Proteasome inhibition induces <Emphasis Type="Italic">hsp30</Emphasis> and <Emphasis Type="Italic">hsp70</Emphasis> gene expression as well as the acquisition of thermotolerance in <Emphasis Type="Italic">Xenopus laevis</Emphasis> A6 cells

Previous studies have shown that inhibiting the activity of the proteasome leads to the accumulation of damaged or unfolded proteins within the cell. In this study, we report that proteasome inhibitors, lactacystin and carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132), induced the accumulation of ubiquitinated proteins as well as a dose- and time-dependent increase in the relative levels of heat shock protein (HSP)30 and HSP70 and their respective mRNAs in Xenopus laevis A6 kidney epithelial cells. In A6 cells recovering from MG132 exposure, HSP30 and HSP70 levels were still elevated after 24 h but decreased substantially after 48 h. The activation of heat shock factor 1 (HSF1) may be involved in MG132-induced hsp gene expression in A6 cells since KNK437, a HSF1 inhibitor, repressed the accumulation of HSP30 and HSP70. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. Immunocytochemical studies determined that HSP30 was localized primarily in the cytoplasm of lactacystin- or MG132-treated cells. In some cells treated with higher concentrations of MG132 or lactacystin, we observed in the cortical cytoplasm (1) relatively large HSP30 staining structures, (2) colocalization of actin and HSP30, and (3) cytoplasmic areas that were devoid of HSP30. Lastly, MG132 treatment of A6 cells conferred a state of thermotolerance such that they were able to survive a subsequent thermal challenge.     

Inhibition of the ubiquitin-proteasome pathway induces differential heat-shock protein response in cardiomyocytes and renders early cardiac protection

The effects of proteasome inhibition (PI) on heat-shock protein (HSP) expression in cardiomyocytes were investigated. Neonatal rat cardiomyocytes were incubated with MG132 (0.1-10 microM) for 1 h. Induction of various HSPs was determined by real-time PCR and Western blotting. PI induced a 2- to 3-fold increase in HSP27, HSP60, and HSP90, and a 18-fold increase in HSP70 mRNA expression, whereas HSP40 levels were unaffected. Western blotting revealed increased protein expression for HSP70 after PI. Similar results were obtained with MG262. HSP induction correlated with enhanced survival of neonatal cardiomyocytes after sublethal heat stress in XTT testing. In papillary muscles, pretreatment with MG132 (10 microM, 90 min) was associated with enhanced recovery of the contractile parameters after a 40-min hypoxia. In these proof-of-principle experiments, we show that PI induces differential heat-shock response in cardiomyocytes, accompanied by enhanced cell survival and functional recovery after various forms of stress.     

Identification of HSP90 as a new GABARAPL1 (GEC1)-interacting protein

GABARAPL1 belongs to the small family of GABARAP proteins (including GABARAP, GABARAPL1 and GABARAPL2/GATE-16), one of the two subfamilies of the yeast Atg8 orthologue. GABARAPL1 is involved in the intracellular transport of receptors, via an interaction with tubulin and GABA(A) or kappa opioid receptors, and also participates in autophagy and cell proliferation. In the present study, we identify the HSP90 protein as a novel interaction partner for GABARAPL1 using GST pull-down, mass spectrometry and coimmunoprecipitation experiments. GABARAPL1 and HSP90 partially colocalize in MCF-7 breast cancer cells overexpressed Dsred-GABARAPL1 and in rat brain. Moreover, treatment of MCF-7 cells overexpressed FLAG-GABARAPL1-6HIS with the HSP90 inhibitor 17-AAG promotes the GABARAPL1 degradation, a process that is blocked by proteasome inhibitors such as MG132, bortezomib and lactacystin. Accordingly, we demonstrate that HSP90 interacts and protects GABARAPL1 from its degradation by the proteasome.     

Treatment with the Proteasome Inhibitor MG132 during the End of Oocyte Maturation Improves Oocyte Competence for Development after Fertilization in Cattle

