Research

Research Areas

The research target of our laboratory is lipid. Lipids are defined as water insoluble biomolecules, and organisms have a huge number of lipid molecules. Energy source, biological membrane formation, and lipid mediator function are famous as three major functions of lipids. However, our laboratory is aiming to explore new research areas of lipids, not these well-known functions. In particular, we are focusing on the barrier function of lipids. The barrier present on the body surface (epidermis and tear film) prevents invasion of pathogens, allergens, and toxic compounds, and allergens as well as water evaporation from inside the body (Figure 1).Only restricted numbers of lipids act as barrier lipids: acylceramides in the epidermis and meibum lipids in the tear film. Both of them are very long-chain lipids and are highly hydrophobic. Impairment of the "barrier function" of lipids can lead to several disorders such as infections, ichthyosis, atopic dermatitis, and dry skin in the epidermis and dry eye and corneal disorders in the eyes.

Sphingolipids are one of the membrane lipids present in eukaryotes and are known as multifunctional lipids. The hydrophobic backbone of sphingolipid is ceramide. One of the ceramide species acylceramide has "barrier function". Our laboratory has identified a number of genes involved in the synthesis and degradation of sphingolipids (Figure 2). In addition, our laboratory is conducting research on the physiological and pathological roles, metabolism, and dynamics of various lipids.

Lipids are extremely rich in diversity compared with other biomolecules such as amino acids, nucleic acids, and sugars. Mammalian lipids are classified into glycerolipids, sphingolipids, and cholesterol, which can be further divided into a great number of subclasses depending on differences in the polar head groups. Such diversity is the most remarkable in sphingolipids 1, 2). Over a hundred sphingolipids (glycosphingolipids) exist that differ in terms of their sugar chains. In addition, lipid diversity is not restricted to the polar head groups, but also exists in fatty acid moieties. Considering all permutations of differences in chain-length, degree of unsaturation, and modification status of the fatty acid portions of lipids, there could be hundreds of thousands of lipids. This ultra-diversity of lipids has been created through evolution. Given that lipids originally functioned only as building blocks of membranes, relatively small varieties of lipids might have been sufficient, as in modern-day bacteria, which do not have sphingolipids or cholesterol. However, when eukaryotes diversified and became multicellular during the course of evolution, the need for an ultra-diversity of lipids might have arisen to fulfill organism-, tissue-, and cell-specific functions and to respond to environmental changes. For example, the stratum corneum of the epidermis contains over 1,000 ceramide species to form the permeability barrier3, 4) (Press release).

The research of our laboratory is especially focused on sphingolipids, which are multifunctional and highly diverse. Ceramide, the hydrophobic backbone of sphingolipids, is composed of a long-chain base and an amide-linked fatty acid. Sphingolipids are involved in a variety of physiological functions such as skin barrier formation, nerve action, glucose tolerance, recognition of bacterial toxins and viruses, immunity, blood vessel formation, and osteogenesis, and are related to several pathologies such as cutaneous disorders, metabolic syndrome, neuronal diseases, and cancer1, 2).

We have identified and analyzed many sphingolipid metabolic genes (Figure 2). Most notably, we have succeeded in determining the entire sphingolipid degradation pathway via sphingosine 1-phosphate (S1P) and in identifying the genes involved1, 6-9). S1P is well known as a lipid mediator that exists in plasma at concentrations of hundreds of nanomolar, and it plays pivotal roles in the vascular and immune systems10). Its function in the immune system has already been utilized in a clinical application, and the immunomodulator fingolimod (development code: FTY720) is used as a therapeutic agent for multiple sclerosis11). Besides its function as a lipid mediator, S1P is also important as a metabolic intermediate of the sphingolipid degradation pathway. This function is conserved in organisms ranging from yeasts to humans and has an older origin than its function as a lipid mediator. Sphingosine, the precursor of S1P, is the major long-chain base in mammals and exists ubiquitously. On the other hand, phytosphingosine, which contains a hydroxy group at the C4 position, exists in specific tissues such as the skin, small intestine, and kidney. We have revealed the phytosphingosine metabolic pathway, where phytosphingosine is converted to pentadecanoic acid, an odd-numbered fatty acid, via 2-hydroxypalmitic acid12, 13). In addition, we have identified the yeast MPO1 (2-hydroxy fatty acid dioxygenase) and mammalian HACL2 (2-hydroxyacyl-CoA lyase) as genes involved in the fatty acid α-oxidation12, 13). 4,14-Sphingadiene is a unique long-chain base characterized by a cis double bond at C14 position. We identified FADS3 as the gene responsible for introducing the C14 double bond14).

