Inhibition of 20 S and 26 S Proteasome Activity by Lithium Chloride

Lithium affects several enzymatic activities, however, the molecular mechanisms of lithium actions are not fully understood. We previously showed that LiCl interacts synergistically with all-trans-retinoic acid to promote terminal differentiation of WEHI-3B D+ cells, a phenomenon accompanied by the recovery of the retinoid-induced loss of retinoic acid receptor α protein pools. Here, we demonstrate the effects of LiCl on proteasome-dependent degradation of retinoic acid receptor α proteins. LiCl alone, or in combination with all-trans-retinoic acid, increased cellular levels of ubiquitinated retinoic acid receptor α and markedly reduced chymotryptic-like activity of WEHI-3B D+ 20 S and 26 S proteasome enzymes. Neither KCl nor all-trans-retinoic acid affected enzyme activity, whereas NaCl produced a modest reduction at relatively high concentrations. In addition, LiCl inhibited 20 S proteasome chymotryptic-like activity from rabbits but had no effect on tryptic-like activity of the 26 S proteasome. This effect has significant consequences in stabilizing the retinoic acid receptor α protein levels that are necessary to promote continued differentiation of leukemia cells in response to all-trans-retinoic acid. In support of this concept, combination of proteasome inhibitors β-clastolactacystin or benzyloxycarbonyl-Leu-Leu-Phe with all-trans-retinoic acid increased differentiation of WEHI-3B D+ cells in a manner that was analogous to the combination of LiCl and all-trans-retinoic acid.

Several mammalian enzymes are known targets of lithium including inositol monophosphatase, glycogen synthase kinase-3, and several phosphomonoesterases; however, the mechanism(s) by which lithium affects cellular events remains unclear (1). Pharmacological doses of lithium cause stabilization of bipolar disorder, developmental defects, subclinical hypothyroidism, and increased neutrophil production (2)(3)(4). Lithium has been proposed to impede G-protein-coupled signaling cascades because of a depletion of intracellular inositol by direct inhibition of inositol monophosphatase (5). Lithium also inhibits glycogen synthase kinase, which can lead to signaling of the wnt pathway through ␤-catenin (6). In addition, we have shown previously that lithium chloride, in combination with all-trans-retinoic acid (ATRA), 1 causes synergistic induction of the differentiation of WEHI-3B D ϩ myelomonocytic leukemia cells and reverses the down-regulation of retinoic acid receptor ␣ protein (RAR␣) produced by ATRA (7,8). Recently, the down-regulation of RAR␣ by ATRA has been reported to occur in acute promyelocytic leukemia and in breast cancer cells by specific targeting of the receptors to the proteasome-dependent degradation pathway (9,10). These observations suggested that lithium may exert some of its effects by targeting a component of the proteasomedependent degradation system.
The implication of proteasome-dependent degradation mediating important cellular events has been the subject of numerous studies to date (for example, see Refs. [11][12][13]. Proteasomes are ubiquitous, multicatalytic complexes responsible for nonlysosomal, ubiquitin-dependent proteolytic activity (14). They are highly conserved polysubunit complexes organized into a 20 S catalytic core or a 26 S complex containing the catalytic core plus an associated regulatory complex (15). Both 20 S and 26 S proteasome particles exhibit at least five different proteolytic activities (16,17). The proteasome plays important roles in cell cycle regulation and differentiation; thus, the degradation of cyclins and cyclin-dependent kinases (p21 and p27) as well as transcription factors (NF-B and IB) and tumor suppressors (p53) occur via the ubiquitin-proteasome pathway (18). During the differentiation of hematopoietic cells, changes in subcellular distribution, subunit composition, and enzymatic activity of proteasomes have been reported (19 -22). In malignant hematopoietic cells and breast cancer cells, concentrations of proteasomes have been reported to be abnormally high and localized in the nucleus of leukemia cells (23). Thus, proteasome regulation is fundamental to the maintenance of normal cellular processes. Indeed, proteasome inhibitors activate apoptosis in human HL60 leukemia cells (24), and proteasome inhibitors have recently shown antitumor activity, with the potent boronic acid proteasome inhibitor PS-341 currently undergoing a clinical trial (25,26). Recently, Gianni et al. (27) have shown that the c-Abl tyrosine kinase inhibitor STI571 enhances retinoid-induced differentiation of acute promyelocytic leukemia (APL) NB4 cells, and that regulation of the proteasome plays a role in this process.
