Activated α2-Macroglobulin Binding to Human Prostate Cancer Cells Triggers Insulin-like Responses

Ligation of cell surface GRP78 by activated α₂-macroglobulin (α₂M*) promotes cell proliferation and suppresses apoptosis. α₂M*-treated human prostate cancer cells exhibit a 2–3-fold increase in glucose uptake and lactate secretion, an effect similar to insulin treatment. In both α₂M* and insulin-treated cells, the mRNA levels of SREBP1-c, SREBP2, fatty-acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, and Glut-1 were significantly increased together with their protein levels, except for SREBP2. Pretreatment of cells with α₂M* antagonist antibody directed against the carboxyl-terminal domain of GRP78 blocks these α₂M*-mediated effects, and silencing GRP78 expression by RNAi inhibits up-regulation of ATP citrate lyase and fatty-acid synthase. α₂M* induces a 2–3-fold increase in lipogenesis as determined by 6-[¹⁴C]glucose or 1-[¹⁴C]acetate incorporation into free cholesterol, cholesterol esters, triglycerides, free fatty acids, and phosphatidylcholine, which is blocked by inhibitors of fatty-acid synthase, PI 3-kinase, mTORC, or an antibody against the carboxyl-terminal domain of GRP78. We also assessed the incorporation of [¹⁴CH₃]choline into phosphatidylcholine and observed similar effects. Lipogenesis is significantly affected by pretreatment of prostate cancer cells with fatostatin A, which blocks sterol regulatory element-binding protein proteolytic cleavage and activation. This study demonstrates that α₂M* functions as a growth factor, leading to proliferation of prostate cancer cells by promoting insulin-like responses. An antibody against the carboxyl-terminal domain of GRP78 may have important applications in prostate cancer therapy.     

GRP78 effectively protect hypoxia/reperfusion‐induced myocardial apoptosis via promotion of the Nrf2/HO‐1 signaling pathway

Myocardial infarction is a major cause of death worldwide. Despite our understanding of the pathophysiology of myocardial infarction and the therapeutic options for treatment have improved substantially, acute myocardial infarction remains a leading cause of morbidity and mortality. Recent findings revealed that GRP78 could protect myocardial cells against ischemia reperfusion injury‐induced apoptosis, but the exact function and molecular mechanism remains unclear. In this study, we aimed to explore the effects of GRP78 on hypoxia/reperfusion (H/R)‐induced cardiomyocyte injury. Intriguingly, we first observed that GRP78 overexpression significantly protected myocytes from H/R‐induced apoptosis. On mechanism, our work revealed that GRP78 protected myocardial cells from hypoxia/reperfusion‐induced apoptosis via the activation of the Nrf2/HO‐1 signaling pathway. We observed the enhanced expression of Nrf2/HO‐1 in GRP78 overexpressed H9c2 cell, while GRP78 deficiency dramatically antagonized the expression of Nrf2/HO‐1. Furthermore, we found that blocked the Nrf2/HO‐1 signaling by the HO‐1 inhibitor zinc protoporphyrin IX (Znpp) significantly retrieved H9c2 cells apoptosis that inhibited by GRP78 overexpression. Taken together, our findings revealed a new mechanism by which GRP78 alleviated H/R‐induced cardiomyocyte apoptosis in H9c2 cells via the promotion of the Nrf2/HO‐1 signaling pathway.     

Reduced survivin expression and tumor cell survival during chronic hypoxia and further cytotoxic enhancement by the cyclooxygenase-2 inhibitor celecoxib

Hypoxia is a characteristic feature of advanced solid tumors and may worsen prognosis. The development of tumor-targeted and hypoxia-inducible gene therapy vectors holds promise to selectively deliver and express suicidal or cytotoxic genes in hypoxic regions of tumors. In this regard, the promoter of the survivin gene, which encodes an anti-apoptotic protein that is strongly expressed in tumor tissue, has received attention because of its supposed inducibility by hypoxia. However, in our present study we demonstrate that treatment of various tumor cell lines with chronic hypoxia or with the hypoxia-mimetic CoCl₂ does not result in increased expression of survivin, but rather strongly suppresses this gene’s activity. In contrast, expression of glucose-regulated protein 78 (GRP78/Bip) is substantially elevated under chronic hypoxia in vitro and in hypoxic areas of tumor tissue in vivo. Although tumor cells in general exhibit increased chemoresistance under hypoxic conditions, we found that hypoxic glioblastoma cells are more sensitive to killing by the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib, and this effect is reflected by further decreased expression of survivin. Intriguingly, 2,5-dimethyl-celecoxib (DMC), a close structural analog of celecoxib that lacks the ability to inhibit COX-2, is able to potently mimic the anti-tumor effects of its parent compound, indicating that inhibition of COX-2 is not involved in these processes. Taken together, our results caution against the use of survivin-based promoters to target hypoxic areas of tumors, but favor constructs that include the strongly hypoxia-inducible GRP78 promoter. In addition, our data introduce celecoxib as a drug with increased cytotoxicity against hypoxic tumor cells.     

