Clusterin mRNA Levels Research Articles

Dynamics of clusterin protein expression in the brain and plasma following experimental traumatic brain injury

Progress in the preclinical and clinical development of neuroprotective and antiepileptogenic treatments for traumatic brain injury (TBI) necessitates the discovery of prognostic biomarkers for post-injury outcome. Our previous mRNA-seq data revealed a 1.8–2.5 fold increase in clusterin mRNA expression in lesioned brain areas in rats with lateral fluid-percussion injury (FPI)-induced TBI. On this basis, we hypothesized that TBI leads to increases in the brain levels of clusterin protein, and consequently, increased plasma clusterin levels. For evaluation, we induced TBI in adult male Sprague-Dawley rats (n = 80) by lateral FPI. We validated our mRNA-seq findings with RT-qPCR, confirming increased clusterin mRNA levels in the perilesional cortex (FC 3.3, p < 0.01) and ipsilateral thalamus (FC 2.4, p < 0.05) at 3 months post-TBI. Immunohistochemistry revealed a marked increase in extracellular clusterin protein expression in the perilesional cortex and ipsilateral hippocampus (7d to 1 month post-TBI), and ipsilateral thalamus (14d to 12 months post-TBI). In the thalamus, punctate immunoreactivity was most intense around activated microglia and mitochondria. Enzyme-linked immunoassays indicated that an acute 15% reduction, rather than an increase in plasma clusterin levels differentiated animals with TBI from sham-operated controls (AUC 0.851, p < 0.05). Our findings suggest that plasma clusterin is a candidate biomarker for acute TBI diagnosis.

High clusterin (CLU) mRNA expression levels in tumors of colorectal cancer patients predict a poor prognostic outcome

ObjectivesClusterin (CLU) is a multifunctional intra-/extra-cellular molecular chaperone with indications of serving as a promising prognostic biomarker for colorectal cancer (CRC). Several studies have examined the potential prognostic value of the CLU protein in CRC; however, our research follows an alternative approach, focusing on the CLU mRNA expression. Design and methodsTotal RNA from 172 cancerous tissue specimens and 39 paired non-cancerous ones was isolated and 2 μg of this were subjected to reverse transcription with an oligo-dT primer. The single stranded DNA, which was synthesized, was amplified with an in-house developed highly sensitive and precise qPCR method, using specific pair of primers for the CLU molecule. Finally, an extensive biostatistical analysis took place for the assessment of the results. ResultsPatients with tumors expressing high CLU mRNA levels had a higher probability for poor outcome (relapse and death), comparing to those with CLU mRNA-negative tumors. This association between CLU mRNA expression status and both disease-free survival (DFS) and overall survival (OS) is evident in Cox regression analysis and is also depicted in the Kaplan-Meier survival curves. Consistently, the aforementioned associations and the CLU mRNA expression levels are significantly enhanced as CRC tumors progress from TNM stage I to IV, further supporting the functional implication of CLU in tumorigenesis. ConclusionsHigh CLU mRNA levels in CRC tumors can act as a new adverse prognostic biomarker of DFS and OS for CRC, independent of clinicopathological and biological features of the patient.

The effects of Vitis vinifera L. phenolic compounds on a blood-brain barrier culture model: Expression of leptin receptors and protection against cytokine-induced damage.

