FIH-1 Disrupts an LRRK1/EGFR Complex to Positively Regulate Keratinocyte Migration
2014; Elsevier BV; Volume: 184; Issue: 12 Linguagem: Inglês
10.1016/j.ajpath.2014.08.014
ISSN1525-2191
AutoresHan Peng, Nihal Kaplan, Wending Yang, Spiro Getsios, Robert M. Lavker,
Tópico(s)Cell Adhesion Molecules Research
ResumoFactor inhibiting hypoxia-inducible factor 1 (FIH-1; official symbol HIF1AN) is a hydroxylase that negatively regulates hypoxia-inducible factor 1α but also targets other ankyrin repeat domain–containing proteins such as Notch receptor to limit epithelial differentiation. We show that FIH-1 null mutant mice exhibit delayed wound healing. Importantly, in vitro scratch wound assays demonstrate that the positive role of FIH-1 in migration is independent of Notch signaling, suggesting that this hydroxylase targets another ankyrin repeat domain–containing protein to positively regulate motogenic signaling pathways. Accordingly, FIH-1 increases epidermal growth factor receptor (EGFR) signaling, which in turn enhances keratinocyte migration via mitogen-activated protein kinase pathway, leading to extracellular signal–regulated kinase 1/2 activation. Our studies identify leucine-rich repeat kinase 1 (LRRK1), a key regulator of the EGFR endosomal trafficking and signaling, as an FIH-1 binding partner. Such an interaction prevents the formation of an EGFR/LRRK1 complex, necessary for proper EGFR turnover. The identification of LRRK1 as a novel target for FIH-1 provides new insight into how FIH-1 functions as a positive regulator of epithelial migration. Factor inhibiting hypoxia-inducible factor 1 (FIH-1; official symbol HIF1AN) is a hydroxylase that negatively regulates hypoxia-inducible factor 1α but also targets other ankyrin repeat domain–containing proteins such as Notch receptor to limit epithelial differentiation. We show that FIH-1 null mutant mice exhibit delayed wound healing. Importantly, in vitro scratch wound assays demonstrate that the positive role of FIH-1 in migration is independent of Notch signaling, suggesting that this hydroxylase targets another ankyrin repeat domain–containing protein to positively regulate motogenic signaling pathways. Accordingly, FIH-1 increases epidermal growth factor receptor (EGFR) signaling, which in turn enhances keratinocyte migration via mitogen-activated protein kinase pathway, leading to extracellular signal–regulated kinase 1/2 activation. Our studies identify leucine-rich repeat kinase 1 (LRRK1), a key regulator of the EGFR endosomal trafficking and signaling, as an FIH-1 binding partner. Such an interaction prevents the formation of an EGFR/LRRK1 complex, necessary for proper EGFR turnover. The identification of LRRK1 as a novel target for FIH-1 provides new insight into how FIH-1 functions as a positive regulator of epithelial migration. CME Accreditation Statement: This activity ("ASIP 2014 AJP CME Program in Pathogenesis") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity ("ASIP 2014 AJP CME Program in Pathogenesis") for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity ("ASIP 2014 AJP CME Program in Pathogenesis") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("ASIP 2014 AJP CME Program in Pathogenesis") for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. The asparaginyl hydroxylase factor-inhibiting hypoxia-inducible factor 1α (FIH-1; official symbol HIF1AN) was originally identified as a protein that interacts with and inhibits the activity of hypoxia-inducible factor 1α (HIF-1α) in the C-terminal transactivation domain1Mahon P.C. Hirota K. Semenza G.L. FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity.Genes Dev. 2001; 15: 2675-2686Crossref PubMed Scopus (1174) Google Scholar, 2Lando D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. Bruick R.K. FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor.Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1264) Google Scholar by coupling the oxidative decarboxylation of 2-oxoglutarate to the hydroxylation of HIF-1α.3Cockman M.E. Webb J.D. Kramer H.B. Kessler B.M. Ratcliffe P.J. Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.Mol Cell Proteomics. 2009; 8: 535-546Crossref PubMed Scopus (122) Google Scholar Significantly, proteins containing the ankyrin repeat domain, such as Notch, are other substrates for FIH-1.3Cockman M.E. Webb J.D. Kramer H.B. Kessler B.M. Ratcliffe P.J. Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.Mol Cell Proteomics. 2009; 8: 535-546Crossref PubMed Scopus (122) Google Scholar Only recently has FIH-1 been recognized to have pleiotropic roles in maintaining epithelial homeostasis.4Peng H. Hamanaka R.B. Katsnelson J. Hao L.L. Yang W. Chandel Lavker R.M. MicroRNA-31 targets FIH-1 to positively regulate corneal epithelial glycogen metabolism.FASEB J. 2012; 26: 3140-3147Crossref PubMed Scopus (51) Google Scholar, 5Peng H. Kaplan N. Hamanaka R.B. Katsnelson J. Blatt H. Yang W. Hao L. Bryar P.J. Johnson R.S. Getsios S. Chandel Lavker R.M. microRNA-31/factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation.Proc Natl Acad Sci U S A. 2012; 109: 14030-14034Crossref PubMed Scopus (101) Google Scholar For example, FIH-1 negatively regulates glycogen metabolism in corneal epithelium in a HIF-1α–independent manner via the direct involvement of the Akt/glycogen synthase kinase 3β signaling pathway.4Peng H. Hamanaka R.B. Katsnelson J. Hao L.L. Yang W. Chandel Lavker R.M. MicroRNA-31 targets FIH-1 to positively regulate corneal epithelial glycogen metabolism.FASEB J. 2012; 26: 3140-3147Crossref PubMed Scopus (51) Google Scholar Furthermore, in epidermal and corneal epithelial keratinocytes, FIH-1 was shown to act as a negative regulator of differentiation via a coordinate decrease in Notch signaling.5Peng H. Kaplan N. Hamanaka R.B. Katsnelson J. Blatt H. Yang W. Hao L. Bryar P.J. Johnson R.S. Getsios S. Chandel Lavker R.M. microRNA-31/factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation.Proc Natl Acad Sci U S A. 2012; 109: 14030-14034Crossref PubMed Scopus (101) Google Scholar What is not clear in these studies is whether FIH-1 affects other signaling pathways known to influence keratinocyte growth, differentiation, and migration. For example, the regulation of Notch 1 activity by FIH-15Peng H. Kaplan N. Hamanaka R.B. Katsnelson J. Blatt H. Yang W. Hao L. Bryar P.J. Johnson R.S. Getsios S. Chandel Lavker R.M. microRNA-31/factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation.Proc Natl Acad Sci U S A. 2012; 109: 14030-14034Crossref PubMed Scopus (101) Google Scholar raises the possibility of cross-talk with the epidermal growth factor receptor (EGFR)-signaling pathway, since EGFR signaling has been shown to be a negative regulator of Notch 1 gene expression and activity in keratinocytes.6Kolev V. Mandinova A. Guinea-Viniegra J. Hu B. Lefort K. Lambertini C. Neel V. Dummer R. Wagner E.F. Dotto G.P. EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer.Nat Cell Biol. 2008; 10 ([erratum in: Nat Cell Biol 2013;15:124]): 902-911Crossref PubMed Scopus (169) Google Scholar Once EGF binds to the EGFR, numerous signaling pathways are activated that impact on cell proliferation, migration, differentiation, and survival.7Olayioye M.A. Neve R.M. Lane H.A. Hynes N.E. The ErbB signaling network: receptor heterodimerization in development and cancer.EMBO J. 2000; 19: 3159-3167Crossref PubMed Google Scholar, 8Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3596) Google Scholar, 9Yarden Y. Sliwkowski M.X. Untangling the ErbB signalling network.Nat Rev Mol Cell Biol. 2001; 2: 127-137Crossref PubMed Scopus (5782) Google Scholar With respect to the skin, EGFR impacts on epidermal and hair follicle development, keratinocyte proliferation, survival, cancer, and immune homeostasis.10Mascia F. Denning M. Kopan R. Yuspa S.H. The black box illuminated: signals and signaling.J Invest Dermatol. 