Clusterin (3. Apoptosis)

It has been shown that clusterin expression is only confined to surviving cells following the induction of apoptosis in vitro, suggesting that it is involved in cell survival rather than death ^Ref . On the basis of its elevated expression in apoptotic tissues, it was originally proposed that the protein might be casually involved in apoptosis. In contrast to the earlier notion, clusterin expression is not enhanced, but rather is down-regulated in the cells undergoing apoptosis and that its expression in the apoptotic tissue is restricted to the vital neighboring cells. These results led to the proposal that rather than being a cell death gene, clusterin is a cell survival gene, exerting a protective function on the surviving bystander cells ^Ref . It was suggested that clusterin gene induction in the vital cells is produced by signaling molecules that are generated by the apoptotic cells and that apoptotic processes ^Ref . Exposure of vital fibroblasts to apoptotic vesicles, disrupted vital cells, and trypsin-treated membrane remnants induces clusterin mRNA. Moreover, lipid vesicles consisting of phosphatidylserine (PtSer) and dimyristoylphos­phatidylcholi­ne (PC), but not liposomes with PC alone nor with dimyristoylphos­phatidylethano­lamine or phosphatidic acid, did elevate clusterin mRNA level. These results suggest that, apart from mediating the endocytic uptake of the apoptotic vesicles, PtSer also serves as a trigger to stimulate the expression of genes that might be involved in the cellular clearance process ^Ref .

Intracellular clusterin inhibits apoptosis by interfering with Bax activation in mitochondria. Intriguingly, in contrast to other inhibitors of Bax, clusterin specifically interacts with conformation-altered Bax in response to chemotherapeutic drugs. This interaction impedes Bax oligomerization, which leads to the release of cytochrome c from mitochondria and caspase activation. Moreover, we also find that clusterin inhibits oncogenic c-Myc-mediated apoptosis by interacting with conformation-altered Bax. Clusterin promotes c-Myc-mediated transformation in vitro and tumour progression in vivo. Taken together, these results suggest that the elevated level of clusterin in human cancers may promote oncogenic transformation and tumour progression by interfering with Bax pro-apoptotic activities ^Ref .

Biological effects of intracellular clusterin were studied and it was observed observed that clusterin forms containing the alpha-chain region strongly accumulated in an ubiquitinated form in juxtanuclear aggregates meeting the main criterions of aggresomes and leading to profound alterations of the mitochondrial network. The viability of cells transfected by intracellular forms of clusterin was improved by overexpression of Bcl-2, and caspase inhibition was capable of rescuing cells expressing clusterin, which presented an altered mitochondrial permeability. It is propose that, although it might be an inherently pro-survival and anti-apoptotic protein expressed by cells under stress in an attempt to protect themselves, clusterin can become highly cytotoxic when accumulated in the intracellular compartment ^Ref .

Confocal microscopy demonstrates that the nuclear accumulation of clusterin is coincident with DNA fragmentation. These data suggest that, at least in secretory epithelial cells, retrograde transport from the Golgi to the ER of a nonglycosylated, uncleaved isoform and the subsequent translocation of clusterin to the nucleus occur in dying cells ^Ref .

Accumulation of clusterin mRNA, whose rate of expression has been associated to the processes of programmed cell death induced by dexameasone in rat thymocytes, preceded the appearance of DNA degradation, starting to increase as early as 30 min after steroid injection, and maintained higher than controls until 8 hrs ^Ref .

A number of inducible leucocyte models of apoptosis was examined, including glucocorticoid and calcium ionophore induced thymocyte death, ’aged’ neutrophils and cytotoxic T cells, and it was found that, in these situations, cell death is not associated with up-regulation of clusterin gene expression. The finding that clusterin is not expressed in all cells undergoing apoptosis would suggest that this molecule is not critical to the mechanism of cell death. It does, however, provide the first example of a readily detectable marker which is differentially expressed in cells undergoing apoptosis and adds further weight to the argument that apoptosis is not a uniform phenomenon, but is dependent on the nature of the cells involved and the means of induction ^Ref . Another study reports that the expression of clusterin mRNA was confined to cells present in the thymic medulla, concentrated mainly around Hassal‘s bodies raising the possibility that the secreted protein is involved in the disposal of cell debris resulting from thymocyte apoptosis ^Ref . The expression of this gene in the rat thymus after the induction of thymocyte programmed cell death (PCD) by in vivo dexamethasone administration was studied. Northern blot analysis of clusterin mRNA 2, 4, 6 and 8 hr after dexamethasone administration in a total of 21 rats revealed no modification in the level of clusterin gene expression. In situ hybridization demonstrated that clusterin gene expression is macroscopically confined to the medullary region of the thymus, and that this distribution is not modified by dexamethasone administration. These results strongly suggest that in the rat,clusterin gene expression is not associated with the programmed cell death of thymocytes following in vivo dexamethasone administration ^Ref . Rat thymocytes were treated with dexamethasone and clusterin mRNA expression was analyzed by semi-quantitative RT-PCR before and after induction of apoptosis. Interestingly, neither the treatment with dexamethasone in vitro nor triggering of apoptosis in vivo up-regulated clusterin expression, opposing the view that clusterin is involved in apoptotic processes ^Ref .