The medicinal properties of grapes (Vitis vinifera L.) are well known since ancient times. Ethnobotanical grape preparations, like the Ayurvedic Darakchasava are used as cardiotonic and for the treatment of cardiovascular diseases. Dried grape products are also applied in Iranian traditional medicine for memory problems, which are linked to the pathology of brain microvessels, a special part of the cardiovascular system. The anti-inflammatory and protective effects of these traditional preparations on the cardiovascular system are related to their bioactive phenolic compounds. The blood-brain barrier (BBB), formed by brain capillaries, is not only involved in inflammatory and other diseases of the central nervous system, but also in many systemic diseases with an inflammatory component. Dietary obesity is a systemic chronic inflammatory condition in which the peripheral and central vascular system is affected. Among the cerebrovascular changes in obesity defective leptin transport across the BBB related to central leptin resistance is observed. Our aim was to study the protective effects of grape phenolic compounds epicatechin (EC), gallic acid (GA) and resveratrol (RSV) and grape-seed proanthocyanidin-rich extract (GSPE) on a cytokine-induced vascular endothelial inflammation model. Using a culture model of the BBB we investigated cytokine-induced endothelial damage and changes in the expression of leptin receptors and leptin transfer. For the BBB model, primary cultures of rat brain endothelial cells, glial cells and pericytes were used in co-culture. Cells were treated by tumor necrosis factor-α (TNF-α) and interleukin-1 β (IL-1β) (10 ng/ml each) to induce damage. Cell toxicity was evaluated by the measurement of impedance. The expression of leptin receptors was assessed by RT-qPCR and western blot. The production of reactive oxygen species (ROS) and nitric oxide (NO) were detected by fluorescent probes. GSPE (10 μg/ml), EC (10 μM), GA (1 μM) or RSV (10 μM) did not change the viability of brain endothelial cells. The gene expression of the short leptin receptor isoform, Ob-Ra, was up-regulated by GSPE, EC and RSV, while the mRNA levels of Lrp2 and clusterin, clu/ApoJ were not affected. The tested compounds did not change the expression of the long leptin receptor isoform, Ob-Rb. RSV protected against the cytokine-induced increase in albumin permeability of the BBB model. GSPE and EC exerted an antioxidant effect and GSPE increased NO both alone and in the presence of cytokines. The cytokine-induced nuclear translocation of transcription factor NF-κB was blocked by GSPE, GA and RSV. Cytokines increased the mRNA expression of Lrp2 which was inhibited by EC. RSV increased Ob-Ra and Clu in the presence of cytokines. Cytokines elevated leptin transfer across the BBB model, which was not modified by GSPE or RSV. Our results obtained on cell culture models confirm that natural grape compounds protect vascular endothelial cells against inflammatory damage in accordance with the ethnopharmacological use of grape preparations in cardiovascular diseases. Furthermore, grape compounds and GSPE, by exerting a beneficial effect on the BBB, may also be considered in the treatment of obesity after validation in clinical trials.

Increased clusterin levels after myocardial infarction is due to a defect in protein degradation systems activity

Clusterin (CLU) is induced in many organs after tissue injury or remodeling. Recently, we show that CLU levels are increased in plasma and left ventricle (LV) after MI, however, the mechanisms involved are not yet elucidated. On the other hand, it has been shown that the activity of the protein degradation systems (PDS) is affected after MI with a decrease in ubiquitin proteasome system (UPS) and an increase in macroautophagy. The aim of this study was to decipher if the increased CLU levels after MI are in part due to the alteration of PDS activity. Rat neonate cardiomyocytes (NCM) were treated with different modulators of UPS and macroautophagy in order to decipher their role in CLU expression, secretion, and degradation. We observed that inhibition of UPS activity in NCM increased CLU mRNA levels, its intracellular protein levels (p-CLU and m-CLU) and its secreted form (s-CLU). Macroautophagy was also induced after MG132 treatment but is not active. The inhibition of macroautophagy induction in MG132-treated NCM increased CLU mRNA and m-CLU levels, but not s-CLU compared to NCM only treated by MG132. We also demonstrate that CLU can be degraded in NCM through proteasome and lysosome by a macroautophagy independent pathway. In another hand, CLU silencing in NCM has no effect either on macroautophagy or apoptosis induced by MG132. However, the overexpression of CLU secreted isoform in H9c2 cells, but not in NCM decreased apoptosis after MG132 treatment. Finally, we observed that increased CLU levels in hypertrophied NCM and in failing human hearts are associated with proteasome inhibition and macroautophagy alteration. All these data suggest that increased CLU expression and secretion after MI is, in part, due to a defect of UPS and macroautophagy activities in the heart and may have a protective effect by decreasing apoptosis induced by proteasome inhibition.

Effects of Calcium Oxalate on Expression of Clusterin and Lower Urinary Tract Symptoms in Prostatitis and Benign Prostatic Hyperplasia Patients with Calculi.

