RegulationofGap Junction Protein,Connexins in Immortalized Human Keratinocytes Cells(HaCaTcells)

Intercellular communication for function of cells and tissues is very important as the intracellular communication system. Gap junctions are ordered arrays of intercellular plasma membrane channels that directly link the cytosols of two adjoining cells. Gap junctional intercelluar communication (GJIC) is an important mechanism controlling cellular homeostasis, proliferation, and differentiation. A family of gap junction proteins, the connexins(Cx), has been identified and comprises at least 20 members. Middle ear cholesteatoma is characterized by the presence of a squamous epithelium invading in the middle ear cavity, and is one of the most prevalent forms of otological diseases. The histologic features of cholesteatomas are similar to hyperproliferation and hyperkeratosis of the skin. The mechanism of intercellular communication in cholesteatomas remains unknown. Human middle ear cholesteatomas showed more increased expression of Cx43 and Cx26 than retroauricular skins. In another study for making in vitro 3 dimensional model of cholesteatomas, there was some difference of epithelial characteristics between a spontaneously immortalized epidermal cell line (HaCaT) and cultured cholesteatoma cells. If it is possible to control the intercellular communication system of cholesteatoma, in other words, if we inhibit the Cx expression of cholesteatoma, it will be able to inhibit the advancement of cholesteatoma. If we can increase the Cx expression in the in-vitro model that uses the HaCaT cells, we will be able to make a more useful in vitro model that is similar to the cholesteatoma. Under such hypotheses, the objectives of this study are to identify the expression of gap junction in HaCaT cells and to control the expression of gap junction proteins using various substances. From clinical point of view, easily applicable substances such as H₂O₂, acetic acid, dexamethasone, all-trans-retinoic acid(RA), and green tea extract epicatechin (EC) and epigallocatechin gallate(EGCG) were used for this study. These substances were first applied on the HaCaT cell line according to their concentration. After they were cultured for 24 hours, the cytotoxicity was analyzed by quantitatively assessing the degree of cell growth, and reverse transcription-polymerase chain reaction(RT-PCR). Western blot and immunocytochemistry staining were performed to analyze the change of Cx production and expression. In results, Cx26, Cx30, Cx43, and Cx31 in that order were expressed well in HaCaT cell line. After 24-hour culture, the substances that interfered the cell growth were acetic acid, H₂O₂, and 100 μM EGCG . Through RT-PCR, substances that up-regulated the expression Cx26 mRNA were 50 ㎍/㎖ EGCG, EC, and RA, and the substances that down-regulated Cx26 were acetic acid, H₂O₂, 5 and 100 ㎍/㎖ EGCG. In Western blot, 50 ㎍/㎖ EGCG up-regulated Cx26 protein, and acetic acid, H₂O₂, and dexamethasone down-regulated it, while 5ug/ml EGCG, EC, and RA showed little changes compared with the control. Immunocytochemistry of HaCaT cells showed decreased expression and abnormal location of Cx26 and Cx43 under acetic acid, H₂O₂, and 50 ㎍/㎖ EGCG. It also showed up-regulated or equal immunostaining of Cx26 and Cx43 under 5 ㎍/㎖ EGCG, EC, and RA and very similar staining in case of dexamethasone compairing with those in control. In conclusions, the gap junction proteins in HaCaT cells can be easily controlled by substances. From the results of this study, acetic acid and H₂O₂ are considered to be useful substances to inhibit the advancement of middle ear cholesteatomas, and RA, EC, and EGCG are thought to be useful when we are making in vitro cholesteatoma model using HaCaT cells.

Author dc:creator

• 김, 병철

Contributors dc:contributor

• 정, 연훈
• 대학원 의학과
• 199824350

• 진주종
• HaCaT 세포주
• 간극결합
• Connexins
• Aceticacid
• H₂O₂
• Dexamethasone
• All-transretinoicacid
• Epicatechin
• Epigallocatechingallate
• Gap junctions
• Cholesteatoma

Language dc:language

ko

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000000665
000000000665

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/1498