Amy S. Lee

The glucose-regulated proteins: stress induction and clinical applications

A protective mechanism used by cells to adapt to stress of the endoplasmic reticulum (ER) is the induction of members of the glucose-regulated protein (Grp) family. The induction of mammalian Grp proteins in response to ER stress involves a complex network of regulators and novel mechanisms. The elucidation of Grp function and regulation opens up new therapeutic approaches to diseases associated with ER stress and cancer.

GRP78 Induction in Cancer: Therapeutic and Prognostic Implications

Cancer cells adapt to chronic stress in the tumor microenvironment by inducing the expression of GRP78/BiP, a major endoplasmic reticulum chaperone with Ca(2+)-binding and antiapoptotic properties. GRP78 promotes tumor proliferation, survival, metastasis, and resistance to a wide variety of therapies. Thus, GRP78 expression may serve as a biomarker for tumor behavior and treatment response. Combination therapy suppressing GRP78 expression may represent a novel approach toward eradication of residual tumors. Furthermore, the recent discovery of GRP78 on the cell surface of cancer cells but not in normal tissues suggests that targeted therapy against cancer via surface GRP78 may be feasible.

ER chaperones in mammalian development and human diseases.

The field of endoplasmic reticulum (ER) stress in mammalian cells has expanded rapidly during the past decade, contributing to understanding of the molecular pathways that allow cells to adapt to perturbations in ER homeostasis. One major mechanism is mediated by molecular ER chaperones which are critical not only for quality control of proteins processed in the ER, but also for regulation of ER signaling in response to ER stress. Here, we summarized the properties and functions of GRP78/BiP, GRP94/gp96, GRP170/ORP150, GRP58/ERp57, PDI, ERp72, calnexin, calreticulin, EDEM, Herp and co‐chaperones SIL1 and P58IPK and their role in development and diseases. Many of the new insights are derived from recently constructed mouse models where the genes encoding the chaperones are genetically altered, providing invaluable tools for examining the physiological involvement of the ER chaperones in vivo.

Stress induction of GRP78/BiP and its role in cancer.

GRP78, also referred to as BiP, is a central regulator of endoplasmic reticulum (ER) function due to its roles in protein folding and assembly, targeting misfolded protein for degradation, ER Ca(2+)-binding and controlling the activation of trans-membrane ER stress sensors. Further, due to its anti-apoptotic property, stress induction of GRP78 represents an important pro-survival component of the unfolded protein response. GRP78 is induced in a wide variety of cancer cells and cancer biopsy tissues. Recent progress, utilizing overexpression and siRNA approaches, establishes that GRP78 contributes to tumor growth and confers drug resistance to cancer cells. The discovery of GRP78 expression on the cell surface of cancer cells further leads to the development of new therapeutic approaches targeted against cancer, in particular, hypoxic tumors where GRP78 is highly induced. Progress has also been made in understanding how Grp78 is induced by ER stress. The identification of the transcription factors interacting with the ER stress response element leads to the discovery of multiple pathways whereby mammalian cells can sense ER stress and trigger the transcription of Grp78. In addition, advances have been made in understanding how Grp78 expression is regulated in the context of chromatin modification. This review summarizes the transcriptional regulation of Grp78, the molecular basis for the cytoprotective function of GRP78 and its role in cancer progression, drug resistance and potential future cancer therapy.

Peroxisomes are signaling platforms for antiviral innate immunity.

Peroxisomes have long been established to play a central role in regulating various metabolic activities in mammalian cells. These organelles act in concert with mitochondria to control the metabolism of lipids and reactive oxygen species. However, while mitochondria have emerged as an important site of antiviral signal transduction, a role for peroxisomes in immune defense is unknown. Here, we report that the RIG-I-like receptor (RLR) adaptor protein MAVS is located on peroxisomes and mitochondria. We find that peroxisomal and mitochondrial MAVS act sequentially to create an antiviral cellular state. Upon viral infection, peroxisomal MAVS induces the rapid interferon-independent expression of defense factors that provide short-term protection, whereas mitochondrial MAVS activates an interferon-dependent signaling pathway with delayed kinetics, which amplifies and stabilizes the antiviral response. The interferon regulatory factor IRF1 plays a crucial role in regulating MAVS-dependent signaling from peroxisomes. These results establish that peroxisomes are an important site of antiviral signal transduction.

Mammalian stress response: induction of the glucose-regulated protein family

The glucose-regulated protein family consists of a set of stress-inducible proteins localized in the endoplasmic reticulum. Since their discovery in 1977, significant advances in our understanding of their structure, function and regulation have been made. Recent findings concerning the physiological roles played by the glucose-regulated proteins, and their regulations at the transcriptional, post-transcriptional, translational and post-translational levels are summarized.

