Extracellular Heat Shock Protein 70 Research Articles

Diversity of extracellular HSP70 in cancer: advancing from a molecular biomarker to a novel therapeutic target.

Heat shock protein 70 (HSP70) is a highly conserved protein functioning as a "molecular chaperone", which is integral to protein folding and maturation. In addition to its high expression within cells upon stressful challenges, HSP70 can be translocated to the cell membrane or released from cells in free form or within extracellular vesicles (EVs). Such trafficking of HSP70 is also present in cancer cells, as HSP70 is overexpressed in various types of patient samples across a range of common malignancies, signifying that extracellular HSP70 (eHSP70) can serve as a tumor biomarker. eHSP70 is involved in a broad range of cancer-related events, including cell proliferation and apoptosis, extracellular matrix (ECM) remodeling, epithelial-mesenchymal transition (EMT), angiogenesis, and immune response. eHSP70 can also induce cancer cell resistance to various treatments, such as chemotherapy, radiotherapy, and anti-programmed death-1 (PD-1) immunotherapy. Though the role of eHSP70 in tumors is contradictory, characterized by both pro-tumor and anti-tumor effects, eHSP70 serves as a promising target in cancer treatment. In this review, we comprehensively summarized the current knowledge about the role of eHSP70 in cancer progression and treatment resistance and discussed the feasibility of eHSP70 as a cancer biomarker and therapeutic target.

Extracellular Heat Shock Protein 70 Increases the Glucocorticoid Receptor and Dual-Specificity Phosphatase 1 via Toll-like Receptor 4 and Attenuates Inflammation in Airway Epithelial Cells.

Heat shock protein 70 (HSP70) regulates the ligand binding of the glucocorticoid receptor (GR). In asthma patients, heat treatment increased both the GR expression and secretion of extracellular HSP70 (eHSP70) by bronchial epithelial cells (EC). The objective of this study was to assess the effects of eHSP70 on GR expression and the GR-dependent regulation of immune response in human bronchial ECs. Cells were treated with either eHSP70 or transfected with an expression vector for intracellular HSP70 (iHSP70). Ribonucleic acid (RNA) and protein levels were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Interleukin (IL-6 and IL-8) secretion was determined by enzyme linked immunosorbent assay (ELISA). The overexpression of iHSP70 decreased, while eHSP70 increased GR expression. In addition, eHSP70 increased the expression of the GR target dual-specificity phosphatase 1 (DUSP-1). In doing so, eHSP70 reduced the tumor growth factor (TGF)-β1-dependent activation of extracellular signal-regulated kinase (Erk)-1/2 and cyclic AMP response element binding protein (CREB) and the secretion of IL-6 and IL-8. Blocking the GR or Toll-like receptor 4 (TLR4) counteracted all eHSP70-induced effects. This study demonstrates a novel anti-inflammatory effect of eHSP70 by the signaling cascade of TLR4-GR-DUSP1, which inhibits TGF-β1-activated pro-inflammatory ERK1/2-CREB signaling and cytokine secretion. The findings suggest that eHSP70 might present a novel non-steroidal therapeutic strategy to control airway inflammation in asthma.

Increased eHSP70-to-iHSP70 ratio disrupts vascular responses to calcium and activates the TLR4-MD2 complex in type 1 diabetes

AimsVascular dysfunction is a clinical hallmark of diabetes. While various pathways drive vascular alterations in diabetes, many gaps persist in understanding this process. Heat-shock protein 70 (HSP70) has a long-recognized role in diabetes, but the contributions of HSP70 to the diabetic vasculature remain largely unknown. Main methodsWe determined the systemic and local (aorta) levels of HSP70 in control (CTL) and streptozotocin (STZ)-induced diabetic rats. Functional studies were conducted in a wire myograph in the presence or absence of a pharmacological inhibitor for HSP70 (VER155008). Calcium (Ca2+) dynamics was indirectly evaluated as a function of change in force development in vehicle and VER-treated vessels, as well as in the presence of inhibitors for voltage-dependent and -independent plasmalemmal Ca2+ channels. Furthermore, mimicking the extracellular diabetic environment, we exposed aortic rings to serum from CTL and STZ-induced animals, which contains higher levels of HSP70, as well as to purified recombinant HSP70. Then, we performed functional studies following the modulation of Toll-like receptor 4 (TLR4) and its co-adaptor MD2, which interact with HSP70. Key findingsHSP70 plays a dual role in diabetes-induced vascular dysfunction: intracellular (i)HSP70 affects Ca2+ handling mechanisms, and extracellular (e)HSP70 modulates the TLR4-MD2 complex. SignificanceThese newly discovered roles of HSP70 push forward the field of vascular biology and open research avenues for other diseased states associated with altered vascular responses.

