Abstract
c-Jun NH2-terminal protein kinase (JNK) and p38 are stress-activated mitogen-activated protein kinases (MAPK) that are phosphorylated by various stimuli. It has been reported that the loss of desmoglein (DSG) 3, a desmosomal transmembrane core molecule, in keratinocytes impairs cell–cell adhesion accompanied by p38 MAPK activation. To understand the biological role of DSG3 in desmosomes and its relationship with stress-activated MAPKs, we established DSG3 knockout keratinocytes (KO cells). Wild-type cells showed a linear localization of DSG1 to cell–cell contacts, whereas KO cells showed a remarkable reduction despite the increased protein levels of DSG1. Cell–cell adhesion in KO cells was impaired over time, as demonstrated by dispase-based dissociation assays. The linear localization of DSG1 to cell–cell contacts and the strength of cell–cell adhesion were promoted by the pharmacological inhibition of JNK. Conversely, pharmacological activation of JNK, but not p38 MAPK, in wild-type cells reduced the linear localization of DSG1 in cell–cell contacts. Our data indicate that DSG1 and DSG2 in KO cells cannot compensate for the attenuation of cell–cell adhesion strength caused by DSG3 deficiency and that JNK inhibition restores the strength of cell–cell adhesion by increasing the linear localization of DSG1 in cell–cell contacts in KO cells. Inhibition of JNK signaling may improve cell–cell adhesion in diseases in which DSG3 expression is impaired.
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The datasets generated and analyzed during the current study are available from the corresponding authors on reasonable request.
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Acknowledgements
The authors would like to thank Editage for English proofreading.
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This study was supported in part by a Grant-in-Aid for Scientific Research (C) (No. 22K09921) from the Ministry of Education, Culture, Sports, Science, and Technology in Japan.
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T.In. conceived and planned the experiments. T.In. and T.Is. established DSG3 KO cells. S.O. carried out all experiments with help from T.O., Y.I., T.M., and T.In., and T.In. contributed to the interpretation of the results. Y.I. analyzed data of dispase-based dissociation assay. All authors read and approved the final manuscript. T.In. wrote the manuscript with input from all authors and supervised the project.
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Supplementary Fig. 1
Negative controls for immunofluorescence. Confluent WT (a, b) and KO (c, d) cells were cultured for one day (a, c) or three days (b, d) in the presence of 1.2 mM calcium. Confluent KO cells were cultured for 6 h in a high-calcium medium with a vehicle (e), 20 μM BIRB 796 (f), or 20 μM SP600125 (g). Confluent WT cells were cultured for 6 h in a high-calcium medium with a vehicle (h), 100 nM anisomycin (i), 100 nM anisomycin plus 20 μM BIRB 796 (j), or 100 nM anisomycin plus 20 μM SP600125 (k). They were incubated with a mixture of anti-mouse and anti-rabbit immunoglobulin conjugated with Alexa 488 or Alexa 568, omitting the primary antibodies. Nuclei were stained with DAPI, and merged images are shown. No specific immunofluorescence was observed. Scale bar: 20 μm. Supplementary file1 (TIF 8886 KB)
Supplementary Fig. 2
