Clusterin Isoforms Research Articles

Retraction: Cell detachment and apoptosis induction of immortalized human prostate epithelial cells are associated with early accumulation of a 45 kDa nuclear isoform of clusterin.

Retraction: Cell detachment and apoptosis induction of immortalized human prostate epithelial cells are associated with early accumulation of a 45 kDa nuclear isoform of clusterin.

Clusterin is increased in morphologically normal cardiomyocytes in lymphocytic myocarditis.

Clusterin is increased in morphologically normal cardiomyocytes in lymphocytic myocarditis.

Deregulated Clusterin as a Marker of Bone Fragility: New Insights into the Pathophysiology of Osteoporosis.

Clusterin (CLU) is a secreted heterodimeric glycoprotein expressed in all organism fluids as well as in the intracellular matrix that plays key roles in several pathological processes. Its recent involvement in muscle degeneration of osteoporotic patients led to investigation of the role of CLU in bone metabolism, given the biochemical and biomechanical crosstalk of the bone–muscle unit. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis of CLU expression was performed in both osteoblasts and Peripheral Blood Mononuclear Cells (PBMCs) from osteoporotic patients (OP) and healthy individuals (CTR). Furthermore, immunohistochemical analysis on femoral head tissues and enzyme-linked immunosorbent assay (ELISA) in plasma samples were performed to investigate CLU expression pattern. Finally, genotyping of CLU rs11136000 polymorphism has also been performed by qRT-PCR assays to explore a possible association with CLU expression levels. Data obtained showed a significantly increased expression level of secreted CLU isoform in PBMCs and osteoblasts from OP patients. Immunohistochemical analysis confirms the increased expression of CLU in OP patients, both in osteocytes and osteoblasts, while plasma analysis reveals a statistically significant decrease of CLU levels. Unfortunately, no functional association between CLU expression levels and the presence of CLU rs11136000 polymorphism in OP patients was found. These data suggest a potential role played by CLU as a potential biomarker for the diagnosis and prognosis of OP progression.

In silico structural analysis of secretory clusterin to assess pathogenicity of mutations identified in the evolutionarily conserved regions

Clusterin (CLU) is a secreted glycoprotein, heterodimeric in nature, and is expressed in a wide variety of tissues and body fluids such as serum and plasma. CLU has also been known to be a promising biomarker for cell death, malignancy, cancer progression, and resistance development. However, the lack of a CLU crystal structure obstructs understanding the possible role of reported mutations on the structure, and the subsequent effects on downstream signaling pathways and cancer progression. Considering the importance of crystal structure, a model structure of the pre-secretory isoform of CLU was built to predict the effect of mutations at the molecular level. Ab initio model was built using RaptorX, and loop refinement and energy minimization were carried out with ModLoop, ModRefiner, and GalaxyWeb servers. The cancer associated mutational spectra of CLU was retrieved from the cBioPortal server and 117 unique missense mutations were identified. Evolutionarily conserved regions and pathogenicity of mutations identified in CLU were analyzed using ConSurf and Rhapsody, respectively. Furthermore, sequence and structure-based mutational analysis were carried out with iSTABLE, DynaMut and PremPS servers. Molecular dynamics simulations were carried out with GROMACS for 50 ns to determine the stability of the wild type and mutant protein structures. A dynamically stable model structure of pre-secretory CLU (psCLU) which has high concurrence with the sequence based secondary structure predictions has been explored. Changes in the intra-atomic interactions and folding pattern between wild type and mutant structures were observed. To our conclusion, eleven mutations with the highest structural and functional significance have been predicted to have pathogenic and deleterious effects. Communicated by Ramaswamy H. Sarma

Contribution of the Transcription Factors Sp1/Sp3 and AP-1 to Clusterin Gene Expression during Corneal Wound Healing of Tissue-Engineered Human Corneas.