Sonosensitized Aggregation-Induced Emission Dots with Capacities of Immunogenic Cell Death Induction and Multivalent Blocking of Programmed Cell Death-Ligand 1 for Amplified Antitumor Immunotherapy

Sonosensitized Aggregation-Induced Emission Dots with Capacities of Immunogenic Cell Death Induction and Multivalent Blocking of Programmed Cell Death-Ligand 1 for Amplified Antitumor Immunotherapy

The Role of Extracellular HSP70 in the Function of Tumor-Associated Immune Cells

Simple SummaryThe intracellular heat shock protein 70 (HSP70) is essential for cells to respond to stress, for instance, by refolding damaged proteins or inhibiting apoptosis. However, in cancer, HSP70 is overexpressed and can translocate to the extracellular milieu, where it emerged as an important modulator of tumor-associated immune cells. By targeting the tumor microenvironment (TME) through different mechanisms, extracellular HSP70 can trigger pro- or anti-tumorigenic responses. Therefore, understanding the pathways and their consequences is crucial for therapeutically targeting cancer and its surrounding microenvironment. In this review, we summarize current knowledge on the translocation of extracellular HSP70. We further elucidate its functions within the TME and provide an overview of potential therapeutic options.Extracellular vesicles released by tumor cells (T-EVs) are known to contain danger-associated molecular patterns (DAMPs), which are released in response to cellular stress to alert the immune system to the dangerous cell. Part of this defense mechanism is the heat shock protein 70 (HSP70), and HSP70-positive T-EVs are known to trigger anti-tumor immune responses. Moreover, extracellular HSP70 acts as an immunogen that contributes to the cross-presentation of major histocompatibility complex (MHC) class I molecules. However, the release of DAMPs, including HSP70, may also induce chronic inflammation or suppress immune cell activity, promoting tumor growth. Here, we summarize the current knowledge on soluble, membrane-bound, and EV-associated HSP70 regarding their functions in regulating tumor-associated immune cells in the tumor microenvironment. The molecular mechanisms involved in the translocation of HSP70 to the plasma membrane of tumor cells and its release via exosomes or soluble proteins are summarized. Furthermore, perspectives for immunotherapies aimed to target HSP70 and its receptors for cancer treatment are discussed and presented.

Effect of a post-hatch lipopolysaccharide challenge in Turkey poults and ducklings after a primary embryonic heat stress

The effect of embryonic thermal manipulation on the post-hatch immune response to a lipopolysaccharide (LPS) challenge was studied in Pekin ducklings and turkey poults. Commercial duck and turkey eggs were distributed among four treatments: SS-Control (37.5 °C from embryonic day [ED] 1 to 25); SS-LPS (37.5 °C from ED1 to 25 + LPS at D0 [hatch]); HH-LPS (38 °C from ED1 to 25 + LPS at D0; SH-LPS (37.5 °C from ED1 to 10 and 38 °C from ED 11 to 25 + LPS at D0). At ED16 and ED24, the egg shell temperature of the duck and turkey eggs in the HH and SH treatments were higher (P ≤ 0.01) than the SS treatment. Ducklings and poults in the HH treatment had the lowest yolk free body weight at hatch (P ≤ 0.05). At 24, 48, and 72 h post-LPS injection, ducklings and poults in the HH-LPS treatment had significantly reduced BW compared with the SS-Con treatments (P ≤ 0.05). Ducklings and poults in the SH-LPS and HH-LPS treatments had increased plasma heat shock protein 70 (HSP70) and lower splenic HSP70 mRNA amounts than the SS-LPS treatments at 24, and 48 h post-challenge (P ≤ 0.05). At 48 and 72 h, macrophage nitric oxide (NO) production in ducklings and poults in the SH-LPS and HH-LPS treatments was lower than in the SS-LPS treatments (P ≤ 0.05). Ducklings and poults in the SH-LPS treatment had increased thymocyte proliferation compared to the SS-LPS treatment at 24, 48 and 72 h (P ≤ 0.05). At 24 h, ducklings in the SH-LPS treatment had increased splenic IL-10 and reduced IFNγ and IL-6 mRNA abundance. However, both ducklings and poults in the HH-LPS treatment had increased IFNγ, and IL-10 mRNA abundance compared to the SS-LPS treatment (P ≤ 0.05). At 48 h, SH-LPS ducklings and poults had lower splenic IL-10 mRNA abundance (P ≤ 0.05) while the HH-LPS treatment resulted in comparable splenic IL-10 mRNA compared to the SS-LPS treatment (P ≥ 0.05). Ducklings and poults in the SH-LPS treatment had increased thymic and splenic CD8+/CD4+ ratios at 24 h versus the SS-LPS treatment (P ≤ 0.05). In conclusion, embryonic thermal manipulation from ED11-25 increased extracellular HSP70 release, thymocyte proliferation and IL-10 but decreased splenic HSP70 and IFNγ mRNA amounts at 24 h post-LPS injection. This suggests that mild heat stress during the later stages of incubation could potentially prime the embryonic immune system thereby enhances the immune response as earlier than 24 h to eliminate the inflammatory response without affecting the growth performance by increase the extracellular release of HSP70 in both ducklings and poults. Continuous exposure to the small increase in temperature from ED 1–25 (HH) caused an imbalance between pro (IFNγ)- and anti-inflammatory cytokines(IL-10) which affects hatchling responses to an inflammatory challenge and increased mortality. The amount of extracellular HSP70 could potentially play an important role in modulating the immune response against inflammatory challenges.