BackgroundProstatic calculi are common in urological treatments. Our major purpose in the present study was to explore the occurrence and composition of prostatic calculi, and investigate the effect of calcium oxalate (CaOx) on clusterin expression and lower urinary tract symptom (LUTS) in prostatitis and benign prostatic hyperplasia (BPH) patients with calculi.Material/MethodsFrom December 2016 to January 2017, a total of 79 prostatitis patients aged more than 50 years were enrolled. The patients were divided into 3 groups: group A had small calculi (discrete, small echoes); group B had large calculi (large masses of multiple echoes, much coarser), and group C had no calculi. Immunohistochemical analysis was performed to evaluate the tissue scores. The clusterin expression was detected by quantitative real-time CR (qRT-PCR), Western blot, and immunofluorescence.ResultsAccording to multifactor analysis, age was significantly associated with prostatic calculus. The composition of prostatic calculus was an independent factor of LUTS. The clusterin expression was elevated in group B. The mRNA and protein levels of clusterin in prostatitis and BPH patients with stones were obviously higher than those in prostatitis and BPH patients without stones. CaOx enhanced clusterin expression in a dose-dependent manner.ConclusionsLarge prostatic calculi were associated with LUST. Furthermore, CaOx enhanced clusterin expression, leading to large prostatic calculi. These results may provide a therapeutic strategy for prostatitis and BPH.

Lipocalin-2 derived from adipose tissue mediates aldosterone-induced renal injury

Increased lipocalin-2 levels are not only the sensitive biomarkers but also causally contribute to the pathogenesis of acute and chronic renal injuries. Mice without lipocalin-2 are protected from the development of chronic kidney diseases. However, the source of lipocalin-2 contributing to the pathological elevation of this molecule in blood and urine during renal injury remain largely unknown. The present study demonstrated that adipose tissue-derived lipocalin-2 represented a major pathological form of this molecule causing inflammatory and fibrotic damages in kidney of mice treated with aldosterone. Compared to sham controls, wild type mice subjected to four-week treatment with aldosterone/uninephrectomy/salt (ANS) showed elevated circulating and urinary lipocalin-2, accompanied by glomerular and tubular injuries in kidney. The same treatment was not able to augment the circulating levels of lipocalin-2 in mice with selective depletion of the Lcn2 alleles in adipose tissues (Adipo-LKO). Despite the increased Lcn2 mRNA expression in kidney and lipocalin-2 protein levels in urine, Adipo-LKO mice were protected from ANS-induced tubular injuries and fibrotic renal damages. Data from acute studies confirmed that aldosterone treatment mainly promoted the urinary excretion of lipocalin-2 originated from adipose tissue; the latter acted to inhibit glomerular expression of Wilms tumor-1 (Wt-1) and enhanced the mRNA levels of clusterin (Clu) and kidney injury marker (KIM)-1. In the presence of aldosterone, acute treatment with recombinant lipocalin-2 variants significantly induced the mRNA expressions of both Clu and kidney injury molecule-1 (Kim-1). Moreover, chronic treatment with R81E, a variant form of lipocain-2, led to renal injuries in mice lacking the Lcn2 alleles, resembling those observed in wild type mice treated with ANS. Collectively, the results demonstrated that lipocalin-2 derived from adipose tissue caused ANS-induced renal injuries.

Alisol A 24-Acetate and Alisol B 23-Acetate Induced Autophagy Mediates Apoptosis and Nephrotoxicity in Human Renal Proximal Tubular Cells.

Two natural compounds alisol A 24-acetate (24A) and alisol B 23-acetate (23B) are abundant in Rhizoma alismatis. In the present study, we evaluated the induction of 24A and 23B on apoptosis and possible nephrotoxicity of human renal proximal tubular (HK-2) cells by activating autophagy and also explored its regulation on PI3K/Akt/mTOR signaling pathway. Presently, Clusterin, Kim-1, and TFF-3 were considered to be new bioindicators of nephrotoxicity. Interestingly, the protein expression and mRNA levels of Clusterin, Kim-1 and TFF-3 could be significantly increased by 23B and 24A in vivo and in vitro. Furthermore, cell apoptosis could be triggered by 23B and 24A via significantly decreasing the protein expression and mRNA levels of Bcl-2 and Bcl-xl. Autophagy of HK-2 cells could be induced by both 23B and 24A via significantly enhancing the ratio of LC3II/LC3I, the protein expression of Beclin-1 as well as the mRNA levels of LC3 and Beclin-1. Meanwhile, PI3K/Akt/mTOR signaling pathway could be inhibited by these two compounds. An autophagy inhibitor, 3-methyladenine, could partially reverse cell viability and conversely change the ratio of LC3II/LC3I and the protein expression of Bcl-2 and Kim-1. Thus this study helped to understand that 23B and 24A induced autophagy resulted in apoptosis and nephrotoxicity through inhibiting PI3K/Akt/mTOR signaling pathway, facilitating further studies for nephrotoxicity induced by these two compounds and could be beneficial for safe use of Rhizoma alismatis in clinic.

