Characterization and functional consequences of underexpression of clusterin in rheumatoid arthritis

We previously compared by microarray analysis gene expression in rheumatoid arthritis (RA) and osteoarthritis (OA) tissues. Among the set of genes identified as a molecular signature of RA, clusterin (clu) was one of the most differentially expressed. In the present study we sought to assess the expression and the role of CLU (mRNA and protein) in the affected joints and in cultured fibroblast-like synoviocytes (FLS) and to determine its functional role. Quantitative RT-PCR, Northern blot, in situ hybridization, immunohistochemistry, and Western blot were used to specify and quantify the expression of CLU in ex vivo synovial tissue. In synovial tissue, the protein was predominantly expressed by synoviocytes and it was detected in synovial fluids. Both full-length and spliced isoform CLU mRNA levels of expression were lower in RA tissues compared with OA and healthy synovium. In synovium and in cultured FLS, the overexpression of CLU concerned all protein isoforms in OA whereas in RA, the intracellular forms of the protein were barely detectable. Transgenic overexpression of CLU in RA FLS promoted apoptosis within 24 h. We observed that CLU knockdown with small interfering RNA promoted IL-6 and IL-8 production. CLU interacted with phosphorylated IkappaBalpha. Differential expression of CLU by OA and RA FLS appeared to be an intrinsic property of the cells. Expression of intracellular isoforms of CLU is differentially regulated between OA and RA. We propose that in RA joints, high levels of extracellular CLU and low expression of intracellular CLU may enhance NF-kappaB activation and survival of the synoviocytes.     

Clusterin enhances AKT2-mediated motility of normal and cancer prostate cells through a PTEN and PHLPP1 circuit

Clusterin (CLU) is a chaperone-like protein with multiple functions. sCLU is frequently upregulated in prostate tumor cells after chemo- or radiotherapy and after surgical or pharmacological castration. Moreover, CLU has been documented to modulate the cellular homolog of murine thymoma virus akt8 oncogene (AKT) activity. Here, we investigated how CLU overexpression influences phosphatidylinositol 3′-kinase (PI3K)/AKT signaling in human normal and cancer epithelial prostate cells. Human prostate cells stably transfected with CLU were broadly profiled by reverse phase protein array (RPPA), with particular emphasis on the PI3K/AKT pathway. The effect of CLU overexpression on normal and cancer cell motility was also tested. Our results clearly indicate that CLU overexpression enhances phosphorylation of AKT restricted to isoform 2. Mechanistically, this can be explained by the finding that the phosphatase PH domain leucine-rich repeat-containing protein phosphatase 1 (PHLPP1), known to dephosphorylate AKT2 at S474, is markedly downregulated by CLU, whereas miR-190, a negative regulator of PHLPP1, is upregulated. Moreover, we found that phosphatase and tensin homolog (PTEN) was heavily phosphorylated at the inhibitory site S380, contributing to the hyperactivation of AKT signaling. By keeping AKT2 phosphorylation high, CLU dramatically enhances the migratory behavior of prostate epithelial cell lines with different migratory and invasive phenotypes, namely prostate normal epithelial 1A (PNT1A) and prostatic carcinoma 3 (PC3) cells. Altogether, our results unravel for the first time a circuit by which CLU can switch a low migration phenotype toward a high migration phenotype, through miR-190-dependent downmodulation of PHLPP1 expression and, in turn, stabilization of AKT2 phosphorylation.     

Nuclear clusterin accumulation during heat shock response: implications for cell survival and thermo-tolerance induction in immortalized and prostate cancer cells

Clusterin (CLU), whose role is still debated, is differentially regulated in several patho-physiological processes and invariably induced during apoptosis. In heat shock response, CLU is considered a stress-inducible, pro-survival/cyto-protective factor via an HSE element present in his promoter. In both human prostate PNT1A and PC-3 epithelial cells we found that apoptotic stimuli induced nuclear localization of CLU (nCLU), and that overexpression of nCLU is pro-apoptotic. We show here that CLU time-course accumulation kinetic is different from that of HSP70 in these cells, thus other factor(s) might mediate HSF-1 activation and CLU expression. Sub-lethal heat shock inhibited the secretion of CLU (sCLU), leading to increased cytoplasm accumulation of CLU (cCLU) in association to cell survival. At difference, lethal heat stress caused massive accumulation of pro-apoptotic nCLU in cells dying by caspase-3-dependent apoptosis. Double heat stress (sub-lethal heat shock followed by recovery and lethal stress) induced HSP70 and thermo-tolerance in PNT1A cells, but not in PC-3 cells. In PNT1A cells, CLU secretion was inhibited and cCLU was accumulated, suggesting that cCLU might be pro-survival, while in PC-3 cells accumulation of nCLU was concomitant to caspase-3 induction and PARP activation instead. Thus, CLU expression/sub-cellular localization is strictly related to cell fate. In particular, nCLU and physiological levels of HSP70 affected cell survival in an antagonistic fashion. Prevalence of heat-induced nCLU, not allowing PC-3 cells to cope with heat shock, could be the rational explaining why malignant cells are more sensitive to heat when delivered by minimally invasive procedures for ablation of localized prostate cancer.     