Salivary Stress Biomarkers During the Lake Placid Ironman® Ultraendurance Event

Biomarkers such as salivary IgA (sIgA) have been established as valid, reliable, and non-invasive stress markers. sIgA concentrations have been reported to decrease following periods of high physiological stress, such as that experienced during ultra-endurance events. Heat shock protein 70 (HSP70), a molecular chaperone, has been assessed primarily as a plasma/serum stress biomarker. The role of HSP70 in saliva, how it responds to extremely stressful exercise events and its correlation to changes in sIgA remain unclear. PURPOSE: To test the hypothesis that salivary HSP70 can be used as a salivary stress biomarker correlated to sIgA during an ultraendurance event. METHODS: Thirty-three subjects competing in the Lake Placid Ironman triathlon participated (all data, mean±SD: 38±8 yrs, 178.4±8.9 cm, 76.3±10.4 kg, 10.8±3.8% body fat, finish time 708±90 min). Environmental symptoms questionnaires (ESQ) were administered before (PRE) and 1 hour after the race (POST). Hydration status was assessed via urine specific gravity (USG). Saliva samples were collected PRE, POST, and 1 day post-race (AMPOST) and analyzed for IgA (Salimetrics) and HSP70 (Enzo Life Sciences) by ELISA according to manufacturer instructions. Significant differences among time points were analyzed by repeated measures ANOVA and LSD post hoc tests. RESULTS: Subjects experienced significant stress with completion of the race (708±90 min finish time, ESQ 1hPOST (20±8) vs. PRE (5±3, p<0.05), POST RPE 18±2). sIgA was decreased POST (41.71 ±20.46 μg·ml-1) vs. PRE (48.88 ±17.20 μg·ml-1, p=0.05) and vs. AMPOST (37.76 ±27.02 μg·ml-1, p=0.006). Salivary HSP70 was increased POST (3.91±3.29 ng·ml-1) vs. PRE (1.68±1.93 ng·ml-1 p=0.0002). CONCLUSIONS: Salivary HSP70 was detectable using a commercially available ultrasensitive HSP70 ELISA. Extracellular HSP70 in the oral cavity may be a non-invasive marker of stress during an ultraendurance event and is correlated with more common salivary stress marker sIgA.

Cell-surface HSP70 associates with thrombomodulin in endothelial cells.

