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Proteasomal Degradation of Retinoblastoma-Related p130 during Adipocyte Differentiation
Biochemical and Biophysical Research Communications 290, 1066 –1071 (2002) doi:10.1006/bbrc.2001.6291, available online at http://www.idealibrary.com on
Proteasomal Degradation of Retinoblastoma-Related p130 during Adipocyte Differentiation Audra M. Prince, Julie S. May, Gregory R. Burton, Robert E. Lyle, and Robert E. McGehee, Jr. 1 Department of Pediatrics, Division of Neonatology, University of Arkansas for Medical Sciences and Arkansas Children’s Hospital, Little Rock, Arkansas 72205
Received December 12, 2001
Within 24 h of hormonally stimulated 3T3-L1 adipocyte differentiation, there are dramatic changes in the protein levels of p130 and p107, two members of the retinoblastoma tumor suppressor gene family. Designated the “p103:p107” switch, this alteration is characterized by a rapid and transient drop in p130 protein levels accompanied by a transient increase in both p107 mRNA and protein levels. Using protease inhibitors, the specific proteolytic pathway involved in degradation of p130 was examined. Treatment of cells with N-acetyl-leu-leu-norleucinal, an inhibitor that blocks proteolytic activity of type I calpain and the 26S proteasome, resulted in a complete block in the degradation of p130 protein, as well as adipocyte differentiation, suggesting that one of these pathways is involved in regulating p130 protein levels. Similar analysis with lactacystin, a specific inhibitor of the 26S proteasome, also resulted in a complete block in both differentiation and p130 degradation. Furthermore, both inhibitors blocked the increase in p107 protein levels normally observed on Day 1, suggesting that the p130:p107 switch is required for adipocyte differentiation and one of the early molecular events involved in activating the p130:p107 switch is the specific degradation of p130 by the 26S proteasome. © 2002 Elsevier Science (USA)
Key Words: adipocyte; differentiation; 3T3-L1 cells; p130; p107; 26S proteasome; lactacystin.
Obesity has been recognized for centuries as a significant health concern, however, recent studies have provided strong evidence that obesity serves as a serious risk factor for numerous diseases such as diabetes mellitus, coronary artery disease, arthritis and cancer (1, 2). Despite a wealth of recent knowledge concerning 1
To whom correspondence and reprint requests should be addressed at Department of Pediatrics, Division of Neonatology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 512-Neo, Little Rock, AR 72205. Fax: (501) 603-1425. E-mail: [email protected]. 0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
the molecular mechanisms involved in adipocyte differentiation (3– 6), critical steps involved in the proliferation and ultimate terminal differentiation of adipocytes, the major cellular component of adipose tissue, remain unclear. Established in vitro cellular models of adipocyte differentiation such as the 3T3-L1 and F442A preadipocyte cell lines have served a central role in increasing our understanding of the molecular regulation of adipogenesis. The ability of these preadipocyte cells to undergo complete differentiation into mature adipocytes under appropriate hormonal stimulation has been well described (reviewed in 3, 4). Upon reaching confluence, growing 3T3-L1 cells become contact inhibited and arrest at the G 1/S boundary (4 – 6). Following hormonal stimulation, the cells undergo an early stage of requisite mitotic clonal expansion during the first 24 h of differentiation, followed by permanent cell cycle withdrawal and subsequent terminal differentiation (4 – 6). It has been previously demonstrated that within the first 24 h (i.e., clonal expansion) of 3T3-L1 adipocyte differentiation, there is dramatic regulation in two members of the retinoblastoma family of proteins, p130 and p107 (5, 6). These proteins play a significant role in cell cycle through regulation of the E2F family of transcription factors (7–9). The alteration in expression levels of these two proteins during differentiation has been designated the p130:p107 switch (5, 6). In quiescent postconfluent Day 0 preadipocytes there are significant levels of p130, with little or no detectable levels of p107. During the first 24 h of hormonal induction (Day 1), the p130:p107 switch becomes activated by rapid changes in the levels of these two proteins. This switch is characterized by a significant decrease in the levels of p130 accompanied by a marked increase in p107 levels. Following clonal expansion (Days 2– 4), the switch resets, so that in quiescent Day 4 terminally differentiated adipocytes the levels of these proteins begin returning to levels similar to those observed at Day 0. Activation of the p130:p107 switch is directly correlated with the mitotic clonal expansion phase.
