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Circulation Research. 2006;99:465-467
doi: 10.1161/01.RES.0000241060.26659.d3
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(Circulation Research. 2006;99:465.)
© 2006 American Heart Association, Inc.

Editorials

Toward Solving the Riddle

The Enigma Becomes Less Mysterious

Joel S. Karliner

From the Cardiology Section, Veterans Affairs Medical Center, San Francisco, and the Department of Medicine and the Cardiovascular Research Institute, University of California, San Francisco.

Correspondence to Joel S. Karliner, MD, VA Medical Center (111C5), 4150 Clement St, San Francisco, CA 94121. E-mail joel.karliner{at}med.va.gov



See related article, pages 468–476


Key Words: sphingosine kinase-1 • sphingosine 1-phosphate • cardioprotection • enzyme regulation • four and a half LIM domain 2 (FLH2)

"It is a riddle wrapped in a mystery inside an enigma."1

The mythical Greek Sphinx posed riddles to passers-by. Those who could not answer met their demise until Oedipus solved the riddle and the Sphinx self-destructed. Ultimately the terms sphinx and enigma became associated and in 1884 J.L.W. Thudichum, known as the "Father of Neurochemistry," named the chemical backbone of sphingolipids for their enigmatic "Sphinx-like" properties. Recently, studies of sphingolipid actions have achieved increasing importance in understanding the pathophysiology of acute ischemia/reperfusion injury through studies of sphingosine kinase (SphK) and its end-product, sphingosine 1-phosphate (S1P). Activation of SphK is the final and rate-limiting enzymatic step in the synthesis of S1P, which is an intracellular and extracellular signaling molecule that regulates many important cellular processes including growth, survival, differentiation, cytoskeletal rearrangements, motility, angiogenesis, and calcium mobilization. In contrast, the sphingosine precursor ceramide is a growth-inhibiting lipid implicated in differentiation and apoptosis. These findings have led to the so-called "sphingolipid rheostat" hypothesis, which proposes that the relative levels of these lipids are important determinants of cell fate.2

It is now accepted that many of the actions of S1P are mediated by a family of G protein–coupled S1P receptor isoforms, termed S1P1–5. With regard to the cardiovascular system, platelets store and release S1P, which is known to regulate endothelial function and signaling. There is a substantial gradient between intracellular and extracellular concentrations of S1P. In human serum estimates of S1P concentration range from 0.5 to 1 µmol/L or more, whereas intracellular levels vary among tissues but are much lower possibly due in part to export of S1P or SphK (Figure). Also implicated in the regulation of S1P levels are degradation by both S1P lyase and dephosporylation by S1P phosphohydrolase. About 60% of S1P in serum is bound to high-density lipoprotein.


Figure 1
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  		Regulation and function of SphK1 and SphK2. Shown in schematic form are some of the negative and positive regulators of SphK1 activity either present in the heart or derived experimentally. As noted in the text, activation of SphK1 in the heart is dependent on {alpha}PKC, which is activated by GM1 and by ischemic and some forms of pharmacologic preconditioning. SphK1 activity can be affected by interacting proteins such as FHL2. The fate and function of SphK1 may follow several pathways, which may vary among cell types. The enzyme can be transported to the plasma membrane in a PKC-dependent manner which results in S1P release to the extracellular space.24 An alternative or complementary explanation is that an isoform of SphK1 itself is exported from the cell and thereby could be responsible for substantial extracellular S1P synthesis.25 SphK1 also generates intracellular S1P which may act within the cell or be exported. The notion of export of intracellular S1P to the cell surface or extracellular space is consistent with a recent report that provided evidence for the extracellular release of S1P, thus supporting a role of S1P as an autocrine/paracrine physiological messenger.26 SphK2 is proapoptotic and can also generate S1P. The location of this S1P generation may determine its effect within the cell and promote either proapoptotic or antiapoptotic effects.12 The mechanism of this location-based effect has not been determined.

