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 that ZO-1 induced in the presence of 27OHChol is mainly localized on the cell surface. Collectively, these findings suggest that ZO-1 may regulate proliferation by sequestering the proteins involved in proliferation in the cytoplasm, in the presence of 27OHChol.
ZO proteins are multi-domain scaffolds that bind directly to various types of proteins, in addition to having scaffolding functions in organizing gap junction complexes, via the PDZ (PSD-95/Discs-large/ZO-1) domain, SH3 domain, and guanylate kinase domain (14). ZO-1 can bind with protein kinases in the cytoplasm. After binding with the serine/threonine-protein kinase MRCKβ, the ZO-1/MRCKβ complex regulates migration at the leading edge of migrating cells (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 (20–22). Therefore, the results of this study suggest the possibility that the upregulated ZO-1 protein may be involved in cell migration, via interactions with protein kinases during inflammation or immune responses.
In summary, we demonstrated that 27OHChol upregulates ZO-1 using ER-to-Golgi body transport system via PI3K, ERK, and src in monocytic cells, which is impaired by the drugs suppressing DC differentiation. These results also cause us to question the biological functions of the upregulated protein. Further investigations are needed to understand whether ZO-1 plays roles in migration and the regulation of cell proliferation during the process of DC differentiation.