Clusterin (CLU) is a secreted heterodimeric glycoprotein thatcan be produced almost ubiquitously in mammalians tissues.1Its gene expression is subjected to complex regulation and canchange enormously according to different stimuli.2 Cloned andidentified as the most potently induced gene in the regressingrat ventral prostate following androgen-ablation,3 CLU wasalmost simultaneously characterized and isolated by differentresearch groups working in widely divergent areas.2 CLU iscoded by a single copy gene, located on chromosome 8.4 Thegene codes for an initial precursor peptide glycosylated andcleaved into two a and b chains of 40 kDa each, held togetherby a unique five disulfide bond motif in the extracellular matureform.1 This secreted form of CLU has been suggested to act asa molecular chaperone following stress-induced injury,5clearing extracellular debris.6 However, it has been reportedthe existence of an inactive, cytoplasmic form of CLUproduced by alternative splicing that is converted by ionizingirradiation to a truncated mature nuclear isoform,7 which bindsthe Ku70/Ku80 complex in cell-free systems8 inhibiting cellgrowth and survival7 probably by a caspase-3-independentmechanism.9 Other alternative CLU isoforms, produced eitherby exon skipping10 or by post-translational modificationsactivated by apoptosis,11,12 were recently described. Thesedifferent isoforms of CLU have been suggested to beantiapoptotic6,13 or proapoptotic.7,10,12,14–16These controversial reports on the role of CLU might berelated to specific proteomic profiles that are produced bydifferent apoptotic stimuli (i.e. the general protein pattern ofCLU and the relative ratio between different CLU isoforms).This might explain why CLU has been involved in a plethora ofpathophysiological processes, including cell–cell and cell–matrix adhesion, cell differentiation, transformation, aging17,18and cancer,19 but its biological role still remains to be clearlyestablished. Reports suggesting that CLU may be a potentialtumor suppressor gene include the finding that CLU suppressesNF-kB activity and the metastatic phenotype ofneuroblastoma cells.20 We have previously reported that CLUoverexpression inhibits cell cycle progression of simian virus40(SV40)-immortalized human prostate PNT2 and PNT1Aepithelial cells.21To further assess the role of CLU in apoptotic processes wehave studied its expression pattern during the regulation ofcalcium homeostasis. Ca2þ is an important regulator ofapoptosis and cell survival.22,23,24 Both pathological increaseof Ca2þ concentration in the cytosol compartment byinophores25 and depletion of intracellular Ca2þ stores maytrigger apoptosis by disrupting intracellular architecture andallowing effectors to gain access to their substrates.24,26,27Activation of apoptotic endonucleases28 eliciting DNA cleavageand chromatin condensation has beenwell documented.29,30,31A tight buffering of intracellular Ca2þ is required for normalgrowth. In fact, apoptosis can be induced by Ca2þ mobilizationfrom intracellular pools,23,24,27 chelation of intracellular Ca2þwith 1,2-bis-(2-aminophenoxy)ethane-N,N,N1,N1-tetra-aceticacid tetra-acetoxymethyl ester (BAPTA-AM)28,29 or removalof extracellular Ca2þ.32 Intracellular Ca2þ deficiency regulatesgene expression22,26 and induces apoptosis through caspasesactivation.23,27,33 It has been shown that extracellular Ca2þdepletion reduces expression of CLU mRNA, but its proteomicprofile has not been studied.26 Thus, we decided to study theeffect of Ca2þ depletion in prostate cells and its effect on CLUprotein expression and localization.We compared the cell proliferation rate of PNT1A cellsgrown under standard culture medium (RPMI plus 10% foetalbovine serum, FBS), keratinocyte serum-free completemedium34 (KSFM, which has subphysiological concentrationsof Ca2þ, 0.1 mM), or KSFM supplemented with 1.8mM Ca2þ(Figure 1a). Under these condition, as previously reported,35KSFM caused a marked inhibition of cell growth rate whencompared to standard culture conditions, inducing anoikis(cell detachment followed by chromatin condensation). Additionof either 1.8mMCa2þ or the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-flu-oromethylketone (z-VAD-fmk, 1 mM)to KSFM medium completely rescued PNT1A cells andprevented cell detachment and anoikis induction (Figure 1a).After 8 days of culture in KSFM medium supplemented with z-VAD-fmk, cells reached a density similar to that observedunder standard conditions (Figure 1a). In PNT1A cells CLUexpression was detected by Western blot assay with acommercially available mouse monoclonal antibody fromUpstate Biotechnology (clone 41D). The prevailing form ofCLU in the cells consists of a 65 kDa intracellular CLUprecursor that is progressively converted to low-molecularweight and secreted CLU forms.36 Upon KSFM incubation,the 65 kDa CLU precursor is rapidly converted to a 45 kDaCLU isoform that accumulates in parallel to poly(ADP-ribose)polymerase (PARP) activation (Figure 1b, KSFM 8-day, lane3). Ca2þ administration blocked both the accumulation of the45 kDa isoform and PARP activation (Figure 1b, KSFMþ Ca2þ, 2 and 8 days, lanes 4 and 5, respectively). 2 days ofz-VAD-fmk administration did not prevent the accumulationof the 45 kDa CLU isoform (Figure 1b, KSFMþz-VAD, lane6). However, 8 days of treatment with z-VAD-fmk restored aCLU proteomic profile similar to that induced by Ca2þadministration (Figure 1b, KSFMþz-VAD, lane 7). To assesswhether intracellular Ca2þ depletion would per se causeapoptosis, we cultured PNT1A cells in the presence ofBAPTA-AM, a well-known intracellular Ca2þ blocker26 thatcan trigger activation of caspases and apoptosis.23,28,29,33Cell proliferation was assessed by crystal violet stainingmethod in the presence of 20 mM BAPTA-AM. As soon as4 h after treatment, cells growth was significantly inhibitedreaching about 55 and 78% inhibition at 24 and 48 h (Po0.01,data not shown), respectively, in strict association withchromatin condensation and fragmentation, cell roundingand detachment, hallmarks of anoikis (see Figure 1e, RPMIþ BAPTA-AM). Consistently with the hypothesis that changesin the intracellular availability of Ca2þ could trigger anoikisin PNT1A cells, a switch to the 45 kDa isoform of CLU wasalready observed 4 h after the beginning of BAPTA-AMadministration, a time when caspase-9 activation started tobe evident (Figure 1c). At 24–48 h-time of BAPTA-AMadministration, when the decrease in cell proliferation wasmassive (data not shown), PNT1A cells showed the highestlevel of 45 kDa CLU isoform accumulation (Figure 1c,RPMIþBAPTA-AM, lanes 4 and 5).Immunocytochemical analysis of PNT1A cells showedsubcellular localization of CLU under different experimentalconditions. We compared the commercially available mousemonoclonal anti-CLU antibody and an affinity-purified polyclonalanti-human CLU antibody (a-324) that we have raisedagainst an epitope located on the a-subunit of human CLUprotein at position 324–341 (Accession Number X14723, cfr.legend to Figure 1). Experiments were conducted in parallelwith cells grown in standard RPMI or in KSFM medium(Figure 1d). Western blot analysis using the a-324 antibodyconfirmed the detection of the 45 kDa nuclear CLU isoform incells grown in KSFM medium (data not shown). Understandard conditions, CLU was detectable in the cytoplasm(Figure 1d, RPMI 2 and 8 days). Conversely, CLU wasdetected in the nucleus already 2 days after KSFM-growth(Figure 1d, KSFM). Detection of CLU in the nucleus precededcell morphological changes. After 8 days in KSFM medium,nuclear CLU accumulated into apoptotic bodies (Figure 1d,KSFM). The comparison between the two antibodies showedthat a-324 gave better results when compared to thecommercially available mouse monoclonal anti-CLU antibody(Figure 1d, compare a-324 and Upstate). When KSFM wassupplemented with 1.8mM Ca2þ, CLU was clearly localizedin the cytoplasm (Figure 1e, KSFMþCa2þ, 2 and 8 days),and cells growth rates (Figure 1a) and cell cycle progression(Figure 1e, subpanels 1 and 2) were identical to that observedunder standard growth conditions (2 days RPMI). As expected,BAPTA-AM administration caused a dramatic reductionin cell growth (data not shown) and viability (Figure 1e,RPMIþBAPTA-AM, subpanels 1 and 2). Fluorescenceactivated cell sorting (FACS) analysis revealed a progressiveloss of the G0/G1 fraction in concomitance to an increaseof the subdiploid