Dobutamine mediates cytoprotection by induction of heat shock protein 70 in vitro

Aims

Dobutamine is cytoprotective when applied before a subsequent stress. However, the underlying molecular mechanism is unknown. Dobutamine also inhibits nuclear factor (NF)-κB in human T lymphocytes. Other inhibitors of NF-κB induce a so-called heat shock response. We hypothesized that dobutamine mediates protection from apoptotic cell death by the induction of a heat shock response.

Main methods

Jurkat T lymphoma cells were preincubated with dobutamine (0.1, 0.5 mM) before the induction of apoptosis (staurosporine, 2 μM). DNA-binding of heat shock factor (HSF)-1 was analyzed by electrophoretic mobility shift assay, mRNA-expression of heat shock protein (hsp)70 and hsp90 by Northern Blot, activity of caspase-3 by fluorogenic caspase activity assay and cleavage of pro-caspase-3 by Western Blot. Apoptosis was assessed by flow cytometry after annexin V-fluorescein isothiocyanate staining. Hsp70 and hsp90 were inhibited using N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolaetam and 17-allylamino-17-demethoxygeldana-mycin, respectively. All data are given as median and 25/75% percentile.

Key findings

Pre-incubation with dobutamine inhibited staurosporine-induced annexin V-fluorescence (28 [20–32] % vs. 12 [9–15] % for dobutamine 0.1 mM and 7 [5–12] % for dobutamine 0.5 mM, p < 0.001), cleavage of pro-caspase-3 as well as caspase-3-like activity (0.46 [0.40–0.48] vs. 0.32 [0.27–0.39] for Dobutamine 0.1 mM and 0.20 [0.19–0.23] for Dobutamine 0.5 mM, p < 0.01). Dobutamine induced DNA-binding of HSF-1 and mRNA-expression of hsp70 and hsp90. While inhibition of Hsp90 had no effect, inhibition of Hsp70 increased the number of annexin V-positive cells (33 [32–36] % vs. 18 [16–24] %) and caspase-3-like activity (0.21 [0.19–0.23] vs. 0.16 [0.13–0.17], p < 0.05).

Significance

Dobutamine protects from apoptotic cell death via the induction of Hsp70.     

The effect of dietary lipid manipulation on murine splenic lymphocytes apoptosis and heat shock protein over expression

In this study, we kept BALB/c mice on a hyperlipidic diet for 120 days and then assessed the predisposition to apoptosis and the appearance of heat shock protein (Hsp) on splenic lymphocytes. By immunoblot analysis, bands corresponding to Hsp 60 and Hsp 70 in cells from mice kept on a saturated fatty acid diet showed a greater expression already after 1 month while two other bands, which correspond to Hsp 25 and Hsp 27, were slightly present after 1 month of treatment. In cells from mice kept on a diet rich in unsaturated fatty acid, there was a marked expression of Hsp 25 and Hsp 27 after only 30 days of treatment, which was maintained constant for up to 4 months; while for bands corresponding to Hsp 60 and Hsp 70, a significant minor signal was only detectable after 2-4 months from the beginning of the treatment. Splenic lymphocytes from animals kept on a lipidic diet containing saturated fatty acids were more susceptible to death by apoptosis, while cells of animals treated with unsaturated fatty acid were shown to be more resistant.     

Intracellular localization of Xenopus small heat shock protein, hsp30, in A6 kidney epithelial cells

Small heat shock proteins (shsps) are molecular chaperones that are inducible by environmental stress. In this study, immunocytochemical analysis and laser scanning confocal microscopy revealed that the shsp family, hsp30, was localized primarily in the cytoplasm of Xenopus A6 kidney epithelial cells after heat shock or sodium arsenite treatment. Heat shock-induced hsp30 was enriched in the perinuclear region with some immunostaining in the nucleus but not in the nucleolus. In sodium arsenite-treated cells hsp30 was enriched towards the cytoplasmic periphery as well as showing some immunostaining in the nucleus. At higher heat shock temperatures (35 degrees C) or after 10 microM sodium arsenite treatment, the actin cytoskeleton displayed some disorganization that co-localized with areas of hsp30 enrichment. Treatment of A6 cells with 50 microM sodium arsenite induced a collapse of the cytoskeleton around the nucleus. These results coupled with previous studies suggest that stress-inducible hsp30 acts as a molecular chaperone primarily in the cytoplasm and may interact with cytoskeletal proteins.     

