27-Hydroxycholesterol induces expression of zonula occludens-1 in monocytic cells via multiple kinases pathways

Zonula occludens (ZO)-1, a tight-junction protein (TJP), is expressed in dendritic cells (DCs) but not in monocytes, and 27-hydroxycholesterol (27OHChol) drives the differentiation of monocytes into DCs. Because the effects of 27OHChol on ZO-1 are not yet clearly defined, we investigated whether 27OHChol induces expression of the TJP. The treatment of human THP-1 monocytic cells with 27OHChol resulted in the elevated transcript levels of ZO-1 but not of ZO-2 or -3. 27OHChol increased the total amount of ZO-1 protein in the cells as well as its level on the cells surface. Cholesterol, however, did not influence expression of ZO-1. And, the expression of ZO-1 protein was mediated by endoplasmic reticulum (ER)-to-Golgi body transport system. Pharmacological kinase inhibition with LY294002 (a PI3K inhibitor), U0126 (a MEK/ERK inhibitor), or PP2 (a Src family kinase inhibitor) resulted in impaired ZO-1 expression at both transcript and protein levels. Drugs that are reported to suppress DC differentiation also inhibited 27OHChol-mediated expression and the localization of ZO-1, indicating the coincidence of ZO-1 upregulation and DC differentiation. These results suggest that ZO-1 is differentially expressed while monocytes differentiate into DCs in the presence of 27OHChol via pathways in which distinct signaling molecules are involved.


Effects of 27OHChol on ZO-1 expression in monocytic cells. To examine whether 27OHChol
affected the ZO-1 levels in monocytes, we analyzed the expression of ZO family transcripts using RT-PCR and quantitative real-time PCR in THP-1 cells. Among the ZO-1 family, 27OHChol elevated the transcript levels of ZO-1, but not of ZO-2 or -3, and cholesterol did not influence any ZO-1 family transcript levels ( Fig. 1A and Supplementary data 1). In agreement with the results of the RT-PCR and real-time PCR, levels of ZO-1 protein increased with 27OHChol treatment, but not with cholesterol (Fig. 1B). The percentage of cells expressing ZO-1 on the surface increased from 2.7 to 18.7% following treatment with 27OHChol. Cholesterol, however, did not alter the ZO-1 immunoreactivity of the cells (Fig. 1C). We also determined the localization of ZO-1. The immunoreactivity of ZO-1 protein was localized on the cell surface, as visualized via confocal microscopy (Supplementary data 3A). These results suggest that ZO-1 protein is upregulated in the presence of 27OHChol.
We investigated the effects of 27OHChol concentrations on ZO-1 levels. Expression of ZO-1 increased in a dose-dependent manner upon treatment with 27OHChol. 27OHChol, up to a concentration of 2.5 µg/ml, elevated the transcript ( Fig. 2A), protein (Fig. 2B), and immunoreactivity levels of ZO-1 (Fig. 2C). We also determined the time-course effects of 27OHChol. The ZO-1 transcripts and protein levels and ZO-1 immunoreactivity

Involvement of endoplasmic reticulum (ER)-Golgi-transport in ZO-1 expression.
We investigated roles of the ER-to-Golgi transport system in the expression of ZO-1 by using BFA. The level of ZO-1 protein on monocytic cells, which was induced by 27OHChol, was downregulated by the treatment with BFA (Fig. 4B). BFA, however, did not affect the transcript levels of ZO-1 (Fig. 4A). The immunoreactivity of ZO-1 (green) detected on cell surface after stimulation with 27OHChol was reduced by BFA (Supplementary data 3B). These results indicated that ZO-1 protein is transported onto cell surface of monocytes via by the ER-to-Golgi transport system.

Effects of drugs suppressing DC differentiation on ZO-1 expression. DC differentiation was sup-
pressed by drugs such as cyclosporine A (CsA), diclofenac (Df), and dexamethasone (Dx) 8,12,13 . Therefore, we investigated the correlation between ZO-1 and DC differentiation by assessing the effects of these drugs on ZO-1 expression. The levels of ZO-1 transcripts, which were elevated following stimulation with 27OHChol, decreased in the presence of Dx (Fig. 6A). In agreement with the results of the RT-PCR and real-time PCR, Dx also suppressed the expression of ZO-1 protein (Fig. 6B). The expressed ZO-1 protein was confirmed using flow cytometry (Fig. 6C). The percentage of ZO-1 expression on the cell surface increased from 2.8 to 21.9% following treatment with 27OHChol, and this was reduced to 1.4%, 1.0%, and 1.0% by 0.01, 0.1, 1 μM of Dx, respectively. When visualized, ZO-1 protein (green fluorescence) was localized on the surface after stimulation with 27OHChol, but the fluorescence was barely detected in the presence of Dx (Supplementary data 3D). CsA and Df also affected the 27OHCHol-induced expression of ZO-1 at transcript and protein levels (Supplementary data 4). The three drugs did not cause cytotoxicity at the concentrations that impaired expression of ZO-1 (Supplementary data 2B). These results suggest that expression of ZO-1 coincides with DC differentiation in a milieu in which 27OHChol is abundant.