peak (Figure 1e, RPMIþBAPTA-AM,subpanels 1 and 2). When cells were incubated in KSFMþ z-VAD-fmk for 2 days, CLU was detected into the nuclei(Figure 1e, KSFM þ z-VAD, 2 days), but DNA fragmentationwas blocked (Figure 1e, KSFMþz-VAD, subpanels 1 and 2).At 8-day culturing, clusterin was almost completely detectedinto the cytoplasm (Figure 1e, KSFMþz-VAD, 8 days), atime when cells were completely rescued and cell growth ratewas very similar to controls and significantly higher than thatobserved in KSFM alone (Figure 1a). Anoikis induction and PARP activation were blocked at 8-day KSFM incubationwith z-VAD-fmk (Figure 1b and e), suggesting that nucleartranslocation of 45 kDa clusterin primes cells to apoptosis.To directly demonstrate that nuclear localization of CLU caninduce anoikis, we transfected PNT1A cells with an expressionvector containing a shortened CLU cDNA (intra-CLU)that starts from nucleotide 152 and therefore lacks thehydrophobic secretion signal sequence. Following transienttransfection, PNT1A cells showed a detectable suppressionof cell proliferation (data not shown) and decreased viability inassociation to a progressive accumulation of intra-CLU(49 kDa, Figure 1f, top). Intra-CLU accumulated in the nucleiof about 75% of transfected cells (Figure 1f, bottom). Notably,expression of nuclear CLU induced a significant increase incaspase-3 activity (Figure 1g), DNA fragmentation (Figure 1h)and cell detachment. Nuclear CLU caused cell death byactivation of caspase-dependent apoptosis. In fact, z-VADfmksignificantly rescued cells from anoikis and DNAfragmentation (Figure 1g and h, intra-CLUþz-VAD versusmock).We report here that calcium (Ca2þ) deprivation causestranslocation of a 45 kDa CLU isoform to the nucleus in humanprostate epithelial cells, leading to inhibition of cell proliferationand caspase-cascade-dependent anoikis. Our data showthat accumulation of nuclear CLU caused induction ofapoptosis and anoikis in prostate cells, and that conditionsrestoring normal cell growth and survival also cause relocalizationof CLU in the cytoplasm. In fact, early beforeinduction of anoikis, we observed translocation of a 45 kDaCLU isoform in the cell nuclei that was prevented by Ca2þ orz-VAD-fmk supplementation, leading to cell rescue. Importantly,transient overexpression of an intracellular, notsecreted, form of CLU accumulating into the nucleus causedcell growth inhibition and anoikis in the absence of Ca2þdeprivation. This effect was also caspase-dependent. In anycondition studied, cell demise was strictly associated withtranslocation of CLU to the nucleus. Taken together, thesedata suggest that anoikis-death by Ca2þ deprivation isaccompanied by nuclear translocation of a 45 kDa CLU andthat a nuclear only form of CLU appears to recapitulate deathby Ca2þ deprivation, suggesting a putative causative linkbetween Ca2þ depletion, CLU nuclear translocation and celldeath. CLU intracellular trafficking can now be linked to Ca2þsignalling pathway, resulting in regulation of survival andproliferation of androgen-dependent prostate epithelial cells.Moreover, the data here presented and previous resultsshowing that CLU is downregulated during prostate cancerprogression19,37 support the hypothesis that CLU is a tumorsuppressorgene for the prostate. The finding that calreticulin,an intracellular Ca2þ-binding protein, is regulated by androgens,38 and that inhibition of spontaneous and androgeninducedprostate growth by a vitamin D analog involvesCLU,39 also supports the hypothesis that a causative linkbetween these pathways may exist.Identification of the molecular mechanisms involved in theproduction and processing of intra-CLU will help in the futurethe molecular characterization of prostate cancer, one of themost diffuse and elusive type of cancer.
|Data di pubblicazione:||2005|
|Titolo:||Ca2+ depletion induces nuclear clusterin, a novel effector of apoptosis in immortalized human prostate cells|
|Autori:||AE Caccamo; M. Scaltriti; A. Caporali; D. D'Arca; A. Corti; D. Corvetta; A. Sala; S. Bettuzzi|
|Digital Object Identifier (DOI):||10.1038/sj.cdd.4401491|
|Appare nelle tipologie:||Articolo su rivista|
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