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. 2010 Nov;51(11):3185-95.
doi: 10.1194/jlr.M006445. Epub 2010 Aug 1.

Regulation of ABCG1 Expression in Human Keratinocytes and Murine Epidermis

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Free PMC article

Regulation of ABCG1 Expression in Human Keratinocytes and Murine Epidermis

Yan J Jiang et al. J Lipid Res. .
Free PMC article

Abstract

ABCG1, a member of the ATP binding cassette superfamily, facilitates the efflux of cholesterol from cells to HDL. In this study, we demonstrate that ABCG1 is expressed in cultured human keratinocytes and murine epidermis, and induced during keratinocyte differentiation, with increased levels in the outer epidermis. ABCG1 is regulated by liver X receptor (LXR) and peroxisome proliferator-activated receptor-δ (PPAR-δ) activators, cellular sterol levels, and acute barrier disruption. Both LXR and PPAR-δ activators markedly stimulate ABCG1 expression in a dose- and time-dependent fashion. PPAR-γ activators also increase ABCG1 expression, but to a lesser degree. In contrast, activators of PPAR-α, retinoic acid receptor, retinoid X receptor, and vitamin D receptor do not alter ABCG1 expression. In response to increased intracellular sterol levels, ABCG1 expression increases, whereas inhibition of cholesterol biosynthesis decreases ABCG1 expression. In vivo, ABCG1 is stimulated 3-6 h after acute barrier disruption by either tape stripping or acetone treatment, an increase that can be inhibited by occlusion, suggesting a potential role of ABCG1 in permeability barrier homeostasis. Although Abcg1-null mice display normal epidermal permeability barrier function and gross morphology, abnormal lamellar body (LB) contents and secretion leading to impaired lamellar bilayer formation could be demonstrated by electron microscopy, indicating a potential role of ABCG1 in normal LB formation and secretion.

