The hepatitis B virus X protein elevates cytosolic calcium signals by modulating mitochondrial calcium uptake - PubMed (original) (raw)

The hepatitis B virus X protein elevates cytosolic calcium signals by modulating mitochondrial calcium uptake

Bei Yang et al. J Virol. 2012 Jan.

Abstract

Chronic hepatitis B virus (HBV) infections are associated with the development of hepatocellular carcinoma (HCC). The HBV X protein (HBx) is thought to play an important role in the development of HBV-associated HCC. One fundamental HBx function is elevation of cytosolic calcium signals; this HBx activity has been linked to HBx stimulation of cell proliferation and transcription pathways, as well as HBV replication. Exactly how HBx elevates cytosolic calcium signals is not clear. The studies described here show that HBx stimulates calcium entry into cells, resulting in an increased plateau level of inositol 1,4,5-triphosphate (IP3)-linked calcium signals. This increased calcium plateau can be inhibited by blocking mitochondrial calcium uptake and store-operated calcium entry (SOCE). Blocking SOCE also reduced HBV replication. Finally, these studies also demonstrate that there is increased mitochondrial calcium uptake in HBx-expressing cells. Cumulatively, these studies suggest that HBx can increase mitochondrial calcium uptake and promote increased SOCE to sustain higher cytosolic calcium and stimulate HBV replication.

PubMed Disclaimer

Figures

Fig 1

The plateau level of IP3-linked cytosolic calcium signal is increased in HBx-expressing cells. (A) ATP-induced calcium response in HepG2 cells derives from calcium mobilization from the intracellular calcium pool. ATP-induced calcium responses were measured in normal ECM buffer and calcium-free ECM buffer. Representative traces of calcium responses in both buffers are shown. The amplitude (initial spike, marked with an arrow) of the calcium response in calcium-free buffer was similar to the calcium amplitude in normal ECM buffer. The plateau of the calcium responses (marked with an arrow) differed under the two conditions. Amplitudes of ATP-induced calcium signals are summarized graphically in the right panel. The amplitude of the calcium spike was calculated as the changed Fura-2 ratio at the peak versus the basal level (_R_peak − _R_basal). The data represent the means ± SE and are taken from at least three experiments from different cell preparations. (B) Forty-eight hours after transfection, HBx expression was detected via Western blot analysis. (C) Representative traces of calcium responses to maximal ATP stimulation in HBx-expressing and control cells. Cells were stimulated with 100 μM ATP, and the calcium spike from each single cell was recorded as described in Materials and Methods. Right panels show the amplitude and plateau level of calcium spikes in HBx-expressing and control cells. The amplitude of the calcium spike was determined as described above. The plateau of the calcium spike was reached at 150 s after the peak in most cells. The plateau level was calculated as the percentage of the amplitude at 150 s after the peak [(_R_150S after peak − _R_basal)/amplitude]. HBx significantly increased the plateau level of the [Ca2+]c spike (P = 0.004). The data represent the means ± SE and are taken from at least three experiments from different cell preparations.

Fig 2

HBx increases the plateau level of the IP3-linked cytosolic calcium signal in the context of HBV replication. HepG2 cells were transfected with HBV or HBx-deficient HBV (*7) expression plasmids. Twenty-four hours after transfection, IP3-linked cytosolic calcium signals were measured after ATP stimulation. (A) Representative traces of calcium responses to 100 μM ATP stimulation in HBV- and *7-expressing cells. The calcium spike from each cell was recorded as described in Materials and Methods. (B) The amplitude and plateau level of the calcium spikes in HBV- and *7-expressing cells. The amplitude and plateau were calculated as described for the Fig. 1 studies. HBx significantly increased the plateau level of the [Ca2+]c spike (P = 0.03) in the context of HBV replication. The data represent the means ± SE and are taken from at least three experiments from different cell preparations.

Fig 3

Increased HBx-induced calcium entry is required for the increased plateau level of IP3-linked calcium signals in HBx-expressing cells. (A) The calcium response to maximal ATP stimulation in calcium-free buffer in HBx-expressing (n = 25) or control (n = 43) cells. Data are average calcium responses from three experiments from different cell preparations. (B) ATP-induced calcium entry across the plasma membrane. Calcium entry across the plasma membrane was induced by the addition of 1 mM or 2 mM CaCl2 to the calcium-free buffer. (C) ATP-induced calcium entry in HBx-expressing and control cells. Amplitude was calculated as the changed Fura-2 ratio at the peak versus the basal level; time to amplitude is the time required to reach the calcium peak after CaCl2 addition. The initial rate of calcium entry was calculated as the amplitude divided by the time to amplitude. The rate of calcium entry was significantly increased in HBx-expressing cells (P = 0.0014). Representative traces of ATP-induced calcium entry are shown. The data represent the means ± SE and are taken from at least three experiments from different cell preparations.