In addition to the known enzymes affected by lithium, we demonstrate in this report that both the 20 S and 26 S forms of mammalian proteasomes are targets of lithium inhibition. Because the proteasome enzyme is intricately involved in the maintenance of many cellular processes, lithium chloride provides a unique method of regulation. An example of the signif-icance of this action is seen in the differentiation of WEHI-3B D ϩ leukemia cells, where LiCl prevents the degradation of RAR␣ protein pools produced by ATRA that are critical to promoting retinoid-induced leukemia cell differentiation (8). Because the structure and function of RAR␣ are critical to the success of ATRA in the therapy of APL, which is often characterized by only short term remission rates and the development of retinoid resistance (28), the prevention of receptor loss by LiCl may allow more complete cell differentiation and longer remissions in APL patients.

EXPERIMENTAL PROCEDURES
Cell Culture and Differentiation-WEHI-3B D ϩ cells were cultured in suspension in McCoy's 5A-modified medium supplemented with 15% fetal calf serum. Differentiation was induced with ATRA and LiCl at a cell density of 5 ϫ 10 4 cells/ml. The degree of differentiation was assessed 72 h post-induction and was measured by the capacity of cells to reduce nitro blue tetrazolium following TPA stimulation as described previously (8). In some experiments, cells were incubated with ␤-clastolactacystin (BioMol, Plymouth Meeting, PA) or benzyloxycarbonyl-Leu-Leu-Phe to induce differentiation.
Immunoprecipitation and Western Blotting-RAR␣ was immunoprecipitated from 100 g of cell lysates prepared in radioimmune precipitation buffer with 2 g of anti-RAR␣ antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 12 h followed by incubation with Protein A/G Plus-agarose beads for 2 h. Immunoprecipitated proteins were washed with phosphate buffered saline and then separated by electrophoresis on a 10% SDS-polyacrylamide gel followed by transfer to nitrocellulose. Ubiquitin-containing proteins were identified by Western blot analysis using mouse anti-ubiquitin antibody (Santa Cruz Biotechnology) followed by ECL detection (Amersham Pharmacia Biotech UK, Ltd., Little Chalfont, Buckinghamshire, UK). RAR␣ and ubiquitinated RAR␣ species were quantified by densitometric scanning of blots and analysis using ImageQuant software.
The proteolytic activity of WEHI-3B D ϩ 20 S and 26 S proteasomes was determined by the measurement of fluorescence generated from chymotryptic cleavage of the fluorogenic substrate Suc-Leu-Leu-Val-Leu-AMC (Bachem, King of Prussia, PA). Proteasomes (35 ng/l) were incubated at 37°C with 2.5-25 M substrate in 20 mM Tris-HCl, pH 8.0, 0.5 mM EDTA buffer in a total reaction volume of 100 l. Release of fluorescent AMC was measured using a Molecular Devices SpectraMax GeminiXS spectrafluorometer at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Inhibitors were added to the enzyme before the addition of substrate. Fluorescence release was measured for 60 min at 1-min intervals. Purified rabbit 20 S proteasome (CalBiochem, La Jolla, CA) activity was measured at 1.2 ng/l as a positive control. The 50% inhibitory concentration (IC 50 ) was determined from plots of the ratio of velocities with and without inhibitor at 0 -10 min versus inhibitor concentration.
Measurement of proteasomal tryptic-like activity was measured as above using the substrate Boc-Leu-Arg-Arg-AMC at 100 M.