A proteomic approach for identifying cellular proteins interacting with erythropoietin in recombinant Chinese hamster ovary cells

Identification of the cellular proteins interacting with incompletely folded and unfolded forms of erythropoietin (EPO) in recombinant CHO (rCHO) cells leads to better insight into the possible genetic manipulation approaches for increasing EPO production. To do so, a pull‐down assay was performed with dual‐tagged (N‐terminal GST‐ and C‐terminal hexahistidine‐tagged) EPO expressed in E. coli as bait proteins and cell lysates of rCHO cells (DG44) as prey proteins. Cellular proteins interacting with dual‐tagged EPO were then resolved by two‐dimensional gel electrophoresis (2DE) and identified by MALDI‐TOF MS/MS. A total of 27 protein spots including glucose‐regulated protein 78 (GRP78) were successfully identified. Western blot analysis of GRP78 confirmed the results of the MS analyses. Taken together, a pull‐down assay followed by a proteomic approach is found to be an efficient means to identify cellular proteins interacting with foreign protein in rCHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The 78 kDa glucose-regulated protein (GRP78/BIP) is expressed on the cell membrane,is released into cell culture medium and is also present in human peripheral circulation

In human rabdomiosarcoma cells (TE671/RD) chronic exposure to 500 nM thapsigargin (a powerful inhibitor of the endoplasmic reticulum Ca²⁺-ATPases) resulted in the induction of the stress protein GRP78/BIP. Making use of the surface biotinylation method, followed by the isolation of the GRP78 using ATP-agarose affinity chromatography, it was found that a fraction of the thapsigargin-induced GRP78 is expressed on the cell surface. The presence of GRP78 on the membrane of thapsigargin-treated cells was confirmed by fractionation of cell lysates into a soluble and a membrane fraction, followed by Western blot analysis with an anti-GRP78 antibody. It was also found that conspicuous amounts of GRP78 are present in the culture medium collected from thapsigargin-treated cultures. This extracellular GRP78 originates mostly by an active release from intact cells and does not result solely from the leakage of proteins from dead cells. Moreover, small amounts of circulating, free GRP78 and naturally-occurring anti-GRP78 autoantibodies were detected in the peripheral circulation of healthy human individuals.     

Ligation of prostate cancer cell surface GRP78 activates a proproliferative and antiapoptotic feedback loop: a role for secreted prostate-specific antigen

GRP78, a well characterized chaperone in the endoplasmic reticulum, is critical to the unfolded protein response. More recently, it has been identified on the cell surface, where it has many roles. On cancer cells, it functions as a signaling receptor coupled to proproliferative/antiapoptotic and promigratory mechanisms. In the current study, we demonstrate that ligation of prostate cancer cell surface GRP78 by its natural ligand, activated α(2)-macroglobulin (α(2)M*), results in a 2-3-fold up-regulation in the synthesis of prostate-specific antigen (PSA). The PSA is secreted into the medium as an active proteinase, where it binds to native α(2)M. The resultant α(2)M·PSA complexes bind to GRP78, causing a 1.5-2-fold increase in the activation of MEK1/2, ERK1/2, S6K, and Akt, which is coupled with a 2-3-fold increase in DNA and protein synthesis. PSA is a marker for the progression of prostate cancer, but its mechanistic role in the disease is unclear. The present studies suggest that PSA may be involved in a signal transduction-dependent feedback loop, whereby it promotes a more aggressive behavior by human prostate cancer cells.     