Overexpression of uncoupling protein 4 promotes proliferation and inhibits apoptosis and differentiation of preadipocytes

Uncoupling proteins are a family of mitochondrial proteins involved in energy metabolism. We previously showed that uncoupling protein 4 (UCP4) is differentially expressed in omental adipose tissue in diet-induced obese and normal rats. However, the effect of UCP4 on adipocytes is unclear. In this work, we established a stable preadipocyte cell line overexpressing UCP4 to observe the direct effect of UCP4 on adipocytes. Cells overexpressing UCP4 showed significantly attenuated differentiation of preadipocytes into adipocytes. During differentiation, expression of adipogenesis-associated markers such as fatty acid synthetase, peroxisome proliferator-activated receptor gamma, CCAAT enhancer binding protein alpha, adipocyte lipid binding protein and lipoprotein lipase were downregulated. Preadipoctes expressing UCP4 grew faster and more of them stayed in S phase compared to control cells. In addition, UCP4 overexpression protected preadipocytes from apoptosis induced by serum deprivation. Our results demonstrate that overexpression of UCP4 can promote proliferation and inhibit apoptosis and differentiation of preadipocytes.     

Heat shock protein responses to aging and proteotoxicity in the olfactory bulb

The olfactory bulb is one of the most vulnerable brain regions in age‐related proteinopathies. Proteinopathic stress is mitigated by the heat shock protein (Hsp) family of chaperones. Here, we describe age‐related decreases in Hsc70 in the olfactory bulb of the female rat and higher levels of Hsp70 and Hsp25 in middle and old age than at 2–4 months. To model proteotoxic and oxidative stress in the olfactory bulb, primary olfactory bulb cultures were treated with the proteasome inhibitors lactacystin and MG132 or the pro‐oxidant paraquat. Toxin‐induced increases were observed in Hsp70, Hsp25, and Hsp32. To determine the functional consequences of the increase in Hsp70, we attenuated Hsp70 activity with two mechanistically distinct inhibitors. The Hsp70 inhibitors greatly potentiated the toxicity of sublethal lactacystin or MG132 but not of paraquat. Although ubiquitinated protein levels were unchanged with aging in vivo or with sublethal MG132 in vitro, there was a large, synergistic increase in ubiquitinated proteins when proteasome and Hsp70 functions were simultaneously inhibited. Our study suggests that olfactory bulb cells rely heavily on Hsp70 chaperones to maintain homeostasis during mild proteotoxic, but not oxidative insults, and that Hsp70 prevents the accrual of ubiquitinated proteins in these cells.

     

Gene networks involved in apoptosis induced by hyperthermia in human lymphoma U937 cells

To define the molecular mechanisms that mediate hyperthermia-induced apoptosis, we performed microarray and computational gene expression analyses. U937 cells, a human myelomonocytic lymphoma cell line, were treated with hyperthermia at 42 °C for 90 min and cultured at 37 °C. Apoptotic cells (∼15%) were seen 6 h after hyperthermic treatment, and elevated expression of heat shock proteins (HSPs) including Hsp27, Hsp40, and Hsp70 was detected, following the activation of heat shock factor-1. Of the 54,675 probe sets analyzed, 1334 were upregulated and 4214 were downregulated by >2.0-fold in the cells treated with hyperthermia. A non-hierarchical gene clustering algorithm, K-means clustering, demonstrated 10 gene clusters. The gene network U1 or U2 that was obtained from up-regulated genes in cluster I or IX contained HSPA1B, DNAJB1, HSPH1, and TXN or PML, LYN, and DUSP1, and were mainly associated with cellular compromise, and cellular function and maintenance or death, and cancer, respectively. In the decreased gene cluster II, the gene network D1 including CCNE1 and CEBPE was associated with the cell cycle and cellular growth and proliferation. These findings will provide a basis for understanding the detailed molecular mechanisms of apoptosis induced by hyperthermia at 42 °C in cells.     

