Upregulation Of Hsp70 Research Articles

Hsp70 inducer, 17-allylamino-demethoxygeldanamycin, provides neuroprotection via anti-inflammatory effects in a rat model of traumatic brain injury.

Traumatic brain injury (TBI) is the predominant cause of mortality in young adults and children living in China. TBI induces inflammatory responses; in addition, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 are important pro-inflammatory cytokines. Considering the observation that Hsp-70 overexpression can exert neuroprotection, identifying a drug that is able to induce the upregulation of Hsp70 has the potential to be a promising therapy for the treatment of neurological diseases. Thus, the present study assessed the clinical effectiveness of an anticancer drug and Hsp70 activator, 17-allylamino-demethoxygeldanamycin (17-AAG), to evaluate its potential as a treatment for patients with TBI. The aim of present study was to determine the neuroprotective effects of 17-AAG following trauma and to investigate the underlying mechanisms of action. To establish rat models, rats were subjected to a controlled cortical impact injury and randomly divided into vehicle or 17-AAG groups. In the 17-AAG group, rats were administered with an intraperitoneal injection of 17-AAG (80 mg/kg) immediately following the establishment of TBI. The motor function was measured using Neurologic Severity Score, and neuronal death was evaluated using immunofluorescence. The expression levels of GLT-1, Bcl-2 and Hsp-70 were detected by western blot analysis and the expression levels of inflammatory cytokines were quantified using ELISA. The present study determined that 17-AAG significantly reduced brain edema and motor neurological deficits (P<0.05), in addition to increasing neuronal survival. The aforementioned findings are associated with a downregulation of the expression levels of pro-inflammatory cytokines TNF-α, IL-1β and IL-6. Conversely, no significant changes of glutamate transporter-1 expression were observed. The present results suggest that 17-AAG treatment may provide a neuroprotective effect by reducing inflammation following TBI.

Hsp60 exerts a tumor suppressor function by inducing cell differentiation and inhibiting invasion in hepatocellular carcinoma.

Heat shock protein 60 (Hsp60), a typical mitochondrial chaperone, is associated with progression of various cancers. However, its expression and significance in hepatocellular carcinoma (HCC) remain largely unclear. In the present study, the mRNA and protein expression of Hsp60 in HCC tissues were detected by quantitative RT-PCR (n=24), western blot (n=7), and immunohistochemical staining (n=295), respectively. The correlation between Hsp60 expression and clinicopathological characteristics of HCC patient was also analyzed. Meanwhile, the influence of Hsp60 on malignant phenotype of HCC cells was further investigated. We found that expression of Hsp60 was significantly downregulated in HCC tissues compared to peritumor tissues. Hsp60 expression was significantly correlated with serum alpha -foetoprotein (AFP) level and tumor differentiation grade. Moreover, high Hsp60 expression cancer/pericancer (C/P) ratio was associated with a better overall survival rate (P=0.035, n=295). The prognostic implication of Hsp60 in HCC was further confirmed in another cohort of 107 HCC patients (P=0.027). Up-regulation of Hsp60 remarkably induced the cell differentiation and inhibited the invasive potential of HCC in vitro and in vivo. Intriguingly, the down-regulation of Hsp60 significantly impaired mitochondrial biogenesis. Although more data are required to clarify the underling mechanism responsible for function of Hsp60, our results suggested that the effect of Hsp60 on differentiation and invasion of HCC cells might be associated with mitochondrial biogenesis. Collectively, our findings indicated that Hsp60 exerted a tumor suppressor function, and might serve as a potential therapeutic target in the treatment of HCC.

Design, synthesis and pharmacological evaluation of 4,5-diarylisoxazols bearing amino acid residues within the 3-amido motif as potent heat shock protein 90 (Hsp90) inhibitors

A structure-based medicinal chemistry optimization was conducted on the clinical Hsp90 inhibitor diarylisoxazole 3. Several series of new compounds were designed and synthesized by incorporating diversified amino acid derivatives with various lengths to the 3-amido motif of the isoxazole scaffold. Compound 14j was identified to have high Hsp90 binding potency (14 nM) and antiproliferative activity against H3122 human lung cancer and BT-474 breast cancer cells. Treatment of 14j with H3122 cell led to degradation of client protein ALK, reduction of downstream phosphorylation of AKT and ERK, and up-regulation of Hsp70. Molecular docking suggested that the terminal valine moiety and the ethyleneglycol linker in compound 14j formed additional apolar and polar interaction network with a number of amino acid residues.

