Sphingosine-l-Phosphate Inhibits PDGF-induced Chemotaxis of Human Arterial Smooth Muscle Cells: Spatial and Temporal Modulation of PDGF Chemotactic Signal Transduction

Activation of the PDGF receptor on human arterial smooth muscle cells (SMC) induces migration and proliferation via separable signal transduction pathways. Sphingosine-l-phosphate (Sph-l-P) can be formed following PDGF receptor activation and therefore may be implicated in PDGF-receptor signal transduction. Here we show that Sph-l-P does not significantly affect PDGF-induced DNA synthesis, proliferation, or activation of mitogenic signal transduction pathways, such as the mitogen-activated protein (MAP) kinase cascade and PI 3-kinase, in human arterial SMC. On the other hand, Sph-l-P strongly mimics PDGF receptor-induced chemotactic signal transduction favoring actin filament disassembly. Although Sph-l-P mimics PDGF, exogenously added Sph-l-P induces more prolonged and quantitatively greater PIP2 hydrolysis compared to PDGF-BB, a markedly stronger calcium mobilization and a subsequent increase in cyclic AMP levels and activation of cAMP-dependent protein kinase. This excessive and prolonged signaling favors actin filament disassembly by Sph-l-P, and results in inhibition of actin nucleation, actin filament assembly and formation of focal adhesion sites. Sph-l-P-induced interference with the dynamics of PDGF-stimulated actin filament disassembly and assembly results in a marked inhibition of cell spreading, of extension of the leading lamellae toward PDGF, and of chemotaxis toward PDGF. The results suggest that spatial and temporal changes in phosphatidylinositol turnover, calcium mobilization and actin filament disassembly may be critical to PDGF-induced chemotaxis and suggest a possible role for endogenous Sph-l-P in the regulation of PDGF receptor chemotactic signal transduction. T rIE accumulation of smooth muscle cells (SMC) 1 during formation and progression of atherosclerotic lesions and in restenosis after angioplasty is due to a combination of proliferation and directed migration of the cells from the media into and within the intimal layer of the artery wall (for review see Ross, 1993). Initiation of both these events is most likely mediated by a number of regulatory polypeptides that are present in the lesion, such as Address all correspondence to Karin E. Bornfeldt, Department of Pathology, SM-30, University of Washington, School of Medicine, Seattle, WA 98195. Tel.: (206) 543-8523. Fax: (206) 685-3018. 1. Abbrevtations used in this paper. DMS, N,N-dimethyl sphingosine; FAK, focal adhesion kinase; IBMX, 3-isobutyl-l-methylxanthine; IP1, inositol monophosphate; MAP kinase, mitogen-activated protein kinase; PDGF-BB, PDGF B-chain homodimer; PDS, plasma-derived serum: PI, phosphatidylinositol; PIP2, phosphatidylinositol bisphosphate" PKA. cAMP-dependent protein kinase; PLC?, phospholipase C~/; SMC. smooth muscle cell; Sph-l-P, sphingosine-l-phosphate. PDGF (Ross et al., 1990; Ferns et al., 1991; Jawien et al., 1992). Both proliferation and directed migration (chemotaxis) of arterial SMC are markedly stimulated by PDGF B-chain homodimer (PDGF-BB), and inhibition of PDGF in vivo partially blocks SMC accumulation following balloon injury of a normal vessel (Grotendorst et al., 1981; Ferns et al., 1990; Raines et al., 1990). After binding of PDGF-BB to its cell surface receptors, the receptor dimerizes and becomes autophosphorylated intracellularly on a number of tyrosine residues that act as docking sites for molecules containing SH2 (src-homology 2 domains). These include enzymes such as phospholipase C~, (PLC~/), phosphatidylinositol 3-kinase (PI 3-kinase), rasGTPase activating protein, the tyrosine phosphatase Syp, and members of the Src family, as well as linker molecules such as growth factor-receptor bound protein 2 and Src homology and collagen protein (Shc; for review see Claesson-Welsh, 1994). A number of different signal transduction pathways are thus induced after PDGF receptor activation. The direct impact of individual signal © The Rockefeller University Press, 0021-95251951071193/14 $2.00 The Journal of Cell Biology, Volume 130, Number 1, July 1995 193-206 193 on D ecem er 3, 2017 jcb.rress.org D ow nladed fom transduction pathways on specific biological functions are, in most cases, not well understood. Emerging results indicate that the intraceUular signaling pathways from the PDGF receptors leading to directed migration may be different from those leading to proliferation (Rrnnstrand et al., 1992; Kashishian and Cooper, 1993; Bornfeldt et al., 1994). In human arterial SMC, PDGF-stimulated phosphatidylinositol (PI)-turnover (mediated through activation of PLC'y) correlates with directed migration (Bornfeldt et al., 1994). Migration of SMC on type I collagen toward PDGF-BB also requires functional et2131 integrins (Skinner et al., 1994). Activation of the MAP kinase cascade through growth factor-receptor bound protein 2 and ras'GTP formation, on the other hand, correlates with the magnitude of proliferation, and does not appear to be required for migration of SMC (Bornfeldt et al., 1994). Recently, a role for sphingolipids in growth factor signal transduction has been proposed (for review see Hakomori, 1990; Kolesnick and Golde, 1994). One of the sphingolipid metabolites that levels can be altered after growth factor receptor stimulation is sphingosine-l-phosphate (Sph-l-P). Sph-l-P can be formed in vivo by phosphorylation of sphingosine by sphingosine kinase (for review see Hakomori, 1990), and is degraded mainly by sphingosine-phosphate lyase, which cleaves Sph-l-P to a fatty aldehyde and ethanolamine-phosphate (for review see van Veldhoven and Mannaerts, 1993). Sph-l-P has recently been suggested as a PDGF receptor signaling molecule (Olivera and Spiegel, 1993), and yet exogenously added Sph-l-P inhibits migration of some cell types through an unknown mechanism (Sadahira et al., 1992). This apparent discrepancy prompted us to further study the effect of Sph-l-P on PDGF-induced signal transduction pathways involved in proliferation versus migration of human SMCs. Here we show that Sph-l-P does not share the effects of PDGF-BB on mitogenic signal transduction. Instead, Sph1-P selectively mimics PDGF receptor signaling events favoring actin filament disassembly, thereby inhibiting actin filament assembly, formation of focal adhesion sites and PDGF-induced migration and chemotaxis. Spatial and temporal differences between PDGF and Sph-l-P signaling may be critical to its interference with PDGF-induced migration. Materials and Methods