Comparative analysis of human and rat S1P5 (edg8): differential expression profiles and sensitivities to antagonists

doi:10.1016/S0006-2952(02)01289-3

Biochemical Pharmacology

Volume 64, Issue 8, 15 October 2002, Pages 1243-1250

https://doi.org/10.1016/S0006-2952(02)01289-3 Get rights and content

Abstract

Five guanine nucleotide-binding protein-coupled receptors (S1P_1–5) for the lysophospholipid mediator sphingosine 1-phosphate (S1P) have thus far been described. Whereas tissue distribution and functional properties of the human S1P_1–4 genes are well characterized, only limited functional and expression data are available for S1P₅, todate. Northern blot analysis indicated that human S1P₅ (hS1P₅) is an alternatively spliced gene, with a 5.4-kb transcript that is predominantly expressed in peripheral tissues, and a 2.4-kb transcript expressed in brain, spleen, and peripheral blood leucocytes. In contrast, rat S1P₅ (rS1P₅) was exclusively detected in brain and skin. Expression of hS1P₅ and rS1P₅ in mammalian CHO-K1 or HEK293 cells conferred onto the cells the ability to mobilize intracellular calcium as determined by a functional Fluorometric Imaging Plate Reader assay, when challenged with S1P and dihydro S1P, respectively. Applying a lipid library with 200 bioactive lipids in a functional Fluorometric Imaging Plate Reader assay did not reveal additional agonists. However, both receptors exhibited differential sensitivity towards the S1P- and lysophosphatidic acid-receptor antagonist, suramin: rS1P₅-mediated intracellular calcium mobilization was partly inhibited by suramin (ic₅₀: 5800 μM), whereas hS1P₅ was completely antagonized (ic₅₀: 130 μM). Both receptors were sensitive towards inhibition with the related drug (8,8′-(carbonylbis(imino-3,1-phenylene))bis(1,3,5-naphthalenetrisulfonic acid)) but ic₅₀ values differed significantly (340 μM for hS1P₅, 4000 μM for rS1P₅). In addition, rS1P₅ displayed antiproliferative effects in transfected CHO-K1 and HEK293 cells in contrast to hS1P₅. Taken together, our data imply that differences between hS1P₅ and rS1P₅ will be an important point to be considered in the development of selective receptor antagonists.

Introduction

The lysolipid phosphate mediators, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), have attracted increasing attention as modulators of a variety of important biological functions [1], [2], [3], [4] and their list of biological activities is continuously growing.

S1P has been implicated in cell proliferation, modulation of cell motility [5], [6], induction/suppression of apoptosis [7], [8], in vitro and in vivo angiogenesis [9], tumor invasiveness [10], [11], platelet activation [12], and neurite retraction [13]. Cellular signaling by S1P involves activation of PLCβ and subsequent intracellular Ca²⁺ release [14], [15], activation of MAP-kinases [16], activation of inward rectifying K⁺-channels [17], [18], and inhibition and/or activation of adenylyl cyclase [14].

Both LPA and S1P are recognized to signal cells through a set of G protein-coupled receptors (GPCRs), formerly known as endothelial differentiation gene (edg) receptors. This family of GPCRs currently comprises eight members and, on the basis of their activating ligand, can be classified into two major groups: S1P_1–5 [S1P₁ (edg1), S1P₂ (edg5), S1P₃ (edg3), S1P₄ (edg6), S1P₅ (edg8)] are stimulated by S1P [19], [20], [21], [22], [23], LPA_1–3 [LPA₁ (edg2), LPA₂ (edg4), LPA₃ (edg7)] preferentially interact with LPA [24], [25].

Assignment of specific biological functions to certain receptor subtypes is hampered by: (1) the overlapping expression of S1P/LPA receptors [26], [27]; (2) activation of multiple, and in part redundant, signal transduction pathways [14], [15], [16], [26]; (3) incomplete selectivity of their activating ligands [28], [29]; and (4) poorly developed medicinal chemistry as specific antagonists for dissecting the pharmacology of individual subtypes are not yet available.

An important step to shed more light on the biological roles of the individual receptor subtypes would be to identify the complete set of receptors that respond to S1P and LPA and then to characterize the specific signaling and pharmacological properties.

Rat S1P₅ (rS1P₅) has recently been identified as the fifth S1P-responsive GPCR. It was originally cloned from rat pheochromocytoma cells, as a nerve growth factor regulated GPCR (nrg-1) [30] exhibiting greatest similarity to the family of S1P_1–4 receptors. rS1P₅ is expressed in spleen and adult brain white matter [23]. Malek et al.[28] demonstrated that rS1P₅ is coupled to G proteins of the G_i/o- and G₁₂-class, and inhibits activation of extracellular regulated kinase (ERK) in CHO cells. Much less is known about the human orthologue of rS1P₅. Im et al.[31] recently published preliminary expression and functional studies of human S1P₅ (hS1P₅). hS1P₅ is expressed in brain and peripheral tissues as determined by Northern blot analysis (multiple tissue dot blot); however, dot blot analysis revealed neither the transcript size nor the existence of tissue specific splice variants. In addition, low-level signals were detected in many tissues, making it hard to distinguish specific expression from background. Given the differential expression patterns of hS1P₅ and rodent S1P₅ receptors, we reasoned that additional pharmacological/biochemical differences may exist between the species homologues.

