Eya1 controls cell polarity, spindle orientation, cell fate and Notch signaling in distal embryonic lung epithelium

Developmental Biology and Regenerative Medicine Program, Saban Research Institute, Childrens Hospital Los Angeles, Keck School of Medicine of University of Southern California, 4661 Sunset Boulevard, Los Angeles, CA 90027, USA.
Development (Impact Factor: 6.46). 04/2011; 138(7):1395-407. DOI: 10.1242/dev.058479
Source: PubMed


Cell polarity, mitotic spindle orientation and asymmetric division play a crucial role in the self-renewal/differentiation of epithelial cells, yet little is known about these processes and the molecular programs that control them in embryonic lung distal epithelium. Herein, we provide the first evidence that embryonic lung distal epithelium is polarized with characteristic perpendicular cell divisions. Consistent with these findings, spindle orientation-regulatory proteins Insc, LGN (Gpsm2) and NuMA, and the cell fate determinant Numb are asymmetrically localized in embryonic lung distal epithelium. Interfering with the function of these proteins in vitro randomizes spindle orientation and changes cell fate. We further show that Eya1 protein regulates cell polarity, spindle orientation and the localization of Numb, which inhibits Notch signaling. Hence, Eya1 promotes both perpendicular division as well as Numb asymmetric segregation to one daughter in mitotic distal lung epithelium, probably by controlling aPKCζ phosphorylation. Thus, epithelial cell polarity and mitotic spindle orientation are defective after interfering with Eya1 function in vivo or in vitro. In addition, in Eya1(-/-) lungs, perpendicular division is not maintained and Numb is segregated to both daughter cells in mitotic epithelial cells, leading to inactivation of Notch signaling. As Notch signaling promotes progenitor cell identity at the expense of differentiated cell phenotypes, we test whether genetic activation of Notch could rescue the Eya1(-/-) lung phenotype, which is characterized by loss of epithelial progenitors, increased epithelial differentiation but reduced branching. Indeed, genetic activation of Notch partially rescues Eya1(-/-) lung epithelial defects. These findings uncover novel functions for Eya1 as a crucial regulator of the complex behavior of distal embryonic lung epithelium.

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    • "Eya1 is a multifunctional protein and a possible target of RTK signaling [35]. Furthermore, Eya1 has recently been shown to be involved in controlling cell polarity, cell fate, orientation of the mitotic spindle and notch signaling [36]. Hence, these mutations may compromise some other unknown or some or all of these recently identified functions of Eya1. "
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    ABSTRACT: Mutations in the human EYA1 gene have been associated with several human diseases including branchio-oto (BO) and branchio-oto-renal (BOR) syndrome, as well as congenital cataracts and ocular anterior segment anomalies. BOR patients suffer from severe malformations of the ears, branchial arches and kidneys. The phenotype of Eya1-heterozygous mice resembles the symptoms of human patients suffering from BOR syndrome. The Eya1 gene encodes a multifunctional protein that acts as a protein tyrosine phosphatase and a transcriptional coactivator. It has been shown that Eya1 interacts with Six transcription factors, which are also required for nuclear translocation of the Eya1 protein. We investigated the effects of seven disease-causing Eya1 missense mutations on Eya1 protein function, in particular cellular localization, ability to interact with Six proteins, and protein stability. We show here that the BOR-associated Eya1 missense mutations S454P, L472R, and L550P lead to enhanced proteasomal degradation of the Eya1 protein in mammalian cells. Moreover, Six proteins lead to a significant stabilization of Eya1, which is caused by Six-mediated protection from proteasomal degradation. In case of the mutant L550P, loss of interaction with Six proteins leads to rapid protein degradation. Our observations suggest that protein destabilization constitutes a novel disease causing mechanism for Eya1.
    Full-text · Article · Jan 2014 · PLoS ONE
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    • "Instead we propose a model where Nubp1 acts as a molecular switch to maintain cell polarity and proper centrosomal behavior. Both cellular processes seem to be essential for proper lung development as shown by recent studies on cell division orientation and asymmetric cell division during lung branching morphogenesis although the molecular outcome of this cellular function remains poorly understood [25], [26]). The Nubp1 mutant described here provides another powerful tool to shed new insights into aforementioned processes and to dissect the interconnections between cellular behaviors and lung morphogenesis. "
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    ABSTRACT: The lung is a complex system in biology and medicine alike. Whereas there is a good understanding of the anatomy and histology of the embryonic and adult lung, less is known about the molecular details and the cellular pathways that ultimately orchestrate lung formation and affect its health. From a forward genetic approach to identify novel genes involved in lung formation, we identified a mutated Nubp1 gene, which leads to syndactyly, eye cataract and lung hypoplasia. In the lung, Nubp1 is expressed in progenitor cells of the distal epithelium. Nubp1(m1Nisw) mutants show increased apoptosis accompanied by a loss of the distal progenitor markers Sftpc, Sox9 and Foxp2. In addition, Nubp1 mutation disrupts localization of the polarity protein Par3 and the mitosis relevant protein Numb. Using knock-down studies in lung epithelial cells, we also demonstrate a function of Nubp1 in regulating centrosome dynamics and microtubule organization. Together, Nubp1 represents an essential protein for lung progenitor survival by coordinating vital cellular processes including cell polarity and centrosomal dynamics.
    Preview · Article · Sep 2012 · PLoS ONE
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    • "Western blotting and immunoprecipitation were performed using ZO-1, occludin, claudin1, E-cadherin and Eya1 antibodies described before (Nunbhakdi-Craig et al., 2002; El-Hashash et al., 2011a), and standard protocols, as described (Tefft et al., 2002; Tefft et al., 2005; Buckley et al., 2005; del Moral et al., 2006a; del Moral et al., 2006b; El-Hashash et al., 2011a; El-Hashash et al., 2011b). Briefly, for immunoprecipitation, E14.5 lung or MLE15 (grown for 3 days in NC medium) cells were lysed in RIPA buffer, centrifuged and the supernatant containing ,1 mg protein was pre-cleared by incubation with rabbit IgG and protein A/G agarose, then centrifuged. "
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    ABSTRACT: Little is known about the regulatory mechanisms underlying lung epithelial tight junction (TJ) assembly, which is inextricably linked to the preservation of epithelial polarity, and is highly coordinated by proteins that regulate epithelial cell polarity such as aPKCζ. We recently reported that Eya1 phosphatase functions through aPKCζ-Notch1 signaling to control cell polarity in the lung epithelium. Here, we have extended these observations to TJ formation to demonstrate that Eya1 is crucial for the maintenance of TJ protein assembly in the lung epithelium, probably by controlling aPKCζ phosphorylation levels, aPKCζ-mediated TJ protein phosphorylation and Notch1-Cdc42 activity. Thus, TJs are disassembled after interfering with Eya1 function in vivo or during calcium-induced TJ assembly in vitro. These effects are reversed by reintroduction of wildtype Eya1 or partially inhibiting aPKCζ in Eya1siRNA cells. Moreover, genetic activation of Notch1 rescues Eya1(-/-) lung epithelial TJ defects. These findings uncover novel functions for Eya1-aPKCζ-Notch1-Cdc42 as a crucial regulatory mechanism of TJ assembly and polarity of the lung epithelium, providing a conceptual framework for future mechanistic and translational studies in this area.
    Full-text · Article · Jun 2012 · Journal of Cell Science
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