Transcriptional Profiling after Lipid Raft Disruption in Keratinocytes Identifies Critical Mediators of Atopic Dermatitis Pathways

Abstract

Lipid rafts are cholesterol-rich cell signaling platforms, and their physiological role can be explored by cholesterol depletion. To characterize transcriptional changes ongoing after lipid raft disruption in epidermal keratinocytes, a cell type that synthesizes its cholesterol in situ, we performed whole-genome expression profiling. Microarray results show that over 3,000 genes are differentially regulated. In particular, IL-8, urokinase-like plasminogen activator receptor, and metalloproteinases are highly upregulated after cholesterol extraction. Quantitative reverse transcriptase PCR validation and protein release measurements demonstrate the physiological relevance of microarray data. Major enriched terms and functions, determined by Ingenuity Pathways Analysis, identify cholesterol biosynthesis as a major function, illustrating the specificity of keratinocyte response toward cholesterol depletion. Moreover, the inflammatory skin disorder atopic dermatitis (AD) is identified as the disease most closely associated with the profile of lipid raft-disrupted keratinocytes. This finding is confirmed in skin of AD patients, in whom transcript levels of major lipid raft target genes are similarly regulated in lesional atopic skin, compared with non-lesional and normal skin. Thus, lipid raft disruption evokes typical features of AD, thereby suggesting that lipid raft organization and signaling could be perturbed in atopic keratinocytes.

Unraveling Sterol-dependent Membrane Phenotypes by Analysis of Protein Abundance-ratio Distributions in Different Membrane Fractions Under Biochemical and Endogenous Sterol Depletion

“…Therefore m␤cd is suitable for detecting false positive cholesterol protein interactions in DRM studies (14 -19). Proteins depleted on m␤cd treatment are finally considered to be sterol dependent (15)(16)(17). To compare the m␤cd treatment for disturbing the sterol distribution in the L o fraction, we studied the sterol biosynthesis deficient mutant smt1.…”

Filaggrin-2

“…Filaggrin -2 expression is even more downregulated than that of pro -fi laggrin in lipid raftdisrupted keratinocytes, a situation that is described as resemblance of AD concerning its transcriptional profi le [ 68 ]. At the protein level, a downregulation has been observed in nonlesional and even more in lesional skin samples of AD patients [ 64 ].…”

Effect of Cholesterol Depletion On Interleukin-8 Production in Human Respiratory Epithelial Cells

“…17 What is interesting is that patients with genetic differences tend to be susceptible to both this condition and other related allergic diseases. 24,25 Such large-scale genome profiling and genetic experiments have confirmed that the disruption of lipid rafts can lead to inflammatory disease under certain circumstances.…”

“…Atopic dermatitis has even been shown to occur in parallel with keratinocytes disrupted by lipid rafts. 17 We hypothesize that inflammatory diseases of the airway are influenced by airway epithelial cells with disrupted lipid rafts, similar to keratinocytes in atopic dermatitis.…”

“…14 It is therefore presumed to change the structure and function of the cell membrane by disrupting lipid rafts. [15][16][17] A stock solution of 10% MβCD in phosphate-buffered saline (PBS) was stored at 4°C. This solution was used at concentrations of 0.5, 1, and 2% (v/v).…”