The unfolded protein response (UPR) is an evolutionarily conserved mechanism to allow cells to adapt to stress targeting the endoplasmic reticulum (ER). Induction of ER chaperone GRP78/BiP increases protein folding capacity; as such it represents a major survival arm of UPR. Considering the central importance of the UPR in regulating cell survival and death, evidence is emerging that cells evolve feedback regulatory pathways to modulate the key UPR executors, however, the precise mechanisms remain to be elucidated. Here, we report the fortuitous discovery of GRP78va, a novel isoform of GRP78 generated by alternative splicing (retention of intron 1) and alternative translation initiation. Bioinformatic and biochemical analyses revealed that expression of GRP78va is enhanced by ER stress and is notably elevated in human leukemic cells and leukemia patients. In contrast to the canonical GRP78 which is primarily an ER lumenal protein, GRP78va is devoid of the ER signaling peptide and is cytosolic. Through specific knockdown of endogenous GRP78va by siRNA without affecting canonical GRP78, we showed that GRP78va promotes cell survival under ER stress. We further demonstrated that GRP78va has the ability to regulate PERK signaling and that GRP78va is able to interact with and antagonize PERK inhibitor P58IPK. Our study describes the discovery of GRP78va, a novel cytosolic isoform of GRP78/BiP, and the first characterization of the modulation of UPR signaling via alternative splicing of nuclear pre-mRNA. Our study further reveals a novel survival mechanism in leukemic cells and other cell types where GRP78va is expressed.

Traditionally, GRP78 is regarded as protective against hypoxia and nutrient starvation prevalent in the microenvironment of solid tumors; thus, its role in the development of hematologic malignancies remains to be determined. To directly elucidate the requirement of GRP78 in leukemogenesis, we created a biallelic conditional knockout mouse model of GRP78 and PTEN in the hematopoietic system. Strikingly, heterozygous knockdown of GRP78 in PTEN null mice is sufficient to restore the hematopoietic stem cell population back to the normal percentage and suppress leukemic blast cell expansion. AKT/mTOR activation in PTEN null BM cells is potently inhibited by Grp78 heterozygosity, corresponding with suppression of the PI3K/AKT pathway by GRP78 knockdown in leukemia cell lines. This is the first demonstration that GRP78 is a critical effector of leukemia progression, at least in part through regulation of oncogenic PI3K/AKT signaling. In agreement with PI3K/AKT as an effector for cytosine arabinoside resistance in acute myeloid leukemia, overexpression of GRP78 renders human leukemic cells more resistant to cytosine arabinoside-induced apoptosis, whereas knockdown of GRP78 sensitizes them. These, coupled with the emerging association of elevated GRP78 expression in leukemic blasts of adult patients and early relapse in childhood leukemia, suggest that GRP78 is a novel therapeutic target for leukemia. (Blood. 2012;119(3):817-825) IntroductionOne of the most frequently mutated tumor suppressor genes in human cancer is PTEN (phosphatase and tension homolog deleted on chromosome 10), which encodes for a nonredundant, plasmamembrane lipid phosphatase that antagonizes the phosphatidylinositol-3-kinase (PI3K) signaling pathway. 1,2 On loss of PTEN, the PI3K/AKT signaling pathway is activated, leading to promotion of cell survival, proliferation, and angiogenesis, as well as activation of the mTOR and S6 kinases, resulting in enhanced protein translation commonly observed in cancers. 3 PTEN also has a role in the maintenance of the hematopoietic stem cells (HSCs), as shown by ablation of PTEN function in adult HSCs through crossing of polyinosine-polycytidine (pIpC)-inducible Mx-1-Cre transgenic mouse line 4 with a Pten flox/flox (Pten f/f ) mouse line. 5 Induced Cre expression in postnatal mice exhausted normal HSCs and promoted excessive proliferation of leukemogenic stem cells, resulting in the development of myeloproliferative disorders (MPDs) and eventually leukemia. 6,7 These studies further showed that the mTOR inhibitor rapamycin effectively suppressed growth of the leukemogenic stem cells and prevented exhaustion of normal HSCs. Furthermore, recent studies have shown that PTEN is down-regulated by BCR-ABL in leukemic stem cells, and PI3K and AKT play critical roles in BCR-ABL-induced leukemia in mice. 8,9 Thus, inhibition of the PTEN/AKT/mTOR pathway not only can suppress solid tumor growth but could also represent a novel therapeutic strategy against stem cell disorders that are leukemogenic in nature.GRP78, also ref...

