Identification of G0S2 as an NF-κB-dependent downstream target of the TNFα pathway
To identify transcriptional targets downstream of TNFα, we used high-density cDNA microarrays to compare the expression profiles of human primary foreskin fibroblasts (PFFs) treated in the presence or absence of TNFα. Of the 33,000 genes represented on the microarray, 138 showed increased expression and 27 showed decreased expression by 4-fold or more upon TNFα treatment (Supplementary Table S1
). As expected, this approach identified numerous genes known to be induced by TNFα including TNFAIP2
), as well as VCAM1
) and PTSG2
(also known as COX2
The gene most highly induced following TNFα treatment was G0/G1 switch 2
, which encodes a protein of unknown function. Northern blot analysis indicated that GOS2
is widely expressed in normal human tissues and is present at particularly high levels in peripheral blood, skeletal muscle and heart (Supplementary Fig. S1
To verify the results of the microarray experiment, we analyzed GOS2
mRNA and protein levels in PFFs following TNFα treatment. The northern blot of (left panel) confirms that G0S2
was transcriptionally up-regulated in PFFs following TNFα treatment. Quantitative RT-PCR (qRT-PCR) analysis indicated that G0S2
induction could be detected as early as 1 hour and reached maximal levels by 4 hours following TNFα treatment (Supplementary Fig. S2
). Immunoblot analysis showed that, like mRNA levels, G0S2 protein levels were also up-regulated in PFFs following TNFα treatment (, right panel).
Figure 1 G0S2 is an NF-κB-dependent downstream target of TNFα that encodes a mitochondrial protein. A, G0S2 expression was analyzed by northern blot (left) or immunoblotting (right) in TNFα-treated PFFs. GAPDH and tubulin were monitored (more ...)
To determine whether TNFα-mediated induction of G0S2
was dependent upon NF-κB, we monitored G0S2
expression in cells infected with an adenovirus expressing a super repressor form of IκB (Ad-IκB-SR), which blocks NF-κB activation (16
). The RT-PCR analysis of shows that G0S2
induction by TNFα was abrogated in PFFs expressing Ad-IκB-SR but not a control adenovirus expressing LacZ (Ad-LacZ). Collectively, these results indicate that G0S2
is induced by TNFα through activation of NF-κB.
TNFα also induced expression of cIAP1
, two known NF-κB target genes (18
), with kinetics comparable to that of G0S2
, whereas expression of another reported NF-κB target gene, Bcl-xL
), was not induced by TNFα treatment (Supplementary Fig. S2
G0S2 is a mitochondrial protein
As a first step toward elucidating the function of G0S2, we determined its subcellular localization. H1299 non-small cell lung cancer cells were transfected with a plasmid encoding a C-terminal hemagglutinin (HA)-tagged version of G0S2 (G0S2-HA) and the protein was detected by indirect immunofluorescence using an α-HA antibody. (top left panel) shows that G0S2-HA displayed a staining pattern that was characteristic of the mitochondria, which was confirmed by co-localization with the mitochondrial dye Mitotracker (, top middle and right panels). The localization pattern was not a consequence of the C-terminal epitope tag, as an N-terminal enhanced green fluorescent protein (EGFP)-tagged version of G0S2 also localized to the mitochondria (, bottom panels). Biochemical fractionation experiments and subsequent immunoblot analysis using an α-G0S2 antibody confirmed that endogenous G0S2 was present in the mitochondria ().
G0S2 directly interacts with the anti-apoptotic protein Bcl-2
The mitochondrial localization of G0S2, in conjunction with the well-established role of NF-κB in apoptosis, prompted us to test whether G0S2 interacts with one or more members of the Bcl-2 family. HEK293 embryonic kidney cells were co-transfected with plasmids expressing G0S2-HA and a FLAG-tagged Bcl-2 family member. G0S2-HA was immunoprecipitated using an α-HA antibody and the immunoprecipitate was analyzed for the Bcl-2 family member by immunoblotting with an α-FLAG antibody. shows that of the proteins tested, only Bcl-2 was readily detectable in the G0S2 immunoprecipitate. Significantly weaker interactions were also detected between G0S2 and the anti-apoptotic proteins Bcl-xL and Mcl-1, which were evident only upon overexposure of the autoradiogram (data not shown).
Figure 2 G0S2 directly interacts with the anti-apoptotic protein Bcl-2. A, HEK293 cells were co-transfected with plasmids expressing G0S2-HA and a FLAG-tagged Bcl-2 protein. G0S2 was immunoprecipitated using an α-HA antibody, and the immunoprecipitate (more ...)
To confirm the association between G0S2 and Bcl-2, HeLa cells were infected with Ad-G0S2 (in which G0S2 was epitope-tagged at the C-terminus with HA), and G0S2 was immunoprecipitated from the mitochondrial fraction using an α-HA antibody and the immunoprecipitate was analyzed for endogenous Bcl-2 using an α-Bcl-2 antibody. shows that Bcl-2 was present in the immunoprecipitate from Ad-G0S2-infected cells but not from control cells infected with Ad-LacZ. In vitro GST pull-down assays using purified GST-tagged Bcl-2 and in vitro translated 35S-methionine labeled G0S2 confirmed that the interaction between G0S2 and Bcl-2 was direct ().
