Lithium Stabilizes the CCAAT/Enhancer-binding Protein {alpha} (C/EBP{alpha}) through a Glycogen Synthase Kinase 3 (GSK3)-independent Pathway Involving Direct Inhibition of Proteasomal Activity*

Minsub Shim and Robert C. Smart {ddagger}

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.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CCAAT/enhancer-binding protein {alpha} (C/EBP{alpha}), a basic leucine zipper transcription factor, is involved in mitotic growth arrest and differentiation. Given that numerous proteins involved in cell cycle regulation are degraded via the ubiquitin-proteasome system, we examined whether the C/EBP{alpha} protein is degraded via a proteasomal mechanism. In cycloheximide-treated BALB/MK2 keratinocytes we found that C/EBP{alpha} is a short-lived protein with a half-life of ~1 h. Treatment with proteasome inhibitors, MG-132 or lactacystin, blocked the degradation of the C/EBP{alpha} protein. Higher molecular weight species of ubiquitinated C/EBP{alpha} were detected in BALB/MK2, and in vitro studies confirmed that C/EBP{alpha} is degraded by the proteasome in an ATP- and ubiquitin-dependent manner. GSK3 is a known C/EBP{alpha} kinase and treatment of keratinocytes with LiCl, an inhibitor of GSK3 resulted in: (i) a 5-fold increase in C/EBP{alpha} protein levels, (ii) increased electrophoretic mobility of C/EBP{alpha}, and (iii) no increase in C/EBP{alpha} mRNA levels suggesting that GSK3-mediated phosphorylation of C/EBP{alpha} may target it for proteasomal degradation. However, a mutant C/EBP{alpha} containing T to A mutations in the GSK3 phosphorylation sites (T222A and T226A) retained its response to LiCl, and additional pharmacological inhibitors of GSK3 did not alter C/EBP{alpha} levels indicating the effects of LiCl on C/EBP{alpha} are GSK3-independent. LiCl treatment of BALB/MK2 cells inhibited C/EBP{alpha} degradation and produced a 6-fold increase in the half-life of C/EBP{alpha} protein. In vitro studies revealed that LiCl inhibited proteasome activity and the ensuing degradation of C/EBP{alpha}. These results demonstrate C/EBP{alpha} is degraded via a ubiquitin-dependent proteasomal pathway, and LiCl stabilizes C/EBP{alpha} through a GSK3-independent pathway involving direct inhibition of proteasome activity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The CCAAT/enhancer-binding protein {alpha} (C/EBP{alpha})1 is a member of the basic leucine zipper (bZIP) class of transcription factors. There are six members of the C/EBP family (C/EBP{alpha}, C/EBP{beta}, C/EBP{delta}, C/EBP{epsilon}, C/EBP{gamma}, and C/EBP{zeta}) (1, 2, 3). The C-terminal region of C/EBP contains a basic domain that is responsible for binding specific DNA sequences and a leucine zipper domain that functions in dimerization (4). C/EBP{alpha} can form homodimers as well as heterodimers with other members of the C/EBP family. The N-terminal region of C/EBP{alpha} contains three transactivation elements and an additional highly conserved region (CR4) that is thought to have a regulatory function. C/EBP{alpha} is highly expressed in liver, fat, lung, peripheral leukocytes, epidermis, intestine, and skeletal muscle (5, 6, 7). In numerous cell types, including preadipocytes (8, 9), various myeloid cells (10, 11), hepatocytes (12), and keratinocytes (6, 13), C/EBP{alpha} is involved in the regulation of mitotic growth arrest and/or differentiation. Consistent with this, C/EBP{alpha}-null mice display cell proliferation defects in liver and lung (14, 15). C/EBP{alpha} also plays a key role in energy homeostasis. C/EBP{alpha}-null mice die shortly after birth because of altered hepatic glucose and glycogen metabolism and also display defects in white adipose tissue differentiation (14).

