From the Cell Signaling and Cancer Group, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695-7633
Received for publication, February 27, 2003
, and in revised form, March 24, 2003.
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ABSTRACT |
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INTRODUCTION |
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The importance of C/EBP in the regulation of growth arrest and differentiation is exemplified by recent studies in which dominant-negative mutations in C/EBP
were found to be associated with human acute myeloid leukemia (16, 17). Such mutations in C/EBP
are thought to result in a differentiation block of the granulocytic blasts and have implicated C/EBP
as a tumor suppressor gene. The growth and differentiation regulatory functions of C/EBP
are complex and multifaceted. For example, C/EBP
has been proposed to regulate p21 expression (18, 19) and interact with retinoblastoma (Rb) family proteins (20, 21). C/EBP
has been shown to directly repress E2F function through its physical associations with E2F, and this repression is necessary for growth arrest and adipocyte and granulocyte differentiation (22). However, recent studies indicate that C/EBP
can block growth independent of its DNA binding and transcriptional activity by forming a complex with cdk2 and cdk4, thereby blocking cyclin-cdk interactions and cell cycle progression (23). Thus, it appears that in addition to its DNA binding/transcription factor activity, C/EBP
can modulate growth arrest and differentiation by protein-protein interactions with cell cycle regulatory proteins independent of its transcription activity. Therefore, the identification of cellular processes that impinge on the regulation of C/EBP
protein stability may be critical in understanding the regulation and/or deregulation of C/EBP
-induced growth arrest and differentiation.
Ubiquitin-proteasome degradation system plays an important role in the degradation of cellular proteins, which are involved in regulating various cellular processes, including cell cycle regulation, differentiation, and apoptosis (24, 25). Recent studies have indicated that GSK3 phosphorylates a number of cell cycle regulatory proteins including p21 (26), -catenin (27), cyclin D (28), and c-Myc (29) and in doing so targets these molecules for proteasomal degradation. Lithium, an inhibitor of GSK3 (30, 31), blocked the proteasomal degradation of these proteins. C/EBP
can be phosphorylated by GSK3 on Thr222 and Thr226, and this phosphorylation can be blocked by lithium (32, 33). However, the functional significance of GSK3-mediated phosphorylation is not known, nor is it known whether C/EBP
is degraded via a proteasomal pathway or whether GSK3 or lithium treatment can alter C/EBP
protein degradation/stability. While the effect of lithium on the inhibition of proteasomal degradation of proteins that are GSK3 substrates has generally been attributed to its inhibition of GSK3, lithium also inhibits a number of other cellular enzymes and more recently lithium has been shown to a be an inhibitor of chymotryptic activity of both the 20 S and 26 S proteasome (34). Therefore, we have examined whether C/EBP
is degraded via a proteasomal mechanism and investigated the role of GSK3 and lithium on C/EBP
protein stability. We demonstrate that C/EBP
is degraded via an ubiquitin-dependent proteasomal pathway and that lithium stabilizes the C/EBP
protein through a GSK3-independent pathway involving direct inhibition of proteasomal activity.
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EXPERIMENTAL PROCEDURES |
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Transfection and Lithium TreatmentBALB/MK2 keratinocytes (a gift from Dr. Weissman, University of North Carolina, Chapel Hill, NC) were plated at 2.5 x 105 cells/60-mm culture dish in Ca2+-free EMEM supplemented with 8% Chelex-treated fetal bovine serum, 4 ng of hEGF per ml, and 0.05 mM calcium chloride. Two days after plating, BALB/MK2 keratinocytes were transfecetd with 2 µg of pcDNA3-C/EBP and 12 µg of lipofectin in 2 ml of serum-free EMEM containing 4 ng of hEGF per ml, and 0.05 mM calcium chloride according to the manufacturer's protocol. Twenty-four hours following transfection, cells were refed with EMEM supplemented with 8% Chelex-treated fetal bovine serum, 4 ng of hEGF per ml, and 0.05 mM calcium chloride, and incubated with 20 mM lithium chloride for 24 h.
