The nucleocytoplasmic translocation of HINT1 regulates the maturation of cell density

In normal epithelial cells on tissue culture dishes, contact inhibition typically progresses with a reduction in cell size after cell–cell contact. This transition involves actin cytoskeleton reorganization from stress fibers (SFs) to a cortical network, stabilizing cell shape and strengthening connections. However, the regulatory signaling pathways remain unclear. We identified histidine triad nucleotide-binding protein 1 (HINT1), also known as protein kinase C inhibitor 1 (PKCI-1), as a regulator for monolayer maturation. At low density, HINT1 localizes in the nucleus and binds to open chromatin. As density increases, exportin 1 drives HINT1 translocation to the cytoplasm. Forced cytoplasmic localization of HINT1 reduces phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) at Ser167/170, sites specifically targeted by PKC and involved in regulating SF formation. MARCKS phosphorylation also decreases naturally at high density. Although cells can form a monolayer without HINT1, its presence is required for full cell confinement and mature monolayer formation. Thus, HINT1 plays a dual role: acting as a nuclear transcriptional coregulator at low density and acting as a cytoplasmic SF modulator at high density.