Cholesterol metabolite, 5-cholesten-3β-25-diol-3-sulfate, promotes hepatic proliferation in mice

Abstract

Oxysterols are well known as physiological ligands of liver X receptors (LXRs). Oxysterols, 25-hydroxycholesterol (25HC) and 27-hydroxycholesterol as endogenous ligands of LXRs, suppress cell proliferation via LXRs signaling pathway. Recent reports have shown that sulfated oxysterol, 5-cholesten-3β-25-diol-3-sulfate (25HC3S) as LXRs antagonist, plays an opposite direction to oxysterols in lipid biosynthesis. The present report was to explore the effect and mechanism of 25HC3S on hepatic proliferation in vivo. Following administration, 25HC3S had a 48 h half life in the circulation and widely distributed in mouse tissues. Profiler™ PCR array and RTqPCR analysis showed that either exogenous or endogenous 25HC3S generated by overexpression of oxysterol sulfotransferase (SULT2B1b) plus administration of 25HC significantly up-regulated the proliferation gene expression of Wt1, Pcna, cMyc, cyclin A, FoxM1b, and CDC25b in a dose-dependent manner in liver while substantially down-regulating the expression of cell cycle arrest gene Chek2 and apoptotic gene Apaf1. Either exogenous or endogenous administration of 25HC3S significantly induced hepatic DNA replication as measured by immunostaining of the PCNA labeling index and was associated with reduction in expression of LXR response genes, such as ABCA1 and SREBP-1c. Synthetic LXR agonist T0901317 effectively blocked 25HC3S-induced hepatic proliferation.

Conclusions: 25HC3S may be a potent regulator of hepatocyte proliferation and oxysterol sulfation may represent a novel regulatory pathway in liver proliferation via inactivating LXR signaling.

Fig. 1. Pharmacokinetics and tissue biodistribution of radioactivity following intravenous injection of [³H]-25HC3S and 25HC3S in mice

Each point represents one animal.

Fig. 2. Expression levels of cell cycle-related genes in response to 25HC3S in mouse liver tissues

Mice were treated with 25HC3S at different concentrations as indicated in Section 2.3 for 48 h. Relative mRNA levels of FoxM1b (A), CDC25b (B), Cyclin A (C), and C-myc (D) were analyzed by RTqPCR at the end of the treatment. The results are shown as mean ± S.D. (n=3-5/group) ^*P<0.05 vs. mRNA expression at 0 mg/kg concentration.

Fig. 3. Effect of exogenous 25HC3S on PCNA labeling index in mouse liver tissues

A and B show representative photomicrographs from PCNA-stained liver sections of vehicle (PBS 10% ethanol, 48 h) group (A), and 25HC3S (5 mg/kg, 48 h) group (B). And immunoreaction was observed as strong reaction (short arrow) or weak reaction (long arrow). PCNA labeling index obtained from liver sections of each group was quantitatively analyzed (C). The results are shown as mean ± S.D. (n=3-5/group) ^*P<0.05 vs. Vehicle.

Fig. 4. Effect of endogenous 25HC3S on PCNA labeling index in mouse liver tissues

Mice were infected with either Ad-Control or Ad-SULT2B1b (1×10⁸ pfu) in the presence or absence of 25HC (25 mg/kg) as indicated in Section 2.3 for 5 d. PCNA labeling index obtained from liver sections of each group were analyzed at the end of the treatment. The results are shown as mean ± S.D. (n=3-5/group) ^*P<0.05 vs. corresponding Ad-Control, #P<0.05 vs. Ad-SULT2B1b and vehicle co-treatment.

Fig. 5. Effect of exogenous 25HC3S on LXR activity and its target gene expressions in mouse liver tissues

Mice were treated with vehicle or 25HC3S (5 mg/kg) as indicated in Section 2.3 for 48 h. LXRα, SREBP-1c, ABCA1, and PCNA proteins were detected by western blot at the end of the treatment (A). Western blot data were quantitatively normalized to ß-actin (B). The results are shown as mean ± S.D. (n=3-5/group) ^*P <0.05 vs. Vehicle.

Fig. 6. Effect of endogenous 25HC3S on LXR activity and its target gene expressions in mouse liver tissues

Mice were infected for 5 d with either Ad-Control or Ad-SULT2B1b (1×10⁸ pfu) in the presence or absence of 25HC (25 mg/kg) as indicated in Section 2.3. LXRα, SREBP-1c, ABCA1, and PCNA proteins were detected by western blot at the end of the treatment (A). Western blot data were quantitatively normalized to ß-actin (B). The results are shown as mean ± S.D. (n=3-5/group) ^*P<0.05 vs. Ad-Control and vehicle co-treatment.

Fig. 7. Effect of LXR activation on 25HC3S-induced proliferation

Mice were treated with either vehicle or 25HC3S (5 mg/kg) in the presence or absence of T0901317 (5 mg/kg) as indicated in Section 2.3 for 48 h. LXRα, SREBP-1c, ABCA1, and PCNA proteins were detected by western blot at the end of the treatment (A). Western blot data were quantitatively normalized to ß-actin (B). The results are shown as mean ± S.D. (n=3-5/group) ^*P<0.05 vs. Vehicle.