2012; 132: 811-819Crossref PubMed Scopus (27) Google Scholar EGFR signaling also plays a prominent role in epidermal and corneal epithelial migration and wound repair. For example, in the epidermis, EGFR signaling has been shown to promote keratinocyte migration and wound repair.11Repertinger S.K. Campagnaro E. Fuhrman J. El-Abaseri T. Yuspa S.H. Hansen L.A. EGFR enhances early healing after cutaneous incisional wounding.J Invest Dermatol. 2004; 123: 982-989Crossref PubMed Scopus (141) Google Scholar Likewise, corneal perturbations activate the EGFR and downstream Ras-Raf-Mek-Erk1/2 (Ras, Raf, mitogen-activated protein kinase kinase, extracellular signal–regulated kinase 1/2) and phosphoinositide 3 kinase–Akt signaling cascades, which are required for efficient wound healing and are attenuated in patients with diabetic keratopathies.12Xu K. Yu F.S. Impaired epithelial wound healing and EGFR signaling pathways in the corneas of diabetic rats.Invest Ophthalmol Vis Sci. 2011; 52: 3301-3308Crossref PubMed Scopus (109) Google Scholar, 13Xu K.P. Li Y. Ljubimov A.V. Yu F.S. High glucose suppresses epidermal growth factor receptor/phosphatidylinositol 3-kinase/Akt signaling pathway and attenuates corneal epithelial wound healing.Diabetes. 2009; 58: 1077-1085Crossref PubMed Scopus (138) Google Scholar, 14Zieske J.D. Takahashi H. Hutcheon A.E. Dalbone A.C. Activation of epidermal growth factor receptor during corneal epithelial migration.Invest Ophthalmol Vis Sci. 2000; 41: 1346-1355PubMed Google Scholar The activation of EGFR also commences endocytic trafficking, whereby the receptor is either packaged in lysosomes for degradation or recycled to the cell surface.15Maxfield F.R. McGraw T.E. Endocytic recycling.Nat Rev Mol Cell Biol. 2004; 5: 121-132Crossref PubMed Scopus (1541) Google Scholar, 16Sorkin A. von Zastrow M. Endocytosis and signalling: intertwining molecular networks.Nat Rev Mol Cell Biol. 2009; 10: 609-622Crossref PubMed Scopus (880) Google Scholar Endosomal trafficking is essential for establishing the extensiveness of the EGF-mediated signal, and thus much attention has been directed toward understanding the steps involved in the movement of the EGFR from the cell surface to cytoplasmic vesicles, such as the endosome, multivesicular body, and lysosome.16Sorkin A. von Zastrow M. Endocytosis and signalling: intertwining molecular networks.Nat Rev Mol Cell Biol. 2009; 10: 609-622Crossref PubMed Scopus (880) Google Scholar, 17Madshus I.H. Stang E. Internalization and intracellular sorting of the EGF receptor: a model for understanding the mechanisms of receptor trafficking.J Cell Sci. 2009; 122: 3433-3439Crossref PubMed Scopus (122) Google Scholar, 18Rush J.S. Quinalty L.M. Engelman L. Sherry D.M. Ceresa B.P. Endosomal accumulation of the activated epidermal growth factor receptor (EGFR) induces apoptosis.J Biol Chem. 2012; 287: 712-722Crossref PubMed Scopus (66) Google Scholar Recently, leucine-rich repeat kinase 1 (LRRK1) was recognized as a key regulator of EGFR endosomal trafficking.19Hanafusa H. Ishikawa K. Kedashiro S. Saigo T. Iemura S. Natsume T. Komada M. Shibuya H. Nara A. Matsumoto K. Leucine-rich repeat kinase LRRK1 regulates endosomal trafficking of the EGF receptor.Nat Commun. 2011; 2: 158Crossref PubMed Scopus (70) Google Scholar, 20Ishikawa K. Nara A. Matsumoto K. Hanafusa H. EGFR-dependent phosphorylation of leucine-rich repeat kinase LRRK1 is important for proper endosomal trafficking of EGFR.Mol Biol Cell. 2012; 23: 1294-1306Crossref PubMed Scopus (22) Google Scholar Specifically, it is believed that LRRK1 forms a complex with activated EGFR through an interaction with growth factor receptor–bound protein 2 and that this complex is internalized in early endosomes.19Hanafusa H. Ishikawa K. Kedashiro S. Saigo T. Iemura S. Natsume T. Komada M. Shibuya H. Nara A. Matsumoto K. Leucine-rich repeat kinase LRRK1 regulates endosomal trafficking of the EGF receptor.Nat Commun. 