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British Journal of Radiology (2005) Supplement_27, 106-113
© 2005 British Institute of Radiology
doi: 10.1259/bjr/18105113
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British Journal of Radiology Supplement_27 (2005),106-113 © 2005 The British Institute of Radiology

Full Paper

Clusterin proteins: stress-inducible polypeptides with proposed functions in multiple organ dysfunction

S Araki, MD, PhD 1 S Israel, MS 1 K S Leskov, PhD 1 T L Criswell, PhD 1 M Beman, BS 1 D Y Klokov, PhD 1 L Sampalth, BTECH 1 K E Reinicke, BS 1,3 E Cataldo, BS 3 L D Mayo, PhD 1,2 and D A Boothman, PhD 1,2,3,3

Departments of 1 Radiation Oncology, 2 Pharmacology and 3 Biochemistry, Laboratory of Molecular Stress Responses, Case School of Medicine and the Case Comprehensive Cancer Center, Case Western Reserve University, 2103 Cornell Road WRB 3-531 North, Cleveland, OH 44106-7285, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Severe trauma (e.g. whole body ionising radiation (IR)), leads to multiple organ dysfunction (MOD) and death. Although not completely understood, proposed triggering events of MOD include reactive oxygen species (ROS), released Ca2+ from endoplasmic reticulum (ER) stores, cell debris resulting from apoptosis and/or necrosis, and liberation of cytokines. Little is known about the defence mechanisms that protect against events that trigger MOD. We propose that the secreted form of clusterin (sCLU) is a major protective factor against trauma-induced MOD. sCLU is induced by IR and by other cytoprotective agents. Interestingly, this secreted protein is cytoprotective in a variety of cell systems following various agents. We demonstrate that sCLU is induced by thapsigargin (TG), a sarcoplasmic-endoplasmic reticulum Ca2+ ATPase (SERCA) pump inhibitor, which causes Ca2+ release from the ER. sCLU induction was delayed, with the earliest increase in sCLU levels detected at 18–24 h post IR, and peak levels noted at 72–96 h. IR and TG exposures resulted in similar induction kinetics, wherein transcription and translation of sCLU occurred with nearly identical temporal responses. Human CLU promoter activity mimicked induction of sCLU protein levels (p<0.01). Interestingly, cells pre-treated with BAPTA-AM, an intracellular Ca2+ chelator, did not prevent sCLU induction or lethality after TG or IR exposures. In fact, high dose exposures of BAPTA-AM induced sCLU responses. Thus, sCLU induction is triggered by ROS, changes in Ca2+ homeostasis and lipid peroxidation, factors prominent in triggering MOD. Future studies should directly examine the role(s) of sCLU in preventing MOD, which may be very important in comtermeasures to MOD in accident victims.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Multiple organ failure (MOF), or multiple organ dysfunction (MOD), may ensue following many different traumas, including sepsis, whole body burns, or high doses of whole body ionising radiation (IR). MOD emerged as a new syndrome approximately 25 years ago. Despite intensive investigation, it remains the leading cause of late post-injury deaths in intensive care units. By the 1930s, loss of blood volume was recognised as the primary cause of traumatic shock. In the early 1970s, a new phenomenon termed "shocked lung" was recognised as a primary cause of late deaths [1]. The application of advanced organ support (mechanical ventilators, inotropic support, total parenteral nutrition, haemodialysis) became widespread, and the number of patients dying of isolated pulmonary failure drastically decreased. It was then recognised that a new syndrome, MOD, was a major factor leading to late post-injury death. In the mid 1980s, it was reported that post-injury MOD could occur without infection [2–4].

MOD has recently been reviewed from a historical perspective [5, 6], and a multiple hit theory was proposed [7, 8]. MOD occurs due to persistent vital organ perfusion deficiencies, with many aetiologies, and carries a mortality that varies from 30% to 100% depending on the organs involved [9]. Death due to such traumas (mostly sepsis and burns) that cause MOD is a major problem in the USA, ranking first in hospital days and overall life-years lost. Furthermore, many of these patients are young [7].