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Furthermore, disruption of the p130:p107 switch has been shown to both inhibit mitotic clonal expansion and terminal differentiation in 3T3-L1 cells (10). Interestingly, p130 and p107 protein levels appear to be regulated by different mechanisms (5, 6). The increase in p107 protein levels is accompanied by a similar increase in its mRNA level, suggesting a transcriptional mechanism. However, p130 mRNA levels are constitutively expressed throughout differentiation in the face of significant changes in its protein level, suggesting regulation through posttranscriptional mechanism(s). Based on these observations, we were interested in investigating the mechanism(s) regulating the dramatic and transient decrease in p130 protein levels during early 3T3-L1 adipocyte differentiation. A likely posttranscriptional mechanism for the decrease in p130 protein levels is degradation of the p130 protein. Recently, in an elegant set of experiments, it was demonstrated that calpain plays a role during 3T3-L1 adipocyte differentiation (11). These experiments demonstrated that calpain was involved in the transcriptional activation of CCAAT/enhancer binding protein ␣, a transcription factor required for terminal differentiation of adipocytes (11). These results were in part elucidated through the use of two protease inhibitors, N-acetyl-Leu-Leu-norleucinal (ALLN) which inhibits both calpain and the 26S proteasome, and calpastatin, a calpain-specific inhibitor. Additional evidence demonstrated that the inhibitors had to be administered early (within the first 24 h) in the differentiation process to effectively arrest adipocyte differentiation (11). Proteolysis plays an important role in cell cycle and differentiation in numerous tissues. Calpain has been shown to be involved in the differentiation of numerous cells, including myocytes and osteoblasts (12, 13). Many regulatory cell cycle proteins are mediated through proteolysis, for example, cyclins A, E, and B1 levels are regulated by the 26S proteasomal pathway and cyclin D1 has recently been shown to be regulated by calpain (14 –17). This evidence, coupled with the observation that the p130:p107 switch and cell cycle reentry is required for 3T3-L1 adipocyte differentiation, suggest that either calpain and/or the 26S proteasomal protease system may play a role in the posttranscriptional mechanism of p130 degradation. In the current study, utilizing both specific and nonspecific protease inhibitors, we provide evidence that the degradation of p130 is predominantly mediated by the 26S proteasome. METHODS Cell culture, adipocyte differentiation, and protease inhibitor treatment. 3T3-L1 cells (American Type Culture Collection, Rockville, MD) were grown to confluence in standard growth media consisting of Dulbecco’s modified Eagle medium (DMEM) supplemented with
10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin (each from Gibco BRL, Gaithersburg, MD) at 37°C in a 5% CO 2 atmosphere. Postconfluent cells were induced to differentiate by treatment with 1.7 M insulin, 0.5 M dexamethasone and 0.5 mM isobutylmethylxanthine [collectively designated IDX (each from Sigma, St. Louis, MO)] as previously described (3, 18). Briefly, confluent preadipocytes (Day 0) were treated with standard growth media described above supplemented with IDX for 3 days. After 3 days, the media was replaced by media supplemented with 1.7 M insulin only. Typically by day 4, ⬎95% of the cells had differentiated into adipocytes as determined by Oil Red O detectable lipid accumulation as described below. For inhibitor experiments, cells were stimulated to differentiate as described above, in the presence or absence of 26 M N-acetyl-Leu-Leu-norleucinal [ALLN (Sigma, St. Louis, MO)] resuspended in 100% ethanol (control cells were treated with an equal volume of 100% ethanol). Additional cells were treated with 5 M clasto-lactacystin -lactone [lactacystin (Calbiochem, La Jolla, CA)] resuspended in dimethyl sulfoxide [DMSO (controls cells were treated with DMSO alone)]. Oil Red O detection of lipid accumulation. Adipocyte differentiation was typically monitored by staining cell cultures with Oil Red O as previously described (10). Briefly, cells were washed gently with PBS, fixed in 10% formalin, washed in 50% isopropyl alcohol, and stained 10 min in a 60% (v/v) saturated Oil Red O/H 2O solution. Plates were rewashed in 50% isopropyl alcohol, counterstained with Mayer’s hematoxylin, overlayed with glycerin jelly and photographed. Whole cell extracts and Western blots. 3T3-L1 cells in 60-mm culture dishes were rinsed 3 times with ice cold PBS, scraped into 0.5 ml of PBS and microcentrifuged at 14,000 rpm for 1 min. Cell pellet was resuspended in 0.5 ml ice cold RIPA buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P 40, 0.5% deoxycholate, 0.1% SDS, 5 mM NaF, and 0.1 mg/ml PMSF). Following shearing through a 20 gauge needle, extracts were incubated on ice 30 min and subsequently centrifuged at 14,000 rpm at 4°C for 20 min. Whole cell extracts were quantitated by the Bio-Rad protein Assay (Bio-Rad Laboratories, Hercules, CA) and stored at ⫺80°C. Western blot analysis was performed on 50 g whole cell extracts prepared from differentiating cells at the indicated times as previously described (19, 20). Blots were incubated with a 1:200 dilution of the primary antibody and the secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Bio-Rad, Richmond, CA) diluted 1:2000. Immune complexes were detected by chemiluminescence with the CDP-Star system (NEN Life Sciences, Boston, MA). Primary antibodies used included anti-p107 #sc-318 and anti-p130 #sc-317 (Santa Cruz Biotechnology, Inc.).
RESULTS Effects of N-Acetyl-Leu-Leu-Norleucinal (ALLN) on p130/p107 Expression and Adipocyte Differentiation Previous results indicated that inhibition of adipocyte differentiation by ALLN was only observed if ALLN was administered during the first 24 h of hormonal stimulation, a time coincident with activation of the p130:p107 switch and clonal expansion (11). To determine if ALLN treatment disrupted the normal expression pattern of p130 and p107 protein levels during adipocyte differentiation, Day 0 3T3-L1 preadipocytes were stimulated to differentiate with insulin, dexamethasone and isobutylmethylxanthine (collectively designated IDX) in the presence or absence of ALLN as described under Methods. Whole cell extracts
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FIG. 1. Effects of N-acetyl-Leu-Leu-norleucinal (ALLN) on p130/ p107 expression and adipocyte differentiation. (A) Whole cell extracts were prepared from 3T3-L1 cells at days 0, 1, and 2 of differentiation in the presence or absence of 26 M ALLN as indicated. Fifty micrograms of each cellular extract were separated by SDS– polyacrylamide gel electrophoresis, transferred to nitrocellulose, and analyzed by Western blot as described under Methods. Molecular weights (not shown) for p130/p107 were 130/107 kDa, respectively. (B) ALLN-induced inhibition of lipid accumulation on Day 4 of 3T3-L1 adipocyte differentiation. Photomicrographs of Day 4 3T3-L1 cells fixed and stained with Oil-Red-O and counterstained with Mayer’s hemotoxylin (magnification ⫽ 200⫻). Cells were treated for 4 days with either unsupplemented media (left) or media supplemented with standard IDX hormonal differentiation cocktail in the absence (center) or presence of ALLN (right).