The importance of C/EBP{alpha} in the regulation of growth arrest and differentiation is exemplified by recent studies in which dominant-negative mutations in C/EBP{alpha} were found to be associated with human acute myeloid leukemia (16, 17). Such mutations in C/EBP{alpha} are thought to result in a differentiation block of the granulocytic blasts and have implicated C/EBP{alpha} as a tumor suppressor gene. The growth and differentiation regulatory functions of C/EBP{alpha} are complex and multifaceted. For example, C/EBP{alpha} has been proposed to regulate p21 expression (18, 19) and interact with retinoblastoma (Rb) family proteins (20, 21). C/EBP{alpha} 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{alpha} 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{alpha} 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{alpha} protein stability may be critical in understanding the regulation and/or deregulation of C/EBP{alpha}-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), {beta}-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{alpha} 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{alpha} is degraded via a proteasomal pathway or whether GSK3 or lithium treatment can alter C/EBP{alpha} 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{alpha} is degraded via a proteasomal mechanism and investigated the role of GSK3 and lithium on C/EBP{alpha} protein stability. We demonstrate that C/EBP{alpha} is degraded via an ubiquitin-dependent proteasomal pathway and that lithium stabilizes the C/EBP{alpha} protein through a GSK3-independent pathway involving direct inhibition of proteasomal activity.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Fetal bovine serum, trypsin, human recombinant epidermal growth factor (hEGF), lipofectin, and Tris-glycine gels were purchased from Invitrogen. Ca2+-free Eagle's minimal essential medium (EMEM) was purchased from BioWhittaker. The enhanced chemiluminescence (ECL) reagents were purchased from PerkinElmer Life Sciences. Horseradish peroxidase-linked donkey anti-rabbit IgG was purchased from Amersham Biosciences. Ubiquitin, HeLa S-100 fraction, energy regeneration system (ERS), ubiquitin-aldehyde, MG-132, and lactacystin were purchased from Boston Biochem. Cycloheximide and monoclonal anti-FLAG antibody were purchased from Sigma. Bio-Rad protein assay reagent was purchased from Bio-Rad. Construction of pcDNA3-C/EBP{alpha} has been previously described (13). {lambda}-phosphatase was purchased from New England Biolabs. GSK3 inhibitor SB216763 and SB415286 were from GlaxoSmithKline. pCMV-FLAG expression vector was a kind gift from Dr. Jun Tsuji (North Carolina State University, Raleigh, NC). Rabbit polyclonal anti-ubiquitin antibody, rabbit polyclonal anti-C/EBP{alpha} antibody, rabbit polyclonal anti-p53 antibody, and protein A/G-plus agarose were purchased from Santa Cruz Biotechnology.

Transfection and Lithium Treatment—BALB/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{alpha} 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 Lysates—Nuclear 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 Analysis—Equal 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{alpha} (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 Assay—BALB/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{alpha} 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{alpha} Protein by Phosphatase Treatment— BALB/MK2 cells were transfected with pcDNA3-C/EBP{alpha}, 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 {lambda}-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 Analysis—Total RNA was isolated from untransfected or pcDNA3-C/EBP{alpha}-transfected BALB/MK2 cells using Promega's SV total RNA isolation kit. C/EBP{alpha} cDNA was labeled with [{alpha}-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{alpha} Degradation by Proteasomal Inhibitors— BALB/MK2 cells were transfected with pcDNA3-C/EBP{alpha}. 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 Assay—Degradation 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{alpha}-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{alpha} Stability—BALB/MK2 cells were transfected with pcDNA3-C/EBP{alpha} 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/EBPa—For the detection of ubiquitinated C/EBP{alpha} protein in the cell, BALB/MK2 keratinocytes were plated on a 60-mm culture dish and transfected with pCMV-FLAG-C/EBP{alpha}. 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 {beta}-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{alpha} 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{alpha} was immunoprecipitated using anti-FLAG antibody. Immunoprecipitated proteins were boiled in 2x SDS sample buffer, electrophoresed, and analyzed by Western blot.

