Madison K C, Sando G N, Howard E J, True C A, Gilbert D, Swartzendruber D C, Wertz P W
Department of Dermatology, University of Iowa College of Medicine, Iowa City, USA.
J Investig Dermatol Symp Proc. 1998 Aug;3(2):80-6. doi: 10.1038/jidsymp.1998.19.
Although lamellar granules are critical to the formation of the epidermal permeability barrier and are a known marker of late keratinocyte differentiation, very little is known about the physiologic regulators of lamellar granule assembly and extrusion. Ceramide glucosyltransferase (CGT), the enzyme responsible for the synthesis of lamellar granule glucosylceramides (GlcCer; the precursors of the stratum corneum ceramides), is localized to the Golgi apparatus in other cell types. We have found that CGT is induced during keratinocyte culture differentiation coincident with increased GlcCer content and the appearance of lamellar granules. In this study we show that the differentiation-related CGT induction is likely mediated at the transcriptional level. In addition, all-trans retinoic acid, a well-known inhibitor of keratinocyte differentiation, prevents the appearance of lamellar granules and decreases culture CGT activity and GlcCer content without affecting sphingomyelin or total lipid content, indicating a specific inhibition of this enzymatic pathway. These data show a direct relationship between CGT activity and epidermal differentiation, suggesting that regulation of CGT expression is a critical part of epidermal barrier generation. The differentiation dependence of CGT activity, the key role of this Golgi-localized enzyme in epidermal GlcCer synthesis, and our previous finding that ceramides are converted to GlcCer in the Golgi apparatus in keratinocyte cultures, strongly suggest a Golgi origin for lamellar granules. In contrast to CGT, the activity of the lysosomal enzymes acid lipase and glucocerebrosidase is less clearly related to epidermal differentiation and the appearance of lamellar granules, although both enzymes show striking colocalization and enrichment in a subcellular lamellar granule fraction derived from pig epidermis. Acid lipase activity in the lamellar granule fraction was found to contain primarily a small lysosomal form of the enzyme, whereas total acid lipase secreted by keratinocyte cultures was found to contain a mannose-6-phosphorylated large prelysosomal form as well as a small lysosomal form. That secreted acid lipase activity is derived from both prelysosomal and lysosomal compartments suggests there may be multiple pathways by which lysosomal enzymes are secreted from keratinocytes. The combined secretion of lipid and lysosomal enzymes from lamellar granules places these organelles in the category of "dual-function" specialized secretory vesicles described in certain other cell types. Electron microscopic images of lamellar granules show shapes consistent with cross-sections of tubules or buds from tubules in addition to vesicles. These images provide evidence for the involvement of trans-Golgi network tubules and/or buds in lamellar granule synthesis and secretion.
尽管板层颗粒对于表皮通透屏障的形成至关重要，并且是晚期角质形成细胞分化的已知标志物，但关于板层颗粒组装和分泌的生理调节因子却知之甚少。神经酰胺葡萄糖基转移酶（CGT）是负责合成板层颗粒葡糖神经酰胺（GlcCer；角质层神经酰胺的前体）的酶，在其他细胞类型中定位于高尔基体。我们发现，在角质形成细胞培养分化过程中CGT被诱导，同时GlcCer含量增加且出现板层颗粒。在本研究中，我们表明与分化相关的CGT诱导可能在转录水平介导。此外，全反式维甲酸是一种众所周知的角质形成细胞分化抑制剂，可阻止板层颗粒的出现，并降低培养物中CGT活性和GlcCer含量，而不影响鞘磷脂或总脂质含量，表明对该酶促途径有特异性抑制作用。这些数据表明CGT活性与表皮分化之间存在直接关系，提示CGT表达的调节是表皮屏障形成的关键部分。CGT活性对分化的依赖性、这种高尔基体定位酶在表皮GlcCer合成中的关键作用，以及我们之前的发现即角质形成细胞培养物中神经酰胺在高尔基体中转化为GlcCer，强烈提示板层颗粒起源于高尔基体。与CGT相反，溶酶体酶酸性脂肪酶和葡糖脑苷脂酶的活性与表皮分化和板层颗粒出现的关系不太明确，尽管这两种酶在源自猪表皮的亚细胞板层颗粒部分中显示出明显的共定位和富集。发现板层颗粒部分中的酸性脂肪酶活性主要包含该酶的一种小的溶酶体形式，而角质形成细胞培养物分泌的总酸性脂肪酶被发现包含一种甘露糖-6-磷酸化的大的前溶酶体形式以及一种小的溶酶体形式。分泌的酸性脂肪酶活性源自前溶酶体和溶酶体区室，这表明角质形成细胞分泌溶酶体酶可能存在多种途径。板层颗粒中脂质和溶酶体酶的联合分泌使这些细胞器属于某些其他细胞类型中描述的“双功能”特殊分泌囊泡类别。板层颗粒的电子显微镜图像显示，除了囊泡外，其形状与小管或小管芽的横截面一致。这些图像为反式高尔基体网络小管和/或芽参与板层颗粒合成和分泌提供了证据。
