Lipid signaling, broadly defined, refers to any biologicalcell signaling event involving alipid messenger that binds a protein target, such as areceptor,kinase orphosphatase, which in turn mediate the effects of these lipids on specific cellular responses. Lipid signaling is thought to be qualitatively different from other classical signaling paradigms (such asmonoamineneurotransmission) because lipids can freelydiffuse throughmembranes (seeosmosis). One consequence of this is that lipid messengers cannot be stored invesicles prior to release and so are oftenbiosynthesized "on demand" at their intended site of action. As such, many lipid signaling molecules cannot circulate freely in solution but, rather, exist bound to special carrier proteins inserum.
Ceramide (Cer) can be generated by the breakdown ofsphingomyelin (SM) bysphingomyelinases (SMases), which areenzymes that hydrolyze thephosphocholine group from thesphingosine backbone. Alternatively, thissphingosine-derivedlipid (sphingolipid) can be synthesized from scratch (de novo) by the enzymesserine palmitoyl transferase (SPT) andceramidesynthase inorganelles such as theendoplasmic reticulum (ER) and possibly, in themitochondria-associated membranes (MAMs) and theperinuclear membranes. Being located in the metabolic hub, ceramide leads to the formation of othersphingolipids, with the C1hydroxyl (-OH) group as the major site of modification. A sugar can be attached toceramide (glycosylation) through the action of the enzymes, glucosyl or galactosylceramidesynthases.[1] Ceramide can also be broken down by enzymes calledceramidases, leading to the formation ofsphingosine,[2][3] Moreover, a phosphate group can be attached to ceramide (phosphorylation) by the enzyme,ceramidekinase.[4] It is also possible to regenerate sphingomyelin from ceramide by accepting aphosphocholine headgroup fromphosphatidylcholine (PC) by the action of an enzyme calledsphingomyelinsynthase.[5] The latter process results in the formation ofdiacylglycerol (DAG) from PC.[citation needed]
Ceramide contains twohydrophobic ("water-fearing") chains and a neutral headgroup. Consequently, it has limited solubility in water and is restricted within theorganelle where it was formed. Also, because of its hydrophobic nature, ceramide readily flip-flops across membranes as supported by studies in membrane models and membranes from red blood cells (erythrocytes).[6] However,ceramide can possibly interact with other lipids to form bigger regions called microdomains which restrict its flip-flopping abilities. This could have immense effects on thesignaling functions of ceramide because it is known thatceramide generated by acidic SMase enzymes in the outer leaflet of an organelle membrane may have different roles compared toceramide that is formed in the inner leaflet by the action of neutral SMase enzymes.[7]
Ceramide mediates many cell-stress responses, including the regulation of programmed cell death (apoptosis)[8] and cell aging (senescence).[9] Numerous research works have focused interest on defining the directprotein targets of action of ceramide. These include enzymes calledceramide-activated Ser-Thrphosphatases (CAPPs), such asproteinphosphatase 1 and 2A (PP1 and PP2A), which were found to interact with ceramide in studies done in a controlled environment outside of a living organism (in vitro).[10] On the other hand, studies in cells have shown that ceramide-inducing agents such astumor necrosis factor-alpha α (TNFα) andpalmitate induce the ceramide-dependent removal of a phosphate group (dephosphorylation) of theretinoblastomagene product RB[11] and the enzymes,protein kinases B (AKT protein family) and C α (PKB and PKCα).[12] Moreover, there is also sufficient evidence which implicatesceramide to the activation of thekinase suppressor of Ras (KSR),[13] PKCζ,[14][15] andcathepsin D.[16]Cathepsin D has been proposed as the main target forceramide formed in organelles calledlysosomes, making lysosomal acidic SMase enzymes one of the key players in the mitochondrial pathway ofapoptosis. Ceramide was also shown to activatePKCζ, implicating it to theinhibition ofAKT, regulation of the voltage difference between the interior and exterior of the cell (membrane potential) and signaling functions that favor apoptosis.[17]Chemotherapeutic agents such asdaunorubicin andetoposide[18][19] enhance thede novo synthesis ofceramide in studies done on mammalian cells. The same results were found for certain inducers ofapoptosis particularly stimulators ofreceptors in a class of lymphocytes (a type of white blood cell) calledB-cells.