High Mobility Group Protein B1 and its downstream elements, syntaxins, contribute to temporal tight junction assembly...

While the epidermis is a stratified epithelium undergoing continuous turnover, tight junctions (TJs), which are critical barrier structures, form transiently and exclusively within specific cells of the upper stratum. The cytoplasmic-to-membrane translocation of ZO-1, a scaffold protein of TJs, accompanies the assembly of TJs. Previously, we demonstrated that a secreted subset of the nuclear protein High Mobility Group Protein B1 (HMGB1) and the type IV membrane protein epimorphin/syntaxin2 (Stx2) impede, whereas the Stx2 paralogue syntaxin3 (Stx3) promotes, the membrane translocation of ZO-1 in HaCaT keratinocytes. In this study, we observed that HMGB1-knockout (HMGB1-KO) increases membrane-localized ZO-1 in only a restricted subset of cells, accompanied by downregulation of both Stx2 and Stx3. Inducible overexpression of exogenously introduced Stx3 significantly accelerates the membrane localization of ZO-1 in most HMGB1-KO cells, accompanied by upregulation of the PRSS3 gene product mesotrypsin, another supportive element for TJ formation, indicating that nuclear HMGB1 abundance regulates TJ assembly, at least partially, through the downregulation of these syntaxins independent of its extracellular secretion. Given that HMGB1, Stx2, Stx3, and mesotrypsin are all known to be transiently extruded into the extracellular space, these observations elucidate a regulatory mechanism underlying the spatiotemporal formation of TJs by these pleiotropic proteins and provide valuable insights into potential therapeutic strategies for inflammatory skin conditions characterized by compromised barrier function.