Targeting HSP70 and GRP78 in canine osteosarcoma cells in combination with doxorubicin chemotherapy.

Heat shock proteins (HSPs) are molecular chaperones subdivided into several families based on their molecular weight. Due to their cytoprotective roles, these proteins may help protect cancer cells against chemotherapy-induced cell death. Investigation into the biologic activity of HSPs in a variety of cancers including primary bone tumors, such as osteosarcoma (OSA), is of great interest. Both human and canine OSA tumor samples have aberrant production of HSP70. This study assessed the response of canine OSA cells to inhibition of HSP70 and GRP78 by the ATP-mimetic VER-155008 and whether this treatment strategy could sensitize cells to doxorubicin chemotherapy. Single-agent VER-155008 treatment decreased cellular viability and clonogenic survival and increased apoptosis in canine OSA cell lines. However, combination schedules with doxorubicin after pretreatment with VER-155008 did not improve inhibition of cellular viability, apoptosis, or clonogenic survival. Treatment with VER-155008 prior to chemotherapy resulted in an upregulation of target proteins HSP70 and GRP78 in addition to the co-chaperone proteins Herp, C/EBP homologous transcription protein (CHOP), and BAG-1. The increased GRP78 was more cytoplasmic in location compared to untreated cells. Single-agent treatment also revealed a dose-dependent reduction in activated and total Akt. Based on these results, targeting GRP78 and HSP70 may have biologic activity in canine osteosarcoma. Further studies are required to determine if and how this strategy may impact the response of osteosarcoma cells to chemotherapy.

Hsp90 and hsp47 appear to play an important role in minnow Puntius sophore for surviving in the hot spring run-off aquatic ecosystem.

Changes in the expression of a number of hsp genes in minnow Puntius sophore collected from a hot spring run-off (Atri hot spring in Odisha, India; 20o09'N 85°18'E, 36-38°C) were investigated to study the upper thermal acclimation response under heat stress, using same species from aquaculture ponds (water temperature 27°C) as control. Expression of hsp genes was analyzed in both groups using RT-qPCR, which showed up-regulation of hsp90 (2.1-fold) and hsp47 (2.5-fold) in hot spring run-off fishes, whereas there was no alteration in expression of other hsps. As the fish inhabit the hot spring run-off area for very long duration, they could have adapted to the environment. To test this hypothesis, fishes collected from hot spring run-off were divided into two groups; one was heat-shocked at 41°C/24h, and the other was acclimatized at 27°C/24h. Up-regulation of all the hsps (except hsp78) was observed in the heat-shocked fishes, whereas expression of all hsps was found to be down-regulated to the basal level in fishes maintained at 27°C/24h. Pathway analysis showed that the expressions of all the hsps except hsp90 are regulated by the transcription factor heat shock factor 1 (Hsf1). This study showed that hsp90 and hsp47 play an important role in Puntius sophore for surviving in the high-temperature environment of the hot spring run-off. Additionally, we show that plasticity in hsp gene expression is not lost in the hot spring run-off population.

Spironolactone Prevents Endothelial Nitric Oxide Synthase Uncoupling and Vascular Dysfunction Induced by β-Adrenergic Overstimulation

Sustained stimulation of β-adrenoceptors (β-ARs) and activation of renin–angiotensin–aldosterone system are common features of cardiovascular diseases with rising sympathetic activation, including essential hypertension, myocardial infarction, and heart failure. In this study, we investigated the role of AT1 receptor and mineralocorticoid receptor (MR) in the vascular alterations caused by β-AR overstimulation. β-AR overstimulation with associated cardiac hypertrophy and increased vasoconstrictor response to phenylephrine in aorta were modeled in rats by 7-day isoproterenol treatment. The increased vasoconstrictor response to phenylephrine in this model was blunted by the MR antagonist spironolactone, but not by the AT1 receptor antagonist losartan, despite the blunting of cardiac hypertrophy with both drugs. Spironolactone, but not losartan, restored NO bioavailability in association with lower endothelial nitric oxide synthase–derived superoxide production, increased endothelial nitric oxide synthase dimerization, and aortic HSP90 upregulation. MR genomic and nongenomic functions were activated in aortas from isoproterenol-treated rats. Isoproterenol did not modify plasma levels of MR ligands aldosterone and corticosterone but rather increased perivascular adipose tissue–derived corticosterone in association with increased expression of 11β-hydroxysteroid dehydrogenase type 1. The anticontractile effect of aortic perivascular adipose tissue was impaired by β-AR overstimulation and restored by MR blockade. These results suggest that activation of vascular MR signaling contributes to the vascular dysfunction induced by β-AR overstimulation associated with endothelial nitric oxide synthase uncoupling. These findings reveal an additional explanation for the protective effects of MR antagonists in cardiovascular disorders with sympathetic activation.

