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.

     

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.     

Characterization and Mechanism of Stress-induced Translocation of 78-Kilodalton Glucose-regulated Protein (GRP78) to the Cell Surface

Glucose-regulated protein (GRP78)/BiP, a major chaperone in the endoplasmic reticulum, is recently discovered to be preferably expressed on the surface of stressed cancer cells, where it regulates critical oncogenic signaling pathways and is emerging as a target for anti-cancer therapy while sparing normal organs. However, because GRP78 does not contain classical transmembrane domains, its mechanism of transport and its anchoring at the cell surface are poorly understood. Using a combination of biochemical, mutational, FACS, and single molecule super-resolution imaging approaches, we discovered that GRP78 majorly exists as a peripheral protein on plasma membrane via interaction with other cell surface proteins including glycosylphosphatidylinositol-anchored proteins. Moreover, cell surface GRP78 expression requires its substrate binding activity but is independent of ATP binding or a membrane insertion motif conserved with HSP70. Unexpectedly, different cancer cell lines rely on different mechanisms for GRP78 cell surface translocation, implying that the process is cell context-dependent.     

GRP78 and Cripto form a complex at the cell surface and collaborate to inhibit transforming growth factor beta signaling and enhance cell growth

Cripto is a multifunctional cell surface protein with important roles in vertebrate embryogenesis and the progression of human tumors. While Cripto has been shown to modulate multiple signaling pathways, its binding partners do not appear to fully explain its molecular actions. Therefore, we conducted a screen aimed at identifying novel Cripto-interacting proteins. This screen led to our identification of glucose-regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone that is also expressed at the surfaces of tumor cells. Here we demonstrate that Cripto and GRP78 interact at the cell surfaces of multiple cell lines and that their interaction is independent of prior association within the ER. Interestingly, short hairpin RNA knockdown of endogenous GRP78 resulted in enhanced transforming growth factor β (TGF-β) signaling, indicating that like Cripto, GRP78 inhibits this pathway. We further show that when coexpressed, GRP78 and Cripto collaborate to antagonize TGF-β responses, including Smad phosphorylation and growth inhibition of prostate cancer cells grown under anchorage-dependent or -independent conditions. Finally, we provide evidence that cells coexpressing GRP78 and Cripto grow much more rapidly in soft agar than do cells expressing either protein individually. Together, our results indicate that these proteins bind at the cell surface to enhance tumor growth via the inhibition of TGF-β signaling.     

Glucose regulated proteins 78 and 75 bind to the receptor for hyaluronan mediated motility in interphase microtubules

The receptor for hyaluronan mediated motility (RHAMM), which is a hyaluronan-binding protein, is a centrosomal and microtubal protein. Here, we have identified two RHAMM-binding proteins, glucose regulated protein (GRP) 78 and GRP75, using co-immunoprecipitation analysis. These two proteins directly bound to glutathione-S-transferase-RHAMM fusion proteins. By double immunostaining, GRP78 and GRP75 colocalized with RHAMM in interphase microtubules, but were separated in mitotic spindles. Prevention of microtubule polymerization by TN-16 and vincristine sulfate induced RHAMM overexpression without a significant change in GRP78/75. Taken together, GRP78/75 and RHAMM complexes may stabilize microtubules in the interphase, associated with a downregulation of RHAMM. These results reveal a new biochemical activity of RHAMM.     

Glucose regulated protein 78 promotes cell invasion via regulation of uPA production and secretion in colon cancer cells

Glucose regulated protein 78 (GRP78) is frequently highly expressed in tumor cells, contributing to the acquisition of several phenotypic cancer hallmarks. GRP78 expression is also positively correlated with tumor metastasis, and promotes hepatocellular carcinoma cell invasion via increasing cell motility, however, other mechanisms involving the prometastatic roles of GRP78 remain to be elucidated. Here we report that forced GRP78 expression promotes colon cancer cell migration and invasion through upregulating MMP-2, MMP-9 and especially uPA production. These effects of GRP78 are mediated by enhancing the activation of β-catenin signaling. Interestingly, we identify that GRP78 interacts with uPA both in the cells and in the culture medium, suggesting that GRP78 protein is likely to directly facilitate uPA secretion via protein-protein interaction. Taken together, our findings demonstrate for the first time that besides stimulation of cell motility, GRP78 can act by increasing proteases production to promote tumor cell invasion. [BMB Reports 2014; 47(8): 445-450]     

Hematopoietic stem cells are regulated by Cripto, as an intermediary of HIF-1α in the hypoxic bone marrow niche