Preparation of Nuclear and Whole Cell LysatesNuclear extracts were prepared as previously described by Schreiber et al. (35). For the preparation of whole cell lysates, cells were washed with cold phosphate-buffered saline, harvested by scraping, collected by brief centrifugation. Cells were lysed in lysis buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin, 100 µg/ml leupeptin, 1 mM sodium orthovanadate, 0.6% Nonidet P-40) by sonication, and then one-tenth volume of 5 M NaCl was added. The incubation mixture was vortexed, incubated for 15 min on ice, and centrifuged (14,000 x g, 10 min, 4 °C). Supernatants were stored at 80 °C until use. Protein concentration was determined by the Bio-Rad protein assay reagent.
Western Blot AnalysisEqual amounts of protein were precipitated by adding equal volume of 20% trichloroacetic acid and washed with acetone (20 °C). Protein samples were solubilized and boiled in SDS sample buffer for 2 min and then separated by SDS-PAGE. The separated proteins were transferred to an Immobilon-P membrane (Millipore). Following incubation in blocking buffer (phosphate-buffered saline with 1% bovine serum albumin, 5% nonfat dry milk, and 0.1% Tween-20) for1hat room temperature, the membranes were probed for 2 h at room temperature with rabbit polyclonal IgG raised against C/EBP (Santa Cruz Biotechnology). The membranes were washed and then probed with a horseradish peroxidase-linked secondary antibody for 1 h at room temperature. Detection was made with an enhanced chemiluminescence reagent followed by exposure of the membrane to film.
Luciferase AssayBALB/MK2 keratinocytes were plated at 1 x 105 cells/well in a 12-well culture plate. Two days after plating, BALB/MK2 keratinocytes were transfected in triplicate with 100 ng of pcDNA3-C/EBP and 400 ng of the specified C/EBP-dependent promoter/reporter plasmid as described in the text and 3 µg of lipofectin in 0.5 ml of serum-free EMEM according to the manufacturer's protocol. Forty-eight hours later, cells were harvested, and the luciferase activity was determined by using the luciferase assay kit (Promega). Protein concentration was determined with the Bio-Rad protein assay reagent.
Dephosphorylation of C/EBP Protein by Phosphatase Treatment BALB/MK2 cells were transfected with pcDNA3-C/EBP
, and 48 h following transfection, nuclear extracts were isolated as described above, except that the phosphatase inhibitor (sodium orthovanadate) was omitted. 50 µg of nuclear extracts were incubated with 400 units of
-phosphatase in 50 µl of phosphatase buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% Brij 35, and 2 mM MnCl2) at 30 °C for 1 h. The reaction was stopped by adding equal volume of 20% trichloroacetic acid. Precipitated proteins were washed with acetone (20 °C), solubilized and boiled in SDS sample buffer for 2 min. Protein samples were separated by SDS-PAGE and analyzed by Western blotting.
Northern Blot AnalysisTotal RNA was isolated from untransfected or pcDNA3-C/EBP-transfected BALB/MK2 cells using Promega's SV total RNA isolation kit. C/EBP
cDNA was labeled with [
-32P]dCTP by using Ready-To-Go labeling beads (Amersham Biosciences). RNA was electrophoresed on agarose gel containing formaldehyde, transferred to zeta-probe GT membrane (BioRad), and UV cross-linked. Blots were incubated at 65 °C in hybridization buffer (0.25 M Na2HPO4, pH 7.2, 7% SDS) and sequentially washed with washing buffer 1 (20 mM Na2HPO4, pH 7.2, 5% SDS) and washing buffer 2 (20 mM Na2HPO4, 1% SDS) at room temperature. Films were exposed to membrane at 80 °C and developed.