a b s t r a c tLittle is known of the genetic diversity of Toxoplasma gondii circulating in wildlife. In the present study wild animals, from the USA were examined for T. gondii infection. Tissues of naturally exposed animals were bioassayed in mice for isolation of viable parasites. Viable T. gondii was isolated from 31 animals including, to our knowledge for the first time, from a bald eagle (Haliaeetus leucocephalus), five gray wolves (Canis lupus), a woodrat (Neotoma micropus), and five Arctic foxes (Alopex lagopus). Additionally, 66 T. gondii isolates obtained previously, but not genetically characterised, were revived in mice. Toxoplasma gondii DNA isolated from these 97 samples (31 + 66) was characterised using 11 PCR-restriction fragment length polymorphism (RFLP) markers (SAG1, 5 0 -and 3 0 -SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico). A total of 95 isolates were successfully genotyped. In addition to clonal Types II, and III, 12 different genotypes were found. These genotype data were combined with 74 T. gondii isolates previously characterised from wildlife from North America and a composite data set of 169 isolates comprised 22 genotypes, including clonal Types II, III and 20 atypical genotypes. Phylogenetic network analysis showed limited diversity with dominance of a recently designated fourth clonal type (Type 12) in North America, followed by the Type II and III lineages. These three major lineages together accounted for 85% of strains in North America. The Type 12 lineage includes previously identified Type A and X strains from sea otters. This study revealed that the Type 12 lineage accounts for 46.7% (79/169) of isolates and is dominant in wildlife of North America. No clonal Type I strain was identified among these wildlife isolates. These results suggest that T. gondii strains in wildlife from North America have limited diversity, with the occurrence of only a few major clonal types. Ó

Hepatocellular carcinoma (HCC) is a prototype of inflammation-related cancer, harboring M1-like and M2-like tumor-associated macrophages. M1 macrophages are thought to be tumoricidal, but some studies report its pro-tumor role. The programmed cell death-ligand (PD-L) 1 expressed in HCC cells is a critical checkpoint molecule to mediate immune escape of HCC. The PD-L1 expression in HCC cells is inducible. In the present study, we ask whether M1 macrophages induce the expression of PD-L1 in HCC cells. First, an association between M1 macrophage infiltration and PD-L1 expression in HCC tissues was determined by bioinformatics and immunohistochemistry experiments. The enrichment score of M1 macrophages was correlated to PD-L1 expression in 90 HCC samples from GEO database. Besides, infiltration of CD68+HLA-DR+ M1-like macrophages correlated with PD-L1 expression level in HCC cells. Moreover, M1-conditioned media was prepared from M1 macrophages derived from THP-1 cell, RAW264.7 cell or murine bone marrow. These supernatants induced expression of PD-L1 in HCC cells. Furthermore, inflammatory cytokine IL-1β in the supernatants was identified to account for the inducible PD-L1 expression by siRNA assay and receptor blockade assay. Additionally, transcription factor p65 and IRF1 in the HCC cells were revealed by CHIP assay to mediate the inducible PD-L1 expression. All the results demonstrate that M1 macrophages induced expression of PD-L1 in HCC cells, supporting the pro-tumor role of M1 macrophages.

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.Anti-estrogen therapy represents a major advance in the treatment of estrogen receptor-positive breast cancer that can produce significant clinical responses and delay of progression. However, their efficacy is limited by intrinsic and acquired therapeutic resistance (1, 2). To overcome this limitation, it is important to understand the resistance mechanisms and identify new therapeutic targets. Estrogen is required for the proliferation of estrogen receptor-positive breast cancer cells (3). When subjected to estrogen starvation, exposure to anti-estrogens, or treatment with aromatase inhibitors, significant apoptosis of breast cancer cells was observed. A key regulator of apoptosis is the B-cell lymphoma (BCL) 2 -2 family of proteins which has been reported to localize to the membranes of various organelles (4). Pro-survival members of the BCL-2 family such as BCL-2, share three or four of the conserved domains known as BCL-2 homology (BH) regions. BCL-2 and BCL-XL lower the Ca 2ϩ store in the endoplasmic reticulum and antagonize the pro-apoptotic function of BAX to promote cell survival (5). Pro-apoptotic members such as BAX and BAK share two or three BH domains. Upon activation, BAX translocates to mitochondria and initiates the release of cytochrome c into the cytosol (6). A third group of apoptotic regulators...