Preclinical Study of AUY922, a Novel Hsp90 Inhibitor, in the Treatment of Esophageal Adenocarcinoma.

To assess the efficacy of heat-shock protein 90 (Hsp90) inhibitor, NVP-AUY922-AG (AUY922), in the treatment of esophageal adenocarcinoma (EAC) in vitro and in vivo. EAC is a leading cause of cancer death, and current treatment options are limited. Hsp90, a chaperone protein that regulates several oncoproteins, is upregulated in EAC, and may be a novel target for therapy. In vitro, EAC cell lines were utilized to evaluate AUY922, alone and in combination with 5-fluorouracil (5-FU) and cisplatin. BrdU ELISA and flow cytometry were used to assess proliferation and measure apoptosis, respectively. Western blot and RT-PCR were performed to quantitate Hsp90 pathway expression. In vivo, esophagojejunostomy was performed on rats and treatment animals received AUY922 32 to 40 weeks postoperatively. Drug efficacy was evaluated with magnetic resonance imaging (MRI), endoscopic biopsy, gross histological evaluation, and Hsp90 pathway expression. In vitro, AUY922 demonstrated antiproliferative activity in both cell lines and showed enhanced efficacy with cisplatin and 5-FU. Western Blot and RT-PCR demonstrated downregulation of CDK1 and CDK4 and upregulation of Hsp72. In vivo, AUY922 showed decrease in tumor volume in 36.4% of rats (control = 9.4%), increase in 9.1% (control = 37.5%), and stable disease in 54.5% (control = 43.7%). Necropsy confirmed the presence of EAC in 50% of treatment animals and 75% of control animals. mRNA expression, pre- and posttreatment, demonstrated significant downregulation of MIF, Hsp70, Hsp90β, and CDK4, and upregulation of Hsp72. AUY922 exhibits antitumor efficacy in vitro and in vivo for EAC, suggesting the need for human clinical trials.

Heat stress-induced phosphorylation of FoxO3a signalling in rat skeletal muscle.

A recent study demonstrated that FoxO3a was directly induced by the overexpression of Hsp72 in rat soleus muscle. However, whether heat stress treatment induces FoxO3a phosphorylation in rat skeletal muscle remains unclear. This study examined the effects of heat stress on the regulation of the FoxO3a signalling pathway in rat skeletal muscle. Thirty-two male Wistar rats (15weeks old) were randomly assigned into two groups; sedentary control group (Sed, n=8) and experimental group (n=24). After an overnight fast, one leg of each rat (HS leg) in the experimental group was immersed in hot water (43°C) for 30min, and the soleus and plantaris muscles in both legs were removed immediately (0min), 30min, 60min, or 24h after the heat stress (n=6 each group). The contralateral, non-heated leg in the experimental group served as an internal control (CT leg). Heat stress treatment resulted in a significant increase in FoxO3a phosphorylation (Ser253) in the soleus and plantaris muscles of heat-stressed legs after 24h. Hsp72 expression in heat-stressed legs was significantly higher at 60min and 24h in these muscles. Activation of the PTEN/Akt and MEK/ERK pathways was also observed in these muscles immediately after stress, but not at 24h. There were no differences in FoxO1 and AMPKα phosphorylation in either muscle. Heat stress in rat skeletal muscle induces phosphorylation of FoxO3a signalling, and it may be related to Hsp72 upregulation, and the activation of the PTEN/Akt and MEK/ERK pathways.