Regulation of heat shock-induced apoptosis by sensitive to apoptosis gene protein

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Sensitive to apoptosis gene (SAG) protein, a novel zinc RING finger protein that protects mammalian cells from apoptosis by redox reagents, is a metal chelator and a potential reactive oxygen species scavenger, but its antioxidant properties have not been completely defined. In this report, we demonstrate that modulation of SAG expression in U937 cells regulates heat shock-induced apoptosis. When we examined the protective role of SAG against heat shock-induced apoptosis with U937 cells transfected with the cDNA for SAG, a clear inverse relationship was observed between the amount of SAG expressed in target cells and their susceptibility to apoptosis. We also observed a significant decrease in the endogenous production of reactive oxygen species and oxidative DNA damage in SAG-overexpressed cells compared to control cells on exposure to heat shock. In addition, transfection of PC3 cells with SAG small interfering RNA markedly decreased the expression of SAG, enhancing the susceptibility of heat shock-induced apoptosis. Taken together, these results indicate that SAG may play an important role in regulating the apoptosis induced by heat shock presumably through maintaining the cellular redox status.     

Potential roles of hepatic heat shock protein 25 and 70i in protection of mice against acetaminophen-induced liver injury

The aim of the present study was to assess the contribution of the level of expression of heat shock protein 25 (HSP25), 60 (HSP60), 70 (HSC70) and 70i (HSP70i) in mouse livers after a lethal dose of acetaminophen (APAP) to their survival. We examined changes in survival ratio, plasma APAP level and alanine aminotransferase (ALT) activity, and hepatic reduced glutathione (GSH), HSP25, HSP60, HSC70 and HSP70i levels following treatment of mice with APAP (500 mg/kg, p.o.). The plasma APAP level increased rapidly, and reached a maximum 0.5 h after APAP treatment. Hepatic GSH decreased rapidly, and was almost completely depleted 1 h after APAP treatment. Plasma ALT activity, an index of liver injury, significantly increased from 3 h onwards after APAP treatment. The survival ratios 9 h, 24 h and 48 h after APAP treatment were 96%, 38% and 36%, respectively. We found a remarkable difference in the patterns of hepatic HSP25 and HSP70i induction in mice that survived after APAP treatment. HSP70i levels increased from 1 h onwards after APAP treatment in a time-dependent manner, and reached a maximum at 9 h. In contrast, HSP25 could be detected just 24 h after APAP treatment, and maximal accumulation was observed at 48 h. Other HSPs examined were unchanged. Notably, the survival ratio dropped by only 2% after HSP25 expression. Recently, a novel role for HSP25 as an anti-inflammatory factor was suggested. We have already shown that 48-h treatment with APAP induces severe centrilobular necrosis with inflammatory cell infiltration in mouse livers. Taken together, the level of expression of hepatic HSP25 may be a crucial determinant of the fate of mice exposed to APAP insult.     

Overexpression of manganese superoxide dismutase promotes the survival of prostate cancer cells exposed to hyperthermia

It has been hypothesized that exposure of cells to hyperthermia results in an increased flux of reactive oxygen species (ROS), primarily superoxide anion radicals, and that increasing antioxidant enzyme levels will result in protection of cells from the toxicity of these ROS. In this study, the prostate cancer cell line, PC-3, and its manganese superoxide dismutase (MnSOD)-overexpressing clones were subjected to hyperthermia (43°C, 1 h). Increased expression of MnSOD increased the mitochondrial membrane potential (MMP). Hyperthermic exposure of PC-3 cells resulted in increased ROS production, as determined by aconitase inactivation, lipid peroxidation, and H₂O₂ formation with a reduction in cell survival. In contrast, PC-3 cells overexpressing MnSOD had less ROS production, less lipid peroxidation, and greater cell survival compared to PC-3 Wt cells. Since MnSOD removes superoxide, these results suggest that superoxide free radical or its reaction products are responsible for part of the cytotoxicity associated with hyperthermia and that MnSOD can reduce cellular injury and thereby enhance heat tolerance.     

