Cancer Letters

Cancer Letters

Volume 194, Issue 1, 8 May 2003, Pages 81-89
Cancer Letters

Overexpression of the ‘silencer of death domain’, SODD/BAG-4, modulates both TNFR1- and CD95-dependent cell death pathways

https://doi.org/10.1016/S0304-3835(03)00009-0Get rights and content

Abstract

The involvement of SODD/BAG-4 was studied in TNFR1 signaling, using SODD/BAG-4- overexpressing HeLa cells as a cellular model. Stable transfection of a SODD/BAG-4 cDNA leads to increased levels of the full-length 70-kDa protein and additional C-terminal fragments as well as altered expression of BAG-1. Concomitantly, the protein amounts of both the TNFR1 and, unexpectedly, the CD95 receptors are elevated. These biochemical changes are paralleled and confirmed by a reduced cellular sensitivity to treatment with extracellular TNFα and CD95 ligand. Moreover, ATP depletion by oligomycin is less toxic in SODD/BAG-4 overexpressing clones. The radiosensitivity is reduced in some, but not in all of these clones. Our results indicate that SODD/BAG-4 is not only a silencer of TNFR1 but also a modulator of CD95 activity, regulating the balance of both death-promoting and -depressing signals.

Introduction

The silencer of death domain, SODD/BAG-4, belongs to a family of anti-apoptotic proteins of the BAG family that were originally identified by their interaction with Bcl-2 [1]. BAG proteins are regulatory proteins that link molecular chaperones with cell signaling pathways. All of them possess a conserved region of approximately 45 amino acids at the C-terminal end, the so-called BAG domain, with 40–60% sequence homology among the BAG proteins. This domain constitutes the binding regions for Hsp70 and Bcl-2. The best characterized BAG proteins are the BAG-1 proteins, which modulate the chaperone activity of Hsp70/Hsc70 through conformational changes and thus alter its biological or biochemical activity. BAG-1 proteins exist in different isoforms arising through alternative mRNA splicing [2], [3], [4]. They all contain a ubiquitin-like domain in their N-terminal end and are highly variable. BAG-1 isoforms differ in their subcellular distribution, and they are candidate proto-oncogenes. In various cancer cells, the upregulation of BAG-1 proteins correlates with the malignant potential of the respective tumors [5], [6], [7].

The BAG-4 proteins were identified as so-called silencers of death domain, SODD, that bind to the tumor necrosis factor receptor 1 (TNFR1) or the death receptor3 (DR3) [8]. In non-stimulated cells, SODD/BAG-4 retains TNFR1 in an inactive, monomeric state. Binding of the TNF ligand dissociates SODD/BAG-4 and allows trimerization of the receptor and the recruitment of death domain containing adapter proteins for the activation of the signaling cascade [8]. It was recently shown that TNFR1 is an ATPase, which is regulated by SODD/BAG-4 [9]. The authors propose that SODD/BAG-4 is a nucleotide exchange factor that promotes disassembly of the TNFR1 complex, since ATP binding induces a conformational change of the receptor and leads to the monomeric state with the silenced death signal. They suggest that Hsc70 and TNFR1 compete for binding to SODD/BAG-4. In contrast to BAG-1, the SODD/BAG-4 protein is devoid of an ubiquitin-like domain or alternative in-frame translational start sites [10].

A potential role of SODD/BAG-4 in carcinogenesis has been proposed based on increased mRNA levels in cancer tissues; e.g. pancreatic cancers, which are resistant to TNFα-mediated apoptosis, show markedly elevated SODD/BAG-4 mRNA in comparison to normal control tissues [11]. Also, differential regulation of SODD/BAG-4 may be responsible for reduced TNFα-induced apoptosis in neonatal lymphocytes [12].

In recent experiments we could demonstrate that SODD/BAG-4 is involved in radiation-induced cellular resistance in various cell lines [13], [14]. Following low-dose fractionated γ-irradiation, a 42-kDa SODD/BAG-4 protein is overexpressed in the resistant, but not in the parental cells. In the present study, stable transfection and overexpression of the mouse homolog of SODD/BAG-4 in a HeLa cell model allowed us to establish and further analyze a role of this regulatory protein in both TNFR1 and CD95 cell death signaling.

Section snippets

Cell transfection

HeLa cells were stably transfected with an expression vector harboring the mouse SODD cDNA (pcDNA-SODD, kindly provided by R. Endres, Klinikum r.d.Isar, TUM, Muenchen) or the respective control vector. Eight micrograms of DNA were electroporated into 5×106 cells, using a Biorad gene pulser (750 V, 25 μF, 200 Ohm, 0.4-cm cuvettes) and cells were selected for stable uptake of expression plasmids by growth in 0.3 mg/ml G418 (Calbiochem) for 2–3 weeks. Individual subclones were isolated for further

Overexpression of SODD/BAG-4 is accompanied by expression of additional C-terminal SODD/BAG-4 fragments

Human HeLa cells were stably transfected with the mouse SODD/BAG-4 cDNA, which shares 84.5% identity to the human homolog. In particular, the C-terminal BAG domain (amino acids 403–449) is 100% identical between the two homologs.

In order to quantitate the expression levels of the SODD/BAG-4 protein in SODD-transfected vs. control cells, cytoplasmic extracts were probed with anti-SODD-H300 polyclonal antibody, which recognizes epitopes in the carboxyterminal half between amino acids 158–457. In

Discussion

The molecular and cellular functions of SODD/BAG-4 have so far been attributed to a regulatory role in TNFR1 signaling. Our results in HeLa cells demonstrate that SODD/BAG-4 is not only a silencer and regulator of TNFR1 but also a modifier of CD95. Both receptors are overexpressed in the SODD/BAG-4 transfected HS cells and concomitantly exhibit increased binding to this regulatory protein. The high SODD/BAG-4 levels protect against TNFα-induced cell death as well as against CD95-L-triggered

Acknowledgements

We thank Mrs H. Pfeifer for excellent technical assistance and Robert Endres from the Klinikum Rechts der Isar (TUM), Muenchen, for providing the SODD/BAG-4 cDNA vector.

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