Syntaxin 4 Mediates NF-κB Signaling and Chemokine Ligand Expression via Specific Interaction With IκBβ

A striking feature of type 1 diabetes (T1D) etiology is that β-cell dysfunction during “pre–type 1 diabetes” precedes overt β-cell failure (1). However, it is not known how β-cell dysfunction begins. A majority of studies have implicated the loss of first-phase glucose-stimulated insulin secretion (GSIS) as the most prominent feature of early prediabetes (2–5). In the β-cell, the rate-limiting step in insulin secretion is determined by the abundance of soluble N-ethylmaleimide–sensitive fusion protein–attachment protein receptor (SNARE) proteins. The SNARE proteins syntaxin 1A (STX1A) and syntaxin 4 (STX4) facilitate GSIS, along with their partners VAMP2 or VAMP8 (6,7) and SNAP25 (8). Identifying the detailed molecular mechanisms underlying pre-T1D can reveal new strategies for preventing or reversing the progression to overt T1D.

Previously, our laboratory demonstrated that STX4 enrichment in human and mouse islets improves β-cell function and prevents streptozotocin-induced hyperglycemia and β-cell death in mice via decreased activation/nuclear localization of nuclear factor-κB (NF-κB) (9). We demonstrated that three NF-κB–dependent chemokine genes, CXCL9, CXCL10, and CXCL11, were elevated upon stimulation with inflammatory cytokines in human β-cells and suppressed when STX4 was overexpressed (9). However, the detailed mechanism(s) by which STX4 attenuates NF-κB signaling remains unknown.

During the progression to T1D, NF-κB activation in islets occurs in response to immune-mediated cytokine release. For example, proinflammatory cytokines such as IL-1β, TNFα, and IFNγ are released by macrophages and T lymphocytes, which progressively invade the islets (10). This immune invasion, resulting in insulitis, selectively destroys the insulin-producing β-cells. Studies using human and rodent islets have shown that this cytokine release during insulitis damages the β-cells (11,12), reduces insulin content (13), and leads to β-cell dysfunction and apoptosis (11,12). The cytotoxic effects of IL-1β and TNFα are thought to be largely mediated by NF-κB activation, which leads to production of nitric oxide (NO) and expression of chemokines, whereas IFNγ is thought to activate stress kinases via STAT1 (14).

In the absence of inflammatory stimuli, the NF-κB inhibitory proteins, IκBβ and IκBα, are found complexed with NF-κB in the cytoplasm (15,16). IL-1β and TNFα then trigger two events: 1) IκBα and IκBβ are phosphorylated by the kinases IKKα and IKKβ, respectively, targeting them for proteasomal degradation via the ubiquitin/proteasome pathway, and releasing free NF-κB to translocate to the nucleus (17–20), and 2) residual IκBβ:NF-κB complexes are translocated to the nucleus, after which IκBβ is released and NF-κB binds and transactivates DNA (21). TNFα stimulation causes a transient activation of NF-κB and a transient loss of the NF-κB regulatory protein IκBα, with no effect on IκBβ (22), whereas IL-1β results in degradation of both IκBα and IκBβ (21,22) in non-β cell types. In β-cells, such differential regulation of IκBα and IκBβ by individual cytokines has yet to be evaluated.

The objective of this study was to determine the mechanistic signaling events linking STX4 enrichment and downregulation of NF-κB activity in islet β-cells challenged with the proinflammatory cytokines affiliated with progression to T1D.