Molecular studies of the immunological effects of the sevoflurane preconditioning in the liver and lung in a rat model of liver ischemia/reperfusion injury

Sevoflurane has been shown to improve ischemia/reperfusion injury (IRI) through several mechanisms, including amelioration of inflammatory response. However, there haven't been any studies considering the potential role of the complement system in sevoflurane-mediated amelioration of ischemia/reperfusion injury. Our purpose was to investigate the molecular mechanisms involved in sevoflurane preconditioning in liver and lung injury induced by liver ischemia-reperfusion (LIR), giving emphasis to the immunological mechanisms. In order to do that, fifty male Wistar rats were randomly allocated in five groups (n=10 each): Animals in group LIR received ketamine and xylazine and were then subjected to ischemia of the right and median hepatic lobe for 45 min and reperfusion for 6h. Group SEVO/LIR received sevoflurane and then LIR was induced, as in group LIR. Animals in group SHAM/LIR were anesthetized with ketamine and xylazine and then laparotomy followed. Group SHAM/SEVO received sevoflurane for 30 min and then laparotomy followed. Finally, in group VEN, animals only received ketamine and xylazine. Our results showed that sevoflurane preconditioning significantly improved liver-biochemical tests (decreased Alanine transaminase (ALT), Alkaline phosphatase (ALP), Aspartate transaminase (AST) and Alkaline phosphatase (ALP) levels) and limited inflammatory cell infiltration in BALF. Additionally, compared with the LIR group, the reduction in plasma C3 was significantly reduced in the SEVO/LIR group. No significant differences were observed in histological examination in the liver and lung. Immunostaining of the liver for Intracellular Adhesion Molecule 1 (ICAM1) however, showed a decrease in ICAM1 levels in the SEVO/LIR group. In the lung, sevoflurane seemed to exert no effect in ICAM1 levels. Caspase 3 (CASP3) levels in the liver and the lung also appeared unaffected by sevoflurane preconditioning. In the SEVO/LIR group, ICAM1 mRNA expression was significantly reduced in the liver. No statistical significantly differences were observed in Complement component 3 (C3), Complement component 5 (C5) and Clusterin (CLU) mRNA levels in the liver or the lung tissue. Summarizing, sevoflurane preconditioning seems to ameliorate LIR-induced injury in the rats, mediated by mechanisms that include ICAM1 and complement C3 down regulation.

The E-box-like sterol regulatory element mediates the insulin-stimulated expression of hepatic clusterin

Clusterin (also known as apolipoprotein J) is a highly conserved glycoprotein involved in various biological processes, including attenuation of complement activity, sperm maturation, apoptosis, and reverse lipid transport. Although clusterin is reportedly associated with metabolic diseases, the metabolic regulation of clusterin expression is largely unknown. We investigated the effect of insulin on hepatic clusterin expression and its underlying mechanisms. Insulin increased the mRNA and protein levels of clusterin in primary hepatocytes and hepatoma cell lines. Serial deletion and mutant analysis of the clusterin promoter demonstrated that insulin-stimulated transactivation is mediated via a non-canonical E-box (NCE-box) motif in the proximal upstream region. Interestingly, sterol regulatory element binding protein-1c (SREBP-1c) co-transfection showed the same transactivation pattern as insulin stimulation in serial deletion and mutant promoter analysis. In contrast, co-transfection with a dominant negative form of SREBP-1c inhibited insulin-stimulated clusterin expression. Furthermore, insulin increased the recruitment of SREBP-1c to the NCE-box of the clusterin promoter region. Taken together, our results suggest that an NCE-box within the clusterin promoter is necessary for insulin-stimulated hepatic expression of clusterin via SREBP-1c.

