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Proc. Natl. Acad. Sci. USA Vol. 93, pp. 1967-1971, March 1996 Physiology

Protein degradation and increased mRNAs encoding proteins of the ubiquitin-proteasome proteolytic pathway in BC3H1 myocytes require an interaction between glucocorticoids and acidification (skeletal muscle/proteolysis/gene expression/metabolic acidosis)

YUKA ISOZAKI, WILLIAM E. MITCH, BRIAN K. ENGLAND,

AND

S. Russ PRICE

Renal Division, Emory University School of Medicine, Atlanta, GA 30322

Communicated by Vernon R. Young, Massachusetts Institute of Technology, Cambridge, MA, December 1, 1995 (received for review June 25, 1995)

units (8-10). Metabolic acidosis increases not only ATPdependent proteolysis in muscle but also the levels of mRNAs encoding proteins of the ATP-dependent ubiquitin-proteasome pathway (7). A potential mediator of muscle proteolysis in acidosis is glucocorticoids since we found that proteolysis was not stimulated in muscles of adrenalectomized rats unless they were given glucocorticoids (4). Interpretation of these results is complicated because other factors that could regulate protein turnover and gene expression, including insulin (11), prostaglandins (12), and cytokines (13-16), could change in adrenalectomized rats. We studied cultured BC3H1 myocytes to identify relationships among acidification, glucocorticoids, proteolysis, and the ubiquitin-proteasome pathway without the complexities present in adrenalectomized rats. These cells were studied because they do not rapidly acidify the medium unlike C2, C2/C12, or L6 myotubes (unpublished data), and we found that acidification of the medium stimulates protein degradation in BC3H1 myocytes (17). In those experiments, we could not link excess glucocorticoids to stimulation of proteolysis. To elucidate whether there is an interaction between acidification and glucocorticoids, we used three strategies. First, we determined whether BC3H1 myocytes express functional glucocorticoid receptors. Next, we studied protein degradation in cells maintained in medium with 1% serum to reduce the exposure to glucocorticoids and to minimize the influence of other hormones and cytokines that might stimulate protein degradation. Finally, we determined whether changes in mRNAs for ubiquitin and subunits of the proteasome induced by acidification require glucocorticoids.

In rats and humans, metabolic acidosis stimABSTRACT ulates protein degradation and glucocorticoids have been implicated in this response. To evaluate the importance of glucocorticoids in stimulating proteolysis, we measured protein degradation in BC3H1 myocytes cultured in 12% serum. Acidification accelerated protein degradation but dexamethasone did not augment this response. To reduce the influence of glucocorticoids and other hormones and cytokines in 12% serum that could mediate proteolysis, we studied BC3H1 myocytes maintained in only 1% serum. Acidification of the medium or addition of dexamethasone at pH 7.4 did not significantly increase protein degradation, while acidification plus dexamethasone accelerated proteolysis. The steroid receptor antagonist RU 486 prevented this proteolytic response. Acidification of the medium with 1% serum did increase the mRNAs for ubiquitin and the C2 proteasome subunit, but when dexamethasone was added the mRNAs were increased significantly more. The steroid-receptor antagonist RU 486 suppressed this response to the addition of dexamethasone but the mRNAs remained at the levels measured in cells at pH 7.1 alone. Thus, acidification alone can increase the mRNAs of the ubiquitin-proteasome proteolytic pathway, but both acidosis and glucocorticoids are required to stimulate protein degradation. Since these changes occur without adding cytokines or other hormones, we conclude that the proteolytic response to acidification requires glucocorticoids.

