Abstract
Vitamin A, which is found in serum, is known to affect keratinocyte proliferation, epidermal differentiation, and keratinization. In mice, stratified epithelia in the oral cavity, esophagus, and forestomach are keratinized; however, these epithelia are not keratinized in humans. Several studies have reported that three-dimensional (3D) cultures of human keratinocytes in serum-containing medium could form keratinized epithelia. Here, we evaluated the effects of serum on the morphology, expression, and localization of differentiation markers and tight junction proteins, and paracellular permeability in 3D cultures of mouse keratinocytes. We found that only 0.1% calcium-depleted serum inhibited keratinization and induced a change in the expression of differentiation marker proteins from loricrin to keratin 4; the inhibition of retinoic acid receptor-mediated signaling reversed these changes. Furthermore, the serum reduced claudin-1 protein expression and prevented its localization at occludin-positive spots on the surface of 3D cultures. On the other hand, the serum increased the protein expression of claudin-4, occludin, zonula occludens-1, and E-cadherin. These changes may contribute to the reduction of the transepithelial electrical resistance by approximately half. In conclusion, mouse keratinocytes derived from the epidermis formed non-keratinized structures in 3D cultures in response to vitamin A in serum. The results suggest that retinoic acid receptor-mediated signaling may be inhibited in the mouse epithelia in the oral cavity, esophagus, and forestomach as well as the epidermis, leading to the keratinization of these epithelia.
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Acknowledgements
This study 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_1741_MOESM1_ESM.tif
Antibody specificities examined by immunofluorescence localization in oral mucosal epithelium obtained from the lower jaws of pigs. Cryosections were double stained with anti-K4 and anti-LOR antibodies. Arrows indicate the boundary between non-keratinized lingual alveolar epithelium (the left-hand side) and keratinized lingual gingiva (the right-hand side). K4 signals (green) were observed in almost all layers in the non-keratinized stratified epithelium (b). A higher magnification of the lower layers of non-keratinized stratified epithelium revealed that K4 signals are faint in the basal layer (d). K4 signals gradually decreased from the upper layers to the basal layer on the right-side of the arrow (b). Contrary to the K4 signals, LOR signals (red) appeared in the upper layer that corresponds to the granular layer on the right-side of the arrow (b’, c’). Merged images are shown in a′′–d”. Nuclei were stained with DAPI (blue). The scale bar in b” is applied to a–a′′ and b–b”: 50 μm. The scale bar in d” is applied to c–c” and d–d”: 20 μm (TIF 29411 KB)
418_2018_1741_MOESM2_ESM.tif
The raw data obtained from the immunoblotting shown in Fig. 7. COCA cultures were airlifted for 2 weeks in the presence of 0%, 0.1%, 1%, or 10% ch-FBS and analyzed by immunoblotting. Cells were lysed, fractionated by SDS-PAGE, and transferred onto PVDF membranes. Immunoblotting was performed using antibodies against CLDN1, CLDN4, occludin, ZO-1, E-cadherin, and K4. Membranes were reprobed after stripping the primary and secondary antibodies. Lysates obtained from COCA 3D cultures in 0% (lane 1), 0.1% (lane 2), 1% (lane 3), and 10% (lane 4) ch-FBS were used. The observed band sizes (indicated by arrowheads) are as follows: CLDNs 1 and 4, ~20 kDa; occludin, 59 kDa; ZO-1, ~225 kDa; E-cadherin, ~135 kDa; K4, 57 kDa (TIF 8974 KB)
418_2018_1741_MOESM3_ESM.tif
Controls for immunofluorescence in COCA 3D cultures. COCA cells seeded on insert filters were airlifted for 2 weeks in the presence of 0% (a, a’, a”), 0.1% (b, b’, b”), 1% (c, c’, c”), and 10% (d, d’, d”) ch-FBS. COCA cells seeded on insert filters were airlifted for 1 week in the presence of 1% (e, e’, e”) ch-FBS and for another week with 0.2 μM BMS 493 (f, f’, f”). Cryosections were incubated with BSA-PBS in place of primary antibodies and then secondary antibodies were added (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–f. Images derived from Alexa 568 (red) are shown in a’–f’. Nuclei were stained with DAPI (blue). Merged images are shown in a”–f”. No specific signals were observed in these controls. Scale bar: 20 μm (TIF 29214 KB)
418_2018_1741_MOESM4_ESM.tif
Antibody specificities examined by immunofluorescence localization in MDCK II cells. Cells were double stained with mouse anti-occludin (b, c, d, e) and either rabbit anti-CLDN1 (b’), anti-CLDN4 (c’), anti-ZO-1 (d’), or anti-E-cadherin (e’) antibodies. In the control (a–a”), BSA-PBS was used in place of primary antibodies. Merged images are shown in a”–e”. Nuclei were stained with DAPI (blue). Occludin was localized at the apical junctions with CLDN1, CLDN4, and ZO-1, but E-cadherin was localized in the lateral cell membrane below the apical junctions. Some nuclei were heavily or weakly stained with anti-CLDN4 (c’) or anti-ZO-1 antibody (d’), respectively. Scale bar: 20 μm (TIF 23817 KB)
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Ozaki, A., Otani, T., Kitagawa, N. et al. Serum affects keratinization and tight junctions in three-dimensional cultures of the mouse keratinocyte cell line COCA through retinoic acid receptor-mediated signaling. Histochem Cell Biol 151, 315–326 (2019). https://doi.org/10.1007/s00418-018-1741-2
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DOI: https://doi.org/10.1007/s00418-018-1741-2