Lack of causal relationship between clusterin expression and photoreceptor apoptosis in light-induced retinal degeneration

Induction of apoptosis in the retina leads to cellular death by molecular mechanisms that are not well understood. Clusterin expression is increased in tissues undergoing apoptosis, including retinal neurodegenerative states, but the causal relationships remain to be clarified. To gain insight into clusterin's role in photoreceptor apoptosis, the cellular distribution of clusterin mRNA was compared with the pattern of apoptotic nuclear labelling in a rat model of light-induced retinal degeneration. In control retinal sections, clusterin mRNA was localized to the retinal pigment epithelium cells, photoreceptor inner segments, inner nuclear layer, and ganglion cell layer. Clusterin expression decreased in photoreceptors and retinal pigment epithelium cells, which progressively degenerated, and increased in preserved inner nuclear layer, in proportion to the duration of light exposure in both cyclic light- and dark-reared animals. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor death, but may relate to cytoprotective functions.     

Clusterin overexpression is responsible for the anti-apoptosis effect in a mouse neuroblastoma cell line,B103

The functional role of clusterin in apoptosis was examined using flow cytometry. Clusterin cDNA was transfected into the mouse neuroblastoma cell line, B103, in order to determine if clusterin overexpression inhibits apoptosis. The increased clusterin expression level in the B103 cells tended to suppress the apoptotic index. This suggests an association of clusterin gene expression with apoptosis inhibition. These results support the conclusion that clusterin expression in B103 cells has an anti-apoptotic influence.     

Clusterin mRNA expression in apoptotic and activated rat thymocytes

Clusterin is a 75-80 kDa heterodimeric glycoprotein,that is produced in most tissues but which exact biological role is still not clear.Pwarticularly,its role in protection or promotion of apoptosis is heavily disputed,since data supporting both views have been reported in several independent sutdies.To clarify this issue,and also to determine whether clusterin expression itself might be affected by apoptosis,in the present study,rat thymocytes were treated with dexamethasone,-a synthetic glucocorticoid that elicits apoptosis in thymocytes-,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.On the other hand,a new clusterin mRNA isoform was detected and isolated,whose expression ws restricted to freshly isolated thymocytes.This novel isoform lacks the post-translational proteolytic cleavage site and is therefore predicted to encode a monomeric protein.The biological function under normal circumstances,however,will nedd further investigations for clarification.While apoptosis could not modulate clusterin expression,activation of thymocytes with concanavalin A and interleukin-2 resulted in up-regulation of clusterin mRNA level,indicating that clusterin expression is rather under the control of cell activation-mediated rather than apoptosis-induced signals.     

Clusterin protects granulosa cells from apoptotic cell death during follicular atresia

Clusterin expression is associated with programmed cell death (apoptosis) in many cell types but its exact role has not yet been defined. This study was carried out to determine the cellular localization of clusterin in the ovary and its functional role in the apoptotic cell death of ovarian follicles. A homogenous population of healthy and atretic follicles was obtained by treating immature rats with pregnant mare serum gonadotropin (PMSG). Apoptotic cell death was evaluated by TUNEL. Clusterin expression in the healthy and atretic follicles was examined by immunohistochemical and Western blot analyses, and gene expression was examined by Northern blot analysis. Clusterin protein and its mRNA are only expressed in granulosa cells of atretic follicles obtained from PMSG-treated rats on day 5 of the treatment. Healthy follicles from PMSG-treated rats on day 2 of the treatment do not express clusterin. Theca and stroma cells of both healthy and atretic follicles showed no signs of apoptosis and did not express clusterin. Withdrawal of trophic support from granulosa cells in cultures to induce apoptosis resulted in a dramatic increase in the levels of clusterin and its mRNA compared to cells cultured in serum-supplemented medium. In an attempt to establish the functional role of clusterin in the apoptotic cell death of ovarian follicles, the biosynthesis of clusterin in granulosa cells of healthy follicles was blocked by treatment of cells with antisense oligonucleotide to its cDNA. Treatment of granulosa cells with the antisense oligonucleotide resulted in an increase in the apoptotic cell death compared to the control. These findings indicate that depletion of clusterin can lead to the programmed cell death in ovary, suggesting a functional role for this protein in follicular atresia.     

