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Molecular and Cellular Biology, June 2003, p. 4035-4045, Vol. 23, No. 12
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.12.4035-4045.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Division of Basic Biomedical Sciences, School of Medicine, University of South Dakota, Vermillion, South Dakota 57069
Received 25 November 2002/ Returned for modification 13 January 2003/ Accepted 17 March 2003
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ABSTRACT |
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INTRODUCTION |
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Oligodendrocytes are the myelinating cells of the central nervous system. Upon differentiation, oligodendrocytes express many myelin-specific proteins, among which is myelin basic protein (MBP). MBP is one of the major protein components of the myelin sheath that surrounds axons to ensure the rapid conduction of nervous impulses.
Oligodendrocytes progress through a series of stages, changing from proliferative, migratory progenitor cells to mature, postmitotic, myelin-membrane-producing cells. The differentiation of oligodendrocytes is accompanied by a striking increase in the level of p27Kip1 protein (37). This accumulation of p27Kip1 causes cell cycle arrest in oligodendrocyte progenitor cells (16), alters the responses of cells to mitogens, and initiates differentiation (11). Miskimins et al. showed previously that p27Kip1 has a role in MBP gene expression in oligodendrocytes, as increased levels of p27Kip1 in CG4 oligodendrocytes dramatically enhance MBP promoter activity (22).
The CG4 oligodendrocyte cell line is derived from primary cultures of rat oligodendrocyte progenitor cells. These cells proliferate in the presence of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (FGF2). Upon withdrawal of PDGF and FGF2, CG4 cells differentiate into mature oligodendrocytes (21). These cells are also capable of myelinating axons after transplantation into myelin-deficient rat brain (13). In this study, CG4 oligodendrocytes were used to investigate the role of p27Kip1 in the activation of the MBP promoter. We show that a conserved GC box located in the proximal region of the MBP promoter is necessary for p27Kip1 to stimulate expression. Increased levels of p27Kip1 during differentiation lead to increased levels of binding of transcription factor Sp1 to this element, contributing to the activation of the MBP promoter. Our data also show that the overexpression of p27Kip1 results in a decrease in Sp1 degradation rates, leading to increased levels of Sp1 promoter binding activity and subsequent activation of the MBP promoter. The results presented here reveal a novel mechanism of p27Kip1-mediated activation of gene transcription.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemicals and plasmids. Roscovitine was obtained from Calbiochem (San Diego, Calif.). Fascaplysin was purchased from Alexis Biochemicals (San Diego, Calif.). The MBP-luciferase constructs were made by PCR with oligonucleotides that hybridized to the 5' end of the MBP promoter and ended at the sequences indicated in Fig. 1 and a common 3' oligonucleotide ending at +30. The PCR fragments were inserted upstream of the luciferase gene in the SmaI site of pGL3Basic (Fig. 1). The p27Kip1 expression plasmid contains the entire mouse p27Kip1 cDNA amplified by PCR and inserted downstream of the cytomegalovirus promoter in pcDNA3.1 (Hygro) (Invitrogen, Carlsbad, Calif.). The Sp1 expression plasmid CMV-Sp1 was a gift from R. Tjian.
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Western blotting. Cells in 35-mm dishes were rinsed with PBS and lysed by the addition of sodium dodecyl sulfate (SDS) sample buffer (2.5 mM Tris-HCl [pH 6.8], 2.5% SDS, 100 mM dithiothreitol [DTT], 10% glycerol, 0.025% pyronin Y). Equal amounts of protein were separated on SDS-polyacrylamide gels. Proteins were transferred to Immobilon P membranes (Millipore, Bedford, Mass.) by using a Bio-Rad Trans-blot apparatus and transfer buffer (48 mM Tris, 39 mM glycine, 0.0375% SDS, 20% methanol). Membranes were processed as previously described (22). Detection was carried out by using a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) and Super Signal chemiluminescent substrate (Pierce, Rockford, Ill.). The primary antibodies used were anti-p27Kip1 (Transduction Laboratories, Lexington, Ky.; 1:2,500), anti-Sp3 (Santa Cruz Biotechnology; 1:2,500), anti-Sp1 (Santa Cruz Biotechnology;1:1,000), and anti-ß-actin (Sigma, St. Louis, Mo.; 1:5,000).