2011; 2: 158Crossref PubMed Scopus (70) Google Scholar The mechanism by which LRRK1 regulates EGFR transport is from early to late endosomes.19Hanafusa H. Ishikawa K. Kedashiro S. Saigo T. Iemura S. Natsume T. Komada M. Shibuya H. Nara A. Matsumoto K. Leucine-rich repeat kinase LRRK1 regulates endosomal trafficking of the EGF receptor.Nat Commun. 2011; 2: 158Crossref PubMed Scopus (70) Google Scholar LRRK1 protein kinase is one of the ROCO proteins, which contain a GTPase-like domain [Ras of complex proteins (Roc)] and a C-terminal of Roc (COR) domain.21Bosgraaf L. Van Haastert P.J. Roc, a Ras/GTPase domain in complex proteins.Biochim Biophys Acta. 2003; 1643: 5-10Crossref PubMed Scopus (268) Google Scholar ROCO proteins have a series of leucine-rich repeats and/or ankyrin repeats, with LRRK1 containing six N-terminal ankyrin repeats.22Korr D. Toschi L. Donner P. Pohlenz H.D. Kreft B. Weiss B. LRRK1 protein kinase activity is stimulated upon binding of GTP to its Roc domain.Cell Signal. 2006; 18: 910-920Crossref PubMed Scopus (102) Google Scholar This latter aspect of LRRK1 is noteworthy since, as mentioned above, proteins with ankyrin repeat domains are potential substrates for FIH-1.3Cockman M.E. Webb J.D. Kramer H.B. Kessler B.M. Ratcliffe P.J. Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.Mol Cell Proteomics. 2009; 8: 535-546Crossref PubMed Scopus (122) Google Scholar Thus, FIH-1 has the potential to directly interact with LRRK1, which could impact on EGFR signaling. Here we show that ectopic expression of FIH-1 in keratinocytes increases phosphorylation of the EGFR, which positively affects keratinocyte migration via stimulation of the mitogen-activated protein kinase pathway, resulting in an increase in phosphorylated ERK1/2. Such enhanced migration is independent of Notch signaling. Moreover, our studies reveal that FIH-1 interacts with LRRK1 and prevents the formation of an EGFR/LRRK1 complex necessary for proper EGFR turnover.19Hanafusa H. Ishikawa K. Kedashiro S. Saigo T. Iemura S. Natsume T. Komada M. Shibuya H. Nara A. Matsumoto K. Leucine-rich repeat kinase LRRK1 regulates endosomal trafficking of the EGF receptor.Nat Commun. 2011; 2: 158Crossref PubMed Scopus (70) Google Scholar The identification of LRRK1 as a substrate for FIH-1 provides new insight into how FIH-1 functions as a positive regulator of epithelial migration. Thus, the breadth of FIH-1 epithelial biology is considerably larger than previously realized. The FIH-1 null mice were generated by breeding the FIHflox mouse with the Ella-Cre transgenic mouse.23Zhang N. Fu Z. Linke S. Chicher J. Gorman J.J. Visk D. Haddad G.G. Poellinger L. Peet D.J. Powell F. Johnson R.S. The asparaginyl hydroxylase factor inhibiting HIF-1alpha is an essential regulator of metabolism.Cell Metab. 2010; 11: 364-378Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar Wild-type (WT) mice (C57BL/6) were purchased from Charles River Laboratories. For skin wound healing assays, mice were first anesthetized, and the area assigned for wounding was shaved. Two 3-mm full-thickness punch wounds were generated on the dorsal skin. Wounds were imaged by a Nikon camera (Nikon Corp., Tokyo, Japan) at 3 and 5 days postwounding, and the sizes of wounds were analyzed using ImageJ software version 1.49d (NIH, Bethesda, MD). To monitor the histological process of wound healing, another cohort of mice was used and sacrificed at 2 days postwounding, and the wound was excised and fixed. A series of sections (N = 4; 50 microns away from each other) were stained with hematoxylin and eosin, and the distances were measured by ImageJ:Epithelialgapclosure=(Widthofwound-epithelialgap)Widthofwound.