Maturation of the oocyte involves nuclear and cytoplasmic changes that include post-translational processing of proteins. The objective was to investigate whether inhibition of proteasomes during maturation would alter competence of the bovine oocyte for fertilization and subsequent development. Cumulus-oocyte complexes were cultured in the presence or absence of the proteasomal inhibitor MG132 from either 0–6 h or 16–22 h after initiation of maturation. Treatment with MG132 early in maturation prevented progression to meiosis II and reduced fertilization rate and the proportion of oocytes and cleaved embryos that became blastocysts. Conversely, treatment with MG132 late in maturation improved the percentage of oocytes and cleaved embryos that became blastocysts without affecting nuclear maturation or fertilization rate. Optimal results with MG132 were achieved at a concentration of 10 µM – effects were generally not observed at lower or higher concentrations. Using proteomic analysis, it was found that MG132 at the end of maturation increased relative expression of 6 proteins and decreased relative expression of 23. Among those increased by MG132 that are potentially important for oocyte competence are GAPDH, involved in glycolysis, TUBA1C, needed for organellar movement, and two proteins involved in protein folding (P4HB and HYOU1). MG132 decreased amounts of several proteins that exert anti-apoptotic actions including ASNS, HSP90B1, PDIA3 and VCP. Another protein decreased by MG132, CDK5, can lead to apoptosis if aberrantly activated and one protein increased by MG132, P4HB, is anti-apoptotic. Finally, the pregnancy rate of cows receiving embryos produced from oocytes treated with MG132 from 16–22 h of maturation was similar to that for control embryos, suggesting that use of MG132 for production of embryos in vitro does not cause a substantial decrease in embryo quality.     

Extracellular heat shock protein HSP90β secreted by MG63 osteosarcoma cells inhibits activation of latent TGF-β1

Transforming growth factor-beta 1 (TGF-β1) is secreted as a latent complex, which consists of latency-associated peptide (LAP) and the mature ligand. The release of the mature ligand from LAP usually occurs through conformational change of the latent complex and is therefore considered to be the first step in the activation of the TGF-β signaling pathway. So far, factors such as heat, pH changes, and proteolytic cleavage are reportedly involved in this activation process, but the precise molecular mechanism is still far from clear. Identification and characterization of the cell surface proteins that bind to LAP are important to our understanding of the latent TGF-β activation process. In this study, we have identified heat shock protein 90 β (HSP90β) from the cell surface of the MG63 osteosarcoma cell line as a LAP binding protein. We have also found that MG63 cells secrete HSP90β into extracellular space which inhibits the activation of latent TGF-β1, and that there is a subsequent decrease in cell proliferation. TGF-β1-mediated stimulation of MG63 cells resulted in the increased cell surface expression of HSP90β. Thus, extracellular HSP90β is a negative regulator for the activation of latent TGF-β1 modulating TGF-β signaling in the extracellular domain.     

The proteasome inhibitor MG132 protects against acute pancreatitis

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 x 100 microug/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 IkappaB degradation and subsequent activation of nuclear factor-kappaB (NF-kappaB). MG132 also increased HSP72 expression. Induction of HSP72 and inhibition of NF-kappaB 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.     

Is Mongolian gerbil really adequate host animal for study of Helicobacter pylori infection-induced gastritis and cancer?

BACKGROUND: Many researches have been published to understand the pathogenesis and mechanism of Helicobacter pylori (Hp)-associated diseases, including gastritis followed by gastric cancer, using Mongolian gerbil (MG) model because Hp could be hardly inoculated in other animal species. The aim of this study was to evaluate the induction ability of heat shock protein (HSP70) and protective ability in the gastric mucosa of MG comparing with those of Sprague-Dawley (SD) rats, since HSP70 is a key molecule known to be involved in important biological activities such as apoptosis, carcinogenesis, and cytoprotection from cytotoxic damage. MATERIALS AND METHODS: Basal expression level and induction ability of gastric mucosal HSP70 were evaluated by immunoblotting and densitometric analysis in MG and SD rats before and after HSP-induction by zinc l-carnosine, gastric HSP70 inducer, administration. Mucosal protective ability against water-immersion stress-induced mucosal lesion was also compared. RESULTS: Basal expression level of HSP70 was not significantly different between MG and SD rats. However, HSP70-induction by zinc derivatives was not observed in MG. Mucosal lesion induced by water-immersion stress was significantly severe in MG compared with SD rats. CONCLUSIONS: MG might be special (not ordinary) animal, in which HSP70-induction was absent and has extremely poor mucosal protective ability in view of HSP-dependent cytoprotection in the gastric mucosa. Our results may suggest that MG is not an adequate animal to evaluate the effect of Hp-infection-associated gastric inflammation followed by development of gastric cancer.     

Sulforaphane attenuates postnatal proteasome inhibition and improves spatial learning in adult mice

Proteasomes are known to degrade proteins involved in various processes like metabolism, signal transduction, cell-cycle regulation, inflammation, and apoptosis. Evidence showed that protein degradation has a strong influence on developing neurons as well as synaptic plasticity. Here, we have shown that sulforaphane (SFN) could prevent the deleterious effects of postnatal proteasomal inhibition on spatial reference and working memory of adult mice. One day old Balb/c mice received intracerebroventricular injections of MG132 and SFN. Sham received an equal volume of aCSF. We observed that SFN pre-administration could attenuate MG132 mediated decrease in proteasome and calpain activities. In vitro findings revealed that SFN could induce proteasomal activity by enhancing the expression of catalytic subunit-β5. SFN pre-administration prevented the hippocampus based spatial memory impairments during adulthood, mediated by postnatal MG132 exposure. Histological examination showed deleterious effects of MG132 on pyramidal neurons and granule cell neurons in DG and CA3 sub-regions respectively. Furthermore, SFN pre-administration has shown to attenuate the effect of MG132 on proteasome subunit-β5 expression and also induce the Nrf2 nuclear translocation. In addition, SFN pre-administered mice have also shown to induce expression of pCaMKII, pCreb, and mature/pro-Bdnf, molecules which play a crucial role in spatial learning and memory consolidation. Our findings have shown that proteasomes play an important role in hippocampal synaptic plasticity during the early postnatal period and SFN pre-administration could enhance the proteasomal activity as well as improve spatial learning and memory consolidation.     

