Dengue virus entry into liver (HepG2) cells is independent of hsp90 and hsp70

Recently, several stress-related proteins including GRP78, hsp70, and hsp90 have been implicated as dengue virus receptors in various cell types, with hsp90/70 being implicated as a receptor complex in monocytes and macrophages, while GRP78 has been implicated as a liver cell expressed dengue virus receptor. To assess whether the hsp90/70 complex plays a role in the internalization of the dengue viruses into liver cells, we undertook infection inhibition studies with lipopolysaccharide and antibodies directed against both hsp70 and hsp90, individually and in combination. No inhibition of any dengue serotype was seen in the presence of lipopolysaccharide or antibodies directed against either hsp70 or hsp90 either singly or in combination. A moderate inhibition of dengue virus serotype 2 entry into liver cells was observed in the presence of antibodies directed against GRP78. These results confirm a proposed role for GRP78 as a dengue virus serotype 2 receptor protein and suggest that the recently identified hsp90/70 complex does not play a role in dengue virus internalization into liver cells.     

Growth factors acting via tyrosine kinase receptors induce HSP90 alpha gene expression.

Hsp90 is a heat-shock protein constitutively expressed in most cells. Besides regulation by thermal stress, the expression of hsp90 is also positively regulated by developmental and mitogenic stimuli. The effect of serum and insulin on protein and hsp90 alpha-mRNA levels has been studied in the chicken hepatoma cell line DU249. The culture of cells in serum-free medium resulted in a decrease of hsp90 alpha-mRNA level. A transient increase was observed at 6-9 h after serum restimulation. The expression of hsp90 gene was also increased by insulin alone in a dose-dependent manner and was maximum between 6 and 9 h treatment. The insulin induced increase of hsp90 alpha-mRNA was suppressed by cycloheximide (10 micrograms/ml) but not by an inhibitor of DNA synthesis, demonstrating that this induction requires protein neosynthesis. In serum starved cells, other growth factors (IGF1, EGF and bFGF) showed a positive effect on hsp90 alpha-mRNA level which took place before DNA synthesis with the same time-course as that of insulin. With PDGF, the induction of hsp90 alpha-mRNA occurred earlier. The time interval between the maximum of hsp90 alpha-mRNA induction and that of DNA synthesis was the same for all growth factors studied. From these results, we conclude that growth factors acting via tyrosine kinase receptors up-regulate hsp90 alpha-mRNA level in a DNA synthesis independent manner, possibly in late G1.     

Pharmacologic and genetic inhibition of hsp90-dependent trafficking reduces aggregation and promotes degradation of the expanded glutamine androgen receptor without stress protein induction

The molecular chaperone hsp90 has emerged as an important therapeutic target in cancer and neurodegenerative diseases, including the polyglutamine expansion disorders, because of its ability to regulate the activity, turnover and trafficking of many proteins. For neurodegenerative disorders associated with protein aggregation, the rationale has been that inhibition of hsp90 by geldanamycin and related compounds activates heat shock factor 1 (HSF1) to induce the production of the chaperones hsp70 and hsp40 that promote disaggregation and protein degradation. However, we show here that geldanamycin blocks the development of aggregates of the expanded glutamine androgen receptor (AR112Q) of Kennedy disease in Hsf1(-/-) mouse embryonic fibroblasts where these chaperones are not induced. Geldanamycin is additionally known to inhibit hsp90-dependent protein trafficking and to promote proteasomal degradation of client proteins. Overexpression of the hsp90 cochaperone p23 also promotes AR112Q degradation, and inhibits both AR trafficking and AR112Q aggregation without altering levels of hsp70 or hsp40. The hsp90-dependent trafficking mechanism has been defined, and it is shown that key immunophilin (IMM) components of the trafficking machinery are present in polyglutamine aggregates in cell and mouse models of Kennedy disease. Our results indicate that inhibition of the hsp90-dependent trafficking mechanism prevents aggregation of the expanded glutamine androgen receptor, thereby opening a variety of novel therapeutic approaches to these neurodegenerative disorders.     