Genetics of clusterin isoform expression and Alzheimer's disease risk

The minor allele of rs11136000 within CLU is strongly associated with reduced Alzheimer's disease (AD) risk. The mechanism underlying this association is unclear. Here, we report that CLU1 and CLU2 are the two primary CLU isoforms in human brain; CLU1 and CLU2 share exons 2-9 but differ in exon 1 and proximal promoters. The expression of both CLU1 and CLU2 was increased in individuals with significant AD neuropathology. However, only CLU1 was associated with the rs11136000 genotype, with the minor "protective" rs11136000T allele being associated with increased CLU1 expression. Since CLU1 and CLU2 are predicted to encode intracellular and secreted proteins, respectively, we compared their expression; for both CLU1 and CLU2 transfected cells, clusterin is present in the secretory pathway, accumulates in the extracellular media, and is similar in size to clusterin in human brain. Overall, we interpret these results as indicating that the AD-protective minor rs11136000T allele is associated with increased CLU1 expression. Since CLU1 and CLU2 appear to produce similar proteins and are increased in AD, the AD-protection afforded by the rs11136000T allele may reflect increased soluble clusterin throughout life.     

Nicotinamide phosphoribosyl transferase/pre-B cell colony-enhancing factor/visfatin is required for lymphocyte development and cellular resistance to genotoxic stress

Nicotinamide phosphoribosyl transferase (Nampt)/pre-B cell colony-enhancing factor (PBEF)/visfatin is a protein displaying multiple functional properties. Originally described as a cytokine-like protein able to regulate B cell development, apoptosis, and glucose metabolism, this protein also plays an important role in NAD biosynthesis. To gain insight into its physiological role, we have generated a mouse strain expressing a conditional Nampt allele. Lack of Nampt expression strongly affects development of both T and B lymphocytes. Analysis of hemizygous cells and in vitro cell lines expressing distinct levels of Nampt illustrates the critical role of this protein in regulating intracellular NAD levels. Consequently, a clear relationship was found between intracellular Nampt levels and cell death in response to the genotoxic agent MNNG (N-methyl-N'-nitro-N-nitrosoguanidine), confirming that this enzyme represents a key regulator of cell sensitivity to NAD-consuming stress secondary to poly(ADP-ribose) polymerases overactivation. By using mutant forms of this protein and a well-characterized pharmacological inhibitor (FK866), we unequivocally demonstrate that the ability of the Nampt to regulate cell viability during genotoxic stress requires its enzymatic activity. Collectively, these data demonstrate that Nampt participates in cellular resistance to genotoxic/oxidative stress, and it may confer to cells of the immune system the ability to survive during stressful situations such as inflammation.     

Interleukin 6 signaling regulates promyelocytic leukemia protein gene expression in human normal and cancer cells

Tumor suppressor PML is induced under viral and genotoxic stresses by interferons and JAK-STAT signaling. However, the mechanism responsible for its cell type-specific regulation under non-stimulated conditions is poorly understood. To analyze the variation of PML expression, we utilized three human cell types, BJ fibroblasts and HeLa and U2OS cell lines, each with a distinct PML expression pattern. Analysis of JAK-STAT signaling in the three cell lines revealed differences in levels of activated STAT3 but not STAT1 correlating with PML mRNA and protein levels. RNAi-mediated knockdown of STAT3 decreased PML expression; both STAT3 level/activity and PML expression relied on IL6 secreted into culture media. We mapped the IL6-responsive sequence to an ISRE(-595/-628) element of the PML promoter. The PI3K/Akt/NFκB branch of IL6 signaling showed also cell-type dependence, being highest in BJ, intermediate in HeLa, and lowest in U2OS cells and correlated with IL6 secretion. RNAi-mediated knockdown of NEMO (NF-κ-B essential modulator), a key component of NFκB activation, suppressed NFκB targets LMP2 and IRF1 together with STAT3 and PML. Combined knockdown of STAT3 and NEMO did not further promote PML suppression, and it can be bypassed by exogenous IL6, indicating the NF-κB pathway acts upstream of JAK-STAT3 through induction of IL6. Our results indicate that the cell type-specific activity of IL6 signaling pathways governs PML expression under unperturbed growth conditions. As IL6 is induced in response to various viral and genotoxic stresses, this cytokine may regulate autocrine/paracrine induction of PML under these pathophysiological states as part of tissue adaptation to local stress.     