Heat shock protein 20 promotes sirtuin 1-dependent cell proliferation in induced pluripotent stem cells

BACKGROUNDHeat shock proteins (HSPs) are molecular chaperones that protect cells against cellular stresses or injury. However, it has been increasingly recognized that they also play crucial roles in regulating fundamental cellular processes. HSP20 has been implicated in cell proliferation, but conflicting studies have shown that it can either promote or suppress proliferation. The underlying mechanisms by which HSP20 regulates cell proliferation and pluripotency remain unexplored. While the effect of HSP20 on cell proliferation has been recognized, its role in inducing pluripotency in human-induced pluripotent stem cells (iPSCs) has not been addressed.AIMTo evaluate the efficacy of HSP20 overexpression in human iPSCs and evaluate the ability to promote cell proliferation. The purpose of this study was to investigate whether overexpression of HSP20 in iPSCs can increase pluripotency and regeneration.METHODSWe used iPSCs, which retain their potential for cell proliferation. HSP20 overexpression effectively enhanced cell proliferation and pluripotency. Overexpression of HSP20 in iPSCs was characterized by immunocytochemistry staining and real-time polymerase chain reaction. We also used cell culture, cell counting, western blotting, and flow cytometry analyses to validate HSP20 overexpression and its mechanism.RESULTSThis study demonstrated that overexpression of HSP20 can increase the pluripotency in iPSCs. Furthermore, by overexpressing HSP20 in iPSCs, we showed that HSP20 upregulated proliferation markers, induced pluripotent genes, and drove cell proliferation in a sirtuin 1 (SIRT1)-dependent manner. These data have practical applications in the field of stem cell-based therapies where the mass expansion of cells is needed to generate large quantities of stem cell-derived cells for transplantation purposes. CONCLUSIONWe found that the overexpression of HSP20 enhanced the proliferation of iPSCs in a SIRT1-dependent manner. Herein, we established the distinct crosstalk between HSP20 and SIRT1 in regulating cell proliferation and pluripotency. Our study provides novel insights into the mechanisms controlling cell proliferation that can potentially be exploited to improve the expansion and pluripotency of human iPSCs for cell transplantation therapies. These results suggest that iPSCs overexpressing HSP20 exert regenerative and proliferative effects and may have the potential to improve clinical outcomes.     

Mediators and mechanisms of heat shock protein 70 based cytoprotection in obstructive nephropathy

Urinary heat shock protein 70 (Hsp70) is rapidly increased in patients with clinical acute kidney injury, indicating that it constitutes a component of the endogenous stress response to renal injury. Moreover, experimental models have demonstrated that Hsp70 activation is associated with the cytoprotective actions of several drugs following obstruction, including nitric oxide (NO) donors, geranylgeranylacetone, vitamin D, and rosuvastatin. Discrete and synergistic effects of the biological activities of Hsp70 may explain its cytoprotective role in obstructive nephropathy. Basic studies point to a combination of effects including inhibition of apoptosis and inflammation, repair of damaged proteins, prevention of unfolded protein aggregation, targeting of damaged protein for degradation, and cytoskeletal stabilization as primary effectors of Hsp70 action. This review summarizes our understanding of how the biological actions of Hsp70 may affect renal cytoprotection in the context of obstructive injury. The potential of Hsp70 to be of central importance to the mechanism of action of various drugs that modify the genesis of experimental obstructive nephropathy is considered.     