The localization of DSG1 decreases in cell–cell contacts of KO cells cultured for one and three days. Confluent KO cells (clone 207-46) were cultured for one (a–f) and three (g–l) days in the presence of 1.2 mM calcium. Cells were double-stained with antibodies against either DSG1 (a, g, green) and E-cadherin (Ecad) (b, h, red) or DSG3 (d, j, green) and DSG2 (e, k, red). Merged images are shown in c, f, i, and l. Nuclei were stained with DAPI (c, f, i, l, blue). After one day of culture, some cell clusters growing on top of the KO cell monolayer (hereafter referred to as surface cells) showed weak to moderate DSG1 staining in the cytoplasm and cell–cell contacts (asterisks in a), while cells that adhered to the glass slides (hereafter referred to as basal cells) showed weak DSG1 staining (a). Arrowheads indicate cell–cell contacts between basal cells that had faint staining for DSG1 (a) but intense staining for Ecad (b). Two single cells (arrows in a) on top of the KO cell monolayer that were not in contact with other surface cells showed very strong DSG1 staining in the cytoplasm (a). DSG3 staining was not detected in KO cells (d). DSG2 staining in cell–cell contacts (e) was heterogeneous and weak. After three days of culture, the area of surface cell clusters increased, and surface cells showed weak to strong DSG1 staining in the cytoplasm (g). Occasionally, some gaps (asterisks in g–i) were observed between basal cells. These gaps were visible thorough three surface cells with weak to moderate cytoplasmic DSG1 staining (outlined by arrowheads in g). DSG3 staining was not detected in KO cells (j). Compared to one day of culture, DSG2 staining in cell–cell contacts was slightly stronger (k). Scale bar: 20 μm. Supplementary file2 (TIF 15810 KB)
Supplementary Fig. 3
BIRB 796 and SP600125 inhibited the phosphorylation (activation) of p38 MAPK and JNK, respectively, in KO cells treated with anisomycin. Confluent KO cells (clone 207-4) were cultured for 0.5 h in a high-calcium medium with a vehicle (control), 100 nM anisomycin (AM), AM plus 20 μM BIRB 796 (BIRB), or AM plus 20 μM SP600125 (SP). Total cell lysates were separated by SDS-PAGE, followed by western blotting with antibodies to phospho-p38 MAPK (P-p38), p38 MAPK (p38), phospho-JNK (P-JNK), JNK and actin. P-JNK was not detected in the control KO cells. AM increased the immunoreactivity of both P-p38 and P-JNK compared with the control. In AM-treated KO cells, BIRB 796 or SP600125 specifically inhibited the phosphorylation of p38 or JNK, respectively. Supplementary file3 (TIF 677 KB)
Supplementary Fig. 4
Linear localization of DSG1 to cell–cell contacts is promoted by JNK inhibition in KO cells. Confluent KO cells (clone 207-46) were cultured for 6 h in a high-calcium medium with a vehicle (a), 20 μM BIRB 796 (b), or 20 μM SP600125 (c). Cells were stained with an antibody against DSG1. In control (a), negative, faint, or zipper-like (arrows) immunoreactivity for DSG1 was detected in cell–cell contacts. BIRB 796 treatment (b) promoted zipper-like (arrows) and linear (arrowheads) localization of DSG1 to cell–cell contacts. SP600125 treatment (c) promoted the linear (arrowheads) localization of DSG1 to cell–cell contacts. Scale bar: 10 μm. Supplementary file4 (TIF 3947 KB)
Supplementary Fig. 5
The inhibition of JNK restores cell–cell adhesion in KO cells. Confluent WT cells were cultured for 24 h in the presence of calcium. Confluent KO cells (clone 207-46) were cultured for 24 h in the presence of calcium or calcium plus 20 μM SP600125 (SP). Cell monolayers were detached from 24-well plates by dispase, transferred to 1.5 ml tubes, vortexed, and transferred to 24-well plates. Representative images of cell monolayers subjected to dispase-based dissociation assays are shown. The number of cell monolayer fragments was counted (n = 4). Compared to the control, the number of KO cell monolayer fragments was reduced by 78.1% with SP treatment. Statistical significance was set as *p < 0.05, **p < 0.01, and ***p < 0.001. ns: not significant. Supplementary file5 (TIF 20591 KB)
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Ogawa, S., Ishii, T., Otani, T. et al. JNK inhibition enhances cell–cell adhesion impaired by desmoglein 3 gene disruption in keratinocytes. Histochem Cell Biol 161, 345–357 (2024). https://doi.org/10.1007/s00418-023-02264-8
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DOI: https://doi.org/10.1007/s00418-023-02264-8