Glucose regulated protein 78: A critical link between tumor microenvironment and cancer hallmarks

Glucose regulated protein 78 (GRP78) has long been recognized as a molecular chaperone in the endoplasmic reticulum (ER) and can be induced by the ER stress response. Besides its location in the ER, GRP78 has been found to be present in cell plasma membrane, cytoplasm, mitochondria, nucleus as well as cellular secretions. GRP78 is implicated in tumor cell proliferation, apoptosis resistance, immune escape, metastasis and angiogenesis, and its elevated expression usually correlates with a variety of tumor microenvironmental stresses, including hypoxia, glucose deprivation, lactic acidosis and inflammatory response. GRP78 protein acts as a centrally located sensor of stress, which feels and adapts to the alteration in the tumor microenvironment. This article reviews the potential contributions of GRP78 to the acquisition of cancer hallmarks based on intervening in stress responses caused by tumor niche alterations. The paper also introduces several potential GRP78 relevant targeted therapies.     

黄芪注射液对网腔钙结合蛋白基因沉默阿霉素损伤心肌细胞内质网应激伴侣蛋白GRP78,GRP94 mRNA的作用

目的：研究黄芪注射液对网腔钙结合蛋白（calumenin）基因沉默阿霉素损伤心肌细胞内质网应激伴侣蛋白GRP78,GRP94 mRNA的作用。方法：实验将体外培养的1~3 d乳鼠心肌细胞分为5组：对照组、模型组（正常细胞+3 mg/L阿霉素）、calumenin基因沉默模型组（慢病毒感染细胞+3 mg/L阿霉素）、黄芪组1（正常细胞+3 mg/L阿霉素+200 g/L黄芪）、黄芪组2（慢病毒感染细胞+3 mg/L阿霉素+200 g/L黄芪）。构建慢病毒-calumenin质粒,转染乳鼠培养心肌细胞,采用实时荧光定量分析（real-time PCR）检测各组心肌细胞calumenin及内质网应激伴侣蛋白GRP78、GRP94 mRNA表达。结果：①与对照组比较,模型组心肌细胞calumenin mRNA表达减少（P<0.05）,而calumenin基因沉默模型组及黄芪组2心肌细胞calumenin mRNA表达明显减少（P<0.01）;与模型组比较,黄芪组1心肌细胞calumenin mRNA表达增加（P<0.05）;与calumenin基因沉默模型组比较,黄芪组2心肌细胞calumenin mRNA表达明显增加（P<0.01）。②与对照组相比较,模型组及calumenin基因沉默模型组心肌细胞内质网应激伴侣蛋白GRP78、GRP94 mRNA表达明显增多（P<0.01）;与模型组比较,黄芪组1心肌细胞GRP78、GRP94 mRNA表达明显减少（P<0.01）;与calumenin基因沉默模型组比较,黄芪组2心肌细胞内质网应激伴侣蛋白GRP78、GRP94 mRNA表达明显减少（P<0.01）。结论：①阿霉素损伤可引起心肌细胞calumenin表达减少。②Calumenin可缓解阿霉素损伤所诱导心肌细胞内质网应激。黄芪注射液可抑制阿霉素损伤所诱导心肌细胞内质网应激,这种作用可能系通过calumenin介导实现的。     

The endoplasmic reticulum stress protein GRP94, in addition to BiP, associates with unassembled immunoglobulin chains.

The molecular chaperone BiP/GRP78 associates with various polypeptides in the endoplasmic reticulum, including immunoglobulin chains. We now show, using chemical cross-linking, that another endoplasmic reticulum stress protein, GRP94, associates with newly synthesized immunoglobulin light and heavy chains. We demonstrate the presence of ternary complexes composed of immunoglobulin chains, BiP and GRP94. Because both BiP and GRP94 associate far less with fully assembled immunoglobulin than with unassembled subunits, our data suggest that GRP94, like BiP, functions as a molecular chaperone. The presence of both BiP and GRP94 in the same complex further suggests that the two stress proteins work in concert during the folding and assembly of immunoglobulins.     