Fig 4

The CRAC channel is responsible for the increased plateau of the IP3-linked calcium spike in HBx-expressing cells. (A) The Orai1E106A dominant-negative mutant efficiently blocks TG-induced SOCE in HepG2 cells. TG-induced SOCE was recorded, and representative traces are shown. Amplitude of SOCE was calculated as the changed Fura-2 ratio at the peak versus the basal level. The data represent the means ± SE and are taken from at least three experiments from different cell preparations. (B) Blocking the CRAC channel inhibits the increased plateau of the calcium spike in HBx-expressing cells. HBx, OraiE106A-expressing cells, pcDNA3.1(−), OraiE106A-expressing cells, HBx, pIRES vector control-expressing cells, and pcDNA3.1(−), pIRES vector control-expressing cells were identified by CFP. The single-cell calcium response to maximal ATP stimulation in these cells was recorded. Amplitude and plateau levels were defined as for Fig. 1C. Representative traces are shown, and all data represent the means ± SE and are taken from at least three experiments from different cell preparations.

Fig 5

SOCE is required for HBV replication in HepG2 cells. (A) Fifty micromolar 2-APB efficiently blocks TG-induced SOCE in HepG2 cells. Representative traces of SOCE are shown. Amplitude of SOCE represents the means ± SE and is taken from at least three experiments from different cell preparations. (B) SOCE inhibitors reduce HBV replication in HepG2 cells. APB, La3+, or vehicle controls (DMSO) were added immediately after pGEMHBV transfection, and 96 h after transfection, cells were collected and HBV replication was assessed (see Materials and Methods). Cell viability under each condition was assayed by using trypan blue; no difference between vehicle control-treated and APB- or La3+-treated cells was noted (not shown). Cell lysates were also used to detect HBcAg and tubulin expression via Western blot analysis. RC, relaxed circle; DL, double-stranded linear; SS, single stranded. The results shown are representative samples from four independent experiments. (C) Orai1 can be efficiently knocked down by Orai1 siRNAs in HepG2 cells. Forty-eight and seventy-two hours after siRNA transfection, the Orai1 expression level was detected by Western blot analysis. The anti-Orai1 antibody detects the ∼50-kDa Orai1 protein; additional bands between 35 and 50 kDa can also be detected and are thought to represent different glycosylated forms of Orai1. “Neg” denotes the scrambled, negative-control siRNAs. (D) Orai1 knockdown can block SOCE in HepG2 cells. Forty-eight hours after siRNA transfection, SOCE was measured in Orai1 siRNA-transfected and control siRNA-transfected cells. Data shown are representative average SOCE responses from Orai1 siRNA-transfected cells and negative-control siRNA-transfected cells from three independent experiments. (E) HBV replication is reduced in Orai1-knockdown cells. Twelve hours after siRNA transfection, cells were transfected with pGEMHBV. HBV replication, tubulin, and HBcAg were assayed 72 h and 96 h after pGEMHBV transfection. The results shown are representative samples from three independent experiments.

Fig 6

Blocking mitochondrial calcium uptake inhibits the increased plateau level of IP3-linked calcium spikes in HBx-expressing HepG2 cells. HBx-expressing or control cells were pretreated with CCCP and oligomycin, antimycin and oligomycin, or DMSO (vehicle control) for 5 min, and then calcium responses to ATP stimulation were recorded. (A) The HBx-induced elevation of the plateau of IP3-linked calcium spikes is inhibited by blocking mitochondrial calcium uptake. The plateau levels of calcium spikes in HBx-expressing and control cells with different treatments are shown. Amplitude and plateau levels were defined as for Fig. 1C. (B) Representative traces are shown. The data represent the means ± SE and are taken from at least three experiments with different cell preparations.

Fig 7

Increased mitochondrial calcium uptake in HBx-expressing cells. (A) Mitochondrial loading of Rhod-2. Analysis by confocal microscopy demonstrated colocalization of Rhod-2 and Mitotracker green. (B) Confirmation of mitochondrial calcium response. One hundred micromolar ATP was added to stimulate the first mitochondrial calcium response. Cells were then washed with ECM buffer for 5 min and incubated with 10 μM CCCP and 5 μg/ml oligomycin or ECM buffer for 5 min. One hundred micromolar ATP was then added again to stimulate the second mitochondrial calcium response. (C) Increased mitochondrial calcium uptake in HBx-expressing cells. The mean mitochondrial calcium response in HBx-expressing and control cells is shown. HBx significantly increased the amplitude of the mitochondrial calcium responses compared to results for control cells. Amplitude of mitochondrial response was calculated as the level of Rhod-2 _F_1/_F_0 at the peak. The data represent the means ± SE and are taken from at least three experiments from different cell preparations.

References

1. Berridge MJ. 2009. Inositol trisphosphate and calcium signalling mechanisms. Biochim. Biophys. Acta 1793(6):933–940 - PubMed
1. Berridge MJ. 1993. Inositol trisphosphate and calcium signalling. Nature 361:315–325 - PubMed
1. Berridge MJ, Bootman MD, Lipp P. 1998. Calcium—a life and death signal. Nature 395:645–648 - PubMed
1. Berridge MJ, Bootman MD, Roderick HL. 2003. Calcium signalling: dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4:517–529 - PubMed
1. Berridge MJ, Lipp P, Bootman MD. 2000. The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell Biol. 1:11–21 - PubMed

The hepatitis B virus X protein elevates cytosolic calcium signals by modulating mitochondrial calcium uptake - PubMed (original) (raw)