were prepared from cells and the level of p107 and p130 proteins determined by Western blotting (Fig. 1A). The first three lanes demonstrate in IDX-only treated cells that activation of the p130:p107 switch is intact with high levels of p130 and barely detectable levels of p107 protein in Day 0 preadipocytes. On Day 1, the switch is activated, evidenced by the fall in p130 expression as well as the increase in p107 expression (compare lanes 1 and 2, Fig. 1A). However, in Day 1 cells treated with IDX and 26 M ALLN, the levels of both p130 and p107 are virtually unchanged from those observed at Day 0, i.e., the p130:p107 switch was blocked (compare lanes 1 and 4, Fig. 1A). By Day 2 in IDX and ALLN-treated cells, there is a small amount of p107 evident along with a slight reduction in p130, potentially due to degradation or decreased stability of the ALLN peptide. On Day 4, cells were fixed and
stained with Oil Red O to assess cytoplasmic triglyceride accumulation, a well-described marker for differentiation in the 3T3-L1 cell model. As expected, Day 4 IDX-only treated cells completely differentiated as assessed by Oil Red O (Fig. 1B, center panel). In agreement with published results (11), Day 4 cells treated with IDX and ALLN failed to differentiate and were virtually indistinguishable from nontreated cells (compare right panel to left panel, Fig. 1B). The inhibitory effect of ALLN on adipocyte differentiation was demonstrated to be reversible, in that cells whose differentiation was arrested by treatment with ALLN were fully capable of complete differentiation when reinduced with IDX (11). The reversible nature of the ALLN effect was interesting, because if the p130: p107 switch is critical to differentiation, then the effects of ALLN on the p130:p107 switch should be reversible as well. To examine this, additional groups of cells were treated either with IDX, IDX and 26 M ALLN, along with another group that were reinduced with IDX after having been subjected to four days of treatment with IDX and ALLN (Fig. 2). Consistent with results described above, the p130:p107 switch is activated in the presence of IDX, an effect that is blocked in the presence of ALLN (Fig. 2, lanes 1–3). However, IDX-only treatment in a group of cells that were pretreated for four days with ALLN resulted in full activation of the switch (Fig. 2, lane 4). As an additional control, reinduction of a group of cells in the continued presence of ALLN resulted in a continued
FIG. 2. Reversible effects of ALLN on p130/p107 protein expression. Whole cell extracts were prepared from 3T3-L1 cells at days 0 and 1 of differentiation and 50 g of each cellular extract was separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose, and analyzed by Western blot as described under Methods. Molecular weights (not shown) for p130/p107 were 130/107 kDa, respectively. One group of cells were treated with either IDX alone or IDX ⫹ 26 M ALLN (lanes 2 and 3). An additional group of cells were treated with IDX and 26 M ALLN for 4 days and subsequently reinduced with either IDX alone or IDX ⫹ 26 M ALLN (last two lanes).
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block in activation of the p130:p107 switch (Fig. 2, lane 5). These results strongly suggest that one of the mechanism(s) of action by which ALLN inhibits adipocyte differentiation occurs by preventing activation of the p130:p107 switch. In the presence of ALLN, the switch is blocked and differentiation fails to proceed, if however, ALLN is removed, the switch in the expression pattern of these two proteins occurs and differentiation proceeds. Effects of Lactacystin on p130/p107 Expression and Adipocyte Differentiation The inhibitory effects of ALLN exerted on both adipocyte differentiation and the p130:p107 switch were dramatic. However, it is important to note that ALLN is not a specific inhibitor of the calpain protease system, but has potent inhibitory effects on proteolysis mediated through the 26S proteasome system as well (21). Therefore, to determine which of these pathways are involved in the specific degradation of p130, the effects of lactacystin, a specific inhibitor of the 26S proteasome (22, 23), were tested. 3T3-L1 preadipocytes were stimulated to differentiate with IDX in the presence or absence of 5 M lactacystin. Whole cell extracts were prepared from treated and/or differentiating cells and the effects of lactacystin on the p130:p107 switch were examined by Western blot. Lactacystin completely blocked the degradation of p130 protein typically associated with Day 1 of differentiation (Fig. 3A, compare Day 1 lanes, 2 and 5). In contrast, lactacystin inhibited upregulation of p107, but not nearly as completely as ALLN (compare results to Figs. 1A and 2). It appears that lactacystin delayed the maximal induction of p107 protein levels by 24 h, because by Day 2, levels of p107 protein are as high as Day 1 levels in IDX-only treated controls, suggesting that both the calpain and the 26 proteasome may be involved in regulating the p107 arm of the p130:p107 switch. Not only did lactacystin block the degradation of p130 protein, it also completely inhibited adipocyte differentiation as assessed by Oil Red O lipid accumulation (Fig. 3B). These results provide strong evidence that p130 proteolytic degradation is mediated by the 26S proteasome and not by calpain, and further establishes that activation of the p130:p107 switch is one the early events during 3T3-L1 adipogenesis. DISCUSSION Proteolysis continues to gain strength as an important mediator of cellular differentiation in many different cell types (12–17). The current study provides further evidence that proteolysis serves an important role in the differentiation of adipocytes. The calciumdependent protease, calpain, was the first proteolytic system to be functionally implicated in the regulation
FIG. 3. Effects of clasto-lactacystin -lactone (lactacytin) on p130/p107 expression and adipocyte differentiation. (A) Whole cell extracts were prepared from 3T3-L1 cells at days 0, 1, 2, and 3 of differentiation in the presence or absence of 5 M lactacystin as indicated. Fifty micrograms of each cellular extract were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose, and analyzed by Western blot as described under Methods. Molecular weights (not shown) for p130/p107 were 130/107 kDa, respectively. (B) Lactacystin-induced inhibition of lipid accumulation on Day 4 of 3T3-L1 adipocyte differentiation. Photomicrographs of Day 4 3T3-L1 cells fixed and stained with Oil-Red-O and counterstained with Mayer’s hemotoxylin (magnification ⫽ 200⫻). Cells were treated for 4 days with either unsupplemented media (left) or media supplemented with standard IDX hormonal differentiation cocktail in the absence (center) or presence of lactacystin (right).
