Prosurvival Bcl-2 proteins stabilize pancreatic mitochondria and protect against necrosis in experimental pancreatitis

Abstract

Acinar cells in pancreatitis die through apoptosis and necrosis, the roles of which are different. The severity of experimental pancreatitis correlates directly with the extent of necrosis and inversely, with apoptosis. Apoptosis is mediated by the release of cytochrome c into the cytosol followed by caspase activation, whereas necrosis is associated with the mitochondrial membrane potential (ΔΨm) loss leading to ATP depletion. Here, we investigate the role of Bcl-2 proteins in apoptosis and necrosis in pancreatitis. We found up-regulation of prosurvival Bcl-2 proteins in pancreas in various experimental models of acute pancreatitis, most pronounced for Bcl-xL. This up-regulation translated into increased levels of Bcl-xL and Bcl-2 in pancreatic mitochondria. Bcl-xL/Bcl-2 inhibitors induced ΔΨm loss and cytochrome c release in isolated mitochondria. Corroborating the results on mitochondria, Bcl-xL/Bcl-2 inhibitors induced ΔΨm loss, ATP depletion and necrosis in pancreatic acinar cells, both untreated and hyperstimulated with CCK-8 (in vitro pancreatitis model). Together Bcl-xL/Bcl-2 inhibitors and CCK induced more necrosis than either treatment alone. Bcl-xL/Bcl-2 inhibitors also stimulated cytochrome c release in acinar cells leading to caspase-3 activation and apoptosis. However, different from their effect on pronecrotic signals, the stimulation by Bcl-xL/Bcl-2 inhibitors of apoptotic responses was less in CCK-treated than control cells. Therefore, Bcl-xL/Bcl-2 inhibitors potentiated CCK-induced necrosis but not apoptosis. Correspondingly, transfection with Bcl-xL siRNA stimulated necrosis but not apoptosis in the in vitro pancreatitis model. Further, in animal models of pancreatitis Bcl-xL up-regulation inversely correlated with necrosis, but not apoptosis. Results indicate that Bcl-xL and Bcl-2 protect acinar cells from necrosis in pancreatitis by stabilizing mitochondria against death signals. We conclude that Bcl-xL/Bcl-2 inhibition would aggravate acute pancreatitis, whereas Bcl-xL/Bcl-2 up-regulation presents a strategy to prevent or attenuate necrosis in pancreatitis.

Introduction

Acinar cell death is a major pathological response of acute pancreatitis; in particular, parenchymal necrosis is a major cause of severe complications and mortality in human pancreatitis [1], [2]. In models of acute pancreatitis acinar cells die through both necrosis and apoptosis. The severity of experimental pancreatitis correlates directly with the extent of necrosis and inversely, with apoptosis [2], [3], [4], [5], [6], [7], [8]. Thus, elucidating the mechanisms that mediate acinar cell death in pancreatitis is important for understanding the mechanism of this disease and is of clinical relevance.

Mechanisms underlying these major forms of cell death are different [9], [10], [11], [12], although they both involve mitochondria. Apoptosis is mediated by the release of cytochrome c from mitochondria into the cytosol. Once in cytosol, cytochrome c causes activation of specific cysteine proteases, the caspases (e.g., the major effector caspase-3), which execute apoptotic cell death [9], [10], [11], [12], [13]. On the other hand, necrosis is mediated by the loss of mitochondrial membrane potential (ΔΨm). Which ultimately leads to depletion of cellular ATP and necrosis [9], [11], [12], [14]. Depolarization is mediated by opening of the mitochondrial permeability transition pore (PTP), a multi-subunit complex formed by proteins residing in both inner and outer mitochondrial membrane. PTP opening is associated with swelling of mitochondrial matrix and consequent rupture of the outer mitochondrial membrane [11], [15], [16], which allows the release of cytochrome c. Recent data on mice lacking cyclophilin D [15], [17] show, however, that cytochrome c can be released independent of PTP, through the channels in the outer mitochondrial membrane [9], [11], [15], [16], [17]. We have recently showed [18], [19], [20] that in isolated pancreatic mitochondria PTP mediates loss of ΔΨm but not cytochrome c release.

Bcl-2 family proteins are important regulators of cell death, particularly apoptosis [9], [11], [21]. They act through regulating of mitochondrial outer membrane permeabilization, which mediates cytochrome c release into cytosol [9], [11], [21]. Much less is known on the role of Bcl-2 proteins in the regulation of mitochondrial depolarization leading to necrosis [22], [23].

Bcl-2 proteins are subdivided into 3 groups on the basis of their Bcl-2 homology (BH) domains. The “prosurvival” members, such as Bcl-2 itself and Bcl-xL, contain four BH domains (BH1–BH4). The “pro-apoptotic” members, such as Bax and Bak, contain three BH domains; and the “BH3-only” proapoptotic proteins, such as Bad, Puma and Noxa, only contain the BH3 domain.

Each of the 3 groups of the Bcl-2 family proteins has specific functional roles in the regulation of apoptosis [9], [11], [13], [24]. In particular, the pro-apoptotic Bax and Bak form channels in the outer mitochondrial membrane through which cytochrome c is released into the cytosol (this permeability system remains poorly characterized) [13], [25]. The BH3-only proteins facilitate Bax/Bak channel formation, and thus cytochrome c release and apoptosis [9], [11]. On the other hand, the prosurvival Bcl-xL and Bcl-2 inhibit apoptosis by sequestering BH3-only proteins (as well as Bax and Bak) [9], [11]. Bcl-2 can also block PTP opening, thus preventing loss of ΔΨm and subsequent necrosis [22], [23]. Small-molecule pharmacological inhibitors of the prosurvival Bcl-xL and Bcl-2 have recently been developed and became a valuable tool to study the roles of these proteins [26], [27], [28].

We and others showed that cytochrome c release and mitochondrial depolarization occur and mediate acinar cell death in pancreatitis [5], [29], [30]. However, there is little known on the roles of Bcl-2 proteins in apoptotic and necrotic cell death in pancreatitis [2], [31].

Here, we measured changes in the levels of various Bcl-2 proteins in models of acute pancreatitis and found marked up-regulation of the prosurvival protein Bcl-xL (as well as Bcl-2) in both total pancreatic tissue and pancreatic mitochondria. Using pharmacological Bcl-xL/Bcl-2 inhibitors and Bcl-xL knockdown with Bcl-xL siRNA transfection, we assessed the role of Bcl-xL and Bcl-2 in the regulation of ΔΨm, cytochrome c release and subsequent necrosis and apoptosis in isolated pancreatic mitochondria, intact pancreatic acinar cells and in acinar cells hyperstimulated with CCK-8, the experimental system considered in vitro model of acute pancreatitis [5], [7], [29], [32], [33], [34], [35], [36].

The results indicate that by preventing mitochondrial depolarization and subsequent ATP depletion, Bcl-xL and Bcl-2 protect acinar cells in pancreatitis against necrosis (rather than apoptosis). They suggest that Bcl-xL/Bcl-2 inhibition, which is applied in clinical trials to stimulate apoptotic death of cancer cells, would likely increase necrosis and thus the severity of acute pancreatitis. By contrast, Bcl-xL/Bcl-2 up-regulation or stabilization may represent a promising strategy to prevent or attenuate necrosis in pancreatitis.