Although we had previously shown that exogenous TG2 decreases HIF activity in human cell lines, in this study we demonstrate for the first time that endogenous TG2 also suppresses HIF signaling in rat primary neurons and in a human neuroblastoma cell line giving strong physiological relevance to our findings. We also show that TG2 must localize to the nucleus to suppress HIF activity and that TG2 is recruited to the HRE preinitiation complex. Further, nuclear localization, but not activity, of TG2 is essential for protection against OGD-induced cell death. In addition to these findings, we demonstrate that the catalytic domain of TG2 is required for its interaction with HIF1β; however this domain is neither necessary nor sufficient for TG2 to suppress HIF activity. On the other hand, the C-terminal β-barrel 2 domain does appear to be essential for TG2 to suppress HIF activity. These data suggest that the binding of HIF1β and TG2 and the ability of TG2 to suppress HIF activity are independent events.
HIF is composed of two subunits: a constitutively expressed β subunit (HIF1β), and the oxygen sensing HIFα subunit [52
]. The HIF1α and HIF2α are structurally similar in their DNA binding and dimerization domains but differ in their transactivation domains and regulate unique target genes [53
]. In our previous report we demonstrated that overexpressed TG2 suppressed both HIF1 and HIF2 dependent transcription [7
]. In this study, we examined the effects of TG2 on expression levels of both HIF1 and HIF2 targets in SH-SY5Y cells. ENO1 [53
], and BNIP3 [55
] are primarily targets for HIF1α, while EPO is preferentially regulated by HIF2α [56
]. VEGF can be regulated by both HIF1α and HIF2α [53
]. EPO and VEGF were highly upregulated in response to hypoxia, whereas ENO1 and BNIP3 were upregulated to a lesser extent. These results suggest that HIF2α may play a more prominent role in HIF signaling in SH-SY5Y cells, which is a neuroblastoma cell line. This is in line with a previous report which demonstrated the relevance of HIF2α in neuroblastoma models [57
]. Nonetheless the hypoxiainduced expression of all HIF responsive genes we have examined thus far significantly increased when TG2 is knocked down. Overall, these results clearly show that endogenous TG2 modulates the transcription of HIF target genes. We examined both prodeath (BNIP3) and prosurvival (EPO and VEGF) genes in this study in order to see if TG2 has a selective effect. From our limited set of genes, we could not detect any selective suppression which could tip the balance of survival and death in one direction; therefore the biological significance of TG2 mediated HIF suppression is not clear in the context of ischemic cell death.
The most straightforward mechanism for this suppression would be direct regulation of the transcriptional machinery forming on the HRE bearing promoters. The results of the ChIP assay () clearly show that endogenous TG2 is recruited to the HRE spanning portions of HIF target genes in response to hypoxia in SH-SY5Y cells. The recruitment was only observed in hypoxic conditions, which strongly indicates that this recruitment has functional outcomes. Furthermore, we failed to detect any TG2 on the Noxa promoter in either hypoxic or normoxic conditions, which argues for specificity and functionality. An important question is whether the conformation of TG2 is important for the recruitment to promoters. Although we lack direct evidence, the likely answer is no; the deletion constructs (except the ones that lacked the β-barrel2 domain), which without question lack the native open or closed conformation of the full length protein, still suppressed HIF activity. If the recruitment to the promoter is necessary for HRE suppression, this observation clearly shows that β-barrel2, not the conformation of the protein is crucial in this phenomenon.
The presence of TG2 in preinitiation complexes has also been shown in other models. For example, in the presence of mutant huntingtin, TG2 localizes to the promoters for cytochrome c and PGC-1α [32
]. Although TG2 is at HRE containing promoters, the exact mechanism by which TG2 is repressing HIF activity is still being investigated. At this point we speculate that TG2 is either mediating the recruitment of certain corepressors or preventing the recruitment of certain coactivators. If TG2 is suppressing HIF activity at the promoter of HIF responsive genes, an inverse correlation between the nuclear TG2 amounts and the HIF activity should be expected. The results shown in strongly indicate that there is indeed an inverse correlation. Whether there is a relationship between the TG2 - HIF1β interaction and TG2-mediated HIF suppression has been a fundamental question for our studies. To this end, we have demonstrated that the HIF1β interacting domain of TG2 is within its catalytic core domain (). Further, our results suggest that the interaction between HIF1β and TG2 is not required for TG2 to suppress HIF-dependent transcription (). The failure of TG2 to influence another HIF1β dependent pathway, namely xenobiotic responsive transcription (Figure C4
), supports the conclusion that the TG2 - HIF1β interaction is not essential per se
for transcriptional repression. However, this interaction might have other physiological roles. For example we have preliminary evidence suggesting that this interaction might be important for the nuclear accumulation of TG2 in hypoxia (data not shown). The question whether HIF1β binding affects in situ
transamidating activity of TG2 is an interesting one. However, given that the transamidating activity of TG2 is not crucial for HRE suppression, this question was not central to our studies and we did not examine this possibility.
