Restoration of calcium-induced differentiation potential and tight junction formation in HaCaT keratinocytes by functional attenuation of overexpressed high mobility group box-1 protein

Abstract

HaCaT cells have been widely used as undifferentiated epidermal keratinocytes, since these non-tumorigenic cells can be readily maintained in conventional medium and partly retain epidermal differentiation potential upon stimulation with high concentration of calcium. In contrast to primary epidermal keratinocytes, however, these cells never form tight junction (TJ), a specific structure in highly differentiated keratinocytes, solely by the differentiation stimulation. Here, we show that HaCaT cells secrete a considerable amount of high mobility group box-1 protein (HMGB1), one of major inflammatory mediator, which appeared to be responsible, at least in part, for such aberrant differentiation response. So far, inhibition of c-Jun N-terminal kinase (JNK) in high calcium medium has been supposed to be the only way to induce TJ formations in HaCaT cells; however, SP600125, a potent inhibitor of JNK showed cytostatic effects and clearly attenuated epidermal differentiation and stratification. In contrast, dipotassium glycyrrhizate (GK2), a soluble analogue of HMGB1blocker Glycyrrhizin, down-regulated interferon-b, a typical inflammatory cytokine induced by secreted HMGB1, and accelerated differentiation responses to the calcium treatment in these cells. In addition, GK2treatmenrt resulted in the formation of double cell layers in cultured HaCaT cells, where the stratified upper cells transiently accumulated TJ proteins at the cell-cell contact sites. These results highlight the importance of attenuation of secreted HMGB1-signals in cultured HaCaT cells for studies of functional keratinocytes.

Direct inhibition of the first PDZ domain of ZO-1 by glycyrrhizin is a possible mechanism of tight junction opening of Caco-2 cells

“…8B). 37–39 In contrast, we demonstrated GL's TJ-opening activity, which is apparently in opposition to its reported protective function (Fig. 8A).…”

PRSS3/mesotrypsin as a putative regulator of the biophysical characteristics of epidermal keratinocytes in superficial layers

“…The induction of G1 arrest and cell cycle exit account for the observed effects on cell proliferation. In terms of differentiation, although HaCaT cells maintained for a long period in conventional high-calcium medium retain their differentiation potential 39 , 40 and mesotrypsin diminishes the abundance of the differentiation marker Pro-FLG (Fig. 3 ), the expression of canonical differentiation markers, including TGase1, Loricrin, and Involucrin, remains unaffected (Fig.…”

Virus-host interaction: investigating novel transcription factors involved in coupling HPV life cycle and epithelial differentiation

“…Despite the diverse biological functions of PAX6, HMGB1, and NFE2, data concerning the involvement of these TFs in the context of keratinocyte differentiation is still very limited. To our knowledge, the link between HMGB1 and cell differentiation was evidenced in only one study which demonstrated that the functional ablation of secreted HMGB1 results in aberrant HaCaT differentiation [45]. Additionally, NFE2 was shown in several studies to activate antioxidant enzymes to protect skin keratinocytes against oxidative damage during differentiation [46][47][48].…”