The proteasome inhibitor MG132 protects against acute pancreatitis

doi:10.1016/j.freeradbiomed.2005.06.003

Free Radical Biology and Medicine

Volume 39, Issue 9, 1 November 2005, Pages 1142-1151

https://doi.org/10.1016/j.freeradbiomed.2005.06.003 Get rights and content

Abstract

The cell-permeant MG132 tripeptide (Z-Leu-Leu-Leu-aldehyde) is a peptide aldehyde proteasome inhibitor that also inhibits other proteases, including calpains and cathepsins. By blocking the proteasome, this tripeptide has been shown to induce the expression of cell-protective heat shock proteins (HSPs) in vitro. Effects of MG132 were studied in an in vivo model of acute pancreatitis. Pancreatitis was induced in male Wistar rats by injecting 2 × 100 μg/kg cholecystokinin octapeptide intraperitoneally (ip) at an interval of 1 h. Pretreating the animals with 10 mg/kg MG132 ip before the induction of pancreatitis significantly inhibited IκB degradation and subsequent activation of nuclear factor-κB (NF-κB). MG132 also increased HSP72 expression. Induction of HSP72 and inhibition of NF-κB improved parameters of acute pancreatitis. Thus MG132 significantly decreased serum amylase, pancreatic weight/body weight ratio, pancreatic myeloperoxidase activity, proinflammatory cytokine concentrations, and the expression of pancreatitis-associated protein. Parameters of oxidative stress (GSH, MDA, SOD, etc.) were improved in both the serum and the pancreas. Histopathological examinations revealed that pancreatic specimens of animals pretreated with the peptide demonstrated milder edema, cellular damage, and inflammatory activity. Our findings show that simultaneous inhibition of calpains, cathepsins, and the proteasome with MG132 prevents the onset of acute pancreatitis.

Section snippets

Experimental protocol

For the in vivo studies male Wistar rats (provided by the Animal Center of the University of Szeged) weighing 250–300 g were used. The animals were kept at a constant room temperature with a 12-h light–dark cycle and were allowed free access to water and standard laboratory chow (Biofarm, Zagyvaszántó, Hungary). The animal experiments performed in this study were approved by the Animal Care Committee of the university and complied with the European Communities Council Directive of 24 November

Results

The following results show the effects of the MG132 tripeptide on the laboratory and histological parameters of CCK-induced pancreatitis. MG132 without any CCK injections did not significantly affect the measured parameters compared to controls (thus results are not shown).

Discussion

NF-κB plays a central role in inducing severe inflammatory diseases like acute pancreatitis [2], [3], [4], [5]. Activation of NF-κB can be inhibited by blocking the degradation of its inhibitor protein IκB. Inhibitors of IκB degradation have been shown to attenuate the severity of experimental acute pancreatitis [19], [20], [21], [22]. The tripeptide (Z-Leu-Leu-Leu-aldehyde) named MG132 is a potent inhibitor of a wide range of proteases, including calpains, cathepsins, and the proteasome [9],

Acknowledgments

This study was supported by the National Research Foundation (OTKA), Grants T30735 and T042589.

