Hepatitis B virus downregulates vitamin D receptor levels in hepatoma cell lines, thereby preventing vitamin D-dependent inhibition of viral transcription and production

Abstract

Background: Vitamin D is a key immune-modulator that plays a role in the innate and adaptive immune systems. Certain pathogens impair the immune defense by downregulating the vitamin D receptor (VDR) pathway. Low serum levels of vitamin D are associated with increased hepatitis B virus (HBV) replication. Our study aimed to assess the in-vitro relationship between HBV production and Vitamin D signaling pathway and to explore the associated mechanism(s).

Methods: HBV transcription and replication was evaluated by qRT-PCR of the HBV-RNA and covalently closed circular DNA (cccDNA). Furthermore, we have transfected the 1.3 X HBV-Luc plasmid to the cells and measured the Luciferase activity using Luminometer. Vitamin D signaling pathway activation was evaluated by measuring the expression levels of VDR, CYP24A1, Tumor necrosis factor α (TNFα) and cathelicidin (CAMP) by qRT-PCR. All assays were performed on HepG2.2.15, HepG2, and HepAD38 cells treated with or without Vitamin D active metabolite: calcitriol.

Results: Calcitriol did not suppress HBV transcription, cccDNA expression or HBV RNA levels in HepG2.2.15 cells. However, VDR transcript levels in HepG2.215 cells were significantly lower compared to HepG2 cells. Similar results were obtained in HepAD38 cell where VDR expression was down-regulated when HBV transcript level was up-regulated. In addition, calcitriol induced VDR-associated signaling, resulting in upregulation of CYP24A1, TNFα and CAMP expression level in HepG2 cells but not in the HepG2.2.15 cells.

Conclusions: These findings indicate that VDR expression is downregulated in HBV-transfected cells, thereby preventing vitamin D from inhibiting transcription and translation of HBV in vitro. HBV might use this mechanism to avoid the immunological defense system by affecting both TNFα and CAMP signaling pathways.

Keywords: Downregulation; Hepatitis B virus (HBV); Immune system; Vitamin D; Vitamin D receptor (VDR).

Fig. 1

Treatment with increasing concentrations of Calcitriol does not affect the transcription and expression levels of HBV genes and cccDNA. HepG2 cells were transfected with the 1.3 X HBV-Luc plasmid. 24 h later, the cells were treated with increasing concentrations of Calcitriol or EtOH, as a control. After an additional 24 h, the cells were harvested and analyzed for Luciferase activity (a). HBV-RNA (b), HBX (c) and cccDNA (d) were measured using RT-PCR. Results are expressed as mean ± standard deviation

Fig. 2

VDR transcript and protein levels were lower in HepG2–2.15 compared to HepG2 cells. HepG2.2.15 and HepG2 cells were harvested and the levels of VDR RNA (a) and protein (b) were measured using RT-PCR and western blot analysis, respectively (p < 0.001). The western blot presented in the figure is a representative of three different experiments; all of these experiments were calculated in the quantification. Results are expressed as mean ± standard deviation

Fig. 3

VDR transcript levels after the induction of HBV expression in HepAD38 cells. HepAD38 cell were cultured in the presence of 0.3 μg/ml tretracyline. In the next stage, HepAD38cells were washed and the medium was replaced by tetracycline free medium. HBV (a) and VDR (b) transcripts levels were measured using RT-PCR. VDR protein levels (c) were measured using western blot analysis. The western blot presented in the figure is a representative of three different experiments; all of these experiments were calculated in the quantification. Results are expressed as mean ± standard deviation

Fig. 4

Up-regulation in CYP24A1 expression level as a result of calcitriol treatment is attenuated in HBV-transfected cells. HepG2.2.15 and HepG2 cells were treated with either 1 nM or 10 nM of Calcitriol or with EtOH, as a control. 24 h later, the cells were harvested and CYP24A1 transcript levels were measured using RT-PCR. (HepG2 vs HepG2 HepG2.2.15 (1 nM) p = 0.048, HepG2 vs HepG2 HepG2.2.15 10 nM p = 0.028). Results are expressed as mean ± standard deviation

Fig. 5

TNFα and CAMP expression levels were significantly lower in Calcitriol-treated HepG2 HepG2.2.15 as compared with HepG2 cells. HepG2.2.15 and HepG2 cells were treated with 10 nM Calcitriol or EtOH, as a control. 24 h later, the cells were harvested and TNFα (a) and CAMP (b) levels were measured by RT-PCR. (p = 0.023 and p = 0.0373, respectively). Results are expressed as mean ± standard deviation