To assess the relationship between cell proliferation and cell death, the mRNA accumulation of ornithine decarboxylase (ODC) and clusterin were measured in human peripheral blood lymphocytes (HPBL) 2–6 hours after stimulation with phytohemagglutinin (PHA). ODC is the rate limiting enzyme of polyamines biosynthesis and its early induction in mitogen-stimulated lymphocytes has been reported. On the other hand, clusterin, a glycoprotein present in most mammalian tissues, is induced in classical models of apoptosis, such as dexamethasone-treated thymocytes. Indeed, a consistent amount of clusterin mRNA in quiescent HPBL, an early and progressive decrease of clusterin mRNA and a parallel increase of ODC mRNA accumulation, were observed, in PHA-stimulated HPBL, suggesting that concomitant repression of clusterin and induction of ODC genes contribute for the cell entering the cell cycle ^Ref .

A single administration of the inhibitor of protein synthesis cycloheximide results in the occurrence of apoptosis in rat liver. The presence of intracellular apoptotic bodies was detected as early as 2 hours after treatment. No evidence of cell necrosis could be observed by histologic and biochemical analysis. Apoptosis was followed by an increased expression of clusterin RNA, a gene whose activity has been associated to apoptotic cell death in involuting rat prostate.the finding of in vivo induction of apoptosis in nonproliferating cells by an inhibitor of protein synthesis, together with the rapidity and synchrony in the occurrence of cell death make this model potentially useful for the analysis of the kinetics of the apoptotic cycle and in exploring some of the mechanisms of regulation of genes possibly involved in this type of cell death ^Ref .

In the ovary, PMSG treatment alone (48 h), and in combination with hCG, dramatically reduced clusterin mRNA to 12–27% of controls (P less than 0.01). Clusterin levels were not elevated until 7 days after hCG; levels then remained constant through day 14 of pseudopregnancy. Since luteal progesterone secretion begins to diminish 5–7 days after hCG, the increased expression of Clusterin on day 7 may be related to the initiation of the regression/de­generation of luteal cells which occurs during luteolysis. Thus, this study demonstrates that alterations in Clusterin expression by the ovary may precede or occur simultaneously with cellular events initiating luteolysis and suggests a role for this glycoprotein as an early marker for luteolysis and implicates it in yet another instance of programmed cell death ^Ref .

Using three different models of PCD: (a) ultraviolet B (UV-B) irradiation of human U937, HeLa, and A431 cell lines, (b) in vitro aging of human peripheral blood neutrophils (PMNs), and © dexamethasone-induced cell death of the human lymphoblastoid cell line CEM-C7, it was shown that the classical morphological and biochemical features of PCD observed did not correlate with an increase, but with either a marked decrease or an absence of clusterin gene expression as assessed by northern blot analysis. In situ hybridization of U937 and A431 cells after UV-B irradiation revealed, in addition, that only morphologically normal cells that are surviving continue to express the clusterin gene. These results demonstrate that in the human myeloid, lymphoid, and epithelial cell types studied, clusterin gene expression is not a prerequisite to their death by apoptosis. In addition, and most interestingly, in situ hybridization of U937 and A431 cells revealed that only surviving cells express the clusterin gene after the induction of PCD, thus providing novel evidence suggesting that clusterin may be associated with cell survival within tissues regressing as a consequence of PCD ^Ref .