of adipocyte differentiation (11). These investigators demonstrated that hormonally induced 3T3-L1 adipocyte differentiation in the presence of both specific and nonspecific inhibitors completely blocked differentiation. In addition, the same study revealed that the inhibitors blocked transcriptional activation of the CCAAT/enhancer binding protein ␣ (C/EPB␣) gene, that encodes the well described transcription factor required for terminal adipocyte differentiation. The inhibitors used in that study included ALLN (a nonspecific protease inhibitor) and calpastatin, a calpain-specific inhibitor. The inhibitory effects of ALLN were duplicated with calpastatin, and thus provided strong evidence for the role of calpain in adipocyte differentiation. Based on the calpain work, it was anticipated that the effects of ALLN on the p130:p107 switch would also be calpain-mediated. The degradation of p130 on Day 1 is so dramatic, it was anticipated that an inducible system would be responsible for it. However, calpain
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activity assays using in vitro fluorescent microscopy on Days 0 and 1 revealed that not only was the activity not inducible, but the amount of activity at Day 1 was slightly less than that observed at Day 0 (data not shown). This result was in agreement with the previous study that demonstrated the levels of both calpain protein and mRNA were not inducible, but also fell during the same time period (11). It was this observation that initially led us to speculate that a proteolytic system other than calpain may be involved in p130 degradation. If another system was potentially involved in regulating p130 degradation, the most likely candidate became the 26S proteasome because ALLN is known to inhibit both calpain and the 26S proteasome (24). The availability of lactacystin, a protease inhibitor specific to the 26S proteasome, allowed a straightforward approach to investigate involvement of the 26S proteasome. Lactacystin inhibits proteasome activity through covalent modification of a highly conserved aminoterminal threonine. It was a logical inhibitor for our study in that it has been shown to completely inhibit the proteasome without any significant effects on calpain (23). The results from these experiments provide strong evidence that p130 degradation is mediated by the 26S proteasome and not calpain during 3T3-L1 adipocyte differentiation. The 26S proteasome is a predominant mechanism reported in the degradation of many proteins in eukaryotic cells (23, 25, 26). Many regulatory proteins have been demonstrated as substrates for the 26S proteasome including mitotic G 1 and S phase cyclins, c-Jun, IB␣, and p53 (26). The degradation of regulatory proteins has since generated increased interest in characterizing the structure and function of the 26S proteasome. The 26S proteasome complex consists of a core 20S subunit and two 19S regulatory subunits. The core 20S subunit includes a threonine active site where peptidase activity occurs, and the 19S subunits contain the substrate binding and ATPase domains (23, 27). Most protein substrates for the proteasome are targeted to the enzyme complex by the attachment of multiple ubiquitin chains. The 19S subunits normally recognize the ubiquitinated substrates and translocate them to the 20S core unit where breakdown of the protein occurs. Despite numerous attempts in the current study to immunoprecipitate ubiquitinated p130, either through p130 immunoprecipitations followed by ubiquitin Western blots or the reciprocal experiments, we were never able to detect polyubiquitinated forms of p130 (data not shown). Initially, this was puzzling but certainly not unprecedented, as ubiquitin-independent targeting to the proteasome has now been demonstrated for several proteins. Ornithine decarboxylase was one of the first examples of a protein shown to be degraded by the 26S proteasome in a ubiquitinindependent fashion (26, 28, 29). Since then, other proteins have been added to the list of those degraded
by the 26S proteasome in an ubiquitin-independent manner including c-Jun and IB␣ (26, 30, 31). While not conclusive, the inability to detect ubiquitinated forms of p130, combined with its protection from degradation in the presence of lactacystin, suggest that it may be targeted to the 26S proteasome through similar ubiquitin-independent process(es). Further investigations will be necessary to conclusively determine if this is the case. Lactacystin is an irreversible inhibitor that covalently modifies the core 20S subunit of the 26S proteasome (23). Previously, we had demonstrated that tumor necrosis factor-␣ permanently disrupted the p130:p107 switch leading to a complete inhibition of differentiation (10). An interesting result of the ALLN experiments was that its effects on adipocyte differentiation were reversible when removed and the cells were reinduced to differentiate. The effects of ALLN on the p130:p107 switch were also reversible, in that the inhibitor did not necessarily disrupt the switch, but it simply arrested its activation. Subsequent removal of the inhibitor, followed by reinduction of differentiation with IDX led to normal activation of the switch and progression to terminal adipocyte differentiation. This result is exciting and provides further evidence for the hypothesis that the p130:p107 switch is required for 3T3-L1 adipocyte differentiation, i.e., if activation of the switch is blocked, differentiation can not proceed, however, if the switch is activated, differentiation proceeds. These experiments also provided us with a little more insight into the molecular mechanisms that regulate the p130:p107 switch, especially the p107 arm. Normally on Day 1 of differentiation there is a significant increase in p107 protein levels. This increase is completely blocked in the presence of ALLN. However, in the presence of lactacystin, the increase in p107 protein on Day 1 is only partially blocked. Through another line of investigation we have initiated mapping experiments on the p107 promoter and have preliminary evidence (not shown) that p130 inhibits transcriptional activation of the p107 gene. We have hypothesized that the degradation of p130 on Day 1 of differentiation removes this inhibition allowing transcriptional activation of the p107 gene. These current protease inhibitor experiments reveal that other mechanisms are also involved in regulating p107, because when the degradation of p130 is completely blocked by lactacystin, there is still some activation of p107 expression. However, when both the 26S proteasome and the calpain protease are inhibited with ALLN, expression of p107 is completely blocked. These results have been highly consistent throughout numerous repetitions, and suggest that both protease systems may be involved in regulating p107. Calpain has already been demonstrated to be involved in the upregulation of C/EBP␣ (11), and it should be interesting to determine how it is involved in p107 regulation as well.
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Currently, the regulation and role of the p130:p107 switch in 3T3-L1 adipocyte differentiation is largely unknown. In all cases where it has been examined, blocking or perturbing the expression of these two proteins (known to be important in cell cycle and differentiation), have led to a block in adipocyte differentiation in the 3T3-L1 model (10, and the current study). Furthermore, the transient switch in expression of these proteins in this cell line are specific to the differentiation process, because they are regulated differently when simply proliferating (5). Continued investigations into the mechanisms of regulation of these proteins should provide an increased understanding of the early molecular events associated with adipocyte differentiation.
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14. 15. 16.