Since the discovery of radioactive materials by Becquerel over 100 years ago, people have used radioactivity as an energy source, a defensive weapon, and for diagnostic and therapeutic medical tools. However, hazards in the form of nuclear power plant accidents, the threat of nuclear war, "terrorist dirty bombs" and the potential for radiation injuries from improper use of radioactive isotopes and IR exist. Death from radiation injury is never caused by injury to a single organ, but takes the form of the accumulation and interaction of severe injury to several vital physiological systems [10]. But what leads to MOF or MOD?

Efficacious treatment of MOD in patients can only succeed if the cause and triggering events of this deathly syndrome are elucidated. Likely factors that could trigger MOD after IR or other traumas include: (1) increased production of reactive oxygen species (ROS) leading not only to "waves" of calcium (Ca2+) release but also causing DNA breaks, with this mechanism also triggering a death response; (2) changes in Ca2+ flux in severely injured cells, leading to massive apoptotic responses, faulty phagocytosis, damage to neighbouring cells and generation of cell debris that circulates throughout tissues of the body; (3) increased production of stress-inducible cytokines, such as transforming growth factor-{beta}1 (TGF-{beta}1) and tumour necrosis factor-{alpha} (TNF-{alpha}), which can cause bystander effects in neighbouring cells and tissues of the body; and (4) increased necrotic cell death due to severe damage to one organ, which then results in overproduction of cytokines and cell debris, causing subsequent distal responses in many organs of the body. Many of the above factors are undoubtedly interconnected, and we proposed the following sequence of events that would stimulate MOD after various traumas to the body.

  1. Whole body exposure to trauma (especially IR) causes extensive ROS.
  2. Increases in intracellular ROS leads to "waves" of Ca2+ release from the endoplasmic reticulum (ER), and subsequent uptake into intracellular organelles (e.g. mitochondria).
  3. Massive uptake of Ca2+ by mitochondria, or other loss of balanced Ca2+ homeostasis in the ER, leads to mitochondrial permeability changes and release of cytochrome c (or other factors such as SMAC/DIABLO or AIF) from the mitochondrial inner membrane. Released cytochrome c causes activation of apaf-1 and caspase-9 (or other death effectors), which triggers cell death (apoptotic and/or necrotic) responses.
  4. Massive tissue apoptotic and/or necrotic responses, especially in localised areas of an organ, lead to extensive cell debris and/or production of cytokines that begin to circulate throughout the tissue and body. These localised areas of cell debris may also inhibit normal phagocytic responses that would otherwise clear apoptotic cells through normal "turnover" processes in each organ.
  5. Cytokines and cell debris from these areas of localised or general cell death tissue responses ultimately lead to distal apoptotic and necrotic responses in organs throughout the body that have not otherwise received lethal doses of a given traumatic agent (e.g. IR). MOD ensues.

In this paper, we focus on clusterin (CLU) gene products and their potential role(s) in MOD. We propose that production and regulation of CLU proteins play major roles in cell survival and death decision responses after various traumatic insults. Our discussion will focus on CLU protein responses after low compared with high doses of IR, and we will discuss CLU production as a response to IR in the context of its possible functions in MOD.

The CLU gene and gene products have been implicated both in cell death and survival responses. Recent work by our lab [11–16], as well as by others [17–21], has begun to clarify these apparently opposing (and confusing) functions of CLU gene products. Recent data have indicated that stress-induced expression of the CLU gene gives rise to at least two protein forms, secretory CLU (sCLU) glycoproteins and a cytoplasmically localised, apparently non-glycosylated, ~49 kDa, pre-nuclear CLU (pnCLU) protein (Figure 1Go). Overexpression of the sCLU protein in cells can provide resistance to various agents, including IR [22–24]. Our recent data using a small interfering RNA (siRNA) knockdown strategy to specifically eliminate sCLU without affecting other CLU protein forms demonstrated enhanced lethality to subsequent IR exposures (Criswell et al., JBC, in press). Thus, sCLU expression can be cytoprotective and responsive to extremely low, non-lethal doses of IR (e.g. 0.1–0.5 Gy) [11, 13, 15, 16]. The sCLU-mediated mechanism of cytoprotection has been linked to the apparent ability of sCLU to act as a molecular chaperone, wherein the secreted protein can bind to, and clear, cell debris [19, 25–27].