A study study was carried out in an attempt to establish the function of clusterin in programmed cell death using tumor necrosis factor (TNF) alpha-induced cytotoxicity in LNCaP cells as the model system. LNCaP is an androgen-sensitive, human prostatic cancer cell line that responds to TNF in culture by undergoing programmed cell death, as determined by the loss of cell number, failure to exclude trypan blue, detection of DNA fragmentation, and increased release of previously incorporated [3H]thymidine. Immunocytochemical staining for clusterin was weak but evident in LNCaP cells. Following treatment with TNF alpha, there was a time-dependent increase in clusterin staining, the intensity of which peaked at 2 h and declined thereafter. Clusterin staining in LNCaP cells was undetectable prior to the onset of DNA fragmentation at 6 h of TNF treatment. This observation indicated that TNF-induced cell death in LNCaP cells was characterized by an initial transient elevation of clusterin, followed by a period of clusterin depletion that preceded cell death. Transfection of LNCaP with a 21-base oligonucleotide antisense to clusterin resulted in a significant increase in cell death that was sequence specific and was accompanied by a reduction in clusterin biosynthesis. These findings supported the concept that clusterin depletion, rather than its expression, was associated with cell death. Finally, stable transfection and subsequent overexpression of clusterin in LNCaP cells resulted in resistance to the cytotoxic effect of TNF. These results have provided evidence to indicate that clusterin plays a role in the protection of TNF-induced cell death in LNCaP cells ^Ref .

LNCaP cells are highly sensitive to the cytotoxic effect of TNF, while PC3 cells are resistant to TNF at 24 hr. Cells were cultured in the presence or absence of TNF (10 ng/ml). LNCaP cells were treated with varying concentrations of exogenous SGP-2, while PC3 cells were treated with antisera to SGP-2 with and without exogenous SGP-2. Following a 24-hr treatment, cultures were assessed by counting of cell number and by the trypan blue exclusion assay. Western blot analysis of conditioned media revealed that PC3 secreted more SGP-2 than did LNCaP. The sensitivity to TNF in LNCaP cells was reduced by the addition of exogenous SGP-2. PC3 cells became sensitive to TNF when SGP-2 antibody was added to the culture. The effect of SGP-2 antibody on PC3 cells was reversed by the addition of exogenous SGP-2 to the culture. CONCLUSIONS: these results suggest that SGP-2 can act as an extracellular mediator of anti-TNF-induced cytotoxicity ^Ref .

High levels of clusterin expression, about 0.2 pg clusterin secreted per cell per 48 h period, specifically protected L929 cells from TNFalpha-mediated cytotoxicity, while low expression (about 4 fg/cell/48 h) had no effect. However, clusterin expression did not provide transfected L929 cells with protection against death mediated by colchicine, staurosporine or azide. High level expression of clusterin in transfected L929 cells also potentiated the cytotoxicity of TGFbeta. It had previously been shown that exposure of L929 cells to TGFbeta provides protection against TNFalpha. This showed that this protective effect is not additive to that of clusterin expression. One interpretation of this data is that it suggests that clusterin and TGFbeta may act via a common mechanism to provide protection against the cytotoxicity of TNFalpha. These results indicate that an intracellular action of clusterin protein is responsible for protection against TNFalpha cytotoxicity. Exposure to TNFalpha induces an increase in the level of cell-associated clusterin and specifically in the level of a novel clusterin molecule, which when analyzed under reducing conditions by SDS/PAGE and immunoblotting appears as two closely spaced bands at about 36 and 38.5 kDa. When analyzed under the same conditions, the normal form of intracellular clusterin, which is present with or without exposure to TNFalpha, appears as two poorly resolved bands at about 43–45 kDa. Since the novel form of clusterin is also expressed in cells exposed to TGFbeta, colchicine, staurosporine, and azide, it may result from toxin-induced disruption of processes of normal cellular protein production ^Ref .

A double-stable prostatic cell line was established for inducible clusterin by using the Tet-On gene expression system. 50% of the cells over expressing clusterin escaped from TNF and actinomycine D induced cell death. Moreover, the incubation of MLL cells with conditioned medium containing the secreted clusterin or the supplementation of purified clusterin in the extracellular medium decreased significantly the TNF-induced apoptosis. This extracellular action implicates megalin, the putative membrane receptor for clusterin to mediate survival. Indeed clusterin over expression up regulated the expression of megalin and induced its phosphorylation in dose dependent manner. We interestingly showed that clusterin over expression is associated with the up-regulation of the phosphorylation of Akt. Activated Akt induced the phosphorylation of Bad and caused a decrease of cytochrome c release. These results enable us to pinpoint one mechanism by which secreted clusterin favours survival in androgen independent prostate cancer cells, implicating its receptor megalin and Akt survival pathway ^Ref .

Apoptotic elimination of intestinal cells following irradiation has been studied in the small and large intestine of the rat, and correlated with the level of expression and localization of clusterin mRNA. Clusterin was moderately expressed in normal intestine where only small levels of apoptosis were found. After irradiation,ho­wever,there was a temporal correlation between an increased apoptotic index and increased clusterin expression. Localization of clusterin mRNA by in situ hybridization identified extensive labelling in the lower part (Paneth cell region) of small intestinal crypt, whereas epithelial cells in the large intestine were diffusely labelled. Clusterin expression was not localized over apoptotic cells and its role may be as a cell protection factor for surviving cells, as had been suggested by others. Clusterin may also be involved in remodelling of the intestinal crypt after radiation damage, a process that includes altering cell-to-cell contact, apoptosis, and sloughing of the dead cells from the intestinal villi. These results do show a close temporal link between apoptosis and clusterin expression, and, as such, expression of the gene may be a useful indicator of presence of apoptosis in the irradiated intestine ^Ref .