Isthmin targets cell-surface GRP78 and triggers apoptosis via induction of mitochondrial dysfunction

Isthmin (ISM) is a secreted 60-kDa protein that potently induces endothelial cell (EC) apoptosis. It suppresses tumor growth and angiogenesis in mice when stably overexpressed in cancer cells. Although αvβ5 integrin serves as a low-affinity receptor for ISM, the mechanism by which ISM mediates antiangiogenesis and apoptosis in ECs remain to be fully resolved. In this work, we report the identification of cell-surface glucose-regulated protein 78 kDa (GRP78) as a high-affinity receptor for ISM (K_d=8.6 nM). We demonstrated that ISM-GRP78 interaction triggers apoptosis not only in activated ECs but also in cancer cells expressing high level of cell-surface GRP78. Normal cells and benign tumor cells tend to express low level of cell-surface GRP78 and are resistant to ISM-induced apoptosis. Upon binding to GRP78, ISM is internalized into ECs through clathrin-dependent endocytosis that is essential for its proapoptotic activity. Once inside the cell, ISM co-targets with GRP78 to mitochondria where it interacts with ADP/ATP carriers on the inner membrane and blocks ATP transport from mitochondria to cytosol, thereby causing apoptosis. Hence, ISM is a novel proapoptotic ligand that targets cell-surface GRP78 to trigger apoptosis by inducing mitochondrial dysfunction. The restricted and high-level expression of cell-surface GRP78 on cancer cells and cancer ECs make them uniquely susceptible to ISM-targeted apoptosis. Indeed, systemic delivery of recombinant ISM potently suppressed subcutaneous 4T1 breast carcinoma and B16 melanoma growth in mice by eliciting apoptosis selectively in the cancer cells and cancer ECs. Together, this work reveals a novel ISM-GRP78 apoptosis pathway and demonstrates the potential of ISM as a cancer-specific and dual-targeting anticancer agent.     

Overexpression of GRP78 enhances survival of CHO cells in response to serum deprivation and oxidative stress

Chinese hamster ovary (CHO) cells are regarded as one of the most commonly used mammalian hosts, which decreases the productivity due to loss in culture viability. Overexpressing antiapoptosis genes in CHO cells was developed as a means of limiting cell death upon exposure to environmental insults. Glucose‐regulated protein 78 (GRP78) is traditionally regarded as a major ER chaperone that participates in protein folding and other cell processes. It is also a potent antiapoptotic protein and plays a critical role in cell survival, proliferation, and metastasis. In this study, the impact of GRP78 on CHO cells in response to environmental insults such as serum deprivation and oxidative stress was investigated. First, it was confirmed that CHO cells were very sensitive to environmental insults. Then, GRP78 overexpressing CHO cell line was established and exposed to serum deprivation and H₂O₂. Results showed that GRP78 engineering increased the viability and decreased the apoptosis of CHO cells. The survival advantage due to GRP78 engineering could be mediated by suppression of caspase‐3 involved in cell death pathways in stressed cells. Besides, GRP78 engineering also enhanced yields of antibody against transferrin receptor in CHO cells. GRP78 should be a potential application in the biopharmaceutical industries.     

Involvement of androgen receptor and glucose-regulated protein 78 kDa in human hepatocarcinogenesis

Previous studies demonstrated that androgen receptor (AR) is expressed in human hepatocellular carcinoma (HCC), one of the male-dominant diseases. Glucose-regulated protein 78 kDa (GRP78/Bip), which has a role in cancer development, is one of the androgen response genes in prostate cell lines. The aim of this study was to investigate the impact of AR on endoplasmic reticulum (ER)-stress signaling in human hepatoma. AR and GRP78 expressions were examined in human liver tissue panels. Human hepatoma cells stably expressing short hairpin RNA targeting AR and cells over-expressing AR were generated. The expressions of ER-stress molecules and AR were measured by real-time RT-PCR and Western blotting. The effect of AR on ER-stress responsive gene expression was examined by reporter assay. Strong positive correlation between AR mRNA and GRP78 mRNA was observed in stage I/II-HCCs. AR enhanced ER-stress responsive element activities and GRP78 expression, and regulated ER-stress response in hepatocytes. Sorafenib strongly induced significant apoptosis in HepG2 cells by the inhibition of AR and inhibition of the downstream GRP78. AR seems a co-regulator of GRP78 especially in earlier-stage HCC. AR plays a critical role in controlling ER-stress, providing new therapeutic options against HCC.     