[20] Regulation of thede novo synthesis of ceramide bypalmitate may have a key role indiabetes and themetabolic syndrome. Experimental evidence shows that there is substantial increase ofceramide levels upon addingpalmitate.Ceramide accumulation activates PP2A and the subsequent dephosphorylation and inactivation ofAKT,[21] a crucial mediator in metabolic control andinsulinsignaling. This results in a substantial decrease ininsulin responsiveness (i.e. to glucose) and in the death of insulin-producing cells in the pancreas calledislets of Langerhans.[22] Inhibition of ceramide synthesis in mice via drug treatments or gene-knockout techniques prevented insulin resistance induced byfatty acids,glucocorticoids orobesity.[23]
An increase inin vitro activity of acid SMase has been observed after applying multiple stress stimuli such asultraviolet (UV) and ionizing radiation, binding of death receptors andchemotherapeutic agents such asplatinum,histone deacetylase inhibitors andpaclitaxel.[24] In some studies, SMase activation results to its transport to theplasma membrane and the simultaneous formation of ceramide.[24]
Ceramide transfer protein (CERT) transports ceramide from ER to theGolgi for the synthesis of SM.[25] CERT is known to bindphosphatidylinositol phosphates, hinting its potential regulation viaphosphorylation, a step of the ceramide metabolism that can be enzymatically regulated byprotein kinases andphosphatases, and byinositollipid metabolic pathways.[26] Up to date, there are at least 26 distinct enzymes with varied subcellular localizations, that act on ceramide as either asubstrate or product. Regulation of ceramide levels can therefore be performed by one of theseenzymes in distinct organelles by particular mechanisms at various times.[27]
Sphingosine (Sph) is formed by the action ofceramidase (CDase) enzymes on ceramide in thelysosome. Sph can also be formed in the extracellular (outer leaflet) side of theplasma membrane by the action of neutral CDase enzyme. Sph then is either recycled back to ceramide or phosphorylated by one of thesphingosine kinase enzymes, SK1 and SK2.[28] The productsphingosine-1-phosphate (S1P) can be dephosphorylated in the ER to regeneratesphingosine by certain S1Pphosphatase enzymes within cells, where the salvaged Sph is recycled toceramide.[29]Sphingosine is a single-chainlipid (usually 18 carbons in length), rendering it to have sufficient solubility in water. This explains its ability to move between membranes and to flip-flop across a membrane. Estimates conducted at physiological pH show that approximately 70% of sphingosine remains in membranes while the remaining 30% is water-soluble.[30] Sph that is formed has sufficient solubility in the liquid found inside cells (cytosol). Thus, Sph may come out of thelysosome and move to the ER without the need for transport via proteins or membrane-enclosed sacs calledvesicles. However, its positive charge favors partitioning inlysosomes. It is proposed that the role of SK1 located near or in the lysosome is to ‘trap’ Sph viaphosphorylation.[31]
Since sphingosine exertssurfactant activity, it is one of the sphingolipids found at lowest cellular levels.[31] The low levels of Sph and their increase in response to stimulation of cells, primarily by activation ofceramidase by growth-inducing proteins such asplatelet-derived growth factor andinsulin-like growth factor, is consistent with its function as asecond messenger. It was found that immediatehydrolysis of only 3 to 10% of newly generatedceramide may double the levels of Sph.[31] Treatment of HL60 cells (a type of leukemia cell line) by a plant-derived organic compound calledphorbol ester increased Sph levels threefold, whereby the cells differentiated into white blood cells calledmacrophages. Treatment of the same cells by exogenous Sph causedapoptosis. A specificprotein kinase phosphorylates 14-3-3, otherwise known assphingosine-dependent protein kinase 1 (SDK1), only in the presence of Sph.[32]
Sph is also known to interact with protein targets such as theprotein kinase H homologue (PKH) and the yeast protein kinase (YPK). These targets in turn mediate the effects of Sph and its related sphingoid bases, with known roles in regulating theactincytoskeleton,endocytosis, thecell cycle andapoptosis.[33] It is important to note however that thesecond messenger function of Sph is not yet established unambiguously.[34]