The rabbit polyclonal antibody against STX4 used for immunoblot of rat STX4 was custom-made as previously described (9); the rabbit polyclonal STX4 antibody used for coimmunoprecipitation was obtained from Chemicon/Millipore (cat. no. AB5330; Temecula, CA). The mouse monoclonal antibodies against tubulin (cat. no. T5168) and STX4 (used for immunoblot of human [h]STX4 [cat. no. 610439]) were purchased from Sigma-Aldrich (St. Louis, MO) and BD Biosciences (San Jose, CA), respectively. Mouse monoclonal antibodies against IκBα (cat. no. 4814), IκBβ (cat. no. 8635), and rabbit polyclonal NF-κB1 (p105/p50; cat. no. 3035) were purchased from Cell Signaling Technology (Danvers, MA). Goat anti-rabbit horseradish peroxidase (cat. no. 172-1019) and anti-mouse horseradish peroxidase (cat. no. 172-1011) secondary antibodies were purchased for immunoblotting (Bio-Rad Laboratories, Hercules, CA). Enhanced chemiluminescence and ECL Prime reagents were purchased from GE Healthcare (cat. no. RPN2106). Proinflammatory cytokines were purchased from ProSpec-Tany TechnoGene (East Brunswick, NJ): human IL-1β, cat. no. CYT208; rat IL-1β, cat. no. CYT394; human TNFα, cat. no. CYT223; rat TNFα, cat. no. CYT393; human IFNγ, cat. no. CYT206; and rat IFNγ, cat. no. CYT359. BI605906 (cat. no. 5300) was from Tocris (Minneapolis, MN), and KT5823 (cat. no. K1388), epoxomicin (cat. no. E3652), and PDTC (ammonium pyrrolidinedithiocarbamate, cat. no. P8765) were obtained from Sigma-Aldrich. hSTX4 (amino acids 1–275, cat. no. LS-G1884) and IκBβ recombinant proteins (cat. no. MBS145178) were purchased from LSBio and MyBioSource, respectively.

Human EndoC-βH1 cells obtained from Dr. Roland Stein (Vanderbilt University, Nashville, TN) were cultured as previously described (23). INS-1 832/13 cells were provided by Dr. Christopher Newgard (Duke University Medical Center, Durham, NC) and were cultured as previously described (24); these studies used cells that were between passages 52 and 68. INS-1 832/13 cells were transfected with plasmid DNA using the cytomegalovirus promoter with a 3′ flag tag (pCMV-FLAG) or pCMV-human (h)STX4 plasmid DNAs with Lipofectamine 2000 Transfection Reagent (cat. no. 11668030; Thermo Fisher Scientific, Waltham, MA). Human EndoC-βH1 cells were transduced with Ad-RIP-STX4 or Ad-RIP-control adenovirus (multiplicity of infection = 100) for 2 h, washed with PBS, and incubated in Connaught Medical Research Laboratories (CMRL)-1066 medium for 48 h. Next, the cells were treated with cytokines (10 ng/mL TNFα, 100 ng/mL IFNγ, 5 ng/mL IL-1β), as previously described (25), individually or together as a cytokine cocktail, for 1 h, 16 h, or 24 h as indicated in the text and figure legends. For evaluation of IκBα and IκBβ levels, the cells were harvested in 1% Nonidet P-40 (NP-40) lysis buffer, nuclei and cell debris pelleted and removed by microcentrifugation for 5 min, and the resulting cleared detergent lysates used for immunoblotting. To isolate nuclear fractions, the NE-PER Nuclear and Cytoplasmic Extraction Kit (cat. no. 78833; Thermo Fisher Scientific) was used. For pharmacologic studies, INS-1 832/13 cells were treated with epoxomicin (proteasome inhibitor, 10 μmol/L), KT5823 (PKG inhibitor [PKGi], 1–5 μmol/L), BI605906 (IKKβ inhibitor [IKKβi], 10 μmol/L), or PDTC (NF-κB inhibitor, 250 μmol/L) for 30 min or 1 h prior to cytokine exposure as described in the figure legends.

Human islets were obtained from the Integrated Islet Distribution Program and from the City of Hope Islet Core (donor information in Supplementary Table 1). Upon arrival, human islets were first allowed to recover in CMRL medium for 2 h, and then islets were handpicked under a dissecting microscope to yield a purity of >95%. Intact islets were then gently dissociated in TrypLE cell dissociation reagent (cat. no. 12605036; Thermo Fisher Scientific), and then dissociated cells were plated on low-adherence cell culture plates (cat. no. 3473; Corning, Corning, NY) overnight and subsequently transfected with Lipofectamine 2000 with either pCMV-FLAG or pCMV-hSTX4 for 48 h, followed by addition of cytokines as described in the figure legends. Human islets were transduced with Ad-RIP-STX4 or Ad-RIP-control adenovirus (multiplicity of infection = 100) for 1 h, washed with PBS, and incubated with CMRL-1066 medium for 48 h, as previously described (9), followed by incubation with cytokines for 1 h.