GRP78 up-regulation is associated with androgen receptor status, Hsp70-Hsp90 client proteins and castrate-resistant prostate cancer

GRP78/BiP is a key member of the molecular chaperone heat shock protein (Hsp) 70 family. It has a critical role in prostate cancer (PC) including Pten loss‐driven carcinogenesis, but the molecular basis of this remains unclear. We investigated the effect of GRP78 and its putative client proteins, including androgen receptor (AR) in clinical PC. Expression of GRP78 and key Hsp70–hsp90 client proteins (HER2, HER3, AR and AKT) were studied in an incidence tissue microarray (TMA) of prostate cancer. The relationship of GRP78 and AR was further tested in in vitro cell models (LNCaP and its derived LNCaP‐CR subclone) and a matched TMA of hormone‐naïve (HNPC) and castrate‐resistant prostate cancer (CRPC). In vitro and in vivo expression of GRP78 and client proteins were assessed by western blotting and immunohistochemistry, respectively, using the weighted histoscore method. Significant co‐expression of GRP78, pAKT, HER2, HER3 and AR was observed in PC. Abnormal AR, GRP78 and pAKT expression have significant impact on patient survival. GRP78 expression in AR⁺ tumours was significantly higher than in AR^? tumours. In keeping with our clinical data, activation of AR by dihydrotestosterone (DHT) potently activated GRP78 expression in both LNCaP and LNCaP‐CR cells. For the first time, using a matched HNPC and CRPC TMA, enhanced cytoplasmic and membranous GRP78 expression was observed in CRPC. Future prospective studies are therefore warranted to validate GRP78 as prognostic marker and therapeutic target, in the context of the AR and pAKT status. In summary, GRP78 is co‐expressed with Hsp70–hsp90 client proteins. Up‐regulated expression of AR and GRP78 expression in untreated prostate cancer predicts a less favourable outcome. This points to the importance of understanding in the molecular interaction among AR, GRP78 and AKT. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Heat shock protein 70 associations with myelin basic protein and proteolipid protein in multiple sclerosis brains

Heat shock proteins (hsp) are known to facilitate the generation of specific immune responses by chaperoning proteins and peptides involved in T cell activation. Hsp have been shown to be strikingly elevated in multiple sclerosis (MS) lesions. The unique chaperonin properties of hsp70 have allowed identification of immunogenic proteins bound to it by the ex vivo demonstration of hsp associations with proteins implicated in the immune response. We have investigated the association of hsp70 with myelin basic protein (MBP), myelin proteolipid protein (PLP) and myelin oligodendrocyte protein (MOG) in MS and control brain tissue. In co-immunoprecipitation experiments, in all samples of MS brains examined (n = 3), but not control brain tissue (n = 3), direct association of MBP with hsp70, but not with hsp90, was found. In some MS brain samples, association between PLP and hsp70 was also seen. In similar co-immunoprecipitation experiments on brain tissue obtained from mice with experimental autoimmune encephalomyelitis (n = 5) induced by immunization with PLP peptide, specific association of hsp70 with PLP and MBP was found. Using surface plasmon resonance we demonstrated specific binding of hsp70 with MBP in vitro. Analysis of the amounts of MBP bound to hsp70 yielded a molecular ratio of MBP binding to hsp70 at 6.5:1. MBP complexed with hsp70 was taken up at significantly higher rates by antigen-presenting cells than MBP alone and enhanced MBP-specific immune responses. These results indicate that hsp70 specifically associates with MBP in MS brain tissue. This association might be relevant to the enhanced immune recognition of MBP in MS.     

ATM deficiency induces oxidative stress and endoplasmic reticulum stress in astrocytes

ATM kinase, the product of the ataxia telangiectasia mutated (Atm) gene, is activated by genomic damage. ATM plays a crucial role in cell growth and development. Here we report that primary astrocytes isolated from ATM-deficient mice grow slowly, become senescent, and die in culture. However, before reaching senescence, these primary Atm(-/-) astrocytes, like Atm(-/-) lymphocytes, show increased spontaneous DNA synthesis. These astrocytes also show markers of oxidative stress and endoplasmic reticulum (ER) stress, including increased levels of heat shock proteins (HSP70 and GRP78), malondialdehyde adducts, Cu/Zn superoxide dismutase, procaspase 12 cleavage, and redox-sensitive phosphorylation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). In addition, HSP70 and ERK1/2 phosphorylation are upregulated in the cerebella of ATM-deficient mice. This increase in ERK1/2 phosphorylation is seen primarily in cerebellar astrocytes, or Bergmann glia, near degenerating Purkinje cells. ERK1/2 activation and astrogliosis are also found in other parts of the brain, for example, the cortex. We conclude that ATM deficiency induces intrinsic growth defects, oxidative stress, ER stress, and ERKs activation in astrocytes.     

