Osmotic and hypoxic induction of the complement factor C9 in cultured human retinal pigment epithelial cells: Regulation of VEGF and NLRP3 expression

Variants of complement factor genes, hypoxia and oxidative stress of the outer retina, and systemic hypertension affect the risk of age-related macular degeneration. Hypertension often results from the high intake of dietary salt that increases extracellular osmolarity. We determined the effects of extracellular hyperosmolarity, hypoxia, and oxidative stress on the expression of complement genes in cultured (dedifferentiated) human RPE cells and investigated the effects of C9 siRNA and C9 protein on RPE cells. Hyperosmolarity was induced by adding 100 mM NaCl or sucrose to the culture medium. Hypoxia was induced by culturing cells in 1% O or by adding the hypoxia mimetic CoCl . Oxidative stress was induced by adding H O . Gene and protein expression levels were determined with real-time RT-PCR, western blot, and ELISA analyses. The expression of the nuclear factor of activated T cell 5 (NFAT5) and complement factor (C9) was knocked down with siRNA. Extracellular hyperosmolarity, hypoxia, and oxidative stress strongly increased the transcription of the C9 gene, while the expression of the C3, C5, CFH, and CFB genes was moderately altered or not altered at all. Hyperosmolarity also induced a moderate increase in the cytosolic C9 protein level. The hyperosmotic C9 gene expression was reduced by inhibitors of the p38 MAPK, ERK1/2, JNK, and PI3K signal transduction pathways and of the transcription factors STAT3 and NFAT5. The hypoxic C9 gene expression was reduced by a STAT3 inhibitor. The knockdown of C9 with siRNA decreased the hypoxic vascular endothelial growth factor (VEGF) and NLRP3 gene expression, the hypoxic secretion of VEGF, and the hyperosmotic expression of the NLRP3 gene. Exogenous C9 protein inhibited the hyperosmotic expression of the C9 gene, the hypoxic and hyperosmotic VEGF gene expression, and the hyperosmotic expression of the NLRP3 gene. Both C9 siRNA and C9 protein inhibited inflammasome activation under hyperosmotic conditions, as indicated by the decrease in the cytosolic level of mature IL-1β. The expression of the C9 gene in cultured RPE cells is highly induced by extracellular hyperosmolarity, hypoxia, and oxidative stress. The data may support the assumption that C9 gene expression may stimulate the expression of inflammatory (NLRP3) and angiogenic growth factors (VEGF) in RPE cells. Extracellular C9 protein may attenuate this effect, in part via negative regulation of the C9 mRNA level.