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MG132 is a cell permeable peptide aldehyde that reversibly antagonizes chymotrypsin-like proteasome reactions [70]. However, MG132, and other peptide aldehydes, may have off-target effects, such as calpains and lysosomal cysteine proteases, like cathepsins [71]. Furthermore, MG132 is rapidly metabolized by liver cytochrome P450 3A enzymes [72].
Impairing autophagy disrupts transforming growth factor beta 1 (TGFβ1) signalling and epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC). Since autophagy and proteasome-mediated degradation are interdependent, we investigated how prolonged downregulation of proteasomal catalytic activity affected TGFβ1-dependent signalling and EMT. Proteasome-dependent degradation was inhibited in A549 and H1299 NSCLC cells using MG132 and lactacystin, which are reversible and irreversible proteasome inhibitors, respectively. We observed that inhibiting proteasomal activity for 24 h decreased TGFβ-dependent nuclear accumulation of Smad2/3. Time course studies were then carried out to characterize the time frame of this observation. Short-term (< 8 h) proteasome inhibition resulted in increased receptor regulated Smad (R-Smad) phosphorylation and steady-state TGFβ receptor type II (TGFβRII) levels. However, prolonged (8–24 h) proteasome inhibition decreased TGFβ1-dependent R-Smad phosphorylation and steady-state TGFβRI and TGFβRII levels. Furthermore, proteasome inhibition blunted TGFβ-dependent E- to N-Cadherin shift, stress fiber formation, and increased cellular apoptosis via the TAK-1-TRAF6-p38 MAPK pathway. Interestingly, proteasome inhibition also increased autophagic flux, steady-state microtubule-associated protein light chain 3B-II and active uncoordinated 51-like autophagy activating kinase 1 levels, and co-localization of lysosomes with autophagy cargo proteins and autophagy-related proteins. Finally, we observed that proteasome inhibition increased TGFβRII endocytosis and trafficking to lysosomes and we conclude that prolonged proteasome inhibition disrupts TGFβ signalling outcomes through altered TGFβ receptor trafficking.
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Consequently, blocking PSMD4-dependent GPX4 degradation inhibits trypsin-enhanced ferroptosis in acinar cells. In fact, administration of the proteasome inhibitor MG132 prevents the onset of acute pancreatitis in mice,76,77 supporting the notion that the proteasomal degradation pathway is one of the therapeutic targets of pancreatitis. The depletion of pancreatic Gpx4 does not affect the normal development and physiological function of the pancreas, but exacerbates acute or chronic pancreatitis caused by pathologic (eg, cerulein) or clinically relevant stressors (eg, alcohol).32
Pancreatitis is characterized by acinar cell death and persistent inflammation. Ferroptosis is a type of lipid peroxidation-dependent necrosis, which is negatively regulated by glutathione peroxidase 4. We studied how trypsin, a serine protease secreted by pancreatic acinar cells, affects the contribution of ferroptosis to triggering pancreatitis.
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Supraphysiological doses of trypsin (500 or 1000 ng/mL) alone did not trigger significant cell death in 266-6 cells and mouse primary pancreatic acinar cells, but did increase the sensitivity of these cells to ferroptosis upon treatment with cerulein, L-arginine, alcohol, erastin, or RSL3. Proteasome 26S subunit, non–adenosine triphosphatase 4–dependent lipid peroxidation caused ferroptosis in pancreatic acinar cells by promoting the proteasomal degradation of glutathione peroxidase 4. The drug screening campaign identified the antipsychotic drug olanzapine as an antioxidant inhibiting ferroptosis in pancreatic acinar cells. Mice lacking pancreatic Gpx4 developed more severe pancreatitis after cerulein infection or ethanol feeding than control mice. Conversely, olanzapine administration protected against pancreatic ferroptotic damage and experimental pancreatitis in Gpx4-deficient mice.
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Inhibition of the proteasome may be an underlying mechanism of protection during the course of AP. Several proteasome inhibitors have been found and it has been demonstrated that its peptide aldehyde predecessors (such as MG-132) could protect against AP [40]. While, we chose bortezomib in our study because it is much more water soluble, potent and selective than MG132.
The proteasome is involved in the activation of NF-κB and can regulate the progression of inflammatory diseases. However, the role of proteasome in acute pancreatitis (AP) has not been demonstrated. In this study, we first observed that the protein level and activity of proteasome 20S were increased significantly in pancreatic injury tissues after caerulein-induced mild acute pancreatitis (MAP) induction, which was in consistent with the expression of the NF-κB nucleoprotein and positively correlated with the severity of AP. Then, bortezomib, a classical proteasome inhibitor, was used to intervene the progression of MAP in mice. The results showed that bortezomib administration reduced the serum amylase and lipase levels and mitigated histopathological manifestation of pancreatic injury in mice. Meanwhile, bortezomib decreased the expression of NF-κB p65 nucleoprotein as well as total proteasome 20S protein, and inhibited the activity of 20S in pancreatic tissues. In addition, we found that bortezomib could protect pancreatic acinar cell against necrosis and mitigate the severity of AP in a severe acute pancreatitis model induced by sodium taurocholate hydrate. Taken together, our study for the first time confirmed that the proteasome participated in the pathogenesis of AP and its inhibitor bortezomib could protect against AP in mice.
Injury-induced platelet-derived growth factor receptor-α expression mediated by interleukin-1β (IL-1β) release and cooperative transactivation by NF-κB and ATF-4. IL-1β facilitates HDAC-1/2 dissociation from promoter
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The use of inhibitors in our studies was pivotal in the identification of NF-κB in the transcriptional control of a PDGF receptor. MG132 has been widely used in cancer research (45, 46) and acute pancreatitis protection (47). BAY 11-7082, like MG132, has been used to define a causal role for NF-κB in diseases such as cancer (48), renal failure (49), and the regulation of stress signal transduction pathways (50).
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The proteosome inhibitor bortezomib is used in the treatment of patients with multiple myeloma. Proteosomes are responsible for the degradation of I-κB, the inhibitory protein of transcription factor nuclear factor kappa B (Nf-κB). The heat shock protein (HSP) inducing effect of bortezomib is also documented. The aim of our work was to test the anti-inflammatory effect of bortezomib in cholecystokinin-octapeptide (CCK-8)-induced acute pancreatitis. Male Wistar rats were divided into three groups (n = 8 in each). Group P received an i.p. injection of physiological saline (p.s.) 60 min. before the induction of acute pancreatitis by three hourly s.c. injections of 100 µg/kg CCK-8. Group BP received 1 mg/kg bortezomib dissolved in p.s. 1 h previous to pancreatitis induction. Group C was treated with the vehicle (p.s.). Animals were exsanguinated 4 h after the last injection of CCK-8. Bortezomib pre-treatment significantly reduced the pancreatic weight/body weight ratio, and improved the histology by decreasing the extent of vacuolization and infiltration. Bortezomib pre-treatment inhibited I-κBβ degradation, and induced the synthesis of HSP72. The results confirmed the anti-inflammatory effect of bortezomib in acute experimental pancreatitis. This effect of the drug is presumably mediated by the inhibition of Nf-κB activation and induction of HSP synthesis.
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Original ContributionThe proteasome inhibitor MG132 protects against acute pancreatitis