Nuclear extracts. Nuclear extracts were made as previously described (1) with the following modifications. Cells grown in 100-mm dishes were harvested by scraping, and the pellets were resuspended in 500 µl of buffer A (10 mM HEPES-KOH [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride [PMSF]). The cells were allowed to swell on ice for 10 min, vortexed (to break the cells), and centrifuged at 14,000 x g for 10 s. The pellets were resuspended in 200 µl of buffer C (20 mM HEPES-KOH [pH 7.9], 1.5 mM MgCl2, 25% glycerol, 0.2 mM EDTA, 420 mM NaCl, 0.2 mM PMSF, 0.5 mM DTT) and incubated on ice for 30 min. Cell debris was removed by centrifugation at 14,000 x g for 15 min at 4°C, and the supernatant fraction was microdialyzed against buffer D2 (20 mM HEPES [pH 7.9], 250 mM sucrose, 50 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 5 mM MgCl2, 0.1 mM ZnCl2, 0.5 mM PMSF). The protein concentration was determined as described above, and samples were stored at -70°C.
Electrophoretic mobility shift assays (EMSAs).
Double-stranded oligonucleotides were end labeled with [
-32P]ATP (NEN, Boston, Mass.) by using T4 polynucleotide kinase (MBI Fermentis, Hanover, Md.). Approximately 40 ng of 32P-labeled oligonucleotide (106 cpm) was incubated for 15 min on ice with 5 µg of nuclear extract in buffer D2 plus poly(dI-dC) (2 µg) in a total volume of 50 µl. When included as competitors, unlabeled oligonucleotides were added in excess to the nuclear extract together with the labeled probe. For supershift experiments, the labeled probe was incubated with the nuclear extract on ice for 15 min, followed by an additional incubation for 15 min with various amounts of antibodies. Complexes were resolved on 3.5 or 5% nondenaturing polyacrylamide gels in 0.25x Tris-borate-EDTA (0.022 M Tris, 0.022 M boric acid, and 0.5 mM EDTA). Gels were dried and subjected to autoradiography. All oligonucleotide probes were used in double-stranded form. They consisted of mouse MBP promoter sequences between positions -105 and -52 corresponding to the wild-type sequence or containing the mutation detailed in Fig. 1, in which the putative Sp1/Sp3 transcription factor binding site has been mutated. Double-stranded oligonucleotides containing the sequence 5'-CCCGGGTGACGTCACGGGGA-3' and its complement were used as nonspecific competitor DNAs, while oligonucleotides containing the sequence 5'-GCTATGACCGCCCCATGAT-3', which harbors the GC box in the MBP promoter, were used as specific competitors for Sp1/Sp3 binding. Antibodies used for supershift assays were obtained from Santa Cruz Biotechnology. The specificities were as follows: Sp1 (PEP 2; sc-59), Sp2 (K-20; sc-643), Sp3 (D-20; sc-644), and Sp4 (V-20; sc-645).
Site-specific mutagenesis. Site-specific mutagenesis of the MBP promoter was carried out by using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.) according to the manufacturer's protocol. Primers for mutation of the GC box were the same oligonucleotides as those used for EMSAs. Transformation was done by using One-Shot competent Escherichia coli cells (Invitrogen). The purified plasmid was subjected to sequencing to confirm the mutation.
Pulse-chase labeling. Cells were grown in 100-mm dishes and transfected with p27Kip1 plasmid or control plasmid as described above. Following transfection, the cells were left in GM or switched to DM. At 2 days after transfection, the cells were washed twice with PBS. The cells were placed in methionine-cysteine-free medium for 1 h, after which 35S-labeled methionine-cysteine was added to 60 µCi/ml (Trans 35S label; ICN). After 1 h, the cells were washed three times with methionine-cysteine-free DMEM and incubated in GM or DM supplemented with 1 mM unlabeled methionine-cysteine. At various times, the cells were washed twice with PBS and lysed for 30 min at 4oC in 1 ml of cell lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1% Tween 20, 1 mM DTT, 10 mM ß-glycerophosphate, 0.1 mM sodium orthovanadate, 10 µg of leupeptin/ml, 2 µg of aprotinin/ml, 100 µM PMSF). Lysates were collected by scraping and centrifuged, and the supernatants were precleared by incubation with protein A-conjugated agarose beads (Calbiochem). For immunoprecipitation, protein A-conjugated magnetic beads (Dynal, Lake Success, N.Y.; 30 µl) were incubated with 6 µg of anti-Sp1 antibody for 3 h, followed by two washes with cell lysis buffer. Antibody-conjugated beads were incubated with 1.3 mg of proteins from the precleared extracts overnight. The beads were collected by magnetic separation and washed four times with radioimmunoprecipitation buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.8], 0.5% deoxycholic acid, 0.1% SDS). After the addition of SDS sample buffer to each sample, the samples were heated for 5 min and loaded onto SDS-7% polyacrylamide gels. The gels were dried, and the bands were visualized by autoradiography. Autoradiographs were subjected to densitometry to estimate the level of Sp1 and to determine the half-life of the protein.