Proteomic analysis identified N-cadherin, clusterin, and HSP27 as mediators of SPARC (secreted protein, acidic and rich in cysteines) activity in melanoma cells

Secreted protein, acidic and rich in cysteines (SPARC) is a secreted protein associated with increased aggressiveness of different human cancer types. In order to identify downstream mediators of SPARC activity, we performed a 2-DE proteomic analysis of human melanoma cells following antisense-mediated downregulation of SPARC expression. We found 23/504 differential spots, 15 of which were identified by peptide fingerprinting analysis. Three of the differential proteins (N-cadherin (N-CAD), clusterin (CLU), and HSP27) were validated by immunoblotting, confirming decreased levels of N-CAD and CLU and increased amounts of HSP27 in conditioned media of cells with diminished SPARC expression. Furthermore, transient knock down of SPARC expression in melanoma cells following adenoviral-mediated transfer of antisense RNA confirmed these changes. We next developed two different RNAs against SPARC that were able to inhibit in vivo melanoma cell growth. Immunoblotting of the secreted fraction of RNAi-transfected melanoma cells confirmed that downregulation of SPARC expression promoted decreased levels of N-CAD and CLU and increased secretion of HSP27. Transient re-expression of SPARC in SPARC-downregulated cells reverted extracellular N-CAD, CLU, and HSP27 to levels similar to those in the control. These results constitute the first evidence that SPARC, N-CAD, CLU, and HSP27 converge in a unique molecular network in melanoma cells.     

Identification of genotoxic stress in human cells by fluorescent monitoring of p53 expression.

The tumor suppressor protein p53 is induced upon DNA damage essentially by post-translational regulatory mechanisms, which lead to a substantial increase of p53 levels. To exploit this essential property of p53, we developed a novel reporter system for monitoring accumulation and subcellular translocation of p53 protein, which is able to function as a simple test for detecting mutagenic and genotoxic stress in human cells. For this purpose, we constructed a plasmid with a specific translational TP53::EGFP gene fusion and selected stable transfected clones in the human cell line HEK293, in which p53 is functionally stabilized due to the expression of the transgenic adenoviral E1A oncoproteins. HEK293-TP53::EGFP clones may be used as a living cell system for monitoring not only of the induction of p53 protein in the cell, but also of its subcellular localization. Using this human reporter cell system, we examined levels of p53 by fluorescence microscopy and by FACS analysis following treatment with several classes of genotoxic and carcinogenic compounds. All tested DNA damaging agents caused a significant increase of intracellular p53-EGFP levels in a concentration-dependent manner. On the other hand, non-genotoxic carcinogens and stress conditions that cannot damage DNA were not able to induce p53-EGFP accumulation. The induction effect caused by genotoxic stress was found to be dependent on the endogenous p53 status, because it was not observed in p53-deficient cell lines. This corroborates the notion that p53 may be used as an universal sensor for genotoxic stress and demonstrates the usefulness of HEK293-p53-EGFP cells as a reporter system for identification of mutagens and genotoxic carcinogens in human cells by means of visualizing and monitoring intracellular p53 levels and localization.     