Reduction of HSP70 and HSC70 Heat Shock mRNA Induction by Pentobarbital After Transient Global Ischemia in Gerbil Brain

Abstract: The effect of pentobarbital on the induction of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs after transient global ischemia in gerbil brains was investigated by in situ hybridization using cloned cDNA probes selective for each mRNA species. In sham control brains, HSP70 mRNA was scarcely present, whereas HSC70 mRNA was present in most cell populations. After a 5-min occlusion of bilateral common carotid arteries, HSP70 and HSC70 mRNAs were induced together in several cells and were especially dense in hippocampal dentate granule cells at 3 h, but the strong hybridization of the mRNAs continued only in hippocampal CA1 cells by 2 days. At 7 days after the ischemia, CA1 neuronal cell death was apparent, and the HSP70 mRNA disappeared and HSC70 mRNA content returned to the sham level, except for in the CA1 cells. Pretreatment with pentobarbital (40 mg/kg, i.p.) greatly reduced or inhibited the induction of HSP70 and HSC70 mRNAs at both early (3-h) and late (2-day) phases after ischemia. The drug also prevented CA1 cell death at 7 days along with the maintenance of expression of HSC70 mRNA at the sham control level. Hypothermic effects of pentobarbital were noted at 30 and 60 min after the reperfusion, whereas at 2 h there was no statistical significance between the control and drug-treated groups. The great reduction of HSP70 and HSC70 mRNA induction at both early and late phases after ischemia suggests that pentobarbital reduces intra- and/or postischemic stress and may protect CA1 cells from ischemic damage. These effects of the drug may be mainly due to its specific action rather than its hypothermic effects.     

Sodium arsenite and cadmium chloride induction of proteasomal inhibition and HSP accumulation in Xenopus laevis A6 kidney epithelial cells

Sodium arsenite (NA) and cadmium chloride (CdCl(2)) are relatively abundant environmental toxicants that have multiple toxic effects including carcinogenesis, dysfunction of gene regulation and DNA and protein damage. In the present study, treatment of Xenopus laevis A6 kidney epithelial cells with concentrations of NA (20-30 μM) or CdCl(2) (100-200 μM) that induced HSP30 and HSP70 accumulation also produced an increase in the relative levels of ubiquitinated protein. Actin protein levels were unchanged in these experiments. In time course experiments, the levels of ubiquitinated protein and HSPs increased over a 24h exposure to NA or CdCl(2). Furthermore, treatment of cells with NA or CdCl(2) reduced the relative levels of proteasome chymotrypsin (CT)-like activity compared to control. Interestingly, pretreatment of cells with the HSP accumulation inhibitor, KNK437, prior to NA or CdCl(2) exposure decreased the relative levels of ubiquitinated protein as well as HSP30 and HSP70. A similar finding was made with ubiquitinated protein induced by proteasomal inhibitors, MG132 and celastrol, known to induce HSP accumulation in A6 cells. However, the NA- or CdCl(2)-induced decrease in proteasome CT-like activity was not altered by KNK437 pretreatment. This study has shown for the first time in poikilothermic vertebrates that NA and CdCl(2) can inhibit proteasomal activity and that there is a possible association between HSP accumulation and the mechanism of protein ubiquitination.     

Sphingosine 1-Phosphate Regulates Heat Shock Protein 27 Induction by a p38 MAP Kinase-Dependent Mechanism in Aortic Smooth Muscle Cells

In an aortic smooth muscle cell line, A10 cells, we investigated the effect of sphingosine 1-phosphate on the induction of heat shock protein 27 (HSP27), a low-molecular-weight heat shock protein. Sphingosine 1-phosphate significantly induced the accumulation of HSP27 in a pertussis toxin-sensitive manner. The effect was dose-dependent in the range between 0.1 and 30 microM. Sphingosine 1-phosphate stimulated an increase in the levels of mRNA for HSP27. Sphingosine 1-phosphate stimulated both p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase activation. PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase, did not affect sphingosine 1-phosphate-stimulated HSP27 induction. In contrast, SB203580, an inhibitor of p38 MAP kinase, reduced sphingosine 1-phosphate-induced HSP27 induction. SB203580 reduced the levels of mRNA for HSP27 induced by sphingosine 1-phosphate. These results indicate that sphingosine 1-phosphate stimulates the induction of HSP27 via p38 MAP kinase activation in aortic smooth muscle cells.     

