Glucose regulates protein catabolism in ras-transformed fibroblasts through a lysosomal-dependent proteolytic pathway.

Transformed cells are exposed to heterogeneous microenvironments, including low D-glucose (Glc) concentrations inside tumours. The regulation of protein turnover is commonly impaired in many types of transformed cells, but the role of Glc in this regulation is unknown. In the present study we demonstrate that Glc controls protein turnover in ras-transformed fibroblasts (KBALB). The regulation by Glc of protein breakdown was correlated with modifications in the levels of lysosomal cathepsins B, L and D, while autophagic sequestration and non-lysosomal proteolytic systems (m- and mu-calpains and the zeta-subunit of the proteasome) remained unaffected. Lactacystin, a selective inhibitor of the proteasome, depressed proteolysis, but did not prevent its regulation by Glc. The sole inhibition of the cysteine endopeptidases (cathepsins B and L, and calpains) by E-64d [(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester] was also not sufficient to alter the effect of Glc on proteolysis. The Glc-dependent increase in proteolysis was, however, prevented after optimal inhibition of lysosomal cysteine and aspartic endopeptidases by methylamine. We conclude that, in transformed cells, Glc plays a critical role in the regulation of protein turnover and that the lysosomal proteolytic capacity is mainly responsible for the control of intracellular proteolysis by Glc.

Glucose controls cathepsin expression in Ras-transformed fibroblasts.

Overexpression and altered trafficking of cathepsins have been associated with the malignant properties of tumors and transformed cells. A characteristic phenotype of transformed cells is also a profound deviation in their metabolism (aerobic glycolysis, glutaminolysis) which enables them to adapt to extreme nutritional conditions. However, whether the altered metabolism may change the expression of proteinases involved in malignancy has not been determined. Herein we present evidences in Kirsten-virus-transformed 3T3 fibroblasts (KBALB) that D-glucose selectively increases active forms of cathepsins L, B, and D, without altering other lysosomal nonproteolytic hydrolases (beta-D-glucosaminidase, acid phosphatase, beta-D-glucuronidase, and beta-D-galactosidase). D-Glucose did not modify mRNA levels for cathepsin B or L and did not affect secretion of pro-cathepsin L. However, D-glucose enhanced strongly the amount of the mature forms of cathepsins B and L, without altering their preferential distribution to light endosomal fractions. Induction by d-glucose of intracellular mature cathepsins B and L required a high growth density of KBALB cells and was reproduced in BALB/3T3 fibroblasts stably transfected with a constitutively activated form of Ras. d-Glucose induction of active cathepsins however was not observed in nontransformed BALB/3T3. D-Mannose, in contrast to nonmetabolized sugars (D-galactose, or L-glucose), caused a similar increase in lysosomal cathepsin activities in dense KBALB cells. The D-glucose analogue, 3-O-methyl-D-glucose, which is transported but not further metabolized, did not reproduce the d-glucose effects. Our findings indicate that, dependent on the nutrient supply and as a consequence of their altered metabolism, transformed cells may modulate the production of active proteinases implicated in malignant progression.

Developmental control of cathepsin B expression in bovine fetal muscles.

Expression of lysosomal cysteine proteinases was studied during fetal calf muscle development. The peptide cleaving activities of cathepsins B and L decreased strongly from 80 to 250 days of fetal age. This decrease in cathepsin activities occurred similarly in three muscles exhibiting different metabolic and contractile properties in adult animals. Cathepsin B from adult or fetal muscle revealed similar enzymatic properties, but presented a five- to sixfold lower concentration in adult muscle as indicated by active-site titration with L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guani din o)butane. During fetal growth, decreases in muscle cathepsin B specific activity and active enzyme concentration were associated with a parallel drop of cathepsin B mRNA levels. Bovine cathepsin B is encoded by two different transcripts resulting from alternative polyadenylation [Mordier, S. B., Béchet, D. M., Roux, M. P., Obled, A., and Ferrara, M. (1995) Eur. J. Biochem. 229, 35-44]. As revealed by ribonuclease protection assays, the two mRNAs encoding cathepsin B declined similarly during fetal muscle growth. This study indicates that lysosomal proteinases in skeletal muscle are under developmental control. The decrease of muscle cathepsins during fetal development appears sufficient to account for the low levels of these enzymes in adult muscles. In fetuses, high activities of lysosomal cysteine proteinases might be important for remodeling muscles during early development.

The structure of the bovine cathepsin B gene. Genetic variability in the 3' untranslated region.

Cathepsin B is a cysteine proteinase which plays an important role in lysosomal proteolysis. We report here the characterization of a 15-kbp genomic clone of the bovine cathepsin B gene. Bovine preprocathepsin B is encoded by nine exons from translational initiation to stop codon. The last exon is sandwiched between Alu-like short interspersed nuclear elements. Alternate use of polyadenylation sites generates three transcripts encoding bovine cathepsin B. In all tissues tested, 3' end cleavage was found to occur in equal proportion at the first and second polyadenylation site, producing transcripts of 2.6-kb. Polyadenylation at the third polyadenylation site generates a 3.2-kb mRNA, only expressed at low levels and in a developmental and tissue-specific manner. Due to micro-heterogeneities between genomic and cDNA clones, sequence polymorphism was investigated in the trailer region. DNA sequencing of PCR-amplified genomic fragments revealed that, in contrast to the protein-encoding region, genetic variability exists in the 3' untranslated region. Polymorphism in the trailer was confirmed in cathepsin B mRNA by ribonuclease-protection assays. We finally emphasize that while exon-exon boundaries in mature cathepsin B are well conserved from nematodes to mammals, exons also tend to correspond to discrete units of cathepsin B structure.