Influenza A virus nucleoprotein induces apoptosis in human airway epithelial cells: implications of a novel interaction between nucleoprotein and host protein Clusterin

Apoptosis induction is an antiviral host response, however, influenza A virus (IAV) infection promotes host cell death. The nucleoprotein (NP) of IAV is known to contribute to viral pathogenesis, but its role in virus-induced host cell death was hitherto unknown. We observed that NP contributes to IAV infection induced cell death and heterologous expression of NP alone can induce apoptosis in human airway epithelial cells. The apoptotic effect of IAV NP was significant when compared with other known proapoptotic proteins of IAV. The cell death induced by IAV NP was executed through the intrinsic apoptosis pathway. We screened host cellular factors for those that may be targeted by NP for inducing apoptosis and identified human antiapoptotic protein Clusterin (CLU) as a novel interacting partner. The interaction between IAV NP and CLU was highly conserved and mediated through β-chain of the CLU protein. Also CLU was found to interact specifically with IAV NP and not with any other known apoptosis modulatory protein of IAV. CLU prevents induction of the intrinsic apoptosis pathway by binding to Bax and inhibiting its movement into the mitochondria. We found that the expression of IAV NP reduced the association between CLU and Bax in mammalian cells. Further, we observed that CLU overexpression attenuated NP-induced cell death and had a negative effect on IAV replication. Collectively, these findings indicate a new function for IAV NP in inducing host cell death and suggest a role for the host antiapoptotic protein CLU in this process.     

Clusterin is a short half‐life,poly‐ubiquitinated protein,which controls the fate of prostate cancer cells

The Clusterin (CLU) gene produces different forms of protein products, which vary in their biological properties and distribution within the cell. Both the extra‐ and intracellular CLU forms regulate cell proliferation and apoptosis. Dis‐regulation of CLU expression occurs in many cancer types, including prostate cancer. The role that CLU plays in tumorigenesis is still unclear. We found that CLU over‐expression inhibited cell proliferation and induced apoptosis in prostate cancer cells. Here we show that depletion of CLU affects the growth of PC‐3 prostate cancer cells. Following siRNA targeting all CLU mRNA variants, all protein products quickly disappeared, inducing cell cycle progression and higher expression of specific proliferation markers (i.e., H3 mRNA, PCNA, and cyclins A, B1, and D) as detected by RT‐qPCR and Western blot. Quite surprisingly, we also found that the turnover of CLU protein is very rapid and tightly regulated by ubiquitin–proteasome mediated degradation. Inhibition of protein synthesis by cycloheximide showed that CLU half‐life is less than 2 h. CLU protein products were found poly‐ubiquitinated by co‐immuniprecipitation. Proteasome inhibition by MG132 caused stabilization and accumulation of all CLU protein products, including the nuclear form of CLU (nCLU), and committing cells to caspase‐dependent death. In conclusion, proteasome inhibition may induce prostate cancer cell death through accumulation of nCLU, a potential tumor suppressor factor. J. Cell. Physiol. 219: 314–323, 2009. © 2009 Wiley‐Liss, Inc.     

Up-regulated lnc-SNHG1 contributes to osteosarcoma progression through sequestration of miR-577 and activation of WNT2B/Wnt/β-catenin pathway

Long noncoding RNA small nucleolar RNA host gene 1 (lnc-SNHG1) was reported to play an oncogenic role in the progression of cancers. However, the roles of SNHG1 and its molecular mechanism in osteosarcoma (OS) cells are largely unknown. In present study, we found that the expression of SNHG1 was up-regulated in OS tissues and cell lines. OS patients with the high SNHG1 expression were positively correlated with tumor size, TNM stage and lymph node metastasis. In addition, SNHG1 overexpression promoted cell proliferation, cell migration and EMT process in U2OS and MG63 cells and tumor growth in vivo. Furthermore, we also found that miR-577 could act as a ceRNAof SNHG1 in OS cells and the promotion of OS progression induced by lnc-SNHG1 overexpression required the inactivity of miR-577. Besides, we identified that WNT2B acted as a target of miR-577, and WNT2B played the oncogenic role in OS cells by activating Wnt/β-catenin pathway. In short, our study suggested that lnc-SNHG1 could promote OS progression via miR-577 and WNT2B. The lnc-SNHG1/miR-577/WNT2B/Wnt/β-catenin axis regulatory network might provide a potential new therapeutic strategy for OS treatment.     