Metabolic conditions causing loss of lean body mass raise questions about how these disturbances activate mechanisms that change protein metabolism. For example, when metabolic acidosis is present, there is evidence for excessive protein catabolism. Metabolic acidosis causes infants and children to grow poorly (1, 2), and Reaich et al. (3) showed that induction of metabolic acidosis in normal adults by administration of ammonium chloride accelerated catabolism of both protein and branched-chain amino acids (BCAAs). Experimental metabolic acidosis in rats reduces growth and stimulates whole body proteolysis and BCAA catabolism (4-6). Even though these reports document that acidosis stimulates protein catabolism, they do not identify which tissues exhibit accelerated proteolysis nor do they elucidate cellular mechanisms causing proteolysis. May et al. (4) showed that acidosis accelerates protein degradation in rat muscle, and there is evidence that this catabolic response involves activation of the ATP-dependent, ubiquitin-proteasome proteolytic pathway (7). This proteolytic system requires ATP for conjugation of ubiquitin to lysines in proteins targeted for degradation. ATP is also required for degradation of the protein substrates by the 26S proteasome, a multicatalytic enzyme complex composed of multiple sub-

MATERIALS AND METHODS Dulbecco's modified Eagle's medium (DMEM; low glucose with glutamine), 0.05% trypsin-treated EDTA, penicillin/ streptomycin (104 units of penicillin-G per ml and 104 ,tg of streptomycin sulfate per ml), LipofectAmine, and Opti-MEM were obtained from GIBCO. Collagen was purchased from Worthington, dexamethasone was from Elkins-Sinn (Cherry Hill, NJ), and RU 486 ((1113,17(3)-11-[4-(dimethylamino)phe-

nyl]-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one}, a steroid receptor antagonist, was a gift of A. Heidland (Wurzberg, Germany). L-[14C]Phenylalanine was obtained from ICN. CPSR-1 sera replacement and other chemicals were obtained from Sigma. Cell Culture. BC3H1 myocytes (passages 13-20) were grown to confluence in collagen-coated wells containing DMEM with penicillin/streptomycin and 12% CPSR-1 serum in a 90% 02/10% CO2 atmosphere (pH 7.4). Initially, cells were differentiated by serum deprivation and protein degradation was measured as the release of L-[14C]phenylalanine from prela-

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Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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Proc. Natl. Acad. Sci. USA 93 (1996)

Physiology: Isozaki et al.

calculated to derive the rate of protein degradation, expressed as the slope of the logio of percentage radioactivity remaining x 103 versus time (17, 20). Northern Blot Hybridization. Total RNA in myocytes was isolated using TriReagent (Molecular Research Center, Cincinnati) and separated by electrophoresis in a 1% agarose gel containing formaldehyde. After transfer to a Nytran nylon membrane (Schleicher & Schuell) and cross-linking by UV, the RNA blots were hybridized with 32P-labeled cDNA probes for ubiquitin (23), subunit C2 of the proteasome (24), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (25) as described (7). mRNAs were quantified by densitometry, and the results were corrected for differences in RNA loading based on the density of GAPDH mRNA. Statistical Analyses. Results are reported as means ± SEM and were compared by analysis of variance and the Bonferroni test for multiple comparisons. Results were considered significant at P < 0.05.