The present study was set out to address the following questions: (i) is hS1P₅ an ubiquitously or specifically expressed gene, (ii) how many splice variants exist for hS1P₅, and how are their tissue expression pattern, (iii) are the different expression patterns of hS1P₅ and rS1P₅ reflected in different sensitivities to agonists or antagonists, and (iv) does hS1P₅ resemble its rat counterpart in the antiproliferative activity upon expression in mammalian cell lines?

Section snippets

Sources of materials

S1P, dhS1P, suramin, and fatty acid-free BSA were from Sigma. The lipid library was from Biomol Research Laboratories, Inc. NF023 was ordered from Tocris Cookson, CHO-K1 cells were obtained from the American Type Culture Collection, cell culture media and sera from Gibco BRL, the calcium fluorescent dye Fluo4 and pluronic acid from Molecular Devices, rat Northern blot membrane from Origene, human Northern blot membrane and the GC-melt PCR kit from Clontech. The expression vector PSPT18, the

Expression of hS1P₅ and rS1P₅

Im et al.[31] recently showed, via dot blot analysis, that hS1P₅ is widespread in peripheral tissues and brain regions. To get more detailed information about the transcript size and putative splice variants, we examined the expression pattern of hS1P₅ by Northern blot analysis (Fig. 1A). A prominent band migrating at 5.5 kilobases (kb) was seen in skeletal muscle, heart, and kidney, while lower abundance of RNA was observed in liver and placenta; no signal was detected in brain, thymus, spleen,

Discussion

Recently, hS1P₅ has been discovered as the fifth member of the S1P receptor family [31]. Whereas many S1P receptors are ubiquitously expressed [27], [37], [38], hS1P₅ displays a more specific expression pattern. Northern blot analysis of this study has revealed two new and interesting aspects of its expression: (1) hS1P₅ is mainly expressed in the cardiovascular system, as opposed to rS1P₅, that is exclusively expressed in brain, spleen, and skin; (2) hS1P₅ exists as two splice variants with

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Cyclical and dose-dependent responses of adult human mature oligodendrocytes to fingolimod
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We documented similar lack of responses with 1 and 2 days of fingolimod-phosphate treatment of rat matured OLGs (data not shown), suggesting species-specific differences in responsiveness to fingolimod. However, disparities between rodent and human OLG responses may be attributable to differences in S1P receptor affinities for the ligand,47 receptor levels and coupling to associated signaling pathways, and sample age/cell maturity. Membrane retraction in human mature OLGs was associated with increased phosphorylation of myosin light chain II, indicative of RhoA GTPase signaling.45
Fingolimod is a sphingosine-1-phosphate (S1P) analogue that has been used in clinical trials as a systemic immunomodulatory therapy for multiple sclerosis. Fingolimod readily accesses the central nervous system, raising the issue of its direct effects on neural cells. We assessed the effects of active fingolimod on dissociated cultures of mature, myelin-producing oligodendrocytes (OLGs) derived from adult human brain. Human OLGs express S1P receptor transcripts in relative abundance of S1P5>S1P3>S1P1, with undetectable levels of S1P4. Low doses of fingolimod (100 pmol/L to 1 nmol/L) induced initial membrane elaboration (2 days), subsequent retraction (4 days), and recurrence of extension with prolonged treatment (8 days). Higher doses (10 nmol/L to 1 μmol/L) caused the opposite modulation of membrane dynamics. Retraction was rescued by co-treatment with the S1P3/S1P5 pathway antagonist, suramin, and was associated with RhoA-mediated cytoskeletal signaling. Membrane elaboration was mimicked using the S1P1 agonist SEW2871. Fingolimod rescued human OLGs from serum and glucose deprivation-induced apoptosis, which was reversed with suramin co-treatment and mimicked using an S1P5 agonist. High doses of fingolimod induced an initial down-regulation of S1P5 mRNA levels relative to control (4 hours), subsequent up-regulation (2 days), and recurrent down-regulation (8 days). S1P1 mRNA levels were inversely regulated compared with S1P5. These results indicate that fingolimod modulates maturity- and species-specific OLG membrane dynamics and survival responses that are directly relevant for myelin integrity.
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Comparative analysis of human and rat S1P5 (edg8): differential expression profiles and sensitivities to antagonists

Abstract

Introduction

Section snippets

Sources of materials

Expression of hS1P5 and rS1P5

Discussion

Curr. Opin. Cell. Biol.

Pharmacol. Ther.

Trends Pharmacol. Sci.

Trends Pharmacol. Sci.

Blood

J. Biol. Chem.

Cell

Blood

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Gene

J. Biol. Chem.

J. Biol. Chem.

Mol. Cell Neurosci.

J. Biol. Chem.

Comparative analysis of human and rat S1P₅ (edg8): differential expression profiles and sensitivities to antagonists

Expression of hS1P₅ and rS1P₅