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  		Figure 1. Synthesis of secretory and nuclear clusterin proteins. (a) Synthesis of secretory clusterin (sCLU) protein forms is depicted. Full length clusterin (CLU) mRNA is translated with a leader polypeptide that targets the protein to endoplasmic reticulum (ER). The ER polypeptide is cleaved and a 60 kDa pre-secretory clusterin (psCLU) protein is produced. As the protein is transported to the Golgi complex, it is cleaved into {alpha} and {beta} polypeptides, which are heavily glycosylated and form a heterodimeric complex via five disulfide bonds. Ultimately, an ~80 kDa protein is excreted from the cell. (b) Intracellular CLU protein is produced from a truncated, alternatively spliced nuclear clusterin (nCLU) mRNA, which lacks the ER-targeting domain [14]. Translation of the nCLU mRNA results in ~49 kDa pre-nuclear clusterin (pnCLU) protein. This 49 kDa pnCLU is dormant in the cytoplasm, presumably by hiding the nuclear localisation site (NLS) within the C-terminal portion of the protein through homodimerisation or self-folding of the protein; pnCLU contains two coiled-coil domains that interact and cause dormancy. After significant stress (>1 Gy), pnCLU is "activated" and altered post-translationally, forming an ~55 kDa cell death protein. Expression of the C-terminus of this nCLU protein was sufficient to cause cell death [14].

 
Thus, vital neighbouring cells may assist in protecting against MOD by expressing specific "protective" proteins. Bach et al [18] showed that incubation of cells with cell debris resulted in sCLU protein induction in fibroblasts that were known to function as non-professional phagocytes. sCLU stimulated uptake of apoptotic vesicles into non-professional phagocytes, suggesting that this inducible protein might act as a molecular adapter, targeting cell debris to specific cell surface receptors for internalisation. Data from Humphreys et al [19] suggested that, like the small heat shock proteins, sCLU may act in a chaperone-like manner during cellular stress and bind to hydrophobic regions of partly unfolded proteins. This binding thereby "solubilizes" cell debris, protecting neighbouring cells from the cytotoxic consequences of protein precipitation. However, sCLU is also known to bind directly to two other external receptor complexes on cells: the TGF-{beta}1 RI and RII receptors, and the glycoprotein 330 (GP330)/megalin receptor [28–30]. Possible functions of sCLU binding to these receptors have not been elucidated. A role for sCLU in regulating TGF-{beta}1 signalling is also being explored (Araki et al, submitted).

Understanding the regulation of sCLU production after stress is vital to eventually comprehending the overall role of this protein in the cell, before or after stress. Recent data strongly suggest that while synthesis of CLU proteins is induced by various non-toxic levels of traumatic agents (e.g. IR), regulation of this gene is also negatively regulated by wild-type p53 [15]. Interestingly, little is known about the factors and signal transduction processes that are responsible for the dramatic up-regulation of this protein after various stress responses. We demonstrate in this paper that ER stress responses, and specifically changes in Ca2+ homeostasis, can lead to the up-regulation of sCLU expression.