Ultrastructural stability under high temperature or intensive light stress conferred by a small heat shock protein in cyanobacteria

The role and sub-cellular localization of the small heat shock protein HspA under stress conditions was investigated comparing the cyanobacterium Synechococcus strain ECT16-1, which constitutively expresses HspA, with the reference strain ECT. The ultrastructure of ECT cells under elevated temperature or intensive light stress exhibited severe damage including aggregation of cytosol and disordered thylakoid membranes, but in ECT16-1 cells these ultrastructural changes were much less conspicuous. Immunocytochemical studies showed that the main localization of HspA in the ECT16-1 cells shifted from the thylakoid area to the cytoplasm, then back to thylakoid area during the heat stress. Expression of HspA stabilized the morphology of nucleoids. The results are discussed, in particular with respect to the unique property of HspA to associate with thylakoid membranes.     

Antisense oligonucleotide to the 70-kDa heat shock cognate protein inhibits synthesis of myelin basic protein

Transfection of rat oligodendrocytes with an oligonucleotide sequence complementary to the mRNA encoding the initial ten amino acids of the rat 70-kDa heat shock cognate protein (HSC70) resulted in a rapid (within 24 h) and significant reduction in HSC70 synthesis (69% of control cells transfected with sense oligonucleotide). A further decrease to approximately 44% of controls was detected after 2 days. At that time, HSC70 protein content fell to approximately 49% of controls, and a significant reduction in the synthesis of myelin basic protein (MBP) was first detected (66% of controls). After 5 days, HSC70 synthesis returned to control levels. As HSC70 protein content recovered, so did the synthesis of MBP. Throughout the 5-day experimental period, only minor changes were detected in cell morphology, overall pattern of protein synthesis and the synthesis and content of proteolipid protein (PLP) and the pi isoenzyme of glutathione-S-transferase (pi). These data show that when HSC70 protein content is sufficiently reduced by antisense oligonucleotide, synthesis of MBP (but not PLP or pi) is correspondingly down-regulated, and provide evidence consistent with the role of HSC70 as a chaperone for MBP. Special issue dedicated to Dr. Marion E. Smith.     

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.     

Over-expression of a small heat shock protein,sHSP17.7, confers both heat tolerance and UV-B resistance to rice plants

Exposure of rice (Oryza sativa L.) seedlings to a high temperature (42°C) for 24 h resulted in a significant increase in resistance to UV-B damage. UV-B resistance was enhanced in parallel with the period of heat treatment. sHSP17.7 was isolated from heated rice seedlings, and the influence of rice sHSP17.7 expression on the viability of E. coli under heat-shock conditions was assessed. After heating, the survival rate of sHSP17.7 cells was 2-fold higher than that of the control cells. The molecular chaperone activity of sHSP17.7 was investigated using catalase as a substrate. Recombinant sHSP17.7 had heat-stable chaperone properties that were capable of protecting stressed catalase from precipitation. sHSP17.7 was overexpressed in the rice cultivar Hoshinoyume, by Agrobacterium-mediated transformation, under the control of a CaMV 35S promoter. Transgenic rice plants with increased levels of sHSP17.7 protein exhibited significantly increased thermotolerance compared to untransformed control plants. The level of increased thermotolerance was correlated with the level of increased sHSP17.7 protein in the transgenic plants. The transgenic rice plant with the highest constitutive expression of sHSP17.7 had significantly greater resistance to UV-B stress than untransformed control plants. Increase in the degree of resistance of transgenic plants to UV-B was accompanied by an increase in production of sHSP17.7 protein.     

