Aya Komiya

Activation of TLR2 Enhances Tight Junction Barrier in Epidermal Keratinocytes

The epidermis has developed physical and immunological barriers that prevent infiltration of deleterious chemicals and pathogens. As a first step to understanding the relationship between these barriers, we investigated whether TLR2 activation functionally alters tight junctions (TJs) in cultured human keratinocytes. Stimulation with peptidoglycan, a ligand for TLR2, elevated the TJ-associated barrier in the space of 3 h. The increase in TJ-associated barrier function due to peptidoglycan stimulation was suppressed by the knockdown of TLR adaptor MyD88 or the pretreatment with TLR2-neutralizing Ab, indicating that TLR2 activation enhanced TJ-associated barrier. One and 3 h after peptidoglycan stimulation, expression levels of the TJ proteins occludin, claudin-1, claudin-4, and ZO-1 were unchanged. However, immunoprecipitation studies demonstrated that the association of phospho-atypical protein kinase Cζ/ι, crucial for TJ biogenesis, with occludin was increased. Significantly, inhibition of atypical protein kinase Cζ/ι activity completely blocked the immediate elevation of the TJ-associated barrier. Finally, peptidoglycan was applied to the stratum corneum surface of a human skin equivalent, and the TJ barrier was evaluated. In the space of 3 h after the stimulation, the amount of intercellular tracer in the stratum corneum incubated from the dermal side was reduced, indicating that the TJ barrier is strengthened via TLR2 activation. Taken together, our findings indicated that infiltration of pathogens into the epidermis immediately enhanced TJ function via TLR2 signaling. Furthermore, the dynamically controlled TJs in skin are considered fundamental in preventing further invasion of pathogens and maintaining cutaneous barrier homeostasis.

Impaired tight junctions obstruct stratum corneum formation by altering polar lipid and profilaggrin processing.

BACKGROUND The stratum corneum (SC) is a well-known structure responsible for the cutaneous barrier. Tight junctions (TJs) function as a paracellular barrier beneath the SC and are involved in the cutaneous barrier. It remains unclear how TJs are involved in the cutaneous barrier. OBJECTIVE In order to clarify the role of TJs in the cutaneous barrier, we investigated skin equivalent models with disrupted TJ barriers focusing on the SC. METHODS Skin equivalents with disrupted TJ barriers were established using GST-C-CPE, a peptide with specific inhibitory action against specific claudins. The changes of the SC barrier in the skin equivalents with disrupted TJ barriers were investigated and compared with control skin equivalents. RESULTS An outside-to-inside skin barrier assay revealed a defective SC barrier in skin equivalents with disrupted TJ barriers. A detailed examination of the SC revealed an increase in the pH of the SC in the skin equivalent with disrupted TJ barriers. An electron microscopy showed the failure of lamellar structures to mature and the failure of keratohyalin granules to degrade in the skin equivalents with disrupted TJ barriers. A thin layer chromatography analysis showed an increase in polar lipids and a decrease in non-polar lipids. A western blot analysis showed an increase in filaggrin dimer and trimer and a decrease in filaggrin monomer. CONCLUSION We found that disrupted TJs obstructed the SC formation responsible for the cutaneous barrier. Our study indicates the possibility that impaired TJ barriers affect polar lipids and profilaggrin processing by disturbing the pH condition of the SC.