Site-directed Mutagenesis—Threonine to alanine mutations on Thr222 and Thr226 of pcDNA3-C/EBP{alpha} (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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
C/EBP{alpha} Is a Short-lived Protein—Numerous proteins involved in regulation of the cell cycle are short-lived proteins that are degraded via an ubiquitin-dependent proteasomal pathway. Because C/EBP{alpha} protein is involved in the regulation of mitotic growth arrest and/or differentiation, we examined the half-life of the C/EBP{alpha} protein and whether proteasomal inhibitors can increase the half-life of C/EBP{alpha}. BALB/MK2 keratinocytes were transfected with pcDNA3-C/EBP{alpha}, and 46 h later cells were treated with cycloheximide in the presence or absence of a proteasomal inhibitor. Cells were collected at 0, 0.5, 1, and 2 h after cycloheximide treatment, and C/EBP{alpha} protein levels were determined by Western blotting. As shown in Fig. 1A, treatment of BALB/MK2 keratinocytes with proteasome inhibitor, MG-132, blocked the degradation of C/EBP{alpha}. Treatment of BALB/MK2 cells with lactacystin, a highly specific proteasome inhibitor (36), also blocked degradation of C/EBP{alpha} (Fig. 1B). Densitometric analysis of these blots revealed that the C/EBP{alpha} protein has a half-life of ~1 h (Fig. 1C). These results demonstrate that C/EBP{alpha} is a short-lived protein and suggest that C/EBP{alpha} is degraded via a proteasomal mechanism.



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FIG. 1.
C/EBP{alpha} is degraded via the proteasome. A and B, BALB/MK2 keratinocytes were transfected with pcDNA3-C/EBP{alpha}, and 46 h later were treated with cycloheximide with/without MG-132 or lactacystin (LCN). Whole cell lysates were prepared at the indicated times, and equal amounts of protein were subjected to Western blot analysis. C, densitometric analysis of results from A and B expressed as % of time 0. (n = 3/time point, average coefficient of variation <15%, r2 = 0.93).

 

C/EBP{alpha} Is Ubiqutinated and Is a Proteasome Substrate— Because proteasome substrates are often polyubiquitinated before their degradation, we examined whether C/EBP{alpha} is ubiquitinated in BALB/MK2 cells. BALB/MK2 keratinocytes were transfected with FLAG-tagged C/EBP{alpha}, 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{alpha}-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{alpha}-transfected cell lysates, and treatment of cells with MG-132 further increased ubiquitin-reactive higher molecular weight forms of C/EBP{alpha}. The membrane was stripped and reprobed with an antibody to C/EBP{alpha}. As shown in Fig. 2A (right panel), high molecular weight immunoreactive forms of C/EBP{alpha} were detected. To provide additional evidence for the proteasomal-mediated degradation of C/EBP{alpha}, we examined whether that C/EBP{alpha} 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{alpha} 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{alpha}. While C/EBP{alpha} was not degraded in the absence of ATP and ubiquitin, its electrophoretic mobility was increased suggesting that C/EBP{alpha} is a substrate for cellular phosphatases in vitro. In order to examine whether C/EBP{alpha} is ubiquitinated in vitro, FLAG-tagged C/EBP{alpha} 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{alpha} 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{alpha} were detected and MG-132 further increased ubiquitin-immunoreactive higher molecular weight forms of C/EBP{alpha}. Similar results were obtained when membrane was stripped and reprobed with an antibody to C/EBP{alpha} (Fig. 2C, right panel). Collectively, these results demonstrate that the C/EBP{alpha} protein is degraded via an ubiquitin-dependent proteasomal pathway.