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RESULTS |
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GC box sequences can be bound by a group of transcription factors belonging to the Sp1 family (8, 35). Indeed, there is evidence that one member of this family, Sp1, can bind to sequences in the MBP promoter (2, 39). In order to determine whether any member(s) of the Sp1 family of transcription factors is involved in forming either complex 1 or complex 2, supershift assays were performed. The results show that complex 1 is most efficiently supershifted by an anti-Sp1 antibody. An anti-Sp3 antibody completely supershifts complex 2 (Fig. 3D). A weak supershifted band is observed when anti-Sp2 antibody is used. A fourth member of the Sp1 transcription factor family, Sp4, does not appear to be involved in the formation of any of the complexes (data not shown). Additionally, the complexes are not supershifted by antibodies to other transcription factors, such as anti-c-Fos or anti-c-Jun (data not shown).
Increased expression of p27Kip1 leads to an increase in binding to the region of the MBP promoter between -105 and -52 (Fig. 3A), and this binding appears to involve Sp1, Sp2, and Sp3. In order to ascertain which of these Sp1 family members is involved in the increased binding to the MBP promoter, supershift assays were performed with extracts from CG4 cells transfected with either an empty vector or a plasmid encoding p27Kip1. With anti-Sp1 antibody, there is a significant increase in the level of the supershifted band in extracts from cells transfected with the p27Kip1 expression construct (Fig. 4). In contrast, there appears to be no major change in the level of the supershifted band with antibody to Sp2 or Sp3 (Fig. 4). These data indicate that the expression of p27Kip1 causes an increase in Sp1 transcription factor binding to the GC box in the MBP promoter.
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The expression of Sp1 has an effect on MBP promoter activity similar to that of p27Kip1. The above data suggest that increased p27Kip1 levels lead to increased binding of Sp1 to the MBP promoter and that this binding results in activation of the promoter. Thus, we would expect that increased expression of Sp1 in CG4 cells would have an effect on the MBP promoter similar to that of increased expression of p27Kip1. To test this idea, CG4 cells were transfected with the MBP-luciferase construct containing the full-length MBP promoter (extending to -1323) together with an Sp1 expression plasmid, a p27Kip1 expression plasmid, or both. When the MBP-luciferase construct is transfected with either the Sp1 expression construct or the p27Kip1 expression construct, there is an increase in luciferase activity over that seen with transfection with the control plasmid (Fig. 6A). The level of the increase is similar when either Sp1 or p27Kip1 is cotransfected. In fact, transfection of both expression constructs with the MBP-luciferase construct did not lead to higher levels of luciferase activity than did transfection of either the Sp1 or the p27Kip1 expression construct alone (Fig. 6A), suggesting that these proteins act through the same pathway. Increased expression of Sp1 and p27Kip1 in transfected cells was confirmed by Western blotting (Fig. 6B). Note that cells transfected with the p27Kip1 expression construct also had higher levels of Sp1 protein, demonstrating that the observed increase in Sp1 binding (Fig. 4) reflects an increase in Sp1 protein levels induced by elevated levels of p27Kip1.
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Increased Sp1 levels are a specific response to increased p27Kip1 levels. In most cells, including oligodendrocytes, p27Kip1 plays a role in cell cycle regulation through its function as a CKI. Since our data show that the overexpression of p27Kip1 leads to an increase in Sp1 protein levels, it was of interest to determine whether this increase is a response to the cell cycle regulatory function of p27Kip1 or is mediated in a different manner. To address this question, CG4 cells were treated with chemical inhibitors of Cdk activity. For these studies, we used roscovitine, a Cdk2 inhibitor, and fascaplysin, an inhibitor of Cdk4. Using bromodeoxyuridine labeling of S-phase cells, we have demonstrated that the concentrations of roscovitine and fascaplysin used in these studies are sufficient to inhibit the division of CG4 cells (data not shown). Treatment of cells transfected with the -105 MBP-luciferase construct indicates that neither roscovitine nor fascaplysin can enhance MBP promoter activity (Fig. 7A). Additionally, when nuclear extracts made from CG4 cells treated with either roscovitine or fascaplysin are used in EMSAs, the level of binding and the pattern of complexes formed are unaltered compared to those seen with untreated cells (data not shown). Further, there is no increase in endogenous Sp1 levels in cells treated with either roscovitine or fascaplysin (Fig. 7B). Thus, it appears that simply stopping the cell cycle is not sufficient to induce increased Sp1 levels and MBP promoter activity. These results are consistent with previous results showing that cell cycle arrest in the absence of increased p27Kip1 levels does not lead to activation of the MBP promoter (22).
