Contribution of Tight Junction Proteins to Ion, Macromolecule, and Water Barrier in Keratinocytes

Abstract

Tight junctions (TJs) form a selective barrier for ions, water, and macromolecules in simple epithelia. In keratinocytes and epidermis, TJs were shown to be involved in individual barrier functions. The absence of the TJ protein claudin-1 (Cldn1) in mice results in a skin-barrier defect characterized by lethal water loss. However, detailed molecular analyses of the various TJ barriers in keratinocytes and the contribution of distinct TJ proteins are missing. Herein, we discriminate TJ-dependent paracellular resistance from transcellular resistance in cultured keratinocytes using the two-path impedance spectroscopy. We demonstrate that keratinocyte TJs form a barrier for Na(+), Cl(-), and Ca(2+), and contribute to barrier function for water and larger molecules of different size. In addition, knockdown of Cldn1, Cldn4, occludin, and zonula occludens-1 increased paracellular permeabilities for ions and larger molecules, demonstrating that all of these TJ proteins contribute to barrier formation. Remarkably, Cldn1 and Cldn4 are not critical for TJ barrier function for water in submerged keratinocyte cultures. However, Cldn1 influences stratum corneum (SC) proteins important for SC water barrier function, and is crucial for TJ barrier formation for allergen-sized macromolecules.

Epidermal tight junctions in health and disease

“…It is puzzling that reduced expression of cldn-1 in keratinocytes did not show an effect on water barrier as measured by a modified Ussing chamber, 30 neither in human primary keratinocytes (siRNA mediated K D ; 83 C/-8% knockdown) nor in mouse keratinocytes isolated from Cldn-1 homozygous knock-out (KO) mice. 24 This was highly surprising, because Cldn-1 KO mice die within the first day of birth due to high transepidermal water loss. 11 Interestingly, no abnormal water loss was observed in heterozygous Cldn-1 KO mice.…”

“…24 While, only cldn-1 and ZO-1 were indispensible for the barrier for larger molecules (40 kDa). 24 In skin explants, treatment with ochratoxin A resulted in a loss of the TJ barrier for a 557 Da tracer and was associated with reductions in cldn-4 expression. 29 However, it is not clear whether ochratoxin A is specific for cldn-4, or whether it may also affect expression/localization of other barrier related proteins.…”

“…Na C , Cl ¡ , Ca 2C ). 24 Knockdown experiments in cultured keratinocytes have shown that cldn-1, cldn-4, occludin and ZO-1 contribute to the TJ barrier for Na C , Cl ¡ , Ca 2C and 4 kDa FITC Dextran. 24 While, only cldn-1 and ZO-1 were indispensible for the barrier for larger molecules (40 kDa).…”

“…24 Knockdown experiments in cultured keratinocytes have shown that cldn-1, cldn-4, occludin and ZO-1 contribute to the TJ barrier for Na C , Cl ¡ , Ca 2C and 4 kDa FITC Dextran. 24 While, only cldn-1 and ZO-1 were indispensible for the barrier for larger molecules (40 kDa). 24 In skin explants, treatment with ochratoxin A resulted in a loss of the TJ barrier for a 557 Da tracer and was associated with reductions in cldn-4 expression.…”

“…Concerning the exact localization of the functional TJ barrier, it has been suggested that it is localized in human and murine skin in the mid layers of the SG 3,11,21 ; although other studies have found TJ barrier to be at the uppermost layer of the SG. 15 Using cultured keratinocytes, it was shown that, TJs form a barrier to water 24 and molecules of different size ( [24][25][26][27][28] ) as well as ions (e.g. Na C , Cl ¡ , Ca 2C ).…”