In contrast to the secreted forms of the protein, intracellular CLU protein products are produced in the cell from alternative splicing of the CLU gene, wherein the ER-targeting polypeptide is eliminated. An alternatively spliced form of the CLU mRNA, which may be stress induced, was recently isolated, characterised and sequenced by our laboratory [14]. Translation of this truncated CLU mRNA resulted in an ~49 kDa protein that appears to be inactive and largely cytoplasmic, as observed by confocal analyses of the endogenous protein, or following green fluorescence protein (GFP)-tagged nuclear CLU (nCLU) proteins made following transient or stable transfections [14]. This pre-nuclear form of CLU, pnCLU, remains dormant in the cytoplasm owing to the protein's coiled-coil domains that possibly allow homodimerisation or self-folding, hiding nuclear localisation sites required for transport into the nucleus [14]. However, pnCLU becomes activated after a certain degree of stress (e.g. after >1 Gy of IR), and mature 55 kDa nCLU then causes cell death in a poorly described process [13]. The potential role of CLU protein forms in stress and responses that determine MOD will be discussed.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture
MCF-7:WS8 (MCF-7) breast cancer cells were grown and maintained in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS) under a 90% air–10% CO2 humidified atmosphere at 37°C, as described previously [31]. MCF-7 cells were stably integrated with a pGL3-1403 base pair (bp) human CLU-promoter fused with the firefly luciferase reporter gene, hereafter referred to as 1403 MCF-7 cells [15, 16] and maintained in RPMI containing 5% FBS and 400 µg ml of G418 under the same conditions as described above. Cells were routinely monitored for, and found free of, mycoplasma contamination [33].

Cell treatments
Log-phase MCF-7 cells were either mock-treated or exposed to various doses of IR or thapsigargin (TG) (Sigma Chemical Co., St Louis, MO) as indicated and previously described [13, 34], with or without BAPTA-AM as described [34]. Cells were treated with IR using a 60Co source, as previously described [13].

Survival assays
Survival of mock-, IR- or TG-treated MCF-7 cells were assessed by colony-forming ability assays, as described previously [32, 35–37].

Western blot analyses
Mock-treated or cytotoxic agent-treated cells were extracted for total whole cell protein levels using standard procedures. Proteins were separated by standard 8–10% SDS-PAGE and probed for steady-state protein levels of CLU and other proteins using standard Western blot analyses, as described previously [13, 15, 16, 32, 35–37]. Antibodies to sCLU (B5) were purchased from Santa Cruz (Biotech, Inc., La Jolla, CA) as described previously [13–15].

CLU promoter activity measurements
All luciferase assays were performed using the Luciferase Assay System (Promega, Inc., Madison, WI) as described by the manufacturer. Briefly, 1403 MCF-7 cells were seeded in 6-well plates at approximately 50% confluency. Cells were treated with the indicated dose(s) of BAPTA-AM and/or TG [34]. Cells were then harvested at various times (24 h, 48 h, 72 h and 96 h). Each dose/time point was completed in triplicate, and Student's t-tests were used for statistical analyses.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
A common feature of all the traumatic agents discussed above known to cause MOD is that they all create ROS and result in dramatic changes in Ca2+ homeostasis. These changes in Ca2+ are usually accompanied by release of this divalent cation into the cytosol. We theorize that ROS (created by IR, burns or sepsis) damages specific Ca2+ transporters, channels that regulate and sequester Ca2+ and prevent free Ca2+ accumulation in the cytosol that would otherwise poison key organelles (e.g. mitochondria) and kill the cell. Since we believe that increases in sCLU are a defence mechanism against trauma, we theorized that altered Ca2+ "homeostasis" and ER stress responses were key intracellular signals that trigger CLU gene expression responses.

To investigate the roles of ER Ca2+ release and stress in the stimulation of CLU gene expression, we examined the effects of TG on CLU promoter activity and endogenous changes in sCLU levels by Western blot analyses. TG is a known inhibitor of the sarcoplasmic-endoplasmic reticulum Ca2+ ATPase (SERCA) pump that sequesters Ca2+ into the ER. We also used {beta}-lapachone, a cytotoxic agent that kills via altered Ca2+ responses in an NAD(P)H:quinone oxidoreductase-1 (NQO1)-dependent fashion [34]. We also examined the effects of the Ca2+ chelator, BAPTA-AM, which may suppress TG effects in some cells at low doses, but which can cause Ca2+ influx from the outside and elicit cell death responses at high doses; BAPTA-AM enters the cell, its AM ester is cleaved, and the charged BAPTA chelator is irreversibly inserted into cells after pre-loading.

