A novel miR-371a-5p-mediated pathway, leading to BAG3 upregulation in cardiomyocytes in response to epinephrine, is lost in Takotsubo cardiomyopathy.

Molecular mechanisms protecting cardiomyocytes from stress-induced death, including tension stress, are essential for cardiac physiology and defects in these protective mechanisms can result in pathological alterations. Bcl2-associated athanogene 3 (BAG3) is expressed in cardiomyocytes and is a component of the chaperone-assisted autophagy pathway, essential for homeostasis of mechanically altered cells. BAG3 ablation in mice results in a lethal cardiomyopathy soon after birth and mutations of this gene have been associated with different cardiomyopathies including stress-induced Takotsubo cardiomyopathy (TTC). The pathogenic mechanism leading to TTC has not been defined, but it has been suggested that the heart can be damaged by excessive epinephrine (epi) spillover in the absence of a protective mechanism. The aim of this study was to provide more evidence for a role of BAG3 in the pathogenesis of TTC. Therefore, we sequenced BAG3 gene in 70 TTC patients and in 81 healthy donors with the absence of evaluable cardiovascular disease. Mutations and polymorphisms detected in the BAG3 gene included a frequent nucleotide change g2252c in the BAG3 3'-untranslated region (3'-UTR) of Takotsubo patients (P<0.05), resulting in loss of binding of microRNA-371a-5p (miR-371a-5p) as evidenced by dual-luciferase reporter assays and argonaute RNA-induced silencing complex catalytic component 2/pull-down assays. Moreover, we describe a novel signaling pathway in cardiomyocytes that leads to BAG3 upregulation on exposure to epi through an ERK-dependent upregulation of miR-371a-5p. In conclusion, the presence of a g2252c polymorphism in the BAG3 3'-UTR determines loss of miR-371a-5p binding and results in an altered response to epi, potentially representing a new molecular mechanism that contributes to TTC pathogenesis.

BAG3 regulates formation of the SNARE complex and insulin secretion.

Insulin release in response to glucose stimulation requires exocytosis of insulin-containing granules. Glucose stimulation of beta cells leads to focal adhesion kinase (FAK) phosphorylation, which acts on the Rho family proteins (Rho, Rac and Cdc42) that direct F-actin remodeling. This process requires docking and fusion of secretory vesicles to the release sites at the plasma membrane and is a complex mechanism that is mediated by SNAREs. This transiently disrupts the F-actin barrier and allows the redistribution of the insulin-containing granules to more peripheral regions of the ß cell, hence facilitating insulin secretion. In this manuscript, we show for the first time that BAG3 plays an important role in this process. We show that BAG3 downregulation results in increased insulin secretion in response to glucose stimulation and in disruption of the F-actin network. Moreover, we show that BAG3 binds to SNAP-25 and syntaxin-1, two components of the t-SNARE complex preventing the interaction between SNAP-25 and syntaxin-1. Upon glucose stimulation BAG3 is phosphorylated by FAK and dissociates from SNAP-25 allowing the formation of the SNARE complex, destabilization of the F-actin network and insulin release.

BAG3 controls angiogenesis through regulation of ERK phosphorylation.

BAG3 is a co-chaperone of the heat shock protein (Hsp) 70, is expressed in many cell types upon cell stress, however, its expression is constitutive in many tumours. We and others have previously shown that in neoplastic cells BAG3 exerts an anti-apoptotic function thus favoring tumour progression. As a consequence we have proposed BAG3 as a target of antineoplastic therapies. Here we identify a novel role for BAG3 in regulation of neo-angiogenesis and show that its downregulation results in reduced angiogenesis therefore expanding the role of BAG3 as a therapeutical target. In brief we show that BAG3 is expressed in endothelial cells and is essential for the interaction between ERK and its phosphatase DUSP6, as a consequence its removal results in reduced binding of DUSP6 to ERK and sustained ERK phosphorylation that in turn determines increased levels of p21 and p15 and cell-cycle arrest in the G1 phase.

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.

Ferritin heavy chain (FHC) is up-regulated in papillomavirus-associated urothelial tumours of the urinary bladder in cattle.

The up-regulation of ferritin heavy chain (FHC) is reported in six papillary and in four invasive urothelial tumours of the urinary bladder of cattle grazing on mountain pastures rich in bracken fern. All tumours contained sequence of bovine papillomavirus type-2 (BPV-2) as determined by polymerase chain reaction (PCR) analyses and validated by direct sequencing of the amplified products. The oncoprotein E5 was also detected in these tumours by immunoprecipitation and by immunofluorescence and laser scanning confocal microscopy. Expression of FHC was evaluated by western blot analysis, reverse transcriptase (RT) PCR, real-time RT-PCR and immunohistochemistry. The oligonucleotide sequence of the bovine ferritin amplicons was identical to that of human ferritin. Nuclear overexpression of p65, an important component of nuclear factor kappaB (NF-kappaB) transcription factors, was also observed. These findings suggest that FHC up-regulation may be mediated by activation of NF-kappaB and that in turn this may be related to the resistance of bovine papillomavirus type-2 (BPV-2) infected urothelial cells to apoptosis.

Activation of BAG3 by Egr-1 in response to FGF-2 in neuroblastoma cells.