beled cells (17). In other experiments, 1% serum was used and the medium was changed every 3 days. Protein degradation and levels of mRNAs were measured 12 days postconfluence in differentiated cells maintained in 1% CPSR-1. Transient Transfection. Transient transfection of BC3H1 myocytes with LipofectAmine was performed using 2 ,tg of DNA per 35-mm well. Cells were cotransfected with (i) p59RATLUC, a plasmid containing upstream a minimal rat angiotensinogen promoter and containing multiple glucocorticoid-responsive elements linked to the firefly luciferase gene (18); and (ii) pSV-,B-galactosidase (Promega), a plasmid containing the f3-galactosidase gene. Cells were maintained in 12% CPSR-1 and 6 hr before harvest, 100 nM dexamethasone and/or 100 nM RU 486 were added (when incubated together, RU 486 was added 1 hr before dexamethasone). Luciferase activity (19) was expressed after correcting for differences in transfection efficiency by dividing luciferase activities by the respective p-galactosidase activity and protein content. Protein Degradation. Protein degradation in differentiated BC3H1 myocytes grown on 35-mm plates was measured after labeling cells by incubating with 0.5 ,uCi of L-[14C]phenylalanine per well (1 Ci = 37 GBq) for 3 days (17). To study the impact of acidification, hydrochloric acid was added and the medium pH was confirmed after equilibration in 90% 02/10% CO2. The experimental medium contained DMEM, 1% CPSR-1 serum, 2 mM unlabeled phenylalanine (to minimize reutilization of L-[14C]phenylalanine released) with or without 50 nM dexamethasone, and/or a 10-fold molar excess (500 nM) of RU 486 (added 1 hr before dexamethasone). After a 2-hr chase (to remove L-[14C]phenylalanine released from short-lived proteins), the medium was replaced with 3.0 ml of fresh experimental medium (20-22) and 0.3-ml aliquots were sequentially removed over 48 hr. Trichloroacetic acid [10% (vol/vol) final concentration] was added to remove protein and radioactivity was measured in each aliquot. At the end of the experiment, cells were solubilized in 2 ml of 1% SDS and the remaining cell protein-associated radioactivity was measured. The total radioactivity released plus that remaining in cells represent the initial L-['4C]phenylalanine in cell proteins. The percentage radioactivity remaining in cells versus time was

RESULTS England et al. (17) showed that protein degradation in BC3H1 myocytes incubated in 12% serum is accelerated by acidification, but dexamethasone did not increase this response. To evaluate why acidification-induced proteolysis in BC3H1 myocytes did not increase with glucocorticoids (4), we investigated whether BC3H1 myocytes express functional glucocorticoid receptors. BC3H1 myocytes were transiently transfected with a luciferase reporter gene under control of a minimal rat angiotensinogen promoter and multiple upstream glucocorticoid responsive elements (18). Dexamethasone increased luciferase activity 2-fold (8.64 ± 0.41 light units per unit of 13-galactosidase per mg of protein vs. 17.03 ± 3.5 in dexamethasone-treated cells; P < 0.05). The response to glucocorticoids was prevented by an equimolar amount of RU 486 added 1 hr before dexamethasone (7.94 ± 0.83; P < 0.05 vs. cells treated with dexamethasone alone). Similar results were obtained when cells were maintained in medium containing 1% serum. We conclude that BC3H1 myocytes express a low level of glucocorticoid receptors compared to L6 myotubes

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pH FIG. 1. RU 486 prevents stimulation of protein degradation by acidification plus dexamethasone in BC3H1 cells. Cell were incubated with or without 50 nM dexamethasone (DEX) and 500 nM RU 486. Results are from one experiment with six wells per group and are reported as the mean + SEM of the negative slope (x 103) of logio of the percentage of L-[14C]phenylalanine remaining in cells per hr. By analysis of variance, acidification plus glucocorticoids stimulate protein degradation; RU 486 prevented the increase in proteolysis. *, P < 0.01 vs. other groups.