Exposure of log-phase MCF-7 cells to TG for 1 h caused a dose-dependent loss of colony-forming ability, with an estimated 50% lethal dose (LD50) of 8.5 nM (Figure 2aGo). Interestingly, addition of 3 µM BAPTA-AM, which was reported to prevent short-term lethality induction (apoptosis) in MCF-7 cells [38], did not block TG toxicity as measured by clonogenic survival assays. TG was able to induce endogenous protein levels at as low as 2 nM (Figure 2bGo) under the same conditions as shown in Figure 2aGo. Thus, as reported with IR exposures [13] and as shown in Figure 2bGo that sCLU was induced by 5 Gy IR, treatment of MCF-7 cells with TG for 1 h resulted in dramatic increases in 60 kDa sCLU and 40 kDa psCLU levels between 24 h and 96 h. Thus, induction of sCLU levels occurred at non-lethal doses of TG, as previously reported with IR [11, 13].



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  		Figure 2. Clusterin (CLU) gene expression is induced by changes in endoplasmic reticulum stress, caused by thapsigargin (TG) or high dose BAPTA-AM, agents that cause extensive changes in Ca2+ homeostasis. (a) Changes in survival (measured by colony-forming ability; see Materials and Methods section) in mock-treated cells compared with cells that were pre-treated with 3 µM BAPTA-AM. (b) Changes in endogenous 60 kDa pre-secretory clusterin (psCLU) as well as ~40 kDa secretory clusterin (sCLU) levels were monitored in untreated (no treatment, NT) or TG-exposed (2–20 nM) MCF-7 cells. Whole cell protein extracts were prepared at 24 h or 72 h and probed for CLU protein responses by standard Western blot procedure as described previously [11, 13]. Induction of endogenous protein levels was observed by as little as 2 nM TG, which did not cause a significant lethality response (see (a)). Cells harvested 24 h and 72 h after 5 Gy ionising radiation (IR) were used as a positive control. All experiments were performed three or more times, and representative Western blots are shown.

 
To investigate whether CLU induction after TG in MCF-7 cells occurred at the transcriptional, post-translational or post-transcriptional levels, we examined the activity of the 1403 bp human CLU promoter using the firefly luciferase reporter, as described previously [15, 16]. MCF-7 cells containing a stably integrated CLU promoter–luciferase reporter construct that showed identical responses to cytotoxic stress as monitored by changes in endogenous CLU promoter levels were used [15, 16]. Increases in CLU promoter activities after exposure (Figure 3Go) were observed only after 10 nM TG. Thus, CLU promoter activity did not exactly mimic changes in endogenous psCLU and sCLU levels (Figure 3Go) in terms of dose, but did mimic endogenous CLU protein responses with respect to time (CLU promoter responses increased at 24 h, with peak levels observed between 72 h and 96 h post treatment). Although these results appear to suggest that endogenous CLU protein induction occurs post-transcriptionally in addition to transcriptional CLU promoter activity at higher doses of TG, we have previously demonstrated that assessing CLU promoter activity using a standard luminometer as shown in Figure 3Go is not accurate at low doses [15]. Subsequently, using new real-time bioimaging technology and longer integration times, we showed that CLU promoter responses after various cytotoxic stresses (including low dose TG treatment) correlate well with endogenous CLU protein responses. Thus, CLU protein induction after cell stress (e.g. TG exposure) appears to be due to increased transcriptional responses and increased CLU promoter activity.