In order to reduce the need for donor corneas, understanding of corneal wound healing and development of an entirely tissue-engineered human cornea (hTECs) is of prime importance. In this study, we exploited the hTEC to determine how deep wound healing affects the transcriptional pattern of corneal epithelial cells through microarray analyses. We demonstrated that the gene encoding clusterin (CLU) has its expression dramatically repressed during closure of hTEC wounds. Western blot analyses confirmed a strong reduction in the expression of the clusterin isoforms after corneal damage and suggest that repression of CLU gene expression might be a prerequisite to hTEC wound closure. Transfection with segments from the human CLU gene promoter revealed the presence of three regulatory regions: a basal promoter and two more distal negative regulatory regions. The basal promoter bears DNA binding sites for very potent transcription factors (TFs): Activator Protein-1 (AP-1) and Specificity protein-1 and 3 (Sp1/Sp3). By exploiting electrophoretic mobility shift assays (EMSA), we demonstrated that AP-1 and Sp1/Sp3 have their DNA binding site overlapping with one another in the basal promoter of the CLU gene in hCECs. Interestingly, expression of both these TFs is reduced (at the protein level) during hTEC wound healing, thereby contributing to the extinction of CLU gene expression during that process. The results of this study contribute to a better understanding of the molecular mechanisms accounting for the repression of CLU gene expression during corneal wound healing.

Novel nucleolar localization of clusterin and its associated functions in human oral cancers: An in vitro and in silico analysis.

Clusterin (CLU), a multifunctional chaperonic glycoprotein associated with diverse cellular functions has been shown to act as an oncogene or tumour suppressor gene in different cancers, implying a dual role in tumorigenesis. Here, we investigated the expression of CLU isoforms, their subcellular localization and functional significance in oral cancer cells. Significant downregulation of secretory CLU (sCLU) transcripts was observed in oral cancer cell lines and tumours versus normal cells while the nuclear CLU (nCLU) transcripts were undetectable. We demonstrated for the first time the nucleolar localization of sCLU, its response to different nucleolar stresses and association with cajal bodies post nucleolar stress. Functionally, knockdown of CLU revealed its negative association with ribosome biogenesis implying a possible tumour suppressor like role in oral cancers. Further, loss of sCLU in these cells also resulted in altered nuclear morphology and shrunken tubulin filaments. In addition, the levels of nucleolar Nucleophosmin 1(NPM1) and Fibrillarin, known to regulate nuclear morphology were downregulated indicating a possible role of sCLU in their stabilization. Further, an in silico docking approach to gain insights into the interaction of sCLU with nucleolar proteins NPM1, Fibrillarin, UBF and Nucleolin, revealed the involvement of a conserved region comprising of amino acid residues 140-155 of sCLU β-chain, specifically via the Phe152 residue in hydrophobic interactions with these client nucleolar proteins indicating a possible stabilizing or regulatory role of sCLU. SIGNIFICANCE OF THE STUDY: This is the first study to demonstrate the nucleolar localization of sCLU and its associated functions in oral cancer cells. Downregulation of sCLU in oral cancer tissues and cell lines, and its negative association with ribogenesis suggest its tumour suppressor like role in oral cancers. The possible role of sCLU in stabilization or regulation of different nucleolar proteins thereby impacting their functions is also implicated.

Cell Culture Experiments Reveal that High S100B and Clusterin Levels may Convey Hypoxia-tolerance to the Hooded Seal (Cystophora cristata) Brain

While the brain of most mammals suffers from irreversible damage after only short periods of low oxygen levels (hypoxia), marine mammals are excellent breath-hold divers that have adapted to hypoxia. In addition to physiological adaptations, such as large oxygen storing capacity and strict oxygen economy during diving, the neurons of the deep-diving hooded seal (Cystophora cristata) have an intrinsic tolerance to hypoxia. We aim to understand the molecular basis of this neuronal hypoxia tolerance.Previously, transcriptomics of the cortex of the hooded seal have revealed remarkably high expression levels of S100B and clusterin (apolipoprotein J) when compared to the ferret, a non-diving carnivore. Both genes have much-debated roles in hypoxia and oxidative stress. Here, we evaluated the effects of S100B and of two isoforms of clusterin (soluble and nucleus clusterin) on the survival, metabolic activity and the amount of reactive oxygen species (ROS) in HN33 neuronal mouse cells exposed to hypoxia and oxidative stress. S100B and soluble clusterin had neuroprotective effects, with reduced ROS-levels and retention of normoxic energy status of cells during both stress conditions. The protective effects of nucleus clusterin were restricted to hypoxia. S100B and clusterin showed purifying selection in marine and terrestrial mammals, indicating a functional conservation across species. Immunofluorescence revealed identical cellular distributions of S100B and clusterin in mice, ferrets and hooded seals, further supporting the functional conservation. Taken together, our data suggest that the neuroprotective effects of all three proteins are exclusively facilitated by their increased expression in the brain of the hooded seal.