Inhibition of C/EBP Degradation by Proteasomal Inhibitors BALB/MK2 cells were transfected with pcDNA3-C/EBP
. Forty-six hours following transfection, cells were incubated with either 25 µM MG-132 or 10 µM lactacystin for 30 min prior to the addition of 50 µg/ml cycloheximide. Lysates were prepared at the indicated time points and subjected to Western blot analysis. MG-132 was prepared as a 25 mM stock solution in Me2SO, and lactacystin was prepared as a 5 mM stock solution in Me2SO. Control experiments were carried out with Me2SO.
In Vitro Proteasomal Degradation AssayDegradation reactions were carried out in a final volume of 10 µl and contained 30 µg of S-100 fraction as the source of ubiquitin-proteasomal system components. The reaction mixtures contained 50 mM Tris-HCl, pH 7.6, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM ATP along with nuclear extracts from pcDNA3-C/EBP-transfected BALB/MK2 cells as a proteasomal substrate (125 ng/reaction) supplemented with the ERS and 1 mM ubiquitin. For the inhibition of proteasomal activity, 25 µM MG-132 or 10 mM LiCl was included. Reactions were incubated at 37 °C for 2 h and were terminated by boiling after the addition of an equal volume of 2x SDS sample buffer. The reaction products were resolved by 10% SDS-PAGE and subjected to Western blot analysis.
Effect of Li+ on C/EBP StabilityBALB/MK2 cells were transfected with pcDNA3-C/EBP
and treated with 20 mM lithium chloride as described above. Twenty-two hours after the lithium treatment, cells were incubated with 50 µg/ml cycloheximide. Cells were harvested at various time points, and extracts were subjected to Western blot analysis followed by densitometric analysis.
Detection of Ubiquitinated C/EBPaFor the detection of ubiquitinated C/EBP protein in the cell, BALB/MK2 keratinocytes were plated on a 60-mm culture dish and transfected with pCMV-FLAG-C/EBP
. Twenty-four hours later transfected cells were treated with 20 mM LiCl for 24 h. For MG-132 treatment, cells were incubated with 25 µM MG-132 for 2 h. Forty-eight hours following transfection, cells were washed with ice-cold phosphate-buffered saline, lysed in ice-cold immunoprecipitation buffer (20 mM Hepes pH 7.4, 150 mM NaCl, 12.5 mM
-glycerophosphate, 1.5 mM MgCl2, 2 mM EGTA, 10 mM NaF, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 mM N-ethylmaleimide, and Mini Complete protease inhibitor tablet from Roche Applied Science) and rocked for 20 min at 4 °C. N-ethylmaleimide was included to inhibit isopeptidase activity. The lysed cells were scraped and centrifuged at 14,000 x g for 10 min at 4 °C. Anti-FLAG monoclonal antibody (1:150) and protein A/G-agarose plus were added to cleared lysates and rocked overnight at 4 °C. Immunoprecipitates were centrifuged at 2500 x g and washed three times with ice-cold lysis buffer. Immunoprecipitated proteins were boiled in 2x SDS sample buffer, electrophoresed, and analyzed by Western blot. In vitro ubiquitination reactions were carried out in a final volume of 15 µl and contained 40 µg of the S-100 fraction. The reaction mixtures contained 50 mM Tris-HCl, pH 7.6, 5 mM MgCl2, 1 mM dithiothreitol, 5 mM ATP along with nuclear extracts from pCMV-FLAG-C/EBP
transfected BALB/MK2 cells as a substrate (2 µg/reaction) supplemented with ERS, 1 mM ubiquitin, and 10 µM ubiquitin-aldehyde. To inhibit proteasomal activity, 25 µM MG-132 was included in reaction mixture. Reactions were incubated at 37 °C for 2 h and were terminated by adding 5 µl of 4% SDS. Reaction mixture was diluted 130 µl of immunoprecipitation buffer and FLAG-C/EBP
was immunoprecipitated using anti-FLAG antibody. Immunoprecipitated proteins were boiled in 2x SDS sample buffer, electrophoresed, and analyzed by Western blot.