PFT-α inhibits antibody-induced activation of p53 and pro-apoptotic signaling in 1-LN prostate cancer cells

Antibodies against the COOH-terminal domain of cell surface GRP78 induce apoptosis in cancer cell lines via activation of p53 signaling. We now have studied the effects of PFT-α, an inhibitor of p53-mediated apoptotic pathways, on anti-GRP78 antibody-induced activation of p53 and pro-apoptotic signaling in 1-LN prostate cancer cells. Pretreatment of 1-LN cancer cells with this agent significantly inhibited antibody or doxorubicin-induced upregulation of p53. Concomitantly, PFT-α treatment prevented down regulation of ERK1/2 activation by either antibody or doxorubicin. Likewise, PFT-α prevented increases in the pro-apoptotic proteins BAD, BAK, BAX, PUMA, and NOXA as well as activation of caspases-3, -7, and -9. We conclude that antibody-induced apoptosis in prostate cancer cells is mediated predominantly by p53 using the mitochondrial pathway of apoptosis.     

Binding of Anti-GRP78 Autoantibodies to Cell Surface GRP78 Increases Tissue Factor Procoagulant Activity via the Release of Calcium from Endoplasmic Reticulum Stores

The increased risk of venous thromboembolism in cancer patients has been attributed to enhanced tissue factor (TF) procoagulant activity (PCA) on the surface of cancer cells. Recent studies have shown that TF PCA can be modulated by GRP78, an endoplasmic reticulum (ER)-resident molecular chaperone. In this study, we investigated the role of cell surface GRP78 in modulating TF PCA in several human cancer cell lines. Although both GRP78 and TF are present on the cell surface of cancer cells, there was no evidence of a stable interaction between recombinant human GRP78 and TF, nor was there any effect of exogenously added recombinant GRP78 on cell surface TF PCA. Treatment of cells with the ER stress-inducing agent thapsigargin, an inhibitor of the sarco(endo)plasmic reticulum Ca²⁺ pump that causes Ca²⁺ efflux from ER stores, increased cytosolic [Ca²⁺] and induced TF PCA. Consistent with these findings, anti-GRP78 autoantibodies that were isolated from the serum of patients with prostate cancer and bind to a specific N-terminal epitope (Leu⁹⁸–Leu¹¹⁵) on cell surface GRP78, caused a dose-dependent increase in cytosolic [Ca²⁺] and enhanced TF PCA. The ability to interfere with cell surface GRP78 binding, block phospholipase C activity, sequester ER Ca²⁺, or prevent plasma membrane phosphatidylserine exposure resulted in a significant decrease in the TF PCA induced by anti-GRP78 autoantibodies. Taken together, these findings provide evidence that engagement of the anti-GRP78 autoantibodies with cell surface GRP78 increases TF PCA through a mechanism that involves the release of Ca²⁺ from ER stores. Furthermore, blocking GRP78 signaling on the surface of cancer cells attenuates TF PCA and has the potential to reduce the risk of cancer-related venous thromboembolism.     

Administration of Cripto in GRP78 overexpressed human MSCs enhances stem cell viability and angiogenesis during human MSC transplantation therapy

Objectives

The purpose of this study was to explore the effectiveness of concurrent GRP78 overexpression combined with Cripto on hMSC proliferation and migration both in vitro and in vivo. Specifically, we explored whether the treatment enhances effectiveness of hMSC transplantation in ischaemic tissue.

Materials and methods

Human MSCs obtained from human adipose tissue were cultured in α‐minimum essential medium (Hyclone, Logan, UT, USA) supplemented with 10% (v/v) foetal bovine serum (Hyclone), 100 U mL^?1 penicillin and 100 μg mL^?1 streptomycin. Murine hindlimb ischaemic model was generated with 8‐week‐old male nude BALB/c mice (Biogenomics, Seoul, Korea) maintained under a 12‐h light/dark cycle following the established protocol with minor modification. Cellular injection was performed no later than 3 hour after surgery. Lipofectamine transfection, single‐cell cultivation assay, transwell assay, scratch wound‐healing migration assay, immunohistochemistry and western blotting assays were performed.