Heat shock protein-70 (HSP70) is crucial for proteostasis and displays cell-protective effects. Meanwhile, enhanced levels of cell-surface (cs) and secreted HSP70 paradoxically associate with pathologic cardiovascular conditions. However, mechanisms regulating csHSP70 pool are unknown. We hypothesized that total and csHSP70 expressions are modulated by hemodynamic forces, major contributors to endothelial pathophysiology. We also investigated whether thrombomodulin, a crucial thromboresistance cell-surface protein, is a csHSP70 target. We used proteomic/western analysis, confocal microscopy, and cs-biotinylation to analyze the pattern and specific characteristics of intracellular and csHSP70. HSP70 interaction with thrombomodulin was investigated by confocal colocalization, en face immunofluorescence, proximity assay, and immunoprecipitation. Thrombomodulin activity was assessed by measured protein C activation two-step assay. Our results show that csHSP70 pool in endothelial cells (EC) exhibits a peculiar cluster-like pattern and undergoes enhanced expression by physiological arterial-level laminar shear stress. Conversely, total and csHSP70 expressions were diminished under low shear stress, a known proatherogenic hemodynamic pattern. Furthermore, total HSP70 levels were decreased in aortic arch (associated with proatherogenic turbulent flow) compared with thoracic aorta (associated with atheroprotective laminar flow). Importantly, csHSP70 co-localized with thrombomodulin in cultured EC and aorta endothelium; proximity ligation assays and immunoprecipitation confirmed their physical interaction in EC. Remarkably, immunoneutralization of csHSP70 enhanced thrombomodulin activity in EC and aorta ex vivo. Overall, proatherogenic hemodynamic forces promote reduced total HSP70 expression, which might implicate in disturbed proteostasis; meanwhile, the associated decrease in cs-HSP70 pool associates with thromboresistance signaling. Cell-surface HSP70 (csHSP70) expression regulation and csHSP70 targets in vascular cells are unknown. We showed that HSP70 levels are shear stress-modulated and decreased under proatherogenic conditions. Remarkably, csHSP70 binds thrombomodulin and inhibits its activity in endothelial cells. This mechanism can potentially explain some deleterious effects previously associated with high extracellular HSP70 levels, as csHSP70 potentially could restrict thromboresistance and support thrombosis/inflammation in stress situations.

Association of extracellular heat shock protein 70 and insulin resistance in type 2 diabetes; independent of obesity and C-reactive protein

Despite few studies on intracellular heat shock protein70, the clinical association between insulin resistance and extracellular heat shock protein70 (eHSP70) is not well studied. In the current study, we examined the association between homeostatic model assessment-insulin resistance (HOMA-IR) and eHSP70 in patients with type 2 diabetes (T2DM) and healthy controls. A total of 145 patients with T2DM and 41 matched healthy controls were selected. Patients and controls were divided based on waist circumference (WC) to two groups, and eHSP70 was compared between them. The association between HOMA-IR and eHSP70 was examined using regression models adjusted for age, high-sensitive C-reactive protein (hs-CRP), and central obesity as confounding factors. While eHSP70 and hs-CRP were significantly correlated with HOMA-IR in patients with T2DM (p = 0.032, 0.025, respectively), there was no correlation between eHSP70 and HOMA-IR in the control group. Extracellular HSP70 and hs-CRP were not correlated in healthy controls. But a significant association appeared between eHSP70 and hs-CRP in patients with T2DM (p = 0.05). Both BMI and WC were not correlated with eHSP70 in both groups. Extracellular HSP70 was positively associated with HOMA-IR in patients with T2DM, independent from hs-CRP and obesity. We also showed eHSP70 levels remained unchanged through increase in BMI or WC in patients with T2D and in healthy controls. Our findings suggest that eHSP70 may contribute to the pathogenesis of T2DM by increasing insulin resistance.

Heat shock protein 70: A promising therapeutic target for myocardial ischemia-reperfusion injury.

Acute myocardial infarction is a major cause of death worldwide. The most important therapy for limiting ischemic injury and infarct size is timely and efficient myocardial reperfusion treatment, which may instead induce cardiomyocyte necrosis due to myocardial ischemia-reperfusion (I/R) injury. Heat shock protein 70 (HSP70), a stress-inducible protein, is overexpressed during myocardial I/R. The induced HSP70 is shown to regulate several intracellular proteins (e.g., transcription factors, enzymes, and apoptosis-related proteins) and signaling pathways (e.g., c-Jun N-terminal kinase pathway and extracellular-signal-regulated kinase 1/2 pathway), forming a complicated network that contributes to reducing reactive oxygen species accumulation, improving calcium homeostasis, inhibiting cellular apoptosis, thereby enhancing the stress adaption of myocardium to I/R injury. In addition, the extracellular HSP70, which is released from injured cardiomyocytes during I/R, acts as a proinflammatory mediator that results in cell death, while the intracellular HSP70 exerts antiinflammatory effects by suppressing proinflammatory signaling pathways. Notably, HSP70 is induced and contributes to the cardioprotection in several types of preconditioning and postconditioning. Meanwhile, it is shown that the cardioprotective effectiveness of preconditioning-induced HSP70 (e.g., hyperthermia preconditioning-induced HSP70) can be impaired by certain pathological conditions, such as hyperlipidemia and hyperglycemia. Thus, we highlight the widespread cardioprotective involvement of HSP70 in preconditioning and postconditioning and elucidate how HSP70-mediated cardioprotection is impaired in these pathological conditions. Furthermore, several therapeutic potentials of HSP70 against myocardial I/R injury and potential directions for future studies are also provided in this review.