The critical role of GRP78 in physiologic and pathologic stress

GRP78 is a major endoplasmic reticulum chaperone as well as a master regulator of the unfolded protein response. In addition to playing an essential role in early embryonic development, recent studies have emerged specifically implicating GRP78 and chaperone integrity in the aging process and age-related diseases. Another exciting discovery is the regulation of GRP78 by insulin/IGF-1 signaling pathways impacting cell proliferation and survival. Mouse models of cancer, in combination with cell culture studies, validate the critical role of GRP78 in tumorigenesis and tumor angiogenesis. Further, these studies demonstrate the ability of GRP78 to suppress oncogenic PI3K/AKT signaling. The discovery of cell surface GRP78, in cancer cells and cells undergoing ER stress, presents a novel therapeutic strategy.     

4-苯基丁酸钠通过抑制内质网应激减轻皮层神经元氧糖剥夺/再灌注损伤

4-苯基丁酸钠（4-phenylbutyric acid,4-PBA）是协助内质网中蛋白质转录后修饰和折叠的分子伴侣,故可减轻非折叠蛋白反应（unfolded protein response,UPR）及其介导的细胞凋亡。既往研究表明,4-PBA可以减轻脑组织的缺血性损伤,但采用原代皮层神经元构建氧糖剥夺/再灌注（oxygen glucose deprivation/reoxygenation, OGD/R）损伤模型,来研究4-PBA对神经元损伤的保护作用及其机制尚未见报道。本文采用原代培养的皮层神经元OGD/R损伤模型,同时给予4-PBA处理,探讨4-PBA对OGD/R诱导的神经元内质网应激（endoplasmic reticulum stress,ERS）的作用及其机制。分别采用MTT、LDH和Hoechst 33342染色法检测神经元存活率、细胞膜完整性和细胞凋亡情况。Western印迹检测ERS标志物葡萄糖调节蛋白78 (glucose regulated protein 78,GRP78),以及肌醇必需酶1（inositol requiring enzyme 1, IRE1）通路相关蛋白质的表达。Western印迹结果显示,在OGD/R后0～48 h,GRP78的表达较对照组明显升高。MTT、LDH漏出率和Hoechst 33342染色法检测显示,4-PBA显著改善OGD/R所导致的神经元存活率下降、LDH漏出率升高和细胞凋亡增加,且具有明显的剂量依赖性。通过Western印迹检测发现,4-PBA显著逆转OGD/R所致GRP78蛋白表达水平的上调。此外,对肌醇必需酶1通路相关蛋白质的检测显示,4-PBA下调氧糖剥夺/再灌注组神经元p IRE1和p JNK的表达,增加抗凋亡蛋白Bcl 2表达。上述研究结果表明,4-PBA在氧糖剥夺/再灌注情况下对神经元具有保护作用,该保护作用可能是通过抑制肌醇必需酶1信号通路介导的非折叠蛋白反应和内质网应激实现的。     

Down-regulation of epidermal growth factor receptor-signaling pathway by binding of GRP78/BiP to the receptor under glucose-starved stress conditions

GRP78/BiP, a molecular chaperone in the endoplasmic reticulum, is induced under such adverse conditions for cell survival as glucose starvation. Induction of GRP78 has been shown to coincide with G1 cell cycle arrest, which is an important cellular defense system. In this study, we investigated involvement of GRP78 in the mechanism of growth arrest by using human epidermoid carcinoma A431 cells. Under a chemical stress condition with 2-deoxyglucose, GRP78 was induced 3–4-fold. In the stressed cells, an underglycosylated form of epidermal growth factor receptor (EGFR) was produced and the mature form was decreased. We found that the molecular chaperone GRP78 in the endoplasmic reticulum formed a stable complex with the underglycosylated EGFR but did not with the mature form. This complex formation occurred specifically under the stress conditions, and the complex was dissociated upon removal of the stress. Treatment of the GRP78-underglycosylated EGFR complex with ATP resulted in a release of the underglycosylated EGFR from GRP78, indicating that the complex could be formed through the chaperone function of GRP78. In accordance with the complex formation with endoplasmic reticulum-resident GRP78, the underglycosylated EGFR could not be translocated to the cell surface. As a result, EGF could not induce expression of cyclin D3, a G1 cyclin, in the stressed cells, whereas it did in non-stressed cells. These results indicated that, in the stressed cells, GRP78 participated in down-regulation of EGF-signaling pathway by forming a stable complex with EGFR and inhibiting EGFR translocation to the cell surface. J. Cell. Physiol. 177:282–288, 1998. © 1998 Wiley-Liss, Inc.     