Clusterin secretion is attenuated by the proinflammatory cytokines interleukin-1β and tumor necrosis factor-α in models of cartilage degradation.

The protein clusterin has been implicated in the molecular alterations that occur in articular cartilage during osteoarthritis (OA). Clusterin exists in two isoforms with opposing functions, and their roles in cartilage have not been explored. The secreted form of clusterin (sCLU) is a cytoprotective extracellular chaperone that prevents protein aggregation, enhances cell proliferation and promotes viability, whereas nuclear clusterin acts as a pro-death signal. Therefore, these two clusterin isoforms may be putative molecular markers of repair and catabolic responses in cartilage and the ratio between them may be important. In this study, we focused on sCLU and used established, pathophysiologically relevant, in vitro models to understand its role in cytokine-stimulated cartilage degradation. The secretome of equine cartilage explants, osteochondral biopsies and isolated unpassaged chondrocytes was analyzed by western blotting for released sCLU, cartilage oligomeric protein (COMP) and matrix metalloproteinases (MMP) 3 and 13, following treatment with the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α. Release of sulfated glycosaminoglycans (sGAG) was determined using the dimethylmethylene blue assay. Clusterin messenger RNA (mRNA) expression was quantified by quantitative real-time polymerase chain reaction. MMP-3, MMP-13, COMP, and sGAG release from explants and osteochondral biopsies was elevated with cytokine treatment, confirming cartilage degradation in these models. sCLU release was attenuated with cytokine treatment in all models, potentially limiting its cytoprotective function. Clusterin mRNA expression was down-regulated 7-days post cytokine stimulation. These observations implicate sCLU in catabolic responses of chondrocytes, but further studies are required to evaluate its role in OA and its potential as an investigative biomarker.

Brain clusterin protein isoforms and mitochondrial localization.

Clusterin (CLU), or apolipoprotein J (ApoJ), is the third most predominant genetic risk factor associated with late-onset Alzheimer's disease (LOAD). In this study, we use multiple rodent and human brain tissue and neural cell models to demonstrate that CLU is expressed as multiple isoforms that have distinct cellular or subcellular localizations in the brain. Of particular significance, we identify a non-glycosylated 45 kDa CLU isoform (mitoCLU) that is localized to the mitochondrial matrix and expressed in both rodent and human neurons and astrocytes. In addition, we show that rodent mitoCLU is translated from a non-canonical CUG (Leu) start site in Exon 3, a site that coincides with an AUG (Met) in human CLU. Last, we reveal that mitoCLU is present at the gene and protein level in the currently available CLU-/- mouse model. Collectively, these data provide foundational knowledge that is integral in elucidating the relationship between CLU and the development of LOAD.

Glyceryl trinitrate‑induced cytotoxicity of docetaxel‑resistant prostatic cancer cells is associated with differential regulation of clusterin.

Metastatic castration resistant prostate cancer (mCRPC) relapse due to acquired resistance to chemotherapy, such as docetaxel, remains a major threat to patient survival. Resistance of mCRPC to docetaxel can be associated with elevated levels of soluble clusterin (sCLU) and growth differentiation factor‑15 (GDF‑15). Any strategies aiming to modulate sCLU and/or GDF‑15 in docetaxel‑resistant prostate cancer cells present a therapeutic interest. The present study reports the cytotoxic effect of a nitric oxide donor, glyceryl trinitrate(GTN), on docetaxel‑resistant mCRPC human cell lines and demonstrates that GTN displays greater inhibition of cell viability toward docetaxel‑resistant mCRPC cells than on mCRPC cells. It is also demonstrated that GTN modulates the level of expression of clusterin(CLU) which is dependent of GDF‑15, two markers associated with docetaxel resistance in prostate cancer. The results indicate that GTN represses the level of expression of the cytoprotective isoform of CLU(sCLU) and can increase the level of expression of the cytotoxic isoform (nuclear CLU) in docetaxel resistant cells. Furthermore, it was observed that GTN differentially regulates the level of the precursor form of GDF‑15 between resistant and parental cells, and that recombinant GDF‑15 can modulate the expression of CLU isoforms and counteract GTN‑induced cytotoxicity in resistant cells. A link was established between GDF‑15 and the expression of CLU isoforms. The present study thus revealed GTN as a potential therapeutic strategy to overcome docetaxel‑resistant mCRPC.

