Regulation of Gap Junctional Intercellular Communication in Human Keratinocyte Cells

Abstract

Background and ObjectivesZZUnlike the normal skin, cholesteatomas characterized by hyperproliferative keratinocytes exhibits up-regulation of connexins (Cxs) and gap junctional intercellular communication (GJIC). Currently, there are no appropriate clinical methods that can inhibit cholesteatoma progression nor are there available optimal in vitro models of cholesteatomas. The objectives of this study were to identify the regulating materials that control GJIC using human keratinocyte cells (HaCaT) and to get preliminary information about how to inhibit cholesteatoma progression with an aim to make in vitro models. Materials and MethodZZAcetic acid (AA), H 2 O 2 , dexamethasone, retinoic acid (RA), or green tea extracts-epicatechin (EC) and epigallocatechin gallate (EGCG) were used for this study. After HaCaT cells were cultured with chemicals for 24 hours, cytotoxicity was quantitatively analyzed by cell counting and Neutral-red uptake test. Reverse transcriptase-polymerase chain reaction, Western blot and immunocytochemistry were performed to analyze the change of Cx expression. GJIC was functionally evaluated with scrape-loading dye transfer (SLDT). ResultsZZAfter the 24-hour culture, H 2 O 2 or EGCG (100 μM) were observed to have interfered with cell growth. In the Western blot, Cx26 and Cx30 showed higher up-regulation by EGCG or dexamethasone, but less down-regulation by AA or H 2 O 2 than the control. In comparison with the control, immunocytochemistry (Cx26, Cx43) showed less expression and abnormal location of Cxs under AA, H 2 O 2 , or 50 μM EGCG than the control, and increased up-regulation or equal expression under 5μM EGCG, EC, RA, or dexamethasone was greater than the control. In SLDT, dye transfer was significantly lower in AA-, H 2 O 2 -, dexamethasone-, or RA-treated cells than in the control cells. EC showed higher dye transfer than the control cells. ConclusionZZThe expression of Cxs and GJIC on human HaCaT keratinocytes can be upor down-regulated by chemicals such as AA, H 2 O 2 , dexamethasone, or EC. These results may be useful information in understanding the progression or inhibition mechanisms of cholesteatomas.