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FIG. 2.
C/EBP{alpha} is ubiquitinated and is a proteasome substrate. A, BALB/MK2 cells were transfected with FLAG-tagged C/EBP{alpha}, and 46 h later cells were treated with 25 µM MG-132 for 2 h. Lysates were immunoprecipitated with FLAG monoclonal antibody, followed by Western blot with ubiquitin antibody (left panel). The membrane was stripped and reprobed with C/EBP{alpha} antibody (right panel). Arrows indicate IgG heavy chain and FLAG-tagged C/EBP{alpha}. B, C/EBP{alpha} was added to reaction mixtures, and degradation was assayed by Western blotting with C/EBP{alpha} antibody. The reaction mixtures contained HeLa S-100 fraction, ubiquitin, ERS, and ATP and were incubated for 2 h at 37 °C except for the 0-h reaction, in which reaction was terminated at t = 0. For inhibition of proteasomal activity, the reaction mixture was incubated with 25 µM MG-132. C, FLAG-tagged C/EBP{alpha} protein was immunoprecipitated with the FLAG monoclonal antibody from in vitro ubiquitination reaction, followed by Western blot with ubiquitin antibody. Membrane was stripped and reprobed with C/EBP{alpha} antibody. Arrows indicate the IgG heavy chain and FLAG-tagged C/EBP{alpha}.

 

Lithium Increases C/EBP{alpha} Protein Levels—Ross et al. (32) showed that GSK3 is a C/EBP{alpha} kinase and phosphorylates Thr222 and Thr226 of C/EBP{alpha}, and lithium treatment can block this phosphorylation. Because phosphorylation by GSK3 is known to target cell cycle regulatory proteins such as p21 (26), {beta}-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{alpha} degradation. BALB/MK2 keratinocytes were transfected with pcDNA3-C/EBP{alpha} and treated with lithium chloride, a known inhibitor of GSK3. Lithium treatment produced a dose-dependent increase in C/EBP{alpha} levels (4–5-fold increase at 20 mM LiCl) as well as an electrophoretically faster migrating form of C/EBP{alpha} (Fig. 3, A and B). This increase in electrophoretic mobility is consistent with lithium inhibiting GSK3-mediated phosphorylation of C/EBP{alpha}. 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{alpha}. However, phosphatase treatment produced a faster migrating form of C/EBP{alpha} than lithium treatment suggesting that there are additional phosphorylation sites within C/EBP{alpha} that are not sensitive to lithium treatment. The effect of lithium on C/EBP{alpha} levels was specific as treatment with sodium, another cationic metal, did not increase C/EBP{alpha} levels (Fig. 3D). In contrast to the effect of lithium on C/EBP{alpha} protein levels, lithium had no effect on C/EBP{alpha} 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{alpha} levels, produced a faster migrating form of C/EBP{alpha} and had no effect on C/EBP{alpha} mRNA levels (data not shown). The fact that lithium treatment produced an increase in C/EBP{alpha} protein but not C/EBP{alpha} mRNA in both C/EBP{alpha}-transfected and untransfected cells indicates that the effects of lithium are promoter-independent (chromatin-embedded C/EBP{alpha} promoter versus the cytomegalovirus promoter of pcDNA3-C/EBP{alpha}) and that the effects of lithium are post-transcriptional.



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FIG. 3.
Effect of lithium on C/EBP{alpha} protein. A, BALB/MK2 keratinocytes were transfected with pcDNA3-C/EBP{alpha}, and 24 h later were treated with 20 mM LiCl. Twenty-four hours later cell lysates were prepared, and equal amounts of protein were subjected to Western blot analysis. B, BALB/MK2 keratinocytes were transfected with pcDNA3-C/EBP{alpha}, and 24 h later were treated with 20 mM LiCl. Twenty-four hours later cell lysates were prepared, and proteins were separated for Western blot analysis. Different amounts of protein were loaded to attempt to equalize the amount of C/EBP{alpha} protein. Lane 1, 3 µg of nuclear extracts from untreated cells; lane 2, 1 µg of nuclear extracts from 20 mM LiCl-treated cells. C, lysates from untreated pcDNA3-C/EBP{alpha}-transfected cells were incubated with {lambda} phosphatase prior to SDS-PAGE. Lane 1, 3 µg of nuclear extracts from untreated cells; lane 2,1 µg of nuclear extracts from 20 mM LiCl-treated cells; lane 3, 10 µg of {lambda}-phosphatase-treated nuclear extracts. D, BALB/MK2 keratinocytes were transfected with pcDNA3-C/EBP{alpha}, and 24 h later were treated with 20 mM LiCl or 20 mM NaCl. Twenty-four hours later cell lysates were prepared, and equal amounts of protein were subjected to Western blot analysis. E, twenty-four hours following transfection with pcDNA3-C/EBP{alpha}, BALB/MK2 cells were treated with 20 mM LiCl and RNA was isolated at the indicated times. 15 µg of total RNA was loaded per each well, and Northern blot analysis was conducted. Membrane was stripped and reprobed for 7 S RNA to confirm equal loading.