Sphingolipids are unique in that their constituent fatty acids are long. Most fatty acids in glycerolipids are long-chain fatty acids (C11–C20), whereas very long-chain fatty acids (VLCFAs; ≥C21), especially C22 and C24 VLCFAs, are rich in sphingolipids. VLCFAs are synthesized on the ER membrane via fatty acid elongation cycle15, 16). Our laboratory is currently investigating the molecular mechanism of VLCFA production.

The sphingolipid backbone ceramide exists ubiquitously in our bodies. However, the cellular levels of ceramide are not very high, since ceramide is typically merely an intermediate of sphingolipid biosynthesis. On the other hand, the epidermis contains exceptionally high levels of ceramides. Furthermore, epidermal ceramides are unique in their structural diversity and chain-length 1, 3, 4). For example, substantial amounts of epidermal ceramides contain ultra long-chain fatty acids (≥C26; ULCFAs). Acylceramides and protein-bound ceramides, which are esterified with linoleic acid or covalently bound to proteins, respectively, exist exclusively in the epidermis1). Ceramides, especially acylceramides and protein-bound ceramides, play essential functions in skin barrier formation, and impairment of their production leads to the skin disorder ichthyosis. On the other hand, changes in the levels, composition, and chain-length of ceramides result in atopic dermatitis. Although the molecular mechanism responsible for producing acylceramide remained unclear for a long time, we have identified the genes involved in acylceramide production, such as fatty acid elongase ELOVL1, fatty acid ω-hydroxylase CYP4F22, transacylase PNPLA1 (Press release), and acyl-CoA synthetase FATP417-20) (Press release). Additionally, we have also elucidated the detailed structure of protein-bound ceramides.

The tear film consists of a lipid layer and an aqueous layer (muco-aqueous layer) . The lipid layer acts as permeability barrier in tears and is important for prevention of dry eye. The constituent lipids of the lipid layer are called meibum lipids, since they are secreted from the meibomian glands on the back of the eyelids. The major components of meibum lipids are cholesteryl esters and wax esters, most of which have a very long-chain. On the other hand, there are also amphiphilic lipids called OAHFAs that play a role in connecting the lipid and aqueous layers. Our laboratory is investigating the molecular mechanisms of meibum lipid production and its role in preventing dry eye. Our laboratory revealed that the synthesis of very long-chain meibum lipids is essential for prevention of dry eye disease21) (Press release). FUrthermore, we have identified acyl-CoA wax alcohol transferases AWAT1 and AWAT2, fatty acid ω-hydroxylase CYP4F22 (Press release), and acyl-CoA reductase FAR2 involved in meibum lipid production, and demonstrated that knockout mice for these genes all exhibit dry eye phenotypes22-24).

