In this study, we investigated potential cell biological and functional effects of IRF5 exon 6 variants. Recent large genetic association studies defined risk and protective IRF5-based haplotypes for human systemic lupus [4
], several of which encode structural variants within IRF5 exon 6. Expression of variant IRF5 isoforms correlate with elevated disease-risk (), with protection from disease, or with neutrality. Although at least 10 IRF5 isoforms have been described in human cells [20
], the four IRF5 isoforms studied here were selected because they differ only in the presence or absence of short insertions within exon 6 and because they may influence the risk of SLE [19
]. Our findings suggest that the alternative splice site-specified IRF5 variant (SV-16) is more potent than the polymorphic exon 6 in/del (in/del-10) for IRF5-driven resistance to apoptosis and promotion of cytokine release. However, in/del-10 co-expression appears capable of neutralizing these effects of SV-16.
Although IRF5 has been characterized as an inducer of Type I Interferon-driven genes, additional key reported functions of IRF5 include the regulation of apoptosis induction [7
] and promotion of pro-inflammatory cytokine secretion [22
]. Nuclear localization sequences within IRF5 are critical for movement of phosphorylated IRF5 from cytoplasm to nucleus, and for IRF5 transactivating capacity [22
], strongly suggesting that major IRF5 functional properties depend upon nuclear translocation.
Our finding that IRF5 variants can act as negative regulators of apoptosis is surprising, since other reports suggest that IRF5 promotes cell death induced by DNA damaging agents [2
]. Potential explanations for the discrepancy between our findings and published reports include cell-type specificity of the apoptotic response and variation in apoptosis-inducing stimuli employed. Barnes et al found that overexpression of human IRF5 in the BJAB lymphoma cell line caused cell-cycle arrest and spontaneous apoptosis [9
]. However, it is not clear whether these overexpression studies employed IRF5 variants carrying or lacking the SV-16 exon 6 regions. Later, Hu et al reported enhanced apoptosis of p53-deficient tumor cells after overexpression of hIRF5 V3 (lacking both SV-16 and in/del-10) [6
]. Our findings are consistent with the latter, since overexpression of hIRF5 V10 (like V3, harboring neither in/del-10 nor SV-16) failed to impair high rates of CPT-11-induced apoptosis observed in IRF5-deficient MEFs (). While Yanai et al studied IRF5−/−
-MEFs, similar to the cells used in experiments reported here, they examined apoptosis in response to viral infection and to gamma irradiation in the presence of overexpressed HA-Ras [2
], rather than chemotherapy (irinotecan)-induced death. Both of the former modes of apoptosis may occur through activation of death receptor signaling cascades, which Hu et al have recently demonstrated are positively regulated by IRF5 [7
]. Thus it is formally possible that IRF5 might serve as an inhibitor of apoptosis induced by topoisomerase I inhibition (irinotecan), while also having ability to promote cell death after DNA damage (such as would occur after irradiation). Our results suggest that the IRF5 SV-16 is required for the protective effect of hIRF5 expression from cell death in MEFs, although the mechanism whereby co-expression of in/del-10 abrogates SV-16 effects remains unclear. Further studies examining the role of IRF5 exon 6 SV-16 and in/del-10 in the apoptosis response of primary innate immune cells to stimuli like viral infection will clarify whether and how much IRF5 exon 6 variants regulate apoptosis relevant to immune-mediated processes.
Overexpresssed IRF5 associates with transactivation of proapoptotic genes such as Bax, Bak and p21 [9
]. This observation is commensurate with our finding that protection from CPT-11-induced apoptosis correlates with reduced IRF5 nuclear translocation (observed in experiments with IRF5−/−
-MEFs expressing the V1 and V2 IRF5 isoforms, , ). The mechanism whereby either IRF5 exon 6 SV-16 or in/del-10 modulates nuclear translocation is not known. A conformational change associated with presence of either insertion may hinder transporter protein access to either of two IRF5 nuclear localization sequences that dynamically regulate IRF5 shuttling between nucleus and cytoplasm [13
]. Lin et al have documented that constitutive cytoplasmic localization of IRF5 depends upon dominant function of an IRF5 nuclear export sequence (NES) [23
]. Interestingly, the N-terminal portion of exon 6 harboring both in/del-10 and SV-16 is located immediately carboxy-terminal to the IRF5 NES [23
]. Thus, the presence of either exon 6 SV-16 or in/del-10 could enhance access of the nuclear exporting transport protein CRM1 to IRF5, and sustain IRF5 presence in the cytoplasm. The observations that the presence of both exon 6 sv-106 and in/del-10, or of neither, is associated with enhanced nuclear localization (), suggest that regulation of IRF5 subcellular localization is multifactorial and complex.
Data from IRF5 structure/function analyses with respect to promotion of cytokine secretion are limited. Mancl et al showed that overexpression of hIRF5 lacking both exon 6 SV-16 and in/del-10 (V3) had diminished capacity to activate a reporter element derived from the IFNα gene promoter [20
]. Recently, Yang et al showed that IRF5 DNA binding capacity is required for LPS-induced IL-6 secretion by a macrophage cell line [24
]. The role of IRF5 exon 6 structural variants in regulating TLR-driven cytokine production has not been examined, however. Our finding that expression of IRF5 carrying the SV-16, but not in/del-10 (V1), results in enhanced LPS-induced IL-6 production in cell culture () may be related to augmentation of reported IRF5 interaction with molecular mediators (e.g. TRAF6, MyD88) of the TLR signaling pathway leading to proinflammatory cytokine production [1
]. Biochemical studies examining the IRF5 structural requirements for protein-protein associations with TLR pathway mediators will shed further light on this potential mechanism.
Expression of an IRF5 exon 6 that is associated with increased risk of SLE (V2; absent SV-16, present in/del-10) correlated with weak IRF5 capacity to prevent CPT-11-induced apoptosis, and no ability to augment TLR-dependent IL-6 production in MEFs (). These findings of comparatively weaker functional effects of in/del-10 relative to SV-16 are compatible with findings of recent genetic studies suggesting that expression of the in/del-10 exon 6 motif in isolation is not functional or deleterious with respect to human SLE risk [5
]. These studies suggest instead that in/del-10 may be in linkage disequilibrium with additional disease risk-modifying polymorphisms of the IRF5 gene. Indeed, a 5 base pair in/del sequence element located 5’ to the first untranslated exon of IRF5 appears to explain the apparent contributions of both in/del-10 and SV-16 to increased risk of SLE [5