Abstract
Keratinocytes in the oral mucosal epithelium, which is a non-keratinized stratified epithelium, are exposed to various stimuli from the oral cavity. JNK and p38 are stress-activated mitogen-activated protein kinases (MAPKs) that are phosphorylated by various stimuli and are involved in the assembly and disassembly of tight junctions (TJs) in keratinocytes. Therefore, we investigated the effects of stress-activated MAPKs on TJs in a mouse keratinocyte cell line during cell–cell junction formation in two-dimensional (2D) cultures or stratification to form non-keratinized epithelium in 3D cultures. In 2D cultures, calcium induced zipper-like staining for ZO-1 at 2 h and string-like staining for ZO-1 at 12 h, which indicated immature and mature cell–cell junctions, respectively. Anisomycin (AM), a JNK and p38 activator, inhibited formation of string-like staining for ZO-1, whereas inhibition of JNK, but not p38, after AM treatment restored string-like staining for ZO-1, although claudins (CLDNs) 4, 6, and 7 did not completely colocalize to ZO-1-positive sites. In 3D cultures, AM treatment for 2 weeks activated only p38, suppressed flattening of the superficial cells, removed CLDN7 from ZO-1-positive spots on the surface of 3D cultures, which represent TJs, and decreased transepithelial electrical resistance. Thus, short-term AM treatment inhibited maturation of cell–cell junctions by JNK, but not p38, activation. p38 activation by long-term AM treatment affected morphology of stratified structures and paracellular permeability, which was increased by CLDN7 removal from TJs. Various chronic stimuli that activate stress-activated MAPKs may weaken the keratinocyte barrier and be involved in TJ-related diseases.
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This work was supported in part by a Grant-in-Aid for Scientific Research (C) (no. 26460285) from the Ministry of Education, Culture, Sports, Science, and Technology in Japan.
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418_2018_1736_MOESM1_ESM.tif
Antibody specifications examined by immnoblotting. Cells were lysed, fractionated by SDS-PAGE, and the protein bands were transferred onto PVDF membranes. Immunoblotting was performed using antibodies against CLDNs 1, 4, 6, and 7, β-actin (A); E-cadherin, rabbit ZO-1, mouse ZO-1, K4 (B); p38, P-p38, ERK, P-ERK, JNK, P-JNK (C); JNK1, JNK2, p38α, p38β, p38γ, and p38δ (D). Membranes were reprobed after stripping primary and secondary antibodies. Membranes were cut along the lines in D. The cell lysates used were as follows: M, MDCK II cells; K, K38; C93, CMT93-II cells; C3D, COCA 3D cultures for 2 weeks; K3D, K38 3D cultures for 2 weeks; M’, MDCK II cells treated with 1 μM AM for 30 min; M’, K38 cells treated with 0.1 μM AM for 30 min; H, HEKa cells; C, COCA. The observed band sizes (arrowheads) were as follows: CLDNs 1, 4, 6, and 7, ~20 kDa; β-actin, 42 kDa; E-cadherin, ~135 kDa; ZO-1 (rabbit) and ZO-1 (mouse), ~225 kDa; K4, 57 kDa; p38 and P-p38, 43 kDa; ERK and P-ERK, 42 (lower band) and 44 (upper band) kDa; JNK, P-JNK, JNK1, and JNK2, 46 (lower band) and 54 (upper band) kDa; p38α, 40 kDa; p38β, 43 kDa; p38γ, 46 kDa; and p38δ, 43 kDa (TIF 21675 KB)
418_2018_1736_MOESM2_ESM.tif
Antibody specifications examined by immunofluorescence localization in MDCK II cells and CMT93-II cells. Cells were double stained with mouse anti-ZO-1 and either rabbit anti-CLDN1 (d–f), anti-CLDN7 (g–i), anti-E-cadherin (j–l), or anti-CLDN6 (v–x) antibodies, or rabbit anti-ZO-1 and either mouse anti- CLDN4 (m–o) or anti- occludin (p–r) antibodies. MDCK II cells (a–r) expressing occludin, E-cadherin, and CLDNs 1, 2, 3, 4, and 7 and CMT 93-II cells (s–x) expressing occludin, E-cadherin, and CLDNs 2, 4, 6, and 7 were used. In the control samples (a–c, s–u), BSA-PBS was used in place of primary antibodies. The green images (d, g, j, m, p, v) show the localization of ZO-1. The red images show the localization of CLDN1 (e), CLDN7 (h), E-cadherin (k), CLDN4 (n), occludin (q), and CLDN6 (w). Merged images are shown in c, f, i, l, o, r, u, and x. Nuclei were stained with DAPI (blue). ZO-1 is localized at the apical junctions with occludin and CLDNs 1, 4, 6, and 7, but E-cadherin is localized in the lateral cell membrane below the apical junctions. Scale bar: 20 μm (TIF 13760 KB)
418_2018_1736_MOESM3_ESM.tif
Controls for immunofluorescence in 2D and 3D cultures. K38 cells seeded on insert filters were airlifted for 2 weeks in the presence of 0.2% DMSO (a–c) or 50 nM AM (d–f). COCA cells seeded on insert filters were airlifted for 2 weeks (g–i). K38 cells (2D cultures) were cultured for 12 h in the presence of 1.2 mM calcium (j–l). Cryosections or 2D-cultured cells were incubated with BSA-PBS in place of the primary antibodies and then, the secondary antibodies (a mixture of anti-mouse and anti-rabbit Ig conjugated with either Alexa 488 or Alexa 568). Images derived from Alexa 488 (green) are shown in a, d, g, and j. Images derived from Alexa 568 (red) are shown in b, e, h, and k. Nuclei were stained with DAPI (blue). Merged images are shown in c, f, i, and l. No specific signals were observed in these controls. Scale bar in i is applied to a–i: 20 μm. Scale bar in l is applied to j–l: 20 μm. (TIF 4732 KB)
418_2018_1736_MOESM4_ESM.tif
Effect of AM on localization of TJ proteins and E-cadherin in K38 2D culture after induction of cell-cell junctions by calcium for 24 h. K38 cells cultured in low calcium FAD medium on glass slides were treated with 1.2 mM calcium and either 0.2% DMSO (A) or 100 nM AM (B) for 24 h. Cells were double stained with antibodies against ZO-1 (a, d, g, j, m, green) and CLDNs 1 (b, red), 4 (e, red), 6 (h, red), or 7 (k, red), or E-cadherin (n, red). Mouse anti-ZO-1 antibody was used in a, g, j, and m while rabbit anti-ZO-1 antibody was used in d. Merged images are shown in c, f, i, l, and o. Nuclei were stained with DAPI (blue). Arrows indicate string-like staining positive for green and red while arrowheads indicate zipper-like staining positive for green but not for red. Scale bars: 20 μm (TIF 18473 KB)
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Nikaido, M., Otani, T., Kitagawa, N. et al. Anisomycin, a JNK and p38 activator, suppresses cell–cell junction formation in 2D cultures of K38 mouse keratinocyte cells and reduces claudin-7 expression, with an increase of paracellular permeability in 3D cultures. Histochem Cell Biol 151, 369–384 (2019). https://doi.org/10.1007/s00418-018-1736-z
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DOI: https://doi.org/10.1007/s00418-018-1736-z