Total RNA was isolated from INS-1 832/13 cells with the QIAGEN RNeasy Plus Mini Kit (QIAGEN, Valencia, CA) and assessed with the QuantiTect SYBR Green RT-PCR kit (QIAGEN) in QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). Sequences for the primers used to detect CXCL9 and hSTX4 are provided in Supplementary Table 2. The fold change was determined relative to the control after normalization to GAPDH.

Plasma membrane (PM) fractions of β-cells were isolated as previously described (26). In brief, 40 h posttransfection, cells were harvested into homogenization buffer and disrupted by 10 strokes through a 27-gauge needle and homogenates were centrifuged at 900g to obtain a postnuclear pellet. The postnuclear pellet was further centrifuged at 113,000g to obtain an interface containing the PM fraction. The interface was centrifuged at 6,000g and purified PM fraction pelleted for resuspension in 1% NP-40 lysis buffer to ensure solubilization of membrane proteins.

Detergent lysates or purified recombinant proteins were combined with anti-STX4 antibody for 2 h at 4°C followed by incubation with Protein G Plus-Agarose for 2 h. The resultant immunoprecipitates were subjected to 12% SDS-PAGE followed by transfer to polyvinylidene fluoride membranes for immunoblotting with use of antibodies described in the legends.

Data were evaluated for statistical significance by use of ANOVA and the Bonferroni post hoc test with GraphPad Prism software, version 8.3.0.

The data sets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request. No applicable resources were generated during this study.

To determine how STX4 suppresses NF-κB nuclear translocation, we evaluated the upstream signaling events leading to NF-κB translocation. Tandem mass tag mass spectroscopy was used to identify changes in protein levels in response to STX4 overexpression in human islet β-cells (via transduction with Ad-RIP-STX4). A top hit identified in the STX4-overexpressing β-cells, relative to that in the control cells, was IκBβ (Supplementary Table 3). Since islets are comprised of many cell types beyond the β-cell, we next evaluated IκBβ levels in cytokine-induced STX4-overexpressing human EndoC-βH1 cells, a surrogate β-cell model with phenotypic characteristics that partially resemble those of primary human islet β-cells. IκBβ levels were found to also be elevated relative to the cytokine-induced control cells (Fig. 1A). This was not specific to human β-cells, as we further observed STX4 overexpression to correlate with elevated IκBβ levels in the widely used rat INS-1 832/13 β-cell line (Fig. 1B). This occurred posttranscriptionally, since mRNA levels of IκBβ were unchanged with STX4 overexpression (Fig. 1B,i); IκBα mRNA was similarly unchanged (Fig. 1B,ii). Rat INS-1 832/13 cells are easily transfected and propagated and responsive to proinflammatory cytokines and, hence, are used in subsequent studies. Activation of NF-κB requires IκB phosphorylation (Ser32/Ser36 in IκBα, Ser19/Ser23 in IκBβ) followed by polyubiquitination, which is carried out by SCFβTrCP-type E3 ligases (27–30). This in turn unmasks the nuclear localization signal, which facilitates the translocation of NF-κB to the nuclei. As anticipated, the proteasomal inhibitor epoxomicin ablated the cytokine-induced loss of IκBβ (Fig. 1C,i), indicating that the cytokine-induced IκBβ loss in INS-1 832/13 cells is due to proteasomal degradation. While cytokine-induced loss of IκBα was also apparent, it was not attenuated by epoxomicin at this time point (Fig. 1C ii), consistent with the known differential temporal regulation of these proteins.

To clarify the effects of STX4 overexpression on the response to cytokine stimulation, we evaluated the levels of IκBβ and IκBα in response to each cytokine individually. In dispersed human islet cells, STX4 overexpression inhibited the cytokine-induced loss of IκBβ levels (Fig. 2A and B) but not IκBα levels (Fig. 2A and C) in response to IL-1β. STX4 overexpression levels of 1.5- to 2.0-fold were sufficient to protect IκBβ (Fig. 2D). In INS-1 832/13 β-cells, we observed that IL-1β and TNFα, when delivered to the cells individually, depleted IκBβ and IκBα levels (Fig. 3). STX4 attenuated the loss of IκBβ (Fig. 3A and B) but not of IκBα (Fig. 3D and E). However, IFNγ did not deplete either IκBβ or IκBα (Fig. 3C and F). These data indicate that the effect of STX4 is selective for IL-1β and TNFα signaling, which drives activation of NF-κB, whereas IFNγ triggers STAT1 activation.

