Journal: bioRxiv

Article Title: The nucleocytoplasmic translocation of HINT1 regulates the maturation of cell density

doi: 10.1101/2025.01.13.632869

Figure Lengend Snippet: Translocation of HINT1 to the cytoplasm inhibits PKC activity (A) MARCKS phosphorylation is dependent on cell density in HEK293A, hsSKM, and MEF cells. Cells were cultured overnight at low and high densities, and the phosphorylation levels of MARCKS were assessed by western blotting using a phospho-MARCKS (Ser167/170) polyclonal antibody. Total MARCKS and actin levels were also measured via western blotting as internal controls. (B) Cytoplasmic forced-expression of HINT1 reduces MARCKS phosphorylation. HEK293A cells were seeded at low density, transfected with either WT HINT1-HA or mutant R24A/K25A HINT1-HA, incubated for 24 hours, and subsequently lysed in SDS sample buffer. Phospho-MARCKS, total MARCKS, and actin levels were analyzed via western blotting. Quantitative analysis of MARCKS phosphorylation levels in HEK293 cells, with or without transfection of WT HINT1-HA or mutant R24A/K25A HINT1-HA. The data was normalized using the band intensity from non-transfected cells. n = 3. Statistical analysis was conducted using paired t-test. * P < 0.05. (C) A proposed model illustrates how HINT1 translocation is regulated by cell density and how this process contributes to the disruption of actin SFs, leading to a rounded, confined cell morphology. At low density, HINT1 predominantly localizes in the nucleus, where it binds to chromatin to regulate gene expression, thereby promoting cell proliferation and development. Cell-cell contact alone is insufficient to induce HINT1 translocation to the cytoplasm. At confluency, cells initiate the export of HINT1 to the cytoplasm, a process mediated by exportin-1. As cell density increases, more HINT1 is actively transported to the cytoplasm. In the cytoplasm, HINT1 inhibits PKC, which influences actin remodeling through various signaling pathways, resulting in a rounded and compact cell morphology. This translocation also suppresses gene expression, leading to a halt in proliferation and promoting CIP. The subcellular localization of HINT1 appears to be cell type-dependent, which may explain why some cells form SFs while others do not in situ .

Article Snippet: Rabbit polyclonal anti- MARCKS (10004-2-Ig) and phospho (Ser167/170)-MARCKS (29145-1-AP) were purchased from Proteintech.

Techniques: Translocation Assay, Activity Assay, Cell Culture, Western Blot, Expressing, Transfection, Mutagenesis, Incubation, Disruption, In Situ