title: Sphingosine-1-phosphate receptor expression in cardiac fibroblasts is modulated by in vitro culture conditions.
abstract: The bioactive molecule sphingosine-1-phosphate (S1P) binds with high affinity to five recognized receptors (S1P(1-5)) to affect various tissues, including cellular responses of cardiac fibroblasts (CFbs) and myocytes. CFbs are essential components of myocardium, and detailed study of their cell signaling and physiology is required for a number of emerging disciplines. Meaningful studies on CFbs, however, necessitate methods for selective, reproducible cell isolations. Macrophages reside within normal cardiac tissues and often are isolated with CFbs. A protocol was therefore developed that significantly reduces macrophage levels and utilizes more CFb-specific markers (discoidin domain receptor-2) instead of, or in addition to, more commonly used cytoskeletal markers. Our results demonstrate that primary isolated, purified CFbs express predominantly S1P(1-3); however, the relative levels of these receptor subtypes are modulated with time and by culture conditions. In coculture experiments, macrophages altered CFb S1P receptor levels relative to controls. Further investigations using known macrophage-secreted factors showed that S1P and H(2)O(2) had minimal effects on CFb S1P(1-3) expression, whereas transforming growth factor-beta1, TNF-alpha, and PDGF-BB significantly altered all S1P receptor subtypes. Lowering FBS concentrations from 10% to 0.1% increased S1P(2), whereas supplementation with either PDGF-BB or Rho-associated protein kinase inhibitor Y-27632 significantly elevated S1P(3) levels. S1P(2) and S1P(3) receptor levels are known to regulate cell migration. Using cells isolated from either normal or S1P(3)-null mice, we demonstrate that S1P(3) is important and necessary for CFb migration. These results highlight the importance of demonstrating CFb culture purity in functional studies of S1P and also identify conditions that modulate S1P receptor expression in CFbs.
authors.affiliation: Department of Bioengineering, University of California San Diego, La Jolla, CA, USA.
authors.forename: Lee K
authors.initials: LK
authors.lastname: Landeen
authors.forename: Nakon
authors.initials: N
authors.lastname: Aroonsakool
authors.forename: Jason H
authors.initials: JH
authors.lastname: Haga
authors.forename: Betty S
authors.initials: BS
authors.lastname: Hu
authors.forename: Wayne R
authors.initials: WR
authors.lastname: Giles
mesh_terms: D000577:Amides / Q000494:pharmacology; D000818:Animals; D000077214:Becaplermin; D002450:Cell Communication; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002633:Chemotaxis; D000070818:Discoidin Domain Receptors; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D008246:Lysophospholipids / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010641:Phenotype; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020574:Proto-Oncogene Proteins c-sis; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D049349:Receptors, Lysosphingolipid / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011975:Receptors, Mitogen / Q000378:metabolism; D012107:Research Design; D044967:Serum / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D000081025:Sphingosine-1-Phosphate Receptors; D013997:Time Factors; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D054460:rho-Associated Kinases
chemical_list: D000577:Amides; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids; D010982:Platelet-Derived Growth Factor; D047428:Protein Kinase Inhibitors; D020574:Proto-Oncogene Proteins c-sis; D011725:Pyridines; D012333:RNA, Messenger; D049349:Receptors, Lysosphingolipid; D011975:Receptors, Mitogen; C501285:S1pr3 protein, mouse; D000081025:Sphingosine-1-Phosphate Receptors; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; C108830:Y 27632; D000077214:Becaplermin; C060506:sphingosine 1-phosphate; D000070818:Discoidin Domain Receptors; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D013110:Sphingosine