Sphingosine-1-phosphate (S1P), like Sph, is composed of a single hydrophobic chain and has sufficient solubility to move between membranes. S1P is formed byphosphorylation ofsphingosine bysphingosine kinase (SK). The phosphate group of the product can be detached (dephosphorylated) to regenerate sphingosine via S1Pphosphatase enzymes or S1P can be broken down by S1Plyase enzymes to ethanolamine phosphate and hexadecenal.[35] Similar to Sph, itssecond messenger function is not yet clear.[34] However, there is substantial evidence that implicates S1P to cell survival,cell migration, andinflammation. Certain growth-inducing proteins such asplatelet-derived growth factor (PDGF),insulin-like growth factor (IGF) andvascular endothelial growth factor (VEGF) promote the formation of SK enzymes, leading to increased levels of S1P. Other factors that induce SK include cellular communication molecules calledcytokines, such astumor necrosis factor α (TNFα) andinterleukin-1 (IL-1),hypoxia or lack of oxygen supply in cells, oxidized low-densitylipoproteins (oxLDL) and severalimmune complexes.[31]
S1P is probably formed at the inner leaflet of the plasma membrane in response to TNFα and other receptor activity-altering compounds calledagonists.[36][37] S1P, being present in low nanomolar concentrations in the cell, has to interact with high-affinity receptors that are capable of sensing their low levels. So far, the only identified receptors for S1P are the high-affinityG protein-coupled receptors (GPCRs), also known as S1P receptors (S1PRs). S1P is required to reach the extracellular side (outer leaflet) of theplasma membrane to interact with S1PRs and launch typical GPCRsignaling pathways.[38][39] However, thezwitterionic headgroup of S1P makes it unlikely to flip-flop spontaneously. To overcome this difficulty, theATP-binding cassette (ABC) transporter C1 (ABCC1) serves as the "exit door" for S1P.[40] On the other hand, thecystic fibrosis transmembrane regulator (CFTR) serves as the means of entry for S1P into the cell.[41] In contrast to its low intracellular concentration, S1P is found in high nanomolar concentrations inserum where it is bound toalbumin andlipoproteins.[42] Inside the cell, S1P can inducecalcium release independent of the S1PRs—the mechanism of which remains unknown. To date, the intracellular molecular targets for S1P are still unidentified.[31]
The SK1-S1P pathway has been extensively studied in relation to cytokine action, with multiple functions connected to effects ofTNFα and IL-1 favoringinflammation. Studies show that knockdown of key enzymes such as S1Plyase and S1P phosphatase increasedprostaglandin production, parallel to increase of S1P levels.[37] This strongly suggests that S1P is the mediator of SK1 action and not subsequent compounds. Research done onendothelial andsmooth muscle cells is consistent to the hypothesis that S1P has a crucial role in regulatingendothelial cell growth, and movement.[43] Recent work on asphingosine analogue, FTY270, demonstrates its ability to act as a potent compound that alters the activity of S1P receptors (agonist). FTY270 was further verified in clinical tests to have roles in immune modulation, such as that onmultiple sclerosis.[44] This highlights the importance of S1P in the regulation oflymphocyte function andimmunity. Most of the studies on S1P are used to further understand diseases such ascancer,arthritis andinflammation,diabetes,immune function andneurodegenerative disorders.[31]
Glucosylceramides (GluCer) are the most widely distributedglycosphingolipids in cells serving asprecursors for the formation of over 200 known glycosphingolipids. GluCer is formed by the glycosylation of ceramide in an organelle calledGolgi via enzymes calledglucosylceramide synthase (GCS) or by the breakdown of complexglycosphingolipids (GSLs) through the action of specifichydrolase enzymes. In turn, certain β-glucosidases hydrolyze these lipids to regenerate ceramide.[45][46] GluCer appears to be synthesized in the inner leaflet of the Golgi. Studies show that GluCer has to flip to the inside of the Golgi or transfer to the site of GSL synthesis to initiate the synthesis of complex GSLs. Transferring to the GSL synthesis site is done with the help of a transport protein known asfour phosphate adaptor protein 2 (FAPP2) while the flipping to the inside of the Golgi is made possible by theABC transporter P-glycoprotein, also known as the multi-drug resistance 1 transporter (MDR1).[47] GluCer is implicated in post-Golgi trafficking and drug resistance particularly tochemotherapeutic agents.[48][49] For instance, a study demonstrated a correlation between cellulardrug resistance and modifications in GluCermetabolism.[50]