Regulation of Clusterin Gene Expression.

Clusterin is a multifunctional glycoprotein whose role in cells has generated a great controversy in recent years. Since its discovery, numerous studies have linked clusterin expression deregulation with various physio-pathological processes such as cancer or Alzheimer's disease. Although the results of such investigations have sometimes been contradictory, mainly due to the dichotomous role of clusterin isoforms, it has been demonstrated that this protein is involved in diverse cellular processes, including apoptosis, cell cycle regulation, DNA repair or the acquisition of cell resistance against multiple conventional therapies. These results, together with the breakthrough of gene therapies, have motivated a great effort to elucidate the importance of clusterin as a potential therapeutic target. However, the understanding of a single gene, with multiple RNA transcripts and several protein isoforms has turned out to be a complex task. In this review, we summarize the studies published to date on factors that can affect clusterin expression and evaluate if a better understanding of this complex gene/protein would be useful to develop new treatment strategies for cancer and other pathologies.

Discovery of gastric cancer specific biomarkers by the application of serum proteomics

Current diagnostic markers for gastric cancer are not sufficiently specific or sensitive for use in clinical practice. The aims of this study are to compare the proteomes of serum samples from patients with gastric cancers and normal controls, and to develop useful tumor markers of gastric cancer by quantitative proteomic analysis. We identified a total of 388 proteins with a ≤1% FDR and with at least two unique peptides from the sera of each group. Among them, 215, 251, and 260 proteins were identified in serum samples of patients in an advanced cancer group, early cancer group, and normal control group, respectively. We selected differentially expressed proteins in cancer patients compared with those of normal controls via semiquantitative analyses comparing the spectral counts of identified proteins. These differentially expressed proteins were successfully verified using an MS-based quantitative assay, multiple reactions monitoring analysis. Four proteins (vitronectin, clusterin isoform 1, thrombospondin 1, and tyrosine-protein kinase SRMS) were shown to have significant changes between the cancer groups and the normal control group. These four serum proteins were able to discriminate gastric cancer patients from normal controls with sufficient specificity and selectivity.

Glycosylation of Human Plasma Clusterin Yields a Novel Candidate Biomarker of Alzheimer's Disease.

Specific glycosylated peptides of clusterin are found associated with hippocampal atrophy. The glycosylation of clusterin from human plasma was comprehensively analyzed and characterized using mass spectrometry (MS)-based glycoproteomics analysis. All six known N-glycosylation sites are covered, three in the alpha subunit (α64N, α81N and α123N) and three in the beta subunit (β64N, β127N, and β147N). More detailed structural characterization of clusterin glycopeptides was also performed, demonstrating the presence of glycosylated peptides and their corresponding glycans. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we have determined the differences in the glycoforms associated at each of the different glycosylation sites in plasma clusterin obtained from subjects of low hippocampal atrophy (n = 13) and high hippocampal atrophy (n = 14). In our pilot study, the β64N site shows the most significant regulations between clinical groups. Eight β64N glycoforms are significantly reduced in patients with high atrophy compared with those with low atrophy, which demonstrates the utility of clusterin isoforms as diagnostic and prognostic Alzheimer's disease (AD) markers. These results provide a novel and robust workflow suitable for rapid verification of specific clusterin glycoforms with utility as AD biomarkers.

Clusterin gene is predominantly regulated by histone modifications in human colon cancer and ectopic expression of the nuclear isoform induces cell death

Clusterin (CLU) is an important glycoprotein involved in various cellular functions. Different reports have mentioned that the two isoforms of CLU; secretary (sCLU) and nuclear (nCLU) have opposite (paradoxical) roles in cancer development. sCLU provides pro-survival signal, whereas nCLU is involved in pro-apoptotic signaling. However, the molecular mechanism of CLU gene regulation is not clear as of yet. We hypothesize that CLU gene is regulated by DNA methylation and histone modifications and clusterin plays an important role in colon cancer. To evaluate the hypothesis, we investigated CLU expression in colon cancer tissues and DNA methylation and histone modification status of CLU gene promoter. It is apparent from immonohistology data that both benign and cancerous (primary and metastasis) formalin fixed paraffin embedded (FFPE) tissue samples exhibit CLU expression. However and interestingly only noncancerous tissue samples show nCLU expression. Ectopic expression of nCLU either by epigenetic modulators or by nCLU transfection is responsible for colon cancer cell death. To clarify the molecular mechanisms for regulation of expression of CLU isoforms, we have analyzed DNA methylation and histone modifications, such as histone H3K9me3, H3K27me3, H3K4me3, and H3K9AcS10P patterns around the CLU promoter. There is no remarkable change in the DNA methylation status upon treatment of the cells by AZA, TSA and SAM. Our findings clearly show that promoter histone H3K9me3 and H3K27me3 marks are elevated in comparison to H3K4me3 and H3K9AcS10P marks in colon cancer cell lines.

