title: α-Melanocyte stimulating hormone (MSH) and prostaglandin E2 (PGE2) drive melanosome transfer by promoting filopodia delivery and shedding spheroid granules: Evidences from atomic force microscopy observation.
abstract: BACKGROUND
  Skin pigmentation is accomplished by production of melanin in melanosome and by transfer of these organelles from melanocytes (MCs) to surrounding keratinocytes (KCs). However, the detailed mechanism is still unknown.
  
  
  OBJECTIVE
  We aimed to investigate the morphological structure changes on human epidermal MCs and KCs, which were either mono-cultured or co-cultured, with or without the treatment of both α-Melanocyte-stimulating hormone (α-MSH) and prostaglandin E2 (PGE2), by atomic force microscopy (AFM) and to provide more direct proofs for process of melanosome transfer.
  
  
  METHODS
  Human epidermal MCs and KCs were isolated and co-cultured with 1:10 ratio in a defined Keratinocyte-serum free medium (K-SFM). After exposure with 100 nM α-MSH or 20 μM PGE2 for 3 days, cells were fixed with 0.5% glutaraldehyde and AFM images of scanning observation were captured by contacting and tapping model under normal atmospheric pressure and temperature.
  
  
  RESULTS
  It showed that human epidermal MCs in culture had secondary or tertiary branches. Except for globular granules structure on the surface of dendrites, some filopodia were protruded on the tips and lateral sides of the dendrites. The administration of α-MSH and PGE2 made not only the dendrites thinner and longer, but also the globular granules more intensive and denser. Many spheroid granules were shed from branches of dendrite and most of them adhered with dense filopodia. Compared with untreated group, the number of filopodia per cell, diameter of filopodia, and shedding spheroid granules per field all increased following α-MSH and PGE2 exposure (P<0.05, n=3). However, many crest-like protrusions, which were distributed homogenously on the surface of mono-cultured KCs, were less changed after α-MSH and PGE2 exposure. In co-culture model, α-MSH and PGE2 increased the number of transferred melanosomes in KCs under laser confocal microscopic examination. Filopodia were observed only on the adhesion area of KCs and MCs in a coiled style by AFM examination. In addition, the number of filopodia per field, diameter of filopodia and shedding spheroid granules per field all increased after the administration of α-MSH and PGE2 (P<0.05, n=3).
  
  
  CONCLUSIONS
  Our data suggest that shedding spheroid granules, filopodia delivery and KC phagocytosis are major mode of melanosome transfer between MCs and KCs. PGE2, as well as α-MSH, drives melanosome transfer by promoting filopodia delivery and numbers of shedding spheroid granules in MCs, but no direct morphological effects on KCs. These findings open a new path in our understanding of MCs-KCs communication regulating pigmentation.
authors.affiliation: Department of Dermatology, the 309th Hospital of Chinese PLA, Beijing, 100091, PR China. Electronic address: melanocytes@163.com.
authors.forename: Hui-Jun
authors.initials: HJ
authors.lastname: Ma
authors.affiliation: Department of Orthopaedics, Heji Affiliated Hospital of Changzhi Medical School, Changzhi City, Shanxi Province, 046000, PR China.
authors.forename: Hui-Yong
authors.initials: HY
authors.lastname: Ma
authors.affiliation: Department of Dermatology, the 309th Hospital of Chinese PLA, Beijing, 100091, PR China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Department of Plastic Surgery, the 309th Hospital of Chinese PLA, Beijing, 100091, PR China.
authors.forename: Peng-Cheng
authors.initials: PC
authors.lastname: Li
authors.affiliation: Department of Dermatology, China Meitan General Hospital, Beijing, 100061, PR China.
authors.forename: Shao-Xia
authors.initials: SX
authors.lastname: Zi
authors.affiliation: Department of Plastic Surgery, the 309th Hospital of Chinese PLA, Beijing, 100091, PR China.
authors.forename: Chi-Yu
authors.initials: CY
authors.lastname: Jia
authors.affiliation: Department of Stomatology, the 309th Hospital of Chinese PLA, Beijing, 100091, PR China. Electronic address: ynan622@sina.com.
authors.forename: Rong
authors.initials: R
authors.lastname: Chen
mesh_terms: D002478:Cells, Cultured; D018920:Coculture Techniques; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D015232:Dinoprostone / Q000008:administration & dosage / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008544:Melanocytes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D020460:Melanosomes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D018625:Microscopy, Atomic Force; D008954:Models, Biological; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D000521:alpha-MSH / Q000008:administration & dosage / Q000378:metabolism*
chemical_list: D000521:alpha-MSH; D015232:Dinoprostone
keywords: Atomic force microscopy; Co-culture; Filopodia delivery; Keratinocytes; Melanocytes; Shedding spheroid granules