The nucleocytoplasmic translocation of HINT1 regulates the maturation of cell density

Cell contact inhibition progresses through three stages: (i) increasing cell density leads to reduced movement while mitosis continues; (ii) a rapid shift to epithelial morphology; and (iii) ongoing division with decreased cell size. This transition involves reorganizing the actin cytoskeleton from stress fibers to a cortical network, stabilizing cell shape, and strengthening cell-cell connections. However, the signaling pathways regulating the final stage remain unclear. We identified histidine triad nucleotide-binding protein 1 (HINT1), also known as protein kinase C inhibitor 1 (PKCI-1), as essential for monolayer maturation. At low density, HINT1 is located in the nucleus, binding to open chromatin. As density increases, exportin-1 relocates HINT1 to the cytoplasm, where it inhibits PKC and remodels the actin cytoskeleton. While monolayer formation can occur without HINT1, its presence is necessary for fully confining cells and achieving a mature monolayer. Additionally, MARCKS phosphorylation decreases in high-density cells, and loss of HINT1 leads to increased cell area, similar to hypertrophic conditions in patients with low HINT1 levels.