In Vitro Translation and Processing of RAR␣-[ 35 S]Methionine-labeled RAR␣ protein was generated by in vitro coupled transcription/ translation from pGem3zf(ϩ)RAR␣ plasmid containing the cDNA of the mouse RAR␣ gene using a rabbit reticulocyte lysate system (Promega, Madison, WI) in the presence of 0.4 mM [ 35 S]methionine (20 Ci) (Amersham Pharmacia Biotech). After completion, the reaction was treated with 10 mM N-ethylmaleimide for 10 min at room temperature followed by the addition of dithiothreitol at a final concentration of 6 mM. The processing of labeled protein was modified from the degradation procedure described by Loda et al. (30). RAR␣-labeling reaction (4.5 l) was incubated at 37°C in degradation buffer (50 mM Tris-HCl, pH 8.0, 5 mM MgCl 2 , 1 mM dithiothreitol, 2 mM ATP, 10 mM creatine phosphokinase, 10 mM creatine phosphate, 5 M ubiquitin) for 30 min in the presence or absence of additional 26 S proteasomes (5 g) partially purified from WEHI-3B D ϩ cells. Degradation reactions were subjected to 10% SDSpolyacrylamide gel electrophoresis followed by phosphorimaging analysis of radiolabeled RAR␣ bands.

RESULTS
Accumulation of Ubiquitinated RAR␣ following Exposure to ATRA and LiCl-In APL cell lines, both PML/RAR␣ and RAR␣ proteins were shown to be down-regulated by ATRA in a proteasome-dependent manner (9), and in MCF7 breast cancer cells, ATRA similarly induced proteasome-dependent degradation of the receptor (10). Therefore, we explored the mechanism by which LiCl maintained steady-state levels of RAR␣ protein by measuring the patterns of ubiquitination of RAR␣ after treatment with LiCl and ATRA, both alone and in combination in WEHI-3B D ϩ cells. Polyubiquitinated RAR␣ species were identified as multiple bands migrating at high molecular masses (Fig. 1A) with the exception of ubiquitinated proteins (between 97 and 66 kDa), which migrated similarly to nonspecific proteins that were detected by RAR␣ antibody. The treatment of cells with 3 M ATRA for 1 h caused a transient 4-fold increase in polyubiquitinated RAR␣ species. Polyubiquitination decreased by 3 h and returned to control levels at 24 h (Fig. 1, A and B). In contrast, LiCl at 5 mM increased the level of polyubiquitinated RAR␣ steadily over a 24-h period resulting in a 5.5-fold increase in polyubiquitinated RAR␣ over untreated cell RAR␣ polyubiquitination. This accumulation was even more pronounced when ATRA was combined with LiCl (Fig. 1C). Accumulated polyubiquitinated bands were similar to the amounts present in the cells treated with the potent proteasome inhibitor ␤-clastolactacystin (Fig. 1C).
The accumulation of polyubiquitinated RAR␣ proteins upon ATRA treatment confirms that the retinoid induces receptor ubiquitination, targeting RAR␣ for proteasomal degradation. LiCl alone maintained increased levels of highly ubiquitinated receptors and, in combination with ATRA, blocked the degradation of ubiquitinated receptors in a manner analogous to that produced by the proteasome inhibitor ␤-clastolactacystin.
Specific Inhibition of Proteasome Chymotryptic-like Activity of WEHI-3B D ϩ Cells by LiCl-The accumulation of polyubiquitinated RAR␣ species produced by treatment with LiCl alone suggested that proteasome-dependent degradation of these receptors was relatively inactive in WEHI-3B D ϩ cells. To gain information on proteasomal activity in these cells, 20 S and 26 S proteasomes were partially purified from WEHI-3B D ϩ cells, and chymotryptic-like activity was assessed. Cleavage of the peptide substrate Suc-Leu-Leu-Val-Leu-AMC by the 20 S proteasome in the presence of 0.035% SDS resulted in fluorescence at 460 nm. 20 S proteasomes from WEHI-3B D ϩ cells displayed activity similar to that of commercially available rabbit 20 S proteasomes (Ͼ95% purity) (Fig. 2A). The presence of increasing concentrations of LiCl decreased the rate of chymotryptic activity of WEHI-3B D ϩ and rabbit 20 S proteasomes (Fig. 2, B and C). LiCl at 2.5 mM significantly reduced peptide cleavage after 1 h by WEHI-3B D ϩ 20 S proteasomes, whereas 10 mM LiCl reduced fluorescence in this system to background levels. A plot of V i /V O versus the concentration of LiCl from 0 -10 min demonstrated an IC 50 for WEHI-3B D ϩ 20 S proteasomes of 4 mM (Fig. 2D). Furthermore, proteasome complexes partially purified from cells pretreated with 5 mM LiCl exhibited decreased activity in the absence of additional LiCl in the in vitro assay (Fig. 2E). LiCl inhibited WEHI-3B D ϩ 26 S proteasomes to a similar extent (Table I), lowering the V max /sec by more than 50% at 2.5 mM.
