By Ashwin Kelkar
What constitutes a cell? In a very general sense, the first successful cell-like structures necessitated a biological barrier that would mediate the flow of molecules into and out of their “bodies.” What developed was the phospholipid bilayer, a semi-permeable membrane intermeshed with various proteins to regulate the passage of important bioactive molecules – ones that regulate cellular responses – in order to initiate various cellular functions.
Because of the relative difficulty of conducting experiments with lipids, they have been thought to only play metabolic and structural roles in the body for many years. It wasn’t until the groundbreaking discovery of the role of diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) in stimulating protein kinase C (PKC) that lipids were also considered bioactive (1).
Enter the sphingolipid, a class of lipids discovered in the late 1800s in brain tissue (2). This new class of lipids was interestingly named after the mythological “Sphinx” for its enigmatic character, but recent research has begun to uncover the important role of sphingolipids as key players in various cellular pathways, including apoptosis, cell division, motility, invasion, and adhesion (2,3). They have since become a major site of investigation for many researchers around the world because slight changes in sphingolipid concentrations can greatly impact the cell cycle, the absence or abundance of which can lead to the development of many pathologies including neuronal disease and cancer. In order to understand the influence sphingolipids can have on cell function, it is important to look past its structural attributions and focus on its diverse, yet specific, functions.
Structurally, sphingolipids are quite different from the typical lipids that are normally found on the lipid bilayer. Rather than a glycerol backbone, sphingolipids have a long alkyl backbone with a 1,3-diol tail. At the 2 position is usually an amine that can form covalent bonds with a fatty-acid chain of varying length. Though these differences are seemingly minimal, they confer great functional differences that allow this class of lipids to play an ever greater role in regulating the cell cycle
Other sphingolipids have other functional groups extending from this 1-position. For example, sphingomyelin has a phosphocholine group attached here. Removal of this phosphocholine results in ceramide formation. Further cleavage by removal of the fatty acid amide linkage produces sphingosine. Both ceramide and sphingosine can be phosphorylated to produce ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P), which have antagonistic effects to their unphosphorylated counterparts (3). A full pathway map is illustrated in Figure 1.
Figure 1 – A schematic of the sphingolipid metabolic pathway is shown above. The various enzymes that mediate interconversion between the various bioactive lipids are localized along with the lipids themselves to various membranes within the cell including the endoplasmic reticulum, golgi body, mitochondrion, and the plasma membrane. Exit from this pathway only occurs with S1P lyase, which cleaves S1P into ethanolamine phosphate and hexadecenal, both of which are used in phospholipid formation and incorporation into the lipid bilayer (3).
Glycosphingolipids are the largest in the sphingolipid family, with carbohydrate moieties extending from the 1-position to form acetal linkages, the largest of which are called gangliosides. Glycosphingolipids were the first sphingolipids to be discovered and were recognized for their importance in maintaining the structural integrity of the myelin sheath surrounding neurons (2).
Sphingolipids were first identified to play a role in neurological pathologies, such as Tay-Sachs and Gaucher’s diseases (2). For example, Tay-Sachs disease is the direct result of ganglioside accumulation in the brain. Gaucher’s disease, another neuropathology associated with sphingolipid metabolism, occurs when glucosylceramide (GlcCer) cannot be broken down and accumulates in various parts of the body, including the central nervous system, causing a massive deterioration in muscle function and in some cases, mental retardation (5).
Specifically, glycosphingolipids act as key structural elements of myelin sheathing. It has been observed that animals lacking galactosylceramide (GalCer) cannot form proper myelin sheaths. Though myelin sheaths are observed, evidence indicates that the resulting nodes of Ranvier in these animals are morphed as a result of the GalCer synthesis gene knockdown (6). Disrupting glycosphingolipid metabolism in mice leads to early axon degeneration and demyelination, causing severe impairment.
These few examples show the necessity of glycosphingolipids and their important role within the nervous system. However, this does not mean that sphingolipids are only necessary in neurons; in other mammalian cell types, they are shown to cluster into lipid rafts within the plasma membrane, specifically on the outer leaflet. It is now believed that glycosphingolipid metabolites can also act as intermediary molecules, passing on messages from outside to inside the cell (6).