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ACKNOWLEDGMENTS This work was supported by research Grant CA78845 from the National Cancer Institute (R.E.M.) and a University of Arkansas for Medical Sciences and Arkansas Children’s Hospital institutional pilot grant (A.M.P.).
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Proteasomal Degradation of Retinoblastoma-Related p130 during Adipocyte Differentiation Audra M. Prince, Julie S. May, Gregory R. Burton, Robert E. Lyle, and Robert E. McGehee, Jr. 1 Department of Pediatrics, Division of Neonatology, University of Arkansas for Medical Sciences and Arkansas Children’s Hospital, Little Rock, Arkansas 72205
Received December 12, 2001
Within 24 h of hormonally stimulated 3T3-L1 adipocyte differentiation, there are dramatic changes in the protein levels of p130 and p107, two members of the retinoblastoma tumor suppressor gene family. Designated the “p103:p107” switch, this alteration is characterized by a rapid and transient drop in p130 protein levels accompanied by a transient increase in both p107 mRNA and protein levels. Using protease inhibitors, the specific proteolytic pathway involved in degradation of p130 was examined. Treatment of cells with N-acetyl-leu-leu-norleucinal, an inhibitor that blocks proteolytic activity of type I calpain and the 26S proteasome, resulted in a complete block in the degradation of p130 protein, as well as adipocyte differentiation, suggesting that one of these pathways is involved in regulating p130 protein levels. Similar analysis with lactacystin, a specific inhibitor of the 26S proteasome, also resulted in a complete block in both differentiation and p130 degradation. Furthermore, both inhibitors blocked the increase in p107 protein levels normally observed on Day 1, suggesting that the p130:p107 switch is required for adipocyte differentiation and one of the early molecular events involved in activating the p130:p107 switch is the specific degradation of p130 by the 26S proteasome. © 2002 Elsevier Science (USA)
Key Words: adipocyte; differentiation; 3T3-L1 cells; p130; p107; 26S proteasome; lactacystin.
Obesity has been recognized for centuries as a significant health concern, however, recent studies have provided strong evidence that obesity serves as a serious risk factor for numerous diseases such as diabetes mellitus, coronary artery disease, arthritis and cancer (1, 2). Despite a wealth of recent knowledge concerning 1
To whom correspondence and reprint requests should be addressed at Department of Pediatrics, Division of Neonatology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 512-Neo, Little Rock, AR 72205. Fax: (501) 603-1425. E-mail: [email protected]. 0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
the molecular mechanisms involved in adipocyte differentiation (3– 6), critical steps involved in the proliferation and ultimate terminal differentiation of adipocytes, the major cellular component of adipose tissue, remain unclear. Established in vitro cellular models of adipocyte differentiation such as the 3T3-L1 and F442A preadipocyte cell lines have served a central role in increasing our understanding of the molecular regulation of adipogenesis. The ability of these preadipocyte cells to undergo complete differentiation into mature adipocytes under appropriate hormonal stimulation has been well described (reviewed in 3, 4). Upon reaching confluence, growing 3T3-L1 cells become contact inhibited and arrest at the G 1/S boundary (4 – 6). Following hormonal stimulation, the cells undergo an early stage of requisite mitotic clonal expansion during the first 24 h of differentiation, followed by permanent cell cycle withdrawal and subsequent terminal differentiation (4 – 6). It has been previously demonstrated that within the first 24 h (i.e., clonal expansion) of 3T3-L1 adipocyte differentiation, there is dramatic regulation in two members of the retinoblastoma family of proteins, p130 and p107 (5, 6). These proteins play a significant role in cell cycle through regulation of the E2F family of transcription factors (7–9). The alteration in expression levels of these two proteins during differentiation has been designated the p130:p107 switch (5, 6). In quiescent postconfluent Day 0 preadipocytes there are significant levels of p130, with little or no detectable levels of p107. During the first 24 h of hormonal induction (Day 1), the p130:p107 switch becomes activated by rapid changes in the levels of these two proteins. This switch is characterized by a significant decrease in the levels of p130 accompanied by a marked increase in p107 levels. Following clonal expansion (Days 2– 4), the switch resets, so that in quiescent Day 4 terminally differentiated adipocytes the levels of these proteins begin returning to levels similar to those observed at Day 0. Activation of the p130:p107 switch is directly correlated with the mitotic clonal expansion phase.
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Furthermore, disruption of the p130:p107 switch has been shown to both inhibit mitotic clonal expansion and terminal differentiation in 3T3-L1 cells (10). Interestingly, p130 and p107 protein levels appear to be regulated by different mechanisms (5, 6). The increase in p107 protein levels is accompanied by a similar increase in its mRNA level, suggesting a transcriptional mechanism. However, p130 mRNA levels are constitutively expressed throughout differentiation in the face of significant changes in its protein level, suggesting regulation through posttranscriptional mechanism(s). Based on these observations, we were interested in investigating the mechanism(s) regulating the dramatic and transient decrease in p130 protein levels during early 3T3-L1 adipocyte differentiation. A likely posttranscriptional mechanism for the decrease in p130 protein levels is degradation of the p130 protein. Recently, in an elegant set of experiments, it was demonstrated that calpain plays a role during 3T3-L1 adipocyte differentiation (11). These experiments demonstrated that calpain was involved in the transcriptional activation of CCAAT/enhancer binding protein ␣, a transcription factor required for