Augmenter of liver regeneration plays a protective role against hydrogen peroxide-induced oxidative stress in renal proximal tubule cells.

Oxidative stress plays an important role in cellular destruction. Augmenter of liver regeneration (ALR) is an anti-apoptotic factor that is expressed in all mammalian cells and functions as an anti-oxidant by stimulating the expression of a secretory isoform of clusterin and inhibiting reactive oxygen species (ROS) generation. Previous work from our group showed that ALR expression is upregulated in acute kidney injury (AKI) rats, and recombinant human ALR reduces tubular injury. In the present study, we used small interfering RNA (siRNA) silencing of ALR to examine its role in H2O2 induced mitochondrial injury and apoptosis. Knockdown of ALR increased ROS levels, reduced mitochondrial membrane potential, and increased the release of mitochondrial proteins and the rate of apoptosis in response to H2O2. In addition, the ratio of Bax/Bcl-2 was increased in siRNA/ALR groups treated with H2O2. These data confirm the protective role of ALR against oxidative stress-induced mitochondrial injury and suggest a potential mechanism underlying the protective role of ALR in AKI.

Protein profile of the seminal plasma of collared peccaries (Pecari tajacu Linnaeus, 1758)

This study was conducted to characterize the major proteins of the peccary seminal plasma, based on the semen samples collected from nine adult and reproductively sound animals. Our approach included the use of two-dimensional electrophoresis followed by Coomassie blue staining and analysis of polypeptide maps with PDQuest Software (Bio-Rad). Proteins were identified by tandem mass spectrometry (LC-MS/MS). We detected 179 protein spots per gel and 98 spots were identified by mass spectrometry, corresponding to 23 different proteins. The combined intensity of those spots accounted for 56.2±6% of the intensities of all spots and 60.9% of the intensities of spots presented in every protein map. Protein spots identified as clusterin represented 19.7±8.3% of the integrated optical densities of all spots detected in the seminal plasma maps. There was a negative association (r=-0.87; P<0.05) between the intensity of a clusterin spot and the percentage of sperm with functional membrane. Spermadhesin porcine seminal plasma protein 1 and bodhesin 2 comprised 5.4±1.9 and 8.8±3.9% of the total intensity of all spots respectively. Many proteins appeared in a polymorphic pattern, such as clusterin (27 spots), epididymal secretory glutathione peroxidase (ten spots), inter-α-trypsin inhibitor (12 spots), and IgG-binding protein (ten spots), among others. In conclusion, we presently describe the major seminal plasma proteome of the peccary, which exhibits a distinct high expression of clusterin isoforms. Knowledge of wild species reproductive biology is crucial for an understanding of their survival strategies and adaptation in a changing environment.

Hypoxia inducible factor-1α directly regulates nuclear clusterin transcription by interacting with hypoxia response elements in the clusterin promoter.

Differential transcription of the clusterin (CLU) gene yields two CLU isoforms, a nuclear form (nCLU) and a secretory form (sCLU), which play crucial roles in prostate tumorigenesis. Pro-apoptotic nCLU and anti-apoptotic sCLU have opposite effects and are differentially expressed in normal and cancer cells; however, their regulatory mechanisms at the transcriptional level are not yet known. Here, we examined the transcriptional regulation of nCLU in response to hypoxia. We identified three putative hypoxia response elements (HREs) in the human CLU promoter between positions −806 and +51 bp. Using a luciferase reporter, electrophoretic gel mobility shift, and chromatin immunoprecipitation assays, we further showed that hypoxia-inducible factor-1α (HIF-1α) bound directly to these sites and activated transcription. Exposure to the hypoxiamimetic compound CoCl2, incubation under 1% O2 conditions, or overexpression of HIF-1α enhanced nCLU expression and induced apoptosis in human prostate cancer PC3M cells. However, LNCaP prostate cancer cells were resistant to hypoxia-induced cell death. Methylation-specific PCR analysis revealed that the CLU promoter in PC3M cells was not methylated; in contrast, the CLU promoter in LNCap cells was methylated. Co-treatment of LNCaP cells with CoCl2 and a demethylating agent promoted apoptotic cell death through the induction of nCLU. We conclude that nCLU expression is regulated by direct binding of HIF-1α to HRE sites and is epigenetically controlled by methylation of its promoter region.