Biotinylation of heat shock protein 70 induces RANTES production in HEK293 cells in a CD40-independent pathway

Biotinylated proteins and peptides have been used as popular ligands for characterization of cell surface receptors by a variety of methods including flow cytometry. The number and the location of biotin moieties incorporated could alter the structural and physicochemical properties of ligands, although biotin is thought to be such a small molecule (244Da) that it is capable of being conjugated to most proteins without affecting their activity. Here, we demonstrate that the biotinylated HSP70 molecule via primary amines bound to epithelium-like HEK 293 cells in a saturable manner whereas the unlabeled counterparts of HSP70 other than mouse Hsp72 do not. This binding was not competed by either HSP70 or the biotin entity itself. Interestingly, the biotinylated HSP70 also elicited the production of CC-chemokine RANTES independent of CD40 signaling. This response occurred regardless of sequence diversity of HSP70 derived from different species, and neither the biotinylated ovalbumin nor the unlabeled HSP70 cross-linked with a biotinylated protein stimulated a significant level of RANTES production which was induced by biotinylated HSP70 itself. Our findings suggest that modification of HSP70 such as biotinylation may function as a biological alarm signal in the innate immune system.     

Methionine protects against hyperthermia-induced cell injury in cultured bovine mammary epithelial cells

The aim of this study was to investigate the effects of methionine on cell proliferation, antioxidant activity, apoptosis, the expression levels of related genes (HSF-1, HSP70, Bax and Bcl-2) and the expression levels of protein (HSP70) in mammary epithelial cells, after heat treatment. Methionine (60 mg/L) increased the viability and attenuated morphological damage in hyperthermia-treated bovine mammary epithelial cells (BMECs). Additionally, methionine significantly reduced lactate dehydrogenase leakage, malondialdehyde formation, nitric oxide, and nitric oxide synthase activity. Superoxide dismutase, catalase, and glutathione peroxidase enzymatic activity was increased significantly in the presence of methionine. Bovine mammary epithelial cells also exhibited a certain amount of HSP70 reserve after methionine pretreatment for 24 h, and the expression level of the HSP70 gene and protein further increased with incubation at 42 °C for 30 min. Compared to the control, the expression of HSF-1 mRNA increased, and there was a significantly reduced expression of Bax/Bcl-2 mRNA and a reduced activity of caspase-3 against heat stress. Methionine also increased survival and decreased early apoptosis of hyperthermia-treated BMECs. Thus, methionine has cytoprotective effects on hyperthermia-induced damage in BMECs.     

Heat shock protein 70 and albuminuria in patients with type 2 diabetes: a matched case control study

Here, we aimed to study serum heat shock protein (HSP) 70 levels in diabetic patients with and without albuminuria. We performed a 1:1 matched case control study on 40 diabetic patients with albuminuria as cases and 40 age, sex, body mass index matched diabetic patients without albuminuria (normoalbuminuria) as controls. Normoalbuminuria was defined as urinary albumin excretion rate <15 mg/12 h, and albuminuria was defined as urinary albumin excretion rate between 100–400 mg/12 h. Patients with albuminuria had a higher HSP70 than controls (0.83 ± 0.50 vs. 0.63 ± 0.06; p = 0.02), while they did not differ in any other studied variables. In ten of the studied pairs, the controls had higher HSP70 levels than cases (reverse relationship). Patients in the “direct relationship group” had higher HbA1c values than the patients in the “reverse relationship group” (8.9 ± 0.3 vs. 7.3 ± 0.6, p = 0.04). Cases in the reverse pairs had a lower low density lipoprotein cholesterol levels than their controls. The odds ratio of HSP70 in the prediction of albuminuria was (28.69 (3.2–250.1), p = 0.002). In conclusion, we have shown an increased HSP70 levels in diabetic patients with albuminuria.     

Hsp70 对低氧大鼠海马DG 区神经细胞p53、p-p38 表达的研究

目的:探讨热休克蛋白70(HSP70)高表达对低氧大鼠海马DG区神经细胞p53、p-p38表达的影响。方法:将大鼠海马DG区神经细胞在41℃温浴1h诱导HSP70高表达,然后低氧培养,Western-blot检测细胞HSP70、p53、p-p38的表达。结果:温浴可以诱导HSP70高表达;HSP70高表达可抑制p53和p-p38表达。结论:HSP70保护低氧诱导的细胞凋亡可能与抑制p53和p-p38表达有关。