In non-β cells, endogenous STX4 is destabilized by phosphorylation of Ser78 (S78) in its Hb helix (Fig. 4A), resulting in the proteasome-dependent degradation of STX4 (31). Consistent with this finding, epoxomicin-treated β-cells contained elevated levels of STX4 (Supplementary Fig. 1). For determination of the role of Ser78 in STX4’s impact on IκBβ and NF-κB signaling, we evaluated the relative capacity of STX4 (wild type [WT]) versus the phosphorylation-resistant mutant (serine to alanine mutant, S78A) enrichment to attenuate IκBβ nuclear localization and cytokine induction of the chemokine ligand CXCL9 in β-cells. Human islets overexpressing STX4 using our Ad-RIP-STX4 adenovirus in the β-cells (driven by rat insulin promoter, RIP, as described previously [9]) showed reduced IκBβ in the nuclear fraction (Fig. 4B); INS-1 832/13 β-cells transfected to express STX4 (WT) showed the same ameliorating effect (Fig. 4C). Notably, the impact of the STX4-S78A mutant is more pronounced than STX4-WT at reducing the nuclear accumulation of IκBβ; p50-NF-κB nuclear accumulation was fully blocked by WT and S78A (Fig. 4C). Both STX4-WT and STX4-S78A overexpression attenuated CXCL9 mRNA expression induced by IL-1β and TNFα (Fig. 4D and E), although STX4-S78A was not significantly different from STX4-WT. Similar results were observed in non-cytokine-treated cells (Supplementary Fig. 2). Neither STX4-WT nor -S78A affected IFNγ-induced CXCL9 (Fig. 4F). Since cytokine-induced NF-κB nuclear localization can induce production of NO via activation of the iNOS promoter (iNOS promoter contains a consensus NF-κB binding site [32]), we determined whether STX4 overexpression attenuates cytokine-induced NO release. Although the oxidant scavenger PDTC attenuated the effects of cytokine stimulation, as expected, no attenuation by STX4 overexpression was detected (Supplementary Fig. 3). These data suggest that STX4 protects β-cells against NF-κB–induced chemokine expression in response to IL-1β or TNFα stimulation but has no impact on NF-κB–induced NO production (33).

For determination of the relative selectivity of STX4-S78A to attenuate the loss of IκBβ versus IκBα in response to individual cytokines, INS-1 832/13 cells were transiently transfected with STX4-WT- or STX4-S78A mutant and challenged with IL-1β, TNFα, or IFNγ. Consistent with its enhanced ability to prevent the nuclear translocation of IκBβ, STX4-S78A was twice as effective at protecting IκBβ from IL-1β–triggered loss compared with STX4-WT (Fig. 5A and B). Like STX4-WT, STX4-S78A failed to prevent the IL-1β–triggered loss of IκBα (Fig. 5A and C). Similar results were seen for TNFα exposure (Fig. 5D–F), and as anticipated, IFNγ did not induce significant losses of IκBβ or IκBα levels (Supplementary Fig. 4).

We next sought to identify the kinase involved in the signaling cascade linking STX4 and IκBβ. Protein kinase G (PKG) has been implicated in the Ser78 phosphorylation of STX4, although not yet definitively demonstrated (31). To address this, we used the PKGi KT5823 to test whether PKG inhibition mimics the phenotype of the STX4-S78A mutant in INS-1 832/13 cells. However, PKGi did not protect IκBβ against cytokine-induced loss in β-cells (Fig. 6A); it also failed to protect IκBα (Fig. 6C). Alternatively, we tested the impact of inhibiting IKKβ. IKKβ is known as an upstream kinase whose phosphorylation of IκBβ marks it for subsequent degradation. This serine-specific IκB kinase subunit (IKKβ), along with another subunit named IKKα, are part of the IKK complex. IKKα and IKKβ associate as homodimers (α + α or β + β) or heterodimers (α + β) and are bound to the regulatory subunit, IKKγ (34–36). As anticipated, the IKKβi BI605906 protected IκBβ from degradation (Fig. 6B), akin to the level of protection afforded by STX4-S78A; by contrast, IKKβi failed to protect IκBα (Fig. 6D). These data validate that IKKβ is a kinase for the phosphorylation and subsequent proteasomal degradation of IκBβ in β-cells and prompted the question as to whether it might be also be a kinase for STX4.