 

Lithium-induced Increase in C/EBP{alpha} Protein Levels Is Accompanied by an Increase in C/EBP{alpha} Transactivation Activity— To ascertain if the lithium-induced increase in C/EBP{alpha} protein was also accompanied by an increase in C/EBP{alpha} transactivation activity we transfected BALB/MK2 keratinocytes with C/EBP{alpha} 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{alpha} 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{alpha} suggesting that lithium induced increases in endogenous C/EBP{alpha} are also accompanied by increases in C/EBP{alpha} 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{alpha}, 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{alpha}, this is not associated with changes in transactivation activity of C/EBP{alpha} protein.



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FIG. 4.
Lithium-induced increase in C/EBP{alpha} levels is accompanied by an increase in C/EBP{alpha} transactivation activity. A, BALB/MK2 keratinocytes were transfected with pcDNA3 and pMGF82 reporter plasmids or with pcDNA3-C/EBP{alpha} and pMGF82 reporter plasmids, and 24 h later were treated with 20 mM LiCl or 20 mM NaCl. Twenty-four hours later cells were lysed with cell culture lysis buffer, and luciferase activity was determined. Luciferase activity was normalized to protein levels, and each value represents the mean ± S.D. of triplicates per treatment. B, C/EBP{alpha} protein levels from the same experiment shown in A. Same blot was differentially exposed to visualize endogenous C/EBP{alpha} and C/EBP{alpha} in pcDNA3-C/EBP{alpha}-transfected cells.

 

Lithium Increases C/EBP{alpha} Protein Levels in a GSK3-independent Manner—To test the idea that lithium produces an increase in C/EBP{alpha} protein by inhibiting GSK3-mediated phosphorylation of C/EBP{alpha} and subsequent proteasomal degradation, we mutated the GSK3 sites in C/EBP{alpha} to produce a mutant protein with T222A and T226A amino acid substitutions. If GSK3-mediated phosphorylation of C/EBP{alpha} targets it for proteasomal degradation then in untreated cells the T222A/T226A C/EBP{alpha} 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{alpha} 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{alpha} protein levels were similar to wild-type protein levels in the untreated cells. Wild-type C/EBP{alpha} 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{alpha}, the effect of lithium on C/EBP{alpha} 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{alpha} protein levels (Fig. 5C).



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FIG. 5.
Effect of lithium on C/EBP{alpha} protein level is GSK3-independent. A, BALB/MK2 keratinocytes were transfected with pcDNA3-C/EBP{alpha} or mutant pcDNA3-C/EBP{alpha} T222A, T226A, and 24 h later were treated with 20 mM LiCl. Twenty-four hours later cell lysates were prepared, and equal amounts of proteins were subjected to Western blot analysis. B, lysates were prepared as described above in A except proteins were separated on an 8% Tris-glycine gel. Different amounts of protein were loaded to attempt to equalize the amount of C/EBP{alpha} protein. Lanes 1 and 3, 3 µg of nuclear extracts from untreated cells; lanes 2 and 4, 1 µg of nuclear extracts from 20 mM LiCl-treated cells. C, BALB/MK2 keratinocytes were transfected with pcDNA3-C/EBP{alpha}, and 24 h later were treated with 20 mM LiCl, 20 µM SB216763, or 40 µM SB415286. Twenty-four hours later cell lysates were prepared, and equal amounts of protein were subjected to Western blot analysis.