Cripto has been known as an embryonic stem (ES)- or tumor-related soluble/cell membrane protein. In this study, we demonstrated that Cripto has a role as an important regulatory factor for hematopoietic stem cells (HSCs). Recombinant Cripto sustained the reconstitution ability of HSCs in vitro. Flow cytometry analysis uncovered that GRP78, one of the candidate receptors for Cripto, was expressed on a subset of HSCs and could distinguish dormant/myeloid-biased HSCs and active/lymphoid-biased HSCs. Cripto is expressed in hypoxic endosteal niche cells where GRP78(+) HSCs mainly reside. Proteomics analysis revealed that Cripto-GRP78 binding stimulates glycolytic metabolism-related proteins and results in lower mitochondrial potential in HSCs. Furthermore, conditional knockout mice for HIF-1α, a master regulator of hypoxic responses, showed reduced Cripto expression and decreased GRP78(+) HSCs in the endosteal niche area. Thus, Cripto-GRP78 is a novel HSC regulatory signal mainly working in the hypoxic niche.     

Roles of GRP78 in physiology and cancer

As one member of 70 kDa heat shock proteins, glucose‐regulated protein 78 (GRP78) participates in protein folding, transportation and degradation. This sort of capacity can be enhanced by stresses under which GRP78 is induced rapidly. Unlike its homologues, GRP78 presents multifaceted subcellular position: When ER retention, it serves as the switch of unfolded protein response; When mitochondrial binding, it directly interacts with apoptotic executors; When cell surface residing, it recognizes extracellular ligands, transducing proliferative signals, especially in certain tumors. The close correlation between GRP78 and neoplasm provides us further insight into the event of carcinogenesis and cancer cell chemoresistance, indicating its prognostic predicting significance and validating potential therapeutics for clinical usage, especially because its small molecular inhibitors are emerging quickly these years. What's more, GRP78‐related signaling may be helpful for clearer understanding of its biological mechanisms. J. Cell. Biochem. 110: 1299–1305, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Mood stabilizing drug lithium increases expression of endoplasmic reticulum stress proteins in primary cultured rat cerebral cortical cells

The mood stabilizing drug lithium is a highly effective treatment for bipolar disorder. Previous studies in our laboratory found that chronic treatment with the mood stabilizing drug valproate in rat brain increased the expression of endoplasmic reticulum (ER) stress proteins GRP78, GRP94 and calreticulin. We report here that in primary cultured rat cerebral cortical cells, expression of GRP78, GRP94 and calreticulin are increased not only by valproate, but also by lithium after chronic treatment for 1 week at therapeutically relevant concentrations. However, two other mood stabilizing drugs carbamazepine and lamotrigine had no effect on expression of GRP78, GRP94 or calreticulin. Chronic treatment with lithium for 1 week increased both mRNA and protein levels of ER stress proteins. In contrast to a classic GRP78 inducer thapsigargin, an inhibitor of the ER Ca2+ -ATPase, chronic treatment with lithium or valproate for 1 week modestly increased GRP78 expression in neuronal cells, had no effect on basal intracellular free Ca2+ concentration and does not induce cell death. These results indicate that lithium and valproate may increase expression of GRP78, GRP94 and calreticulin in primary cultured rat cerebral cortical cells without causing cell damage. These results also suggest that the mechanism of GRP78 increase induced by lithium and valproate may be different from that of thapsigargin.     

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.     

GRP78 protects CHO cells from ribosylation

d-Ribose (Rib), a reactive glycation compound that exists in organisms, abnormally increases in the urine of diabetic patients and can yield large amounts of advanced glycation end products (AGEs), leading to cell dysfunction. However, whether cellular proteins are sensitive to this type of glycation is unknown. In this study, we found that cellular AGEs accumulate in Chinese hamster ovary (CHO) cells with increased Rib concentration and administration time. Mass spectrum analysis of isolated AGE-modified proteins from cell lysates showed that glucose-regulated protein 78?kD (GRP78) is one of the main ribosylated proteins. Co-immunoprecipitation assays further confirmed the interaction between AGEs and GRP78. Compared with d-glucose (Glc), Rib produced much more AGEs in cells. In kinetic studies, the first order rate constant of LDH released from CHO cells incubated with Rib was nearly 8-fold higher than that of Glc, suggesting that Rib is highly cytotoxic. Immunofluorescent co-localization analysis manifested partial superimposition of AGEs and GRP78, which were distributed throughout the endoplasmic reticulum. Western blotting showed that the expression of GRP78 is up-regulated and then down-regulated in CHO cells during Rib treatment. In the presence of Rib, the suppression of GRP78 expression either with transfected siRNA or with the inhibitor (-)-epigallocatechin gallate (EGCG) dramatically increased AGE levels and decreased cell viability compared with these parameters in the control groups. GRP78 overexpression decreased AGE levels and rescued the cells from Rib-induced cytotoxicity. These data indicate that GRP78 plays a role in preventing Rib-induced CHO cell cytotoxicity.     