terminal differentiation of adipocytes (11). These results were in part elucidated through the use of two protease inhibitors, N-acetyl-Leu-Leu-norleucinal (ALLN) which inhibits both calpain and the 26S proteasome, and calpastatin, a calpain-specific inhibitor. Additional evidence demonstrated that the inhibitors had to be administered early (within the first 24 h) in the differentiation process to effectively arrest adipocyte differentiation (11). Proteolysis plays an important role in cell cycle and differentiation in numerous tissues. Calpain has been shown to be involved in the differentiation of numerous cells, including myocytes and osteoblasts (12, 13). Many regulatory cell cycle proteins are mediated through proteolysis, for example, cyclins A, E, and B1 levels are regulated by the 26S proteasomal pathway and cyclin D1 has recently been shown to be regulated by calpain (14 –17). This evidence, coupled with the observation that the p130:p107 switch and cell cycle reentry is required for 3T3-L1 adipocyte differentiation, suggest that either calpain and/or the 26S proteasomal protease system may play a role in the posttranscriptional mechanism of p130 degradation. In the current study, utilizing both specific and nonspecific protease inhibitors, we provide evidence that the degradation of p130 is predominantly mediated by the 26S proteasome. METHODS Cell culture, adipocyte differentiation, and protease inhibitor treatment. 3T3-L1 cells (American Type Culture Collection, Rockville, MD) were grown to confluence in standard growth media consisting of Dulbecco’s modified Eagle medium (DMEM) supplemented with
10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin (each from Gibco BRL, Gaithersburg, MD) at 37°C in a 5% CO 2 atmosphere. Postconfluent cells were induced to differentiate by treatment with 1.7 M insulin, 0.5 M dexamethasone and 0.5 mM isobutylmethylxanthine [collectively designated IDX (each from Sigma, St. Louis, MO)] as previously described (3, 18). Briefly, confluent preadipocytes (Day 0) were treated with standard growth media described above supplemented with IDX for 3 days. After 3 days, the media was replaced by media supplemented with 1.7 M insulin only. Typically by day 4, ⬎95% of the cells had differentiated into adipocytes as determined by Oil Red O detectable lipid accumulation as described below. For inhibitor experiments, cells were stimulated to differentiate as described above, in the presence or absence of 26 M N-acetyl-Leu-Leu-norleucinal [ALLN (Sigma, St. Louis, MO)] resuspended in 100% ethanol (control cells were treated with an equal volume of 100% ethanol). Additional cells were treated with 5 M clasto-lactacystin -lactone [lactacystin (Calbiochem, La Jolla, CA)] resuspended in dimethyl sulfoxide [DMSO (controls cells were treated with DMSO alone)]. Oil Red O detection of lipid accumulation. Adipocyte differentiation was typically monitored by staining cell cultures with Oil Red O as previously described (10). Briefly, cells were washed gently with PBS, fixed in 10% formalin, washed in 50% isopropyl alcohol, and stained 10 min in a 60% (v/v) saturated Oil Red O/H 2O solution. Plates were rewashed in 50% isopropyl alcohol, counterstained with Mayer’s hematoxylin, overlayed with glycerin jelly and photographed. Whole cell extracts and Western blots. 3T3-L1 cells in 60-mm culture dishes were rinsed 3 times with ice cold PBS, scraped into 0.5 ml of PBS and microcentrifuged at 14,000 rpm for 1 min. Cell pellet was resuspended in 0.5 ml ice cold RIPA buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P 40, 0.5% deoxycholate, 0.1% SDS, 5 mM NaF, and 0.1 mg/ml PMSF). Following shearing through a 20 gauge needle, extracts were incubated on ice 30 min and subsequently centrifuged at 14,000 rpm at 4°C for 20 min. Whole cell extracts were quantitated by the Bio-Rad protein Assay (Bio-Rad Laboratories, Hercules, CA) and stored at ⫺80°C. Western blot analysis was performed on 50 g whole cell extracts prepared from differentiating cells at the indicated times as previously described (19, 20). Blots were incubated with a 1:200 dilution of the primary antibody and the secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Bio-Rad, Richmond, CA) diluted 1:2000. Immune complexes were detected by chemiluminescence with the CDP-Star system (NEN Life Sciences, Boston, MA). Primary antibodies used included anti-p107 #sc-318 and anti-p130 #sc-317 (Santa Cruz Biotechnology, Inc.).
RESULTS Effects of N-Acetyl-Leu-Leu-Norleucinal (ALLN) on p130/p107 Expression and Adipocyte Differentiation Previous results indicated that inhibition of adipocyte differentiation by ALLN was only observed if ALLN was administered during the first 24 h of hormonal stimulation, a time coincident with activation of the p130:p107 switch and clonal expansion (11). To determine if ALLN treatment disrupted the normal expression pattern of p130 and p107 protein levels during adipocyte differentiation, Day 0 3T3-L1 preadipocytes were stimulated to differentiate with insulin, dexamethasone and isobutylmethylxanthine (collectively designated IDX) in the presence or absence of ALLN as described under Methods. Whole cell extracts
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FIG. 1. Effects of N-acetyl-Leu-Leu-norleucinal (ALLN) on p130/ p107 expression and adipocyte differentiation. (A) Whole cell extracts were prepared from 3T3-L1 cells at days 0, 1, and 2 of differentiation in the presence or absence of 26 M ALLN as indicated. Fifty micrograms of each cellular extract were separated by SDS– polyacrylamide gel electrophoresis, transferred to nitrocellulose, and analyzed by Western blot as described under Methods. Molecular weights (not shown) for p130/p107 were 130/107 kDa, respectively. (B) ALLN-induced inhibition of lipid accumulation on Day 4 of 3T3-L1 adipocyte differentiation. Photomicrographs of Day 4 3T3-L1 cells fixed and stained with Oil-Red-O and counterstained with Mayer’s hemotoxylin (magnification ⫽ 200⫻). Cells were treated for 4 days with either unsupplemented media (left) or media supplemented with standard IDX hormonal differentiation cocktail in the absence (center) or presence of ALLN (right).