Clusterin抑制人肾近曲小管上皮HK-2细胞的凋亡

Clusterin是一种硫酸糖蛋白.最近研究发现,clusterin具有抗凋亡作用,同时对肾细胞具有保护作用,但抗凋亡的具体机制仍不清楚.为研究clusterin及其不同功能区域在人肾近曲小管上皮HK-2细胞中的抗凋亡作用,构建了含有全长及缺失前导序列的clusterin重组质粒（分别命名为pIRES2-EGFP/cluac和pIRES2-EGFP/clubc）.将重组质粒转染人肾近曲小管上皮HK-2细胞后,检测转染细胞中clusterin的表达及其抗Na₂SeO₃（10μmol/L）诱导的凋亡作用.Western印迹显示,转染pIRES2-EGFP/cluac的HK-2细胞培养上清及细胞裂解液中均可检测到clusterin蛋白表达,但转染pIRES2-EGFP/clubc的HK-2细胞仅在裂解液中检测到clusterin,在培养上清液中未检测到该蛋白表达.流式细胞术检验显示,HK-2 /clubc细胞实验组出现明显凋亡峰,而 HK-2 /cluac细胞组则未见凋亡;两组的凋亡百分率之间也存在显著性差异（P<0.05）.以Cy3标记的Annexin V染色后于荧光显微镜下观察细胞凋亡情况与FCM检测结果基本一致.上述结果证明,clusterin有明显的抑制人肾近曲小管上皮HK-2细胞凋亡的作用;clusterin前导序列是其发挥抗凋亡作用的必需功能区域,提示clusterin抗凋亡作用是通过细胞外途径产生的.     

Ectopic expression of clusterin／apolipoprotein J or Bcl-2 decreases thesensitivity of HaCaT cells to toxic effects of ropivacaine

Local anesthetics inhibit cell proliferation and induce apoptosis in various cell types. Ropivacaine, a unique, novel tertiary amine-type anesthetic, was shown to inhibit the proliferation of several cell types including keratinocytes. We found that Ropivacaine could inhibit the proliferation and induce apoptosis in an immortalized human keratinocyte line,HaCaT, in a dose- and time-dependent manner and with the deprivation of serum. The dose-dependent induction of apoptosis by ropivacaine was demonstrated by DNA fragmentation analysis and the proteolytic cleavage of a caspase-3 substrate—poly (ADP-ribose) polymerase (PARP). In addition, ropivacaine downregulated the expression of clusterin/ apoliporotein J, a protein with anti-apoptotic properties, in a dose-dependent manner, which well correlated with the induction of apoptosis of HaCaT cells. To investigate the role of clusterin/apoliporotein J in ropivacaine-induced apoptosis,HaCaT cells overexpressing clusterin/apoliporotein J were generated and compared to cells expressing the well established anti-apoptotic Bcl-2 protein. Ectopic overexpression of the secreted form of clusterin/apoliporotein J or Bcl-2decreased the sensitivity of HaCaT cells to toxic effects of ropivacaine as demonstrated by DNA fragmentation, the proteolytic cleavage of PARP and by a reduction in procaspase-3 expression. Furthermore, the downregulation of endogenous clusterin/apolipoprotein J levels by ropivacaine suggested that this might be one mechanism by which ropivacaine induced cell death in HaCaT cells. In conclusion, the ability of ropivacaine to induce antiproliferative responses and to suppress the expression of the anti-apoptotic protein clusterin/apolipoprotein J, combined with previously reported anti-inflammatory activity and analgesic property of the drug, suggests that ropivacaine may have potential utility in the local treatment of tumors.     

Clusterin activates survival through the phosphatidylinositol 3-kinase/Akt pathway

Clusterin is, in its major form, a secreted heterodimeric disulfide-linked glycoprotein (75-80 kDa). It was first linked to cell death in the rat ventral prostate after androgen deprivation. Recent studies have demonstrated that overexpression of clusterin in prostatic cells protects them against tumor necrosis factor-alpha (TNFalpha)-induced apoptosis. However the details of this survival mechanism remain undefined. Here, we investigate how clusterin prevents cells from undergoing TNFalpha-induced apoptosis. We established a double-stable prostatic cell line for inducible clusterin by using the Tet-On gene expression system. We demonstrated that 50% of the cells overexpressing clusterin escaped from TNFalpha- and actinomycin D-induced cell death. Moreover we demonstrated that the incubation of MLL cells with conditioned medium containing the secreted clusterin or the supplementation of purified clusterin in the extracellular medium decreased the TNFalpha-induced apoptosis significantly. This extracellular action implicates megalin, the putative membrane receptor for clusterin to mediate survival. Indeed clusterin overexpression up-regulated the expression of megalin and induced its phosphorylation in a dose-dependent manner. We interestingly showed that clusterin overexpression 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 favors survival in androgen-independent prostate cancer cells, implicating its receptor megalin and Akt survival pathway.     

Clusterin inhibits apoptosis by interacting with activated Bax

Clusterin is an enigmatic glycoprotein that is overexpressed in several human cancers such as prostate and breast cancers, and squamous cell carcinoma. Because the suppression of clusterin expression renders human cancer cells sensitive to chemotherapeutic drug-mediated apoptosis, it is currently an antisense target in clinical trials for prostate cancer. However, the molecular mechanisms by which clusterin inhibits apoptosis in human cancer cells are unknown. Here we report that 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, our 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.     

