Secretion Of Heat Shock Proteins Research Articles

Heat-Induced Secretion of Heat Shock Proteins 70 and 90 Does not Affect the Expression of the Glucocorticoid Receptor in Primary Airway Cells in COPD

PurposeThe response to glucocorticoids is hampered in many COPD patients by a yet unknown mechanism. Earlier we reported that short-term heat exposure of primary human bronchial epithelial cells (BEC) and airway smooth muscle cells (ASMC) of asthma patients increased the expression and secretion of extracellular heat shock proteins (eHSPs) resulting in increased expression of glucocorticoid receptor (GR) in BEC and inhibition of ASMC remodeling. The aim of the present study was to assess if the same mechanism is also present in primary airway wall cells of COPD patients.MethodsPrimary BEC and ASMC were established from endobronchial biopsies obtained from COPD patients (n = 73), who participated in the HISTORIC study, an investigator-initiated and driven clinical trial. Secretion and protein expression of HSPs was assessed by ELISA and Western blotting. Expression of total GR, its isoforms GRα and GRβ and toll-like receptor 4 (TLR4) was determined by Western-blotting.ResultsShort heat exposure (65 °C, 10 s) of BEC resulted in a significant increase of the secretion of eHSP70 and eHSP90, while the intracellular protein was not altered. Heat treatment or exposure to eHSP70 or eHSP90 had no effect on the expression of GR and GR-isoforms. However, eHSP70 and eHSP90 significantly reduced the expression of TLR4.ConclusionsThe results of this study indicate that primary airway cells from COPD patients respond differently to heat exposure and extracellular HSP70 or HSP90 than cells from asthma patients regarding the expression of GR and this may explain the reduced response to glucocorticoids in patients with COPD.Trial Registration: ISRCTN11017699

Heat Stress: A Serious Disruptor of the Reproductive Physiology of Dairy Cows.

Global warming is a significant threat to the sustainability and profitability of the dairy sector, not only in tropical or subtropical regions but also in temperate zones where extreme summer temperatures have become a new and challenging reality. Prolonged exposure of dairy cows to high temperatures compromises animal welfare, increases morbidity, and suppresses fertility, resulting in devastating economic losses for farmers. To counteract the deleterious effects of heat stress, cattl e employ various adaptive thermoregulatory mechanisms including molecular, endocrine, physiological, and behavioral responses. These adaptations involve the immediate secretion of heat shock proteins and cortisol, followed by a complex network of disrupted secretion of metabolic and reproductive hormones such as prolactin, ghrelin, ovarian steroid, and pituitary gonadotrophins. While the strategic heat stress mitigation measures can restore milk production through modifications of the microclimate and nutritional interventions, the summer fertility records remain at low levels compared to those of the thermoneutral periods of the year. This is because sustainment of high fertility is a multifaceted process that requires appropriate energy balance, undisrupted mode of various hormones secretion to sustain the maturation and fertilizing competence of the oocyte, the normal development of the early embryo and unhampered maternal-embryo crosstalk. In this review, we summarize the major molecular and endocrine responses to elevated temperatures in dairy cows, as well as the impacts on maturing oocytes and early embryos, and discuss the consequences that heat stress brings about in dairy cattle fertility.

Heat stress stimulates colon cancer cells to secret specific population of extracellular nanovesicles enriched by HSP70 and microRNAs

Background. Heat stress (HS) induces the cellular secretion of heat shock proteins (HSP ) and extracellular nanovesicles (ENVs). The biological link between these phenomena is poorly understood. In the case of colorectal cancer (CRC) cells, the secretion of HSP s and ENV may be involved in the clinical response to intraperitoneal therapy of peritoneal carcinomatosis.Material and Methods. Established colon cancer cell lines COLO 320, HCT 116, HT29 and DLD 1 were used. ENVs were isolated from culture media by differential ultra-centrifugation and analyzed by dynamic light scattering, nanoparticle tracking analysis, atomic force microscopy and flow cytometry. Super-paramagnetic particles (SPMP ) covered by antibodies to the membrane form of Hsp70 were used for isolation and quantification of Hsp70(+) ENVs. Vesicular microRNA was assayed by RT-qPC R.Results. HS induces the secretion of ENVs by CRC cells, the resistance to HS correlates with the activity of HS-induced ENVs secretion. HS induces the secretion of a specific population of ENVs enriched by membrane form Hsp70 (mHsp70). The microRNA content of mHsp70(+) ENVs has qualitative and quantitative features. The concentration of miR-126-3p, -181-5p, -155-5p, -223 is increased in mHSP 70(+) ENVs secreted by three CRC cell lines.Conclusion. HS induces the secretion of mHSP 70(+) ENVs by CRC cells. This phenomenon may be involved in a clinical response to intraperitoneal chemo-hyperthermic perfusion therapy of peritoneal carcinomatosis.