Finally, to determine whether G0S2 directly interacted with Bcl-2 in vivo, we used a fluorescence resonance energy transfer (FRET) assay. Plasmids expressing enhanced cyan fluorescence protein (ECFP) fused to the C-terminus of Bcl-2 or Bad (Bcl-2-ECFP or Bad-ECFP, respectively) and enhanced yellow fluorescence protein (EYFP) fused to the C-terminus of G0S2 (G0S2-EYFP) were transiently transfected into HCT116 colorectal cells in pairwise combinations, and the cell lines were analyzed by FRET. The results of show that a FRET signal was observed in cells expressing Bcl-2-ECFP and G0S2-EYFP, consistent with the results of , but not in cells expressing Bad-ECFP and G0S2-EYFP. Collectively, the results of indicate that G0S2 directly interacts with Bcl-2 both in vitro and in vivo.
G0S2 induces apoptosis in transformed cells dependent upon interaction with Bcl-2
The above findings raised the possibility that G0S2 could have pro- or anti-apoptotic activity. We therefore tested whether ectopic expression of G0S2 could induce apoptosis in transformed cells. H1299 and HCT116 cells were infected with Ad-G0S2 or Ad-LacZ, and 36 hours later stained with Annexin V-PE. shows that in both cell lines expression of Ad-G0S2 induced pronounced apoptosis, whereas little or no cell death was observed in Ad-LacZ or mock-infected cells. G0S2 expression and induction of apoptosis could be detected by 20 hours post-infection and the level of apoptosis increased over 72 hours (Supplementary Fig. S3
Figure 3 G0S2 induces apoptosis in transformed cells dependent upon interaction with Bcl-2. A, H1299 and HCT116 cells were infected with Ad-G0S2 or Ad-LacZ, or mock-infected, and cell death was monitored by Annexin V-PE staining. B, (Top) Schematic representations (more ...)
To examine whether the ability of G0S2 to induce apoptosis and to interact with Bcl-2 were related activities, we sought to generate G0S2 mutants that were abrogated for their ability to interact with Bcl-2. We first delineated the region of G0S2 required for Bcl-2 interaction using a panel of G0S2 deletion mutants in which fragments of G0S2 were fused to the C-terminus of GFP (, top panel). The co-immunoprecipitation experiments of (bottom panel) show that only constructs harboring the central region of G0S2 (amino acids 33 to 67) interacted with Bcl-2.
We next constructed a series of point mutants in the central region by mutating pairs of amino acids to alanine (, top). shows that mutations in amino acids 35 to 46 retained association with Bcl-2 at a level comparable to that of wild-type G0S2 protein. However, mutations in amino acids 50 to 58 reduced interaction with Bcl-2; in particular, the G0S2 mutant derivative in which arginine-57 and aspartic acid-58 were mutated to alanine (R57A, D58A) was dramatically reduced for Bcl-2 interaction.
We then tested whether the G0S2(R57A, D58A) mutant was impaired for apoptotic activity. H1299 cells were infected with an adenovirus expressing wild-type G0S2 or the G0S2(R57A, D58A) mutant, and apoptosis was monitored by staining with Annexin V-PE. shows that the ability of G0S2(R57A, D58A) to induce apoptosis was significantly reduced compared to the wild-type protein. The reduced ability of G0S2(R57A, D58A) to interact with Bcl-2 and induce apoptosis was not a result of mislocalization of the protein, as the mutant protein retained mitochondrial localization (Supplementary Fig. S4
). These results indicate that interaction with Bcl-2 is required for G0S2 to induce apoptosis.
G0S2 promotes apoptosis by preventing Bcl-2/Bax heterodimerization
We next investigated the mechanism by which G0S2 induces apoptosis. Bcl-2 mediates its survival function, at least in part, by heterodimerizing with Bcl-2 family members, such as Bax (20
), and countering their pro-apoptotic activity. The co-immunoprecipitation experiment of shows that addition of G0S2 inhibited the ability of Bcl-2 and Bax to co-immunoprecipitate, suggesting that G0S2 inhibits Bcl-2 function by preventing the formation of protective Bcl-2/Bax heterodimeric complexes.
Figure 4 G0S2 promotes apoptosis by preventing Bcl-2/Bax heterodimerization. A, HEK293 cells were co-transfected with plasmids expressing G0S2-HA, FLAG-Bcl-2 and/or V5-Bax. Bcl-2 was immunoprecipitated with an α-FLAG antibody and the immunoprecipitate (more ...)