Activation of Toll-like Receptor 4 (TLR4) Attenuates Adaptive Thermogenesis via Endoplasmic Reticulum Stress

Adaptive thermogenesis is the cellular process transforming chemical energy into heat in response to cold. A decrease in adaptive thermogenesis is a contributing factor to obesity. However, the molecular mechanisms responsible for the compromised adaptive thermogenesis in obese subjects have not yet been elucidated. In this study we hypothesized that Toll-like receptor 4 (TLR4) activation and subsequent inflammatory responses are key regulators to suppress adaptive thermogenesis. To test this hypothesis, C57BL/6 mice were either fed a palmitate-enriched high fat diet or administered with chronic low-dose LPS before cold acclimation. TLR4 stimulation by a high fat diet or LPS were both associated with reduced core body temperature and heat release. Impairment of thermogenic activation was correlated with diminished expression of brown-specific markers and mitochondrial dysfunction in subcutaneous white adipose tissue (sWAT). Defective sWAT browning was concomitant with elevated levels of endoplasmic reticulum (ER) stress and autophagy. Consistently, TLR4 activation by LPS abolished cAMP-induced up-regulation of uncoupling protein 1 (UCP1) in primary human adipocytes, which was reversed by silencing of C/EBP homologous protein (CHOP). Moreover, the inactivation of ER stress by genetic deletion of CHOP or chemical chaperone conferred a resistance to the LPS-induced suppression of adaptive thermogenesis. Collectively, our data indicate the existence of a novel signaling network that links TLR4 activation, ER stress, and mitochondrial dysfunction, thereby antagonizing thermogenic activation of sWAT. Our results also suggest that TLR4/ER stress axis activation may be a responsible mechanism for obesity-mediated defective brown adipose tissue activation.     

Regulated expression of GRP78 during vasopressin-induced hypertrophy of heart-derived myocytes

Although the development of cellular hypertrophy is widely believed to involve Ca(2+) signaling, potential supporting roles for sequestered Ca(2+) in this process have not been explored. H9c2 cardiomyocytes respond to arginine vasopressin with an initial mobilization of Ca(2+) stores and reduced rates of mRNA translation followed by repletion of Ca(2+) stores, up-regulation of translation beyond initial rates, and the development of hypertrophy. Rates of synthesis of the endoplasmic reticulum (ER) chaperones, GRP78 and GRP94, were found to increase preferentially at early times of vasopressin treatment. Total GRP78 content increased 2- to 3-fold within 8 h after which the chaperone was subject to post-translational modification. Preferential synthesis of GRP78 and the increase in chaperone content both occurred at pM vasopressin concentrations and were abolished at supraphysiologic Ca(2+) concentrations. Co-treatment with phorbol myristate acetate decreased vasopressin-dependent Ca(2+) mobilization and slowed appearance of new GRP78 molecules in response to the hormone, whereas 24 h pretreatment with phorbol ester prolonged vasopressin-dependent Ca(2+) mobilization and further increased rates of GRP78 synthesis in response to the hormone. Findings did not support a role for newly synthesized GRP78 in translational up-regulation by vasopressin. However up-regulation, which does not depend on Ca(2+) sequestration, appeared to expedite chaperone expression. This report provides the first evidence that a Ca(2+)-mobilizing hormone at physiologic concentrations signals increased expression of GRP78. Translational tolerance to depletion of ER Ca(2+) stores, typifying a robust ER stress response, did not accompany vasopressin-induced hypertrophy.     