To determine the specificity of proteasome inhibition by LiCl, the activity was measured in the presence of 1-10 mM NaCl, KCl, or 3-10 M ATRA. No effect was observed on 20 S proteasome activity by the presence of 1-5 mM NaCl (Fig. 3A); however, 10 mM NaCl decreased activity slightly. In contrast, neither KCl nor ATRA significantly affected the rate of chymotryptic-like activity of 20 S proteasomes (Fig. 3, B and C). These observations suggest that LiCl has a unique effect on both WEHI-3B D ϩ and rabbit proteasome activity, and that this inhibitory effect probably is responsible for the observed accumulation of polyubiquitinated RAR␣ species following exposure of WEHI-3B D ϩ cells to LiCl.
LiCl at concentrations as high as 10 mM had no significant effect on the tryptic-like activity of partially purified 26 S  proteasomes from WEHI-3B D ϩ cells (Fig. 4). Thus, LiCl exhibited specificity for inhibition of chymotryptic-like protease activity.
LiCl Inhibits the Direct Proteasomal Degradation of RAR␣ Protein Substrates in Vitro-The combination of ATRA and LiCl acts synergistically to promote the terminal differentiation of WEHI-3B D ϩ cells (8). Because LiCl inhibits proteasome activity, we postulate that at least a portion of the mechanism by which LiCl enhances the differentiation of WEHI-3B D ϩ cells produced by ATRA is attributed to the inhibition of proteasomal-targeted degradation of RAR␣. This action results in the restoration of RAR␣ protein pools depleted by the retinoid. To assess whether RAR␣ is a direct substrate for degradation by the proteasome, we measured the proteolytic cleavage of in vitro translated [ 35 S]methionine-labeled RAR␣ protein by 26 S proteasomes partially purified from WEHI-3B D ϩ cells. The addition of 26 S proteasomes to [ 35 S]methionine-labeled RAR␣ proteins translated by rabbit reticulocyte lysate caused a marked decrease in radiolabel associated with RAR␣ protein (Fig. 5). Only 7.5% of RAR␣ protein remained after exposure to proteasomes. However, in the presence of 5 mM LiCl, degradation by 26 S proteasomes was dramatically reduced, maintaining 80% of control levels of radiolabeled receptor protein. This observation illustrates that RAR␣ proteins are direct substrates of the proteasome, and that LiCl prevents proteolytic degradation in a manner independent of other cellular components.
Induction of WEHI-3B D ϩ Cell Differentiation by Proteasome Inhibitors-To determine whether inhibition of proteasome activity by LiCl in intact cells is involved in the mechanism by which LiCl promotes the differentiation of WEHI-3B D ϩ cells by ATRA, the dependence of retinoidinduced differentiation on proteasome function was assessed.
The extent of terminal differentiation was measured by determining the percentage of nitro blue tetrazolium-positive cells after 3 days of treatment of WEHI-3B D ϩ cells with either ␤-clastolactacystin, an irreversible proteasome inhibitor, or the peptide aldehyde inhibitor benzyloxycarbonyl-Leu-Leu-Phe. Proteasome inhibitors had no significant effect on differentiation when used alone. However, in combination with ATRA, ␤-clastolactacystin at 1 M and 5 M increased the degree of differentiation in a concentration-dependent manner from 27.7 Ϯ 7.5% for the retinoid alone to 46.3 Ϯ 4.9% and 65.0 Ϯ 4.0%, respectively (Table II). A combination of LiCl with ATRA induced 74 Ϯ 6% cell differentiation, a 2.7-fold increase over differentiation induced by ATRA alone, and benzyloxycarbonyl-Leu-Leu-Phe at 1 M also exhibited synergism in combination with ATRA, inducing 62.0 Ϯ 1.5% terminal differentiation. DISCUSSION In the present investigation, we report a new site of action of lithium, demonstrating that LiCl specifically inhibits the chymotryptic-like activity of both the 20 S and 26 S proteasome. An analysis of the rate of ubiquitination of RAR␣ in the WEHI-3B D ϩ leukemia cell system demonstrated transient increases in polyubiquitination of RAR␣ induced by ATRA in WEHI-3B D ϩ cells, which was increased by the addition of LiCl. In addition to preventing the loss of ubiquitinated RAR␣ species, LiCl alone was able to markedly reduce the enzymatic activity of the proteasome from WEHI-3B D ϩ cells. These results suggest that LiCl does not act to enhance ubiquitination but produces an accumulation of ubiquitinated RAR␣ proteins by blocking ubiquitin-dependent degradation. We have also shown that other salts do not affect proteasome activity in a manner analogous to LiCl, suggesting a relatively unique effect on the proteasome by lithium (Fig. 3).