Curcumin inhibits liver cancer by inhibiting DAMP molecule HSP70 and TLR4 signaling.

Curcumin has been revealed to inhibit liver cancer, however, no studies have reported that the mechanism of curcumin's action on liver cancer is related to damage-associated molecular pattern (DAMP) molecules heat shock protein70 (HSP70) and the toll-like receptor 4 (TLR4) signaling. This study aimed to investigate whether the activation of TLR4 signaling by HSP70 could be inhibited by curcumin, thus investigating the possible mechanism of curcumin in the inhibition of liver cancer. Western blotting was used to evaluate the expression of the HSP70 and TLR4 in HepG2 cells and ELISA was used to detect the concentration of HSP70 in cell culture medium. A thermal tolerance HepG2 (HepG2TT) cell model was established to simulate HSP70 accumulation in the microenvironment. A certain concentration of curcumin was co-cultured with HepG2 and HepG2TT cells to observe the changes of HSP70 and TLR4. Our results revealed that heat stress significantly increased the expression of extracellular HSP70 (eHSP70) and TLR4 (P<0.01), but significantly reduced the expression of intracellular HSP70 (P<0.01). Curcumin inhibited proliferation, invasion, and metastasis of HepG2 cells, caused cells to remain in the DNA Sphase, promoted apoptosis, and significantly reduced intracellular HSP70, eHSP70 and TLR4 levels of HepG2TT cells. Following the removal of curcumin, eHSP70 increased again. In summary, our results demonstrated that the antitumor effect of curcumin was related to the inhibition HSP70-TLR4 signaling.

The role of heat shock proteins and co-chaperones in heart failure.

The ongoing contractile and metabolic demands of the heart require a tight control over protein quality control, including the maintenance of protein folding, turnover and synthesis. In heart disease, increases in mechanical and oxidative stresses, post-translational modifications (e.g., phosphorylation), for example, decrease protein stability to favour misfolding in myocardial infarction, heart failure or ageing. These misfolded proteins are toxic to cardiomyocytes, directly contributing to the common accumulation found in human heart failure. One of the critical class of proteins involved in protecting the heart against these threats are molecular chaperones, including the heat shock protein70 (HSP70), HSP90 and co-chaperones CHIP (carboxy terminus of Hsp70-interacting protein, encoded by the Stub1 gene) and BAG-3 (BCL2-associated athanogene 3). Here, we review their emerging roles in the maintenance of cardiomyocytes in human and experimental models of heart failure, including their roles in facilitating the removal of misfolded and degraded proteins, inhibiting apoptosis and maintaining the structural integrity of the sarcomere and regulation of nuclear receptors. Furthermore, we discuss emerging evidence of increased expression of extracellular HSP70, HSP90 and BAG-3 in heart failure, with complementary independent roles from intracellular functions with important therapeutic and diagnostic considerations. While our understanding of these major HSPs in heart failure is incomplete, there is a clear potential role for therapeutic modulation of HSPs in heart failure with important contextual considerations to counteract the imbalance of protein damage and endogenous protein quality control systems.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective'.