The role of MTJ-1 in cell surface translocation of GRP78, a receptor for alpha 2-macroglobulin-dependent signaling

MTJ-1 associates with a glucose-regulated protein of Mr approximately 78,000(GRP78) in the endoplasmic reticulum and modulates GRP78 activity as a chaperone. GRP78 also exists on the cell surface membrane, where it is associated with a number of functions. MHC class I Ags on the cell surface are complexed to GRP78. GRP78 also serves as the receptor for alpha2-macroglobulin-dependent signaling and for uptake of certain pathogenic viruses. The means by which GRP78, lacking a transmembrane domain, can fulfill such functions is unclear. In this study we have examined the question of whether MTJ-1, a transmembrane protein, is involved in the translocation of GRP78 to the cell surface. MTJ-1 and GRP78 coimmunoprecipitated from macrophage plasma membrane lysates. Silencing of MTJ-1 gene expression greatly reduced MTJ-1 mRNA and protein levels, but also abolished cell surface localization of GRP78. Consequently, binding of the activated and receptor-recognized form of alpha2-macroglobulin to macrophages was greatly reduced, and activated and receptor-recognized form of alpha2-macroglobulin-induced calcium signaling was abolished in these cells. In conclusion, we show that in addition to assisting the chaperone GRP78 in protein quality control in the endoplasmic reticulum, MTJ-1 is essential for transport of GRP78 to the cell surface, which serves a number of functions in immune regulation and signal transduction.     

GRP78 的表达与非小细胞肺癌生物学特性及预后的关系

目的:探讨GRP78在非小细胞肺癌和癌旁组织中的表达情况,并研究其与生物学特征及临床预后的关系 方法:收集非小细胞肺癌术后切除标本88例,及其癌旁组织20例作为对照.采用免疫组织化学方法检测GRP78的表达.结果:GRP78在非小细胞肺癌组织和癌旁组织中的表达有统计学差异.GRP78的表达与非小细胞肺癌的临床分期、分化程度有关,而与患者性别、年龄和病理类型无关.非小细胞肺癌中GRP78高表达的患者生存时间短于GRP78低表达的患者.GRP78的表达情况是影响非小细胞肺癌患者手术预后的独立危险因素.结论:非小细胞肺癌患者的GRP78的表达可能与肿瘤细胞的发生及发展有关,GRP78可以作为一个预测非小细胞肺癌患者预后的分子标志物.     

Oxidative stress-mediated aldehyde adduction of GRP78 in a mouse model of alcoholic liver disease: functional independence of ATPase activity and chaperone function

Pathogenesis in alcoholic liver disease (ALD) is complicated and multifactorial but clearly involves oxidative stress and inflammation. Currently, conflicting reports exist regarding the role of endoplasmic reticulum (ER) stress in the etiology of ALD. The glucose-regulated protein 78 (GRP78) is the ER homolog of HSP70 and plays a critical role in the cellular response to ER stress by serving as a chaperone assisting protein folding and by regulating the signaling of the unfolded protein response (UPR). Comprising three functional domains, an ATPase, a peptide-binding, and a lid domain, GRP78 folds nascent polypeptides via the substrate-binding domain. Earlier work has indicated that the ATPase function of GRP78 is intrinsically linked and essential to its chaperone activity. Previous work in our laboratory has indicated that GRP78 and the UPR are not induced in a mouse model of ALD but that GRP78 is adducted by the lipid electrophiles 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE) in vivo. As impairment of GRP78 has the potential to contribute to pathogenesis in ALD, we investigated the functional consequences of aldehyde adduction on GRP78 function. Identification of 4-HNE and 4-ONE target residues in purified human GRP78 revealed a marked propensity for Lys and His adduction within the ATPase domain and a relative paucity of adduct formation within the peptide-binding domain. Consistent with these findings, we observed a concomitant dose-dependent decrease in ATP-binding and ATPase activity without any discernible impairment of chaperone function. Collectively, our data indicate that ATPase activity is not essential for GRP78-mediated chaperone activity and is consistent with the hypothesis that ER stress does not play a primary initiating role in the early stages of ALD.     

Membrane-anchored CD14 is required for LPS-induced TLR4 endocytosis in TLR4/MD-2/CD14 overexpressing CHO cells

Lipopolysaccharide (LPS) induces inflammatory activation through TLR4 (toll-like receptor-4)/MD-2 (myeloid differentiation-2)/CD14 (cluster of differentiation-14) complex. Although optimal LPS signaling is required to activate our innate immune systems against gram-negative bacterium, excessive amount of LPS signaling develops a detrimental inflammatory response in gram-negative bacterial infections. Downregulation of surface TLR4 expression is one of the critical mechanisms that can restrict LPS signaling. Here, we found that membrane-anchored CD14 is required for LPS-induced downregulation of TLR4 and MD-2 in CHO cells. Moreover, pretreatment of the cells with sterol-binding agent filipin reduced LPS-induced TLR4 downregulation, suggesting the involvement of caveolae-mediated endocytosis pathway. Involvement of caveolae in LPS-induced TLR4 endocytosis was further confirmed by immunoprecipitation. Thus, our data indicate that caveolae-dependent endocytosis pathway is involved in LPS-induced TLR4 downregulation and that this is dependent on membrane-anchored CD14 expression.     