References

  1. Kihara A (2016) Synthesis and degradation pathways, functions, and pathology of ceramides and epidermal acylceramides. Prog Lipid Res, 63, 50-69. Review.
  2. Kihara A (2015) Sphingolipid metabolism via sphingosine 1-phosphate and its role in physiology pathology, and nutrition. "Bioactive lipid mediators: current reviews and protocols (Yokomizo T, Murakami M eds.)", Springer Japan, Tokyo. pp 127-138. Review
  3. Kawana M, Miyamoto M, Ohno Y, Kihara A. (2020) Comparative profiling and comprehensive quantification of stratum corneum ceramides in humans and mice by LC-MS/MS. J Lipid Res, 61, 884-895.
  4. Suzuki M, Ohno Y, Kihara A. (2022) Whole picture of human stratum corneum ceramides, including the chain-length diversity of long-chain bases. J Lipid Res, 63, 100235.
    (Press release) (ASBMB (2022) vol.21, No. 10, p18)
  5. Kihara A (2014) Sphingosine 1-phosphate is a key metabolite linking sphingolipids to glycerophospholipids. Biochim Biophys Acta, 1841, 766-772. Review.
  6. Kihara A (2014) Sphingosine 1-phosphate is a key metabolite linking sphingolipids to glycerophospholipids. Biochim Biophys Acta, 1841, 766-772. Review.
  7. Nakahara K, Ohkuni A, Kitamura T, Abe K, Naganuma T, Ohno Y, Zoeller RA, Kihara A (2012) The Sjögren-Larsson syndrome gene encodes a hexadecenal dehydrogenase of the sphingosine 1-phosphate degradation pathway. Mol Cell, 46, 461-471.
  8. Ohkuni A, Ohno Y, Kihara A (2013) Identification of acyl-CoA synthetases involved in the mammalian sphingosine 1-phosphate metabolic pathway. Biochem Biophys Res Commun, 442, 195-201.
  9. Wakashima T, Abe K, Kihara A (2014) Dual functions of the trans-2-enoyl-CoA reductase TER in the sphingosine 1-phosphate metabolic pathway and in fatty acid elongation. J Biol Chem, 289, 24736-24748.
  10. Kihara A, Mitsutake S, Mizutani Y, Igarashi Y (2007) Metabolism and biological functions of two phosphorylated sphingolipids, sphingosine 1-phosphate and ceramide 1-phosphate. Prog Lipid Res, 46, 126-144. Review
  11. Kihara A, Igarashi Y (2008) Production and release of sphingosine 1-phosphate and the phosphorylated form of the immunomodulator FTY720. Biochim Biophys Acta, 1781, 496-502. Review
  12. Kondo N, Ohno Y, Yamagata M, Obara T, Seki N, Kitamura T, Naganuma T, Kihara A (2014) Identification of the phytosphingosine metabolic pathway leading to odd-numbered fatty acids. Nat Commun, 5, 5338.
  13. Kitamura T, Seki N, Kihara A (2017) Phytosphingosine degradation pathway includes fatty acid α-oxidation reactions in the endoplasmic reticulum. Proc Natl Acad Sci USA, 114, E2616-E2623.
  14. Jojima K, Edagawa M, Sawai M, Ohno Y, Kihara A. (2020) Biosynthesis of the anti-lipid-microdomain sphingoid base 4,14-sphingadiene by the ceramide desaturase FADS3. FASEB J, 34, 3318-3335.
  15. Sassa T, Kihara A (2014) Metabolism of very long-chain fatty acids: genes and pathophysiology. Biomol Ther, 22, 83-92. Review.
  16. Kihara A (2012) Very long-chain fatty acids: elongation, physiology and related disorders. J Biochem, 152, 387-395. Review
  17. Sassa T, Ohno Y, Suzuki S, Nomura T, Nishioka C, Kashiwagi T, Hirayama T, Akiyama M, Taguchi R, Shimizu H, Itohara S, Kihara A (2013) Impaired epidermal permeability barrier in mice lacking the Elovl1 gene responsible for very long-chain fatty acid production. Mol Cell Biol, 33, 2787-2796.
  18. Ohno Y, Nakamichi S, Ohkuni A, Kamiyama N, Naoe A, Tsujimura H, Yokose U, Sugiura K, Ishikawa J, Akiyama M, Kihara A (2015) Essential role of the cytochrome P450 CYP4F22 in the production of acylceramide, the key lipid for skin permeability barrier formation. Proc. Natl. Acad. Sci. USA, 112, 7707-7712.
  19. Ohno Y, Kamiyama N, Nakamichi S, Kihara A. (2017) PNPLA1 is a transacylase essential for the generation of the skin barrier lipid ω-O-acylceramide. Nat Commun, 8, 14610. (Press release)
  20. Yamamoto H, Hattori M, Chamulitrat W, Ohno Y, Kihara A. (2020) Skin permeability barrier formation by the ichthyosis-causative gene FATP4 through formation of the barrier lipid ω-O-acylceramide. Proc Natl Acad Sci USA, 117, 2914-2922. (Press release)
  21. Sassa T, Tadaki M, Kiyonari H, Kihara A (2018) Very long-chain tear film lipids produced by fatty acid elongase ELOVL1 prevent dry eye disease in mice. FASEB J,32, 2966-2978. (Press release)
  22. Miyamoto M, Sassa T, Sawai M, Kihara A. (2020) Lipid polarity gradient formed by ω-hydroxy lipids in tear film prevents dry eye disease. eLife, 9, e53582. (Press release)
  23. Sawai M, Watanabe K, Tanaka K, Kinoshita W, Otsuka K, Miyamoto M, Sassa T, Kihara A. (2021) Diverse meibum lipids produced by Awat1 and Awat2 are important for stabilizing tear film and protecting the ocular surface. iScience, 24, 102478.
  24. Otsuka K, Sawai M, Kihara A. (2022) Formation of fatty alcohols—components of meibum lipids—by the fatty acyl-CoA reductase FAR2 is essential for dry eye prevention. FASEB J, 36, e22216.