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  		Figure 3. Thapsigargin (TG) induces clusterin (CLU) promoter activity. Log-phase 1403 MCF-7 cells, which contain a stably integrated CLU promoter–firefly luciferase reporter construct [15], were exposed to various doses of TG (0–12 nM) and monitored for changes in CLU promoter activity at 24 h, 48 h, 72 h and 96 h post treatment. CLU promoter activities were monitored by standard luminometer assays, as described previously [15, 16]. Cells harvested at 24 h, 48 h, 72 h and 96 h after 10 Gy ionising radiation were used as a positive control. Experiments were performed three times in triplicate, and statistical analyses of the data were performed using the Student's t-test. NT, no treatment.

 
Since 3 µM BAPTA-AM has been previously shown to suppress short-term TG-induced apoptosis in MCF-7 cells [38], we examined the effects of pre-treating cells with 3 µM BAPTA-AM (30 min prior to TG exposures) on subsequent TG-induced lethality, as measured by changes in colony-forming ability (see Materials and Methods section). Two studies were performed (Figure 4Go): (1) increasing doses of BAPTA-AM (0–5 µM) were administered with a single 10 nM dose of TG; and (2) cells were pre-treated with BAPTA-AM (3 µM) followed by subsequent increasing doses of TG (0–20 nM, 1 h). Interestingly, administration of BAPTA-AM in either experimental design did not affect TG-induced lethality (Figure 2aGo) or CLU promoter activity (Figure 4Go). Thus, sequestration of TG-released ER Ca2+ in MCF-7 cells by BAPTA-AM did not prevent lethality or CLU gene expression responses. However, the same doses of BAPTA-AM did block {beta}-lapachone cytotoxicity (not shown, [34]). Interestingly, high doses of BAPTA-AM alone elicited cell death (Figure 2aGo) and sCLU promoter induction (Figure 5Go); high dose BAPTA-AM can lead to massive Ca2+ influx from the outside, leading to cell death. These data suggest that massive uptake of Ca2+ by the mitochondria or the loss of balanced Ca2+ homeostasis by BAPTA-AM could lead to activation of proteins such as caspases or other proapoptotic proteins that trigger cell death responses.



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  		Figure 4. Altered Ca2+ homeostasis is sufficient to induce clusterin (CLU) gene expression. Two experiments were performed using 1403 MCF-7 cells: (1) increasing doses of BAPTA-AM (0–20 µM) were administered with a single dose of thapsigargin (TG) (10 nM); and (2) cells were pre-treated with one dose of BAPTA-AM (3 µM) followed by subsequent increasing doses of TG (0–20 nM, 1 h). CLU promoter activity was assessed as described in Figure 3Go and in the Materials and Methods section. Experiments were performed three times in triplicate, and statistical analyses of the data were performed using the Student's t-test. NT, no treatment.

 


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  		Figure 5. BAPTA-AM exposures at high doses cause increased clusterin (CLU) promoter activities. 1403 MCF-7 cells were pre-loaded with vehicle alone (0.1% dimethylsulfoxide (DMSO)) or with BAPTA-AM in vehicle for 30 min, and then mock irradiated or exposed to 5 Gy ionising radiation (IR). CLU promoter activity was then assessed by standard luminometer readings as described in the Materials and Methods section. Experiments were performed three times in triplicate, and statistical analyses of the data were performed using the Student's t-test. At high doses, BAPTA-AM can cause Ca2+ influx and induce CLU promoter activity to a level statistically equivalent to 5 Gy IR. High dose BAPTA-AM exposures also resulted in increases in endogenous CLU protein responses (not shown).