Clusterin produced by Sertoli cells inhibits heat stress-induced apoptosis in the rat testis.

The objectives of this study were to determine whether the inhibition of clusterin expression in rat Sertoli cells enhances heat stress-induced apoptosis. The scrotums of rats were immersed in a water bath of 43 °C for 15 min. Testicular weight and germ cell number markedly decreased after the heat treatment in a time-dependent manner. In contrast, clusterin mRNA and protein expression levels were significantly up-regulated and peaked on day 21. The apoptotic index was markedly increased 1 day after the heat treatment. We then purified Sertoli cells from the rat testes, and an expression vector containing siRNA targeting the clusterin gene was transiently transfected into Sertoli cells. Following exposure to heat stress at 41 °C for 12 h, clusterin mRNA was markedly up-regulated after transfection with the control vector; however, the transfection of siRNA targeting the clusterin resulted in >70% reduction in the expression of clusterin mRNA. Furthermore, the apoptotic index in these Sertoli cells was significantly higher after the treatment with siRNA targeting the clusterin than control, and the most prominent difference was observed within 24 h after the heat treatment. These results suggest that an increase in the secretion of clusterin by Sertoli cells protects the testes from heat stress-induced injury.

Ochratoxin A induces rat renal carcinogenicity with limited induction of oxidative stress responses

Ochratoxin A (OTA) has displayed nephrotoxicity and renal carcinogenicity in mammals, however, no clear mechanisms have been identified detailing the relationship between oxidative stress and these toxicities. This study was performed to clarify the relationship between oxidative stress and the renal carcinogenicity induced by OTA. Rats were treated with 70 or 210μg/kg b.w. OTA for 4 or 13weeks. In the rats administrated with OTA for 13weeks, the kidney was damaged seriously. Cytoplasmic vacuolization was observed in the outer stripe of the outer medulla. Karyomegaly was prominent in the tubular epithelium. Kidney injury molecule-1 (Kim-1) was detected in the outer stripe of the outer medulla in both low- and high-dose groups. OTA increased the mRNA levels of clusterin in rat kidneys. Interestingly, OTA did not significantly alter the oxidative stress level in rat liver and kidney. Yet, some indications related to proliferation and carcinogenicity were observed. A dose-related increase in proliferating cell nuclear antigen (PCNA) was observed at 4weeks in both liver and kidney, but at 13weeks, only in the kidney. OTA down-regulated reactive oxygen species (ROS) and up-regulated vimentin and lipocalin 2 in rat kidney at 13weeks. The p53 gene was decreased in both liver and kidney at 13weeks. These results suggest that OTA caused apparent kidney damage within 13weeks but exerted limited effect on oxidative stress parameters. It implies that cell proliferation is the proposed mode of action for OTA-induced renal carcinogenicity.

Role of an extracellular chaperone, Clusterin in the pathogenesis of Pseudoexfoliation Syndrome and Pseudoexfoliation Glaucoma

Pseudoexfoliation (PEX), an age related disorder is a prominent contributor to secondary glaucoma. Earlier studies have suggested involvement of clusterin in the development of PEX. We designed a case-control study to understand the role of clusterin single nucleotide polymorphisms (SNPs) in PEX and analyzed the role of risk alleles in the disease. Genotyping of SNPs in 136 PEX patients and 89 controls of Indian origin revealed a genetic association between rs2279590 and PEX in Indian population with a p-value of 0.004. The high risk allele “G” at rs2279590 has an effect on clusterin mRNA expression. There was a twofold higher clusterin mRNA level in “GG” genotyped individuals in comparison to “AA” genotyped individuals (p = 0.039). Western blot and immunohistochemistry studies showed an upregulation of Clusterin protein in pseudoexfoliation glaucoma (PXG) affected individuals in both aqueous humor and lens capsules respectively. Together, our results reveal that rs2279590 was found to be associated with PEX in Indian population and the risk allele mediates an allele specific upregulation of the clusterin mRNA. Moreover, upregulation of Clusterin protein in PXG individuals augments further protein deposition.