Exploring the context of diacidic motif DE as a signal for unconventional protein secretion in eukaryotic proteins

Unconventional protein secretion (UPS) is an important phenomenon with fundamental implications to cargo export. How eukaryotic proteins transported by UPS are recognized without a conventional signal peptide has been an open question. It was recently observed that a diacidic amino acid motif (ASP-GLU or DE) is necessary for the secretion of superoxide dismutase 1 (SOD1) from yeast under nutrient starvation. Taking cue from this discovery, we explore the hypothesis of whether the diacidic motif DE, which can occur fairly ubiquitously, along with its context, can be a generic signal for unconventional secretion of proteins. Four different contexts were evaluated: a physical context encompassing the structural order and charge signature in the neighbourhood of DE, two signalling contexts reflecting the presence of either a phosphorylatable amino acid (‘X’ in XDE, DXE, DEX) or an LC3 interacting region (LIR) which can trigger autophagy and a co-evolutionary constraint relative to other amino acids in the protein interpreted by examining sequences across different species. Among the 100 proteins we curated from different physiological or pathological conditions, we observe a pattern in the unconventional secretion of heat shock proteins in the cancer secretome, where DE in an ordered structural region has higher odds of being a UPS signal.

Exploring the context of diacidic motif DE as a signal for unconventional protein secretion in eukaryotic proteins.

Unconventional protein secretion (UPS) is an important phenomenon with fundamental implications to cargo export. How eukaryotic proteins transported by UPS are recognized without a conventional signal peptide has been an open question. It was recently observed that a diacidic amino acid motif (ASP-GLU or DE) is necessary for the secretion of superoxide dismutase 1 (SOD1) from yeast under nutrient starvation. Taking cue from this discovery, we explore the hypothesis of whether the diacidic motif DE, which can occur fairly ubiquitously, along with its context, can be a generic signal for unconventional secretion of proteins. Four different contexts were evaluated: a physical context encompassing the structural order and charge signature in the neighbourhood of DE, two signalling contexts reflecting the presence of either a phosphorylatable amino acid ('X' in XDE, DXE, DEX) or an LC3 interacting region (LIR) which can trigger autophagy and a co-evolutionary constraint relative to other amino acids in the protein interpreted by examining sequences across different species. Among the 100 proteins we curated from different physiological or pathological conditions, we observe a pattern in the unconventional secretion of heat shock proteins in the cancer secretome, where DE in an ordered structural region has higher odds of being a UPS signal.

Cold Atmospheric Plasma Suppresses Ovarian Cancer Cell Activity With Concurrent Secretion Of Heat Shock Protein 27 Affecting Monocyte Fate