The co-chaperone protein, BAG3, which belongs to the BAG protein family, has an established antiapoptotic function in different tumor cell lines. Here we demonstrated that treatment of the human neuroblastoma cell line, SK-N-MC, with fibroblast growth factor-2 (FGF-2) results in induction of BAG3 expression. Induction of BAG3 protein by FGF-2 occurs at the transcriptional level; it requires the extracellular regulated kinase1/2 pathway and is dependent on the activity of Egr-1 upon the BAG3 promoter. Targeted suppression of BAG3 by small-interfering RNA results in dysregulation of cell-cycle progression most notably at S and G(2) phases, which corroborates the decreased level of cyclin B1 expression. These observations suggest a new role for BAG3 in regulation of the cell cycle.

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 hsp90alpha gene, that encodes the inducible form of HSP90, is regulated by nuclear factor-kappaB (NF-kappaB) activity. Indeed, we found that NF-kappaB factors bound to one of the two putative consensus sequences present in the hsp90alpha-flanking region; mutation of such motif hampered the phorbol-myristate-13-acetate-stimulated expression of a luciferase reporter gene under the control of the hsp90alpha promoter. Furthermore, the downmodulation of NF-kappaB (p65) levels by a specific small interfering (si) RNA resulted in reducing the levels of endogenous HSP90alpha protein. These findings disclose a previously unrecognized mechanism that contributes to connect NF-kappaB factors and HSPs in cell defence machinery.

Antiproliferative and pro-apoptotic activity of novel phenolic derivatives of resveratrol.

Gloriosaols A-C, isolated from Yucca gloriosa (Agavaceae), are novel phenolic compounds structurally related to resveratrol. In the present study, we show that gloriosaols possess antiproliferative and pro-apoptotic activity on tumor cells of different histogenetic origin and that their cell growth inhibition potential is higher than that of resveratrol. Despite the close similarities in their structure, gloriosaols A-C exhibited different antiproliferative potency, as the EC(50) ascending order is: gloriosaol C, gloriosaol A, gloriosaol B. Further mechanisms of gloriosaol C cytotoxicity were elucidated in detail in U937 cells, the most sensitive of the cell lines tested. The effect of gloriosaol C on cell growth turned out to be strongly dependent upon the concentration. Gloriosaol C doses lower than the EC(50) value (8 mu-icroM) blocked the cell cycle in G(0)/G(1), with a concurrent decrease in the number of cells in the G(2)/M phases of the cell cycle. At higher doses, this arrest overlaps with the occurrence of apoptosis and necrosis. In the 10-25 microM range of doses, gloriosaol C caused cell death mainly by apoptosis, as measured by hypodiploidia induction, phosphatidyl serine externalization and disruption of mitochondrial transmembrane potential. A switch in the mode of death from apoptosis to necrosis occurred at doses of gloriosaol C higher than 30 microM. Gloriosaol C was found to induce production of reactive species dose-dependently, but also to counteract their elevation in stressed cells. Thus, the different fate of cells, that is cell cycle arrest or cell death, in response to different doses of gloriosaol C might be related to the extent of induced oxidative stress.

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

The activity of NF-kappaB/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 nfkappab or ikappaB genes are documented in some hematologic neoplasias, while in others dysfunction in NF-kappaB/Rel-activating signaling pathways can be recognized. The prosurvival properties of NF-kappaB/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-kappaB/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-kappaB/Rel-blocking approaches have been introduced in antineoplastic strategies. The identification of NF-kappaB/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-kappaB/Rel inhibition. Moreover, comparative analyses of normal hematopoietic progenitors and neoplastic cell sensitivities to inhibitors of NF-kappaB/Rel and their target genes will allow to evaluate the impact of these tools on normal bone marrow.

Oxaliplatin (L-OHP) treatment of human myeloma cells induces in vitro growth inhibition and apoptotic cell death.

Oxaliplatin (L-OHP), a diaminocyclohexane platinum derivative, is an active and well tolerated anticancer drug which is presently used in the treatment of gastrointestinal tumours. Since the efficacy of L-OHP in the treatment of multiple myeloma (MM) has not yet been evaluated, we studied the antiproliferative activity of this compound in vitro in a panel of MM cell lines (XG1, XG1a, U266 and IM-9). We found that L-OHP inhibited the growth of MM cells at therapeutically achievable concentrations (IC(50): 5-10 microM after 24 h of exposure) and was more active than Cisplatin (CDDP) or Carboplatin (CBDCA). The activity of L-OHP was apparently not affected by interleukin-6 (IL-6), the major growth and survival factor of MM cells. We also found that L-OHP induced apoptotic cell death. We demonstrated that the combination of L-OHP with Dexamethasone (Dex) resulted in the enhancement of the anti-myeloma effects. L-OHP and Dex both induced poly adenosine diphosphate (ADP)-ribose polymerase (PARP) cleavage and this induction was enhanced by the combined treatment. L-OHP-induced apoptosis correlated with caspase-3 cleavage, but this correlation could not be demonstrated in Dex-treated cells. Taken together, these in vitro results provide a rationale for the experimental use of L-OHP in the treatment of MM patients and suggest therapeutic combinations of potential value.