Deficiency of Clusterin Exacerbates High-Fat Diet-Induced Insulin Resistance in Male Mice

The present study examined the role of clusterin in insulin resistance in high fat-fed wild-type and clusterin knockout (KO) mice. The plasma levels of glucose and C-peptide and islet size were increased in clusterin KO mice after an 8-week high-fat diet. In an ip glucose tolerance test, the area under the curve for glucose was not different, whereas the area under the curve for insulin was higher in clusterin KO mice. In a hyperinsulinemic-euglycemic clamp, the clamp insulin levels were higher in clusterin KO mice after the high-fat diet. After adjusting for the clamp insulin levels, the glucose infusion rate, suppression of hepatic glucose production, and glucose uptake were lower in clusterin KO mice in the high fat-fed group. The plasma levels of clusterin and clusterin mRNA levels in the skeletal muscle and liver were increased by the high-fat diet. The mRNA levels of the antioxidant enzymes were lower, and the mRNA levels of nicotinamide adenine dinucleotide phosphate oxidase (NOX) 1 and cytokines and protein carbonylation were higher in the skeletal muscle and liver in clusterin KO mice after the high-fat diet. Palmitate-induced gene expressions of NOX1 and cytokines were higher in the primary cultured hepatocytes of clusterin KO mice compared with the wild-type mice. Clusterin inhibited the gene expression and reactive oxygen species generation by palmitate in the hepatocytes and C2C12. AKT phosphorylation by insulin was reduced in the hepatocytes of clusterin KO mice. These results suggest that clusterin plays a protective role against high-fat diet-induced insulin resistance through the suppression of oxidative stress and inflammation.

MP66-08 CLUSTERIN PRODUCED BY SERTOLI CELLS INHIBITS HEAT STRESS-INDUCED APOPTOSIS IN RAT TESTIS

You have accessJournal of UrologyInfertility: Basic Research, Physiology & Pathophysiology1 Apr 2014MP66-08 CLUSTERIN PRODUCED BY SERTOLI CELLS INHIBITS HEAT STRESS-INDUCED APOPTOSIS IN RAT TESTIS Kei Matsushita, Hideaki Miyake, and Masato Fujisawa Kei MatsushitaKei Matsushita More articles by this author , Hideaki MiyakeHideaki Miyake More articles by this author , and Masato FujisawaMasato Fujisawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.2055AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The objectives of this study were to characterize the changes in expression of clusterin that is shown to have a chaperon-like activity and act as a powerful inhibitor against a wide variety of proapoptotic stimuli, in rat testis following heat stress, and to investigate whether the inhibition of clusterin expression in rat Sertoli cells enhances apoptosis induced by heat exposure. METHODS Adult male Sprague-Dawley rats were used in this study. The scrotum of rats were completely immersed in a water bath and exposed to either 43C or 37C as a control for 15 minutes. Rats were then sacrificed on days 1, 4, 7, 14 and 21 after heat treatment. Time-course changes in testicular weight and germ cell number were measured, expression levels of clusterin mRNA and protein were evaluated respectively, and immunohistochemical staining of clusterin and TUNEL assay were also performed. We then purified Sertoli cells from rat testes, and expression vector containing short hairpin RNA (shRNA) targeting rat clusterin gene or control vector alone was transiently transfected into Sertoli cells. Following exposure to heat stress at 41C for 12 hours, changes in clusterin mRNA expression and apoptotic index were analyzed by real-time RT-PCR and TUNEL assay, respectively. RESULTS Both testicular weight and germ cell number in rat testis after heat treatment were markedly reduced in time-dependent manners compared with those after control treatment. To the contrary, expression levels of clusterin mRNA and protein were significantly up-regulated following heat treatment and reached to the highest levels on day 21, while the apoptotic index was highly increased 1 day after heat treatment, and was maintained at higher levels than that after control treatment. In cultured rat Sertoli cells exposed to heat stress, clusterin mRNA was markedly up-regulated in a time-dependent manner after transfection with control vector; however, shRNA targeting clusterin resulted in the inhibition of clusterin mRNA expression by more than 70%. Furthermore, apoptotic index in these Sertoli cells after treatment with shRNA targeting clusterin was significantly higher than that after treatment with control vector, and this difference was most prominent within 24 hours after heat stress. CONCLUSIONS These findings suggest that clusterin secreted by Sertoli cells protect testis from injury induced by heat stress; accordingly, further up-regulation of clusterin expression in the testis following the exposure to heat stress could be a promising approach for the inhibition of heat-induced disruption of spermatogenesis. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e743 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Kei Matsushita More articles by this author Hideaki Miyake More articles by this author Masato Fujisawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Clusterin Plays an Important Role in Clear Renal Cell Cancer Metastasis

