Maria Caterina Turco

BAG3: a multifaceted protein that regulates major cell pathways

Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110–124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is instead expressed in several tumor types. In different tumor contexts, BAG3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. In some tumor types, down-modulation of BAG3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. This review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression.

Review of molecular mechanisms involved in the activation of the Nrf2-ARE signaling pathway by chemopreventive agents

Human exposures to environmental toxicants have been associated with etiology of many diseases including inflammation, cancer, and cardiovascular and neurodegenerative disorders. To counteract the detrimental effect of environmental insults, mammalian cells have evolved a hierarchy of sophisticated sensing and signaling mechanisms to turn on or off endogenous antioxidant responses accordingly. One of the major cellular antioxidant responses is the induction of antioxidative and carcinogen-detoxification enzymes through the cytoplasmic oxidative stress system (Nrf2-Keap1) activated by a variety of natural and synthetic chemopreventive agents. Under normal conditions, Keap1 anchors the Nrf2 transcription factor within the cytoplasm targeting it for ubiquitination and proteasomal degradation to maintain low levels of Nrf2 that mediate the constitutive expression of Nrf2 downstream genes. When cells are exposed to chemopreventive agents and oxidative stress, a signal involving phosphorylation and/or redox modification of critical cysteine residues in Keap1 inhibits the enzymatic activity of the Keap1-Cul3-Rbx1 E3 ubiquitin ligase complex, resulting in decreased Nrf2 ubiquitination and degradation. As a consequence, free Nrf2 translocates into the nucleus and in combination with other transcription factors (e.g., sMaf, ATF4, JunD, PMF-1) transactivates the antioxidant response elements (AREs)/electrophile response elements (EpREs) of many cytoprotective genes, as well as Nrf2 itself. Upon recovery of cellular redox homeostasis, Keap1 travels into the nucleus to dissociate Nrf2 from the ARE. Subsequently, the Nrf2-Keap1 complex is exported out of the nucleus by the nuclear export sequence (NES) in Keap1. Once in the cytoplasm, the Nrf2-Keap1 complex associates with the Cul3-Rbx1 core ubiquitin machinery, resulting in degradation of Nrf2 and termination of the Nrf2/ARE signaling pathway. The discovery of multiple nuclear localization signals (NLSs) and nuclear export signals (NESs) in Nrf2 also suggests that the nucleocytoplasm translocation of transcription factors is the consequence of a dynamic equilibrium of multivalent NLSs and NESs. On the other hand, Keap1 may provide an additional regulation of the quantity of Nrf2 both in basal and inducible conditions. This chapter summarizes the current body of knowledge regarding the molecular mechanisms through which ARE inducers (chemopreventive agents) regulate the coordinated transcriptional induction of genes encoding phase II and antioxidant enzymes as well as other defensive proteins, via the nuclear factor-erythroid 2 (NF-E2-p45)-related factor 2(Nrf2)/(ARE) signaling pathway.

Growth inhibition and synergistic induction of apoptosis by zoledronate and dexamethasone in human myeloma cell lines.

Bisphosphonates (BPs) are commonly used in the treatment of myeloma-associated osteolytic lesions. Recent reports have suggested that BPs may also exert direct antitumor effects on myeloma cells. Here, we show that the treatment of myeloma cell lines with the combination of the potent BP zoledronate and dexamethasone inhibits cell growth and synergistically induces apoptotic cell death, providing a rationale for potential applications in vivo.

NF-κB/Rel-mediated regulation of apoptosis in hematologic malignancies and normal hematopoietic progenitors

The activity of NF-κB/Rel transcription factors can downmodulate apoptosis in normal and neoplastic cells of the hematologic and other compartments, contributing in maintaining neoplastic clone survival and impairing response to therapy. Alterations in nfκb or iκB genes are documented in some hematologic neoplasias, while in others dysfunction in NF-κB/Rel-activating signaling pathways can be recognized. The prosurvival properties of NF-κB/Rel appear to rely on the induced expression of molecules (caspase inhibitors, Bcl2 protein family members, etc.), which interfere with the apoptosis pathway. Constitutive NF-κB/Rel activity in some hematologic malignancies could be advantageous for neoplastic clone expansion by counteracting stress stimuli (consumption of growth factors and metabolites) and immune system-triggered apoptosis; it is furthermore likely to play a central role in determining resistance to therapy. Based on this evidence, NF-κB/Rel-blocking approaches have been introduced in antineoplastic strategies. The identification of NF-κB/Rel target genes relevant for survival in specific neoplasias is required in order to address tailored therapies and avoid possible detrimental effects due to widespread NF-κB/Rel inhibition. Moreover, comparative analyses of normal hematopoietic progenitors and neoplastic cell sensitivities to inhibitors of NF-κB/Rel and their target genes will allow to evaluate the impact of these tools on normal bone marrow.

bag3 gene expression is regulated by heat shock factor 1

BAG3 protein, a member of the BAG co‐chaperones family, sustains cell survival, through its interaction with the heat shock protein (HSP) 70, in a variety of normal and neoplastic cell types. bag3 gene expression is induced by stressful stimuli. Here we report for the first time that two of the three putative heat shock‐responsive elements (HSEs) in bag3 promoter interact with the heat shock factor (HSF) 1 in vitro and in vivo. Furthermore, downmodulation of HSF1 protein levels by specific small interfering (si) RNAs results in reducing BAG3 protein levels, indicating that the transcription factor plays a major role in bag3 gene expression. Because of the anti‐apoptotic role of BAG3 protein, these results disclose a previously unrecognized pathway, through which HSF1 maintains cell survival. J. Cell. Physiol. 215: 575–577, 2008.

