RESUMO
The erythrocyte S1P transporter Mfsd2b is also expressed in the heart. We hypothesized that S1P transport by Mfsd2b is involved in cardiac function. Hypertension-induced cardiac remodeling was induced by 4-weeks Angiotensin II (AngII) administration and assessed by echocardiography. Ca2+ transients and sarcomere shortening were examined in adult cardiomyocytes (ACM) from Mfsd2b+/+ and Mfsd2b-/- mice. Tension and force development were measured in skinned cardiac fibers. Myocardial gene expression was determined by real-time PCR, Protein Phosphatase 2A (PP2A) by enzymatic assay, and S1P by LC/MS, respectively. Msfd2b was expressed in the murine and human heart, and its deficiency led to higher cardiac S1P. Mfsd2b-/- mice had regular basal cardiac function but were protected against AngII-induced deterioration of left-ventricular function as evidenced by ~ 30% better stroke volume and cardiac index, and preserved ejection fraction despite similar increases in blood pressure. Mfsd2b-/- ACM exhibited attenuated Ca2+ mobilization in response to isoprenaline whereas contractility was unchanged. Mfsd2b-/- ACM showed no changes in proteins responsible for Ca2+ homeostasis, and skinned cardiac fibers exhibited reduced passive tension generation with preserved contractility. Verapamil abolished the differences in Ca2+ mobilization between Mfsd2b+/+ and Mfsd2b-/- ACM suggesting that S1P inhibits L-type-Ca2+ channels (LTCC). In agreement, intracellular S1P activated the inhibitory LTCC phosphatase PP2A in ACM and PP2A activity was increased in Mfsd2b-/- hearts. We suggest that myocardial S1P protects from hypertension-induced left-ventricular remodeling by inhibiting LTCC through PP2A activation. Pharmacologic inhibition of Mfsd2b may thus offer a novel approach to heart failure.
RESUMO
MFSD2B has been identified as the exclusive sphingosine-1-phosphate (S1P) transporter in red blood cells (RBC) and platelets. MFSD2B-mediated S1P export from platelets is required for aggregation and thrombus formation, whereas RBC MFSD2B maintains plasma S1P levels in concert with SPNS2, the vascular and lymphatic endothelial cell S1P exporter, to control endothelial permeability and ensure normal vascular development. However, the physiological function of MFSD2B in RBC remains rather elusive despite mounting evidence that the intracellular S1P pool plays important roles in RBC glycolysis, adaptation to hypoxia and the regulation of cell shape, hydration, and cytoskeletal organisation. The large accumulation of S1P and sphingosine in MFSD2B-deficient RBC coincides with stomatocytosis and membrane abnormalities, the reasons for which have remained obscure. MFS family members transport substrates in a cation-dependent manner along electrochemical gradients, and disturbances in cation permeability are known to alter cell hydration and shape in RBC. Furthermore, the mfsd2 gene is a transcriptional target of GATA together with mylk3, the gene encoding myosin light chain kinase (MYLK). S1P is known to activate MYLK and thereby impact on myosin phosphorylation and cytoskeletal architecture. This suggests that metabolic, transcriptional and functional interactions may exist between MFSD2B-mediated S1P transport and RBC deformability. Here, we review the evidence for such interactions and the implications for RBC homeostasis.