Results

Overexpression of GRP78 along with Cripto enhanced hMSC proliferation, migration and invasion. It increased interaction of surface GRP78 receptor with Cripto via JAK2/STAT3 pathway. We confirmed our proposed mechanism by showing that treatment with GRP78 antibody blocks the enhancement in vitro. In vivo, we observed that Cripto induced by the hypoxic environment in hindlimb ischaemic model interacts with the overexpressed GRP78 and increases hMSC proliferation, migration and invasion potentials as well as angiogenesis around transplanted ischaemic site via cytokine secretions.

Conclusions

These results demonstrate supporting evidences that GRP78‐Cripto combination technique offers novel strategy to enhance MSC proliferation, migration and invasion potentials as well as angiogenesis around ischaemic site, ultimately facilitating MSC‐based transplantation therapy in ischaemic conditions.

     

Receptor-Recognized α2-Macroglobulin Binds to Cell Surface-Associated GRP78 and Activates mTORC1 and mTORC2 Signaling in Prostate Cancer Cells

Objective

Tetrameric α₂-macroglobulin (α₂M), a plasma panproteinase inhibitor, is activated upon interaction with a proteinase, and undergoes a major conformational change exposing a receptor recognition site in each of its subunits. Activated α₂M (α₂M*) binds to cancer cell surface GRP78 and triggers proliferative and antiapoptotic signaling. We have studied the role of α₂M* in the regulation of mTORC1 and TORC2 signaling in the growth of human prostate cancer cells.

Methods

Employing immunoprecipitation techniques and Western blotting as well as kinase assays, activation of the mTORC1 and mTORC2 complexes, as well as down stream targets were studied. RNAi was also employed to silence expression of Raptor, Rictor, or GRP78 in parallel studies.

Results

Stimulation of cells with α₂M* promotes phosphorylation of mTOR, TSC2, S6-Kinase, 4EBP, Akt^T308, and Akt^S473 in a concentration and time-dependent manner. Rheb, Raptor, and Rictor also increased. α₂M* treatment of cells elevated mTORC1 kinase activity as determined by kinase assays of mTOR or Raptor immunoprecipitates. mTORC1 activity was sensitive to {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and rapamycin or transfection of cells with GRP78 dsRNA. Down regulation of Raptor expression by RNAi significantly reduced α₂M*-induced S6-Kinase phosphorylation at T389 and kinase activity in Raptor immunoprecipitates. α₂M*-treated cells demonstrate about a twofold increase in mTORC2 kinase activity as determined by kinase assay of Akt^S473 phosphorylation and levels of p-Akt^S473 in mTOR and Rictor immunoprecipitates. mTORC2 activity was sensitive to {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and transfection of cells with GRP78 dsRNA, but insensitive to rapamycin. Down regulation of Rictor expression by RNAi significantly reduces α₂M*-induced phosphorylation of Akt^S473 phosphorylation in Rictor immunoprecipitates.

Conclusion

Binding of α₂M* to prostate cancer cell surface GRP78 upregulates mTORC1 and mTORC2 activation and promotes protein synthesis in the prostate cancer cells.     

Binding of Tissue-type Plasminogen Activator to the Glucose-regulated Protein 78 (GRP78) Modulates Plasminogen Activation and Promotes Human Neuroblastoma Cell Proliferation in Vitro

The glucose-regulated protein 78 (GRP78) is a plasminogen (Pg) receptor on the cell surface. In this study, we demonstrate that GRP78 also binds the tissue-type plasminogen activator (t-PA), which results in a decrease in K_m and an increase in the V_max for both its amidolytic activity and activation of its substrate, Pg. This results in accelerated Pg activation when GRP78, t-PA, and Pg are bound together. The increase in t-PA activity is the result of a mechanism involving a t-PA lysine-dependent binding site in the GRP78 amino acid sequence ⁹⁸LIGRTWNDPSVQQDIKFL¹¹⁵. We found that GRP78 is expressed on the surface of neuroblastoma SK-N-SH cells where it is co-localized with the voltage-dependent anion channel (VDAC), which is also a t-PA-binding protein in these cells. We demonstrate that both Pg and t-PA serve as a bridge between GRP78 and VDAC bringing them together to facilitate Pg activation. t-PA induces SK-N-SH cell proliferation via binding to GRP78 on the cell surface. Furthermore, Pg binding to the COOH-terminal region of GRP78 stimulates cell proliferation via its microplasminogen domain. This study confirms previous findings from our laboratory showing that GRP78 acts as a growth factor-like receptor and that its association with t-PA, Pg, and VDAC on the cell surface may be part of a system controlling cell growth.