Blocking ATP-sensitive potassium channel alleviates morphine tolerance by inhibiting HSP70-TLR4-NLRP3-mediated neuroinflammation

BackgroundLong-term use of morphine induces analgesic tolerance, which limits its clinical efficacy. Evidence indicated morphine-evoked neuroinflammation mediated by toll-like receptor 4 (TLR4) - NOD-like receptor protein 3 (NLRP3) inflammasome was important for morphine tolerance. In our study, we investigated whether other existing alternative pathways caused morphine-induced activation of TLR4 in microglia. We focused on heat shock protein 70 (HSP70), a damage-associated molecular pattern (DAMP), which was released from various cells upon stimulations under the control of KATP channel and bound with TLR4-inducing inflammation. Glibenclamide, a classic KATP channel blocker, can improve neuroinflammation by inhibiting the activation of NLRP3 inflammasome. Our present study investigated the effect and possible mechanism of glibenclamide in improving morphine tolerance via its specific inhibition on the release of HSP70 and activation of NLRP3 inflammasome induced by morphine.MethodsCD-1 mice were used for tail-flick test to evaluate morphine tolerance. The microglial cell line BV-2 and neural cell line SH-SY5Y were used to investigate the pharmacological effects and the mechanism of glibenclamide on morphine-induced neuroinflammation. The activation of microglia was accessed by immunofluorescence staining. Neuroinflammation-related cytokines were measured by western blot and real-time PCR. The level of HSP70 and related signaling pathway were evaluated by western blot and immunofluorescence staining.ResultsMorphine induced the release of HSP70 from neurons. The released HSP70 activated microglia and triggered TLR4-mediated inflammatory response, leading to the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) p65 and the activation of NLRP3 inflammasome. Moreover, anti-HSP70 neutralizing antibody partly attenuated chronic morphine tolerance. The secretion of HSP70 was under the control of MOR/AKT/KATP/ERK signal pathway. Glibenclamide as a classic KATP channel blocker markedly inhibited the release of HSP70 induced by morphine and suppressed HSP70-TLR4-NLRP3 inflammasome-mediated neuroinflammation, which consequently attenuated morphine tolerance.ConclusionsOur study indicated that morphine-induced extracellular HSP70 was an alternative way for the activation of TLR4-NLRP3 in analgesic tolerance. The release of HSP70 was regulated by MOR/AKT/KATP/ERK pathway. Our study suggested a promising target, KATP channel and a new leading compound, glibenclamide, for treating morphine tolerance.

Collaboration of Heat Shock Protein 70 and Stress-induced NKG2D Ligands in the Activation of NK Cells against Tumors

The stress-inducible heat shock protein 70 (HSP70) contributes to the cellular stress response that protects cells from adverse environmental conditions. HSP70 can block apoptosis and other types of cell death induced by various stimuli. In malignant cells, the expression of HSP70 is frequently elevated and some cancers even depend on HSP70 overexpression to maintain their malignant phenotype. Extracellular HSP70 in contrast, provides a danger signal to the immune system and may activate anti-tumor immune responses by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. HSP70-activated NK cells, for example, can attack tumor cells that express HSP70 at the plasma membrane. This feature of HSP70 reminds of other stress-inducible molecules including the major histocompatibility complex (MHC) class I chain-related molecules A (MICA) and B (MICB), which are ligands for activating NK receptor NK group 2, member D (NKG2D) and are induced by cellular and genotoxic stress on many tumor cells. Interestingly, the stress-inducible HSP70 and stressinducible NKG2D ligands can collaborate in tumor immunosurveillance by NK cells. HSP70-activated NK cells can also kill tumor cells that express NKG2D ligands at the plasma membrane instead of HSP70. An adaptive cellular immunotherapy with autologous HSP70-stimulated NK cells may therefore target not only tumors that express HSP70 at the plasma membrane but also those that express NKG2D ligands. Notably, an overexpression of HSP70 in tumors usually fails to protect against apoptosis executed in the granule exocytosis pathway of CTL and NK cells, making immunotherapies attractive to treat HSP70-overexpressing malignancies. Keywords: Heat shock protein 70, NKG2D ligands, NK cells, cytotoxic T lymphocytes, apoptosis, cancer, stress-induced self, tumor immunity.

