Study on the influence of substrate roughness on the development of E-cadherin junction in oral keratinocytes

Abstract

Junctional epithelium (JE) may attach to the substrates with varying levels of roughness from a smooth crown surface to a root surface roughened due to various causes including periodontal treatments such as root planing. Since the physical property of substrates to which the epithelial cells attach may regulate the development of intercellular junctions, the development of intercellular junction between human gingival keratinocytes (HGKs) was studied on the rough substrates. HGKs were cultured on substrates with varying levels of roughness, which were prepared by rubbing hydrophobic polystyrene dishes with silicon carbide papers. The development of intercellular junctions was analyzed using scanning electron microscopy or confocal laser scanning microscopy after immunohistochemical staining of the cells for E-cadherin which organizes adherens junction, one of major intercellular junctions of oral keratinocytes. HGKs developed tight intercellular junctions devoid of wide intercellular gaps on smooth substrates and on rough substrates with low-nanometer dimensions (average roughness [Ra] = 121.3 ± 13.4 nm), although the ECJs of HGKs on rough substrates with low-nanometer dimensions developed later than those of HGKs on smooth substrates. In contrast, HGKs developed short intercellular junctions with wide intercellular gaps on rough substrates with mid- or high-nanometer dimensions (Ra = 505.3 ± 115.3 nm, 867.0 ± 168.6 nm). Notably, the stability of the ECJs was low on the rough substrates, as demonstrated by the rapid destruction of the cell junction following calcium depletion. Cortical actins developed at the cell membrane where ECJ did not develop. JNK activation promoted the development of cortical actins to regulate the development of ECJs in HGKs. In contrast, inhibition of JNK activity promoted ECJ development concomitant with the dissociation of cortical actins regardless of the levels of substrate roughness when HGKs were cultured at high density. These results indicate that the ECJs of HGKs develop slowly or defectively on the rough substrates with nanometer dimensions, and that this effect can be reversed by inhibiting JNK.