As was shown that within these tissues clusterin is expressed in the surviving rather than in the dying cells, and that clusterin gene expression is actually down-regulated in the apoptotic cells, a study examined the expression of the clusterin gene in apoptotic MDCK cells. Cell death was initiated by three different stimuli: application of the steroid hormone antagonist RU 486, activation of protein kinase C by the application of the phorbol ester TPA, and-since clusterin is involved in lipid and cholesterol transport–perturbation of cell membranes by cholesterol. It was shown that clusterin gene expression was repressed in cells undergoing apoptosis in response to the application of RU 486 and TPA, but was unchanged in cells in which apoptosis has been triggered by cholesterol treatment ^Ref .

40 archival radical prostatectomy and/or biopsy specimens of varying grades of prostate cancer were subjected to immunohistochemical SGP-2 staining. The resulting epithelial stains were quantified subjectively on a scale of 1–3 by four independent observers. Benign prostatic epithelial cells from young donors served as controls and showed a consistently weak staining intensity. In contrast, prostate cancer specimens showed varying degrees of staining intensity that correlated with a Gleason pattern (P = 0.006). This correlation supports the hypothesis that protection from apoptotic death may account, in part, for biologically aggressive tumor behavior ^Ref .

Complement, Inflammation and Sepsis

Clusterin/human complement lysis inhibitor (CLI) is incorporated stoichiometrically into the soluble terminal complement complex and inhibits the cytolytic reaction of purified complement components C5b-9 in vitro10. The presence of <10% of the circulating clusterin in its heterodimeric, active form could reduce the rate of complement cytolysis of nucleated cells by 10-fold, and under some conditions by 100-fold or more. This would provide a high level of protection for certain cells and may allow time for action by other inhibitors of complement ^Ref .

However, a conradictory study reports that clusterin gave dose-dependent protection of antibody-coated sheep erythrocytes against complement-mediated lysis by diluted normal human serum. There was a linear relationship between the concentration of clusterin giving 50% protection and the concentration of serum; extrapolation of this to the case of undiluted human serum showed that a clusterin concentration at least two orders of magnitude greater than its physiological plasma concentration would be needed to confer protection against complement-mediated cytolysis under physiological conditions. Physiological concentrations of clusterin did not protect rabbit erythrocytes against alternative complement pathway-mediated lysis using dilute human serum. Exogenous clusterin had no effect on lysis of human erythrocytes triggered by the addition of inulin to autologous human serum. Induction of cell-surface clusterin expression by L929 (murine fibroblast) cells which had been stably transfected with cDNA for human clusterin linked to DNA coding for the 44 C-terminal amino acid residues of CD55 did not protect the cells against complement-mediated lysis by either normal or clusterin-depleted human serum. These data suggest that clusterin may not be a physiologically relevant regulator of complement activation ^Ref .

It seems that clusterin has a particular affinity for membráně attack komplex (MAC) which is associated with immunoglobulin. This observation should help to distinguish between the different forms of MAC, and might indicate that MAC associated with immunoglobulin is essentially in its cytolytically inactive form ^Ref .

The amount of clusterin and vitronectin on MAC in plasma, also referred to as soluble MAC (SMAC) was measured, as well as on MAC bound to CIC (MAC-CIC), using antibody directed to polymerized C9 in systemic lupus erythematosus (SLE) patients. A strong correlation among the quantities of SMAC and MAC-CIC was observed. The amount of both clusterin and vitronectin associated with MAC-CIC was two- to threefold higher in comparison to the SMAC. Patients with high levels of clusterin and vitronectin demonstrated renal involvement. It was hypothesized that these complement regulatory proteins besides regulating the insertion of MAC play other critical roles, in disease pathogenesis ^Ref .

An ELISA method for measuring the binding of clusterin to immunoglobulins was developed. Clusterin purified by IgG affinity chromatography bound to pooled human IgG with a similar affinity (S0.5 5.9 +/- 0.4 micrograms/ml) as clusterin purified by monoclonal antibody chromatography (s0.5 6.1 +/- 0.2 micrograms/ml). The apparent affinity of clusterin for IgG immobilised on ELISA plates increased with increasing concentrations of IgG in the coating solution. Aggregated IgG in solution was a more potent inhibitor of the binding of clusterin to immobilised IgG than was monomer IgG. Clusterin bound to all of the isotypes of human IgG, and to human IgA and IgM, with apparent affinities in the order IgG3 > IgG4 > IgM > IgG1 > IgG2, IgA. Clusterin bound to both the Fab and Fc fragments of human IgG. The clusterin binding site(s) on the Fc do not overlap with those for protein A and Clq ^Ref .