(1) For corneal wounds, mice were first anesthetized, and the application of a rotating diamond burr to the surface of the central cornea resulted in the removal of the corneal epithelium, whereas the limbal epithelium remained intact and then tissues were embedded in paraffin blocks. Animal experiments were approved by the Northwestern University Animal Care and Use Committee. Primary cultures of human epidermal keratinocytes (HEKs) were isolated from neonatal foreskin by Northwestern University Skin Disease Research Center keratinocyte core as described24Getsios S. Simpson C.L. Kojima S. Harmon R. Sheu L.J. Dusek R.L. Cornwell M. Green K.J. Desmoglein 1-dependent suppression of EGFR signaling promotes epidermal differentiation and morphogenesis.J Cell Biol. 2009; 185: 1243-1258Crossref PubMed Scopus (176) Google Scholar and maintained in medium 154 (Cascade BiologicsInc., Portland, OR) containing human keratinocyte growth supplements and 0.07 mmol/L CaCl2. Primary human corneal epithelial keratinocytes (HCEKs) were isolated from cadaver donor corneas provided by Midwest Eye Bank (Ann Arbor, MI) and cultured in CnT-20 media with supplements (CELLnTEC Advanced Cell Systems AG, Bern, Switzerland) on collagen IV–coated plates (BD Biosciences, San Jose, CA). The limbal derived corneal epithelial cell line, hTCEpi,25Robertson D.M. Li L. Fisher S. Pearce V.P. Shay J.W. Wright W.E. Cavanagh H.D. Jester J.V. Characterization of growth and differentiation in a telomerase-immortalized human corneal epithelial cell line.Invest Ophthalmol Vis Sci. 2005; 46: 470-478Crossref PubMed Scopus (230) Google Scholar was grown in keratinocyte serum-free medium with supplements (Invitrogen, Carlsbad, CA) and 0.15 mmol/L CaCl2. For retroviral infections, keratinocytes were transduced with retroviral supernatants produced in Phoenix amphotropic packaging cells, as previously described.24Getsios S. Simpson C.L. Kojima S. Harmon R. Sheu L.J. Dusek R.L. Cornwell M. Green K.J. Desmoglein 1-dependent suppression of EGFR signaling promotes epidermal differentiation and morphogenesis.J Cell Biol. 2009; 185: 1243-1258Crossref PubMed Scopus (176) Google Scholar For lentiviral infections, keratinocytes were transduced with lentiviral supernatants (produced by the Northwestern University Skin Disease Research Center RNA/DNA Delivery Core Facility) for 6 hours and switched to fresh culture medium overnight. To silence gene expression, 20 nmol/L siRNA smart pools targeting at least two different sequences in the FIH-1 and LRRK1 genes or a scrambled negative control (Dharmacon, Inc., Lafayette, CO) were transiently transfected into cells using siRNA transfection reagent (RNAiMAX; Invitrogen), as described.26Lin S. Gordon K. Kaplan N. Getsios S. Ligand targeting of EphA2 enhances keratinocyte adhesion and differentiation via desmoglein 1.Mol Biol Cell. 2010; 21: 3902-3914Crossref PubMed Scopus (39) Google Scholar For immunoprecipitation, protein lysates were prepared in radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, phosphatase inhibitor cocktail (Thermo Fisher Scientific Inc., Rockford, IL), and protease inhibitor cocktail (Thermo Fisher Scientific Inc.)] and subjected to immunoprecipitation, as previously described.27Kaplan N. Fatima A. Peng H. Bryar P.J. Lavker R.M. Getsios S. EphA2/Ephrin-A1 signaling complexes restrict corneal epithelial cell migration.Invest Ophthalmol Vis Sci. 2012; 53: 936-945Crossref PubMed Scopus (35) Google Scholar The supernatant was added with 20 μL of protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and the indicated antibodies and rotated for 2 hours at 4°C. The beads were washed three times with ice-cold phosphate-buffered saline and subjected to immunoblot analysis. Western blot analyses were performed as described previously.5Peng H. Kaplan N. Hamanaka R.B. Katsnelson J. Blatt H. Yang W. Hao L. Bryar P.J. Johnson R.S. Getsios S. Chandel Lavker R.M. microRNA-31/factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation.Proc Natl Acad Sci U S A. 2012; 109: 14030-14034Crossref PubMed Scopus (101) Google Scholar The following antibodies were used: FIH-1 (sc-271780), α-tubulin (sc-23948), EGFR (sc-373746), glyceraldehyde-3-phosphate dehydrogenase (sc-25778; Santa Cruz Biotechnology), LRRK1 (PA5-13868; Thermo Fisher Scientific Inc.), p-tyrosine (05-321; EMD Millipore, Billerica, MA), p-EGFR (2234 and 2237), and p-ERK (4370; Cell Signaling Technology, Inc., Beverly, MA). Paraffin sections were processed for immunohistochemical (IHC) analysis or hematoxylin and eosin staining, as described previously.5Peng H. Kaplan N. Hamanaka R.B. Katsnelson J. Blatt H. Yang W. Hao L. Bryar P.J. Johnson R.S. Getsios S. Chandel Lavker R.M. microRNA-31/factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation.Proc Natl Acad Sci U S A. 2012; 109: 14030-14034Crossref PubMed Scopus (101) Google Scholar For FIH-1 staining, sections were incubated for 1 hour with FIH-1 rabbit polyclonal antibody (1:300; sc-48813; Santa Cruz Biotechnology). Sections were counterstained with hematoxylin to visualize morphology and mounted in Permount (Thermo Fisher Scientific Inc.). Images were obtained using a Zeiss AxioCam HR digital camera mounted on a Zeiss Axioplan 2 bright field microscope system with a Plan-Neofluar 40×/0.75 objective (Carl Zeiss AG, Oberkochen, Germany). AxioVision software version 4.8 (Carl Zeiss AG) was used to acquire the images. Early endosome abundance (EEA) 1 staining was performed as previously described.19Hanafusa H. Ishikawa K. Kedashiro S. Saigo T. Iemura S. Natsume T. Komada M. Shibuya H. Nara A. Matsumoto K. Leucine-rich repeat kinase LRRK1 regulates endosomal trafficking of the EGF receptor.Nat Commun. 2011; 2: 158Crossref PubMed Scopus (70) Google Scholar Briefly, cells grown on culture slides were incubated in the medium without supplements overnight and then treated with Alexa Fluor 647 EGF complex (Life Technologies Corp., Carlsbad, CA) (100 ng/mL) at 4°C for 30 minutes. After washing with phosphate-buffered saline, cells were incubated in the medium without supplements and EGF for the indicated time at 37°C and fixed with 4% paraformaldehyde for 10 minutes. Slides were incubated overnight with an antibody recognizing EEA1 (1:100; 610457; BD Transduction Laboratories, San Jose, CA). After washing, slides were incubated with Alexa 488-linked secondary IgG (Vector Laboratories, Inc, Burlingame, CA). Images were acquired using a laser-scanning confocal microscope imaging system (UV LSM 510 META; Carl Zeiss) with a Plan Apochromat63 chroma/1.4 oil immersion objective. The Pearson coefficient was analyzed by ImageJ. For pharmacological inhibition of FIH-1 activity, Mek-ERK1/2, EGFR, Notch signaling, and endocytosis, the cells were pretreated with 1 mmol/L dimethyloxalylglycine (Santa Cruz Biotechnology) for 2 hours, 10 μmol/L U0126 (Sigma-Aldrich Corp., St. Louis, MO) for 2 hours, 0.1 μmol/L AG1478 (Sigma-Aldrich Corp.) for 2 hours, 10 μmol/L N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (Sigma-Aldrich Corp.) for 16 hours, and 20 μM chloroquine overnight, respectively. Cells were grown to confluence on 12-well plastic dishes, and linear scratch wounds (in triplicate) were generated on the confluent monolayers using a 200-μL pipette tip. Images were obtained at room temperature using a Zeiss AxioCam MR digital camera mounted on a Zeiss Axiovert 40CFL inverted light microscope with an A-plan 10×/0.25 Ph1Var objective (Carl Zeiss AG). AxioVision software was used to acquire the images. The percentage decrease in the wound gaps were calculated using AxioVision computer-assisted image analysis and normalized to the time-0 wounds. To rule out the contribution of proliferation in the sealing of linear scratch wounds, cells were pretreated with 5 μg/mL mitomycin C (EMD BioSciences, Inc., San Diego, CA). All values are expressed as means ± SD. The significance of the differences between two groups was evaluated by an unpaired Student's t-test and two-way analysis of variance test. Increased FIH-1 levels are associated with defects in keratinocyte differentiation and glycogen metabolism.4Peng H. Hamanaka R.B. Katsnelson J. Hao L.L. Yang W. Chandel Lavker R.M. MicroRNA-31 targets FIH-1 to positively regulate corneal epithelial glycogen metabolism.FASEB J. 2012; 26: 3140-3147Crossref PubMed