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Introduction Exosomes are nanograde membrane-bound vesicles secreted from most cell types through the fusion of multivesicular bodies with plasma membranes. Some of these exosomes are well defined, and are known to have immunomodulatory properties as well as play critical roles in intercellular communications. In this study, we characterized the exosomes derived from Toxoplasma gondii and their functions in aspect of immune responses. Methods T. gondii exosomes were isolated and identified using electron microscopy, nanoparticle tracking analysis, and Western blotting. The viability of macrophage RAW264.7 cells affected by exosomes was evaluated using a Cell Counting Kit (CCK-8). Then the uptake of T. gondii exosomes by RAW264.7 cells was detected by labeling with fluorescent dye PKH67. After exosomes stimulation, in vitro the production of interleukin (IL)-12, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10 in RAW264.7 cells were investigated using enzyme-linked immunosorbent assay (ELISA). In immunized BALB/c mice, the antibodies, cytokines as well as the percentage of CD4+ and CD8+ T cells were determined using ELISA and flow cytometric analysis. Protective efficacy was evaluated by challenging intraperitoneally with tachyzoites of T. gondii . Results We successfully isolated and characterized the exosomes derived from T. gondii . Functionally, the viability of macrophage RAW264.7 cells was significantly affected by exosomes at a high concentration (160 μg/mL). The production of IL-12, TNF-α and IFN-γ in macrophage cells were increased, and the level of IL-10 was decreased. Furthermore, BALB/c mice immunized with T. gondii exosomes showed both humoral and cellular immune responses and also exhibited a prolonged survival time. Conclusion T. gondii exosomes could modulate macrophage activation in vitro and trigger humoral and cellular immune responses and partial protection against acute parasite infection in mice, which suggested that exosomes may serve as a potential candidate against toxoplasmosis.

BackgroundToxoplasmosis, caused by an obligate intracellular protozoan parasite Toxoplasma gondii, has been a serious clinical and veterinary problem. Effective DNA vaccines against T. gondii can prevent and control the spread of toxoplasmosis, which is important for both human health and the farming industry. The T. gondii 14-3-3 protein has been proved to be antigenic and immunogenic and was a potential vaccine candidate against toxoplasmosis. In this study, we evaluated the immune responses induced by recombinant plasmids encoding T. gondii surface antigen 1 (SAG1) and 14-3-3 protein by immunizing BALB/c mice intramuscularly.MethodsIn the present study, BALB/c mice were randomly divided into five groups, including three experimental groups (pSAG1, p14-3-3 and pSAG1/14-3-3) and two control groups (PBS and pBudCE4.1), and were immunized intramuscularly three times. The levels of IgG antibodies and cytokine production in mouse sera were determined by enzyme-linked immunosorbent assays (ELISA). Two weeks after the last immunization, all mice were challenged intraperitoneally (i.p.) with 1×104 tachyzoites of T. gondii and the survival time of mice was observed and recorded every day.ResultsMice vaccinated with pSAG1, p14-3-3 or pSAG1/14-3-3 developed high levels of IgG2a and gamma interferon (IFN-γ) and low levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) compared to control groups (PBS or pBudCE4.1), which suggested a modulated Th1 type immune response (P<0.05). After intraperitoneal challenge with 1×104 tachyzoites of T. gondii (RH strain), the survival time of mice in experimental groups was longer than control groups (P<0.05). Mouse immunized with pSAG1/14-3-3 induced a higher level of IgG antibody response and significantly prolonged the survival time when compared with pSAG1 or p14-3-3 (P<0.05).ConclusionsThe study suggested that T. gondii 14-3-3 protein can induce effective immune responses in BALB/c mice and was a novel DNA vaccine candidate against toxoplasmosis, and the immune protective efficacy elicited by SAG1 gene was also demonstrated. Our results also showed multi-gene vaccine significantly enhanced immune responses and protective efficacy and was superior to the single-gene vaccine.