Recent studies have shown that exogenous S1P added to cultured neonatal rat ventricular myocytes was protective against hypoxia-induced cell death.3 These studies were extended to an ex vivo mouse model in which S1P administered via an aortic cannula before ischemia/reperfusion injury improved hemodynamics, reduced creatine kinase release, and diminished infarct size.4 Similar findings were described in the rat.5 It was also found that the monoganglioside GM-1, which activates SphK, was cardioprotective in this system. In the {alpha}PKC null mouse, GM-1 was ineffective, presumably because it recruits {alpha}PKC which in turn is required for SphK activation. Subsequently, chemical inhibition of enzyme activity by dimethylsphingosine (DMS) was used to show that SphK activation mediates ischemic preconditioning in the isolated murine heart.6 In this connection, it was found in more recent work that the effect of DMS is concentration dependent. It is inhibitory at higher concentrations, but actually stimulates SphK activity and is cardioprotective at lower concentrations.7 It has also been shown that S1P induces a prosurvival pathway in adult mouse cardiac myocytes subjected to prolonged hypoxia.8 Based on these observations we proposed a new paradigm implicating SphK activation as an alternative or additional target of {alpha}PKC as part of the cardioprotective mechanism of ischemic preconditioning.6 As further evidence for this hypothesis, a newly developed assay for SphK activity9 was used to show that ischemic preconditioning blunted the decline of enzyme activity during ischemia, and permitted the return of SphK activity to control levels during reperfusion, whereas non-preconditioned hearts continued to exhibit markedly reduced activity.10

Two isozymes of mammalian SphK, SphK1 and SphK2, have been cloned and characterized. The tissue distribution and developmental expression pattern differ between these two major isoforms, and they have different cellular functions. SphK1 is the isoform implicated in cell growth, proliferation, antiapoptosis, and inflammatory responses. In contrast, SphK2, a BH3-only protein, has been described as a growth inhibitor, and is considered to be proapoptotic. Several additional alternatively spliced variants of human SphK 1 and 2 have also been described.11 A recent study showed that not only do Sphk1 and SphK2 have opposing roles in the regulation of cell responses, but also that the location of S1P production may dictate its functions.12 In addition, S1P produced by PKC-dependent translocation of SphK1 to the plasma membrane has been implicated in transactivation of cell surface S1P receptors (Figure).

SphK1 inhibitors, dominant negative SphK1 and small interfering RNA, have been used to study the function of endogenous SphK1. Knockdown of SphK1 by small interfering RNA caused MCF-7 cell cycle arrest and induced apoptosis.13 Endogenous SphK1 is an important regulator of ceramide levels in the cell, and its downregulation results in enhanced ceramide synthesis and its accumulation in the mitochondrion, which may be key in initiating the mitochondrial events leading to cell death.13 Limitations to these studies include the lack of a specific SphK1 inhibitor and the absence of an effect on basal enzyme activity. Moreover, most such studies are performed in cell lines. To circumvent these limitations, investigators have begun to develop SphK1 null mice.14 These mice are fertile, long lived, and without any obvious abnormalities.14 There are no changes in the mRNA levels for the genes encoding enzymes that are known to regulate S1P levels, including S1P lyase, S1P phosphatase, or ceramidase.14 In their SphK1 null mouse model, Allende et al reported a >60% reduction in serum S1P levels, but surprisingly no significant decrease in tissue levels, including brain, heart, kidney, liver, spleen, and testis.14 However, substantial reductions in SphK1 enzyme activity were observed in all of these organs, suggesting a compensatory increase in SphK2 activity.

Accordingly, additional evidence for the role of SphK in cardioprotection was sought by testing the hypothesis that elimination of SphK1 by genetic means would alter the response to ischemia/reperfusion injury and ischemic preconditioning.15 There were no significant differences in baseline cardiac function between wild-type and SphK1 null mouse hearts. However, after global ischemia followed by reperfusion, left ventricular performance was significantly decreased in the SphK1 null hearts. Infarct size and creatine kinase release were significantly increased in the null hearts by ischemia/reperfusion injury. Strikingly, cardioprotection induced by ischemic preconditioning was abolished in the SphK1 null hearts.15 These results provide further evidence in a genetically modified animal that SphK1 is an important lipid kinase mediating cell survival and that this isozyme is indeed required for ischemic preconditioning in the heart.