Site-directed MutagenesisThreonine to alanine mutations on Thr222 and Thr226 of pcDNA3-C/EBP (Thr222-Pro-Pro-Pro-Thr226-Pro-Val-Pro-Ser230-Pro) were introduced using the QuickChange mutagenesis kit (Stratagene) according to the manufacturer's protocol. The following primer was used to generate the threonine to alanine mutations in T222A and T226A: 5'-648GCAGCCTGGCCACCCT(A
G)CGCCGCCGCCG(A
G)CGCC CGTGCCCAGCCCTC694-3'. Nucleotide changes from cDNA sequence are indicated. Specific mutations were confirmed by sequencing.
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RESULTS |
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C/EBP Is Ubiqutinated and Is a Proteasome Substrate Because proteasome substrates are often polyubiquitinated before their degradation, we examined whether C/EBP
is ubiquitinated in BALB/MK2 cells. BALB/MK2 keratinocytes were transfected with FLAG-tagged C/EBP
, and 46 h later cells were left untreated or treated with proteasome inhibitor, MG-132 for 2 h. FLAG immunoprecipitates were prepared from pCMV-FLAG C/EBP
-transfected BALB/MK2 cells by using monoclonal antibodies to FLAG, followed by immunoblot with polyclonal antibody to ubiquitin. As shown in Fig. 2A (left panel), ubiquitin-immunoreactive higher molecular weight forms were detected in FLAG-C/EBP
-transfected cell lysates, and treatment of cells with MG-132 further increased ubiquitin-reactive higher molecular weight forms of C/EBP
. The membrane was stripped and reprobed with an antibody to C/EBP
. As shown in Fig. 2A (right panel), high molecular weight immunoreactive forms of C/EBP
were detected. To provide additional evidence for the proteasomal-mediated degradation of C/EBP
, we examined whether that C/EBP
is degraded via the proteasome and ubiquitinated in a cell-free assay. HeLa S-100 fraction contains proteasome and ubiquitin ligases and can be used to demonstrate whether a protein is degraded via an ubiquitin-proteasome pathway. As shown in Fig. 2B, C/EBP
protein was degraded by the HeLa S-100 fraction in an ATP- and ubiquitin-dependent manner. When MG-132 was included in the assay, it blocked the degradation of C/EBP
. While C/EBP
was not degraded in the absence of ATP and ubiquitin, its electrophoretic mobility was increased suggesting that C/EBP
is a substrate for cellular phosphatases in vitro. In order to examine whether C/EBP
is ubiquitinated in vitro, FLAG-tagged C/EBP
was incubated with HeLa S-100 fraction for 2 h with/without MG-132 in the presence of ubiquitin-aldehyde, an isopeptidase inhibitor. FLAG-tagged C/EBP
was immunoprecipitated from an in vitro ubiquitination mixture and subjected to immunoblot with ubiquitin antibody. As shown in Fig. 2C (left panel), ubiquitin-immunoreactive higher molecular weight forms of C/EBP
were detected and MG-132 further increased ubiquitin-immunoreactive higher molecular weight forms of C/EBP
. Similar results were obtained when membrane was stripped and reprobed with an antibody to C/EBP
(Fig. 2C, right panel). Collectively, these results demonstrate that the C/EBP
protein is degraded via an ubiquitin-dependent proteasomal pathway.