were prepared from cells and the level of p107 and p130 proteins determined by Western blotting (Fig. 1A). The first three lanes demonstrate in IDX-only treated cells that activation of the p130:p107 switch is intact with high levels of p130 and barely detectable levels of p107 protein in Day 0 preadipocytes. On Day 1, the switch is activated, evidenced by the fall in p130 expression as well as the increase in p107 expression (compare lanes 1 and 2, Fig. 1A). However, in Day 1 cells treated with IDX and 26 M ALLN, the levels of both p130 and p107 are virtually unchanged from those observed at Day 0, i.e., the p130:p107 switch was blocked (compare lanes 1 and 4, Fig. 1A). By Day 2 in IDX and ALLN-treated cells, there is a small amount of p107 evident along with a slight reduction in p130, potentially due to degradation or decreased stability of the ALLN peptide. On Day 4, cells were fixed and
stained with Oil Red O to assess cytoplasmic triglyceride accumulation, a well-described marker for differentiation in the 3T3-L1 cell model. As expected, Day 4 IDX-only treated cells completely differentiated as assessed by Oil Red O (Fig. 1B, center panel). In agreement with published results (11), Day 4 cells treated with IDX and ALLN failed to differentiate and were virtually indistinguishable from nontreated cells (compare right panel to left panel, Fig. 1B). The inhibitory effect of ALLN on adipocyte differentiation was demonstrated to be reversible, in that cells whose differentiation was arrested by treatment with ALLN were fully capable of complete differentiation when reinduced with IDX (11). The reversible nature of the ALLN effect was interesting, because if the p130: p107 switch is critical to differentiation, then the effects of ALLN on the p130:p107 switch should be reversible as well. To examine this, additional groups of cells were treated either with IDX, IDX and 26 M ALLN, along with another group that were reinduced with IDX after having been subjected to four days of treatment with IDX and ALLN (Fig. 2). Consistent with results described above, the p130:p107 switch is activated in the presence of IDX, an effect that is blocked in the presence of ALLN (Fig. 2, lanes 1–3). However, IDX-only treatment in a group of cells that were pretreated for four days with ALLN resulted in full activation of the switch (Fig. 2, lane 4). As an additional control, reinduction of a group of cells in the continued presence of ALLN resulted in a continued
FIG. 2. Reversible effects of ALLN on p130/p107 protein expression. Whole cell extracts were prepared from 3T3-L1 cells at days 0 and 1 of differentiation and 50 g of each cellular extract was separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose, and analyzed by Western blot as described under Methods. Molecular weights (not shown) for p130/p107 were 130/107 kDa, respectively. One group of cells were treated with either IDX alone or IDX ⫹ 26 M ALLN (lanes 2 and 3). An additional group of cells were treated with IDX and 26 M ALLN for 4 days and subsequently reinduced with either IDX alone or IDX ⫹ 26 M ALLN (last two lanes).
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block in activation of the p130:p107 switch (Fig. 2, lane 5). These results strongly suggest that one of the mechanism(s) of action by which ALLN inhibits adipocyte differentiation occurs by preventing activation of the p130:p107 switch. In the presence of ALLN, the switch is blocked and differentiation fails to proceed, if however, ALLN is removed, the switch in the expression pattern of these two proteins occurs and differentiation proceeds. Effects of Lactacystin on p130/p107 Expression and Adipocyte Differentiation The inhibitory effects of ALLN exerted on both adipocyte differentiation and the p130:p107 switch were dramatic. However, it is important to note that ALLN is not a specific inhibitor of the calpain protease system, but has potent inhibitory effects on proteolysis mediated through the 26S proteasome system as well (21). Therefore, to determine which of these pathways are involved in the specific degradation of p130, the effects of lactacystin, a specific inhibitor of the 26S proteasome (22, 23), were tested. 3T3-L1 preadipocytes were stimulated to differentiate with IDX in the presence or absence of 5 M lactacystin. Whole cell extracts were prepared from treated and/or differentiating cells and the effects of lactacystin on the p130:p107 switch were examined by Western blot. Lactacystin completely blocked the degradation of p130 protein typically associated with Day 1 of differentiation (Fig. 3A, compare Day 1 lanes, 2 and 5). In contrast, lactacystin inhibited upregulation of p107, but not nearly as completely as ALLN (compare results to Figs. 1A and 2). It appears that lactacystin delayed the maximal induction of p107 protein levels by 24 h, because by Day 2, levels of p107 protein are as high as Day 1 levels in IDX-only treated controls, suggesting that both the calpain and the 26 proteasome may be involved in regulating the p107 arm of the p130:p107 switch. Not only did lactacystin block the degradation of p130 protein, it also completely inhibited adipocyte differentiation as assessed by Oil Red O lipid accumulation (Fig. 3B). These results provide strong evidence that p130 proteolytic degradation is mediated by the 26S proteasome and not by calpain, and further establishes that activation of the p130:p107 switch is one the early events during 3T3-L1 adipogenesis. DISCUSSION Proteolysis continues to gain strength as an important mediator of cellular differentiation in many different cell types (12–17). The current study provides further evidence that proteolysis serves an important role in the differentiation of adipocytes. The calciumdependent protease, calpain, was the first proteolytic system to be functionally implicated in the regulation
FIG. 3. Effects of clasto-lactacystin -lactone (lactacytin) on p130/p107 expression and adipocyte differentiation. (A) Whole cell extracts were prepared from 3T3-L1 cells at days 0, 1, 2, and 3 of differentiation in the presence or absence of 5 M lactacystin as indicated. Fifty micrograms of each cellular extract were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose, and analyzed by Western blot as described under Methods. Molecular weights (not shown) for p130/p107 were 130/107 kDa, respectively. (B) Lactacystin-induced inhibition of lipid accumulation on Day 4 of 3T3-L1 adipocyte differentiation. Photomicrographs of Day 4 3T3-L1 cells fixed and stained with Oil-Red-O and counterstained with Mayer’s hemotoxylin (magnification ⫽ 200⫻). Cells were treated for 4 days with either unsupplemented media (left) or media supplemented with standard IDX hormonal differentiation cocktail in the absence (center) or presence of lactacystin (right).