Mutation of CERKL, a novel human ceramide kinase gene, causes autosomal recessive retinitis pigmentosa (RP26)

Retinitis pigmentosa (RP), the main cause of adult blindness, is a genetically heterogeneous disorder characterized by progressive loss of photoreceptors through apoptosis. Up to now, 39 genes and loci have been implicated in nonsyndromic RP, yet the genetic bases of >50% of the cases, particularly of the recessive forms, remain unknown. Previous linkage analysis in a Spanish consanguineous family allowed us to define a novel autosomal recessive RP (arRP) locus, RP26, within an 11-cM interval (17.4 Mb) on 2q31.2-q32.3. In the present study, we further refine the RP26 locus down to 2.5 Mb, by microsatellite and single-nucleotide polymorphism (SNP) homozygosity mapping. After unsuccessful mutational analysis of the nine genes initially reported in this region, a detailed gene search based on expressed-sequence-tag data was undertaken. We finally identified a novel gene encoding a ceramide kinase (CERKL), which encompassed 13 exons. All of the patients from the RP26 family bear a homozygous mutation in exon 5, which generates a premature termination codon. The same mutation was also characterized in another, unrelated, Spanish pedigree with arRP. Human CERKL is expressed in the retina, among other adult and fetal tissues. A more detailed analysis by in situ hybridization on adult murine retina sections shows expression of Cerkl in the ganglion cell layer. Ceramide kinases convert the sphingolipid metabolite ceramide into ceramide-1-phosphate, both key mediators of cellular apoptosis and survival. Ceramide metabolism plays an essential role in the viability of neuronal cells, the membranes of which are particularly rich in sphingolipids. Therefore, CERKL deficiency could shift the relative levels of the signaling sphingolipid metabolites and increase sensitivity of photoreceptor and other retinal cells to apoptotic stimuli. This is the first genetic report suggesting a direct link between retinal neurodegeneration in RP and sphingolipid-mediated apoptosis.     

Differential regulation of the clusterin gene by Ha-ras and c-myc oncogenes and during apoptosis

Clusterin (ApoJ) is an extracellular glycoprotein expressed during processes of tissue differentiation and regression that involve programmed cell death (apoptosis). Increased clusterin expression has also been found in tumors, however, the mechanism underlying this induction is not known. Apoptotic processes in tumors could be responsible for clusterin gene activation. Alternatively, oncogenic mutations could modulate signal transduction, thereby inducing the gene. We examined the response of the rat clusterin gene to two oncogenes, Ha-ras and c-myc, in transfected Rat1 fibroblasts. While c-myc overexpression did not modify clusterin gene activity, the Ha-ras oncogene produced a seven to tenfold repression of clusterin mRNA; this down-regulation was also observed in the presence of c-myc. Since no induction of the clusterin gene was observed by the two oncogenes, we tested the alternative mechanism involving apoptosis. Growth factor withdrawal induced apoptosis, as shown by DNA degradation and micronuclei formation in the floating cells. Concomittantly we observed a three to tenfold increase in the amount of clusterin mRNA in the adhering cells of Rat1 and the c-myc transformed cell lines, and a weaker induction in the Ha-ras transformed cell line. On the basis of our results, we suggest that clusterin gene induction in the vital cells is produced by signaling molecules that are generated by the apoptotic cells. We conclude that apoptotic processes, not oncogenic mutations, are responsible for increased clusterin expression in tumors.     

Opposite regulation of clusterin and LH receptor in the swine corpus luteum during luteolysis

Luteolysis, which occurs in a cyclical way to remove luteal tissue, may be an example of physiological apoptosis which counterbalances rapid tissue growth after ovulation. Clusterin is a multifunctional glycoprotein expressed in different tissues undergoing apoptosis. In this study we investigated clusterin and LH receptor gene expression during luteolysis as potential regulators of tissue growth and regression. Luteolysis was induced in pregnant sows (45 days) by Cloprostenol (PGF2 alpha analogue) treatment. Clusterin expression increased in the corpora lutea of pregnant sows ovariectomized 0, 6, 12, 24, 48 or 72 (n = 3) h after the luteolytic stimulus; maximum values were observed 24-48 h after the treatment (P < 0.01). An opposite trend between clusterin mRNA expression and markers of luteal function, such as progesterone levels in the corpora lutea and plasma, and LHr mRNA expression levels, was observed; moreover, clusterin expression was positively correlated with the degree of genomic DNA fragmentation, a marker of occurring apoptosis (P < 0.01). This pattern may be important in regulating luteolysis by a switch between luteotrophic and apoptotic stimulus. Our data indicate that P4 levels decrease prior to the increase in clusterin mRNA and the drop in LHr mRNA expression; we may therefore hypothesize a split between functional and structural luteolysis as reported in other species.     

