Nine days of hindlimb suspension resulted in atrophy (55%) and loss of protein (53%) in rat soleus muscle due to a marked elevation in protein breakdown (66%, P < 0.005). To define which proteolytic system(s) contributed to this increase, soleus muscles from unweighted rats were incubated in the presence of proteolytic inhibitors. An increase in lysosomal and Ca 2+-activated proteolysis (254%, P < 0.05) occurred in the atrophying incubated muscles. In agreement with the measurements in vitro, cathepsin B, cathepsins B + L and m-calpain enzyme activities increased by 111%, 92% and 180% (P < 0.005) respectively in the atrophying muscles. Enhanced mRNA levels for these proteinases (P < 0.05 to P < 0.001) paralleled the increased enzyme activities, suggesting a transcriptional regulation of these enzymes. However, the lysosomal and Ca 2+-dependent proteolytic pathways accounted for a minor part of total proteolysis in both control (9%) and unweighted rats (18%). Furthermore the inhibition of these pathways failed to suppress increased protein breakdown in unweighted muscle. Thus a non-lysosomal Ca 2+-independent proteolytic process essentially accounted for the increased proteolysis and subsequent muscle wasting. Increased mRNA levels for ubiquitin, the 14 kDa ubiquitin-conjugating enzyme E2 (involved in the ubiquitylation of protein substrates) and the C2 and C9 subunits of the 20 S proteasome (i.e. the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates) were observed in the atrophying muscles (P < 0.02 to P < 0.001). Analysis of C9 mRNA in polyribosomes showed equal distribution into both translationally active and inactive mRNA pools, in either unweighted or control rats. These results suggest that increased ATP-ubiquitin-dependent proteolysis is most probably responsible for muscle wasting in the unweighted soleus muscle.
In mammals, plasma concentration of amino acids is affected by nutritional or pathological conditions. It has been well established that nutrients, and particularly amino acids, are involved in the control of gene expression. Here we examined the molecular mechanisms involved in the regulation of CHOP (a CCAAT/enhancerbinding protein [C/EBP]-related gene) expression upon amino acid limitation. We have previously shown that regulation of CHOP mRNA expression by amino acid concentration has both transcriptional and posttranscriptional components. We report the analysis of cis-and trans-acting elements involved in the transcriptional activation of the human CHOP gene by leucine starvation. Using a transient expression assay, we show that a cis-positive element is essential for amino acid regulation of the CHOP promoter. This sequence is the first described that can regulate a basal promoter in response to starvation for several individual amino acids and therefore can be called an amino acid response element (AARE). In addition, we show that the CHOP AARE is related to C/EBP and ATF/CRE binding sites and binds in vitro the activating transcription factor 2 (ATF-2) in starved and unstarved conditions. Using ATF-2-deficient mouse embryonic fibroblasts and an ATF-2-dominant negative mutant, we demonstrate that expression of this transcription factor is essential for the transcriptional activation of CHOP by leucine starvation. Altogether, these results suggest that ATF-2 may be a member of a cascade of molecular events by which the cellular concentration of amino acids can regulate mammalian gene expression.
In mammals, plasma concentrations of amino acids are affected by nutritional or pathological conditions. Here we examined the role of amino acid limitation in regulating the expression of CHOP, a CCAAT/enhancer binding protein (C/EBP)-related gene. CHOP protein is capable of interacting with other C/EBPs to modify their DNA binding activities and may function as a negative regulator of these transcription factors. Our data show that leucine limitation in human cell lines leads to induction of CHOP mRNA and protein in a dose-dependent manner. CHOP mRNA induction is rapidly reversed by leucine replenishment. Elevated mRNA levels result from both an increase in the rate of CHOP transcription and an increase in the CHOP mRNA stability. Using a transient expression assay, we show that a promoter fragment, when linked to a reporter gene, is sufficient to mediate the regulation of CHOP expression by leucine starvation in HeLa cells. In addition, we found that decreasing amino acid concentration by itself can induce CHOP expression independently of a cellular stress due to protein synthesis inhibition. Moreover, CHOP expression is induced at leucine concentrations in the range of those observed in blood of protein-restricted animals suggesting that amino acids can participate, in concert with hormones, in the regulation of gene expression.
We have developed ribozymes (Rz) that inhibit BRCA1 expression in order to study the role of this gene in chemosensitivity. Two Rz, targeting positions 358 or 5282 of the BRCA1 mRNA, were cloned into the retroviral vector LXSN and lipofected into the breast cancer cell-line HBL100. We obtained 79 ± 99% inhibition of BRCA1 expression, as determined by real-time quantitative PCR and by Western blotting. Decreased expression of BRCA1 led to sensitivity to the DNA damaging agents cisplatin and etoposide, resistance to the microtubule-interfering agents (MIA) taxol and vincristine. The molecular mechanism of resistance to MIA was investigated further by determining the status of the JNK pathway. We found that JNK1 expression was elevated, while JNK2 expression was decreased in Rz-expressing clones compared to controls. We have quanti®ed the mRNA levels of BRCA1, JNK1, 2, MEK-4, -7 and c-jun after treatment with MIA. Vincristine treatment of control cells resulted in transcriptional repression of BRCA1, while the JNK1, 2, MEK-4, -7 and c-jun genes were induced. In Rz-treated cells, only JNK1 and MEK-4 were expressed and none was induced after MIA treatment. We then studied the phosphorylation of c-jun, a downstream eector of the JNK pathway. We observed a strong increase in phosphorylated c-jun after MIA treatment of the control cells but not in BRCA1-Rz treated cells, suggesting inhibition of the JNK pathway. These results show that the BRCA1-JNK pathway is involved in the cytotoxic response to MIA treatment, and inhibition of BRCA1 leads to transcriptional modi®cations of the JNK pathway. Oncogene (2001) 20, 6597 ± 6606.
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