Effects of HYS-32 on Caveolin-3 and Connexin43 Expression in Rat Astrocytes

• Main Authors: Chuan-Hui Chang, 張荃惠
• Other Authors: Jiahn-Chun Wu
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/78208326089532225804

碩士 === 國立陽明大學 === 解剖學及細胞生物學研究所 === 102 === HYS-32, an analogue of combretastatin A-4 (CA-4) containing a cis-stilbene moiety, has previously been demonstrated to delay Cx43 degradation and upregulate both connexin43 (Cx43) expression and gap junction intercellular communication (GJIC) in rat astrocytes. In this study, we investigated the signaling pathway involved in the HYS-32-induced upregulation of Cav-3 and Cx43 and the mechanism involved in rat primary astrocytes. Double-label immunofluorescence microscopy shown that HYS-32 (5μM) time-dependently increased colocalization of Cx43 and Cav-3 immunoreactive patches at cell-cell contact sites. In a time course study, HYS-32 upregulated Cx43 protein levels in astrocytes 3-24 h after treatment, whereas the expression of Cav-3 increased at 6 h and sustained for up to 24 h. The Cav-3 and Cx43 protein were also increased in a dose-dependent manner following HYS-32 treatment. Furthermore, HYS-32 increased both phosphorylated PKC and phosphorylated ERK levels in a dose-dependent manner in astrocytes. Co-treatment of HYS-32 with PKC inhibitor Gö6976 or ERK inhibitor PD98059 prevented the HYS-32-induced increase in Cav-3 and Cx43 levels. HYS-32 induced an accumulation of Cav-3 and Cx43 in caveolae (caveolin-rich membrane domain), where an increased association of Cav-3 and Cx43 was detected. Co-treatment with HYS-32 and cycloheximide (CHX) delayed Cx43 turnover, whereas disruption of caveolae by methyl--cyclodextrin (MCD) caused dissociation of Cav-3 and Cx43 from caveolae and accelerated their turnover. The HYS-32-induced colocalization of Cx43 and Cav-3 was abolished by MCD. In conclusion, our results suggest that HYS-32 upregulates Cav-3 and Cx43 via PKC-ERK signaling pathway and that the HYS-32-induced Cx43 stabilization in cell membrane, postponed Cx43 turnover, and enhanced GJIC in astrocytes rely on the existence of intact caveolae .