Effects of 4-(3,4-Dimethoxyphenyl)-3-(naphthalen-2-yl)-2(5H)-furanone on Connexin43 Gap Junction in Rat Astrocytes

• Main Authors: Pei-Chun Lin, 林沛君
• Other Authors: Jiahn-Chun Wu
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/4dx4pq

碩士 === 國立陽明大學 === 解剖學及細胞生物學研究所 === 100 === HYS-32, 4-(3,4-dimethoxyphenyl)-3-(naphthalen-2-yl)-2(5H)-furanone compound containing a cis-stilbene moiety, is a new analogue of anti-tumor combretastatin A-4 (CA-4) originally isolated from the South African bush willow tree Combretum caffrum. In addition to its anticancer activity, 2(5H)-furanones also display anti-oxidant and anti-inflammatory properties against certain bacteria and fungi infections. In this study, we investigated the effect of HYS-32 on connexin43 (Cx43) gap junction in rat primary astrocytes and the signaling pathway involved. Immunoblot analyses showed that HYS-32 (5 μΜ) induces a time-dependent increase in Cx43 protein levels up to 24 h after treatment without changing Cx43 mRNA levels. Scrape-loading/dye transfer analyses demonstrated that treatment of HYS-32 for 24 h increases the gap junction intercellular communication (GJIC) in astrocytes. Phase-contrast microscopic images demonstrated that treatment of astrocytes with HYS-32 for 24 h caused cell hypertrophy without changing glial fibrillary acidic protein (GFAP) expression. Double immunofluorescence microscopy showed that treatment of HYS-32 for 24 h caused microtubule coiling and aggregation of Cx43 into large plaques at cell-cell contact sites. However, removal of HYS-32 for 24 h depleted microtubule coiling concurrent with a reduced distribution of Cx43 plaques at the plasma membrane. Furthermore, treatment of cycloheximide (CHX), which blocks de novo protein synthesis, resulted in a decrease in Cx43 half-life, while co-treatment of CHX with HYS-32 rescued the CHX-decreased Cx43 half-life, indicating a delayed protein turnover of Cx43 in HYS-32-treated astrocytes. Moreover, HYS-32 induced protein kinase C (PKC), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal protein kinases (JNK) activation in astrocytes. Co-treatment with PKC inhibitor Go6976 or ERK inhibitor PD98059, but not JNK inhibitor SP600125, was able to prevent the HYS-32-increased Cx43 expression and GJIC function in astrocytes. In conclusion, our results suggest that treatment of HYS-32 in astrocytes causes microtubule coiling concurrent with a reduced Cx43 degradation and an increased accumulation of Cx43 junction plaques at plasma membrane and leads to upregulation of Cx43 expression and GJIC function via PKC and ERK signaling pathways.