 
Ca2+ is an important regulator of apoptosis in many cell models [34]. Intracellular Ca2+ depletion has deleterious effects on cell survival and alters gene expression [39, 40], with resulting changes in Ca2+ homeostasis and uptake into intracellular organelles that plays a key factor in cell signalling and apoptosis. The results presented in this paper clearly show that alterations in intracellular Ca2+ dramatically induce sCLU, presumably as a cytoprotective mechanism to this stress. However, Ca2+ release may not be the triggering event, since administration of the Ca2+ chelator BAPTA-AM did not prevent sCLU induction.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Among the several mechanisms of MOD that have been investigated, extended periods of ischaemia were found to be a major cause of cell death, which impairs organ function. Basic research indicated that ischaemia within viable tissue in a variety of organs was vulnerable to xanthine oxidase-mediated reperfusion injury [41–44]. To explain the mechanism of MOD, a "two-hit model" was proposed in the pathogenesis of MOD (Figure 6Go). The first hit is usually the injury itself, such as a whole body burn. The ensuing hypovolaemic shock, followed by a septic response, are largely considered the most important factors in the genesis of MOD. The large amount of devitalised tissues (dramatically enhanced levels of cell debris), along with the often observed development of invasive infection, frequently constitutes the second hit [10, 45, 46] (Figure 6Go).



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  		Figure 6. Two-hit theory of initiation of multiple organ dysfunction (MOD). The initial trauma to a tissue or organ is considered the initial event. Reactive oxygen species (ROS), lipid peroxidation, released Ca2+ from the endoplasmic reticulum (ER) and liberated cytokines are proposed to mediate cell death reactions in initially affected cells as well as in neighbouring cells and tissue that may or may not have been initially affected by the traumatic stress. The "second hit" is thought to be initiated by activation of inflammatory cells and genesis of cell debris or secondary infections and endotoxaemia. Uncontrolled systemic inflammatory responses (SIRS) and immunological dissonance then initiates MOD in localised tissues and organs, and globally throughout the body. See text for further discussion.

 
Increased production of ROS appears to be a very important factor in MOF. Radiation damage to cells is caused by kinetic energy transferred from particles or photons to existing molecules, causing ionisation of water and forming ROS that are intensified by divalent cation (e.g. iron)-driven Fenton reactions. Superoxide is enzymatically reduced to hydrogen peroxide in the presence of a ubiquitously distributed enzyme, superoxide dismutase (SOD). Thus, endogenous SOD keeps superoxide under very tight control. However, in many disease states, including organ failure, there is an imbalance between the amount of superoxide formed and the ability of SOD enzyme to detoxify ROS, leading to superoxide-driven damage. In the case of MOD, newly-formed ROS can be toxic and may initiate other damaging free radical reactions. For example, lipid peroxidation usually occurs in cells, where the lipid peroxyl radical extracts a hydrogen atom from a neighbouring polyunsaturated fatty acid, converting itself into a lipid peroxidase. The end result of extensive lipid peroxidation is cell death [47].

Lipid peroxidation can incite changes in Ca2+ homeostasis, particularly causing Ca2+ release from the ER in severely injured cells of the tissue. Ca2+ released into the cytoplasm of cells is particularly toxic, causing massive mitochondrial dysfunction and apoptotic responses. Extensive local cell death in a tissue can lead to faulty neighbouring cell phagocytosis, ultimately generating cell debris that circulates throughout the tissue and body. Recently, ROS formation during shock and ischaemia as well as reperfusion have been linked to multiple effects on Ca2+ signalling in both the endothelium and smooth muscle. Phagocytes are engaged in engulfing and killing not only pathogenic microorganisms, but also in the removal of damaged and senescent peripheral blood, and apoptotic and opsonised cells [48]. Since our data clearly demonstrate that Ca2+ release from the ER and subsequent ER stress responses cause sCLU induction (Figures 2b, 3–5GoGoGoGo), we propose that this secreted cytoprotective molecular chaperone represents an important and major defence mechanism against MOD.