Understanding mechanism of sea cucumber Apostichopus japonicus aestivation: Insights from TMT-based proteomic study

Marine invertebrate aestivation is a unique strategy for summer survival in response to hot marine conditions. The sea cucumber, Apostichopus japonicus, is an excellent model marine invertebrate for studies of environmentally-induced aestivation. In the present study, we used a tandem mass tag (TMT)-coupled LC-MS/MS approach to identify and quantify the global proteome expression profile over the aestivation-arousal cycle of A. japonicus. A total of 3920 proteins were identified from the intestine of sea cucumber. Among them, 630 proteins showed significant differential expression when comparing three conditions of sea cucumbers: non-aestivating (active), deep-aestivation (at least 15days of continuous aestivation), and arousal after aestivation (renewed moving and feeding). Sea cucumbers in deep aestivation showed substantial differentially expressed proteins (143 up-regulated and 267 down-regulated proteins compared with non-aestivating controls). These differentially expressed proteins suggested that protein and phospholipid probably are major fuel sources during hypometabolism and a general attenuation of carbohydrate metabolism was observed during deep aestivation. Differentially expressed proteins also provided the first global picture of a shift in protein synthesis, protein folding, DNA binding, apoptosis, cellular transport and signaling, and cytoskeletal proteins during deep aestivation in sea cucumbers. A comparison of arousal from aestivation with deep aestivation, revealed a general reversal of the changes that occurred in aestivation for most proteins. Western blot detection further validated the significant up-regulation of HSP70 and down-regulation of methyltransferase-like protein 7A-like in deep-aestivation. Our results suggest that there is substantial post-transcriptional regulation of proteins during the aestivation-arousal cycle in sea cucumbers.

Antioxidant Properties and Gastroprotective Effects of 2-(Ethylthio)Benzohydrazones on Ethanol-Induced Acute Gastric Mucosal Lesions in Rats.

A series of new 2-(ethylthio)benzohydrazone derivatives (1–6) were prepared and characterised by IR, 1H NMR, and 13C NMR spectroscopy and mass spectrometry. The newly prepared compounds were screened for their in vitro antioxidant activities using free radical scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. Among them, most powerful antioxidant, compound 1 has been selected in order to illustrate anti-ulcer effect on ethanol-induced gastric mucosal lesions in rats. Four groups of Sprague Dawley rats were respectively treated with 10% Tween 20 as ulcer control group, 20 mg/kg omeprazole as reference group, 50 mg/kg and 100 mg/kg compound 1 as experimental animals. Macroscopically, ulcer control group showed extensive hemorrhagic lesions of gastric mucosa compared with omeprazole or compound 1. Rats pre-treated with compound 1 showed increased in gastric pH and gastric mucus. Histologically, ulcer control group showed severe damage to gastric mucosa with edema and leucocytes infiltration of submucosal layer. In immunohistochemical analysis, rats which were pre-treated with compound 1 showed up-regulation of HSP70 and down-regulation of Bax proteins. In conclusion, the gastroprotective effect of compound 1 may be due to its antioxidant activity, and/or due to up-regulation of HSP70 and down-regulation of Bax protein in stained tissue section.

Effects of abnormal temperature and starvation on the internal defense system of the schistosome-transmitting snail Biomphalaria glabrata

Climate change may affect the internal defense system (IDS) of freshwater snails, and as a result their capacity to transmit disease. We examined effects of short-term exposure to supra- and sub-optimal temperatures or starvation on 3 parameters of the IDS of the schistosome-resistant Salvador strain of Biomphalaria glabrata — hemocyte concentrations, cell division in the amebocyte-producing organ (APO), and resistance to infection with Schistosoma mansoni. Adult snails were exposed to 1 of 3 temperatures, 20°C, 27°C (controls), or 33°C, for 1 or 2weeks, with food. A fourth group was maintained at 27°C, but without food. Compared to the controls, starved snails had significantly higher hemocyte counts at both 1 and 2weeks, although mitotic activity in the APO was significantly lower at both time periods. Exposure to 20°C or 33°C for 1 or 2weeks did not affect hemocyte numbers. However, APO mitotic activity in snails exposed to 20°C was significantly higher at both 1 and 2weeks, whereas mitotic activity in snails exposed to 33°C was significantly lower at 1week but normal at 2weeks. None of the treatments altered the resistance phenotype of Salvador snails. In a follow-up experiment, exposure to 33°C for 4–5h, a treatment previously reported to both induce expression of heat shock proteins (Hsps) and abrogate resistance to infection, caused immediate upregulation of Hsp 70 and Hsp 90 expression, but did not alter resistance, and Hsp expression levels returned to baseline after 2weeks at 33°C. Results of this study indicate that abnormal environmental conditions can have both stimulatory and inhibitory effects on the IDS in adult B. glabrata, and that some degree of acclimation to abnormal temperatures may occur.

Pelleted Bone Marrow Derived Mesenchymal Stem Cells Are Better Protected from the Deleterious Effects of Arthroscopic Heat Shock.