Major histocompatibility class one molecule associates with glucose regulated protein (GRP) 78 on the cell surface

Glucose regulated protein 78 (GRP78) is a member of the heat shock protein (hsp) 70 family. It is an endoplasmic reticulum (ER) chaperone, whose function is generally thought to be limited to the structural maturation of nascent glycoproteins. However, recent observations have shown that ER chaperones, such as GRP78, display peptide-binding activity. These peptide-binding activities along with the observation that heat shock proteins associated with peptides can elicit antigen-specific CTL responses suggest additional roles for these proteins. In this study we provide evidence that GRP78 is not only resident in the ER, but also exists on the cell surface. Furthermore, using biochemical and imaging studies we have found that GRP78 associates with MHC class I on the cell surface. Its presence on the cell surface is not dependent on MHC class I expression. In the absence of MHC class I its cell surface expression is upregulated.     

Induction of heat shock proteins in lymphocytes increases with mitogen stimulation

The effect of the growth state of a cell on the ability of hyperthermia to induce the synthesis of heat shock proteins (HSPs) was studied in resting and concanavalin A (ConA)-stimulated lymphocytes. Hyperthermia induced the synthesis of hsp 110, hsp 90, hsc 70, and hsp 70 in both resting and ConA-stimulated lymhocytes, and ConA-treatment induced the synthesis of the hsp 90 and hsc 70 at normal temperature. The induction of the synthesis of hsp 110 and hsp 70 by hyperthermia was 3- to 6-fold higher for lymphocytes cultured with ConA for 12 and 24 h than in non-stimulated lymphocytes. Thus, lymphocytes induced to undergo proliferation showed a greater response to hyperthermia than resting lymphocytes.     

Involvement of endoplasmic reticulum stress in myofibroblastic differentiation of lung fibroblasts

Stress that impairs endoplasmic reticulum (ER) function leads to an accumulation of unfolded or misfolded proteins in the ER (ER stress) and triggers the unfolded protein response (UPR). Recent studies suggest that ER stress is involved in idiopathic pulmonary fibrosis (IPF). The present study was undertaken to determine the role of ER stress on myofibroblastic differentiation of fibroblasts. Fibroblasts in fibroblastic foci of IPF showed immunoreactivity for GRP78. To determine the role of ER stress on α-smooth muscle actin (α-SMA) and collagen type I expression in fibroblasts, mouse and human lung fibroblasts were treated with TGF-β1, and expression of ER stress-related proteins, α-SMA, and collagen type I was analyzed by Western blotting. TGF-β1 significantly increased expression of GRP78, XBP-1, and ATF6α, which was accompanied by increases in α-SMA and collagen type I expression in mouse and human fibroblasts. A chemical chaperone, 4-PBA, suppressed TGF-β1-induced UPR and α-SMA and collagen type I induction. We also showed that TGF-β1-induced UPR was mediated through the reactive oxygen species generation. Our study provides the first evidence implicating the UPR in myofibroblastic differentiation during fibrosis. These findings of the role of ER stress and chemical chaperones in pulmonary fibrosis may improve our understanding of the pathogenesis of IPF.     

The effects of the immunosuppressant rapamycin on the growth of rheumatoid arthritis (RA) synovial fibroblast

RA is a chronic inflammatory disease characterized by mononuclear cell infiltration and the overgrowth of synovial fibroblast. This invasive growth of synovial tissues corresponds with the progressive destruction of articular carilage and bone. Several immunosuppresive agents, such as cyclophosphamide, cyclosporin A and mizoribine, have been clinically used to control disease progresssion, though relatively little is known of their effects on rheumatoid synovium. Rapamycin exhibits a strong immunosuppressive activity by acting on T cell signalling pathways. In the present study we examined the effects of rapamycin on the growth of synovial fibroblast isolated from RA patients. Platelet-derived growth factor (PDGF) is a potent growth factor in synovial fibroblasts isolated from RA patients. PDGF and serum stimulation resulted in a rapid phosphorylation of tyrosine and activation of mitogen-activated protein kinase (MAP kinase), 70-kD-S6 kinase (P70^S6K) and 90-kD-S6 kinase (P90^rsk). Rapamycin, a macrolide immunosuppressant, inhibited completely growth factor-induced synovial fibroblast proliferation and P70^S6K activation. In contrast, tyrosine phosphorylation and activation of MAP kinases and P90^rsk were not influenced by rapamycin treatment. Our data demonstrate that growth factor-mediated P70^S6K activation is closely related to the growth of synovial fibroblast, and suggest the efficacy of rapamycin for controlling synovial hyperplasia in RA.     