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Lithium Increases C/EBP Protein LevelsRoss et al. (32) showed that GSK3 is a C/EBP
kinase and phosphorylates Thr222 and Thr226 of C/EBP
, and lithium treatment can block this phosphorylation. Because phosphorylation by GSK3 is known to target cell cycle regulatory proteins such as p21 (26),
-catenin (27), cyclin D1 (28), and c-Myc (29) for proteasomal degradation, we examined whether treatment with lithium, an inhibitor of GSK3 (30, 31), can block C/EBP
degradation. BALB/MK2 keratinocytes were transfected with pcDNA3-C/EBP
and treated with lithium chloride, a known inhibitor of GSK3. Lithium treatment produced a dose-dependent increase in C/EBP
levels (45-fold increase at 20 mM LiCl) as well as an electrophoretically faster migrating form of C/EBP
(Fig. 3, A and B). This increase in electrophoretic mobility is consistent with lithium inhibiting GSK3-mediated phosphorylation of C/EBP
. To confirm this notion, lysates from untreated cells were incubated with protein phosphatase prior to electrophoresis. As shown in Fig. 3C, phosphatase treatment, like lithium, resulted in a faster migrating form of C/EBP
. However, phosphatase treatment produced a faster migrating form of C/EBP
than lithium treatment suggesting that there are additional phosphorylation sites within C/EBP
that are not sensitive to lithium treatment. The effect of lithium on C/EBP
levels was specific as treatment with sodium, another cationic metal, did not increase C/EBP
levels (Fig. 3D). In contrast to the effect of lithium on C/EBP
protein levels, lithium had no effect on C/EBP
mRNA levels indicating that the effects of lithium are post-transcriptional (Fig. 3E). Lithium treatment of untransfected BALB/MK2 cells also resulted in an increase in the endogenous C/EBP
levels, produced a faster migrating form of C/EBP
and had no effect on C/EBP
mRNA levels (data not shown). The fact that lithium treatment produced an increase in C/EBP
protein but not C/EBP
mRNA in both C/EBP
-transfected and untransfected cells indicates that the effects of lithium are promoter-independent (chromatin-embedded C/EBP
promoter versus the cytomegalovirus promoter of pcDNA3-C/EBP
) and that the effects of lithium are post-transcriptional.
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Lithium-induced Increase in C/EBP Protein Levels Is Accompanied by an Increase in C/EBP
Transactivation Activity To ascertain if the lithium-induced increase in C/EBP
protein was also accompanied by an increase in C/EBP
transactivation activity we transfected BALB/MK2 keratinocytes with C/EBP
and a luciferase reporter gene fused to the C/EBP-dependent myelomonocytic growth factor promoter (MGF82) and then treated these cells with lithium. As shown in Fig. 4A, lithium treatment, but not sodium treatment, resulted in a 10-fold increase in pMGF-82 reporter activity over that observed in cells not treated with lithium. This increase in reporter activity was accompanied by an increase in C/EBP
protein levels (Fig. 4B). In contrast, lithium treatment did not produce an increase in MGF-40 reporter activity, a reporter plasmid that lacks C/EBP sites, demonstrating that C/EBP binding sites are required for the lithium response (data not shown). pMGF-82 reporter activity was also increased in cells treated with lithium but not transfected with C/EBP
suggesting that lithium induced increases in endogenous C/EBP
are also accompanied by increases in C/EBP
transactivation activity. Because lithium is also known to inhibit inositol monophosphatase, we treated keratinocytes with 10 mM myoinositol and lithium together or L-690,330 alone. L-690,330 is a potent inhibitor of inositol monophosphatase. Myoinositol and L-690,330 had no effect on the transactivation activity of C/EBP
, indicating that the observed effect of lithium is not due to inhibition of inositol monophosphatase (data not shown). The above results suggest that while lithium inhibits GSK3-mediated phosphorylation of C/EBP
, this is not associated with changes in transactivation activity of C/EBP
protein.
