Sphingosine-1-phosphate inhibits cell migration and endothelial to mesenchymal cell transformation during cardiac development

Abstract

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid metabolite that exerts important effects on numerous cellular events via cell surface receptors, S1P(1-5). S1P influences differentiation, proliferation, and migration during vascular development. However, the effects of S1P signaling on early cardiac development are not well understood. To address this issue, we examined the expression of S1P regulatory enzymes and S1P receptors during cardiac development. We observed that enzymes that regulate S1P levels, sphingosine kinase and sphingosine-1-phosphate phosphatase, are expressed in the developing heart. In addition, RT-PCR revealed that four of the five known S1P receptors (S1P(1-4)) are also expressed in the developing heart. Next, effects of altered S1P levels on whole embryo and atrioventricular (AV) canal cultures were investigated. We demonstrate that inactivation of the S1P producing enzyme, sphingosine kinase, leads to cell death in cardiac tissue which is rescued by exogenous S1P treatment. Other experiments reveal that increased S1P concentration prevents alterations in cell morphology that are required for cell migration. This effect results in reduced cell migration and inhibited mesenchymal cell formation in AV canal cushion tissue. These data indicate that S1P, locally maintained within a specific concentration range, is an important and necessary component of early heart development.

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“…Quantitative measurements of the C-R length and hearts were taken from printouts of the embryos and hearts. Embryos were fixed, embedded in paraffin, and sectioned as described previously (42). Sections were stained with Harris-modified hematoxylin/eosin Y according to standard procedures (Fisher Scientific, Fair Lawn, NJ).…”

“…Apoptosis was assessed with the in situ cell death detection kit (Roche, Mannheim, Germany) according to the manufacturer's instructions as described previously (42)(43)(44).…”

“…Total RNA was collected from whole embryos or isolated hearts at E10.5 with the RNeasy kit (Qiagen), and cDNA was prepared as described previously (42). All real-time PCRs contained DyNAmo HS SYBR Green 2X Master Mix (Finnzymes, distributed by New England Biolabs, Beverly, MA) with 50 ng of cDNA and 0.5 M each primer, and they were performed at 55°C for 35 cycles with the Opticon 2 DNA Engine (Bio-Rad).…”

“…All real-time PCRs contained DyNAmo HS SYBR Green 2X Master Mix (Finnzymes, distributed by New England Biolabs, Beverly, MA) with 50 ng of cDNA and 0.5 M each primer, and they were performed at 55°C for 35 cycles with the Opticon 2 DNA Engine (Bio-Rad). All PCR primers were designed against mouse sequences including GAPDH (42), all other real-time primers were obtained from SuperArray (Bioscience Corp., Frederick, MD). No DNA contamination was detected in any of the PCRs (data not shown).…”

“…A 1 ARϩ/ϩ mice were used for wholeembryo cultures that were performed as described (42,43,47). Control embryos were exposed to 5% O 2 (balance N 2 ) from 0 to 24 h, followed by 20% O 2 from 24 to 48 h (47).…”