Fibronectin-1 modulated by the long noncoding RNA OIP5-AS1/miR-200b-3p axis contributes to doxorubicin resistance of osteosarcoma cells

Chemoresistance has been an obstacle in the further improvement of 5-year survival rates of osteosarcoma (OS) patients, but the underlying mechanism of chemo-resistance remains unclear. A comprehensive analysis of mRNAs and noncoding RNAs related to OS chemo-resistance could help solve this problem. In the current study, we first identified that fibronectin-1 (FN1), screened by microarray analysis in three paired chemo-resistant and chemo-sensitive OS cell lines, was significantly upregulated in the chemo-resistant OS cell lines and tissues and was related to unfavourable prognosis. Further functional assays revealed that FN1 inhibition greatly increased the sensitivity of OS cells to doxorubicin in vitro and in vivo, whereas FN1 overexpression had the opposite effect. Moreover, mechanistic investigation demonstrated, by a series of assays that included luciferase reporter gene, RNA immunoprecipitation, RNA pull-down and rescue assays, that FN1 expression was regulated by the oncogenic long noncoding RNA (lncRNA) OIP5-AS1 through sponging miR-200b-3p. Thus, these results indicated the role and potential application of the lncRNA OIP5-AS1/miR-200b-3p/FN1 regulatory pathway as a promising target in treatment of OS chemo-resistance.     

Hematopoietic cell kinase enhances osteosarcoma development via the MEK/ERK pathway

Osteosarcoma (OS) is a sarcoma with high rates of pulmonary metastases and mortality. The mechanisms underlying tumour generation and development in OS are not well-understood. Haematopoietic cell kinase (HCK), a vital member of the Src family of kinase proteins, plays crucial roles in cancer progression and may act as an anticancer target; however, the mechanism by which HCK enhances OS development remains unexplored. Therefore, we investigated the role of HCK in OS development in vitro and in vivo. Downregulation of HCK attenuated OS cell proliferation, migration and invasion and increased OS cell apoptosis, whereas overexpression of HCK enhanced these processes. Mechanistically, HCK expression enhanced OS tumorigenesis via the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway; HCK upregulation increased the phosphorylation of MEK and ERK and promoted epithelial-mesenchymal transition, with a reduction in E-cadherin in vitro. Furthermore, HCK downregulation decreased the tumour volume and weight in mice transplanted with OS cells. In conclusion, HCK plays a crucial role in OS tumorigenesis, progression and metastasis via the MEK/ERK pathway, suggesting that HCK is a potential target for developing treatments for OS.     

Development and characterization of SV40 immortalized rat parotid acinar cell lines

Summary Rat parotid salivary gland acinar cells were transfected by CaPO₄ precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 30 clonal cell lines, 2 were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the β-adrenergic agonist (isoproterenol), vasoactive intestinal peptide prostaglandin E₁, and forskolin were effective activators of intracellular cyclic adenosine 3′:5′-cyclic monophosphate production. Phenylephrine, carbamylcholine, and UTP were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without affect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express the SV40 large T antigen. Electron microscopic evaluation documented moderate to high levels of cytodifferentiation with the maintenance of tripartite junctional complexes, cellular polarization, and presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 22 and 36 h, respectively.     

TSSC3 overexpression reduces stemness and induces apoptosis of osteosarcoma tumor-initiating cells

Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents, typically presenting with poor prognosis. Recent studies suggested that tumor initiating cells (T-ICs) drive tumor formation and relapse or metastasis and are relatively resistant to cell death induced by conventional chemo- and radiotherapies. Therefore, the poor prognosis of OS appears to be associated with T-ICs. Here, we enriched T-ICs in OS cell lines and evaluated whether the imprinted gene TSSC3 (tumor-suppressing STF cDNA 3) associated with apoptosis could affect T-ICs in OS. Sarcosphere selection and serial clone-forming unit assays were successfully used to enrich T-ICs from OS cell lines. Enrichment of T-ICs from a malignantly transformed hFOB1.19 osteoblast cell line (MThFOB1.19) indicated that OS T-ICs could originate from differentiated cells, and most of these MThFOB1.19 cells showed stem-like features. TSSC3 was expressed at a low level in T-ICs, while overexpression of TSSC3 could efficiently downregulate the expression of stem cell markers Nanog, Oct4 and Sox2 in T-ICs and decrease the clone formation rate, as well as downregulate tumorigenesis in MThFOB1.19 cells, supporting a suppressive role for TSSC3 in OS T-ICs. Furthermore, overexpression of TSSC3 was found to induce apoptosis of OS T-ICs through increasing cleaved caspase-3 (active form), increasing the release of Cyt c and decreasing pro-caspase-9 (pro-enzyme form), as well as disruption of the mitochondrial membrane potential (ΔΨ). Taken together, our findings provide preliminary evidence that TSSC3 inhibits OS tumorigenicity through reducing stemness and promoting apoptosis of T-ICs. Thus, targeting TSSC3 may be a promising approach to suppressing tumorigenicity in OS.