 

Lithium Increases the Half-life of the C/EBP{alpha} Protein by Direct Inhibition of Proteasome Activity—As the effect of lithium on C/EBP{alpha} protein levels were post-transcriptional and GSK3-independent, we examined whether lithium directly affects the stability of the C/EBP{alpha} protein. To determine whether lithium can alter the stability of the C/EBP{alpha} protein we treated C/EBP{alpha}-transfected cells with cycloheximide in the presence or absence of lithium. As shown in Fig. 6, A and B, lithium treatment inhibited C/EBP{alpha} protein degradation and greatly increased the stability and the half-life of C/EBP{alpha}. In order to determine whether lithium increases ubiquitination of C/EBP{alpha}, we transfected BALB/MK2 keratinocytes with FLAG-tagged C/EBP{alpha}, and 24 h later cells were treated with 20 mM LiCl for 24 h. FLAG immunoprecipitates were prepared from pCMV-FLAG C/EBP{alpha}-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{alpha}. We next examined the effect of lithium on proteasome-mediated degradation of C/EBP{alpha} 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{alpha} in vitro. Collectively these results suggest that lithium increases C/EBP{alpha} 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{alpha} 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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FIG. 6.
Lithium stabilizes C/EBP{alpha} protein by interfering with proteasomal activity. A, BALB/MK2 keratinocytes were transfected with pcDNA3-C/EBP{alpha}, and 24 h later were treated with or without 20 mM LiCl. Forty-six hours post-transfection, cells were treated with 50 µg/ml cycloheximide, and whole cell lysates were prepared at the indicated times, and equal amounts of protein were subjected to Western blot analysis. B, densitometric analysis of results from A expressed as % of time 0. (n = 3/time point, average coefficient of variation <15%, r2 = 0.93 for –Li, r2 = 0.65 for +Li). C, BALB/MK2 cells were transfected with FLAG-tagged C/EBP{alpha}. 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. Lysates were immunoprecipitated with FLAG monoclonal antibody, followed by Western blot with ubiquitin antibody. D, C/EBP{alpha} was added to reaction mixtures, and degradation was assayed by Western blotting with C/EBP{alpha} antibody. The reaction mixtures contained HeLa S-100 fraction, ubiquitin, ERS, and ATP and were incubated for 2 h at 37 °C except for the 0-h reaction, in which the reaction was terminated at t = 0. For inhibition of C/EBP{alpha} degradation, the reaction mixture was incubated with 25 µM MG-132 or 10 mM LiCl.

 


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FIG. 7.
Lithium treatment increases p53 levels in BALB/MK2 cells and blocks p53 degradation by HeLa S-100 fraction. A, BALB/MK2 keratinocytes were transfected with p53 expression vector and 24 h later were treated with 20 mM LiCl. Twenty-four hours later cell lysates were prepared, and equal amounts of protein were subjected to Western blot analysis. B, In vitro p53 proteasomal degradation assay. p53 was added to reaction mixtures, and degradation was assayed by Western blotting with p53 antibody. The reaction mixtures contained HeLa S-100 fraction, ubiquitin, ERS, and ATP and were incubated for 2 h at 37 °C except for the 0-h reaction, in which the reaction was terminated at t = 0. For inhibition of p53 degradation, the reaction mixture was incubated with 25 µM MG-132 or 10 mM LiCl.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study we observed that C/EBP{alpha} is short-lived protein with a half-life of ~1 h and that treatment with proteasome inhibitors, MG-132 or lactacystin, blocks C/EBP{alpha} protein degradation. We provide evidence that C/EBP{alpha} is polyubiquitinated in BALB/MK2 cells, and the results of our in vitro studies demonstrate that C/EBP{alpha} is degraded via the proteasome in an ATP- and ubiquitin-dependent manner. This is the first demonstration that the C/EBP{alpha} transcription factor is degraded via an ubiquitin-proteasomal pathway in mammalian cells. C/EBP{alpha} is an important regulator of cell growth and/or differentiation in numerous cell types. In addition to its function as a transcription factor, C/EBP{alpha} has been reported to negatively regulate cell proliferation through a transcription-independent mechanism in which C/EBP{alpha} forms a complex with cdk2 and cdk4 preventing cyclin/cdk complex formation and cell cycle progression (18). Regulation of C/EBP{alpha} protein levels through an ubiquitin-proteasomal pathway would serve to control both the transcription-dependent and transcription-independent activities of C/EBP{alpha}. Recently C/EBP{alpha}, C/EBP{beta}, C/EBP{delta}, and C/EBP{epsilon} have been shown to undergo sumoylation, and covalent modification of C/EBP{epsilon} by SUMO1 appears to activate C/EBP{epsilon} transactivation function but does not target C/EBP for degradation (39, 40). Thus, the C/EBP{alpha} transcription activity and protein level can be modulated by covalent modification involving sumoylation and ubquitination. Whether proteasomal degradation of C/EBP{alpha} can be regulated in a cell cycle-dependent manner or through C/EBP{alpha} phosphorylation via sites other than GSK3 sites is not known. However, in some tissues/cells C/EBP{alpha} protein levels are often disparate with mRNA levels suggesting C/EBP{alpha} protein levels are regulated at the translation and/or post-translational level (2, 6).

