Caspase-dependent Alterations of Ca2+ Signaling in the Induction of Apoptosis by Hepatitis B Virus X Protein

Abstract

The hepatitis B virus X protein (HBx) is a multifunctional protein, acting on different targets (e.g. transcription factors, cytoplasmic kinases, and mitochondrial proteins) and exerting cellular effects as diverse as stimulation of cell proliferation and apoptosis. In its biological effects, the modulation of cellular Ca 2؉ signals has been proposed to be involved, but the direct assessment of Ca 2؉ homeostasis in HBx-transfected cells has not been carried out yet. In this work, we have employed for this purpose aequorin-based recombinant probes specifically targeted to intracellular organelles and microdomains. Using these probes, we observed that overexpression of HBx enhanced agonist-evoked cytosolic Ca 2؉ signals in HepG2 and HeLa cells, without affecting either the steady state of endoplasmic reticulum Ca 2؉ concentration or the kinetics of Ca 2؉ release. Rather, caspase-3-dependent cleavage of the plasma membrane Ca 2؉ ATPase could be demonstrated, and larger rises were detected in the cytoplasmic rim beneath the plasma membrane. In mitochondria, major morphological (fragmentation and swelling) and functional (reduced Ca 2؉ uptake) alterations were detected in HBx-expressing cells. As to the cellular consequences, we observed that HBx-induced apoptosis was markedly reduced when the alterations in Ca 2؉ signaling (e.g. by loading a Ca 2؉ chelator or preventing PMCA cleavage) or the downstream effects (e.g. by inhibiting mitochondrial permeability transition) were prevented. Overall, these results indicate that HBx perturbs intracellular Ca 2؉ homeostasis, acting on the extrusion mechanisms, and that this effect plays an important role in the control of HBx-related apoptosis.

Bcl-2 and Ca2+ homeostasis in the endoplasmic reticulum

“…However, this is not due to ER Ca 2 þ overload, as the steady-state [Ca 2 þ ] er levels are identical to those of mock-transfected cells. 66 The mechanism leading to cell death is similar to what previously reported in staurosporin-treated neurons: the Ca 2 þ extrusion capacity of the cell is impaired by caspase-3-dependent cleavage of the plasma membrane Ca 2 þ ATPase (PMCA). 67 Indeed in HBx-transfected cells there is caspase-3 activation and PMCA cleavage, and recombinant expression of a noncleavable PMCA mutant both restored the Ca 2 þ -signalling patterns and amplitude to those of control cells and reduced the apoptotic efficiency of the viral protein.…”

“…PMCA represents a very effective target for enhancing Ca 2 þ responses, because it represents the most powerful route allowing the rapid return of [Ca 2 þ ] c to basal level. 66 Along the same lines, the Na þ /Ca 2 þ transporter type 1 is also cleaved by caspase-3 in cerebellar granule neurons undergoing apoptosis. 68 Two final aspects require attention.…”

“…HBx caused, both in HeLa and hepatic cell lines, a dramatic fragmentation and swelling of the mitochondrial network, that was inhibited (together with the apoptotic efficacy) by the PTP blocker cyclosporin A. 66 As to Bax, its primary effect on Ca 2 þ homeostasis was, early upon expression, an increased ER Ca 2 þ loading, with consequent potentiation of mitochondrial Ca 2 þ responses, then the cells progressed into an apoptotic phenotype (that included drastic reductions of the cytosolic and mitochondrial Ca 2 þ responses upon challenging of cells with stimuli causing ER Ca 2 þ release). In this later phase, no difference became detectable between the steady-state [Ca 2 þ ] er levels of controls and Bax-expressing cells.…”

Calcium signalling-dependent mitochondrial dysfunction and bioenergetics regulation in respiratory chain Complex II deficiency

“…Plasma membrane and microsomal fractions were isolated as already described. 18,40 SDS-PAGE was then performed. The signal was revealed using ECL Plus western-blot detection reagent (Amersham Biosciences, Orsay, France).…”

Mitochondria and the central nervous system: searching for a pathophysiological basis of psychiatric disorders

“…Following the initiation of necrosis, mitochondria are overloaded with Ca 2+ , the electrochemical proton gradient collapses, and necrotic cell death is induced. 61 Thus, it seems that alteration of this cellular response (for example, by a tumor or viral proteins) 51,54,62,63 plays a role in the pathogenesis of human disorders. Prolonged permeability transition pore opening leads to a complete collapse of the membrane potential and Ca 2+ release, which results in the complete loss of mitochondrial function and necrotic cell death.…”