Ceramide and C1P
Because of the varying length of the fatty acid functional groups on the sphingosine backbone, many different ceramides are naturally occurring within the context of the body, each with different functionalities (7). Ceramide is formed from the hydrolysis of sphingomyelin via sphingomyelinases (SMase). Ceramide can then be phosphorylated directly by ceramide kinase to generate C1P. Ceramide is neutrally charged and therefore does not have the ability to interact very well outside of the lipid bilayer, but its hydrophobic structure allows it to readily flip-flop between the cytosolic and extracellular faces of membranes. On the other hand, the anionic phosphate group on C1P allows it to interact with hydrophilic substances more readily. These structural properties give ceramide and C1P, as well as the enzymes that synthesize and catabolize them, distinct functional properties and localizations within the cell (3).
Ceramides are most closely associated with the skin and can often be found in beauty products to keep skin healthy. This is due to the fact that ceramides are one of the major components of the water barrier located on the epidermis which prevents excess water transpiration (http://archderm.jamanetwork.com/article.aspx?articleid=527990). There is a clear positive correlation between skin health and ceramide levels, though it is difficult to pinpoint which of the various lipid products is responsible for these conditions due to the sheer amount of compounds necessary for proper skin health (8).
Along with its water barrier functions, ceramide is heavily involved in the stress response in cells, signaling cell death and cell cycle arrest to halt cell aging (7,9). One particular activator of ceramide is tumor necrosis factor-α (TNFα), a protein that plays a major role in the inflammatory response. High palmitate levels as a result of high carbohydrate intake also stimulates ceramide synthesis (3). Activation of ceramide in turn activates second messenger systems that result in self-induced cell death, cell cycle arrest, and a continuation of the inflammatory response. This shows ceramide’s versatility as both a second messenger and downstream effector (7,9).
C1P, on the other hand, has been shown to exert the opposite influences upon the cell. Not only does it inhibit cell death, but it also activates cell proliferation. This two-fold activity results in hyperactivity of cell cycle turnover. C1P is known to inhibit ceramide production by directly binding acid SMase, while also activating phosphatidylinositol 3-kinase (PI3-K). This produces phosphatidylinositol (3,4,5)-triphosphate (PIP3), a direct inhibitor of acid SMase (7). By inhibiting SMase, ceramide production is halted, allowing the cell to continue through the cell cycle and initiate proliferative cellular functions. This dual pathway exemplifies the complexity with which the sphingolipids can be interconverted to implement minute changes within the cell. To add to the intricacy, further experimentation has shown C1P to have both pro- and anti-inflammatory properties, and these properties are dependent on cell type (10).
Sphingosine and S1P
Sphingosine is a metabolite of ceramide via hydrolysis of the fatty acid chain on ceramide, which is catalyzed by one of five ceramidases, each of which is localized to a particular lipid bilayer within the cell. Phosphorylation of sphingosine by one of two sphingosine kinases produces sphingosine-1-phosphate (S1P) (3).
Both S1P and C1P as well as sphingosine and ceramide have similar functionalities due to their structural similarities. This necessitates the question of why the cell would require two separate bioactive lipids to regulate the same functions.
But the functions of S1P extend beyond those that are also exhibited by C1P. A study conducted by Chun et al demonstrated that fingolimod-phosphate, an agonist of S1P receptors, can reduce the symptoms of multiple sclerosis in animal models and is currently undergoing phase 3 clinical trials (11). Moreover, S1P has been linked to angiogenesis and vessel morphogenesis in embryonic development (12).
Since sphingolipids traverse various overlapping biochemical pathways, it is at first difficult to understand why each functionality is distinct and important. It is important to note that these functionalities arose evolutionarily in order to optimize cell survival. These interconnected pathways allow the cell to coordinate and regulate the total positive and negative effectors for cell cycle, death, proliferation, migration, invasion, and many other functions. Thus, the cell can affect minute changes within itself to only slightly alter its major functions. The sphingolipid family therefore orchestrates a much more complex and intricate regulation of cell functionality than the canonical cellular second messenger pathways such as adenylyl cyclase.
Though the various functions of sphingolipids and their metabolites have been briefly discussed and highlighted here, the actual functions of each bioactive lipid are much more extensive and do not end here. Research being conducted every day is opening new doorways to what sphingolipids are capable of accomplishing. owever, it is clear to see that lipids function as more than just structural compounds, but instead have a major and growing role in cell cycle regulation.
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- Chun, H.P. Hartung, Mechanism of action of oral fingolimod (FTY720) in multiple sclerosis. Clin Neuropharmacol.33, 91-101 (2010), doi:10.1097/WNF.0b013e3181cbf825.
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