Objective: Clusterin (CLU) is implicated in regulating clear renal cell carcinoma (CRCC) progression and metastasis, yet the mechanisms are not elucidated. In the present study, we explored the potential role of CLU in CRCC metastasis. Methods: Levels of CLU mRNA and CLU protein were measured by RT-PCR and immunohistochemistry analysis in 22 CRCC with metastasis and 22 without metastasis and 22 samples of normal kidney tissue. After CLU silencing and re-expression, the migration and invasion in vitro and in vivo of Caki-2 cells were determined by wound healing assay, transwell migration assay and pulmonary nodule assay, respectively. The expression of pERK1/2 and MMP-9 were detected by RT-PCR and Western blot assay. Results: We found a significant increase of CLU and CLU mRNA expression in CRCC, and the expression of CLU is strongly correlated in patients with metastatic disease. We discovered that CLU-rich Caki-2 cells displayed higher invasive ability which prompted us to investigate if CLU silencing could reduce the migration and invasion in Caki-2 cells. Compared with the vector-transfected cells, CLU knocked-down (CLUi) cells showed reduced migration and invasion in vitro, as well as decreased metastatic potential in experimental metastasis. Re-expression of CLU in CLUi cells restored the invasive phenotypes. We found that MMP-9 was downregulated in CLUi cells. We also discovered that levels of activated ERK1/2 correlated with the rich expression of CLU and MMP-9. Conclusion: Our data suggest that CLU may regulate aggressive behavior of human CRCC cells through modulating ERK1/2 signaling and MMP-9 expression.

The chaperone protein clusterin may serve as a cerebrospinal fluid biomarker for chronic spinal cord disorders in the dog

Chronic spinal cord dysfunction occurs in dogs as a consequence of diverse aetiologies, including long-standing spinal cord compression and insidious neurodegenerative conditions. One such neurodegenerative condition is canine degenerative myelopathy (DM), which clinically is a challenge to differentiate from other chronic spinal cord conditions. Although the clinical diagnosis of DM can be strengthened by the identification of the Sod1 mutations that are observed in affected dogs, genetic analysis alone is insufficient to provide a definitive diagnosis. There is a requirement to identify biomarkers that can differentiate conditions with a similar clinical presentation, thus facilitating patient diagnostic and management strategies. A comparison of the cerebrospinal fluid (CSF) protein gel electrophoresis profile between idiopathic epilepsy (IE) and DM identified a protein band that was more prominent in DM. This band was subsequently found to contain a multifunctional protein clusterin (apolipoprotein J) that is protective against endoplasmic reticulum (ER) stress-mediated apoptosis, oxidative stress, and also serves as an extracellular chaperone influencing protein aggregation. Western blot analysis of CSF clusterin confirmed elevated levels in DM compared to IE (p < 0.05). Analysis of spinal cord tissue from DM and control material found that clusterin expression was evident in neurons and that the clusterin mRNA levels from tissue extracts were elevated in DM compared to the control. The plasma clusterin levels was comparable between these groups. However, a comparison of clusterin CSF levels in a number of neurological conditions found that clusterin was elevated in both DM and chronic intervertebral disc disease (cIVDD) but not in meningoencephalitis and IE. These findings indicate that clusterin may potentially serve as a marker for chronic spinal cord disease in the dog; however, additional markers are required to differentiate DM from a concurrent condition such as cIVDD.

Blood Clusterin Levels, rs9331888 Polymorphism, and the Risk of Alzheimer's Disease

Variants in the clusterin gene have been associated with Alzheimer's disease (AD) through replicated genome-wide studies, but the underlying mechanisms remain unknown. In this study the association of the AD clusterin common risk polymorphism rs9331888 with blood clusterin levels was tested in 104 AD subjects and 104 healthy controls. Blood clusterin levels were significantly elevated in AD patients (p < 0.05). The rs9331888 AD-risk variant was associated with low clusterin mRNA and protein levels in an allele-dose dependent manner in both groups (p < 0.001). This study indicates that the rs9331888 AD-risk variant is associated with low blood clusterin levels.

