Screening of integrins localized on the surface of human epidermal melanocytes.

Abstract

In vivo, melanocytes occupy three-dimensional (3D) space. Nevertheless, most experiments involving melanocytes are performed in a two-dimensional microenvironment, resulting in difficulty obtaining accurate results. Therefore, it is necessary to construct an artificial in vivo-like 3D microenvironment. Here, as a step towards engineering a precisely defined acellular 3D microenvironment supporting the maintenance of human epidermal melanocytes (HEMs), we examined the types of integrin heterodimers that are expressed transcriptionally, translationally, and functionally in HEMs. Real-time PCR and fluorescent immunoassay analyses were used to elucidate the expression of integrin α and β subunit genes at the transcriptional and translational levels, respectively. The functionality of the presumed integrin heterodimers was confirmed using attachment and antibody-inhibition assays. Among the genes encoding 12 integrin subunits (α1, α2, α3, α4, α5, α6, α7, αV, β1, β3, β5, and β8) showing significantly higher transcription levels, proteins translated from the integrin α2, α4, α5, β1, β3, and β5 subunit genes were detected on the surface of HEMs. These HEMs showed significantly increased adhesion to collagen I, fibronectin, laminin, and vitronectin, and functional blockade of the integrin α2 subunits significantly inhibited adhesion to collagen I, fibronectin, and laminin. In addition, there was no significant inhibition of the adhesion to fibronectin or vitronectin in HEMs with functional blockade of the integrin α4, α5, or αV subunits. These results indicate that the active integrin α2β1 heterodimer and the inactive integrin α4, α5, αV, β3, and β5 subunits are all localized on the surface of HEMs.