Assuntos
Eritrócitos , Lisofosfolipídeos , Humanos , Eritrócitos/metabolismo , Homeostase , Lisofosfolipídeos/metabolismo , Esfingosina/metabolismoRESUMO
Sphingosine 1-phosphate (S1P) is one of the lipid mediators involved in diverse physiological functions. S1P circulates in blood and lymph bound to carrier proteins. Three S1P carrier proteins have been reported, albumin, apolipoprotein M (ApoM) and apolipoprotein A4 (ApoA4). The carrier-bound S1P exerts its functions via specific S1P receptors (S1PR1-5) on target cells. Previous studies showed several differences in physiological functions between albumin-bound S1P and ApoM-bound S1P. However, molecular mechanisms underlying the carrier-dependent differences have not been clarified. In addition, ApoA4 is a recently identified S1P carrier protein, and its functional differences from albumin and ApoM have not been addressed. Here, we compared the three carrier proteins in the processes of S1P degradation, release from S1P-producing cells and receptor activation. ApoM retained S1P more stable than albumin and ApoA4 in the cell culture medium when compared in the equimolar amounts. ApoM facilitated theS1P release from endothelial cells most efficiently. Furthermore, ApoM-bound S1P showed a tendency to induce prolonged activation of Akt via S1PR1 and S1PR3. These results suggest that the carrier-dependent functional differences of S1P are partly ascribed to the differences in the S1P stability, S1P-releasing efficiency and signaling duration.
Assuntos
Lisofosfolipídeos , Proteínas Proto-Oncogênicas c-akt , Humanos , Apolipoproteínas M/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Lisofosfolipídeos/farmacologia , Esfingosina/farmacologia , Proteínas de Transporte/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Albuminas/metabolismoRESUMO
Sphingosine-1-phosphate (S1P) is a potent lipid mediator that is secreted by several cell types. We recently showed that Mfsd2b is an S1P transporter from hematopoietic cells that contributes approximately 50% plasma S1P. Here we report the characterization of compound deletion of Mfsd2b and Spns2, another S1P transporter active primarily in endothelial cells. Global deletion of Mfsd2b and Spns2 (global double knockout [gDKO]) results in embryonic lethality beyond embryonic day 14.5 (E14.5), with severe hemorrhage accompanied by defects of tight junction proteins, indicating that Mfsd2b and Spns2 provide S1P for signaling, which is essential for blood vessel integrity. Compound postnatal deletion of Mfsd2b and Spns2 using Mx1Cre (ctDKO-Mx1Cre) results in maximal 80% reduction of plasma S1P. ctDKO-Mx1Cre mice exhibit severe susceptibility to anaphylaxis, indicating that S1P from Mfsd2b and Spns2 is indispensable for vascular homeostasis. Our results show that S1P export from Mfsd2b and Spns2 is essential for developing and mature vasculature.
Assuntos
Anafilaxia , Proteínas de Membrana/metabolismo , Anafilaxia/metabolismo , Animais , Proteínas de Transporte de Ânions/metabolismo , Transporte Biológico , Células Endoteliais/metabolismo , Homeostase , Lisofosfolipídeos/metabolismo , Camundongos , Esfingosina/metabolismoRESUMO
Sphingosine-1-phosphate (S1P) is a potent lipid mediator that exerts its activity via activation of five different G protein-coupled receptors, designated as S1P1-5. This potent lipid mediator is synthesized from the sphingosine precursor by two sphingosine kinases (SphK1 and 2) and must be exported to exert extracellular signaling functions. We recently identified Mfsd2b as the S1P transporter in the hematopoietic system. However, the sources of sphingosine for S1P synthesis and the transport mechanism of Mfsd2b in erythrocytes remain to be determined. Here, we show that erythrocytes efficiently take up exogenous sphingosine and that a de novo synthesis pathway in part provides sphingosines to erythrocytes. The uptake of sphingosine in erythrocytes is facilitated by the activity of SphK1. By converting sphingosine into S1P, SphK1 indirectly increases the influx of sphingosine, a process that is irreversible in erythrocytes. Our results explain for the abnormally high amount of sphingosine accumulation in Mfsd2b knockout erythrocytes. Furthermore, we show that Mfsd2b utilizes a proton gradient to facilitate the release of S1P. The negatively charged residues D95 and T157 are essential for Mfsd2b transport activity. Of interest, we also discovered an S1P analog that inhibits S1P export from erythrocytes, providing evidence that sphingosine analogs can be used to inhibit S1P export by Mfsd2b. Collectively, our results highlight that erythrocytes are efficient in sphingosine uptake for S1P production and the release of S1P is dependent on Mfsd2b functions.