Discussion
In this study, we demonstrated the increased expression of ZO-1 alone in monocytic cells following exposure to 27OHChol, indicating an induction of ZO-1 in response to an extracellular stimulus of oxidized cholesterol. ZO proteins, comprising ZO-1, ZO-2, and ZO-3, are ubiquitous scaffolding proteins. They are co-localized at junctional sites and can bind with themselves via the PDZ2 domain 14 . They display overlapping, but distinctive, expression patterns. ZO-1 is expressed at cell junctions of cardiac myocytes, but ZO-2 is not expressed in the heart 15 . ZO-2 and ZO-3 are concentrated in epithelial and endothelial tight junctions 14,16 . Although they share functional and structural similarities, ZO proteins display differential regulation and functions under hypercholesterolemic circumstances.
Besides the specific association of ZO-1 with tight junctions, it is involved in the regulation of the cell cycle. This protein has been detected in the nucleus during the proliferation of epithelial cells 17,18 . ZO-1 binds with ZO-1-associated nucleic acid-binding protein (ZONAB), promoting cell proliferation. ZO-1 functions as a suppressor of ZONAB and controls the accumulation of ZONAB in the nucleus by cytoplasmic sequestration; the overexpression of ZO-1 results in reduced nuclear ZONAB accumulation and proliferation 16,18 . We demonstrated   19 . Monocytic cells exposed to 27OHChol differentiate into DCs, which can migrate to inflammatory regions where they accelerate immune responses 7 . We demonstrated that CsA, Df, and Dx impair the ZO-1 expression induced by 27OHChol, and these drugs are well known for their anti-inflammatory and immunosuppressive effects 8,12,13 . Furthermore, the PI3K, ERK, and src kinases play crucial roles in migration [20][21][22] . Therefore, the results of this study suggest the possibility that the upregulated ZO-1 protein may be involved in www.nature.com/scientificreports/ cell migration, via interactions with protein kinases during inflammation or immune responses. Additionally, a study showed that the ZO-1 expression was affected through redox signaling pathway 23 . And, a recent study showed that oxysterol induced biological effects via redox signaling pathway 24 . These studies suggest a relationship of the ZO-1 expression induced by 27OHChol and NOX signals in the monocytic cells. However, the relationship does not well studied yet, and we need further studies about the signaling involvement. Oxysterols exist as lipid-mixture in blood and tissues. A study reviewed that the mixed oxysterols played as immune-modulator in intestinal immunity 25 . To know synergic effects of the oxysterol-mixture, we examined with normal cholesterol and various oxysterols oxygenated by enzymatically (24sOHChol and 27OHChol) and non-enzymatically (7αOHChol, 7βOHChol and 7-ketocholesterol (7 K)). The ZO-1 expression of monocytic cells was only induced by the treatment of 27OHChol and 7αOHChol, and the other oxysterols were not affected. The ZO-1 expression by 7αOHChol was reported 11 . Co-treatment of the other oxysterols with 27OHChol was not showed negatively or positively synergic effects. Co-treatment of 7 K and 27OHChol was showed a toxic effect. These studies indicate that not all oxysterols influence to the TJP expression on monocytic cells.

Materials and methods
Western blot analysis. Cells were lysed with lysis buffer (INTRON Biotechnology, Daejeon, Korea) containing a protease inhibitor cocktail (Sigma-Aldrich). Proteins in the lysates were separated by SDS-PAGE and transferred onto PVDF membranes. The membrane was separated for binding of ZO-1 (about 220 kDa) and β-actin (about 45 kDa) antibodies. After incubating for 1 h with 5% skim milk in 0.1% Tween 20/TBS to block the non-specific binding of primary antibodies, the separated membranes placed in each tray were probed with specific primary antibodies at 4 °C overnight. After three washes with the wash buffer (0.1% Tween 20/TBS) for 15 min each, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Membranes were then washed a further three times using the wash buffer for 15 min each, and bands were detected using the enhanced chemiluminescence (ECL) Western blotting detection system (Thermo Scientific, IL, USA). Chemiluminescence images were captured using an Amersham Imager 600 (GE Healthcare Life Sciences, Pittsburgh, PA, USA).
Immunofluorescence. THP-1 cells were seeded on cover slips (coated with 0.2% gelatin/PBS for 1 h) and stimulated for 48 h with cholesterol or 27OHChol. The cells were fixed for 20 min with 1% paraformaldehyde and incubated for 1 h with a blocking solution of 5% skim milk in PBS. After incubating for 2 h with the primary antibody against ZO-1 diluted in the blocking solution (1:100) at room temperature, the cells were washed twice for 5 min each using PBS. Following incubation for 1 h with secondary antibodies diluted in PBS (1:200) at room temperature in the dark, the cells were washed using PBS. The cover slips were mounted, and the cells were visualized using a confocal microscope (FV1000; Olympus Cor., Tokyo, Japan). www.nature.com/scientificreports/ Flow cytometric analysis. After harvesting by centrifugation at 200×g for 5 min at room temperature, both treated and untreated THP-1 cells were incubated for 2 h with anti-ZO-1 antibodies diluted to 1:100 in FACS buffer (2 mM EDTA and 0.2% BSA in PBS). The cells were washed with cold PBS and incubated for 1 h with fluorescent dye-conjugated secondary antibodies diluted to 1:200 in FACS buffer at 4 °C. After washing with cold PBS, the cells were resuspended in 1% paraformaldehyde in PBS. Subsequently, the cells were analyzed using a flow cytometer (FACS CANTO II; BD Company, NJ, USA).

Statistical analysis.
Statistical analyses were performed using one-way ANOVA, followed by Tukey's multiple comparison tests, using GraphPad Prism (version 5.0; GraphPad software Inc., CA, USA).

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.