title: Angiogenic peptide (AG)-30/5C activates human keratinocytes to produce cytokines/chemokines and to migrate and proliferate via MrgX receptors.
abstract: BACKGROUND
  In addition to their antimicrobial activities, antimicrobial peptides, also known as host defense peptides (HDPs) activate keratinocytes; promote wound healing; and improve the skin barrier. AG-30/5C is a novel angiogenic HDP that activates various functions of fibroblasts and endothelial cells, including cytokine/chemokine production and wound healing.
  
  
  OBJECTIVE
  To investigate whether AG-30/5C activates human keratinocytes and to examine the underlying mechanisms.
  
  
  METHODS
  Production of cytokines/chemokines was assessed by ELISA. Expression of Mas-related G-protein coupled receptors X (MrgXs) in keratinocytes was determined by real-time PCR and Western blot. MAPK and NF-κB activation was analysed by Western blot. Cell migration was assessed by chemotaxis microchamber and in vitro wound closure assay, whereas cell proliferation was analysed using an XTT assay.
  
  
  RESULTS
  We found that AG-30/5C was more efficient than its parent peptide AG-30 in increasing the production of various cytokines/chemokines and promoting keratinocyte migration and proliferation. Furthermore, MrgX3 and MrgX4 receptors were constitutively expressed in keratinocytes at higher levels than MrgX1 and MrgX2, and were up-regulated upon stimulation with TLR ligands. Because MrgX3 and MrgX4 siRNAs suppressed AG-30/5C-mediated cytokine/chemokine production, keratinocyte migration and proliferation, we propose that AG-30/5C utilizes these MrgXs to stimulate keratinocytes. In addition, AG-30/5C-induced activation of keratinocytes was controlled by MAPK and NF-κB pathways, as evidenced by the inhibitory effects of ERK-, JNK-, p38- and NF-κB-specific inhibitors. Indeed, we confirmed that AG-30/5C enhanced phosphorylation of MAPKs and IκB.
  
  
  CONCLUSIONS
  Our findings provide novel evidence that AG-30/5C may be a useful therapeutic agent for wound healing by activating human keratinocytes.
authors.affiliation: Atopy (Allergy) Research Center, Tokyo, Japan; Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Dermatology, Department of Medical Services, Ministry of Public Health, Bangkok, Thailand.
authors.forename: Chanisa
authors.initials: C
authors.lastname: Kiatsurayanon
authors.affiliation: Atopy (Allergy) Research Center, Tokyo, Japan; Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan. Electronic address: francois@juntendo.ac.jp.
authors.forename: François
authors.initials: F
authors.lastname: Niyonsaba
authors.affiliation: Atopy (Allergy) Research Center, Tokyo, Japan; Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
authors.forename: Panjit
authors.initials: P
authors.lastname: Chieosilapatham
authors.affiliation: Atopy (Allergy) Research Center, Tokyo, Japan.
authors.forename: Ko
authors.initials: K
authors.lastname: Okumura
authors.affiliation: Atopy (Allergy) Research Center, Tokyo, Japan; Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
authors.forename: Shigaku
authors.initials: S
authors.lastname: Ikeda
authors.affiliation: Atopy (Allergy) Research Center, Tokyo, Japan.
authors.forename: Hideoki
authors.initials: H
authors.lastname: Ogawa
mesh_terms: D043925:Angiogenesis Inducing Agents / Q000494:pharmacology*; D002465:Cell Movement / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D018925:Chemokines / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000819:agonists / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology*; D010766:Phosphorylation; D034622:RNA Interference; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D018013:Receptors, Neuropeptide / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014162:Transfection; D014945:Wound Healing / Q000187:drug effects*
chemical_list: C576453:AG30-5C peptide; D043925:Angiogenesis Inducing Agents; D018925:Chemokines; D016207:Cytokines; C480039:MRGPRX2 protein, human; C501064:MRGPRX3 protein, human; C000657324:MRGPRX4 protein, human; D016328:NF-kappa B; D009419:Nerve Tissue Proteins; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; C510803:mas-related gene-X1 receptor, human; D020928:Mitogen-Activated Protein Kinases
keywords: Antimicrobial peptide; MAPK; MrgX receptor; NF-κB; Skin; Wound healing