Clusterin mRNA and Protein in Alzheimer's Disease

Clusterin, a multifunctional lipoprotein is expressed in a number of tissues but expression is particularly high in the brain, where it binds to amyloid-β (Aβ), possibly facilitating Aβ transport into the bloodstream. Its concentration in peripheral blood was identified as a potential biomarker for Alzheimer's disease (AD) and predicted retention of (11)C-Pittsburgh Compound B in the temporal lobe. Single-nucleotide polymorphisms in the clusterin gene, CLU, are associated with the risk of developing AD. We measured clusterin mRNA levels in control and AD brains and investigated the relationship of the clusterin protein to soluble, insoluble, and plaque-associated Aβ. Clusterin mRNA levels were unchanged when normalized to GAPDH but modestly increased in the frontal and temporal cortex in AD in relation to NSE and MAP-2. Levels of NSE and MAP-2 mRNA were reduced in the AD frontal cortex. Clusterin protein concentration was unchanged and did not correlate with the amount of Aβ present. In the frontal cortex, clusterin concentration was higher in APOE ε4-negative brains but no effect of APOE was detected in the temporal cortex or thalamus. Overall clusterin mRNA and protein levels are unaltered in the neocortex in AD and clusterin concentration does not reflect Aβ content. The increase in clusterin noted in peripheral blood in AD may reflect increased passage of this chaperone protein across the blood-brain barrier but further work is needed to determine how CLU variants influence the development of AD.

The Conserved Clusterin Gene Is Expressed in the Developing Choroid Plexus Under the Regulation of Notch But Not IGF Signaling in Zebrafish

Recent genome-wide association studies have implicated the clusterin gene in the etiology of Alzheimer's disease. The expression and function of clusterin in the developing brain, however, is poorly understood. In this study, we have characterized the zebrafish clusterin gene and determined its structural conservation, developmental expression, and physiological regulation. The structure of the zebrafish clusterin gene and protein is similar to its human orthologue. Biochemical assays show that zebrafish Clusterin is a secreted protein that cannot bind IGFs. In adult zebrafish, clusterin mRNA is detected in many tissues. In early development, clusterin mRNA becomes detectable at 12 h postfertilization, and its levels gradually increase thereafter. In situ hybridization analysis indicates that clusterin mRNA is specifically expressed in the developing diencephalic and myelencephalic choroid plexus. Among various stresses tested, heat shock, but not hypoxic or ionic stresses, increases the levels of clusterin mRNA. Inhibition of the IGF-I receptor-mediated signaling or overexpression of IGF ligands did not change clusterin mRNA levels. In comparison, inhibition or targeted knockdown of Notch signaling significantly increased clusterin mRNA expression in choroid plexus. These results suggest that clusterin is a marker of choroid plexus in zebrafish, and its expression in the developing choroid plexus is under the regulation of Notch but not IGF signaling.

Deficiency of Clusterin Inhibits Neointimal Hyperplasia After Vascular Injury

Increased clusterin mRNA and protein levels have been detected in various tissues undergoing stress, and we previously reported that clusterin is markedly induced in media and neointima following vascular injury. The present study therefore investigated the impact of clusterin on neointimal hyperplasia following vascular injury. As compared with wild-type mice, clusterin knockout mice (clusterin-KO) demonstrated a significant decrease of the intima/media ratio 4 weeks after cuff placement. Immunohistochemical analysis of injured femoral arteries in clusterin-KO demonstrated the accumulation of p53 in nuclei of neointimal vascular smooth muscle cells (VSMCs). Moreover, VSMCs from either clusterin-KO or rat VSMCs treated with clusterin-short-interfering (si) RNA subjected to static stretch exhibited significantly increased p53 and p21, and increased G1 cell cycle arrest as indicated by flow cytometry compared with VSMCs from wild-type mice. Reduced clusterin expression reduced the proliferation of VSMCs and induced G1 arrest via p53 and p21. Clusterin therefore represents a promising molecular target to limit restenosis after coronary intervention.