The Effect And Mechanism Of Oxysterol Sulfotransferase (SULT2B1b) And Its Product25HC3S On Hepatocyte Proliferation In Vivo And In Vitro

IntroductionCytosolic sulfotransferase (SULT)2Blb catalyzes sulfation of oxysterol and hydroxysterol, including25-hydroxycholesterol (25HC). The main product of this reaction is25-hydroxycholesterol-3-sulfate (25HC3S). It has been reported that SULT2Blb inhibits the activation of oxysterol to Liver X Receptors (LXRs) and25HC3S decreases cellular lipids via suppressing LXR/sterol regulatory element-binding proteins (SREBPs) signaling in THP-1derived macrophages. Furthermore, LXR activation has been identified as anti-proliferative factor in several cellular and animal models. Therefore, the key enzyme for the biosynthesis of25HC3S, SULT2Blb, may play a crucial role in regulating liver proliferation. In the present study, we investigated the effects and mechanisms of SULT2B1b and its product-25HC3S in liver proliferation in mouse liver with or without partial hepatectomy (PH) and in primary rat hepatocyte (PRH).Part Ⅰ. Effect of SULT2Blb on hepatic proliferation in vivo and in vitroSection I:The effect and mechanism of cytosolic sulfotransferase2Blb (SULT2B1b) on proliferation in mouse liver tissues and in primary rat hepatocyte (PRH)Aims:To investigate the effect and the underlying mechanism of SULT2Blb on liver proliferation in vivo and in vitro. Methods:Primary rat hepatocyte (PRH) and C57BL/6mice were infected with adenovirus encoding SULT2B1b. Liver proliferation was determined by measuring the proliferating cell nuclear antigen (PCNA) immunostaining labeling index. The correlation between the SULT2B1b and PCNA expression in mouse liver tissues were determined by double immunofluorescence. Gene expressions were evaluated by quantitative real-time PCR and western blot analysis.Results:SULT2B1b overexpression in mouse liver tissues increased PCNA-positive cells in a dose-and time-dependent manner. The expression of PCNA in mouse liver tissues was almost observed in the SULT2Blb-transgenic cells, while PCNA is undetectable in the cells without SULT2Blb overexpression. Small interference RNA-SULT2B1b significantly inhibited cell cycle regulatory gene expressions in primary rat hepatocytes (PRH). LXR activation by T0901317, effectively suppressed SULT2B1b-induced gene expression both in vivo and in vitro.Conclusions:SULT2B1b may promote hepatocyte proliferation via inactivating oxysterol/LXR signaling in vivo and in vitro.Section Ⅱ:Effect of SULT2Blb on liver regeneration following mouse partial hepatectomy (PH)Aims:To evaluate the effect and mechanism of SULT2B11b on liver regeneration.Methods:C57BL/6mice were performed PH and infected with Ad-SULT2B1b or Ad-control for5days. Liver proliferation was determined by measuring the PCNA immunostaining labeling index. Levels of hepatic oxysterols were analysed by HPLC. Gene expressions were evaluated by quantitative real-time PCR and western blot analysis. Results:C57BL/6mice infected with Ad-SULT2Blb exhibited a faster liver re-growth, taking3days to reach80%of their original liver mass, while control mice took nearly5days to reach this liver mass. Following PH, SULT2B1b overexpression resulted in an apparent reduction of oxysterols coupled with a further down-regulation of the LXR signaling while the expression of proliferative genes were further increased. T0901317, a synthetic agonist of LXR, completely blocked SULT2B1b-induced increases in PCNA protein and decreases in the LXR signaling pathway.Conclusions:Increases in SULT2B1b promote Liver regeneration after PH via inhibiting LXR signaling.Part II. Effect of cholesterol metabolite (25HC3S) on hepatic proliferation in miceAims:To determine the effects of exogenous or endogenous25HC3S on the hepatic proliferation in vivo of C57BL/6mice.Methods:C57BL/6mice were injected with exogenous25HC3S, or infected with adenovirus encoding SULT2B1b, combined by25HC administration to get endogenous25HC3S. The biodistribution of exogenous25HC3S in tissues and the formation of endogenous25HC3S in liver was examined by [3H]-25HC3S radioactivity analysis. Hepatic gene expressions were evaluated by quantitative real-time PCR, cell cycle RT2ProfilerTM PCR array, and western blot analysis. Liver proliferation was determined by measuring the proliferating cell nuclear antigen (PCNA) immunostaining labeling index (LI). Results:Following exogenous administration,25HC3S had a48h half life in circulation and widely distributed in mouse tissues.25HC3S or overexpression of SULT2B1b plus administration of25HC (endogenous25HC3S) significantly up-regulated the proliferation gene expression of Wt1, Pcna, cMyc, cyclin A, FoxM1b, and CDC25b in a dose-dependent manner in liver; substantially down-regulated the expression of cell cycle arrest gene Chek2and apoptotic gene Apaf1. Both exogenous and endogenous administration of25HC3S significantly induced hepatic DNA replication as measured by immunostaining of the PCNA labeling index and associated with reduction in expression of LXRs response genes, such as ABCA1and SREBPlc. LXR activation by synthetic agonist T0901317effectively blocked the25HC3S-induced hepatic proliferation.Conclusions:25HC3S is a potent regulator of proliferation and the oxysterol sulfation may represent a novel regulatory pathway in liver proliferation via inactivating LXR signaling.In summary,25HC sulfation by SULT2B1b may promote hepatic proliferation and regeneration by down-regulation of LXR signaling pathway in vitro PRH and in vivo mouse liver tissues.