that exhibited a 10-fold increase in luciferase activity when dexamethasone was added (data not shown). Unfortunately, L6 myotubes rapidly acidify the medium so we did not study protein degradation in these cells. Next, we examined the effect of the glucocorticoid antagonist RU 486 on protein degradation in normal and acidified BC3H1 myocytes. Acidification of medium containing 12% serum (pH 7.1) stimulated protein degradation (log slope x 103 = 6.06 + 0. 10 at pH 7.4 vs. 6.63 ± 0.06 at pH 7.1; P < 0.05) and 10 ,uM RU 486 blocked the increase in proteolysis (log slope x 103 = 5.27 ± 0.29). However, RU 486 also suppressed protein degradation in pH 7.4 medium (log slope x 103 = 5.50 + 0.15). These ambiguous results do not elucidate a role for glucocorticoids in the proteolytic response to acidification. We calculated that 12% serum contains 1.3 nM cortisol and decided to reduce serum in the medium to 1% throughout differentiation and the experimental periods. In three separate experiments of six wells per group in each experiment, we consistently found that acidification increased protein degradation slightly but the increase was not statistically significant compared to the rate measured in cells incubated in 1% serum at pH 7.4 (Fig. 1). We also found that addition of 50 nM dexamethasone did not stimulate protein degradation in cells incubated at pH 7.4. In contrast, the combination of acidification and 50 nM dexamethasone stimulated proteolysis in all three experiments (Fig. 1; P < 0.01 vs. all groups). To demonstrate involvement of the glucocorticoid receptor, we added the steroid receptor antagonist RU 486. In medium with 1% serum, RU 486 alone did not alter the rate of protein degradation at pH 7.4 or pH 7.1 (Fig. 1), but it blocked the increase in protein degradation measured in cells incubated at pH 7.1 plus dexamethasone. To determine whether differences in intracellular pH could account for the increase in protein degradation and mRNA levels found with the combination of acidification plus dexamethasone, we measured this variable (17). In cells incubated at pH 7.1 and at pH 7.1 with dexamethasone, the intracellular pH (7.06 ± 0.03 and 7.02 + 0.02; n = 8 for each group) did not differ statistically. These values, like the intracellular pH measured in cells incubated at pH 7.4 with dexamethasone (7.27 + 0.03; n = 8), are indistinguishable from the respective values we found earlier in BC3H1 myocytes incubated at pH 7.4 or pH 7.1 (17). We found that metabolic acidosis increases the levels of mRNAs encoding ubiquitin and proteasome subunits in rat muscle (7). To determine whether a similar response occurs in BC3H1 cells, we hybridized RNA from cells incubated for 12 hr with or without dexamethasone at different pH values. As

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FIG. 3. Acidification plus glucocorticoids increase ubiquitin mRNA abundance in BC3H1 myocytes. Densitometry values of ubiquitin mRNA for each experiment were corrected for differences in RNA loading by dividing by the respective GAPDH mRNA values. Data for each group were combined and are reported as means ± SEM (n = 8). By analysis of variance, acidification increases ubiquitin mRNA abundance, while acidification plus dexamethasone (DEX) cause an additional increase. *, P < 0.01 vs. pH 7.4 without dexamethasone; **, P < 0.01 vs. other groups.

found previously (7), two ubiquitin mRNA transcripts, 2.4 and 1.4 kb, were detected (Fig. 2). Densitometry analyses of both ubiquitin transcripts from the four treatment groups revealed that addition of dexamethasone at pH 7.4 did not increase ubiquitin mRNA but acidification alone increased it 26% (P < 0.01 vs. pH 7.4; Fig. 3). The highest value (P < 0.01 vs. all groups) was measured when glucocorticoids and acidification were combined. With the combination, ubiquitin mRNA was 64% and 45% higher than in cells incubated at pH 7.4 without and with dexamethasone, respectively. Ubiquitin mRNA was 29% higher with the combination than in cells incubated at pH 7.1 without exogenous glucocorticoids. **

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FIG. 2. Hybridization of ubiquitin mRNA from BC3H1 cells after incubation with or without dexamethasone at pH 7.4 or pH 7.1. Ten micrograms of total RNA from each of the four groups was analyzed by Northern blot hybridization with ubiquitin and GAPDH cDNAs. Shown are representative lanes from each group incubated at pH 7.4 or pH 7.1 with or without 50 nM dexamethasone (DEX).

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FIG. 4. Acidification plus glucocorticoids increase proteasome C2 subunit mRNA abundance in BC3H1 myocytes. Densitometry values of proteasome C2 subunit mRNA were corrected for differences in loading by dividing by each respective GAPDH mRNA value. Data for each group were combined and are reported as means + SEM (n = 8). By analysis of variance, acidification increases C2 mRNA abundance, while acidification plus dexamethasone (DEX) cause an additional increase. *, P < 0.01 vs. pH 7.4 with and without dexamethasone; **, P < 0.01 vs. other groups.