Introduction: The impact of arthroscopic temperature on joint tissues is poorly understood and it is not known how mesenchymal stem cells (MSCs) respond to the effects of heat generated by the device during the process of arthroscopy assisted experimental cell-based therapy. In the present study, we isolated and phenotypically characterized human bone marrow mesenchymal stem cells (hBMMSCs) from osteoarthritis (OA) patients, and evaluated the effect of arthroscopic heat on cells in suspension and pellet cultures.Methods: Primary cultures of hBMMSCs were isolated from bone marrow aspirates of OA patients and cultured using DMEM supplemented with 10% FBS and characterized for their stemness. hBMMSCs (1 × 106 cells) cultured as single cell suspensions or cell pellets were exposed to an illuminated arthroscope for 10, 20, or 30 min. This was followed by analysis of cellular proliferation and heat shock related gene expression.Results: hBMMSCs were viable and exhibited population doubling, short spindle morphology, MSC related CD surface markers expression and tri-lineage differentiation into adipocytes, chondrocytes and osteoblasts. Chondrogenic and osteogenic differentiation increased collagen production and alkaline phosphatase activity. Exposure of hBMMSCs to an illuminated arthroscope for 10, 20, or 30 min for 72 h decreased metabolic activity of the cells in suspensions (63.27% at 30 min) and increased metabolic activity in cell pellets (62.86% at 10 min and 68.57% at 20 min). hBMMSCs exposed to 37, 45, and 55°C for 120 s demonstrated significant upregulation of BAX, P53, Cyclin A2, Cyclin E1, TNF-α, and HSP70 in cell suspensions compared to cell pellets.Conclusions: hBMMSC cell pellets are better protected from temperature alterations compared to cell suspensions. Transplantation of hBMMSCs as pellets rather than as cell suspensions to the cartilage defect site would therefore support their viability and may aid enhanced cartilage regeneration.

Dual PI3K- and mTOR-inhibitor PI-103 can either enhance or reduce the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in tumor cells: The role of drug-irradiation schedule.

Inhibition of Hsp90 can increase the radiosensitivity of tumor cells. However, inhibition of Hsp90 alone induces the anti-apoptotic Hsp70 and thereby decreases radiosensitivity. Therefore, preventing Hsp70 induction can be a promising strategy for radiosensitization. PI-103, an inhibitor of PI3K and mTOR, has previously been shown to suppress the up-regulation of Hsp70. Here, we explore the impact of combining PI-103 with the Hsp90 inhibitor NVP-AUY922 in irradiated glioblastoma and colon carcinoma cells. We analyzed the cellular response to drug-irradiation treatments by colony-forming assay, expression of several marker proteins, cell cycle progression and induction/repair of DNA damage. Although PI-103, given 24 h prior to irradiation, slightly suppressed the NVP-AUY922-mediated up-regulation of Hsp70, it did not cause radiosensitization and even diminished the radiosensitizing effect of NVP-AUY922. This result can be explained by the activation of PI3K and ERK pathways along with G1-arrest at the time of irradiation. In sharp contrast, PI-103 not only exerted a radiosensitizing effect but also strongly enhanced the radiosensitization by NVP-AUY922 when both inhibitors were added 3 h before irradiation and kept in culture for 24 h. Possible reasons for the observed radiosensitization under this drug-irradiation schedule may be a down-regulation of PI3K and ERK pathways during or directly after irradiation, increased residual DNA damage and strong G2/M arrest 24 h thereafter. We conclude that duration of drug treatment before irradiation plays a key role in the concomitant targeting of PI3K/mTOR and Hsp90 in tumor cells.

Functional proteomic analysis revels that the ethanol extract of Annona muricata L. induces liver cancer cell apoptosis through endoplasmic reticulum stress pathway