Increased production of stress-inducible cytokines, such as TGF-{beta}1 and TNF-{alpha}, are also known to result from traumatic insults. These cytokines can cause "bystander" effects in non-traumatised cells as well as in affected neighbouring cells and tissues of the body. The generation of pro-inflammatory cytokines has several consequences for the microvasculature with respect to blood coagulation and disseminated intravascular coagulopathy (DIC). Vascular endothelial cells may be perturbed by the action of cytokines, such as interleukins (IL-1, IL-6 and IL-8), as well as TNF-{alpha}. These cytokines change the general anticoagulant phenotype of the endothelium into a pro-coagulant phenotype. Consistent with a potential role for this protein as a defence against MOD, the sCLU gene has also been reported to be a downstream gene induced by many of the above cytokines (specifically TGF-{beta}1 and TNF-{alpha}) [49–51]. Increased necrotic cell death due to severe damage to one organ may result in overproduction of cytokines and cell debris and have subsequent distal responses in many organs of the body, leading to MOD.

Thus, we propose that CLU gene regulation is a major factor that determines overall MOD sensitivities in defined organs. Unfortunately, to date the role of CLU protein in these responses has not been investigated in detail. We show in this paper that sequestration of TG-released Ca2+ in MCF-7 cells by BAPTA-AM did not prevent lethality or CLU gene expression responses. In contrast, BAPTA-AM could suppress {beta}-lapachone toxicity responses [32]. To date, our data are consistent with a role for sCLU in protecting cells from damage caused by ROS (Figure 7Go). The CLU gene appears to be induced by extremely low levels/doses of agents (e.g. IR), as previously reported from our lab [13, 15], and the sCLU protein appears to protect cells from low level damage. After low doses of agents, it is proposed that sCLU released from cells can "clear" cell debris from local and distal affected areas, thus preventing organ failure.



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  		Figure 7. Clusterin (CLU) gene products are proposed to be major players in defence and initiation responses to trauma that directly influence multiple organ dysfunction (MOD). CLU gene activation is a molecular switch between life and death. At low doses, secretory clusterin (sCLU) production provides a cytoprotective molecular chaperone defence mechanism in clearing cell debris from traumatised tissue. At higher doses, the pre-nuclear form of CLU (pnCLU, a form of the protein that resides dormant in the cytoplasm of most cells) becomes activated through an as yet uncharacterised post-translational mechanism. Activated nuclear clusterin (nCLU), a pro-death mature 55 kDa protein that causes apoptosis, is formed. Widespread activation of nCLU in organs after MOD-causing traumatic insults then facilitates multiple organ failure. See text for further discussion. ROS, reactive oxygen species.

 
However, the CLU gene also produces an intracellular dormant form of the protein, the pnCLU protein, which is activated by higher doses of IR (>1 Gy) [13, 14]. Activation of pnCLU (49 kDa) to mature nCLU (55 kDa) could be a major cause of the cell death observed after high doses of whole body IR that cause MOD. As shown in Figures 3 and 4, 5GoGoGo Gy or higher (10 Gy) doses of irradiation caused the same CLU promoter induction as that caused by 10 nM TG, which was a completely lethal dose according to Figure 2aGo. Thus, CLU gene expression and the alternative splicing mechanisms after IR-induced damage that produce nCLU compared with sCLU could be an important molecular switch that determines life or death of cells, or protection vs induction of MOD. The role of CLU gene expression in MOD should, therefore, should be thoroughly investigated.


   Acknowledgments
 
The authors are grateful to Mr Andy Bruening for his technical assistance.


   Footnotes
 
This work was supported by the Flow Cytometry Core and Radiation Resources Core of the Case Comprehensive Cancer Center (P30 CA43703). This research was supported by grants from the NIH (NIH/NCI R01 CA-92250) and DOE (DE-FG-022179) to DAB, and DOD pre-doctoral and post-doctoral fellowships (DAMD-17-01-1-0196 DAMD 01-1-0194 and W81 XWH-05-1-0248) to TLC, KSL and KER, respectively. Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Skjoldberg H. Letter: Shocked lung. Ugeskr Laeger 1973;136:41–2.[Medline]
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