Inhibitory Effect Of MG132 Research Articles

Photorhabdus luminescens Tc toxin is inhibited by the protease inhibitor MG132 and activated by protease cleavage resulting in increased binding to target cells.

Photorhabdus luminescens Tc toxins consist of the cell-binding component TcA, the linker component TcB, and the enzyme component TcC. TccC3, a specific isoform of TcC, ADP-ribosylates actin and causes redistribution of the actin cytoskeleton. TccC5, another isoform of TcC, ADP-ribosylates and activates Rho proteins. Here, we report that the proteasome inhibitor MG132 blocks the intoxication of cells by Tc toxin. The inhibitory effect of MG132 was not observed, when the ADP-ribosyltransferase domain of the TcC component was introduced into target cells by protective antigen, which is the binding and delivery component of anthrax toxin. Additionally, MG132 affected neither pore formation by TcA in artificial membranes nor binding of the toxin to cells. Furthermore, the in vitro ADP-ribosylation of actin by the enzyme domain of TccC3 was not affected by MG132. Similar to MG132, several calpain inhibitors blocked the action of the Tc toxin. Proteolytic cleavage of the binding component TcA induced by P.luminescens protease PrtA1 or by collagenase largely increased the toxicity of the Tc toxin. MG132 exhibited no inhibitory effect on the cleaved TcA component. Moreover, binding of TcA to target cells was largely increased after cleavage. The data indicate that Tc toxin is activated by proteolytic processing of the TcA component, resulting in increased receptor binding. Toxin processing is probably inhibited by MG132.

O22-3 - Upregulation of AMP Deaminase in the Vicinity of the SR via Translational Regulation by miR-301b Contributes to Diabetic Cardiomyopathy

Background: We recently reported that ATP loss by upregulated activity of AMP deaminase (AMPD) contributes to afterload-induced diastolic dysfunction in OLETF, type 2 diabetic rats. Here we examined the mechanism underlying the upregulation of AMPD. Methods and Results: Western blot analysis revealed that protein level of 90-kDa full-length AMPD3 was significantly increased in the sarcoplasmic reticulum (SR) fraction (117%) as well as in whole cell lysates (171%) in OLETF compared to its level in LETO, non-diabetic control rats. Levels of AMPD3 mRNA determined by RT-PCR was comparable in OLETF and LETO. Inhibitory effect of MG-132, a proteasome inhibitor, on degradation of the 90-kDa AMPD3 ex vivo was larger in OLETF than in LETO, arguing against suppressed degradation of AMPD3 in OLETF. MicroRNA array analysis revealed downregulation (>50%) of 57 microRNAs in OLETF compared to those in LETO. Transfection of H9c2 cells with an inhibitor of miR-301b, one of the 57 microRNAs, significantly increased level of the 90-kDa AMPD3. Binding of miR-301b to the 3'UTR of AMPD3 mRNA was confirmed by a luciferase reporter assay. Conclusion: Up-regulated AMPD3 translation by reduced miR-301b mediates increased AMPD3 in the diabetic heart. Since ATP generated by SR-associated glycolytic enzymes contributes to Ca2+ uptake by SERCA2a and promote diastolic relaxation, AMPD3 in the vicinity of the SR is a therapeutic target for diabetic cardiomyopathy.

Identification of ATF2 as a transcriptional regulator of renin gene

The cAMP response element (enhCRE) in the distal enhancer regulatory region of renin gene is believed to play a major role in the control of renin transcription. enhCRE binds the CRE-binding protein (CREB), which is the main transcription factor target of cAMP signaling. Using the mouse renin-producing cell line As4.1 we found that activating transcription factor-2 (ATF2) also binds to enhCRE. N-terminal phosphorylation of ATF2, which controls its transactivation, is associated with downregulation of renin gene expression by the cytokine tumor necrosis factor-α (TNFα). The ubiquitin proteasome inhibitor MG132 also phosphorylates ATF2 and inhibits renin expression. Knockdown of ATF2 attenuated the suppression of renin gene expression by MG132, thus demonstrating that ATF2 mediates the inhibitory effect of MG132. In addition, MG132 increased the DNA-binding of ATF2 as well as the ratio of bound ATF2 to CREB. Using ATF2- and CREB-Gal4 fusion protein constructs coupled with luciferase reporter system we showed that ATF2 has a weaker transactivating capacity than CREB. These data suggest that ATF2 represses renin expression by drifting the transcriptional control of renin gene away from CREB. Accordingly, TNFα completely abrogated the cAMP-dependent stimulation of renin gene expression.