Background Ovarian cancer (OC) is a gynecologic tumor with poor prognosis. Despite radical cytoreductive surgery and platinum-based adjuvant systemic treatment, OC relapses in the majority of the cases. Several studies have suggested profound antitumor activity of cold atmospheric plasma (CAP). CAP is an ionized gas that mainly mediates its effects via the release of a variety of reactive oxygen and nitrogen species. Evidence of CAP treatment on OC cells is scarce, and accordingly we investigated their biological responses following exposure to plasma. Materials and Methods We tested the activity of CAP and CAP-treated medium to inactivate OC cell lines, and investigated their secretory products following CAP treatment. Cell proliferation and cell motility was measured. The presence of HSP27 was measured by Western blotting and enzyme-linked immunosorbent assay (ELISA). Supernatants of plasma-treated OC cells were added to the human THP-1 monocytic cell line. Cellular oxidation, viability, growth, morphology, surface marker expression, and cytokine release were monitored over up to 4 days of culture with supernatants of untreated and plasma-treated cancer cells. Results CAP treatment led to an attenuation of OC cell growth and motility. Incubation with CAP-treated cell culture medium gave similar effects. Results were consistent in four OC cell lines. Notably, CAP treatment of OC cells led to a decrease of intracellular HSP27 accompanied by an increase of extracellular HSP27 in the cell culture supernatant. These supernatants did not cause oxidation or blunted cell growth in THP-1 monocytes. Cell morphology was rather unaffected whereas from 12 cell surface markers screened, two (CD271 and CD55) gave a significant increase in plasma-treated OC cell supernatants compared to untreated controls. HSP27 alone led to a decrease of both markers. Likewise, a screening of 13 cytokines release from THP-1 revealed a differential regulation of a number of targets, for example, an upregulation of interleukin 8 in plasma-treated samples compared to untreated controls. Conclusion Plasma treatment was cytotoxic in ovarian cancer cell lines with functional and phenotypical consequences in human monocytes. The role of extracellular HSP27 induced by CAP treatment may represent immunomodulating mechanisms, which will be characterized more detailed in future.

Unconventional Secretion of Heat Shock Proteins in Cancer.

Heat shock proteins (HSPs) are abundant cellular proteins involved with protein homeostasis. They have both constitutive and inducible isoforms, whose expression levels are further increased by stress conditions, such as temperature elevation, reduced oxygen levels, infection, inflammation and exposure to toxic substances. In these situations, HSPs exert a pivotal role in offering protection, preventing cell death and promoting cell recovery. Although the majority of HSPs functions are exerted in the cytoplasm and organelles, several lines of evidence reveal that HSPs are able to induce cell responses in the extracellular milieu. HSPs do not possess secretion signal peptides, and their secretion was subject to widespread skepticism until the demonstration of the role of unconventional secretion forms such as exosomes. Secretion of HSPs may confer immune system modulation and be a cell-to-cell mediated form of increasing stress resistance. Thus, there is a wide potential for secreted HSPs in resistance of cancer therapy and in the development new therapeutic strategies.

Aberrant Expression and Secretion of Heat Shock Protein 90 in Patients with Bullous Pemphigoid

The cell stress chaperone heat shock protein 90 (Hsp90) has been implicated in inflammatory responses and its inhibition has proven successful in different mouse models of autoimmune diseases, including epidermolysis bullosa acquisita. Here, we investigated expression levels and secretory responses of Hsp90 in patients with bullous pemphigoid (BP), the most common subepidermal autoimmune blistering skin disease. In comparison to healthy controls, the following observations were made: (i) Hsp90 was highly expressed in the skin of BP patients, whereas its serum levels were decreased and inversely associated with IgG autoantibody levels against the NC16A immunodominant region of the BP180 autoantigen, (ii) in contrast, neither aberrant levels of circulating Hsp90 nor any correlation of this protein with serum autoantibodies was found in a control cohort of autoimmune bullous disease patients with pemphigus vulgaris, (iii) Hsp90 was highly expressed in and restrictedly released from peripheral blood mononuclear cells of BP patients, and (iv) Hsp90 was potently induced in and restrictedly secreted from human keratinocyte (HaCaT) cells by BP serum and isolated anti-BP180 NC16A IgG autoantibodies, respectively. Our results reveal an upregulated Hsp90 expression at the site of inflammation and an autoantibody-mediated dysregulation of the intracellular and extracellular distribution of this chaperone in BP patients. These findings suggest that Hsp90 may play a pathophysiological role and represent a novel potential treatment target in BP.