Endoplasmic Reticulum Stress-induced Apoptosis MULTIPLE PATHWAYS AND ACTIVATION OF p53-UP-REGULATED MODULATOR OF APOPTOSIS (PUMA) AND NOXA BY p53

Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in the development of multiple diseases. However, the in vivo signaling pathways are still not fully understood. In this report, through the use of genetically deficient mouse embryo fibroblasts (MEFs) and their matched wild-type controls, we have demonstrated that the mitochondrial apoptotic pathway mediated by Apaf-1 is an integral part of ER stress-induced apoptosis and that ER stress activates different caspases through Apaf-1-dependent and -independent mechanisms. In search of the molecular link between ER stress and the mitochondrial apoptotic pathway, we have discovered that in MEFs, ER stress selectively activates BH3-only proteins PUMA and NOXA at the transcript level through the tumor suppressor gene p53. In p53-/- MEFs, ER stress-induced apoptosis is partially suppressed. The p53-independent apoptotic pathway may be mediated by C/EBP homologous protein (CHOP) and caspase-12, as their activation is intact in p53-/- MEFs. In multiple MEF lines, p53 is primarily nuclear and its level is elevated upon ER stress. To establish the role of NOXA and PUMA in ER stress-induced apoptosis, we have shown that, in MEFs deficient in NOXA or PUMA, ER stress-induced apoptosis is reduced. Reversibly, overexpression of NOXA or PUMA induces apoptosis as evidenced by the activation of BAK and caspase-7. Our results provide new evidence that, in MEFs, in addition to PUMA, p53 and NOXA are novel components of the ER stress-induced apoptotic pathway, and both contribute to ER stress-induced apoptosis.

Coordinated regulation of a set of genes by glucose and calcium ionophores in mammalian cells

Abstract Cells respond to glucose starvation and calcium ionophore treatment by increased synthesis of a specific set of proteins localized within the endoplasmic reticulum and the Golgi apparatus. Perturbations of protein glycosylation under these and other adverse physiological conditions may trigger a regulatory mechanism resulting in increased transcription of the genes encoding these proteins.

The Unfolded Protein Response Regulator GRP78/BiP as a Novel Target for Increasing Chemosensitivity in Malignant Gliomas

Poor chemosensitivity and the development of chemoresistance remain major obstacles to successful chemotherapy of malignant gliomas. GRP78 is a key regulator of the unfolded protein response (UPR). As a Ca2+-binding molecular chaperone in the endoplasmic reticulum (ER), GRP78 maintains ER homeostasis, suppresses stress-induced apoptosis, and controls UPR signaling. We report here that GRP78 is expressed at low levels in normal adult brain, but is significantly elevated in malignant glioma specimens and human malignant glioma cell lines, correlating with their rate of proliferation. Down-regulation of GRP78 by small interfering RNA leads to a slowdown in glioma cell growth. Our studies further reveal that temozolomide, the chemotherapeutic agent of choice for treatment of malignant gliomas, leads to induction of CHOP, a major proapoptotic arm of the UPR. Knockdown of GRP78 in glioblastoma cell lines induces CHOP and activates caspase-7 in temozolomide-treated cells. Colony survival assays further establish that knockdown of GRP78 lowers resistance of glioma cells to temozolomide, and, conversely, overexpression of GRP78 confers higher resistance. Knockdown of GRP78 also sensitizes glioma cells to 5-fluorouracil and CPT-11. Treatment of glioma cells with (-)-epigallocatechin gallate, which targets the ATP-binding domain of GRP78 and blocks its protective function, sensitizes glioma cells to temozolomide. These results identify a novel chemoresistance mechanism in malignant gliomas and show that combination of drugs capable of suppressing GRP78 with conventional agents such as temozolomide might represent a novel approach to eliminate residual tumor cells after surgery and increase the effectiveness of malignant glioma chemotherapy.

GRP78/BiP Is Required for Cell Proliferation and Protecting the Inner Cell Mass from Apoptosis during Early Mouse Embryonic Development

ABSTRACT GRP78, also known as BiP, is a central regulator of endoplasmic reticulum (ER) homeostasis due to its multiple functional roles in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. ER stress induction of GRP78/BiP represents a major prosurvival arm of the unfolded protein response (UPR). However, the physiological role of GRP78 in development is not known. Using a transgenic approach, we discovered that the Grp78 promoter is activated in both the trophectoderm and inner cell mass (ICM) of embryos at embryonic day 3.5 via a mechanism requiring the ER stress elements. To reveal the function of the GRP78 in vivo, we created a tri-loxP Grp78 mutant allele, which was further crossed with EIIA-cre to create a knockout allele. The Grp78+/− mice, which express 50% of the wild-type level of the GRP78 protein, are viable. Interestingly, the heterozygous Grp78 cells up-regulate the ER proteins GRP94 and protein disulfide isomerase at both the transcript and protein levels, while other UPR targets such as CHOP and XBP-1 are not affected. Further studies revealed that mouse embryonic fibroblasts from Grp78+/− mice are capable of responding to ER stress. However, Grp78−/− embryos that are completely devoid of GRP78 lead to peri-implantation lethality. These embryos do not hatch from the zona pellucida in vitro, fail to grow in culture, and exhibit proliferation defects and a massive increase in apoptosis in the ICM, which is the precursor of embryonic stem cells. These findings provide the first evidence that GRP78 is essential for embryonic cell growth and pluripotent cell survival.