Abstract

Section snippets

Experimental protocol

Results

Discussion

Acknowledgments

Early events in acute pancreatitis

Baillieres Best Pract. Res., Clin. Gastroenterol.

From acinar cell damage to systemic inflammatory response: current concepts in pancreatitis

Pancreatology

Acute experimental pancreatitis and NF-kappaB/Rel activation

Pancreatology

Acute pancreatitis

Curr. Opin. Gastroenterol.

Pathophysiology of acute pancreatitis

Pancreatology

Inflammatory mediators in acute pancreatitis

J. Pathol.

NF-kappaB and cytokines in pancreatic acinar cells

J. Korean Med. Sci.

Cytokine storm in acute pancreatitis

J. Hepatobiliary Pancreatic Surg.

Proteasome inhibitors: valuable new tools for cell biologists

Trends Cell Biol.

Proteasome inhibition: a new anti-inflammatory strategy

J. Mol. Med.

Nuclear factor kappa B: a pivotal role in the systemic inflammatory response syndrome and new target for therapy

Intensive Care Med.

Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis

J. Clin. Invest.

Cathepsin B inhibition prevents trypsinogen activation and reduces pancreatitis severity

Am. J. Physiol.: Gastrointest. Liver Physiol.

Early events in acute pancreatitis

Clin. Lab. Med.

Selective inhibition of NF-kappaB attenuates the severity of cerulein-induced acute pancreatitis

J. Am. Coll. Surg.

Pyrrolidine dithiocarbamate reduces the severity of cerulein-induced murine acute pancreatitis

Shock

Role of nuclear factor-kappaB, reactive oxygen species and cellular signaling in the early phase of acute pancreatitis

Scand. J. Gastroenterol.

A nuclear import inhibitory peptide ameliorates the severity of cholecystokinin-induced acute pancreatitis

World J. Gastroenterol.

Inhibition of ubiquitin–proteasome pathway-mediated I kappa B alpha degradation by a naturally occurring antibacterial peptide

J. Clin. Invest.

Cerulein upregulates ICAM-1 in pancreatic acinar cells, which mediates neutrophil adhesion to these cells

Am. J. Physiol.: Gastrointest. Liver Physiol.

Dysregulation of the calpain–calpastatin system plays a role in the development of cerulein-induced acute pancreatitis in the rat

Am. J. Physiol.: Gastrointest. Liver Physiol.

Calpain I inhibitor ameliorates the indices of disease severity in a murine model of cerulein-induced acute pancreatitis

Intensive Care Med.

Proinflammatory effects of pancreatic elastase are mediated through TLR4 and NF-κB

Biochem. Biophys. Res. Commun.

Proteasome inhibition leads to a heat-shock response, induction of endoplasmic reticulum chaperones, and thermotolerance

J. Biol. Chem.

Proteasome inhibitors cause induction of heat shock proteins and trehalose, which together confer thermotolerance in Saccharomyces cerevisiae

Mol. Cell. Biol.

Evidence for involvement of the proteasome complex (26S) and NFkappaB in IL-1beta-induced nitric oxide and prostaglandin production by rat islets and RINm5F cells

Diabetes

Inflammatory-mediated repression of the rat ileal sodium-dependent bile acid transporter by c-fos nuclear translocation

Gastroenterology

Aberrant Toll receptor expression and endotoxin hypersensitivity in mice lacking a functional TGF-beta 1 signaling pathway

J. Immunol.

Original Contribution
The proteasome inhibitor MG132 protects against acute pancreatitis