Ethnopharmacological relevanceAnnona muricata L. is used to treat cancer in some countries. Extracts of Annona muricata have been shown to cause apoptosis of various cancer cells in vitro, and inhibit tumor growth in vivo in animal models. However, the molecular mechanisms underlying its anti-cancer and apoptotic effects of the herb remain to be explored. Aim of studyThe study investigated the molecular mechanisms underlying liver cancer cell apoptosis triggered by the ethanol extract of leaves of Annona muricata L. Materials and methodsLiver cancer HepG2 cells were used as experimental model. MTT assay was employed to evaluate cell viability. Flow cytometry and TUNEL assays were performed to confirm apoptosis. We employed functional proteomic analysis to delineate molecular pathways underlying apoptosis triggered by the herbal extract. ResultsWe showed that the extract was able to reduce viability and trigger apoptosis of the cancer cells. Proteomic analysis identified 14 proteins associated with the extract-elicited apoptosis, which included the increased expression levels of HSP70, GRP94 and DPI-related protein 5. Western blot analysis confirmed that the extract did up-regulated the protein levels of HSP70 and GRP94. Results from bioinformatic annotation pulled out two molecular pathways for the extract, which, notably, included endoplasmic reticulum (ER) stress which was evidenced by the up-regulation of HSP70, GRP94 and PDI-related protein 5. Further examinations of typical protein signaling events in ER stress using western blot analysis have shown that the extract up-regulated the phorsphorelation of PERK and eIF2α as well as the expression level of Bip and CHOP. ConclusionOur results indicate that the ethanol extract of leaves of Annona muricata L. causes apoptosis of liver cancer cells through ER stress pathway, which supports the ethnomedicinal use of this herb as an alternative or complementary therapy for cancer.

Up-regulation of Hsp27 by ERα/Sp1 facilitates proliferation and confers resistance to apoptosis in human papillary thyroid cancer cells

17β-estradiol (E2) has been suggested to play a role in the development and progression of papillary thyroid cancer. Heat shock protein 27 (Hsp27) is a member of the Hsp family that is responsible for cell survival under stressful conditions. Previous studies have shown that the 5′-promoter region of Hsp27 gene contains a specificity protein-1 (Spl) and estrogen response element half-site (ERE-half), which contributes to Hsp27 induction by E2 in breast cancer cells. However, it is unclear whether Hsp27 can be up-regulated by E2 and which estrogen receptor (ER) isoform and tethered transcription factor are involved in this regulation in papillary thyroid cancer cells. In the present study, we demonstrated that Hsp27 can be effectively up-regulated by E2 at mRNA and protein levels in human K1 and BCPAP papillary thyroid cancer cells which have more than two times higher level of ERα than that of ERβ. The up-regulation of Hsp27 by E2 is mediated by ERα/Sp1 and ERβ has repressive effect on this ERα/Sp1-mediated up-regulation of Hsp27. Moreover, we showed that the up-regulation of Hsp27 by ERα/Sp1 facilitates proliferation and confers resistance to apoptosis through interaction with procaspase-3. Targeting this pathway may be a potential strategy for therapy of papillary thyroid cancer.

Impact of Silver and Carbon Nanoparticle Exposures on Macrophage Responses to Mycobacterium tuberculosis (M.tb)

Abstract To assess biological risks of nanoparticle (NP) exposures, we examined the effects of silver (Ag) core Ag20-citrate (Ag20) and carbon black (CB) on human host innate immune responses to M.tb, a respiratory pathogen that causes tuberculosis. Human peripheral blood monocyte-derived macrophages (MDMs) were exposed to 1, 10, 25 and 50 μg/mL of Ag20 and CB for 4 and 24 h. At 4 h no significant cytotoxicity was observed with neither of the NPs at none of the concentrations examined relative to unexposed MDMs. At 24 h, viability of the MDMs was reduced by 60–70% and 20% following Ag20 and CB exposure, respectively, at doses ≥25 μg/mL. M.tb-induced host immune responses are mediated by the activation of TLR signalling pathways that culminate in NF-κB activation. Effects of Ag20 and CB (10μg/mL for 4 h, a nontoxic dose) on TLR signalling in M.tb-infected and uninfected MDMs were compared using a human TLR signaling pathway-specific profiler array. Ag20 exposure suppressed the M.tb-induced expression of a subset of NF-κB-mediated target genes (CSF2, CSF3, IFNG, IL1A, IL1B, IL6, IL10, TNFA, NFKB1A). Ag20-mediated suppression of M.tb-induced activation of the TLR-signalling pathway was not due to decreased uptake of M.tb by MDMs or mycobacteriocidal effects of Ag20. Ag20 exposure increased the expression of HSPA1A mRNA that encodes for the stress-induced Hsp72. In contrast, exposure to CB resulted in little effects on M.tb-induced host gene expression indicating that immunosuppressive effects of Ag20 are NP-specific. Since Hsp72 has been shown to suppress NF-κB activation, we propose that Ag20-mediated Hsp72 upregulation contributes to the suppression of M.tb-induced NF-κB activation and host immune responses. Funding NIEHS 5U19 ES019536

Short-term effect of bisphenol-a on oxidative stress responses in Atlantic salmon kidney cell line: a transcriptional study

