Nihal Kaplan1, Sijia Wang2, Junyi Wang3, Wending Yang1, Rosa Ventrella4, Ahmed Majekodunmi5, Bethany E Perez White1, Spiro Getsios6, Brian J Mitchell4, Han Peng7, Robert M Lavker8. 1. Department of Dermatology, Northwestern University, Chicago, IL, 60611, USA. 2. Department of Dermatology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, 510515, China. 3. Department of Dermatology, Northwestern University, Chicago, IL, 60611, USA; Department of Ophthalmology, The Third Medical Center of Chinese PLA General Hospital, Beijing, China. 4. Cell and Developmental Biology, Northwestern University, Chicago, IL 60611, USA. 5. Department of Neurology, Northwestern University, Chicago, IL, 60611, USA. 6. Aspect Biosystems Ltd., Vancouver, BC, Canada. 7. Department of Dermatology, Northwestern University, Chicago, IL, 60611, USA. Electronic address: han-peng@northwestern.edu. 8. Department of Dermatology, Northwestern University, Chicago, IL, 60611, USA. Electronic address: r-lavker@northwestern.edu.
Abstract
PURPOSE: To understand the relationship between ciliogenesis and autophagy in the corneal epithelium. METHODS: siRNAs for EphA2 or PLD1 were used to inhibit protein expression in vitro. Morpholino-anti-EphA2 was used to knockdown EphA2 in Xenopus skin. An EphA2 knockout mouse was used to conduct loss of function studies. Autophagic vacuoles were visualized by contrast light microscopy. Autophagy flux, was measured by LC3 turnover and p62 protein levels. Immunostaining and confocal microscopy were conducted to visualize cilia in cultured cells and in vivo. RESULTS: Loss of EphA2 (i) increased corneal epithelial thickness by elevating proliferative potential in wing cells, (ii) reduced the number of ciliated cells, (iii) increased large hollow vacuoles, that could be rescued by BafA1; (iv) inhibited autophagy flux and (v) increased GFP-LC3 puncta in the mouse corneal epithelium. This indicated a role for EphA2 in stratified epithelial assembly via regulation of proliferation as well as a positive role in both ciliogenesis and end-stage autophagy. Inhibition of PLD1, an EphA2 interacting protein that is a critical regulator of end-stage autophagy, reversed the accumulation of vacuoles, and the reduction in the number of ciliated cells due to EphA2 depletion, suggesting EphA2 regulation of both end-stage autophagy and ciliogenesis via PLD1. PLD1 mediated rescue of ciliogenesis by EphA2 depletion was blocked by BafA1, placing autophagy between EphA2 signaling and regulation of ciliogenesis. CONCLUSION: Our findings demonstrate a novel role for EphA2 in regulating both autophagy and ciliogenesis, processes that are essential for proper corneal epithelial homeostasis.
PURPOSE: To understand the relationship between ciliogenesis and autophagy in the corneal epithelium. METHODS: siRNAs for EphA2 or PLD1 were used to inhibit protein expression in vitro. Morpholino-anti-EphA2 was used to knockdown EphA2 in Xenopus skin. An EphA2 knockout mouse was used to conduct loss of function studies. Autophagic vacuoles were visualized by contrast light microscopy. Autophagy flux, was measured by LC3 turnover and p62 protein levels. Immunostaining and confocal microscopy were conducted to visualize cilia in cultured cells and in vivo. RESULTS: Loss of EphA2 (i) increased corneal epithelial thickness by elevating proliferative potential in wing cells, (ii) reduced the number of ciliated cells, (iii) increased large hollow vacuoles, that could be rescued by BafA1; (iv) inhibited autophagy flux and (v) increased GFP-LC3 puncta in the mouse corneal epithelium. This indicated a role for EphA2 in stratified epithelial assembly via regulation of proliferation as well as a positive role in both ciliogenesis and end-stage autophagy. Inhibition of PLD1, an EphA2 interacting protein that is a critical regulator of end-stage autophagy, reversed the accumulation of vacuoles, and the reduction in the number of ciliated cells due to EphA2 depletion, suggesting EphA2 regulation of both end-stage autophagy and ciliogenesis via PLD1. PLD1 mediated rescue of ciliogenesis by EphA2 depletion was blocked by BafA1, placing autophagy between EphA2 signaling and regulation of ciliogenesis. CONCLUSION: Our findings demonstrate a novel role for EphA2 in regulating both autophagy and ciliogenesis, processes that are essential for proper corneal epithelial homeostasis.
Authors: Jing Gao; Mindy C DeRouen; Chih-Hsin Chen; Michael Nguyen; Ngon T Nguyen; Hiroyuki Ido; Kenji Harada; Kiyotoshi Sekiguchi; Bruce A Morgan; Jeffery H Miner; Anthony E Oro; M Peter Marinkovich Journal: Genes Dev Date: 2008-08-01 Impact factor: 11.361
Authors: Teunis J P van Dam; Julie Kennedy; Robin van der Lee; Erik de Vrieze; Kirsten A Wunderlich; Suzanne Rix; Gerard W Dougherty; Nils J Lambacher; Chunmei Li; Victor L Jensen; Michel R Leroux; Rim Hjeij; Nicola Horn; Yves Texier; Yasmin Wissinger; Jeroen van Reeuwijk; Gabrielle Wheway; Barbara Knapp; Jan F Scheel; Brunella Franco; Dorus A Mans; Erwin van Wijk; François Képès; Gisela G Slaats; Grischa Toedt; Hannie Kremer; Heymut Omran; Katarzyna Szymanska; Konstantinos Koutroumpas; Marius Ueffing; Thanh-Minh T Nguyen; Stef J F Letteboer; Machteld M Oud; Sylvia E C van Beersum; Miriam Schmidts; Philip L Beales; Qianhao Lu; Rachel H Giles; Radek Szklarczyk; Robert B Russell; Toby J Gibson; Colin A Johnson; Oliver E Blacque; Uwe Wolfrum; Karsten Boldt; Ronald Roepman; Victor Hernandez-Hernandez; Martijn A Huynen Journal: PLoS One Date: 2019-05-16 Impact factor: 3.240
Authors: Kristin Gordon; James J Kochkodan; Hanz Blatt; Samantha Y Lin; Nihal Kaplan; Andrew Johnston; William R Swindell; Paul Hoover; Bethanee J Schlosser; James T Elder; Johann E Gudjonsson; Spiro Getsios Journal: J Invest Dermatol Date: 2012-11-29 Impact factor: 8.551