Effects of in vitro heat shock on immune cells in diet-induced obese mice

Obesity has been associated with impaired immune responses and inflammation. The mechanisms underlying these immune disturbances in obesity are not yet clarified. This study investigated the effects of in vitro heat shock (HS) on immune cells from the point of view of thymocyte apoptosis and T-cell mitogen-stimulated splenocyte cytokine production as well as the heat shock protein 70 (HSP70) protein levels in diet-induced obese mice to explore a possible association between the disturbance of T cell immunity and HS response in obesity. Obese mice had increased apoptotic and necrotic thymocytes populations and increased splenocyte cytokine production of both proinflammatory and anti-inflammatory cytokines compared with lean mice. The in vitro HS at 42°C decreased the rate of live cells in thymocytes, and the degree of the decrease was larger in obese mice compared with lean mice. The in vitro HS increased the intracellular and extracellular HSP70 protein levels in thymocytes and splenocytes, while the effects of obesity on the HSP70 protein levels were not obvious. The in vitro HS prior to T cell mitogen stimulation decreased IFN-γ and IL-10 production by mitogen-stimulated splenocytes. This change in cytokine production due to HS was not affected by obesity. The obvious alteration of the HSP70 protein levels and association between cytokine production and the HS response in obesity were not found in this obesity model; however, our results indicate an association between the viability of thymocytes and an altered HS response in obesity and provide evidence that the increase in thymocyte apoptosis and acceleration of thymus involution in obesity could be, in part, due to the alteration of the HS response.

Extracellular HSP70 Activates ERK1/2, NF-kB and Pro-Inflammatory Gene Transcription Through Binding with RAGE in A549 Human Lung Cancer Cells

Background/Aims: Heat shock protein 70 (HSP70) has been recently described with extracellular actions, where it is actively released in inflammatory conditions. Acting as DAMPs (damage associated molecular pattern), extracellular HSP70 (eHSP70) interacts with membrane receptors and activates inflammatory pathways. At this context, the receptor for advanced glycation endproducts (RAGE) emerges as a possible candidate for interaction with eHSP70. RAGE is a pattern-recognition receptor and its expression is increased in several diseases related to a chronic pro-inflammatory state. One of the main consequences of RAGE ligand-binding is the ERK1/2 (extracellular signal–regulated kinases)-dependent activation of NF-kB (nuclear factor kappa B), which leads to expression of TNF-α (tumor necrosis factor alpha) and other cytokines. The purpose of this work is to elucidate if eHSP70 is able to evoke RAGE-dependent signaling using A549 human lung cancer cells, which constitutively express RAGE. Methods: Immunoprecipitation and protein proximity assay were utilized to demonstrate the linkage between RAGE and eHSP70. To investigate RAGE relevance on cell response to eHSP70, siRNA was used to knockdown the receptor expression. Signaling pathways activation were evaluated by western blotting, gene reporter luciferase and real time quantitative PCR. Results: Protein eHSP70 shown to be interacting physically with the receptor RAGE in our cell model. Treatment with eHSP70 caused ERK1/2 activation and NF-κB transactivation impaired by RAGE knockdown. Moreover, the stimulation of pro-inflammatory cytokines expression by eHSP70 was inhibited in RAGE-silenced cells. Finally, conditioned medium of eHSP70-treated A549 cells caused differential effects in monocytes cytokine expression when A549 RAGE expression is inhibited. Conclusions: Our results evidence eHSP70 as a novel RAGE agonist capable of influence the cross-talk between cancer and immune system cells.

Erratum to: Primary glioblastoma multiforme tumors and recurrence: comparative analysis of the danger signals HMGB1, HSP70, and calreticulin

Purpose Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor. Despite improved multimodal therapies, the tumor recurs in most cases. Diverging patient survival suggests great tumor heterogeneity and different therapy responses. Danger signals such as high-mobility group box protein 1 (HMGB1), heat shock protein 70 (HSP70), and calreticulin (CRT) are biomarker candidates, due to their association with tumor progression versus induction of antitumor immune responses. Overexpression of these danger signals has been reported for various types of tumors; however, their role in GBM is still elusive. A direct comparison of their expression in the primary tumor versus the corresponding relapse is still lacking for most tumor entities. Patients and methods We therefore performed an expression analysis by immunohistochemistry of the danger signals HMGB1, HSP70, and CRT in primary tumors and the corresponding relapses of 9 patients with de novo GBM. Results HMGB1 was highly expressed in primary tumors with a significant reduction in the respective relapse. The extracellular HSP70 expression was significantly increased in the relapse compared to the primary tumor. CRT was generally highly expressed in the primary tumor, with a slight increase in the relapse. Conclusion The combination of a decreased expression of HMGB1, an increased expression of extracellular HSP70, and an increased expression of CRT in the relapse seems to be beneficial for patient survival. HMGB1, extracellular HSP70, and CRT could be taken into concerted consideration as potential biomarkers for the prognosis of patients with GBM.

Oncogenic extracellular HSP70 disrupts the gap-junctional coupling between capillary cells.