Although we have observed a pronounced effect of LiCl on the isolated proteasome enzyme, we have also shown a reduction in the activity of proteasomes from cells pretreated with LiCl before purification (Fig. 2). This finding suggests that LiCl may affect the proteasome structure irreversibly. It remains unclear how LiCl affects the proteasome; however, it is possible that LiCl causes spot-denaturation or small conformational changes at the chymotryptic-like active site. Alternatively, LiCl may induce the release of one or more of the subunits of this multisubunit complex.
We have demonstrated that the effect of LiCl on proteasome enzymatic activity is related to its ability to potentiate retinoidinduced cell differentiation. Since activity of proteasomes toward the RAR␣ protein substrate in a system separate from cell-signaling mechanisms was inhibited by LiCl (Fig. 5), we conclude that the effects of LiCl on proteasome activity directly relates to the regulation of the retinoid receptor shown to be key to ligand-induced terminal differentiation.
Evidence to suggest that LiCl affects the proteasome in situ is seen when peptide inhibitors of the proteasome were used in combination with ATRA to differentiate WEHI-3B D ϩ cells. Because the increase in differentiation obtained was similar to that observed with LiCl ϩ ATRA, we conclude that the modulation of ATRA-induced differentiation by LiCl occurs, at least in part, through the inhibition of the proteasome. Interestingly, the degree of differentiation produced by the peptide inhibitors of the proteasome in combination with ATRA was slightly less than that achieved by the LiCl/ATRA combination. The use of proteasome inhibitors as antitumor agents is currently being evaluated as an approach to the therapy of cancer (25,26,31), suggesting that lithium at therapeutic doses may have use in the treatment of malignant diseases other than APL.
The ubiquitin-dependent proteasome has been reported to localize more prominently in the nucleus of leukemia cells as well as be expressed at much higher levels in these neoplastic cells than in normal cells (23). Therefore, proteasome location and function may be implicated in maintaining the proliferative state, and regulation of the proteasome may play a key role in cancer cell growth. Furthermore, one can speculate that in cancerous cells that are not ATRA-responsive but have supranormal levels of proteasome activity, the inhibition of the proteasome by LiCl may reduce cellular proteasome activity levels to those normally found in non-malignant cell types and thereby decrease proliferative activity. In WEHI-3B D ϩ leukemia cells, the disruption of the ATRA-induced targeting of RAR␣ to the proteasome by LiCl appears to be important for promoting optimal terminal differentiation. Thus, upon treatment of WEHI-3B D ϩ cells with the combination of ATRA and LiCl, the accumulation of polyubiquitinated adducts appears to signal the cell to rebuild the pool of RAR␣ protein, and RAR␣ mRNA accumulation occurs (8). The rebuilding of the RAR␣ protein pool may be attributed to an increase in the back reaction of the ubiquitination enzyme system (i.e. ubiquitin hydrolysis), an increase in the rate of mRNA translation, or a combination of both events.
The effects of LiCl on proteasome activity are likely not to be specific to leukemia cells and, therefore, may be beneficial in the differentiation therapy in combination with ATRA of a broad spectrum of cancers. The identification of this new cellular target of lithium may also help answer questions on the mechanism of lithium action implicated in non-cancer-related cellular processes. a Benzyloxycarbonyl-LLF, benzyloxycarbonyl-Leu-Leu-Phe. b LiCl was used at 5 mM, and ATRA was used at 3 M. nd, not determined.