Scopus (51) Google Scholar, 5Peng H. Kaplan N. Hamanaka R.B. Katsnelson J. Blatt H. Yang W. Hao L. Bryar P.J. Johnson R.S. Getsios S. Chandel Lavker R.M. microRNA-31/factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation.Proc Natl Acad Sci U S A. 2012; 109: 14030-14034Crossref PubMed Scopus (101) Google Scholar, 28Peng H. Katsnelson J. Yang W. Brown M.A. Lavker R.M. FIH-1/c-kit signaling: a novel contributor to corneal epithelial glycogen metabolism.Invest Ophthalmol Vis Sci. 2013; 54: 2781-2786Crossref PubMed Scopus (15) Google Scholar Not surprisingly, FIH-1 is normally undetectable in mouse epidermis and corneal epithelium (Figure 1A). In contrast, there is a marked up-regulation of FIH on wounding, particularly in cells at the leading edge of the wound (Figure 1A). This led us to hypothesize that FIH-1 plays a role in epithelial cell migration. To determine whether FIH-1 is required for normal wound healing, we examined the ability of mice with a null mutation in the FIH gene (FIH-1−/−) to heal full-thickness skin wounds. Wound closure was visibly delayed in FIH-1−/− mice beginning 3 days after wound initiation (P < 0.001) (Figure 1B). Wounds were clinically visible in the FIH-1−/− mice 5 days postwounding, whereas wounds were virtually closed in the control mice at this time (Figure 1B). Re-epithelialization, a histological measure of epidermal closure, was less in control mice versus the FIH-1−/− mice on day 2 (P < 0.05) and on day 3 (P = 0.057) after wound initiation (Figure 1C). Taken together, these findings support the idea that FIH-1 enhances re-epithelialization, which contributes, in part, to efficient wound healing. To examine whether FIH-1 has a direct effect on epithelial cell migration, we used a scratch wound assay in a telomerase immortalized human corneal epithelial cell line,25Robertson D.M. Li L. Fisher S. Pearce V.P. Shay J.W. Wright W.E. Cavanagh H.D. Jester J.V. Characterization of growth and differentiation in a telomerase-immortalized human corneal epithelial cell line.Invest Ophthalmol Vis Sci. 2005; 46: 470-478Crossref PubMed Scopus (230) Google Scholar which has high endogenous levels of FIH-1.5Peng H. Kaplan N. Hamanaka R.B. Katsnelson J. Blatt H. Yang W. Hao L. Bryar P.J. Johnson R.S. Getsios S. Chandel Lavker R.M. microRNA-31/factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation.Proc Natl Acad Sci U S A. 2012; 109: 14030-14034Crossref PubMed Scopus (101) Google Scholar Treatment of hTCEpi cells with the cell-permeable FIH inhibitor dimethyloxalylglycine2Lando D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. Bruick R.K. FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor.Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1264) Google Scholar to inhibit hydroxylase activity resulted in a 50% delay in sealing the linear scratch wounds (Figure 2, A and B). This finding suggests that the hydroxylase activity of FIH-1 is important in normal wound healing. As dimethyloxalylglycine is a general hydroxylase inhibitor and could have off-target effects, we also used shFIH-1 to knock down FIH-1 in hTCEpi cells (Figure 2C), and with such treatment, the healed distance was 20% less at 6 hours (Figure 2D). To exclude the possible nonspecific effect of RNA interference, we also used a siRNA smart pool, targeting two different sequences in FIH-1 mRNA. In hTECpi cells, knocking down FIH-1 using this siRNA smart pool also led to a significantly slower wound closure (Figure 2L). To confirm the siRNA results in primary keratinocytes, we used HEKs and noted an even greater delay in sealing scratch wounds when FIH-1 was decreased in these cells (Figure 2, E and F). Notably, HEKs and HCEKs express relatively low levels of FIH-1 and seal wounds slower compared with hTCEpi cell lines. We reasoned that elevating the levels of FIH-1 in HEKs and HCEKs would enhance wound sealing if this hydroxylase were a positive regulator of ep
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