The apoptosis of bovine lens epithelial cells induced by proteasome inhibitor MG132

To investigate the effect of proteasome inhibitor MG132 on the apoptosis of bovine lens epithelial cells (BLECs), the cells were treated with MG132 at different concentrations for 12, 24 and 36 h. The cell viability was analyzed by MTT assay and the effect of MG132 on the apoptosis of BLECs was assessed by flow cytometry (FCM). The results showed that after treatment for the same period, the inhibitory effect of MG132 on BLECs proliferation was enhanced with the increment of the concentration of MG132 (0, 2, 5, 10, mumol/L) (P<0.05). The 50% inhibiting concentration (IC(50)) was 2.03 mumol/L when the BLECs were treated with MG132 for 36 h. MG132 also induced the apoptosis of BLECs obviously. FCM showed that the apoptosis index of the cells treated by MG132 at 2 mumol/L for 12 h was (20.24+/-1.51)%, and that of the control was (0.98+/-0.20)% respectively (P<0.01, n=3). It was concluded that MG132 could lead to apoptosis of BLECs. The decrease of proteasome activity may play an important role in the formation and development of cataract.

Proteasome inhibitors induce osteoclast survival by activating the Akt pathway

Osteoclasts rapidly undergo spontaneous apoptosis when deprived of survival factors. Regulation of osteoclast survival is important to treat bone-related diseases, such as osteoporosis. In this study, we found that the proteasome inhibitors, MG132 and ALLN, significantly inhibited osteoclast apoptosis induced by etoposide, as well as under conditions of survival factor deprivation. MG132 and ALLN inhibited the release of cytochrome c from mitochondria into the cytosol in the absence of survival factors and suppressed the cleavage of pro-caspase-9 and -3 to its active forms induced by etoposide. In addition, MG132 and ALLN enhanced the phosphorylation of Akt and ERK in osteoclasts. However, MG132 and ALLN did not inhibit the cleavage of caspase-9 and -3 in the presence of the phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002, while the inhibitory effect of MG132 and ALLN were intact in presence of the MEK1/2 inhibitor, U0126. LY294002 inhibited the survival of osteoclasts induced by MG132 and ALLN. Taken together, our results have demonstrated that proteasome inhibitors suppressed osteoclast apoptosis under conditions of survival factors deprivation through activation of the PI-3K/Akt pathway.

MG-132 Inhibits Telomerase Activity, Induces Apoptosis and G1 Arrest Associated with Upregulated p27kip1 Expression and Downregulated Survivin Expression in Gastric Carcinoma Cells

Ubiquitin-proteasome pathway (UPP) is the major system for the selective degradation of cellular proteins that play key roles in cellular processes. Previous study indicated that ubiquitin-proteasome inhibitor MG-132 could inhibit growth of some carcinoma. However, anti-carcinoma mechanism of MG-132 is unclear. Our objective was to investigate mechanisms of growth inhibitory effect of MG-132 on gastric carcinoma cells. Gastric carcinoma cell SGC-7901 was treated with ubiquitin-proteasome inhibitor MG-132. Cell growth suppression was evaluated with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. DNA synthesis was evaluated by 3H-thymidine (3H-TdR) incorporation. Activity of telomerase was examined by telomeric repeat amplification protocol (TRAP) PCR-ELISA. Cell cycle and apoptosis were detected by flow cytometry (FCM). DNA fragment analysis was used to confirm the presence of apoptosis. Expression of p27kip1 and survivin was detected using the western blot method. After exposed to MG-132, the growth and value of 3H-TdR incorporation of gastric carcinoma cells were obviously inhibited. TRAP PCR-ELISA showed that light absorption of cells gradually decreased after exposed to 5 μ M of MG-132 for 24, 48, 72 and 96 h (P < 0.01). The percentage of cells at G0/G1 phase was increased and that at S and G2/M phase was decreased (P < 0.01). The ratio of apoptotic cells treated with 5 μ M MG-132 for 96 h was 53.7 ± 6.4%. Agarose electrophoresis showed marked ladders. Moreover, expression of p27kip1 of cells was increased and expression of survivin was decreased. Our results suggest that MG-132 inhibits telomerase activity, induces apoptosis and G1 arrest which is associated with upregulated p27kip1 expression and downregulated survivin expression in gastric carcinoma cells.