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Lithium Increases C/EBP Protein Levels in a GSK3-independent MannerTo test the idea that lithium produces an increase in C/EBP
protein by inhibiting GSK3-mediated phosphorylation of C/EBP
and subsequent proteasomal degradation, we mutated the GSK3 sites in C/EBP
to produce a mutant protein with T222A and T226A amino acid substitutions. If GSK3-mediated phosphorylation of C/EBP
targets it for proteasomal degradation then in untreated cells the T222A/T226A C/EBP
mutant protein should display increased levels compared with wild-type protein. Moreover, in lithium-treated cells, the mutant protein should not be increased. BALB/MK2 keratinocytes were transfected with mutant or wild-type C/EBP
and then treated with lithium. Lithium treatment produced a similar increase in both the wild-type and mutant protein (Fig. 5A). Moreover, T222A/T226A C/EBP
protein levels were similar to wild-type protein levels in the untreated cells. Wild-type C/EBP
protein demonstrated an electrophoretic mobility shift as described above; however the mutant protein did not exhibit an electrophoretic mobility shift suggesting it was no longer a GSK3 substrate (Fig. 5B). These results suggest that while lithium inhibits GSK3-mediated phosphorylation on C/EBP
, the effect of lithium on C/EBP
protein is through a GSK3-independent mechanism. To confirm this notion, we treated cells with GSK inhibitors SB216763 and SB415286, which have been reported to be specific inhibitors of GSK3 (37). Unlike lithium neither SB216763 nor SB 415286 increased C/EBP
protein levels (Fig. 5C).
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Lithium Increases the Half-life of the C/EBP Protein by Direct Inhibition of Proteasome ActivityAs the effect of lithium on C/EBP
protein levels were post-transcriptional and GSK3-independent, we examined whether lithium directly affects the stability of the C/EBP
protein. To determine whether lithium can alter the stability of the C/EBP
protein we treated C/EBP
-transfected cells with cycloheximide in the presence or absence of lithium. As shown in Fig. 6, A and B, lithium treatment inhibited C/EBP
protein degradation and greatly increased the stability and the half-life of C/EBP
. In order to determine whether lithium increases ubiquitination of C/EBP
, we transfected BALB/MK2 keratinocytes with FLAG-tagged C/EBP
, and 24 h later cells were treated with 20 mM LiCl for 24 h. FLAG immunoprecipitates were prepared from pCMV-FLAG C/EBP
-transfected BALB/MK2 cells by using monoclonal antibodies to FLAG, followed by immunoblot with polyclonal antibody to ubiquitin. As shown in Fig. 6C, lithium treatment increased ubiquitin-immunoreactive higher molecular weight forms of C/EBP
. We next examined the effect of lithium on proteasome-mediated degradation of C/EBP
in vitro using HeLa S-100 fraction. As shown in Fig. 6D, the addition of lithium or the proteasome inhibitor, MG-132 to HeLa S-100 fraction directly inhibited the degradation of C/EBP
in vitro. Collectively these results suggest that lithium increases C/EBP
protein levels through direct inhibition of proteasome activity. In support of this finding are recent studies by Rice and Sartorelli (34) demonstrating that LiCl specifically inhibits the chymotryptic-like activity of the 20 S and 26 S proteasome. To extend the role of lithium as a general proteasomal inhibitor, we examined the effect of lithium on p53, because p53 is degraded via the ubiquitin-proteasome pathway (38). Similar to the C/EBP
results, lithium treatment increased p53 levels in BALB/MK2 cells and blocked the in vitro degradation of p53 (Fig. 7, A and B).
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DISCUSSION |
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Lithium has been successfully used as a therapeutic agent for bipolar disorder and has multiple effects on embryonic development, glycogen synthesis, hematopoiesis, and other cellular processes (31). We observed that lithium treatment of BALB/MK2 keratinocytes resulted in a 5-fold increase in the C/EBP protein. Generally, lithium is considered to produce its biological effects through the inhibition of GSK3 or inositol monophosphatase. Because phosphorylation by GSK3 is known to target cell cycle regulatory proteins such as p21 (26),
-catenin (27), cyclin D1 (28), and c-Myc (29) for proteasomal degradation, we examined whether the effect of lithium on C/EBP
levels was mediated through inhibition of GSK3. While GSK3 is a C/EBP
kinase and has been shown to phosphorylate Thr222 and Thr226 of C/EBP
(32), our results with mutant C/EBP
(T222A/T226A) as well as with pharmacological inhibitors of GSK3, SB216763 and SB415286, indicate that the effect of lithium on C/EBP
protein levels is independent of GSK3. Moreover, inhibitors of inositol monophosphatase, unlike lithium treatment, had no effect on C/EBP
transactivation activity, suggesting that the effects of lithium involve cellular targets other than inositol monophosphatase or GSK3.
