EGFにより惹起されるCOS7細胞のマクロピノサイトシスに於ける受容体を介する S1Pシグナルの関与について

概要

INTRODUCTION
Sphingolipids derive their name from the fatty amino alcohol ‘sphingosine’ that constitutes their backbone. Sphingolipid metabolites such as sphingosine 1-phosphate (S1P) and ceramide are bioactive molecules involved in numerous cell signaling pathways to bring about different cellular actions. Cell signaling encompasses the detection, relay and translation, by a cell, of signals in its external and internal environments that culminate into cellular responses. The signaling machinery involves a host of different receptors, adaptor proteins, and enzymes etc. that coordinately and specifically interact and inter-regulate to translate a signal into cellular action. S1P is a phosphorylated product of sphingosine catalyzed by two isoforms of sphingosine kinase (SphK). S1P is a ligand to five S1P specific receptors (S1P₁₋₅R) through which it acts in an autocrine and/or paracrine manner to influence cell action. S1P has also been shown to act on intracellular targets as a second messenger-like mediator. S1P is reversibly catabolized by dephosphorylation through S1P phosphatases to reproduce sphingosine, or degraded irreversibly by S1P lyase into phosphoethanolamine and hexadecenal.

Macropinocytosis is a highly conserved process of non-clathrin mediated endocytosis whereby cells take in gulps of extracellular fluid into large, heterogenous vesicles known as macropinosomes. Macropinocytosis involves Rac 1 controlled actin cytoskeleton remodeling to form membrane ruffles that eventually seal-up into macropinosomes. Cells use macropinocytosis to acquire nutrients, sample their extracellular environment for antigens etc. Growth factors such as the epidermal growth factor (EGF) can induce macropinocytosis in many different cells, although detailed account of the molecular mechanisms underlying EGF-induced macropinocytosis remains incomplete. In a recent study we observed S1P-mediated activation of S1P₁R to be important for subsequent Rac 1 activation and actin polymerization during cargo sorting into multivesicular endosomes.

In the present study, we have shown that the trans-activation of S1P1 receptor (S1P1R) is necessary for EGF-induced macropinocytosis in COS7 cells. Molecular mechanism underlying EGF-induced trans-activation of S1P1R is described and its pathophysiological relevance is discussed herein.

RESULTS
1. Sphingosine Kinase Depletion Impaired EGF-Induced Macropinocytosis
In the experiment to verify effect of EGF treatment on macropinocytosis, EGF caused a robust enhancement of macropinocytosis compared to non-stimulated controls as visualized by confocal microscopy and judged by the uptake of FITC labeled dextran. EGF-induced macropinocytosis was much weaker in SphK1- or SphK2-depleted cells compared to control siRNA-treated cells. A quantitative analysis of fluorescence of uptaked dextran using a flow cytometer showed that EGF-induced enhancement of macropinocytosis (10 fold in control siRNA) became much weaker (1.6 fold and 4 fold) in SphK1- or SphK2-siRNA treatment, respectively. Both siRNAs were verified for their ability to down-regulate each mRNA levels of each isoform and shown to be effective. These results imply that sphingosine kinases may play a promotive role in EGF-induced macropinocytosis in COS7 cells.

2. Expression of Kinase Activity-Competent SphK Restored EGF-Induced Macropinocytosis in Sphingosine Kinase Depleted Cells
To further characterize the role of SphK in macropinocytosis, the effect of expression of mCherry-fused SphK1 and SphK2 was compared with mCherry only in each isoform-depleted COS7 cells. Suppression in EGF-induced macropinocytosis observed in SphK1-depleted cells was almost completely rescued by the expression of wild-type mCherry-SphK1 (mCherry-SphK1(WT)) as compared to mCherry only expression. Importantly, in kinase activity-dead variants of mCherry-SphK1 (mCherry- SphK1(KD)) expressing cells macropinocytosis was not restored. Similar results were obtained in the SphK2-depleted cells. The diminished EGF-induced macropinocytosis in SphK2-depleted cells was overcome by mCherry-SphK2(WT) but not by mCherry- SphK2(KD) expression. These results strongly suggest the importance of the catalytic activity of SphK, i.e., production of S1P, rather than the protein/protein interaction for the EGF-induced enhancement of macropinocytosis in COS7 cells.

3. Requirement for S1PRs in EGF-Induced Macropinocytosis
The finding that catalytic activity of sphingosine kinases is involved in mediating EGF induced macropinocytosis prompted an examination of the mode of actions of S1P in this phenomenon. Since many of the S1P actions are known to be mediated by S1P receptors, we tested the three most ubiquitously expressed receptors, S1P₁₋₃R out of S1P₁₋₅R. S1P₁R, S1P₂R or S1P₃R was depleted using the respective siRNA and EGF- induced macropinocytosis was analyzed by flow cytometry. The results suggested the involvement of S1PRs in EGF-induced macropinocytosis in this cell line, with S1P₁R depletion registering the strongest impairment of EGF-induced macropinocytosis and S1P₂R depletion showing a weaker but significant effect. S1P₃R seems to have a minimal role in this phenomenon. All the siRNAs were verified for their ability to down-regulate mRNA levels of each isotype. These results indicate that receptor-mediated S1P action plays an important role in EGF-induced enhancement of macropinocytosis in COS7 cells.

4. EGF Stimulation Trans activates S1P₁R in a SphK-Dependent manner
To prove the hypothesis that receptor-mediated S1P action is important during EGF-induced enhancement of macropinocytosis, it is essential to show the activation of S1P receptor during EGF stimulation. To address this issue, EGF-induced S1PR transactivation was studied next. S1P₁R was chosen for this experiment since knockdown of this subtype showed the strongest effect in impairing EGF-induced macropinocytosis. To detect S1P₁R activation, a one-molecular FRET tool made up of the FRET pair, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), separately fused in the same S1P₁R molecule was employed. Using cells transiently expressing this reporter probe, EGF stimulation induced conformational changes of S1P₁R, that are consistent with receptor activation, in a manner inhibitable by SphK1 knockdown. This indicates that EGF actually causes S1P1R transactivation through SphK-catalyzed production of S1P, which plays an important role in EGF-induced enhancement of macropinocytosis in COS7 cells.

5. EGF Stimulation Trans activates Rac 1 in a SphK and S1PR-Dependent Manner
Rac 1 activation is essential for membrane ruffling, a precursor event in macropinocytosis. Therefore, to further strengthen the hypothesis that S1P/S1PR signaling plays an important role in EGF-induced enhancement of macropinocytosis, the effect of SphK1 or S1P₁R depletion on EGF-triggered Rac1 activation was examined. Rac1 activation was assessed by a FRET-based, Ras and interacting protein chimeric unit (Raichu), Raichu-Rac1 probe containing YFP and CFP FRET pair fused to the N and C- terminals of the chimera respectively, such that Rac 1 activation causes FRET to occur. In SphK1-depleted Raichu-Rac1 expressing cells Rac1 activation was significantly weaker than in control cells, as denoted by a smaller normalized FRET/CFP ratio for the SphK1 knockdown cell than corresponding controls. Similar results were obtained from S1P₁R knockdown experiment. These results demonstrate that SphK1 and S1P₁R are essential intermediates in the EGF-initiated signaling pathway for macropinocytosis in COS7 cells and that they act upstream of Rac 1 activation.

CONCLUSION
In epidermal growth factor stimulated macropinocytosis in COS7 cells, S1P- mediated S1PR activation is necessary and this event precedes the transactivation of Rac1 which initiates actin cytoskeleton reorganization for membrane ruffling which hallmarks macropinocytosis.