From Humans to Experimental Models: The Cytoprotective Role of Clusterin in the Kidney

Clusterin (CLU) is a chaperone-like protein and has been discovered more than thirty years ago; however, its biological significance is still not fully understood. This review aims to summarize the principal observations of CLU roles related to the kidney. In humans, three or more mRNA isoforms of CLU could be expressed due to different translation start sites, but only two forms of CLU protein, secreted (sCLU, isoform 2) and nuclear (nCLU, isoform 1), have been well characterized, whereas there is only sCLU form in mice. In the biopsies of renal tissue from patients, up regulated CLU expression has been found in rejecting kidney transplants or diseased kidneys, and a lower level of serum CLU is correlated with many types of kidney disease in patients. In mice, a deficiency in CLU expression specifically leads to the phenotype of age-dependent chronic glomerular injury - moderate to severe accumulation of the mesangial matrix, becomes more susceptible to ischemia-reperfusion injury (IRI), negatively impacts renal repair after IRI and worsens renal fibrosis after ureteral obstruction. All these observations may imply the biological significance of CLU for the maintenance of the tissue homeostasis in adult kidneys. However, how CLU protects the kidney from injury or by which extracellular and intracellular pathways mediate the cyto-protection of CLU in the kidney has not been well investigated. Understanding of the cyto-protective activities of CLU in the kidney could lead to the development of novel therapeutic strategies for the prevention and/or treatment of kidney injury or diseases.

Correction: Non-Secreted Clusterin Isoforms Are Translated in Rare Amounts from Distinct Human mRNA Variants and Do Not Affect Bax-Mediated Apoptosis or the NF-κB Signaling Pathway

Correction: Non-Secreted Clusterin Isoforms Are Translated in Rare Amounts from Distinct Human mRNA Variants and Do Not Affect Bax-Mediated Apoptosis or the NF-κB Signaling Pathway

Non-Secreted Clusterin Isoforms Are Translated in Rare Amounts from Distinct Human mRNA Variants and Do Not Affect Bax-Mediated Apoptosis or the NF-κB Signaling Pathway

Clusterin, also known as apolipoprotein J, is expressed from a variety of tissues and implicated in pathological disorders such as neurodegenerative diseases, ischemia and cancer. In contrast to secretory clusterin (sCLU), which acts as an extracellular chaperone, the synthesis, subcellular localization and function(s) of intracellular CLU isoforms is currently a matter of intense discussion. By investigating human CLU mRNAs we here unravel mechanisms leading to the synthesis of distinct CLU protein isoforms and analyze their subcellular localization and their impact on apoptosis and on NF-κB-activity. Quantitative PCR-analyses revealed the expression of four different stress-inducible CLU mRNA variants in non-cancer and cancer cell lines. In all cell lines variant 1 represents the most abundant mRNA, whereas all other variants collectively account for no more than 0.34% of total CLU mRNA, even under stressed conditions. Overexpression of CLU cDNAs combined with in vitro mutagenesis revealed distinct translational start sites including a so far uncharacterized non-canonical CUG start codon. We show that all exon 2-containing mRNAs encode sCLU and at least three non-glycosylated intracellular isoforms, CLU1‑449, CLU21‑449 and CLU34‑449, which all reside in the cytosol of unstressed and stressed HEK‑293 cells. The latter is the only form expressed from an alternatively spliced mRNA variant lacking exon 2. Functional analysis revealed that none of these cytosolic CLU forms modulate caspase-mediated intrinsic apoptosis or significantly affects TNF-α-induced NF-κB-activity. Therefore our data challenge some of the current ideas regarding the physiological functions of CLU isoforms in pathologies.