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{alpha} 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), {beta}-catenin (27), cyclin D1 (28), and c-Myc (29) for proteasomal degradation, we examined whether the effect of lithium on C/EBP{alpha} levels was mediated through inhibition of GSK3. While GSK3 is a C/EBP{alpha} kinase and has been shown to phosphorylate Thr222 and Thr226 of C/EBP{alpha} (32), our results with mutant C/EBP{alpha} (T222A/T226A) as well as with pharmacological inhibitors of GSK3, SB216763 and SB415286, indicate that the effect of lithium on C/EBP{alpha} protein levels is independent of GSK3. Moreover, inhibitors of inositol monophosphatase, unlike lithium treatment, had no effect on C/EBP{alpha} 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{alpha} 6-fold and that lithium directly inhibits proteasome activity and blocks C/EBP{alpha} degradation in vitro. Taken together these results indicate that lithium is increasing C/EBP{alpha} protein levels in BALB/MK2 cells by directly inhibiting proteasomal activity. Overexpression of C/EBP{alpha} 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{alpha} 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, {beta}-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{alpha} protein was accompanied by comparable increases in C/EBP{alpha} transactivation activity. We also observed that the T222A/T226A mutant C/EBP{alpha} protein displayed similar increases in transactivation activity and protein levels as wild-type C/EBP{alpha} in the presence of lithium, indicating that inhibition of GSK3-mediated phosphorylation of C/EBP{alpha} does not significantly alter C/EBP{alpha} transactivation activity. In contrast to C/EBP{alpha}, lithium treatment had little to no effect on C/EBP{beta} protein levels; however, lithium treatment did increase C/EBP{beta} transactivation activity in C/EBP{beta}-transfected keratinocytes.2 This observation is consistent with a recent report where growth hormone-induced dephosphorylation of C/EBP{beta}, presumably mediated by a phosphatidylinositol 3-kinase/AKT/GSK3 pathway resulted in increased C/EBP{beta} 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 {beta}-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{alpha} and peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) 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{alpha} whereas in keratinocytes we observed that lithium treatment produces an increase in C/EBP{alpha} 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{alpha} and PPAR{gamma} thus masking an effect of lithium on C/EBP{alpha} protein stability.

In conclusion our results demonstrate that C/EBP{alpha} is degraded via an ubiquitin-dependent proteasomal pathway and that lithium stabilizes C/EBP{alpha} 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.


    FOOTNOTES
 
* This work was supported by Grant CA 46637 from the NCI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} 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{alpha}, CCAAT/enhancer-binding protein {alpha}; GSK3, glycogen synthase kinase 3; EMEM, Eagle's minimal essential medium; hEGF, human epidermal growth factor; ERS, energy regeneration system. Back

2 R. C. Smart and M. Shim, unpublished manuscript. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Francesco Melandri at Boston Biochem for helpful discussions. We also thank Dr. Yoshiaki Tsuji and Dr. Jun Ninomiya-Tsuji for helpful discussions and the kind gift of plasmids.



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