However, cytochalasin D did not affect cell motility following the BP IgG treatment

However, cytochalasin D did not affect cell motility following the BP IgG treatment. specific split at the lamina lucida and induction of BMZ blistering (37). According to another report, ColXVII mediates the anchorage of basal keratinocytes by regulating cell motility (68). Thus, we speculate that this changes in the adhesion and motility of keratinocytes are involved in the pathogenesis of blistering in patients with BP. As shown in reports (69, 70), IgGs targeting proteins other than ColXVII-NC16a do not detach cells from culture dishes. Interestingly, an IgG targeting the C-terminus of ColXVII neither induced obvious IgG-ColXVII internalization nor had any significant effect on cell detachment. Together with the results of the study showing that IgGs targeting the ColXVII ectodomain fail to reproduce blistering in an animal model (71), the findings from previous studies and our data confirm the pathogenicity of the anti-ColXVII-NC16a antibodies in subjects with BP. Based on the existing literature, the reduction in the cell adhesion observed upon BP IgG stimulation can be accounted Asiatic acid for by ColXVII internalization (43, 72). However, researchers have not clearly decided how ColXVII internalization might influence cell adhesion. In the present study, the BP IgG-induced cell detachment was not directly induced by macropinosome formation, because alterations in actin, the well-known and necessary molecule for macropinosome formation (73), did not completely prevent NHEK detachment. NHEKs disassembled their contacts with neighboring cells Asiatic acid and detached from the culture dish following an incubation with BP IgG. Furthermore, epithelial cell destabilization has also been shown to require a step mediated by the proteasome (74). For this reason, we speculated and confirmed that this BP IgG-induced cell detachment was associated with proteasome activation, and the internalization of the IgG-ColXVII complex probably requires the initial event of proteasome activation. Another interesting aspect of this study was that the Asiatic acid BP IgG treatment increased NHEK motility. Based on the BP IgG-induced cell detachment, we speculate that this BP IgG-induced alterations in cell motility are likely due to a decrease in the cell density. On the other hand, ColXVII has been shown to regulate keratinocyte TNFRSF4 motility, while changes in cell motility following the loss of ColXVII remain controversial (26). Studies using ColXVII-knockdown keratinocytes have reported that the loss of ColXVII reduces lamellipodial stability (75) and induces cell migration mediated by Rac1 (76, 77). Cell migration is usually associated with the remodeling of the actin cytoskeleton. However, cytochalasin D did not affect cell motility following the BP IgG treatment. This discrepancy might be explained by the binding of ColXVII to two different cytoskeleton systems in keratinocytes: actin-associated focal contacts and keratin-associated hemidesmosome compounds (15, 78, 79). Our findings provide a better understanding of the direct effects of BP IgG on keratinocytes by increasing the fragility of the cell membrane, resulting in keratinocyte dysfunction, probably through oncosis. In addition, the BP IgG-induced cellular dysfunction was reversed by Rac1/proteasome inhibition. We believe that our identification of the Rac1/proteasome-mediated signaling pathway provides useful new insights that have improved our understanding of the direct effects of BP IgG on keratinocytes. Author Contributions DT designed the study and wrote the initial draft of the manuscript. XD contributed to data collection and interpretation, and.