Sphingosine-1-phosphate receptor expression in cardiac fibroblasts is modulated by in vitro culture conditions

Abstract

Landeen LK, Aroonsakool N, Haga JH, Hu BS, Giles WR. Sphingosine-1-phosphate receptor expression in cardiac fibroblasts is modulated by in vitro culture conditions. Am J Physiol Heart Circ Physiol 292: H2698 -H2711, 2007. First published March 2, 2007; doi:10.1152/ajpheart.01065.2006.-The bioactive molecule sphingosine-1-phosphate (S1P) binds with high affinity to five recognized receptors (S1P1-5) 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 2O2 had minimal effects on CFb S1P1-3 expression, whereas transforming growth factor-␤1, TNF-␣, and PDGF-BB significantly altered all S1P receptor subtypes. Lowering FBS concentrations from 10% to 0.1% increased S1P2, whereas supplementation with either PDGF-BB or Rho-associated protein kinase inhibitor Y-27632 significantly elevated S1P3 levels. S1P2 and S1P3 receptor levels are known to regulate cell migration. Using cells isolated from either normal or S1P3-null mice, we demonstrate that S1P3 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. migration; macrophage; phenotype SPHINGOSINE-1-PHOSPHATE (S1P) is a biologically active, cell membrane-associated sphingolipid that is secreted by various cells upon activation. S1P binds with high affinity to five distinct G protein-coupled receptors, also referred to as endothelial differentiation gene (EDG) receptors. S1P receptors are ubiquitously expressed on mammalian cells and can affect such cellular responses as proliferation, differentiation, and migration (reviewed in Refs. 1,12,13,39,40,and 45). To characterize S1P regulation, it is therefore necessary to have pure populations of the target cells.Consistently obtaining highly purified cell cultures, which do not include any significant contaminating cell types, remains a considerable challenge when working with primary isolates from organs and tissues. However, this initial step is a requirement for many current molecular and c...

“Inside-Out” Signaling of Sphingosine-1-Phosphate: Therapeutic Targets

“…Conversely, binding of these growth factors to their RTKs can also translocate and activate SphK1 (Pitson et al, 2005), leading to spatially restricted formation of S1P that in turn further activates inside-out signaling . Additionally, some of these RTKs, such as PDGF receptor, can also increase S1P 1 mRNA expression (Landeen et al, 2007), leading to further complex interplay between S1P receptors and RTKs. S1P 2 , like S1P 1 and S1P 3 receptors, is also widely expressed and is the dominant receptor in the vascular smooth muscle cells (Waeber et al, 2004).…”

Implication of sphingosin-1-phosphate in cardiovascular regulation

“…Cardiac fibroblasts make up >50% of the total heart cells and provide a pivotal contribution to cardiac remodeling (80-82). It has been shown that cardiac fibroblasts express S1P1-3 receptors and that the levels of these receptors in cultured cardiac fibroblasts are significantly altered after stimulation by the factors associated with remodeling such as transforming growth factor (TGF)-β1, TNF-α, and platelet-derived growth factor (83). Furthermore, in cultured cardiac fibroblasts, TGF-β stimulates S1P production via SPHK1 and that S1P increases collagen expression via S1P2 receptor; TGF-β-stimulated collagen production is blocked by the inhibition of SPHK1 or S1P2, demonstrating that S1P is a critical mediator in TGF-β-induced fibrogenic effect in cardiac fibroblasts (84).…”

Sphingosine-1-Phosphate as a Potential Target for the Treatment of Myocardial Infarction

“…16 Although S1P1 receptors predominate in cardiac myocytes, S1P3 receptors are the most prevalent subtype in cardiac fibroblasts. 21 Activation of S1P receptors on fibroblasts can mediate migration and proliferation, responses that are necessary for fibrosis and critical to cardiac remodeling. For instance, adult cardiac fibroblast migration in response to growth factors is reduced by S1P3 receptor deletion.…”