The unfolded protein response to endoplasmic reticulum stress in cultured astrocytes and rat brain during experimental diabetes

Oxidative-nitrosative stress and inflammatory responses are associated with endoplasmic reticulum (ER) stress in diabetic retinopathy, raising the possibility that disturbances in ER protein processing may contribute to CNS dysfunction in diabetics. Upregulation of the unfolded protein response (UPR) is a homeostatic response to accumulation of abnormal proteins in the ER, and the present study tested the hypothesis that the UPR is upregulated in two models for diabetes, cultured astrocytes grown in 25 mmol/L glucose for up to 4 weeks and brain of streptozotocin (STZ)-treated rats with diabetes for 1–7 months. Markers associated with translational blockade (phospho-eIF2α and apoptosis (CHOP), inflammatory response (inducible nitric oxide synthase, iNOS), and nitrosative stress (nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase, GAPDH) were not detected in either model. Nrf2 was present in nuclei of low- and high-glucose cultures, consistent with oxidative stress. Astrocytic ATF4 expression was not altered by culture glucose concentration, whereas phospho-IRE and ATF6 levels were higher in low- compared with high-glucose cultures. The glucose-regulated chaperones, GRP78 and GRP94, were also expressed at higher levels in low- than high-glucose cultures, probably due to recurrent glucose depletion between feeding cycles. In STZ-rat cerebral cortex, ATF4 level was transiently reduced at 4 months, and p-IRE levels were transiently elevated at 3 months. However, GRP78 and GRP94 expression was not upregulated, and iNOS, amyloid-β, and nuclear accumulation of GAPDH were not evident in STZ-diabetic brain. High-glucose cultured astrocytes and STZ-diabetic brain are relatively resistant to diabetes-induced ER stress, in sharp contrast with cultured retinal Müller cells and diabetic rodent retina.     

Overexpression of GRP78 mitigates stress induction of glucose regulated proteins and blocks secretion of selective proteins in Chinese hamster ovary cells.

GRP78 is a resident protein of the endoplasmic reticulum (ER) and a member of the glucose regulated protein (GRP) family. Many secretion incompetent proteins are found in stable association with GRP78 and are retained in the ER. Some proteins which are destined for secretion transiently associate with GRP78. To further increase our understanding of the role of GRP78 in secretion, we have stably overexpressed GRP78 in Chinese hamster ovary (CHO) cells and examined the effect on protein secretion and the stress response. GRP78 overexpressing cells treated with tunicamycin or A23187 exhibited a reduced induction of endogenous GRP78 and GRP94 mRNAs compared to wild-type CHO cells. This suggests that GRP78 overexpression either alleviates the stress or is directly involved in signaling stress-induced expression of GRPs. Transient expression of secreted proteins was used to measure secretion efficiency in the GRP78 overexpressing cells. Secretion of von Willebrand factor and a mutant form of factor VIII, two proteins which transiently associate with GRP78, was reduced by GRP78 overexpression. In contrast, secretion of M-CSF, which was not detected in association with GRP78, was unaffected. This indicates that elevated levels of GRP78 may increase stable association and decrease the secretion efficiency of proteins which normally transiently associate with GRP78. These results indicate that one function of GRP78 is selective protein retention in the ER.     