C1q is responsible for the ability of clusterin preparations to promote immune complex formation ^Ref .

The presence of complement regulators such as clusterin in Sjoegren syndrome patients‘ saliva and the high expression of these proteins in inflamed salivary gland tissue followed the inflammatory reaction. These regulators may be involved in protecting the exocrine glands from complement mediated injury ^Ref .

Various S. aureus strains were examined and all appeared to bind clusterin to some extent, while nonpathogenic control strains Bacillus subtilis BR151 and Escherichia coli JM109 did not. Three S. aureus isolates were selected for more detailed study; binding of labeled clusterin was saturable, inhibited in the presence of excess unlabeled clusterin, and prevented by pretreatment of bacteria with proteases. From the saturation binding studies, estimates of the affinity constants for the binding of clusterin to the bacteria ranged from 31 to 57 nM. Addition of clusterin to S. ureus cultures was also found to result in aggregation of the bacterial cells; aggregation was not detected when clusterin was added to B. subtilis Br151 or E. coli JM109 cultures. These results suggest that at least some S. aureus strains possess specific proteinaceous receptors for clusterin. Such receptors may be an important new bacterial virulence determinant for S. aureus, as clusterin has been proposed to have a role in the regulation of complement activity ^Ref .

For clusterin, erythrocytes lysis was measured to reflect the end product of complement activation (membrane attack complex). The complement-induced hemolysis increased when human serum was pre-incubated with clusterin-binding strains, S. epidermidis J9P. The enhancement of hemolysis by J9P decreased when serum was supplemented by exogenous clusterin. The data imply that interaction between coagulase-negative staphylococci and clusterin interferes with one of their physiological functions, complement inhibition.

Vascular leakage and shock are the major causes of death in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). It has been suggested that patients with an elevated level of the free soluble form of dengue virus (DV) nonstructural protein 1 (sNS1) are at risk of developing DHF. To understand the role of sNS1 in blood, a study searched for the host molecule with which NS1 interacts in human plasma by affinity purification using a GST-fused NS1. Clusterin, which naturally inhibits the formation of terminal complement complex (TCC), was identified by mass spectrometry. A recombinant sNS1 produced from 293T cells and sNS1 from DV-infected Vero cells interacted with human Clu. Since an activated complement system reportedly causes vascular leakage, the interaction between sNS1 and Clu may contribute to the progression of DHF ^Ref .

The aim of another study was to investigate potential protective effects of CLU on pulmonary vasculature after in situ polymorphonuclear (PMN) activation in isolated rabbit lungs. The experiments were performed on 24 isolated and ventilated rabbit lungs that were perfused with 200 ml of Krebs-Henseleit10% blood buffer with a constant flow of 150 ml/min in a recirculating system. It was tested whether pretreatment with clusterin (2.5 microg/ml; n = 8) or catalase (CAT, 5000 u/ml; n = 8) before n-formyl-Met-leu-Phe (fMLP; 10(-8) M) injection influenced pulmonary artery pressure (PAP) peak airway pressures (PAW) and edema formation as compared with controls (n = 8). Baseline values of PAP were 9–11 mmHg and PAW 11–13 cm H2O. Application of fMlP resulted in an acute significant (P < 0.01) increase of PAP (48 +/- 29 mmHg) within 2 min in the control group and PAW increased to 35 +/- 7 cm H2O within 30 min. Pretreatment with CLU completely suppressed the PAP and PAW response as a result of the fMLP challenge (P < 0.001), whereas a transient PAW increase up to 27 +/- 15 mmHg was observed after CAT. Complement factor C3a release was suppressed by CAT, whereas clusterin blocked the complement cascade at the level of C5b-9 formation. Moreover, generation of thromboxane A(2) was reduced after clusterin and CAT. Lung edema occurred in the fMLP group but was absent (P < 0.001) after clusterin and CAT treatment. Both clusterin and CAT prevented fMLP-induced lung injury. Stabilizing effects of clusterin, point towards complement regulating features at the level of the terminal complement sequence. Elevated levels of clusterin during inflammation could reflect a compensatory organ protective mechanism. Further studies are required to elucidate the clinical impact of the observed organ-protective properties of clusterin ^Ref .

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