Abstract A74: Tumor secreted Hsp90 initiates tumor growth and the epithelial to mesenchymal transition (EMT) in prostate cancer

Abstract Prostate cancer (PCa) is the most frequently diagnosed malignancy and second most common cause of cancer related lethality in men. Disease mortality is due primarily to metastatic spread, highlighting the urgent need to identify factors involved in this progression. Increased secretion of heat shock protein 90 (Hsp90) has been reported in diverse cancer cells, and secreted, or extracellular Hsp90 (eHsp90) is also found in patient plasma. Although eHsp90 promotes cancer cell motility and invasion and facilitates tumor metastasis, the mechanistic basis for its activity has remained unknown. The goal of this study was to explore whether extracellular Hsp90 (eHsp90) may play a role in PCa progression. To explore this, 3 sets of lineage-related differentially metastatic cell pairs were utilized to evaluate eHsp90 expression and activity. We found that eHsp90 secretion correlated with PCa aggressiveness. To evaluate the functional role of eHsp90, we modulated its expression and activity. Upregulation of eHsp90 was accomplished by exposure of prostate epithelial cells to exogenous Hsp90 protein, or alternatively, cells were transduced with a lentivirus directing the secretion of Hsp90. Blockade of eHsp90 function was accomplished by exposure of cells to anti-Hsp90 antibodies or to a cell-impermeant specific inhibitor of Hsp90. We demonstrate that eHsp90 is a critical factor required for PCa cell motility and for initiation of the epithelial to mesenchymal transition (EMT) genetic program. Moreover, modest increases in eHsp90 were sufficient to induce tumor growth and localized invasion in a subrenal capsule xenograft model of PCa. Finally, eHsp90 was detected in human cancer specimens and its expression was correlated with upregulated expression of pro-invasive genes. Our findings offer insights into the mechanistic basis of eHsp90 in PCa, and further support a putative clinical role for eHsp90 as a potential driver of PCa progression. Citation Format: Michael W. Hance, Krystal Nolan, Udhayakumar Gopal, Jessica E. Bohonowych, Agnieszka Jezierska-Drutel, Carola A. Neumann, Haibo Liu, Garraway P. Isla, Omar E. Franco, Simon W. Hayward, Jennifer S. Isaacs. Tumor secreted Hsp90 initiates tumor growth and the epithelial to mesenchymal transition (EMT) in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A74.

Heat Shock Proteins 27, 60, 70, 90α, and 20S Proteasome in On-Pump Versus Off-Pump Coronary Artery Bypass Graft Patients

The secretion of heat shock protein (HSP) 27, HSP60, HSP70, HSP90alpha, 20S proteasome, and their correlations to proinflammatory cytokine interleukin-6 is unknown in patients undergoing on-pump versus off-pump coronary artery bypass graft (CABG) operation. Forty patients were included in this explorative study (on- versus off-pump CABG, each n = 20). Serum samples were obtained before and 30 minutes, 60 minutes, and 24 hours after CABG operation. Enzyme-linked immunosorbent assay technique was utilized to determine soluble HSP27, 60, 70, and 90alpha, 20S proteasome, and levels of interleukin-6. Serum levels of HSP are increased in patients undergoing on-pump CABG operation as compared with off-pump CABG technique. These differences were highly significant for HSP27, 70, and 90alpha at 60 minutes after initiation of cardiopulmonary bypass (all, p < 0.001). Concentrations of soluble 20S proteasome were increased 24 hours after operation in on- and off-pump CABG patients (p < 0.001) and correlated significantly with the serum content of HSP 27, 70, and 90alpha at 60 minutes after initiation of cardiopulmonary bypass (p < 0.001). No correlation was found when comparing interleukin-6 levels with intravascular leakage of HSP and 20S proteasome after CABG operation. We conclude from our data that the innate immune system is activated owing to spillage of known immune modulatory and apoptosis-associated proteins after CABG operation.

Measuring the secretion of heat shock proteins from cells

Heat shock proteins have been shown to be secreted from a number of cell types. Necrotic cells release heat shock proteins in a passive manner, whereas we, and others, have shown that viable cells secrete Hsp70 and Hsp60 through an active mechanism involving lysosomal vesicles and lipid rafts. This release of Hsp70 and Hsp60 is regulated, for example by being increased by elevated temperature. This article outlines procedures, using Hsp70 as the example, to: ensure the status of cells (viable, apoptotic or necrotic); identify the heat shock protein secreted; and quantify the secreted protein. Hsp70 has previously been quantified by ELISA, but newer methods are now being adopted, such as BIAcore and bead-based assays for use by FACS. These methods have the advantages of being more sensitive and requiring less sample than ELISA. The BIAcore has the potential to analyse Hsp70 ligands and provide affinity constants. The FACS bead assay system can be used to run multiplex assays.