Stabilization of microtubules restores barrier function after cytokine-induced defects in reconstructed human epidermis

Abstract

Background

A variety of human skin disorders is characterized by defects in the epidermal barrier, leading to dehydration, itchiness, and rashes. Previously published literature suggests that microtubule stabilization at the cortex of differentiating keratinocytes is necessary for the formation of the epidermal barrier.

Objectives

We tested whether stabilization of microtubules with paclitaxel or epothilone B can repair barrier defects that were experimentally induced in three-dimensional culture models of epidermis.

Methods

We established two models of defective epidermis in vitro, using three-dimensional cultures of primary human keratinocytes on filter supports: immature reconstructed human epidermis (RHE), and RHE that was compromised by treatment with inflammatory cytokines, the latter mimicking defects seen in atopic dermatitis.

Results

Both paclitaxel and epothilone B promoted keratinocyte differentiation, accumulation of junctional proteins at the cell cortex, and the early appearance of lamellar bodies in immature RHE, whereas destabilization of microtubules by nocodazole had the reverse effect. Moreover, stabilization of microtubules rescued the barrier after cytokine treatment. The rescued barrier function correlated with the restoration of filaggrin and loricrin protein levels, the cortical accumulation of junctional proteins (E-cadherin, β-catenin, and claudin-1), and with the secretion of lamellar bodies.

Conclusions

Our data suggest that the microtubule network is important for the formation of the epidermis, and that stabilization of microtubules promotes barrier formation. Microtubule stabilization may support regeneration of damaged skin, by restoring or improving the barrier.

Introduction

At the interface between the organism and the environment, the skin provides mechanical protection and represents an important barrier against aggressions from the outside and against uncontrolled loss of body fluids. The barrier function is mainly attributed to the epidermis, a stratified epithelium with a basal layer of proliferative cells and multiple suprabasal layers of differentiated keratinocytes. Keratinocytes assemble massive amounts of cytoskeletal filaments that are in part anchored to intercellular junctions, such as desmosomes, tight junctions, and adherens junctions. These elements contribute to the mechanical stability and optimal elasticity of the epithelium. Furthermore, differentiated keratinocytes secrete the content of lamellar bodies – lysosome-related vesicles with a unique lipid and protein composition. This process increases insulation, by providing a hydrophobic barrier.

Because several events during epidermal barrier formation are likely to involve microtubules, we wanted to test whether stabilization of the microtubule network can attenuate or repair epidermal defects as seen in experimental models of atopic dermatitis (AD). This idea was fueled by reports on the reorganization and stabilization of microtubules at the keratinocyte cortex, and on the involvement of microtubule-dependent motor proteins as a prerequisite for the assembly of intercellular junctions such as desmosomes, tight, and adherens junctions [1], [2], [3], [4]. Because cortical anchorage of multiple microtubule-organizing proteins (ninein, Ndel, and Lis1) depends on the desmosomal protein desmoplakin, and because loss of Lis1 causes defects in desmosome assembly and in the barrier of mouse skin [1], [2], we strongly suspect that there is a mutual dependence between cell junctions and microtubule reorganization, and that both are needed to ensure the proper formation and function of the epidermal barrier. In agreement with this, interference with components of intercellular junctions in mouse models provokes skin abnormalities, inflammatory lesions and loss of homeostasis [5], [6], [7], [8], [9], [10]. Also, several comparative transcriptomic analyses of normal versus AD skin, lesional and non-lesional, have shown that the expression of many genes encoding tubulins and microtubule-associated proteins is altered in the diseased skin [11], [12], [13], [14]. Finally, a global proteomic analysis of lamellar body-enriched fractions has identified several proteins involved in microtubule-dependent motility [15], suggesting that lamellar bodies are transported along microtubules.

Our present study indicates a role of microtubules in the skin barrier. In a culture model of immature epidermis, microtubule stabilization increased the expression of epidermal differentiation markers, whereas destabilization had the reverse effect. In reconstructed human epidermis (RHE), we demonstrated that stabilization of microtubules repairs defects induced by Th2 cytokine treatment, such as reduced thickness of the cornified layer, reduced amounts of filaggrin expression, reduction of lamellar body secretion, reduced cortical localization of intercellular junction proteins, and increased transepidermal water loss (TEWL).