Marine sponge depsipeptide increases gap junction length in HTC cells transfected with Cx43–GFP

Connexins are membrane proteins that form GJ (gap junction) channels between adjacent cells. Cx43 (connexin 43), the most widely expressed member of the connexin family, has a rapid turnover rate, and its degradation involves both the lysosomal and ubiquitin-proteasome pathway. The goal of this work was to study the effects of geodiamolides, natural peptides from marine sponge that normally are involved with microfilament disruption, on connexin assembly or degradation in the plasma membrane. HTC (hepatocarcinoma cells) expressing Cx43-GFP (green fluorescent protein) were submitted to treatment with 200 nM geodiamolides A, B, H and I for 2 and 4 h. Microfilament distribution and the presence and size of GJ plaques were evaluated by laser scanning confocal microscopy. Among the four peptides tested, only Geo H (geodiamolide H) statistically enhanced the length of GJ plaques. Geodiamolide A also showed activity in the GJ plaque size; however, its effect was less pronounced. Treatment with Geo H could interfere with the delivery of connexins to the degradation structures, similar to proteasomal pathways, keeping the connexins assembled and accumulating GJ plaques. Further experiments, with the cells treated with Geo H, using the fungal antibiotic BFA (brefeldin A), were performed in order to uncouple events leading to GJ assembly from those related to GJ removal, since BFA is known to block protein trafficking within a fused ER (endoplasmic reticulum)/Golgi compartment. GJ plaques were drastically reduced after BFA/Geo H treatment, thus indicating that Geo H affects mainly the delivery pathway of Cx43 protein.