Given the key role of SphK as a checkpoint in both prosurvival and proapoptotic signaling pathways, learning more about its regulation becomes ever more pertinent. Regulation by ischemic preconditioning and {alpha}PKC, the monoganglioside GM1, and by the site of intracellular localization4,6,12 has already been mentioned. Numerous upstream activators of SphK have been described, including G protein–coupled receptors, small GTPases, tyrosine kinase receptors, proinflammatory cytokines, immunoglobulin receptors, calcium, protein kinase activators, anionic phospholipids, dibutryl cAMP, forskolin, and phosopolipase C (for a review see reference 16). Previous reports have also described protein–protein interactions. Among these interacting proteins are TNF{alpha} receptor–associated factor 2 (TRAF2), SK1-interacting protein (SKIP), platelet endothelial adhesion molecule-1 (PECAM-1), aminocyclase 1, delta-catenin/neural plakophilin-related armadillo repeat protein, and RPK118.17–22 Although none of these have been specifically studied in intact heart preparations, it might be expected that among these molecules, TRAF2, PECAM-1, and SKIP, which are known to be present in heart, vascular tissue, or platelets,17–19 might regulate SphK1 in the heart or blood vessels under conditions of oxidative or other stress.

In the study published in the current issue of Circulation Research,23 Sun et al have identified a LIM-only factor (four and a half LIM domain 2 or FHL2) as an SphK1 interacting protein in the heart. These investigators used a yeast two-hybrid screen with SphK1 as bait and a human adult cardiac cDNA library to identify this novel interacting protein. They demonstrated that FHL2, but not FHL1 or FHL3, coimmunoprecipitated with SphK1 in HEK 293 cells. Using confocal microscopy they showed that SphK1 and FHL2 are co-localized in the cytoplasm of both HEK293 cells and neonatal rat ventricular myocytes. The SphK1 C-terminal portion containing the C5 domain appears to mediate its interaction with FHL2. Transfection of FHL2 in HEK293 cells did not affect SphK1 expression but markedly decreased its activity. In NIH3T3 cells transfection of FHL2 attenuated the antiapoptotic effects of SphK1 whereas a mutant LIM protein was without effect. When neonatal rat ventricular myocytes were treated with the cardioprotective agent endothelin-1, the FHL2 interaction with SphK1 was decreased and SphK activity was increased by 85%. Finally, siRNA-mediated knockdown of FHL2 expression markedly increased SphK1 activity and protected cardiomyocytes from H2O2-induced apoptosis compared with cells treated with control siRNA.

These exciting results identify FHL2 as a new negative regulator of SphK1 activity and provide further evidence for the prosurvival function of SphK1 in cardiac and other cells. Like all creative research, the observations of Sun et al enhance what is known about the function and regulation of SphK1 (Figure) and open new avenues of investigation. Among many unanswered questions are the following: What is the mechanism by which FHL2 inhibits SphK1? How selective is the FHL2-SphK1 interaction? How do FHL2 gene–targeted mouse hearts respond to ischemia/perfusion injury? Can a selective inhibitor of FHL2 be found that could be used to develop a drug in humans that would help to salvage ischemic myocardium?

In summary, the work of Sun et al takes an important step in solving the sphingolipid riddle by removing a layer of mystery from the enigma of sphingosine kinase regulation.


*    Acknowledgments
 
Source of Funding

This work was supported by 1PO1 HL 068738 from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.

Disclosures

None.


*    Footnotes
 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.


*    References
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 *References
 

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Related Article:

FHL2/SLIM3 Decreases Cardiomyocyte Survival by Inhibitory Interaction With Sphingosine Kinase-1
Jianxin Sun, Guijun Yan, Aixia Ren, Bei You, and James K. Liao
Circ. Res. 2006 99: 468-476. [Abstract] [Full Text] [PDF]



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