In addition to their role as building blocks of biological membranes,glycosphingolipids have long attracted attention because of their supposed involvement in cell growth,differentiation, and formation of tumors.[31] The production of GluCer from Cer was found to be important in the growth of neurons or brain cells.[51] On the other hand, pharmacologicalinhibition of GluCer synthase is being considered a technique to avoidinsulin resistance.[52]
Ceramide-1-phosphate (C1P) is formed by the action ofceramide kinase (CK) enzymes on Cer. C1P carry ionic charge at neutral pH and contain two hydrophobic chains making it relatively insoluble in aqueous environment. Thus, C1P reside in the organelle where it was formed and is unlikely to spontaneously flip-flop across membrane bilayers.[31]
C1P activatephospholipase A2 and is found, along with CK, to be a mediator ofarachidonic acid released in cells in response to a protein calledinterleukin-1β (IL-1β) and a lipid-soluble molecule that transports calcium ions (Ca2+) across the bilayer, also known as calciumionophore.[53] C1P was also previously reported to encouragecell division (mitogenic) infibroblasts, blockapoptosis by inhibiting acid SMase inwhite blood cells within tissues (macrophages)[54] and increase intracellular freecalcium concentrations inthyroid cells.[55] C1P also has known roles invesicular trafficking, cell survival,phagocytosis ("cell eating") andmacrophagedegranulation.[56][57]
PIP2 binds directly to ion channels and modulates their activity. PIP2 was shown to directly agonizesInward rectifying potassium channels(Kir).[58] In this regard intact PIP2 signals as a bona fide neurotransmitter-like ligand.[59] PIP2's interaction with many ion channels suggest that the intact form of PIP2 has an important signaling role independent of second messenger signaling.[citation needed]
A generalsecond messenger system mechanism can be broken down into four steps. First, the agonist activates a membrane-bound receptor. Second, the activated G-protein produces a primary effector. Third, the primary effect stimulates the second messenger synthesis. Fourth, the second messenger activates a certain cellular process.
TheG-protein coupled receptors for the PIP2 messenger system produces two effectors,phospholipase C (PLC) andphosphoinositide 3-kinase (PI3K). PLC as an effector produces two different second messengers,inositol triphosphate (IP3) andDiacylglycerol (DAG).
IP3 is soluble and diffuses freely into the cytoplasm. As a second messenger, it is recognized by the inositol triphosphate receptor (IP3R), a Ca2+ channel in theendoplasmic reticulum (ER) membrane, which stores intracellular Ca2+. The binding of IP3 to IP3R releases Ca2+ from the ER into the normally Ca2+-poor cytoplasm, which then triggers various events of Ca2+ signaling. Specifically in blood vessels, the increase in Ca2+ concentration from IP3 releases nitric oxide, which then diffuses into the smooth muscle tissue and causes relaxation.[34]
DAG remains bound to the membrane by itsfatty acid "tails" where it recruits and activates both conventional and novel members of theprotein kinase C family. Thus, both IP3 and DAG contribute to activation of PKCs.[60][61]
Phosphoinositide 3-kinase (PI3K) as an effector phosphorylatesphosphatidylinositol bisphosphate (PIP2) to producephosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 has been shown to activateprotein kinase B, increase binding to extracellular proteins and ultimately enhance cell survival.[34]
See main article onG-protein coupled receptors
LPA is the result ofphospholipase A2 action onphosphatidic acid. The SN-1 position can contain either anester bond or anether bond, withether LPA being found at elevated levels in certain cancers. LPA binds the high-affinityG-protein coupled receptorsLPA1,LPA2, andLPA3 (also known asEDG2,EDG4, andEDG7, respectively).[citation needed]
S1P is present at high concentrations in plasma and secreted locally at elevated concentrations at sites of inflammation. It is formed by the regulatedphosphorylation ofsphingosine. It acts through five dedicated high-affinityG-protein coupled receptors,S1P1 -S1P5. Targeted deletion of S1P1 results in lethality in mice and deletion of S1P2 results in seizures and deafness. Additionally, a mere 3- to 5-fold elevation in serum S1P concentrations induces sudden cardiac death by anS1P3-receptor specific mechanism.
