomas, P.D. (2019). PANTHER version 14: more genomes, a new PANTHER GO-slim and improvements in enrichment analysis tools. Nucleic Acids Res 47, D419–D426.

    Article  CAS  PubMed  Google Scholar 

  • Nelson, W.J., and Nusse, R. (2004). Convergence of Wnt, β-catenin, and cadherin pathways. Science 303, 1483–1487.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Park, H., Yamamoto, H., Mohn, L., Ambühl, L., Kanai, K., Schmidt, I., Kim, K.P., Fraccaroli, A., Feil, S., Junge, H.J., et al. (2019). Integrin-linked kinase controls retinal angiogenesis and is linked to Wnt signaling and exudative vitreoretinopathy. Nat Commun 10, 5243.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Pitulescu, M.E., Schmidt, I., Benedito, R., and Adams, R.H. (2010). Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice. Nat Protoc 5, 1518–1534.

    Article  CAS  PubMed  Google Scholar 

  • Pleiner, T., Tomaleri, G.P., Januszyk, K., Inglis, A.J., Hazu, M., and Voorhees, R.M. (2020). Structural basis for membrane insertion by the human ER membrane protein complex. Science 369, 433–436.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Potente, M., Gerhardt, H., and Carmeliet, P. (2011). Basic and therapeutic aspects of angiogenesis. Cell 146, 873–887.

    Article  CAS  PubMed  Google Scholar 

  • Richard, M., Boulin, T., Robert, V.J.P., Richmond, J.E., and Bessereau, J.L. (2013). Biosynthesis of ionotropic acetylcholine receptors requires the evolutionarily conserved ER membrane complex. Proc Natl Acad Sci USA 110, E1055–E1063.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sabbagh, M.F., Heng, J.S., Luo, C., Castanon, R.G., Nery, J.R., Rattner, A., Goff, L.A., Ecker, J.R., and Nathans, J. (2018). Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells. eLife 7, e36187.

    Article  PubMed  PubMed Central  Google Scholar 

  • Satoh, T., Ohba, A., Liu, Z., Inagaki, T., and Satoh, A.K. (2015). dPob/EMC is essential for biosynthesis of rhodopsin and other multi-pass membrane proteins in Drosophila photoreceptors. eLife 4, e06306.

    Article  PubMed Central  Google Scholar 

  • Savidis, G., McDougall, W.M., Meraner, P., Perreira, J.M., Portmann, J.M., Trincucci, G., John, S.P., Aker, A.M., Renzette, N., Robbins, D.R., et al. (2016). Identification of Zika virus and Dengue virus dependency factors using functional genomics. Cell Rep 16, 232–246.

    Article  CAS  PubMed  Google Scholar 

  • Shurtleff, M.J., Itzhak, D.N., Hussmann, J.A., Schirle Oakdale, N.T., Costa, E.A., Jonikas, M., Weibezahn, J., Popova, K.D., Jan, C.H., Sinitcyn, P., et al. (2018). The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins. eLife 7, e37018.

    Article  PubMed  PubMed Central  Google Scholar 

  • Smallwood, P.M., Williams, J., Xu, Q., Leahy, D.J., and Nathans, J. (2007). Mutational analysis of Norrin-Frizzled4 recognition. J Biol Chem 282, 4057–4068.

    Article  CAS  PubMed  Google Scholar 

  • Stambolic, V., Ruel, L., and Woodgett, J.R. (1996). Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol 6, 1664–1669.

    Article  CAS  PubMed  Google Scholar 

  • Tang, X., Snowball, J.M., Xu, Y., Na, C.L., Weaver, T.E., Clair, G., Kyle, J. E., Zink, E.M., Ansong, C., Wei, W., et al. (2017). EMC3 coordinates surfactant protein and lipid homeostasis required for respiration. J Clin Invest 127, 4314–4325.

    Article  PubMed  PubMed Central  Google Scholar 

  • Tao, L., Zhang, J., Meraner, P., Tovaglieri, A., Wu, X., Gerhard, R., Zhang, X., Stallcup, W.B., Miao, J., He, X., et al. (2016). Frizzled proteins are colonic epithelial receptors for C. difficile toxin B. Nature 538, 350–355.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Taylor, M.R., Kikkawa, S., Diez-Juan, A., Ramamurthy, V., Kawakami, K., Carmeliet, P., and Brockerhoff, S.E. (2005). The zebrafish pob gene encodes a novel protein required for survival of red cone photoreceptor cells. Genetics 170, 263–273.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tian, S., Muneeruddin, K., Choi, M.Y., Tao, L., Bhuiyan, R.H., Ohmi, Y., Furukawa, K., Furukawa, K., Boland, S., Shaffer, S.A., et al. (2018). Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation. PLoS Biol 16, e2006951.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Volkmar, N., Thezenas, M.L., Louie, S.M., Juszkiewicz, S., Nomura, D.K., Hegde, R.S., Kessler, B.M., and Christianson, J.C. (2019). The ER membrane protein complex (EMC) promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis. J Cell Sci 132, jcs223453.

