Abstract
Sphingosine 1-phosphate (S1P) is a key bioactive lipid that regulates a myriad of physiological and pathophysiological processes, including endothelial barrier function, vascular tone, vascular inflammation, and angiogenesis. Various S1P receptor subtypes have been suggested to be involved in the regulation of these processes, whereas the contribution of intracellular S1P (iS1P) through intracellular targets is little explored. In this study, we used the human cerebral microvascular endothelial cell line HCMEC/D3 to stably downregulate the S1P lyase (SPL-kd) and evaluate the consequences on endothelial barrier function and on the molecular factors that regulate barrier tightness under normal and inflammatory conditions. The results show that in SPL-kd cells, transendothelial electrical resistance, as a measure of barrier integrity, was regulated in a dual manner. SPL-kd cells had a delayed barrier build up, a shorter interval of a stable barrier, and, thereafter, a continuous breakdown. Contrariwise, a protection was seen from the rapid proinflammatory cytokine-mediated barrier breakdown. On the molecular level, SPL-kd caused an increased basal protein expression of the adherens junction molecules PECAM-1, VE-cadherin, and β-catenin, increased activity of the signaling kinases protein kinase C, AMP-dependent kinase, and p38-MAPK, but reduced protein expression of the transcription factor c-Jun. However, the only factors that were significantly reduced in TNFα/SPL-kd compared to TNFα/control cells, which could explain the observed protection, were VCAM-1, IL-6, MCP-1, and c-Jun. Furthermore, lipid profiling revealed that dihydro-S1P and S1P were strongly enhanced in TNFα-treated SPL-kd cells. In summary, our data suggest that SPL inhibition is a valid approach to dampenan inflammatory response and augmente barrier integrity during an inflammatory challenge.
Highlights
The blood–brain barrier (BBB) is a unique barrier present in all mammals and is localized at the interface between the blood and the central nervous system (CNS)
In the present study, we demonstrate that the genetic knockdown of SPL in the human cerebral microvIanscthuelaprreesnednottshteuldiayl, cweelldleinmeoHnsCtrMatEeCth/Dat3thaeltgeersnebtiacrrkinerocfkudnocwtionno,f wSPhLicihn othcecuhrusminanacedruebalral mmanincreor.vaUscnudlaerr iennfldaomthmelaiatol rcyelclonlidneitioHnCs,MwEhCi/cDh3noalrtmerasllbyatrrriigegr efruancctoionnti,nwuohuicshanodcculornsgi-nlasatindgual barrier breakdown (Figure 3), SPL-kd demonstrated consolidated barrier function
Increased endothelial permeability is considered a serious complication in many inflammatory diseases or as a factor that contributes to the pathology of neurodegenerative diseases, trauma, tumors, and ischemia. Such endothelial dysfunction may be induced by various factors, for instance, inflammatory cytokines, histamine, thrombin, LPS, vascular endothelial growth factor, and bradykinin [30,31,32]
Summary
The blood–brain barrier (BBB) is a unique barrier present in all mammals and is localized at the interface between the blood and the central nervous system (CNS). To preserve the intracellular sphingolipid balance, which is known as “sphingolipid rheostat”, the cells exploit a series of enzymes that reversibly backconvert S1P to sphingosine, including lipid phosphate phosphatases and S1P phosphatases, or that irreversibly degrade S1P to hexadecenal and ethanolamine-phosphate and thereby terminate signaling The latter is catalyzed by the endoplasmic reticulum-resident enzyme S1P lyase (Sgpl, SPL) [14,15]. We used an SPL knockdown (kd) approach in endothelial cells derived from the BBB, HCMEC/D3, to evaluate the implications of increased intracellular S1P levels on endothelial phenotype markers, responses to inflammatory stimuli, and the consequences on barrier tightness. CCoonnttrrooll HHCCMMEECC//DD33 aanndd SSPPLL--kkdd cceellllss wweerree sseeeeddeedd wwiitthh aa ddeennssiittyy bbeettwweeeenn 2200,,000000 aanndd 5500,,000000 cceellllss//mmLL ttoo ddeetteerrmmiinnee aa cceellll ccoonncceennttrraattiioonn rreessuullttiinngg iinn aa ssttaabbllee,, lloonngg--tteerrmm bbaarrrriieerr ffuunnccttiioonn. SCo+nCvyertssetliym, puolastt-itorne,atdmidenntot wiatfhfeSc1tPb, agrivrieenr 2st4ahbialifttyerinLPeSith+eCr ycet lsltitmypuelsatoiovne,rstihgeniwfichaonltelytiamtteenpuearitoedd L(FPiSgu+reCSy9t-,iSn1d0u).cCedonbvaerrriseerly, depsotasbt-itlrizeaattmionenitn wcoitnhtroSl1PH,CgMivEeCn /D243 hcelalfst,ebruLtPthSis+wCaystosntliymsueleantioanft,ersi1g2n0ifhica(FnitglyuraetsteSn1u1aatnedd SL1P2S). + NoCyefft-einctdoufcpedosbt-atrreriaetrmdeensttawbiitlhizSa1tiPonwains sceoenntrionlSHPCL-MkdECce/lDls3(Fceigllusr, ebsuSt1t1haisndwSas12o)nolrywseitehnSa1fPtearlo1n20e h in(bFoigthurceelSl1li1n,Se1s2()F.igNuoreesffSe9c–tSo1f2p).ost-treatment with S1P was seen in SPL-kd cells (Figure S11,S12) or with S1P alone in both cell lines (Figure S9–S12)
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