calpain-inhibitor-iii and lactacystin

The dysfunction of the proteasome system is implicated in the pathomechanism of several chronic neurodegenerative diseases. Lactacystin (LC), an irreversible proteasome inhibitor, induces cell death in primary cortical neurons, however, the molecular mechanisms of its neurotoxic action has been only partially unraveled. In this study we aimed to elucidate an involvement of the key enzymatic pathways responsible for LC-induced neuronal cell death. Incubation of primary cortical neurons with LC (0.25-50 μg/ml) evoked neuronal cell death in concentration- and time-dependent manner. Lactacystin (2.5 μg/ml; 6.6μM) enhanced caspase-3 activity, but caspase-3 inhibitor, Ac-DEVD-CHO did not attenuate the LC-evoked cell damage. Western blot analysis showed a time-dependent, prolonged activation of MAPK/ERK1/2 pathway after LC exposure. Moreover, inhibitors of MAPK/ERK1/2 signaling, U0126 and PD98052 attenuated the LC-evoked cell death. We also found that LC-treatment resulted in the induction of calpains and calpain inhibitors (MDL28170 and calpeptin) protected neurons against the LC-induced cell damage. Neuroprotective action of MAPK/ERK1/2 and calpain inhibitors were connected with attenuation of LC-induced DNA fragmentation measured by Hoechst 33342 staining and TUNEL assay. However, only MAPK/ERK1/2 but not calpain inhibitors, attenuated the LC-induced AIF (apoptosis inducing factor) release. Further studies showed no synergy between neuroprotective effects of MAPK/ERK1/2 and calpain inhibitors given in combination when compared to their effects alone. The obtained data provided evidence for neuroprotective potency of MAPK/ERK1/2 and calpain, but not caspase-3 inhibition against the neurotoxic effects of LC in primary cortical neurons and give rationale for using these inhibitors in the treatment of neurodegenerative diseases connected with proteasome dysfunction.

Topics: Acetylcysteine; Animals; Apoptosis; Blotting, Western; Butadienes; Calpain; Caspase 3; Caspase Inhibitors; Cells, Cultured; Cerebral Cortex; Cysteine Proteinase Inhibitors; Cytoprotection; Dipeptides; Dose-Response Relationship, Drug; Enzyme Activation; Gestational Age; In Situ Nick-End Labeling; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurons; Neuroprotective Agents; Nitriles; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Kinase Inhibitors; Signal Transduction; Spectrin; Time Factors

p120-catenin contributes to the cadherin-mediated adhesion and aggregation of cells. mu-Calpain was activated and p120-catenin was degraded after 36 h of ischemia in differentiated SH-SY5Y cells. Calpain inhibitors Cbz-Val-Phe-H (MDL28170, 20 microM) and N-acetyl-leucyl-leucyl-norleucinal (ALLN, 20 microM) increased the levels of dephosphorylated p120-catenin, aggregation, and cell survival as detected by reduced LDH release in ischemic cells. However, a proteasome inhibitor lactacystin had no such effects. This is the first report of the calpain-mediated degradation of p120-catenin and an association between the level of dephosphorylated p120-catenin and cell aggregation in ischemic neuronal cells.

Topics: Acetylcysteine; Calpain; Catenins; Cell Adhesion Molecules; Cell Aggregation; Cell Death; Cell Line, Tumor; Delta Catenin; Dipeptides; Humans; Ischemia; Leupeptins; Neuroblastoma; Phosphoproteins

To firmly establish the pathway involved in ligand-induced degradation of the AHR, cell lines derived from mouse rat or human tissues were exposed to inhibitors specific to the proteasome or calpain proteases and exposed to TCDD. The level of endogenous AHR and CYP1A1 protein was then evaluated by quantitative Western blotting. Treatment of cells with the calpain inhibitors: calpeptin, calpain inhibitor III, or PD150606 either individually or in combinations up to 75 microM did not reduce TCDD-induced degradation of the AHR, the induction of endogenous CYP1A1 or the nuclear accumulation of the AHR. The activity of the inhibitors was verified with an in vivo calpain assay. In contrast, exposure of cells to the specific proteasome inhibitors: epoxomicin (1-5 microM), proteasome inhibitor I (5-10 microM) or lactacystin (5-15 microM) completely inhibited TCDD-induced degradation of the AHR. Inhibition of AHR degradation with these compounds did not reduce the induction of endogenous CYP1A1. In addition, exposure of the Hepa-1 line to the various proteasome inhibitors caused an accumulation of the AHR in the nucleus in the absence of TCDD exposure. Finally, Western blot analysis of the DNA bound AHR showed that its molecular mass was unchanged in comparison to the unliganded (cytoplasmic) AHR. Thus, these studies conclusively implicate the proteasome and not calpain proteases in the ligand-induced degradation of the mouse, rat and human AHR and suggest that the pharmacological use of proteasome inhibitors may impact the time course and magnitude of gene regulatory events mediated through the AHR.

Topics: Acetylcysteine; Acrylates; Animals; Cell Line, Tumor; Cell Nucleus; Cysteine Proteinase Inhibitors; Cytochrome P-450 CYP1A1; Dipeptides; Dose-Response Relationship, Drug; Down-Regulation; Drug Antagonism; Drug Combinations; Environmental Pollutants; Humans; Mice; Oligopeptides; Polychlorinated Dibenzodioxins; Rats; Receptors, Aryl Hydrocarbon