BAG3 protein is overexpressed in human glioblastoma and is a potential target for therapy.

Glioblastoma multiforme, which represents 80% of malignant gliomas, is characterized by aggressiveness and high recurrence rates. Despite therapeutic advances, patients with glioblastoma multiforme show a poor survival, and identification of novel markers and molecular targets for therapy is needed. A role for BAG3, a member of the BAG family of HSC/HSP70 co-chaperones, in promoting tumor cell growth in vivo has recently been described. We analyzed BAG3 levels by IHC in specimens from patients affected by brain tumors and we found that BAG3, although negative in normal brain tissues, was highly expressed in astrocytic tumors and increasingly expressed in more aggressive types of cancer; it was particularly high in glioblastomas. Down-regulating BAG3 both in vitro and in vivo in a rat glioblastoma model resulted in increased sensitivity to apoptosis, suggesting that BAG3 is a potential target for novel therapies. Finally, we determined that the underlying molecular mechanism requires the formation of a complex of BAG3, HSP70, and BAX that prevents BAX translocation to mitochondria, thus protecting tumor cells from apoptosis. Our data identify BAG3 as a potential marker of glial brain tumor sensitivity to therapy and thus also an attractive candidate for new molecular therapies.

BAG3 protein regulates stress- induced apoptosis in normal and neoplastic leukocytes

We express our sincere thanks to Dr Junjiro Tsuchiyama for the NK-YS cell line and all medical and nursing staff in the Department of Medicine, Queen Mary Hospital for the provision of expert medical care. CS Chim KY Wong F Loong G Srivastava University Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong; and Department of Pathology, Queen Mary Hospital, The University of Hong Kong, Hong Kong

Evidence for BAG3 modulation of HIV-1 gene transcription†

A family of co‐chaperone proteins that share the Bcl‐2‐associated athanogene (BAG) domain are involved in a number of cellular processes, including proliferation and apoptosis. Among these proteins, BAG3 has received increased attention due to its high levels in several disease models and ability to associate with Hsp70 and a number of other molecular partners. BAG3 expression is stimulated during cell response to stressful conditions, such as exposure to high temperature, heavy metals, and certain drugs. Here, we demonstrate that BAG3 expression is elevated upon HIV‐1 infection of human lymphocytes and fetal microglial cells. Furthermore, BAG3 protein was detectable in the cytoplasm of reactive astrocytes in HIV‐1‐associated encephalopathy biopsies, suggesting that induction of BAG3 is part of the host cell response to viral infection. To assess the impact of BAG3 upregulation on HIV‐1 gene expression, we performed transcription assays and demonstrated that BAG3 can suppress transcription of the HIV‐1 long terminal repeat (LTR) in microglial cells. This activity was mapped to the κB motif of the HIV‐1 LTR. Results from in vitro and in vivo binding assays revealed that BAG3 suppresses interaction of the p65 subunit of NF‐κB with the κB DNA motif of the LTR. Results from binding and transcriptional assay identified the C‐terminus of BAG3 as a potential domain involved in the observed inhibitory effect of BAG3 on p65 activity. These observations reveal a previously unrecognized cell response, that is, an increase in BAG3, elicited by HIV‐1 infection, and may provide a new avenue for the suppression of HIV‐1 gene expression. J. Cell. Physiol. 210: 676–683, 2007.

BAG3 protein regulates cell survival in childhood acute lymphoblastic leukemia cells.

No abstract available.

The activity of hsp90|[alpha]| promoter is regulated by NF-|[kappa]|B transcription factors

Heat-shock proteins (HSP) 90 exert a relevant role in the survival and response to therapy of many neoplastic cell types. Here, we show that the promoter of hsp90α gene, that encodes the inducible form of HSP90, is regulated by nuclear factor-κB (NF-κB) activity. Indeed, we found that NF-κB factors bound to one of the two putative consensus sequences present in the hsp90α-flanking region; mutation of such motif hampered the phorbol-myristate-13-acetate-stimulated expression of a luciferase reporter gene under the control of the hsp90α promoter. Furthermore, the downmodulation of NF-κB (p65) levels by a specific small interfering (si) RNA resulted in reducing the levels of endogenous HSP90α protein. These findings disclose a previously unrecognized mechanism that contributes to connect NF-κB factors and HSPs in cell defence machinery.