Heat shock-initiated apoptosis is accelerated and removal of damaged cells is delayed in the testis of clusterin/ApoJ knock-out mice

The secretion and localization of clusterin in the testis has led to the hypothesis that clusterin plays a role in spermatogenesis. Furthermore, the association of clusterin with apoptosis, cellular injury, disease, and regression of nongonadal tissues has led to the hypothesis that clusterin acts to protect cells from apoptosis or may be involved in tissue remodeling. To investigate the role of clusterin in the testis, we analyzed clusterin knock-out (cluKO) mice to determine the impact of the absence of clusterin on spermatogenesis. Furthermore, we investigated the cellular response to injury caused by methoxyacetic acid (MAA) toxicity and mild heat exposure in the cluKO mice to determine the extent to which clusterin protects against apoptosis or participates in tissue remodeling. We found that cluKO mice were fertile and had essentially normal spermatogenesis with the exception of some incomplete spermiation after stage VIII. No differences in testicular morphology or the incidence of apoptosis in the testis were seen between the cluKO and clusterin wild-type (cluWT) mice after MAA treatment. In contrast, apoptosis was delayed in the cluWT mice compared with the cluKO mice after heat exposure, suggesting that clusterin does have a slight protective effect against apoptosis under some conditions. Also, a dramatic loss of germ cells after heat stress occurred earlier in the cluWT testes than in the cluKO testes. Clusterin is clearly acting in a dual role in that cells can be protected from damage and dead cells can be more easily removed after some types of cellular damage but not after others.     

Expression profiles of apoptosis genes in mammary epithelial cells

To investigate apoptosis in HC11 mammary epithelial cells, we compared the gene expression profiles of actively growing and serum-starved apoptotic cells using a mouse apoptosis gene array and 33P-labeled cDNA prepared from the RNA of the two cultures. Analysis of the arrays showed that expression of several genes such as clusterin, secreted frizzled related protein mRNA (sFRP-1), CREB-binding protein (CBP), and others was higher in the apoptotic cells whereas expression of certain genes including survivin, cell division cycle 2 homolog A (CDC2), and cyclin A was lower. These expression patterns were confirmed by RT-PCR and/or Northern analyses. We compared the expression of some of these genes in the mouse mammary gland under various physiological conditions. The expression levels of genes (clusterin, CBP, and M6P-R) up-regulated in apoptotic conditions were higher at involution than during lactation. On the other hand, genes (Pin, CDC2) downregulated in apoptotic conditions were relatively highly expressed in virgin and pregnant mice. We conclude that certain genes such as clusterin, sFRP-1, GAS1 and CBP are induced in apoptotic mammary epithelial cells, and others are repressed. Moreover, the apoptosis array is an efficient technique for comparing gene expression profiles in different states of the same cell type.     

A novel anti-proliferative property of clusterin in prostate cancer cells

Clusterin is a ubiquitous secretory glycoprotein that is known to suppress certain forms of apoptosis. Since apoptosis and proliferation are two opposing cellular events, it remains unclear if clusterin has any effect on cellular proliferation. The objective of the present study was to examine the effects of clusterin on proliferation in a prostate cancer cell line, LNCaP. We found that clusterin inhibited EGF-mediated proliferation in these cells, as measured by (3)H-thymidine incorporation and by cell counting. Clusterin did not bind with EGF nor did it block phosphorylation of the EGF receptor. Treatment of LNCaP cells with EGF resulted in a transient increase in the expression of both c-Fos and c-Jun. Addition of clusterin to these cultures significantly down-regulated the protein level of c-Fos, but not c-Jun. These results demonstrated a novel biological role for clusterin. Clusterin is not only anti-apoptotic but also anti-proliferative. The anti-proliferative event maybe associated with a down-regulation of c-Fos.     

The Role of Clusterin in Alzheimer’s Disease: Pathways,Pathogenesis, and Therapy

Genetic variation in clusterin gene, also known as apolipoprotein J, has been associated with Alzheimer’s disease (AD) through replicated genome-wide studies, and plasma clusterin levels are associated with brain atrophy, baseline prevalence and severity, and rapid clinical progression in patients with AD, highlighting the importance of clusterin in AD pathogenesis. Emerging data suggest that clusterin contributes to AD through various pathways, including amyloid-β aggregation and clearance, lipid metabolism, neuroinflammation, and neuronal cell cycle control and apoptosis. Moreover, epigenetic regulation of the clusterin expression also seems to play an important role in the pathogenesis of AD. Emerging knowledge of the contribution of clusterin to the pathogenesis of AD presents new opportunities for AD therapy.