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p27Kip1 increases the levels of Sp1 by decreasing its rate of degradation.
The increase in Sp1 levels induced by the increase in p27Kip1 levels could be caused by an increase in the production of Sp1 or by a decrease in its turnover. To test whether p27Kip1 increased the stability of existing Sp1 protein, a pulse-chase experiment was performed. CG4 cells were transfected with a plasmid encoding p27Kip1 or a control plasmid. At 2 days after transfection, the cells were starved for methionine and cysteine for 1 h, after which 35S-labeled methionine-cysteine was added to the medium. After 1 h, the cells were washed and the medium was replaced. The cells were harvested at different times after the medium was changed and were lysed, and Sp1 was immunoprecipitated. Figure 9A shows that the levels of labeled Sp1 at all time points except for the zero time point are higher in cells transfected with p27Kip1. The autoradiograph was subjected to densitometry, and the results were plotted against time (Fig. 9B). The results show that Sp1 in control transfected cells in DM has a half-life of approximately 4 h, while in p27Kip1-transfected cells, it has a half-life of 10 h or more. Additionally, the transfection results predict that the half-life of Sp1 should be longer in differentiating cells than in growing cells, since the endogenous levels of p27Kip1 are higher in differentiating cells. This prediction appears to be correct, since the half-life of cells maintained in GM is shorter (
2 h) than that in cells placed in DM (Fig. 9). Thus, it appears that increases in the levels of p27Kip1 can enhance MBP gene expression through the stabilization of transcription factor Sp1.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous results indicated that the MBP promoter could be activated in response to an increase in p27Kip1 protein levels (22). The response of the MBP promoter to increased p27Kip1 levels could not be mimicked by inhibiting the activities of Cdk2 and Cdk4, as indicated by the results presented here, in which the inhibition of Cdk2 or Cdk4 with chemical inhibitors did not lead to an increase in MBP promoter activity. Additionally, it was shown previously that MBP promoter activity is not increased when other CKIs, such as p21, p57, and p16, are coexpressed with -1323Luc (22). Other studies also indicated that p27Kip1 is a multifunctional protein. In addition to its function as a cell cycle repressor, p27Kip1 plays a role in transcription in other systems. In some cells, p27Kip1 can repress transcription. In anergic T cells, p27Kip1 associates with the c-Jun coactivator JAB1, resulting in defective transactivation of AP-1 and interleukin 2 transcription (6). In mouse fibroblasts, p27Kip1 downregulates the P4 promoter of parvovirus through a cyclic AMP response element that interacts with complexes containing transcription factor E2F (9). In intestinal cells, the overexpression of p27Kip1 enhances sucrase-isomaltase gene expression by activating its promoter, while the overexpression of other CKIs, including p21 and p57, has no significant effect (10). Thus, p27Kip1 has specific effects on gene expression through mechanisms which at present are poorly understood.
By exploring the elements in the MBP promoter that are involved in the response to the increased expression of p27Kip1, we identified a GC box in the proximal region of the MBP promoter that is essential for the response to p27Kip1. Transcription factor Sp1 also was identified as interacting with the GC box. Other regions of the MBP promoter show differential responses to increased p27Kip1 levels. Computer analysis of these regions shows several additional potential Sp1 binding sites upstream of the one explored here. Thus, it is possible that these sites are responsible for the responses of other regions of the MBP promoter to increased p27 levels. Indeed, the increased expression of Sp1 mimics the effect of p27Kip1 on the MBP promoter and, when coexpressed, they activate the MBP promoter to the same level as either does alone. This finding suggests that they are a part of the same pathway of activation. The binding of Sp1 to the GC box is required for the effect of p27Kip1, as the disruption of binding through site-specific mutation of the GC box abolishes the ability of p27Kip1 to activate the MBP promoter. Disruption of the Sp1 binding site leads to a decrease in the basal promoter activity of MBP-luciferase constructs, indicating the importance of this site for promoter activity. However, we believe that the above results indicate a role for the binding of Sp1 to this site in mediating the effect of p27Kip1 on the MBP promoter. This effect of p27Kip1—increasing binding of Sp1 and binding to the GC box—is not simply the result of inhibiting Cdk2 activity, as treatment with chemical inhibitors of Cdk2 (or Cdk4) (Fig. 7) or the expression of p21, p57, or p16 (22) does not lead to increased Sp1 binding.