High levels of circulating heat shock protein 70 (HSP70) are detected in many cancers. In order to explore the effects of extracellular HSP70 on human microvascular endothelial cells (HMEC), we initially used gap-FRAP technique. Extracellular human HSP70 (rhHSP70), but not rhHSP27, blocks the gap-junction intercellular communication (GJIC) between HMEC, disrupts the structural integrity of HMEC junction plaques, and decreases connexin43 (Cx43) expression, which correlates with the phosphorylation of Cx43 serine residues. Further exploration of these effects identified a rapid transactivation of the Epidermal Growth Factor Receptor in a Toll-Like Receptor 4-dependent manner, preceding its internalization. In turn, cytosolic Ca2+ oscillations are generated. Both GJIC blockade and Ca2+ mobilization partially depend on ATP release through Cx43 and pannexin (Panx-1) channels, as demonstrated by blocking activity or expression of channels, and inactivating extracellular ATP. By monitoring dye-spreading into adjacent cells, we show that HSP70 released from human monocytes in response to macrophage colony-stimulating factor, prevents the formation of GJIC between monocytes and HMEC. Therapeutic manipulation of this pathway could be of interest in inflammatory and tumor growth.

Increased expression of heat shock protein 70 in chronic obstructive pulmonary disease

Heat shock protein 70 (HSP70) plays a critical role in the process of inflammation and innate immunity response under environmental stress. This study was to investigate HSP70 expression in the peripheral lung tissues of chronic obstructive pulmonary disease (COPD) patients and in human bronchial epithelial cells (16-HBE) exposed to cigarette smoke extract (CSE). Peripheral lung tissues were collected after lung cancer resection from 26 patients without COPD, 20 with mild COPD and 15 with advanced COPD, classified by lung function criteria. Among these cases, 37 were smokers and 24 non-smokers. Lung tissues were examined for histopathological changes and levels of HSP70 and IL-8. Cultured 16-HBE cells were stimulated with CSE in the absence or presence of HSP70 neutralizing antibody and the expressions of IL-8 and phospho-EGFR protein were determined. Compared to patients without COPD, the levels of HSP70 and IL-8 were significantly increased in the lung tissues of COPD patients and positively correlated with the severity of the disease. The HSP70 expression was significantly higher in current smokers than that in non-smokers. Moreover, CSE-induced HSP70 significantly enhanced IL-8 production and EGFR phosphorylation in 16-HBE cells. The increases in IL-8 and phospho-EGFR were blocked by anti-HSP70 antibody. Our study clarified that increased expression of HSP70 is closely related to COPD disease severity and smoking status. Extracellular HSP70 regulated chemokine productions and EGFR phosphorylation and plays an important role in the CSE-induced inflammatory and innate immunity responses in bronchial epithelia cells.

Elevated levels of mitochondrial mortalin and cytosolic HSP70 in blood as risk factors in patients with colorectal cancer

Mortalin/GRP75 is a ubiquitous mitochondrial chaperone related to the cytosolic heat shock protein 70 (HSP70). It protects cells from senescence and apoptosis and is overexpressed in cancer cells. Cell resistance to complement-dependent cytotoxicity depends on mortalin and during complement attack mortalin is released from cells. Our goal was to determine whether cancer patients have circulating mortalin in blood. The significance of mortalin in blood to survival prospects of colorectal cancer patients was evaluated. Occurrence of extracellular soluble HSP70 (sHSP70) is documented. We developed a sensitive ELISA for mortalin. The association between mortalin level and survival was subjected to the Cox proportional hazards analysis (univariate and multivariate analyses). Mortalin concentration in serum of colorectal cancer patients was 10-214 ng/ml. Survival data of the patients were known from an earlier study of sHSP70 in these samples. Cox regression analysis indicated that high mortalin (>60 ng/ml) is a risk factor for shorter survival. Serum levels of sHSP70 and mortalin in patients were independent variables. Concurrence of high sHSP70 and mortalin was associated with rapid disease progression (HR = 4, 2.04-8.45, p < 0.001). Addition of high sHSP70 and mortalin to a baseline model of age, sex and TNM stage, significantly (p < 0.001) enhanced the risk score to 8 (3.26-20.46). This is the first demonstration of circulating mortalin in cancer patients. Analysis of mortalin in blood, and even more so of mortalin and sHSP70, adds a high prognostic value to the TNM stage and will identify colorectal cancer patients at high risk of poor survival.