PAF is a potent activator of platelet aggregation, inflammation, and anaphylaxis. It is similar to the ubiquitous membranephospholipidphosphatidylcholine except that it contains anacetyl-group in the SN-2 position and the SN-1 position contains anether-linkage. PAF signals through a dedicatedG-protein coupled receptor, PAFR and is inactivated by PAF acetylhydrolase.
The endogenouscannabinoids, orendocannabinoids, are endogenous lipids that activatecannabinoid receptors. The first such lipid to be isolated wasanandamide which is the arachidonoylamide ofethanolamine. Anandamide is formed via enzymatic release from N-arachidonoylphosphatidylethanolamine by theN-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD).[62] Anandamide activates both the CB1 receptor, found primarily in thecentral nervous system, and the CB2 receptor which is found primarily inlymphocytes and the periphery. It is found at very low levels (nM) in most tissues and is inactivated by thefatty acid amide hydrolase. Subsequently, another endocannabinoid was isolated,2-arachidonoylglycerol, which is produced whenphospholipase C releasesdiacylglycerol which is then converted to2-AG bydiacylglycerol lipase.2-AG can also activate bothcannabinoid receptors and is inactivated bymonoacylglycerol lipase. It is present at approximately 100-times the concentration ofanandamide in most tissues. Elevations in either of these lipids causesanalgesia and anti-inflammation and tissue protection during states of ischemia, but the precise roles played by these various endocannabinoids are still not totally known and intensive research into their function, metabolism, and regulation is ongoing. One saturated lipid from this class, often called an endocannabinoid, but with no relevant affinity for the CB1 and CB 2 receptor ispalmitoylethanolamide. This signaling lipid has great affinity for the GRP55 receptor and the PPAR alpha receptor. It has been identified as an anti-inflammatory compound already in 1957, and as an analgesic compound in 1975.Rita Levi-Montalcini first identified one of its biological mechanisms of action, the inhibition of activated mast cells. Palmitoylethanolamide is the only endocannabinoid available on the market for treatment, as a food supplement.
Prostaglandins are formed throughoxidation ofarachidonic acid bycyclooxygenases and otherprostaglandin synthases. There are currently nine knownG-protein coupled receptors (eicosanoid receptors) that largely mediate prostaglandin physiology (although some prostaglandins activatenuclear receptors, see below).
FAHFAs (fatty acid esters of hydroxy fatty acids) are formed in adipose tissue, improve glucose tolerance and also reduce adipose tissue inflammation. Palmitic acid esters of hydroxy-stearic acids (PAHSAs) are among the most bioactive members able to activateG-protein coupled receptors 120.[63] Docosahexaenoic acid ester of hydroxy-linoleic acid (DHAHLA) exert anti-inflammatory and pro-resolving properties.[64]
Retinaldehyde is aretinol (vitamin A) derivative responsible for vision. It bindsrhodopsin, a well-characterizedGPCR that binds all-cisretinal in its inactive state. Upon photoisomerization by aphoton the cis-retinal is converted to trans-retinal causing activation ofrhodopsin which ultimately leads todepolarization of theneuron thereby enablingvisual perception.
See the main article onnuclear receptors
This large and diverse class ofsteroids are biosynthesized fromisoprenoids and structurally resemblecholesterol. Mammalian steroid hormones can be grouped into five groups by the receptors to which they bind:glucocorticoids,mineralocorticoids,androgens,estrogens, andprogestogens.
Retinol (vitamin A) can be metabolized toretinoic acid which activatesnuclear receptors such as theRAR to control differentiation and proliferation of many types of cells during development.[65]
The majority ofprostaglandin signaling occurs viaGPCRs (see above) although certainprostaglandins activate nuclear receptors in thePPAR family. (See articleeicosanoid receptors for more information).