of adipocyte differentiation (11). These investigators demonstrated that hormonally induced 3T3-L1 adipocyte differentiation in the presence of both specific and nonspecific inhibitors completely blocked differentiation. In addition, the same study revealed that the inhibitors blocked transcriptional activation of the CCAAT/enhancer binding protein ␣ (C/EPB␣) gene, that encodes the well described transcription factor required for terminal adipocyte differentiation. The inhibitors used in that study included ALLN (a nonspecific protease inhibitor) and calpastatin, a calpain-specific inhibitor. The inhibitory effects of ALLN were duplicated with calpastatin, and thus provided strong evidence for the role of calpain in adipocyte differentiation. Based on the calpain work, it was anticipated that the effects of ALLN on the p130:p107 switch would also be calpain-mediated. The degradation of p130 on Day 1 is so dramatic, it was anticipated that an inducible system would be responsible for it. However, calpain
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activity assays using in vitro fluorescent microscopy on Days 0 and 1 revealed that not only was the activity not inducible, but the amount of activity at Day 1 was slightly less than that observed at Day 0 (data not shown). This result was in agreement with the previous study that demonstrated the levels of both calpain protein and mRNA were not inducible, but also fell during the same time period (11). It was this observation that initially led us to speculate that a proteolytic system other than calpain may be involved in p130 degradation. If another system was potentially involved in regulating p130 degradation, the most likely candidate became the 26S proteasome because ALLN is known to inhibit both calpain and the 26S proteasome (24). The availability of lactacystin, a protease inhibitor specific to the 26S proteasome, allowed a straightforward approach to investigate involvement of the 26S proteasome. Lactacystin inhibits proteasome activity through covalent modification of a highly conserved aminoterminal threonine. It was a logical inhibitor for our study in that it has been shown to completely inhibit the proteasome without any significant effects on calpain (23). The results from these experiments provide strong evidence that p130 degradation is mediated by the 26S proteasome and not calpain during 3T3-L1 adipocyte differentiation. The 26S proteasome is a predominant mechanism reported in the degradation of many proteins in eukaryotic cells (23, 25, 26). Many regulatory proteins have been demonstrated as substrates for the 26S proteasome including mitotic G 1 and S phase cyclins, c-Jun, IB␣, and p53 (26). The degradation of regulatory proteins has since generated increased interest in characterizing the structure and function of the 26S proteasome. The 26S proteasome complex consists of a core 20S subunit and two 19S regulatory subunits. The core 20S subunit includes a threonine active site where peptidase activity occurs, and the 19S subunits contain the substrate binding and ATPase domains (23, 27). Most protein substrates for the proteasome are targeted to the enzyme complex by the attachment of multiple ubiquitin chains. The 19S subunits normally recognize the ubiquitinated substrates and translocate them to the 20S core unit where breakdown of the protein occurs. Despite numerous attempts in the current study to immunoprecipitate ubiquitinated p130, either through p130 immunoprecipitations followed by ubiquitin Western blots or the reciprocal experiments, we were never able to detect polyubiquitinated forms of p130 (data not shown). Initially, this was puzzling but certainly not unprecedented, as ubiquitin-independent targeting to the proteasome has now been demonstrated for several proteins. Ornithine decarboxylase was one of the first examples of a protein shown to be degraded by the 26S proteasome in a ubiquitinindependent fashion (26, 28, 29). Since then, other proteins have been added to the list of those degraded
by the 26S proteasome in an ubiquitin-independent manner including c-Jun and IB␣ (26, 30, 31). While not conclusive, the inability to detect ubiquitinated forms of p130, combined with its protection from degradation in the presence of lactacystin, suggest that it may be targeted to the 26S proteasome through similar ubiquitin-independent process(es). Further investigations will be necessary to conclusively determine if this is the case. Lactacystin is an irreversible inhibitor that covalently modifies the core 20S subunit of the 26S proteasome (23). Previously, we had demonstrated that tumor necrosis factor-␣ permanently disrupted the p130:p107 switch leading to a complete inhibition of differentiation (10). An interesting result of the ALLN experiments was that its effects on adipocyte differentiation were reversible when removed and the cells were reinduced to differentiate. The effects of ALLN on the p130:p107 switch were also reversible, in that the inhibitor did not necessarily disrupt the switch, but it simply arrested its activation. Subsequent removal of the inhibitor, followed by reinduction of differentiation with IDX led to normal activation of the switch and progression to terminal adipocyte differentiation. This result is exciting and provides further evidence for the hypothesis that the p130:p107 switch is required for 3T3-L1 adipocyte differentiation, i.e., if activation of the switch is blocked, differentiation can not proceed, however, if the switch is activated, differentiation proceeds. These experiments also provided us with a little more insight into the molecular mechanisms that regulate the p130:p107 switch, especially the p107 arm. Normally on Day 1 of differentiation there is a significant increase in p107 protein levels. This increase is completely blocked in the presence of ALLN. However, in the presence of lactacystin, the increase in p107 protein on Day 1 is only partially blocked. Through another line of investigation we have initiated mapping experiments on the p107 promoter and have preliminary evidence (not shown) that p130 inhibits transcriptional activation of the p107 gene. We have hypothesized that the degradation of p130 on Day 1 of differentiation removes this inhibition allowing transcriptional activation of the p107 gene. These current protease inhibitor experiments reveal that other mechanisms are also involved in regulating p107, because when the degradation of p130 is completely blocked by lactacystin, there is still some activation of p107 expression. However, when both the 26S proteasome and the calpain protease are inhibited with ALLN, expression of p107 is completely blocked. These results have been highly consistent throughout numerous repetitions, and suggest that both protease systems may be involved in regulating p107. Calpain has already been demonstrated to be involved in the upregulation of C/EBP␣ (11), and it should be interesting to determine how it is involved in p107 regulation as well.
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Currently, the regulation and role of the p130:p107 switch in 3T3-L1 adipocyte differentiation is largely unknown. In all cases where it has been examined, blocking or perturbing the expression of these two proteins (known to be important in cell cycle and differentiation), have led to a block in adipocyte differentiation in the 3T3-L1 model (10, and the current study). Furthermore, the transient switch in expression of these proteins in this cell line are specific to the differentiation process, because they are regulated differently when simply proliferating (5). Continued investigations into the mechanisms of regulation of these proteins should provide an increased understanding of the early molecular events associated with adipocyte differentiation.
13.
14. 15. 16.
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ACKNOWLEDGMENTS This work was supported by research Grant CA78845 from the National Cancer Institute (R.E.M.) and a University of Arkansas for Medical Sciences and Arkansas Children’s Hospital institutional pilot grant (A.M.P.).
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