葡萄糖调节蛋白GRP78和GRP94在小鼠脑发育过程中的差异表达

为探讨葡萄糖调节蛋白GRP78和GRP94在小鼠脑发育过程中的生物学意义,利用蛋白质免疫印迹、免疫荧光及RNA印迹技术,检测了发育不同时期小鼠脑组织中GRP78、GRP94的表达及分布情况.结果显示:小鼠脑发育过程中GRP78、GRP94的表达在时间和空间上呈现出显著差异,在脑发育的早中期GRP78表达水平高于GRP94,随发育的进程GRP78不断下降而GRP94逐渐升高,至胚胎发育晚期GRP94表达水平高于GRP78.在E16.5的不同脑区,GRP78的表达呈现出从端脑到后脑逐渐递减的“浓度梯度”分布,而GRP94在不同脑区中表达相同.小鼠出生后,二者作为应激蛋白在脑组织中的表达没有明显的差异性.免疫荧光结果显示,GRP78和GRP94在大脑组织中的分布基本相同,神经细胞和神经胶质细胞的细胞质均有分布.这些观察得到的结果提示,GRP78和GRP94与神经细胞分化和脑的形态建成有关,它们分别在脑发育的不同时期起作用.     

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.     

Attenuation of unfolded protein response and apoptosis by mReg2 induced GRP78 in mouse insulinoma cells

Murine regenerating (mReg) genes have been implicated in preserving islet cell biology. Expanding on our previous work showing that overexpression of mReg2 protects MIN6 insulinoma cells against streptozotocin-induced apoptosis, we now demonstrate that mReg2 induces glucose-regulated peptide 78 (GRP78) expression via the Akt–mTORC1 axis and protects MIN6 cells against ER stress induced by thapsigargin and glucolipotoxicity. Activation of mTORC1 activity results from both mReg2-induced increased mTOR phosphorylation as well as increased expression of Raptor and GβL. Inhibition of Akt and mTORC1 blunted the ability of mReg2 to induce GRP78 and attenuate unfolded protein response (UPR). Knockdown of GRP78 sensitized the cells overexpressing mReg2 to UPR without affecting its ability to activate Akt–mTORC1 signaling. Induced expression of mReg2 may protect insulin producing cells from ER stress in diabetes.     

ADP-ribosylation of the molecular chaperone GRP78/BiP

Starvation of mouse hepatoma cells for essential amino acids or glucose results in the ADP-ribosylation of the molecular chaperone BiP/GRP78. Addition of the missing nutrient to the medium reverses the reaction. The signal mediating the response to environmental nutrients involves the translational efficiency. An inhibitor of proteins synthesis, cycloheximide, or reduced temperature, both of which reduce translational efficiency, stimulate the ADP-ribosylation of BiP/GRP78. Inhibition of N-linked glycosylation of proteins results in the overproduction of BiP/GRP78. The over produced protein is not ADP-ribosylated suggesting that this is the functional form of BiP/GRP78. The over produced BiP/GRP78 can, however, be ADP-ribosylated if the cells are starved for an essential amino acid. BiP/GRP78 resides in the lumen of the endoplasmic reticulum where it participates in the assembly of secretory and integral membrane proteins. ADP-ribosylation of BiP/GRP78 during starvation is probably part of a nutritional stress response which conserves limited nutrients by slowing flow through the secretory pathway.     

Anti-KDEL-coated nanoparticles: A promising tumor targeting approach for ovarian cancer?

The purpose of this study was to target ovarian cancer cells by coupling paclitaxel (Tx)-loaded nanoparticles (NPs-Tx) to antibodies against KDEL sequence, able to recognize GRP94 and GRP78 that are located at cell surface in cancer cells whereas they are in the endoplasmic reticulum in healthy cells. Tx-loaded poly (dl-lactic acid) nanoparticles coated with anti-KDEL antibodies (NPs-Tx-KDEL) were successfully prepared and characterized. Interaction between tumor cells and NPs-Tx or NPs-Tx-KDEL was observed by microscopy with fluorescently labeled NPs and the efficacy of the different formulations was compared by a viability assay.