Toll-like receptors (TLRs) play an important role in the recognition of pathogens in keratinocytes. In this study, we investigated whether the differentiation state of HaCaT keratinocytes correlates with the expression of TLR2 and TLR4 genes. The expression levels of TLR2 and TLR4 in a HaCaT differentiation model system were determined using quantitative real-time RT-PCR (Q-RT-PCR) and flow cytometry. The progression of keratinocyte differentiation was monitored by determining the level of involucrin gene expression using Q-RT-PCR. The expression levels of TLR2 and TLR4 increased with the stage of differentiation and there were strong correlations between the expression level of the involucrin gene and those of the TLR2 gene ( r=0.809, P<0.0001) and the TLR4 gene ( r=0.568, P<0.02). Increased cell surface expression of TLR2 and TLR4 was also found in differentiated HaCaT keratinocytes by flow cytometric analysis. Our findings suggest that upregulation of TLR expression during differentiation in keratinocytes could be a part of the differentiation process of keratinocytes and could have biological significance in protecting skin against microbes.

In previous work we described a novel culture technique using a cholera toxin and PMA-free medium (Mel-mix) for obtaining pure melanocyte cultures from human adult epidermis. In Mel-mix medium the cultured melanocytes are bipolar, unpigmented and highly proliferative. Further characterization of the cultured melanocytes revealed the disappearance of c-Kit and TRP-1 and induction of nestin expression, indicating that melanocytes dedifferentiated in this in vitro culture. Cholera toxin and PMA were able to induce c-Kit and TRP-1 protein expressions in the cells, reversing dedifferentiation. TRP-1 mRNA expression was induced in dedifferentiated melanocytes by UV-B irradiated keratinocyte supernatants, however direct UV-B irradiation of the cells resulted in further decrease of TRP-1 mRNA expression. These dedifferentiated, easily accessible cultured melanocytes provide a good model for studying melanocyte differentiation and possibly transdifferentiation. Because melanocytes in Mel-mix medium can be cultured with human serum as the only supplement, this culture system is also suitable for autologous cell transplantation.

In this study, we show that the G0-G1/S phase of HaCaT keratinocyte cell cycle is characterized by D1-type cyclin expression, whereas during the repeated rapid turnover of highly proliferating cells, the expression of cyclins D2 and D3 dominates. Knocking down cyclin D1 mRNA resulted in no change of cell proliferation and morphology, indicating that D2 and D3 cyclins could substitute for D1 in driving the cell cycle. Increased numbers of cyclin D1-expressing keratinocytes were found in the basal layers of the lesional psoriatic epidermis compared to both normal and non-lesional epidermis without increased expression of cyclin D1 mRNA, suggesting a possible dysfunction in the degradation of cyclin D1 protein. We also detected a significant increase in cyclin D2 and D3 mRNA expressions in psoriatic epidermis compared to normal epidermis with no difference in protein expressions. Blocking alpha5-integrin function by a neutralizing antibody in HaCaT keratinocytes downregulated the expression of cyclin D1 mRNA without affecting the expressions of cyclin D2 and D3 indicating a regulatory role for alpha5-integrin in the expression of cyclin D1. Our data suggest a possible role for D-type cyclins in the excessive basal-cell proliferation and perturbed keratinocyte differentiation in the psoriatic epidermis.

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Current data suggest that tissue microenvironment control immune functions. Therefore, understanding the tissue environment in which immune activation occurs will enhance our capability to interfere with abnormal immune pathology. Here, we argue that studying the constitutively abnormal functions of clinically uninvolved psoriatic skin in patients with plaque type psoriasis is very important to better understand psoriasis pathobiology, because non‐lesional skin provides the tissue environment in which the psoriatic lesion develops. A key question in psoriasis is what initiates the abnormal, uncontrolled immune activation in the first place and the answer may lie in the skin. In light of this concept, we summarize abnormalities at the dermal‐epidermal junction region which shows a special “non‐healing‐like” micro‐wound phenotype in the psoriatic non‐lesional skin that may act as a crucial susceptibility factor in the development of the disease.