Both Sp1 and Sp3 binding activities appear to be present in CG4 cells, and both factors are capable of binding the GC box in the MBP promoter. The activation of promoters containing Sp1 binding sites can be regulated by the ratio of Sp1 to Sp3 in a cell at any given time. For example, in primary keratinocytes, Sp3 levels are higher than Sp1 levels. When these cells differentiate, the ratio becomes inverted, suggesting that the transcription of several genes required for cell type specification in keratinocytes can be regulated by a change in the ratio of Sp1 to Sp3 (3). A similar mechanism may occur in oligodendrocyte differentiation. As the cells enter the differentiation pathway, p27Kip1 levels increase and, subsequently, genes involved in the production of myelin are expressed. Both Sp3 and Sp1 are capable of binding the GC box in the MBP promoter. With an increase in p27Kip1 levels, Sp1 levels appear to increase, while there is no apparent change in Sp3 binding to the promoter. Thus, it is possible that the activation of the MBP promoter is achieved by an alteration in the ratio of Sp3 to Sp1.
Increased expression of p27Kip1 leads to increased Sp1 expression and binding to the MBP promoter. Prior to the increase in endogenous p27Kip1 levels, MBP expression is low, suggesting that regulation of the abundance of cellular Sp1 is one mechanism through which MBP transcription may be regulated. Changes in the abundance of Sp1 may occur through the regulation of Sp1 gene expression, mRNA stability, or posttranslational events that result in increased affinity for its binding site or decreased protein turnover. Interestingly, we found that the overexpression of p27Kip1 increases Sp1 levels by decreasing its turnover. The turnover of Sp1 in other systems has been investigated. For example, in the green monkey kidney cell line CV-1, Sp1 is susceptible to a specific protease, SPase, purified from cell nuclear extracts (27). Human Sug1 (also known as p45 or thyroid hormone receptor-interacting protein), an ATPase subunit of the 26S proteasome and a putative transcriptional modulator, is also able to stimulate the proteasome-dependent degradation of Sp1 (34). The role of these or other proteins in p27Kip1-mediated Sp1 stabilization in oligodendrocytes remains to be determined.
Additionally, Sp1 is phosphorylated by a number of protein kinases, including the cyclin A-Cdk2 complex (14), DNA-dependent protein kinase (19), casein kinase II (4), protein kinase A (30), and an unidentified cell cycle-regulated kinase (5). While the phosphorylation of Sp1 is mostly involved in the regulation of its transactivation activity, the phosphorylation of some specific sites also has an effect on its degradation. In a rat pituitary cell line, GH4, sustained stimulation by epidermal growth factor initiates a cascade of phosphorylation events that promotes Sp1 proteolysis and decreases the levels of Sp1 in the nucleus (24). In human tumor cells, blocking epidermal growth factor receptor tyrosine kinase activity upregulates the expression of p27Kip1 (7). These findings raise the possibility that the expression of p27Kip1 may inhibit the phosphorylation of Sp1 at a specific site or sites, leading to its stabilization. The effect of increased p27Kip1 expression on the phosphorylation status of Sp1 in oligodendrocytes remains to be determined.
In addition to phosphorylation, Sp1 can be modified through covalent linkage of the monosaccharide N-acetylglucosamine to serine and threonine residues (O-linked glycosylation). Reduced O-linked glycosylation of Sp1 is associated with increased proteasome susceptibility (17). Through an as-yet-undetermined mechanism, p27Kip1 may function to decrease the level of phosphorylation of Sp1 or increase its level of glycosylation, thus increasing Sp1 stability.
We have shown here a novel mechanism by which p27Kip1 can activate the MBP promoter. Elevated p27Kip1 expression causes an increase in the level of Sp1 protein by decreasing the rate of Sp1 degradation. Sp1 acts as a transcriptional activator of the MBP promoter through binding to the conserved GC box in the proximal region of the MBP promoter. In this way, p27Kip1 enhances MBP promoter activity. The mechanism by which p27Kip1 leads to a decrease in Sp1 degradation remains to be determined.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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) or DM (•) or transfected with the p27Kip1 expression vector (
) were quantitated by densitometry and plotted against the chase time. The zero-hour time point for cells in DM was set to 100%. All values shown are relative to this value.