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Table 1. RU 486 decreases the stimulatory effect of acidification plus dexamethasone on ubiquitin and C2 subunit mRNAs Treatment C2 mRNA Ubiquitin mRNA pH 7.1 alone 1.61 ± 0.05 (4) 1.49 ± 0.05 (4) 1.92 ± 0.07 (8)* 2.20 ± 0.02 (8)t pH 7.1 + dexamethasone 1.66 mmm+0.08 (8) 1.51 ± 0.02 (8) pH 7.1 + dexamethasone + RU 486 Densitometric values of ubiquitin and C2 mRNAs were corrected for differences in RNA loading by dividing by the respective GAPDH mRNA value; data are expressed as means ± SEM. Numbers in parentheses indicate number of independent determinations. By analysis of variance, acidification plus dexamethasone increases the levels of ubiquitin and C2 subunit mRNA above acidification alone and this increase is blocked by addition of RU 486. *P < 0.05. tp < 0.01.

The mRNA of the C2 proteasome subunit exhibited similar changes. Addition of dexamethasone to cells at pH 7.4 did not increase C2 mRNA but acidification alone increased C2 mRNA 45% (P < 0.01 vs. 7.4; Fig. 4). Again, the combination of acidification plus dexamethasone increased C2 mRNA 79% above the value at pH 7.4 without added dexamethasone (P < 0.01) and 24% above acidification alone (P < 0.01). When myocytes were treated similarly for shorter periods (i.e., 1 or 6 hr), these changes in ubiquitin and C2 subunit mRNAs were not detected. To examine whether the influence of acidification on mRNA levels could be separated from those of glucocorticoids, we incubated myocytes for 12 hr in 1% serum (pH 7.1) with dexamethasone plus a 10-fold excess of RU 486. Ubiquitin mRNA in these cells was not different from that measured in cells incubated in 1% serum at pH 7.1 alone, but it was less than that measured at pH 7.1 with dexamethasone (P < 0.05; Table 1). The same results were obtained with the C2 subunit mRNA (P < 0.01; Table 1).

DISCUSSION Our studies of adrenalectomized, acidotic rats suggest that there is an interaction between acidosis and glucocorticoids in stimulating muscle proteolysis (4). Unfortunately, results from adrenalectomized rats are difficult to interpret because there could be changes in other hormones or cytokines that affect protein metabolism. To circumvent endocrine and humoral responses to acidosis that might occur in intact animals, we studied proteolysis in cultured BC3H1. These cells, unlike other muscle cell lines, do not rapidly acidify the medium so we could examine the influence of acidification alone. When BC3H1 myocytes incubated in 12% serum were acidified, protein degradation increased but adding dexamethasone did not increase this response. To explore the apparent difference from results in the rat, we examined whether these cells express functional glucocorticoid receptors and found that they do, albeit at a low level. This led to the conclusion that there is sufficient cortisol in 12% serum to stimulate protein degradation when myocytes are acidified. We determined that dexamethasone-induced stimulation of the luciferase gene could be blocked by an equimolar concentration of RU 486, providing a rationale for using this compound to evaluate the role of glucocorticoids in the responses of BC3H1 myocytes to acidification. To separate the influences of acidification and glucocorticoids, we reduced the exposure of myocytes to glucocorticoids and other hormones and cytokines by maintaining cells chronically in medium containing 1% serum. Under these conditions, neither acidification alone nor dexamethasone alone significantly increased protein degradation in three separate studies of six wells in each experimental group. In contrast, acidification plus dexamethasone significantly increased proteolysis (Fig. 1). The 15% increase in proteolysis contrasts sharply with the maximal 4% increase in protein degradation