Expression of HSP72 in the gastric mucosa is regulated by gastric acid in rats-correlation of HSP72 expression with mucosal protection

BACKGROUND AND AIM: The real mechanism of adaptive cytoprotection in the gastric mucosa is not well established. In the present study, we investigated the effect of acid suppressing agents on a 72-kDa heat shock protein (HSP72) expression, which is known as endogenous cytoprotective factor, in the gastric mucosa. Also, the association of gastric mucosal protective function against HCl-challenge was compared between HSP72-induced and -reduced group. MATERIALS AND METHODS: Expression of HSP72 was measured by Western blotting in the gastric mucosa before and after administration of famotidine or omeprazole. The gastric mucosal protective function against 0.6 N HCl was compared between control group and HSP72-reduced group. Also, the effect of increased expression of gastric HSP72 by additional administration of zinc sulfate or zinc L-carnosine, which is known as HSP72-inducer, on mucosal protective function was studied. RESULTS: HSP72 expression in the gastric mucosa was reduced by acid suppressing agents. The lowest expression level of HSP72 was observed 12 h (famotidine, H2-receptor antagonist) or 48 h (omeprazole, proton pump inhibitor) after administration. The gastric mucosal protective ability against 0.6 N HCl was also reduced when HSP72 expression was decreased by famotidine or omeprazole. This phenomenon was reversed by HSP72 induction by additional administration of zinc derivatives. CONCLUSION: Our results might indicate that the expression of HSP72 in the gastric mucosa is physiologically regulated by gastric acid, and that HSP72 induction could be important in view of mucosal protection especially when HSP72 expression is reduced by administration of acid suppressing agents such as proton pump inhibitor or H2 receptor antagonist.     

Heat-Induced Proteasomic Degradation of HSF1 in Serum-Starved Human Fibroblasts Aging in Vitro

The exposure of human fibroblasts (HF) aging in vitro to heat shock resulted in an attenuated expression of the heat shock-inducible HSP70. When late passage cells were cultured in the continuous presence of serum, we observed a reduced accumulation of the cytoplasmic polyadenylated HSP70 mRNA. The levels of HSF1 activation and nuclear HSP70 mRNA were comparable to those of early passage cells (M. A. Bonelli et al., Exp. Cell Res. 252, 20-32, 1999). When late passage cells were serum-starved overnight, we observed a reduced activation of HSF1 and a decreased level of HSP70 mRNA during heat shock. However, at 37 degrees C the levels of HSF1 differed little between late passage HF and early passage cells, irrespective of the presence of serum. Interestingly, during heat shock a marked decrease in the level and, consequently, in the binding activity of HSF1 was noted only in serum-starved, late passage HF. The decrease in the level of HSF1 was counteracted by back addition of serum to the cells during heat shock. Addition of the specific proteasome inhibitor MG132 blocked a decrease in HSF1 during heat shock, maintaining levels observed in late passage cells and HSF1 activity comparable to that of early passage HF. The recovery of the level and activity of HSF1 observed in late passage HF incubated in the presence of MG132 suggests that heat shock unmasks a latent proteasome activity responsible for HSF1 degradation.     

Mechanism of simvastatin on induction of heat shock protein in osteoblasts

It has recently been reported that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) stimulate bone formation. However, the mechanism of stimulation of bone metabolism by statins is not precisely clarified. In this study, we investigated whether simvastatin induces heat shock protein (HSP) 27, HSP70, and HSP90 in osteoblast-like MC3T3-E1 cells. Simvastatin increased the levels of HSP27 while having little effect on the levels of HSP70 or HSP90. The effect of simvastatin on HSP27 accumulation was dose dependent. Cycloheximide reduced the accumulation. Simvastatin induced an increase in the levels of mRNA for HSP27. Actinomycin D suppressed the mRNA levels. Simvastatin induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase among the MAP kinase superfamily. SB203580 and PD169316, inhibitors of p38 MAP kinase, suppressed the HSP27 accumulation by simvastatin while SB202474, a negative control of p38 MAP kinase inhibitor, had no effect. SB203580 reduced the simvastatin-increased mRNA levels for HSP27. Lovastatin, another statin, also induced the HSP27 accumulation and SB203580 suppressed the HSP27 accumulation. These results strongly suggest that statins such as simvastatin do not stimulate the induction of HSP70 and HSP90, but do stimulate the induction of HSP27 in osteoblasts and that p38 MAP kinase plays a role in this induction.