title: K6PC-5, a sphingosine kinase activator, induces anti-aging effects in intrinsically aged skin through intracellular Ca2+ signaling.
abstract: BACKGROUND
  Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, regulates multiple cellular responses such as Ca(2+) signaling, growth, survival, and differentiation. Because sphingosine kinase (SK) is the enzyme directly responsible for the production of S1P, many factors have been identified that regulate its activity and subsequent S1P levels. To date, there are no reports to demonstrate a chemically induced, direct activation of SK.
  
  
  OBJECTIVE
  Here we have studied the effects of K6PC-5 as a newly synthesized SK activator on fibroblast proliferation in both human fibroblasts and aged mouse skin. To demonstrate that K6PC-5 has S1P-mediated action mechanism in fibroblasts, we have measured SK-dependent intracellular Ca(2+) signaling.
  
  
  METHODS
  Fibroblasts were cultured primarily from human foreskin and were used to study the effect of K6PC-5 and S1P on intracellular Ca(2+) signaling and fibroblast proliferation. Changes in intracellular Ca(2+) were detected by fluorescence with fura-2/AM. To study skin anti-aging effects of K6PC-5, we used intrinsically aged hairless mice (56 weeks old).
  
  
  RESULTS
  K6PC-5 promoted fibroblast proliferation and procollagen production in human fibroblasts significantly. K6PC-5 induced intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in human fibroblasts. Both dimethylsphingosine and dihydroxysphingosine, SK inhibitors, and the transfection of SK1-siRNA blocked the K6PC-5-induced increases in [Ca(2+)](i), an effect independent of the classical PLC/IP(3)-mediated pathway. The K6PC-5-induced [Ca(2+)](i) oscillations were dependent on thapsigargin-sensitive Ca(2+) stores and Ca(2+) entry. Topical application of K6PC-5 for 2 weeks to intrinsically aged hairless mice enhanced fibroblast proliferation, collagen production, and eventually increased dermal thickness (10%). K6PC-5 also promoted specific epidermal differentiation marker proteins, including involucrin, loricrin, filaggrin, and keratin 5, without any alterations on epidermal barrier function.
  
  
  CONCLUSIONS
  These results suggest that K6PC-5 acts to regulate fibroblast proliferation through intracellular S1P production, and can further promote keratinocyte differentiation. We anticipate that the regulation of S1P levels may represent a novel approach for the treatment of skin disorders, including skin aging.
authors.affiliation: Brain Korea 21 Project for Medical Science, Yonsei University, Seoul, Republic of Korea.
authors.forename: Jong-Kyung
authors.initials: JK
authors.lastname: Youm
authors.forename: Hae
authors.initials: H
authors.lastname: Jo
authors.forename: Jeong Hee
authors.initials: JH
authors.lastname: Hong
authors.forename: Dong Min
authors.initials: DM
authors.lastname: Shin
authors.forename: Mi Jung
authors.initials: MJ
authors.lastname: Kwon
authors.forename: Se Kyoo
authors.initials: SK
authors.lastname: Jeong
authors.forename: Byeong Deog
authors.initials: BD
authors.lastname: Park
authors.forename: Eung Ho
authors.initials: EH
authors.lastname: Choi
authors.forename: Seung Hun
authors.initials: SH
authors.lastname: Lee
mesh_terms: D000375:Aging / Q000187:drug effects* / Q000378:metabolism; D000577:Amides / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D000091344:Filaggrin Proteins; D006801:Humans; D008246:Lysophospholipids / Q000037:antagonists & inhibitors / Q000378:metabolism; D008297:Male; D051379:Mice; D008812:Mice, Hairless; D023421:Models, Animal; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011347:Procollagen / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism; D000099157:Sphingosine Kinase
chemical_list: D000577:Amides; D002118:Calcium; D000091344:Filaggrin Proteins; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D011347:Procollagen; D013110:Sphingosine; D000099157:Sphingosine Kinase; C000717249:FLG protein, human; C531172:N-(1,3-dihydroxyisopropyl)-2-hexyl-3-oxodecanamide; C060506:sphingosine 1-phosphate