Apolipoprotein e4 impairs macrophage efferocytosis and potentiates apoptosis by accelerating endoplasmic reticulum stress

Apolipoprotein (apo) E4 is a major genetic risk factor for a wide spectrum of inflammatory metabolic diseases, including atherosclerosis, diabetes, and Alzheimer disease. This study compared diet-induced adipose tissue inflammation as well as functional properties of macrophages isolated from human APOE3 and APOE4 mice to identify the mechanism responsible for the association between apoE4 and inflammatory metabolic diseases. The initial study confirmed previous reports that APOE4 gene replacement mice were less sensitive than APOE3 mice to diet-induced body weight gain but exhibited hyperinsulinemia, and their adipose tissues were similarly inflamed as those in APOE3 mice. Peritoneal macrophages isolated from APOE4 mice were defective in efferocytosis compared with APOE3 macrophages. Increased cell death was also observed in APOE4 macrophages when stimulated with LPS or oxidized LDL. Western blot analysis of cell lysates revealed that APOE4 macrophages displayed elevated JNK phosphorylation indicative of cell stress even under basal culturing conditions. Significantly higher cell stress due mainly to potentiation of endoplasmic reticulum (ER) stress signaling was also observed in APOE4 macrophages after LPS and oxidized LDL activation. The defect in efferocytosis and elevated apoptosis sensitivity of APOE4 macrophages was ameliorated by treatment with the ER chaperone tauroursodeoxycholic acid. Taken together, these results showed that apoE4 expression causes macrophage dysfunction and promotes apoptosis via ER stress induction. The reduction of ER stress in macrophages may be a viable option to reduce inflammation and inflammation-related metabolic disorders associated with the apoE4 polymorphism.     

Novel mechanism of anti-apoptotic function of 78-kDa glucose-regulated protein (GRP78): endocrine resistance factor in breast cancer, through release of B-cell lymphoma 2 (BCL-2) from BCL-2-interacting killer (BIK)

Activation of the intrinsic apoptotic pathway represents a major mechanism for breast cancer regression resulting from anti-estrogen therapy. The BH3-only protein BIK is inducible by estrogen-starvation and anti-estrogen treatment and plays an important role in anti-estrogen induced apoptosis of breast cancer cells. BIK is predominantly localized to the endoplasmic reticulum where it regulates BAX/BAK-dependent release of Ca(2+) from the endoplasmic reticulum stores and cooperates with other BH3-only proteins such as NOXA to cause rapid release of cytochrome c from mitochondria and activate apoptosis. BIK is also known to inactivate BCL-2 through complex formation. Previously, we demonstrated that apoptosis triggered by BIK in estrogen-starved human breast cancer cells is suppressed by GRP78, a major endoplasmic reticulum chaperone. Here we described the isolation of a novel clonal human breast cancer cell line (MCF-7/BUS-10) resistant to long-term estrogen deprivation. These cells exhibit elevated level of GRP78, which protects them from estrogen starvation-induced apoptosis. Our studies revealed that overexpression of GRP78 suppresses apoptosis induced by BIK and NOXA, either alone or in combination. Surprisingly, the interaction of GRP78 with BIK does not require its BH3 domain, which has been implicated in all previous BIK protein interactions. We further showed GRP78 and BCL-2 form independent complex with BIK and that increased expression of GRP78 decreases BIK binding to BCL-2. Our findings provide the first evidence that GRP78 can decrease BCL-2 sequestration by BIK at the endoplasmic reticulum, thus uncovering a potential new mechanism whereby GRP78 confers endocrine resistance in breast cancer.     

GRP94 生物学特点与肿瘤的关系的研究进展

葡萄糖调节蛋白94又叫做内质网蛋白99,是一种内质网分子伴侣蛋白,与HSP90有50%的同源性。GRP94蛋白可以和Ca2+结合具有蛋白伴侣特性,能协助新合成的多肽转位、折叠、寡聚体的组装、降解,抑制错误折叠蛋白的分泌;GRP94还具有抗原呈递的作用,可以作为肿瘤细胞的伴侣蛋白,参与肿瘤细胞的新陈代谢,保护肿瘤细胞免受有害因素的侵害。GRP94可能与人类多种肿瘤的发生有关,其表达的增高可能是肿瘤发生发展的一个重要因素。GRP94在肿瘤组织中高表达提示相关研究者,应用基因手段抑制GRP94的表达可能能够抑制肿瘤细胞的生长、侵袭和转移、增加肿瘤细胞对化疗药物的敏感性等,并且利用GRP94作为一种新的肿瘤治疗的靶分子或介质可能为肿瘤的基因治疗带来更广泛的应用前景。