Expression of lysosomal cathepsin B during calf myoblast-myotube differentiation. Characterization of a cDNA encoding bovine cathepsin B.

Expression of lysosomal cysteine proteinases was studied during fetal calf myoblast-myotube differentiation. Activities of cathepsin B and L, but not cathepsin H, increase during bovine myogenic differentiation. In fetal muscle, cathepsin B and L activities are 2-4-fold orders of magnitude lower than in cultured myoblasts. Active-site titrations of cathepsin B with E-64 nevertheless reveal similar concentrations of active cathepsin B in myoblasts and myotubes, but 5-6-fold lower concentrations in fetal muscle. To specify whether concentrations of cathepsin B are related to levels of cathepsin B transcript, a cDNA clone encoding bovine cathepsin B was isolated and liquid hybridizations were performed with 32P-riboprobes complementary to the mRNA. In agreement with active-site titrations, there is no difference in cathepsin B mRNA levels between cultured myoblasts and myotubes, but lower levels of mRNA are found in fetal muscle. Concentrations of active cathepsin B therefore reflect levels of cathepsin B mRNA. Kinetic studies revealed that the catalytic efficiency (kcat/Km) of cathepsin B is 2-3-fold higher in myotubes than in myoblasts. The increase in cathepsin B activity during calf myoblast-myotube differentiation is thus due to modifications of enzymatic properties, and not of enzyme concentrations. The different catalytic efficiency of cathepsin B in myotubes and myoblasts was related neither to modifications of mRNA size, as revealed by Northern blot analysis, nor to a different Mr of the active enzyme, as revealed by affinity labeling with benzyloxycarbonyl-Tyr(-125I)-Ala-CHN2, but to limited differences in cathepsin B isozymes.

Cimaterol reduces cathepsin activities but has no anabolic effect in cultured myotubes.

The effect of the beta-adrenergic agonist cimaterol on bovine and chicken primary myotubes was assessed. Cimaterol at 10-100 nM concentrations reduced cathepsin B benzyloxy-carbonyl-Arg-Arg-4-methyl-7-coumarylamide hydrolyzing activity, as well as benzyloxycarbonyl-Phe-Arg-4-methyl-7-coumarylamide hydrolysis, which is a substrate for both cathepsin B and cathepsin L. Maximum effect was observed after 6-16 h treatment. Cathepsin H Arg-4-methyl-7-coumarylamide hydrolyzing activity was low and not significantly affected by cimaterol treatment. Despite decreasing cathepsin activities, cimaterol also increased proteolysis rates but induced no detectable effect on protein synthesis rates. These observations suggest that beta-agonists, as a result of a direct action on muscle, can decrease cathepsin activities but that beta-agonist-induced muscle hypertrophy may not be due to a direct effect on muscle cells.

A novel class of inactive steroid-binding sites in female rat liver nuclei.

Nuclear salt extracts from intact female rat liver showed insignificant levels of progesterone-, oestradiol-, testosterone- or dexamethasone-specific binding. However, brief exposure of nuclear extracts to dextran-coated charcoal (DCC) induced binding for all the above classes of steroids. This 'DCC-effect', which was reproduced neither by gel filtration nor by extensive dialysis of the nuclear extract, could not be ascribed to removal of endogenous free or loosely bound steroids. We show that rat liver nuclei contain a class of secondary binding sites (BSII), which exhibit moderate or low affinity for steroid ligands, positive co-operativity, and cross-reaction between classes of steroids. The capacity of BSII sites to bind steroids depends strictly on prior neutralization by DCC of endogenous heat-stable non-dialysable inhibitor(s). The putative roles of these BSII binding sites are discussed in relation to component(s) probably responsible for inhibitory activity.

Oestrogen specific binding sites in bovine muscle nuclei.

A method for preparation of purified bovine diaphragm nuclei was developed and the presence of specific oestradiol binding sites was demonstrated in salt extracts of such muscle nuclei by kinetic, equilibrium and competition studies. Scatchard analysis of [3H]zeranol binding also indicated the presence of high-affinity binding sites in nuclei from female animals (heifers) for this synthetic oestrogenic anabolic agent. Measured levels of specific [3H]oestradiol binding were higher in zeranol-treated steer than in untreated heifer, or steer diaphragm nuclei. A second, lower-affinity oestrogen-binding component was identified using [3H]oestradiol at concentrations greater than 8 nM in all three types of animals. The data suggest that gonadal oestrogens or related anabolic agents might have direct effects on muscle through receptor-like macromolecules.