A prediction of this model is that in the presence of G0S2, Bcl-2 would be unable to carry out its anti-apoptotic function. To test this idea, HeLa cells stably expressing either Bcl-2 (HeLa/Bcl-2) or, as a control, vector (HeLa/Neo), were infected with Ad-G0S2 or Ad-LacZ. shows, as expected, that over-expression of Bcl-2 protected HeLa cells from UV-induced apoptosis (left panel) but failed to protect HeLa cells from G0S2-mediated apoptosis (right panel).
As an alternative experimental approach, we used a co-transfection strategy. In this experiment, apoptosis was monitored using an assay in which luciferase activity serves as a proxy for cell death (12
). H1299 cells were co–transfected with plasmids expressing luciferase and, as an apoptotic stimulus, Bax. shows, as expected, that expression of Bax alone induced cell death, as evidenced by a dramatic reduction in luciferase activity compared to that observed in cells transfected with empty vector. Co-expression of Bcl-2 and Bax prevented the reduction in luciferase activity, consistent with previous studies showing that expression of Bcl-2 can inhibit Bax-induced apoptosis (20
). Significantly, expression of G0S2 together with Bax and Bcl-2 resulted in a level of luciferase activity comparable to that obtained upon expression of Bax alone, indicating the anti-apoptotic activity of Bcl-2 was inhibited. Collectively, the results of indicate that G0S2 induces apoptosis by inhibiting the ability of Bcl-2 to form anti-apoptotic Bcl-2/Bax heterodimers.
G0S2 sensitizes primary cells to undergo apoptosis
The finding that G0S2
could induce apoptosis in transformed cells prompted us to ask whether G0S2 also had a pro-apoptotic effect in primary cells. As has been observed in previous studies (see for example, refs. 16
), we found that treatment of primary fibroblasts, such as PFFs, with TNFα did not induce apoptosis (see below). However, we tested whether TNFα treatment sensitized cells to apoptosis. Toward this end, PFFs were treated with a series of low concentrations of the DNA damaging agent camptothecin, a known inducer of apoptosis, in the presence or absence of TNFα. The results of indicate that TNFα treatment resulted in increased apoptosis, indicating that the net effect of TNFα treatment is pro-apoptotic.
Figure 5 Ectopic expression of G0S2 sensitizes primary cells to apoptosis. A, PFFs were treated with various concentrations of camptothecin in the presence or absence of TNFα, and monitored for apoptosis. B, PFFs stably expressing a non-silencing (NS) (more ...)
To determine whether G0S2 was responsible for the pro-apoptotic activity of TNFα we performed an RNA interference (RNAi) experiment. The results of indicate that shRNA-mediated knockdown of G0S2 (Supplementary Fig. S5
) abrogated the ability of TNFα to increase the level of camptothecin-induced apoptosis. Thus, G0S2
is a TNFα-inducible gene required for sensitizing PFFs to undergo apoptosis.
Finally, we asked whether ectopic expression of G0S2 was sufficient to sensitize PFFs to undergo apoptosis. PFFs were infected with Ad-G0S2 or Ad-LacZ and 24 hours later challenged with various doses of camptothecin. The results of show that ectopic expression of G0S2 resulted in a higher level of apoptosis, whereas little or no cell death was observed in control Ad-LacZ-infected cells. Thus, G0S2 is sufficient to sensitize primary cells to undergo apoptosis in response to DNA damage.
The G0S2 gene is epigenetically silenced in human cancer cell lines
Many genes that negatively regulate cell survival are epigentically silenced in human cancers through promoter hypermethylation (reviewed in ref. 23
). Notably, the G0S2
promoter contains a CpG-rich island (24
), suggesting it may undergo epigenetic silencing. To test this hypothesis, we monitored G0S2
expression in a panel of human cancer cell lines before and after treatment with the demethylating agent 5-aza-2′-deoxycytidine (AZA) and the histone deacetylase inhibitor trichostatin A (TSA). The RT-PCR results of show that G0S2
expression was induced by AZA/TSA treatment in all human cancer cell lines examined, but not in normal PFFs. Cell lines in which G0S2
was highly induced were also analyzed for G0S2 protein by immunoblotting; as expected, in all cases AZA/TSA treatment resulted in a substantial increase in G0S2 protein levels (). These results indicate that the G0S2
gene is epigenetically silenced in a variety of human cancer cell lines.
Figure 6 G0S2 is epigenetically silenced in human cancer cell lines. A, RT-PCR analysis of G0S2 expression in human cancer cell lines and PFFs, in the presence or absence of AZA/TSA treatment. B, Immunoblot analysis in a subset of cell lines. C, qRT-PCR analysis (more ...)
Several of the cell lines in which G0S2
was epigenetically repressed were derived from non-small cell lung cancers. We therefore analyzed whether G0S2
expression was down-regulated in a panel of non-small cell lung tumors. The qRT-PCR results of indicate that G0S2
expression was down-regulated greater than 5-fold in 70% of lung adenocarcinoma samples and 90% of lung squamous cell carcinoma samples analyzed. In addition, a search of the Oncomine cancer profiling database (25
) revealed that G0S2
was frequently down-regulated in a variety of other solid tumors (Supplementary Fig. S6