Bisphenol A (BPA) is regularly detected in aquatic ecosystems due to increased use of products based on polycarbonate plastics and epoxy resins. It migrates from these products directly into rivers and marine waters or indirectly through effluents from wastewater treatment plants and landfilled sites. BPA can affect aquatic organisms both chronically and acutely at sensitive live stages. Despite reports indicating harmful effects of BPA, little is known about its role in oxidative stress responses in fish. In this study, we investigated the transcriptional effect of BPA (0, 1, 10, 100 μM) on an Atlantic salmon kidney (ASK) cell line for 6 h and 24 h by monitoring expression of 11 genes: elongation factor 1-alpha (ef1a), 18S ribosomal RNA (18s), gluthation (gsh), superoxide dismutase (sod), thioredoxin (txd), Salmo salar oxidative stress-responsive serine-rich 1 (oxr), glucose-regulated protein 78 (grp78), heat shock protein 70 (hsp70), sequestosome1 (p62), interleukin-1 beta (il-1beta) and toll-like receptor 8 (tlr8). In general, only the 100 μM concentration treatment altered the mRNA expression. BPA down-regulated the expression of gsh and sod genes for both exposure-times while txd gene was the only down-regulated after 6-h exposure. The up-regulation of genes in the ASK cell line exposed for 6 h was only observed in il-1beta, while the 24-h exposure resulted in the up-regulation of oxr, tlr8, hsp70, p62 and il-1beta genes. The last three genes increased several fold compared to the others. The results showed that BPA exposure at 100 μM imposed oxidative stress on the ASK cell line and longer exposure time involved transcriptional responses of immune-related genes. This may indicate the possible role of BPA-associated oxidative stress in induction of inflammatory response in this macrophage-like cell type.

Compression Induced Stress Response of Nucleus Pulposus Cells in 3D Collagen Gel

Introduction The cause of degenerative disc disease is multi-factorial and is often associated with mechanical loading. However, mechanism of the cells' response to compressive loading is not fully known. It was demonstrated that in mechanical force can induce chondrocytes to upregulate expression of heat shock proteins (HSPs) as a form of cellular stress response.1 Similarly, stretching annulus fibrosus cells was found to upregulate endoplasmic reticulum stress markers.2 However, it is unclear that whether cells in the intervertebral disc (IVD) would experience compressive force as cellular stress in 3D environment. The objective of this study is to investigate the stress response of nucleus pulposus cells (NPCs) during compression in 3D culture environment. Materials and Methods To simulate the condition in the IVD, NPCs were isolated from bovine caudal disc and encapsulated in 3D collagen gel as described previously.3 The culture system was shown to be able to maintain NPCs' phenotype.4 The cell-encapsulated gels were cultured for 4 days and then compressed with different types of loading (static and dynamic loading), different strains (10% - 70%) and different durations (2h - 8h). Samples were retrieved to study the expressions of stress response genes in two groups: Heat Shock Response (HSP70, HSP27, HSP90 and HSF1) and Unfolded Protein Response (GRP78, GRP94, ATF4 and CHOP). Results Encapsulation in 3D collagen gel was found to upregulate HSP70 in NPCs. Compression further induced stress response of NPCs which depended on multiple factors. The upregulation of stress response genes was generally insignificant with increasing loading strain in short duration. However the upregulations increased with longer duration where increasing loading duration has a larger effect in the upregulation of HSP70 and HSP27. When high static strain was applied, the expressions of Heat Shock Response genes were continued to be upregulated with time after the load was removed while Unfolded Protein Response genes were downregulated with incubation duration. The changes in expression with incubation duration show that cellular stress response may play a role in NPC survival and protein homeostasis post-loading. Conclusion The NPCs demonstrated cellular stress response during compression in collagen gel. The expression of stress response genes were mainly affected by loading duration and incubation duration after loading. This study helps us to understand how the NPCs cope with mechanical stress. Acknowledgments This project was funded by TBRS (T12–708/12-N). References Kaarniranta K, Elo M, Sironen R, et al. Hsp70 accumulation in chondrocytic cells exposed to high continuous hydrostatic pressure coincides with mRNA stabilization rather than transcriptional activation. Proc Natl Acad Sci U S A 1998;95(5):2319–2324 Zhang YH, Zhao CQ, Jiang LS, Dai LY. Cyclic stretch-induced apoptosis in rat annulus fibrosus cells is mediated in part by endoplasmic reticulum stress through nitric oxide production. Eur Spine J 2011;20(8):1233–1243 Chan BP, Hui TY, Yeung CW, Li J, Mo I, Chan GC. Self-assembled collagen-human mesenchymal stem cell microspheres for regenerative medicine. Biomaterials 2007;28(31):4652–4666 Yuan M, Leong KW, Chan BP. Three-dimensional culture of rabbit nucleus pulposus cells in collagen microspheres. Spine J 2011;11(10):947–960