Recently, Rice and Sartorelli (34) demonstrated that lithium chloride is a specific inhibitor of the chymotrptic activity of both the 20 S and 26 S proteasome. In BALB/MK keratincytes, we observed that lithium treatment increases the half-life of C/EBP 6-fold and that lithium directly inhibits proteasome activity and blocks C/EBP
degradation in vitro. Taken together these results indicate that lithium is increasing C/EBP
protein levels in BALB/MK2 cells by directly inhibiting proteasomal activity. Overexpression of C/EBP
in BALB/MK2 keratinoytes inhibits cell proliferation (13), and we have observed that lithium treatment of BALB/MK2 cells significantly inhibits thymidine incorporation into DNA.2 Thus, it is possible that the lithium-induced increase in C/EBP
is associated with inhibition in cell proliferation; however, additional studies are required to understand this effect because other cell cycle regulatory proteins such as p21,
-catenin, cyclin D1, and c-Myc are altered by lithium treatment. Recently Mao et al. (41) have shown that lithium treatment of bovine endothelial cells causes the accumulation of the p53 protein without affecting p53 mRNA levels. We have confirmed their findings in BALB/MK2 keratinocytes and have extended these results by showing that lithium directly inhibits proteasomal degradation of p53.
Lithium-induced increases in the C/EBP protein was accompanied by comparable increases in C/EBP
transactivation activity. We also observed that the T222A/T226A mutant C/EBP
protein displayed similar increases in transactivation activity and protein levels as wild-type C/EBP
in the presence of lithium, indicating that inhibition of GSK3-mediated phosphorylation of C/EBP
does not significantly alter C/EBP
transactivation activity. In contrast to C/EBP
, lithium treatment had little to no effect on C/EBP
protein levels; however, lithium treatment did increase C/EBP
transactivation activity in C/EBP
-transfected keratinocytes.2 This observation is consistent with a recent report where growth hormone-induced dephosphorylation of C/EBP
, presumably mediated by a phosphatidylinositol 3-kinase/AKT/GSK3 pathway resulted in increased C/EBP
DNA binding activity (42).
GSK3 has an important role in the Wnt signaling pathway where it along with other proteins regulates the phosphorylation and degradation of -catenin. Recently, Ross et al. (33) have shown that Wnt signaling results in the inhibition of adipogeneisis, and this inhibition of adipogenesis is associated with the loss of expression of C/EBP
and peroxisome proliferator-activated receptor-
(PPAR
) mRNA and protein, two transcription factors that are important in adipogenesis. Importantly, lithium treatment also inhibits adipogenesis (32). Thus, in 3T3-L1 cells, Wnt signaling or lithium treatment (Wnt mimic) results in decreased expression of C/EBP
whereas in keratinocytes we observed that lithium treatment produces an increase in C/EBP
protein levels. This apparent difference could be caused by a differential response between the two cell types, or it is possible that Wnt and lithium treatment block adipogenesis upstream of the expression of C/EBP
and PPAR
thus masking an effect of lithium on C/EBP
protein stability.
In conclusion our results demonstrate that C/EBP is degraded via an ubiquitin-dependent proteasomal pathway and that lithium stabilizes C/EBP
through direct inhibition of proteasome activity in a GSK3-independent manner. In addition to the effect of lithium on inositol monophosphatases and GSK3, the inhibition of proteasome activity by lithium is another mechanism that should be considered when lithium is employed in biological systems.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 919-515-7245; Fax: 919-515-7169; E-mail: rcsmart{at}unity.ncsu.edu.
1 The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein
; GSK3, glycogen synthase kinase 3; EMEM, Eagle's minimal essential medium; hEGF, human epidermal growth factor; ERS, energy regeneration system.
2 R. C. Smart and M. Shim, unpublished manuscript.
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ACKNOWLEDGMENTS |
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REFERENCES |
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