occurring with acidification alone. Since RU 486 blocked the increase in protein degradation stimulated by acidification plus glucocorticoids and since glucocorticoids alone did not increase protein breakdown, we conclude that the proteolytic response requires both acidification and glucocorticoids. Based on results obtained in rats and the increase in protein degradation in BC3H1 myocytes (Fig. 1), we expected to find that the combination of acidification plus glucocorticoids would increase ubiquitin and proteasome subunit mRNAs (7, 26). We found that acidification alone tended to stimulate protein degradation slightly and increased mRNAs of the ubiquitin-proteasome pathway in myocytes. Dexamethasone alone did not increase mRNAs encoding ubiquitin or the C2 proteasome subunit. The largest increase in protein degradation and in ubiquitin and C2 proteasome subunit mRNAs were measured with the combination of acidification and dexamethasone (Figs. 3 and 4). The increase in levels of ubiquitin and C2 subunit mRNAs with acidification alone could mean that even the glucocorticoid concentration in 1 % serum is sufficient to elicit changes in mRNAs. Unfortunately, we could not study BC3H1 myocytes without serum because they express morphologic changes when incubated in zero or stripped, charcoal-treated serum. Regardless, the increase in mRNAs was not blocked by adding a 10-fold excess of RU 486, so we must conclude that acidification alone is capable of increasing ubiquitin and C2 subunit mRNAs but not a measurable increase in proteolysis. Note that an equimolar amount of RU 486 blocked the increase in luciferase expression induced by dexamethasone. Other investigators report that acidification alone can increase the level of mRNAs for glutaminase and phosphoenolpyruvate carboxykinase in cultured cells, so it is possible that protons may increase the mRNAs for ubiquitin and the C2 proteasome subunit (27-30). Our results provide strong evidence that dexamethasone interacts with acidification to increase the mRNAs encoding ubiquitin and the C2 proteasome subunit more than acidification alone (Table 1), and both factors are necessary to elicit a measurable increase in protein degradation (Fig. 1). These resvlts and the fact that the myocytes were kept in only 1% serum indicates that changes in other mediators (e.g., insulin, cytokines, etc.) are not responsible for the increase in protein degradation or mRNA levels stimulated by acidification plus glucocorticoids. We have not uncovered how the combination of acidification and glucocorticoids causes these catabolic responses. Although upstream sequences of several proteasome subunit genes have been described, potential regulatory elements have not been characterized (31, 32). Consequently, we cannot speculate about factors that could regulate expression of the ubiquitin and C2 proteasome subunit genes. Studies of isolated skeletal muscles of fasting, juvenile rats (33) revealed that the increase in protein degradation is associated with higher levels of the mRNAs of ubiquitin and subunits of the proteasome (33, 34). Exploration of the mechanism for this response revealed that starvation did not increase protein degradation or the level of these mRNAs if

Physiology: Isozaki et al. the rats were adrenalectomized but the responses were restored if the starved, adrenalectomized rats were given glucocorticoids. It was concluded that glucocorticoids are responsible for activation of the ATP ubiquitin-dependent proteolytic pathway but the influence of glucocorticoids on these responses in muscles of fed rats was not tested (33). Our results in BC3H1 myocytes suggest that glucocorticoids alone would not be sufficient to stimulate protein degradation or to elevate the mRNAs encoding proteins of the ubiquitin-proteasome pathway. This requirement of acidification plus glucocorticoids is analogous to the requirements we find in rat muscle (4, 26). In summary, acidification of BC3H1 myocytes accelerates protein degradation but only if glucocorticoids are present. After 12 hr of exposure to pH 7.1, there is also a coordinated increase in the abundance of mRNAs that encode proteins involved in the ATP ubiquitin-proteasome proteolytic pathway. Since these studies were performed under conditions that minimize the influence of other hormones and/or factors in serum, they provide strong evidence that glucocorticoids and acidosis act together to elicit a catabolic response in muscle cells. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK37175 and DK45215 (W.E.M.) and a Veterans Administrative Merit Review Grant

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