For determination of the relationship among IKKβ, PKG, STX4, and CXCL9 expression, INS-1 832/13 cells were exposed to cytokine cocktail for 24 h in the presence of IKKβi, PKGi, or PDTC. Cytokine-induced CXCL9 levels were unchanged in the presence of PKGi (Fig. 7A); the oxidant scavenger PDTC was used as a control to show the reversibility of the cytokine-induced CXCL9 expression in the same cells. By contrast, CXCL9 levels were significantly attenuated by IKKβi (Fig. 7B), suggesting specificity of the effect to IKKβ. Cytokine exposure reduces STX4 levels in nondiabetic human islets, and STX4 levels are low in human islets from donors with type 2 diabetes relative to donors without diabetes (7), although the underlying mechanism remains unclear. Here we show that cytokine cocktail exposure also reduced STX4 levels in INS-1 832/13 cells and that this loss could be attenuated by IKKβi (Fig. 7C). Given the similar result that was observed in cells exposed to cytokines and cotreated with epoxomicin (Supplementary Fig. 1), these data suggest that IKKβi may protect STX4 from proteasomal degradation.

Given the ability of IKKβi to preserve STX4 and IκBβ from cytokine-induced degradation, and STX4-S78A to protect IκBβ to the degree afforded by IKKβi, we interrogated the possibility that STX4 associated with IκBβ in β-cells. Human islets transduced with Ad-RIP-control or Ad-RIP-STX4 to express STX4 selectively in β-cells were harvested, and the resultant detergent cell lysates then were used in immunoprecipitation reactions including anti-STX4 antibody or IgG control. Both endogenous and overexpressed STX4 coimmunoprecipitated IκBβ (Fig. 8A). For elimination of the endogenous STX4 component for evaluation of IκBβ association with STX4, INS-1 832/13 rat β-cells were transfected with hSTX4 constructs and a human selective anti-STX4 antibody used for coimmunoprecipitation. Rodent INS-1 832/13 cells transfected with vector only (control) showed no hSTX4 immunoprecipitation (Fig. 8B), validating the preference of the antibody for hSTX4. While the WT version of STX4 did coimmunoprecipitate IκBβ, STX4-S78A showed significantly greater IκBβ association (Fig. 8Bi).

The dominant activated form of NF-κB is comprised of the p50:p65 heterodimer. Since STX4 was determined not to coimmunoprecipitate with the p65 subunit of NF-κB in β-cells (9) (Fig. 8B), we assessed for STX4 interaction with the p50 subunit of NF-κB. Indeed, p50-NF-κB was coimmunoprecipitated by STX4-WT, and similar to the observation of STX4 binding to IκBβ, STX4-S78A coimmunoprecipitated significantly more p50-NF-κB than did STX4-WT (Fig. 8B,ii). Furthermore, IκBβ was coimmunoprecipitated by STX4-WT and STX4-S78A from PM fractions of these cells, indicating the subcellular location of this complex (Fig. 8C). With use of purified recombinant hSTX4 (amino acids 1–275) and IκBβ proteins in interaction assays, STX4 was found to coimmunoprecipitate IκBβ, suggestive of a direct protein-protein interaction (Fig. 8D and Supplementary Fig. 6). These data support a model whereby STX4 binds to IκBβ, preventing the phosphorylation and proteasomal degradation of IκBβ, as well as preventing its translocation with p50-NF-κB into the nucleus.

STX4 has been shown to prevent β-cell inflammation in mice in vivo caused by multiple low-dose streptozotocin administrations (9,37), suggesting STX4 to be candidate factor for intervention, although the mechanism has remained elusive. We demonstrate herein that STX4 mediates NF-κB signaling via regulation of IκBβ and p50-NF-κB. STX4 attenuated the cytokine-induced proteasomal degradation of IκBβ, which likely accounts for, at least in part, the observed reduction in the translocation and transactivation of NF-κB (9). This finding fills the gap in our rudimentary understanding of how STX4 overexpression in human islet β-cells prevents the expression of dominant chemokine ligand genes (9) that are expressed in the islets of T1D humans. Unveiling this new mechanism provides novel avenues for intervention.

