Vimentin is necessary for colony growth of human diploid keratinocytes

Abstract

The role of vimentin (Vim) in diploid epithelial cells is not well known. To understand its biological function, we cultured human epidermal keratinocytes under conditions that support migration, proliferation, stratification and terminal differentiation. We identified a keratinocyte subpopulation that shows a p63(+)/α5β1(bright) phenotype and displays Vim intermediate filaments (IFs) besides their keratin IF network. These cells were mainly located at the proliferative/migratory rim of the growing colonies; but also, they were scarce and scattered or formed small groups of basal cells in confluent stratified epithelia. Stimulation of cells with EGF and wounding experiments in confluent arrested epithelia increased the number of Vim(+) keratinocytes in an extent higher to the expected for a cell population doubling. BrdU labeling demonstrated that most of the proliferative cells located at the migratory border of the colony have Vim, in contrast with proliferative cells located at the basal layer at the center of big colonies which lacked of Vim IFs, suggesting that Vim expression was not solely linked to proliferation. Therefore, we silenced Vim mRNA in the cultured keratinocytes and observed an inhibition of colony growth. Such results, together with long-term cultivation assays which showed that Vim might be associated to pattern formation in cultured epithelia, suggest that Vim expression is essential for a highly motile phenotype, which is necessary for keratinocyte colony growth and possibly for development and wound healing. Vim(+)/p63(+)/α5β1(bright) epithelial cells may play a significant physiological role in embryonic morphogenetic movements; wound healing and other pathologies such as carcinomas and hyperproliferative diseases.

Epithelial cell migration requires the interaction between the vimentin and keratin intermediate filaments

“…We showed that Vim + keratinocytes are mainly located at the periphery of the colony ( Fig. 1A ), and knocking down Vim mRNA, impaired keratinocyte colony growth 13 . Since the growth of a keratinocyte colony depends on the outward migration of the rapidly proliferating cells located in the thin rim of the colony perimeter 18 , and since Vim is involved in cell migration and invasiveness of carcinoma cells 19 , in order to study the role of Vim during diploid epidermal cell migration, we also knocked down Vim mRNA, and determined keratinocyte migration in a specific assay 20 21 , such as the microliter radial monolayer assay (MRMA) described by Berens et al .…”

“…1A ; ref. 13 . Vim filaments (IFs) were located mainly at the bottom of the cell below the nucleus and at the cell leading edge, which is in close contact with the tissue culture substrate ( Fig.…”

“…On the other hand, diploid keratinocytes also show Vim IFs in certain circumstances. We reported recently that a subpopulation of diploid Vim positive (Vim + ) keratinocytes, involved in the growth of the keratinocyte colonies in human epidermal cultures, express the brightest α5β1 integrin and p63 13 that have been considered putative stem cell markers in the epidermis 14 . In this paper we extended our understanding of Vim role in cell migration and we studied its mechanism of action using as a model system the diploid keratinocytes transfected with Vim mutated in the 2B domain.…”

Bioengineering a Human Plasma-Based Epidermal Substitute With Efficient Grafting Capacity and High Content in Clonogenic Cells

“…High proliferation and migration levels of keratinocytes and low differentiation phenotype are related to rapid and efficient wound healing and thus are key characteristics for epidermal substitutes before grafting. We demonstrated in our study that our process induced high proliferation and expression of vimentin (an epithelial-mesenchymal transition marker linked with high cell motility [44][45][46]) (Fig. 4A).…”

First Evidences of Epithelial-Mesenchymal Transition and Cancer Stem-Cell Phenotype Acquisition in Dermo-Epidermal Junction of BPV-Infected Neoplasms

“…Vimentin is one of the most abundant mesenchymal protein expressed in mammals [96]. The VIM is evolutionary conserved [20], for this reason, the anti-vimentin clone V9 antibody has been used for the vimentin detection in bovines [97,98].…”