Upregulation of Hsp70 Protects the Lung Microvascular Endothelial Barrier from Pneumolysin Induced Injury

Pneumonia is a leading cause of death in children and the elderly worldwide, accounting for 15% of all deaths of children under 5 years old. Despite optimal antibiotic therapies and supportive care, mortality remains unacceptably high. Streptococcus pneumonia is one of the most common and most aggressive causes of pneumonia and sepsis. The Gram positive toxin, pneumolysin (PLY), is released from the cytosol of compromised Streptococcus and plays a key role in the development of the extensive permeability edema that causes acute lung injury (ALI). The mechanism by which PLY disrupts the endothelial barrier is not fully understood and greater insight is needed to develop novel therapeutics to counter microvascular permeability and ALI. Previously we found that Hsp90 inhibitors significantly reduce the production of barrier disruptive reactive oxygen species (ROS) via inhibition of NADPH oxidase activity. The suppression of Hsp90 is accompanied by increased expression of Hsp70 and we revealed an unexpected but potent role of Hsp70 in suppressing ROS production. Therefore the goal of the current study was to identify whether the upregulation of Hsp70 is an effective strategy to protect the lung microvascular endothelial barrier from Gram positive bacterial toxins. Overexpression of Hsp70 via adenovirus decreased NOX‐derived ROS and was highly effective at preventing the PLY‐stimulated increases in permeability of human lung microvascular endothelial cells (HLMVEC) and limiting cytotoxicity. To acutely stimulate endogenous Hsp70 expression, we employed geranylgeranylacetone (GGA) an activator of HSF. After 4hrs treatment with GGA, HLMVECs exposed to PLY displayed improved cell viability and enhanced endothelial barrier function as measured by both Electric Cell‐substrate Impedance Sensing (ECIS) and transwell permeability assay compared to controls. In mice, GGA was administered IP and 4hrs later PLY was administered IT to disrupt lung microvascular function. Mice treated with GGA had significantly less Evans Blue Dye extravasation suggesting that the acute upregulation of Hsp70 may be an effective therapeutic approach in the treatment of lung injury associated with pneumonia.Support or Funding InformationNIH, P01HL101902

Transcriptional profiling induced by pesticides employed in organic agriculture in a wild population of Chironomus riparius under laboratory conditions

Copper (Cu) and azadirachtin (AZA-A+B) are pesticides allowed in organic agriculture whose environmental risk and toxicity for aquatic wildlife is only partially known. Reverse Transcription Polymerase Chain Reaction was used to assess the molecular effect of acute and short-term exposure (3, 24h) of Cu (0.01, 0.05, 1, 10, 25mgl−1) and AZA-A+B (0.2, 0.3, 0.4, 0.5, 1mgl−1) on the expression of five candidate genes (hsp70, hsc70, hsp40, hsp10 and cyP450) in a non-target species, Chironomus riparius. Fourth-instar larvae were collected from a mountain stream polluted by agricultural land run-off. All genes were responsive to both pesticides but each gene had a specific response to the different experimental concentrations and exposure times. A few similarities in transcriptional profiling were observed, such as a linear concentration-dependent response of hsp70 after 24h of exposure (at ≥1mgl−1 of Cu and ≥0.2mgl−1 of AZA-A+B) and an up-regulation regardless of the concentration of hsc70 after 24h of exposure (at ≥0mgl−1 of Cu and ≥0.2mgl−1 of AZA-A+B and the up-regulation of hsp70 after 3h of exposure at ~LC50 (Cu-LC50=26.1±2.5mgl−1, AZA-A+B-LC50=1.1±0.2mgl−1). According to the results, hsp40, hsp10 and cyP450 may be defined as pesticide-dependent (i.e., hsp40 and hsp10 seemed to responded mainly to AZA-A+B and cyP450 to Cu), while hsc70 as time-dependent regardless of the pesticide (i.e., hsc70 responded only after 24h of treatment with Cu and AZA-A+B). This study gives new insights on the potential role of the C. riparius's hsps and cyP450 genes as sensitive biomarkers for freshwater monitoring.