How does STX4, a PM-localized exocytosis protein, suppress the NF-κB inflammatory cascade? We originally expected the tandem mass tag mass spectrometry studies to reveal changes in the levels of vesicle trafficking proteins, based on the known role for STX4 in exocytosis. In line with this concept, STX3, also a PM-localized SNARE protein, is involved in trafficking immune mediators in dendritic cells (38,39). STX3 and STX4 have been implicated in facilitating vesicle fusion to promote antibody secretion by plasma cells. However, we found that STX4 in β-cells, also integrated as part of the inflammatory cascade, yet rather as a substrate for IKKβ, in tandem with IκBβ, to selectively modulate IκBβ:NF-κB complexes and downstream signaling.

In addition to phosphorylation of STX4 at S78, STX4 can also be modified by S-nitrosylation in response to cytokine stress (40). The order of addition of these posttranslational modifications, or if they can co-occur on the same STX4 protein, remains to be determined. Nevertheless, STX4-S78A was resistant to cytokine-induced proteasomal degradation, and this has not been reported for nitrosylation-resistant mutants of STX4 (40). STX1 has also been reported to be nitrosylated, yet no affiliated observation of degradation was reported (41). These data are consistent with the concept that Ser78 modification of STX4 is key to our understanding of how and why STX4 levels are substantially reduced in diabetic β-cells and in other cell types subjected to inflammatory stress (7). Overexpression of STX4 increases the pool of available STX4 to compensate for the loss of endogenous STX4 under stress conditions; a nonphosphorylatable form of STX4 (S78A) is more effective at protecting IκBβ. Oddly, STX4-WT and -S78A were equivalently capable of reducing the escalation of CXCL9 induced by IL-1β– or TNFα-induced NF-κB signaling cascade. One possibility is that p65-NF-κB homodimers contributed to the CXCL9 expression: with neither STX4-WT nor -S78A associating with p65-NF-κB, it remains possible that the p65-NF-κB homodimers contributed to the CXCL9 signal, obscuring the ability to detect differences between STX4-WT and -S78A on CXCL9.

The IκB proteins have well-described modes of regulation in the context of NF-κB signaling, and we were surprised to discover a novel mode of IκBβ regulation via STX4. For example, the IκB negative-feedback loop occurs following the NF-κB–mediated transcription of the NFKBIA gene that encodes IκBα, which then generates newly synthesized IκBα that enters the nucleus, dissociates NF-κB from the DNA, and escorts it back to the cytoplasm (42). Such a negative-feedback regulation is also revealed for IκBβ and IκBε, albeit with different kinetics of degradation and resynthesis (42). In addition, IκBβ and IκBα proteins regulate the differential compartmental localization (nuclei vs. cytoplasm) of NF-κB. NF-κB/IκB complexes are sequestered in the cytoplasm because the IκB proteins will otherwise mask the nuclear localization sequence of NF-κB protein through direct protein-protein interactions (15–22), thereby preventing NF-κB from binding to the DNA. IκBα and IκBβ share a characteristic ankyrin repeat motif that is required for their interaction with the Rel homology region of NF-κB that includes DNA binding and dimerization domains and a nuclear localization sequence (15–22). Since our coimmunoprecipitation protocol excluded nuclear material from the lysates, and because STX4 is most abundant at the PM in β-cells (26,43), it is speculated that STX4 interacts with IκBβ and the p50-NF-κB in a manner distinct from how IκBβ associates with the NF-κB (p65:p50) complex. STX4 is renowned as an unusual STX4 isoform, given that it is the only one known to interact directly with F-actin (44) and contains an α-spectrin–like domain in its far N-terminus through which it binds to the actin-severing protein gelsolin (45). These interaction interfaces were identified with use of truncation mutants and peptides derived from STX4. Future studies of STX4 associations in macromolecular complexes with IKKβ, IκBβ, and p50-NF-κB will be required to discern the dynamics of association and dissociation in response to cytokines. In addition, it will be important to determine what features of IκBβ, which are not present in IκBα, confer selective regulation by STX4. For example, although both have the ankyrin repeat domain, IκBβ possesses fewer flexible residues than IκBα—indeed, the fewest of all IκB family members (46). This unique feature of IκBβ is considered to be a primary reason for its differential binding partner specificities (47) and may be important for STX4 interaction.

