Uchida Yoshikazu, Hama Hiroko, Alderson Nathan L, Douangpanya Sounthala, Wang Yu, Crumrine Debra A, Elias Peter M, Holleran Walter M
Department of Dermatology, School of Medicine, University of California, San Francisco, CA, USA.
J Biol Chem. 2007 May 4;282(18):13211-9. doi: 10.1074/jbc.M611562200. Epub 2007 Mar 12.
Ceramides in mammalian stratum corneum comprise a heterogeneous mixture of molecular species that subserve the epidermal permeability barrier, an essential function for survival in a terrestrial environment. In addition to a variation of sphingol species, hydroxylation of the amide-linked fatty acids contributes to the diversity of epidermal ceramides. Fatty acid 2-hydroxylase, encoded by the gene FA2H, the mammalian homologue of FAH1 in yeast, catalyzes the synthesis of 2-hydroxy fatty acid-containing sphingolipids. We assessed here whether FA2H accounts for 2-hydroxyceramide/2-hydroxyglucosylceramide synthesis in epidermis. Reverse transcription-PCR and Western immunoblots demonstrated that FA2H is expressed in cultured human keratinocytes and human epidermis, with FA2H expression and fatty acid 2-hydroxylase activity increased with differentiation. FA2H-siRNA suppressed 2-hydroxylase activity and decreased 2-hydroxyceramide/2-hydroxyglucosylceramide levels, demonstrating that FA2H accounts for synthesis of these sphingolipids in keratinocytes. Whereas FA2H expression and 2-hydroxy free fatty acid production increased early in keratinocyte differentiation, production of 2-hydroxyceramides/2-hydroxyglucosylceramides with longer chain amide-linked fatty acids (> or =C24) increased later. Keratinocytes transduced with FA2H-siRNA contained abnormal epidermal lamellar bodies and did not form the normal extracellular lamellar membranes required for the epidermal permeability barrier. These results reveal that 1) differentiation-dependent up-regulation of ceramide synthesis and fatty acid elongation is accompanied by up-regulation of FA2H; 2) 2-hydroxylation of fatty acid by FA2H occurs prior to generation of ceramides/glucosylceramides; and 3) 2-hydroxyceramides/2-hydroxyglucosylceramides are required for epidermal lamellar membrane formation. Thus, late differentiation-linked increases in FA2H expression are essential for epidermal permeability barrier homeostasis.
哺乳动物角质层中的神经酰胺由多种分子种类组成，它们构成了表皮通透屏障，这是在陆地环境中生存的一项基本功能。除了鞘氨醇种类的变化外，酰胺连接的脂肪酸的羟基化也导致了表皮神经酰胺的多样性。由基因FA2H编码的脂肪酸2 -羟化酶是酵母中FAH1的哺乳动物同源物，它催化含2 -羟基脂肪酸的鞘脂的合成。我们在此评估FA2H是否参与表皮中2 -羟基神经酰胺/2 -羟基葡萄糖神经酰胺的合成。逆转录 - PCR和蛋白质免疫印迹表明，FA2H在培养的人角质形成细胞和人表皮中表达，且随着分化，FA2H的表达和脂肪酸2 -羟化酶活性增加。FA2H - siRNA抑制了2 -羟化酶活性并降低了2 -羟基神经酰胺/2 -羟基葡萄糖神经酰胺水平，表明FA2H参与角质形成细胞中这些鞘脂的合成。虽然FA2H的表达和2 -羟基游离脂肪酸的产生在角质形成细胞分化早期增加，但具有较长链酰胺连接脂肪酸（≥C24）的2 -羟基神经酰胺/2 -羟基葡萄糖神经酰胺的产生在后期增加。用FA2H - siRNA转导的角质形成细胞含有异常的表皮板层小体，且未形成表皮通透屏障所需的正常细胞外板层膜。这些结果表明：1）神经酰胺合成和脂肪酸延长的分化依赖性上调伴随着FA2H的上调；2）FA2H介导的脂肪酸2 -羟基化发生在神经酰胺/葡萄糖神经酰胺生成之前；3）2 -羟基神经酰胺/2 -羟基葡萄糖神经酰胺是表皮板层膜形成所必需的。因此，FA2H表达在分化后期的增加对于表皮通透屏障的稳态至关重要。