TG2 can differentially affect a signaling process in a cell type specific manner. Therefore it is crucial to exercise caution when interpreting data obtained through the use of more than one cell line. In this study we have used several cell models and the amount of nuclear TG2 varies greatly between the cell lines used. However if we manipulate nuclear TG2 amounts using genetic approaches, the outcome is very similar. This observation suggests that the mechanism of HRE suppression by TG2 is conserved among cell types and the amount of TG2 in the nucleus is the variable that determines whether TG2 suppresses HIF signaling or not. This common response among various cell types is informative in this regard. Therefore, using more than one cell line has advantages, as well as drawbacks. In a similar trade-off situation, we had to choose whether to manipulate the nuclear TG2 amounts through mutating the proposed endogenous NLS residues in the protein or by adding exogenous NLS and NES signals. Both approaches have its merits and drawbacks; however, mutating the endogenous NLS sites may have secondary unforeseeable effects on HIF signaling other than changing the localization of the protein. Furthermore, the presence of endogenous NLS sites in TG2 have not been unequivocally confirmed experimentally [32
]. Therefore, the nuclear TG2 amounts were manipulated by exogenous tags in this study. It should be noted that this approach does not allow us to titrate the TG2 amounts in the nucleus; rather the use of NLS and NES tagged constructs allow us to relatively increase or decrease the nuclear TG2 amounts compared to untagged constructs. Since we lack the tools to quantitatively manipulate nuclear TG2 amounts, we are unable to test whether there is a linear relationship between nuclear TG2 amounts and survival in OGD.
The depletion of endogenous TG2 not only upregulated HIF target genes, but also decreased survival of SH-SY5Y cells in response to OGD. However it still needs to be established whether or not there is a causal relationship between the TG2-mediated HIF suppression and TG2-mediated improvement in survival. Considering that TG2 modulates many aspects of cell survival/death processes [19
], it would not be surprising if they were not directly linked. More work is required to clarify the mechanisms by which TG2 protects cells against ischemic cell death. However the data we obtained using HEK-293A cells suggest that the protection conferred by TG2 in OGD and the suppression of HIF activity are separate phenomena. Both wild type and W241A-TG2 with NLS-tags were equally effective in suppressing HRE activity in HEK-293A cells (), although, the W241A mutant version, and not the wild type, protected the cells in OGD (). More importantly, NLS-V5-TG2ΔCAT and NLS-V5-TG2-CAT alone failed to diminish or facilitate OGD-induced cell death in HEK-293A cells (). However, their effect on HIF signaling was dramatically different: while NLS-V5-TG2ΔCAT strongly suppressed HIF activity, NLS-V5-TG2-CAT alone activated HIF (). This result indicates that the two events are not likely to be directly linked. Nonetheless, the extent of the present data does not allow us to rule out the possibility that TG2-dependent suppression of HIF is partly contributing to TG2-mediated protection in OGD.
Although some TG2 constructs protected HEK-293A cells against OGD-induced cell death, two of them, namely non-nuclear R580A () and non-nuclear V5-TG2 CAT (), facilitated it. The R580 residue is located at the center of the guanine nucleotide binding site and has been shown to be indispensable for binding [19
]. Mutation of this residue, therefore, has two important outcomes: the mutant is more likely to exist in an open conformation (and hence certain domains which are buried in the closed conformation are exposed) and it exhibits higher transamidating activity inside the cell [19
]. Therefore, the facilitation of OGD-induced cell death by R580A () could be attributed either to this mutant’s conformation and/or its increased intracellular transamidase activity. Interestingly, the expression of the catalytic core domain without the NLS tag also facilitated OGD-induced cell death (). The catalytic core domain alone has no transamidating activity (), but it might present a potentially “toxic” region of the molecule due to the deletion of the N-terminal and C-terminal domains. Unless R580A-TG2 and the catalytic core domain exert their toxicities through different mechanisms; these data suggest that the conformation, not the disinhibited transamidating activity, of TG2 is facilitating the ODG-induced cell death. This conclusion is also supported by our previous findings which showed that the open conformation of TG2 exacerbated OGD-induced cell death in an immortalized striatal cell model [23