Two different stages of epidermal growth factor (EGF) receptor endocytosis are sensitive to free ubiquitin depletion produced by proteasome inhibitor MG132

Numerous studies implicate proteasomes in the regulation of EGF receptor (EGFR) endocytosis on the basis of the ability of inhibitors to decrease EGFR degradation, but the exact mechanisms remain obscure. We demonstrated that EGFR itself is not a direct target for proteasome, since it is delivered to lysosomes intact. Evidence is presented that the inhibitory effect of MG132 on EGF degradation is due mostly to free ubiquitin depletion resultant from the suppression of proteasomal functioning by MG132. By subcellular fractionation, we show two MG132-sensitive steps in the EGFR degradation pathway: sorting from early (EE) to late (LE) endosomes, and late stage of LE maturation. MG132 treatment resulted in stabilization of EGFR tyrosine phosphorylation and its association with c-Cbl. Nevertheless, ubiquitination of EGFR at late stages of endocytosis was significantly lower than that in control cells. Highly ubiquitinated forms of EGFR demonstrated more sensitivity to MG132 treatment.

Proteasomal Proteolysis in the Adrenergic Induction of Arylalkylamine-N-Acetyltransferase in Rat Pinealocytes

In this study, we investigated the effect of proteasomal inhibition on the induction of arylalkylamine-N-acetyltransferase (AA-NAT) enzyme in cultured rat pinealocytes, using two proteasome inhibitors, MG132 and clastolactacystin beta-lactone (c-lact). Addition of c-lact or MG132 3 h after norepinephrine (NE) stimulation produced a significant increase in AA-NAT protein level and enzyme activity. However, when the proteasome inhibitors were added before or together with NE, significant reductions of the NE-induced aa-nat mRNA, protein, and enzyme activity were observed. A similar inhibitory effect of MG132 on aa-nat transcription was observed when cells were stimulated by dibutyryl cAMP, indicating an effect distal to a post-cAMP step. The inhibitory effect of MG132 on adrenergic-induced aa-nat transcription was long lasting because it remained effective after 14 h of washout and appeared specific for aa-nat because the induction of another adrenergic-regulated gene, MAPK phosphatase-1, by NE was not affected. Time-profile studies revealed that the inhibitory effect of MG132 on NE-stimulated aa-nat induction was detected after 1 h, suggesting accumulation of a protein repressor as a possible mechanism of action. This possibility was also supported by the finding that the inhibitory effect of c-lact on NE-induced aa-nat induction was markedly reduced by cycloheximide, a protein synthesis inhibitor. Together, these results support an important role of proteasomal proteolysis in the adrenergic-mediated induction of aa-nat transcription through its effect on a protein repressor.

Nuclear factor-kappa B is required for tumor necrosis factor-alpha-induced manganese superoxide dismutase expression in human endometrial stromal cells.

We recently found that manganese superoxide dismutase (Mn-SOD) is up-regulated by TNF alpha at the transcription level in human endometrial stromal cells (ESC) and that TNF alpha-induced Mn-SOD expression is mediated by protein kinase C (PKC)-dependent phosphorylation. This study was undertaken to investigate whether nuclear factor-kappa B (NF-kappa B), a transcription factor, is involved in Mn-SOD induction by TNF alpha or PKC in human ESC. Electrophoretic mobility shift assay revealed that TNF alpha (1 ng/ml) and phorbol 12-myristate 13-acetate (TPA; 0.4 micro M), PKC activator, caused marked increases in nuclear NF-kappa B DNA binding activity. Secondly, ESC were incubated with MG132 (proteasome inhibitor) or SN50 (inhibitor of translocation of NF-kappa B into the nucleus) in the presence of TNF alpha or TPA. TNF alpha and TPA significantly increased Mn-SOD activities and Mn-SOD mRNA levels, and those effects were completely inhibited by MG132 and SN50. TNF alpha alone caused no effect on cell viability, but in the presence of MG132, TNF alpha significantly decreased cell viability. This inhibitory effect of MG132 was blocked by simultaneous addition of N-acetyl-L-cysteine, an antioxidant. In conclusion, the present study showed the involvement of NF-kappa B in Mn-SOD induction by TNF alpha or PKC in human ESC. This phenomenon could be a self-defense system of ESC against TNF alpha-mediated oxidative stress.