Our data suggest that the initial STX4 phosphorylation and proteasomal targeting in β-cells may be initiated by IKKβ rather than by PKG. PKG was first evaluated because NF-κB translocation to the nucleus would transactivate iNOS and produce NO, thereby activating guanylate cyclase, the upstream activator of PKG (48). However, despite evidence to show that PKG is activated with cytokine exposure in β-cells, and that this is inhibited by PKGi treatment (Supplementary Fig. 5A), PKGi had no effect on NO release from β-cells (Supplementary Fig. 5B), suggesting that PKG activation is downstream of NO release. While IKKβi had no impact on cytokine-induced NO release (Supplementary Fig. 5C), IKKβi did ablate cytokine-induced CXCL9 expression, whereas PKGi failed to ablate. IKKβi also fully protected STX4 levels from cytokine-induced degradation. Taken together, our findings suggest exquisite specificity of this STX4-regulated IκBβ:NF-κB complex to suppress the proinflammatory cytokine–induced chemokine ligand expression, while the NO release pathway remains intact.

The differential effect of STX4 on chemokine ligand versus NO release may be related to the apparent preferential binding of STX4 with a particular subset of NF-κB complexes. The most abundant activated form of NF-κB is the p50:p65 heterodimer, and it is implicated as the predominant of the NF-κB complexes capable of specific suppression of CXCL9, CXCL10, and CXCL11 in human keratinocytes (49). By contrast, p65:p65 and p50:p50 homodimers directly bind to the NOS2 promoter to regulate iNOS expression in myeloid cells (50). Given that STX4 coimmunoprecipitates with p50 but not p65, and yet STX4 enrichment impacts chemokine ligand expression and not NO production, these data suggest that STX4 may preferentially target the NF-κB p50:p65 heterodimer subset. Furthermore, strategies to dampen IKKβ:IκBβ-mediated NF-κB signaling using STX4 intervention may benefit from mutation of Ser78 to Ala78 in STX4 to provide longer-lasting STX4-mediated effects.

In summary, STX4 plays an unexpected role in suppressing a cytokine-induced NF-κB signaling cascade, which favors the preservation of islet β-cells. Expression of STX4 in human islet β-cells was correlated with a dampening of proinflammatory cytokine-induced IKKβ activation to trigger IκBβ proteasomal degradation, coordinate with a decrease of IκBβ:NF-κB complexes in the nuclear compartment and reduced expression of the T1D-associated chemokine ligand CXCL9. Given the specificity of STX4 regulation for IκBβ, and not IκBα, potentially via IKKβ kinase activity, STX4 has the potential to selectively dampen an immune storm in β-cells without global inactivation of the immune system in all cells. STX4 is also a bona fide SNARE protein that when overexpressed exerts enhanced β-cell function by docking/fusing more insulin granules. Since poorly functioning β-cells are targeted for immune attack, it is possible that STX4 is working in direct events for both β-cell function and β-cell protection.

Acknowledgments. The authors thank Dr. Arthur D. Riggs (City of Hope) for his valuable insights in this project, as well as Drs. Cristiana Perotta (University of Milan) for the STX4-S78A mutant and Roland Stein (Vanderbilt University) for provision of the EndoC-βH1 cell stock. The authors thank Dr. Jinhee Hwang (City of Hope) for assistance with the artwork in Fig. 4A. Nancy Linford (Linford Biomedical Communications, LLC) provided editing assistance.

Funding. This study was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (DK067912, DK112917, and DK102233 to D.C.T.); JDRF (17-2013-454 to D.C.T.); and the Wanek Project to Cure Type 1 Diabetes at the City of Hope. Human islets were supplied by the Southern California Islet Cell Resource Center (City of Hope) and by the Integrated Islet Distribution Program. Research reported in this publication also includes work performed in the City of Hope Islet Core, the Integrated Genomics Core, the Proteomics Core, and the Light Microscopy Core, supported by the National Cancer Institute, National Institutes of Health, under award number P30CA33572.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. R.V., E.O., M.A., and D.C.B. researched data, contributed to discussion, and reviewed and edited the manuscript. R.V. and D.C.T. conceived of the study, contributed to discussion, and wrote, reviewed, and edited the manuscript. All authors read and approved the final version of the manuscript. D.C.T. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.