Regulatory function of hyperosmotic stress-induced signaling cascades in the expression of transcription factors and osmolyte transporters in freshwater Japanese eel primary gill cell culture

In the present study, we investigated the early activation of osmotic stress-related protein kinases, with the aim of characterizing their functional links with downstream effectors (i.e. transcription factors and osmolyte transporters). Freshwater eel primary gill cells were cultured in hypertonic medium (500?mosmol?l^-1) for 6?h. Protein lysates and total RNA were collected for western blotting and quantitative real-time PCR assays. In this study, the osmotic challenge stimulated histone H3 phosphorylation, various signaling pathways (i.e. ERK1/2, p38 MAPK, JNK, CREB, MARCKS and MLCK) and expression of some downstream effectors (i.e. Na⁺/K ⁺-ATPase, TauT and Ostf). Increased phosphorylation of acetylated histone is known to promote chromatin relaxation for global gene transcription, probably leading to the activation of downstream effectors for osmotic responses. In addition, the importance of the p38 MAPK and MLCK pathways in the regulation of the expression of Na⁺/K⁺-ATPase and TauT was demonstrated. Inhibition of the p38 MAPK pathway by SB202190 reduced histone H3 phosphorylation and TauT mRNA expression. Moreover, inhibition of the MLCK pathway by ML-7 decreased the expression level of Na⁺/K ⁺-ATPase but increased the transcript level of TauT. Collectively, the present study reveals possible functional links of osmosensing signaling cascades to the regulation of downstream effectors.