    PubMed  PubMed Central  Google Scholar 

  • Wang, Y., Rattner, A., Zhou, Y., Williams, J., Smallwood, P.M., and Nathans, J. (2012). Norrin/Frizzled4 signaling in retinal vascular development and blood brain barrier plasticity. Cell 151, 1332–1344.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang, Y., Cho, C., Williams, J., Smallwood, P.M., Zhang, C., Junge, H.J., and Nathans, J. (2018). Interplay of the Norrin and Wnt7a/Wnt7b signaling systems in blood-brain barrier and blood-retina barrier development and maintenance. Proc Natl Acad Sci USA 115, E11827.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wideman, J.G. (2015). The ubiquitous and ancient ER membrane protein complex (EMC): tether or not? F1000Research 4, 624.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Xiong, L., Zhang, L., Yang, Y., Li, N., Lai, W., Wang, F., Zhu, X., and Wang, T. (2020). ER complex proteins are required for rhodopsin biosynthesis and photoreceptor survival in Drosophila and mice. Cell Death Differ 27, 646–661.

    Article  CAS  PubMed  Google Scholar 

  • Xu, Q., Wang, Y., Dabdoub, A., Smallwood, P.M., Williams, J., Woods, C., Kelley, M.W., Jiang, L., Tasman, W., Zhang, K., et al. (2004). Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair. Cell 116, 883–895.

    Article  CAS  PubMed  Google Scholar 

  • Yang, Z., Camp, N.J., Sun, H., Tong, Z., Gibbs, D., Cameron, D.J., Chen, H., Zhao, Y., Pearson, E., Li, X., et al. (2006). A variant of the HTRA1 gene increases susceptibility to age-related macular degeneration. Science 314, 992–993.

    Article  CAS  PubMed  Google Scholar 

  • Ye, X., Wang, Y., Cahill, H., Yu, M., Badea, T.C., Smallwood, P.M., Peachey, N.S., and Nathans, J. (2009). Norrin, Frizzled-4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization. Cell 139, 285–298.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang, L., Yang, Y., Li, S., Tai, Z., Huang, L., Liu, Y., Zhu, X., Di, Y., Qu, C., Jiang, Z., et al. (2016a). Whole exome sequencing analysis identifies mutations in LRP5 in Indian families with familial exudative vitreoretinopathy. Genet Testing Mol Biomarkers 20, 346–351.

    Article  CAS  Google Scholar 

  • Zhang, R., Miner, J.J., Gorman, M.J., Rausch, K., Ramage, H., White, J.P., Zuiani, A., Zhang, P., Fernandez, E., Zhang, Q., et al. (2016b). A CRISPR screen defines a signal peptide processing pathway required by flaviviruses. Nature 535, 164–168.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhou, Y., Wang, Y., Tischfield, M., Williams, J., Smallwood, P.M., Rattner, A., Taketo, M.M., and Nathans, J. (2014). Canonical WNT signaling components in vascular development and barrier formation. J Clin Invest 124, 3825–3846.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu, X., Qi, X., Yang, Y., Tian, W., Liu, W., Jiang, Z., Li, S., and Zhu, X. (2020). Loss of the ER membrane protein complex subunit Emc3 leads to retinal bipolar cell degeneration in aged mice. PLoS ONE 15, e0238435.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu, X., Yang, M., Zhao, P., Li, S., Zhang, L., Huang, L., Huang, Y., Fei, P., Yang, Y., Zhang, S., et al. (2021). Catenin α 1 mutations cause familial exudative vitreoretinopathy by overactivating Norrin/β-catenin signaling. J Clin Invest 131.

  • Zudaire, E., Gambardella, L., Kurcz, C., and Vermeren, S. (2011). A computational tool for quantitative analysis of vascular networks. PLoS ONE 6, e27385.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (81970841, 81770950, 81790643, 82000913, and 82071009), the Department of Science and Technology of Sichuan Province (2020JDZH0027 and 2020ZYD037), the CAMS Innovation Fund for Medical Sciences (2019-12M-5-032), the fund for Sichuan Provincial People’s Hospital (2021QN01) and the Chengdu Science and Technology Bureau (2019-YF05-00572-SN).

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Authors and Affiliations

  1. Sichuan Provincial Key Laboratory for Human Disease Gene Study, Department of Medical Genetics, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, 610072, China

    Mu Yang, Shujin Li, Wenjing Liu, Xiao Li, Yunqi He, Yeming Yang, Kuanxiang Sun, Lin Zhang, Wanli Tian, Lixin Duan, Zhenglin Yang & Xianjun Zhu

  2. Research Unit for Blindness Prevention of Chinese Academy of Medical Sciences (2019RU026), Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital, Chengdu, 610072, China

    Mu Yang, Shujin Li, Zhenglin Yang & Xianjun Zhu

  3. Key Laboratory of Tibetan Medicine Research, Chinese Academy of Sciences and Qinghai Provincial Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology, Xining, 810008, China

    Xianjun Zhu

  4. Department of Ophthalmology, Shangqiu First People’s Hospital, Shangqiu, 476000, China

    Xianjun Zhu

  5. Natural Products Research Center, Institute of Chengdu Biology, Sichuan Translational Medicine Hospital, Chinese Academy of Sciences, Chengdu, 610072, China

    Yunqi He, Zhenglin Yang & Xianjun Zhu

  6. College of Life Sciences and Technology, University of Electronic Science and Technology of China, Chengdu, 610072, China

    Huafu Chen & Dezhong Yao

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  1. Mu Yang
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Yang, M., Li, S., Liu, W. et al. The ER membrane protein complex subunit Emc3 controls angiogenesis via the FZD4/WNT signaling axis. Sci. China Life Sci. 64, 1868–1883 (2021). https://doi.org/10.1007/s11427-021-1941-7

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