Figures

Fig. 1.
Fig. 1.
ABCG1 mRNA expression increases during keratinocyte differentiation. Cultured human keratinocytes (CHKs) were cultured in medium containing either 0.03 or 1.2 mM Ca2+ for various periods of time (0, 2, 4, or 7 days). Real-time PCR was performed to measure mRNA levels of ABCG1, involucrin (INV), and cyclophilin (housekeeping gene). Data are expressed as percentage of 0 day control (100%) and presented as mean ± SEM (n = 4). ** P < 0.01; *** P < 0.001 compared with 0 time point.
Fig. 2.
Fig. 2.
ABCG1 mRNA expression is regulated by cellular sterol levels. CHKs were incubated with or without 100 µg/ml LDL in 0.03 mM Ca2+ medium for 24 h (A). Alternatively, cells were incubated with either cholesterol sulfate (CS, 20 µM), or lovastatin (10 µM), or vehicle (DMSO) for 24 h (B). ABCG1 and cyclophilin mRNA levels were determined as described. Data are expressed as percentage of control (100%) and presented as mean ± SEM (n = 4). ** P < 0.01 compared with control.
Fig. 3.
Fig. 3.
Activation of liver X receptor (LXR) and peroxisome proliferator-activated receptor-δ (PPAR-δ) increases ABCG1 mRNA expression. CHKs were incubated in 0.03 mM Ca2+ medium in the presence or absence of various activators of PPAR-α, PPAR-δ, PPAR-γ, RAR, RXR, VDR, or vehicle (ethanol) for 24 h. Note that 9-cis-RA can activate both RXR and RAR. Real-time PCR was performed to measure mRNA levels of ABCG1 and cyclophilin as described. Data are expressed as percentage of control (100%) and presented as mean ± SEM (n = 5). * P < 0.05; ** P < 0.01; *** P < 0.001 compared with control. ATRA, all-trans retinoic acid; TO, TO901317; Clo, clofibrate; WY, WY14643; GW, GW 610742X; Cig, ciglitazone; GI, GI251929. VD3, 1α,25-dihydroxyvitamin D3.
Fig. 4.
Fig. 4.
The LXR activator 22R increases ABCG1 mRNA expression in a dose- and time-dependent manner. CHKs were incubated with 22R at various doses (0, 2.5, 5.0, and 10 µM) or vehicle (ethanol) in 0.03 mM Ca2+ medium for 24 h (A). Alternatively, cells were cultured in the presence of 10 µM 22R or vehicle (ethanol) in the same medium for various periods of time (0, 3, 6, 16, and 24 h) (B). Real-time PCR was performed to measure mRNA levels of ABCG1 and cyclophilin. Data are expressed as percentage of control (100%) and presented as mean ± SEM (n = 4). For the time course study, data are presented as percentage of control (in the absence of 22R) for each matched time point. Similar results were obtained when the experiment was repeated with a different batch of cells. * P < 0.05; ** P < 0.01; *** P < 0.001 compared with control.
Fig. 5.
Fig. 5.
LXR and PPAR-δ activators increase ABCG1 protein expression. CHKs were incubated with either vehicle (ethanol), 22R (10 µM) (A), or GW 610742X (8 µM) (B) in 0.03 mM Ca2+ medium for the indicated periods of time. The whole-cell extract was prepared and subjected to Western blot analysis to determine ABCG1 or GAPDH protein levels as described. Data are expressed as percentage of vehicle control densitometry (100%) and presented as mean ± SEM (n = 4–5). Representative blots are shown. * P < 0.05; *** P < 0.001 compared with control.
Fig. 6.
Fig. 6.
LXR activation increases ABCG1 mRNA and protein levels in mouse epidermis. Hairless mice were topically treated with either vehicle (ethanol) or the LXR activator TO901317 (15 mM) for 3 days, and the epidermis was isolated for measuring ABCG1 and 36B4 (housekeeping gene) mRNAs by real-time PCR (A), or ABCG1 protein levels by Western blot (B). Data are expressed as percentage of vehicle control densitometry (100%) and presented as mean ± SEM (n = 6). A representative blot is shown (B). *** P < 0.001 compared with control.
Fig. 7.
Fig. 7.
Permeability barrier disruption by tape stripping (TS) increases ABCG1 mRNA expression in mouse epidermis. At 0, 1, 3, 6, or 24 h following acute barrier disruption by TS, hairless mouse epidermis was collected and subjected to analysis for ABCG1 and 36B4 mRNAs by real-time PCR. Data are expressed as percentage of nontreatment control (100%) and are presented as mean ± SEM (n = 6–7). The representative graph is shown from two different sets of animals with similar results. * P < 0.05 compared with nontreatment control.
Fig. 8.
Fig. 8.
Permeability barrier disruption by acetone treatment increases ABCG1 mRNA expression in mouse. Following acute barrier disruption by acetone treatment, epidermis was collected at 0, 1, 3, 6, and 24 h and subjected to analysis for ABCG1 and 36B4 mRNAs by real-time PCR. Data are expressed as percentage of control (100%) and presented as mean ± SEM (n = 5). The representative graph is shown from two different sets of animals with similar results. * P < 0.05 compared with control.
Fig. 9
Fig. 9
Abcg1 knockout (KO) mouse epidermis displays abnormalities in the lamellar body (LB) secretory system with impaired lamellar bilayer formation. Skin samples were collected from both Abcg1 KO and wild-type (WT) mice, postfixed with either reduced osmium-tetroxide or ruthenium-tetroxide, and processed for electron microscopy as described in MATERIALS AND METHODS. At the interface of the stratum granulosum (SG) and stratum corneum (SC), abundant, normal-looking LBs are present in WT mouse epidermis (A, insert), and secreted LB contents are processed into lamellar bilayers (B, arrows). In Abcg1 KO mice, however, a large number of LBs are empty (C, arrows), with disrupted lamellar bilayer formation (D, arrows).

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