Aquaporin 9 inhibits hepatocellular carcinoma through up-regulating FOXO1 expression.

Aquaporin 9 (AQP9) is the main aquaglyceroporin in the liver. Few studies have been performed regarding the role of AQP9 in liver cancer. Here we report AQP9 expression and function in liver cancer. We found that AQP9 mRNA and protein levels were reduced in human hepatocellular cancer compared to the para-tumor normal liver tissues. Human hepatoma cell line SMMC7721 expressed low basal levels of AQP9. When AQP9 was overexpressed in SMMC7721 cell line, cell proliferation was inhibited due to cell cycle arrest at G1 phase and increased apoptosis. At the molecular level, AQP9 overexpression decreased the protein levels of phosphatidylinositol-3-kinase (PI3K), leading to reduced phosphorylation of Akt. Subsequently, the protein levels of forkhead box protein O1 (FOXO1) were increased, resulting in down-regulation of proliferating cell nuclear antigen (PCNA) expression and up-regulation of caspase-3 expression. AQP9 overexpression inhibited growth of subcutaneously xenografted liver tumors in nude mice. These findings suggest that AQP9 expression is down-regulated in liver cancer compared to the normal liver tissue and restoration of AQP9 expression can inhibit development of liver cancer.

FOXO1-mediated epigenetic modifications are involved in the insulin-mediated repression of hepatocyte aquaporin 9 expression.

Aquaporin (AQP) 9 transports glycerol and water, and belongs to the aquaglyceroporin subfamily. Insulin acts as a negative regulator of AQP9, and FOXO1 has the ability to mediate the regulatory effects of insulin on target gene expression. The aim of the present study was to determine whether insulin­induced repression of AQP9 involved an epigenetic mechanism. HepG2 human hepatocyte cells were treated with 500 µM insulin for different durations. AQP9 mRNA expression levels were determined by quantitative polymerase chain reaction (qPCR), and histone H3 acetylation, phosphorylation and methylation at the insulin responsive element (IRE) of the AQP9 promoter was assessed using chromatin immunoprecipitation coupled with qPCR. The effects of lentiviral FOXO1 overexpression on AQP9 expression levels and H3 modifications at the AQP9 promoter were also determined. The insulin treatment resulted in a significant and time­dependent reduction in AQP9 mRNA expression levels in HepG2 cells, as compared with untreated cells (P<0.05). In the insulin­treated cells, the levels of H3 acetylation and phosphorylation were significantly reduced (P<0.05), but the level of H3 methylation was increased. Enforced expression of FOXO1 increased AQP9 mRNA and protein expression levels in HepG2 cells. Furthermore, FOXO1 overexpression promoted H3 acetylation and phosphorylation, and reduced H3 methylation at the IRE locus of the AQP9 promoter. These data provide, to the best of our knowledge, the first evidence that insulin­induced transcriptional suppression of AQP9 expression in hepatocytes involves FOXO1­mediated H3 modifications at the IRE locus in the promoter.

[Expression of aquaporin 3 and aquaporin 9 is regulated by oleic acid through the PI3K/Akt and p38 MAPK signaling pathways].

OBJECTIVE: To study the effect of oleic acid (OA) on expression of aquaglyceroporin genes, AQP3 and AQP9, in hepatocyte steatosis and to investigate the underlying molecular mechanisms using an in vitro system. METHODS: HepG2 cells were treated with OA at different concentration to establish in vitro models of nonalcoholic hepatocyte steatosis. The corresponding extents of hepatic steatosis modeling were assessed by oil red O staining and optical density (OD) measurements of the intracellular fat content. The model lines were then treated with inhibitors of the PI3K/Akt and p38 MAPK signaling pathway factors and effects on AQP3/9 expression was measured by real time RT-PCR and western blotting. RESULTS: The fat concentration, indicative of hepatic steatosis, increased in conjunction with increased concentrations of OA (0 less than 250 less than 500 mumol/L). OA exposure also down-regulated AQP3 mRNA and up-regulated AQP9 mRNA levels in a concentration-dependent manner. The most robust changes in expression occurred in response to the 500 mumol/L concentration of OA for both AQP3 (0.47+/-0.18; t = 4.5450, P less than 0.05) and AQP9 (1.57+/-0.21; t = 3.0306, P less than 0.05). Treatment with OA + PI3K pathway inhibitor (LY294004) significantly decreased AQP9 mRNA expression (4.55+/-0.62) as compared to the control group (1.00+/-0.10; t = 9.7909, P less than 0.01), that 500 mumol/L OA group (2.43+/-0.53; t = 4.5018, P less than 0.05), and the LY294002 group (1.90+/-0.16; t = 7.1683, P less than 0.01). Treatment with p38 MAPK pathway inhibitor (SB230580) significantly increased the OA-suppressed level of AQP3 mRNA to the level detected in the control group (1.27+/-0.11; t = 5.7455, P less than 0.01) and decreased the OA-stimulated AQP9 mRNA (0.38+/-0.09; t = 6.5727, P less than 0.01). No significant changes in mRNA expression of AQP3/9 were observed with inhibition of the ERK1/2 and JNK signal transduction pathways. The OA-induced changes in protein expression levels of AQR3 and AQP9 followed a similar trend of the genes. Finally, OA suppressed the level of phosphorylated Akt (from 0.21+/-0.02 to 0.13+/-0.03; t = 3.8431, P less than 0.05) but elevated the level of phosphorylated p38 (from 0.58+/-0.06 to 1.02+/-0.10; t = 12.5289, P less than 0.01). Again, OA treatment produced no significant affect on ERK1/2 and JNK phosphorylation. CONCLUSION: OA down-regulates AQP3 expression by stimulating the p38 MAPK signaling pathway, and up-regulates the AQP9 by blocking the PI3K/Akt pathway and activating the p38 MAPK signaling pathway.