Long-term effect of heat shock protein 60 from Actinobacillus actinomycetemcomitans on epithelial cell viability and mitogen-activated protein kinases

Our previous studies showed that bacterial heat shock protein 60 (hsp60) induces cultured epithelial cell proliferation within 24 h. Here we investigated the long-term effects of heat shock protein 60 isolated from Actinobacillus actinomycetemcomitans on skin keratinocyte (HaCaT cell line) viability and the cell signaling involved. Prolonged incubation in the presence of hsp60 increased the rate of epithelial cell death. The number of viable cells in hsp60-treated culture was 37% higher than the number in the control at 24 h but 27% lower at 144 h. A kinetics study of the effect of hsp60 on the phosphorylation of mitogen-activated protein kinases (MAPKs) involving Western blotting with phospho-specific antibodies showed that in addition to a transient early increase in p38 levels, a second peak appeared in keratinocytes 24 h after the addition of hsp60. In contrast, prolonged incubation with hsp60 caused a decrease in the level of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) compared with that in the controls, possibly as a result of protein phosphatase activity. We found that hsp60 increased the levels of several phosphatases, including MAP-2, which strongly dephosphorylates ERK1/2. Moreover, hsp60 increased the level of tumor necrosis factor alpha (TNF-alpha) in culture medium in a dose-dependent manner. TNF-alpha added to culture showed a cytotoxic effect on epithelial cells, particularly with longer incubation periods. TNF-alpha also induced the phosphorylation of p38. Finally, our results show that bacterial hsp60 inhibited stress-induced synthesis of cellular hsp60. Therefore, several cell behavior changes caused by long-term exposure to bacterial hsp60 may lead to impaired epithelial cell viability.     

Keratinocyte growth factor stimulates alveolar type II cell proliferation through the extracellular signal-regulated kinase and phosphatidylinositol 3-OH kinase pathways

Keratinocyte growth factor (KGF or FGF-7) stimulates alveolar type II cell proliferation, but little is known about the signaling pathways involved. We investigated the role of the ERK (p42/44 mitogen activated protein [MAP] kinase) and phosphatidylinositol 3-OH kinase (PI3 kinase) pathways on alveolar type II cell proliferation and differentiation. Rat type II cells were cultured on tissue culture plastic and Matrigel in the presence or absence of KGF and specific chemical inhibitors PD98059, LY294002, and rapamycin at various concentrations. Proliferation was measured by thymidine incorporation and DNA quantitation, and differentiation was measured by expression of surfactant protein A and alkaline phosphatase. We demonstrate that KGF activates distal effectors of the PI3 kinase pathway, PKB/Akt, and p70S6 kinase, as well as p42/44 MAP kinase proteins. Inhibition of these pathways with PD98059, LY294002, or rapamycin inhibited type II cell proliferation but had no significant effect on differentiation. KGF did not activate the c-Jun kinase or p38 MAP kinase pathways. We conclude that the p42/44 MAP kinase and PI3 kinase pathways are important in regulating alveolar type II cell proliferation in response to KGF.     

Trimethylamine-N-oxide,as a risk factor for atherosclerosis,induces stress in J774A.1 murine macrophages

Purpose

Trimethylamine N-oxide (TMAO) is a biomarker for kidney problems. It has also been introduced as a risk factor for atherosclerosis. The classic risk factors for atherosclerosis trigger cellular and humeral immunoreaction in macrophages through induction of heat shock protein expressions and increased levels of GRP94 and HSP70 are associated with increased atherosclerosis risk. The present study evaluated the possible effect(s) of TMAO on the expression of GRP94 and HSP70 at protein levels.