Absence of GdX/UBL4A protects against inflammatory bowel diseases by regulating NF-κB signaling in DCs and macrophages

Nuclear factor-kappa B (NF-κB) activation is critical for innate immune responses. Here we report that the UBL4A (Ubiquitin-like protein 4A, also named GdX) enhances dendritic cells (DCs) and macrophages (Mφ)-mediated innate immune defenses by positively regulating NF-κB signaling. GdX-deficient mice were resistant to LPS-induced endotoxin shock and DSS-induced colitis. DC- or Mφ-specific GdX-deficient mice displayed alleviated mucosal inflammation, and the production of pro-inflammatory cytokines by GdX-deficient DCs and Mφ was reduced. Mechanistically, we found that PTPN2 (TC45) and PP2A form a complex with RelA (p65) to mediate its dephosphorylation whereas GdX interrupts the TC45/PP2A/p65 complex formation and restrict p65 dephosphorylation by trapping TC45. Our study provides a mechanism by which NF-κB signaling is positively regulated by an adaptor protein GdX in DC or Mφ to maintain the innate immune response. Targeting GdX could be a strategy to reduce over-activated immune response in inflammatory diseases.


46
NF-κB signaling is important in innate immune responses and is mediated by innate suggest that GdX directly regulates p65 rather than the upstream components of the 157 NF-κB signaling pathway.

158
To investigate the effect of GdX on p65, the splenocytes from GdX -/Y mice, 159 challenged with LPS for 16 h, were collected for western blot analyses. The results 160 showed that p65 serine phosphorylation was dramatically decreased in the splenocytes 161 obtained from GdX -/Y mice, compared with that in GdX +/Y mice ( Figure 2E). We then interacted only with TC45 among these kinases and phosphatases ( Figure 3A), suggesting 183 TC45 might associate with the regulation of p65 phosphorylation by GdX. 184 We then questioned whether TC45 could directly interact with p65. Co-IP analyses 185 revealed that Flag-tagged p65 was able to precipitate with HA-tagged TC45 ( Figure 3B Figure 3C). In addition, purified proteins GST-TC45 pulled down 189 Flag-p65, suggesting a direct interaction of TC45 with p65 ( Figure 3D). Furthermore, the 190 interaction of p65 and TC45 was enhanced after TNF-α stimulation in both the 191 over-expression ( Figure 3E) and endogenous conditions ( Figure 3F). Interestingly,when 192 Myc-GdX, HA-TC45 and Flag-p65 were co-expressed together, we observed that To reveal whether GdX-impaired interaction of TC45 and p65 is due to the 207 interaction of GdX with TC45, we recruited a mutant GdX, GdX(L29P), which lacks the 208 ability to interact with TC45 (Wang et al., 2014). IP experiments demonstrated that 209 GdX(L29P) failed to disrupt the interaction of TC45 and p65 ( Figure 3I). Therefore, we 210 conclude that GdX interacts with TC45 and then disrupts the interaction of TC45 with 211 p65. 212 Furthermore, we performed a molecular docking analysis to show the detailed 213 interaction of TC45 with p65 ( Figure 3J) and GdX ( Figure 3K). The results showed that 214 11 the surface of TC45 for the interaction with GdX is the same as it interacts with p65 215 although GdX interacts on a slight shift site. In particular, residue F183 in TC45 faces to 216 the interacting sites of p65 and GdX ( Figure 3J and 3K). Therefore, the interaction of 217 GdX with TC45 occupies F183 and disrupts the interaction of p65 with TC45 ( Figure   218 3-figure supplement 1F). This structure base of interaction explains the mechanism for 219 the exclusive interaction of TC45 with GdX or p65. 220 GdX prolongs p65 phosphorylation 221 Since GdX inhibits TC45 binding to p65, we speculated that the altered p-p65 level 222 by GdX might be due to an altered dephosphorylation process. To test this possibility, we  Given that TC45 interacts with p65, we speculated that TC45 might mediate the comparing lanes 12 to 9), indicating that GdX is unable to regulate the p-p65 level 252 without TC45. These results suggest that GdX regulates the dephosphorylation of p65 via 253 TC45. Taken together, we conclude that GdX-elevated phosphorylation of p65 is due to 254 the interruption of TC45 from binding to p65.

255
Residue Y100 in p65 is critical for TC45 to mediate p65 dephosphorylation 256 13 We next mapped the region for the interaction of p65 with TC45. IP experiments 257 (Figure 5-figure supplement 1A) showed that truncated p65-n6 (where 1-90 amino acids 258 remained) failed to interact with TC45, whereas other truncated forms of p65 maintained 259 strong interactions with TC45 ( Figure 5-figure supplement 1B). These data suggested that 260 the region from amino acid 90 to 170 is essential for p65 to interact with TC45.

261
Since TC45 is a tyrosine phosphatase, we checked the conserved residues in this 262 region and identified two tyrosine residues Y100 and Y152 ( Figure 5A). We speculated 263 that these two tyrosine residues might be critical for TC45-mediated p65 264 dephosphorylation. To examine this hypothesis, we mutated these two residues into 265 phenylalanine (F). Luciferase experiments indicated that p65(Y100F) had a decreased 266 activity on the NF-κB reporter whereas p65(Y152F) remained the same activity as wild 267 type p65 ( Figure 5B, black columns). Interestingly, over-expression of TC45 failed to 268 inhibit p65(Y100F)-induced luciferase activity but remained to inhibit 269 p65(Y152F)-induced activity ( Figure 5B). These results suggest that TC45 inhibits the 270 activity of p65 through Y100. Simultaneously, we observed that over-expression of GdX 271 elevated the reporter activity mediated by p65 and p65(Y152F) but had no effect on 272 p65(Y100F)-induced activation ( Figure 5C). Consistent with these observations, 273 over-expression of TC45 appeared to have no effect on the phosphorylation level of 274 p65(Y100F), which though appeared lower than that of p65 (WT) and p65(Y152F) 275 ( Figure 5D). We further deciphered that p65(Y100F) lost the interaction with TC45 but 276 retained the interaction with p65(Y152F) ( Figure 5E). A molecular structure docking 277 14 analysis suggests that this Y100 forms a link with K118 at TC45 to maintain a surface for 278 the interaction of p65 with TC45 ( Figure 5-figure supplement 1C but not on other phosphatases including PP1, PP4 and WIP1 ( Figure 6A), which implied 286 that TC45 might mediate the dephosphorylation of p65 through PP2A. PP2A is the main 287 source of phosphatase activity in the cell, which has been reported to interact with p65 288 (Yang et al., 2001). We then questioned whether TC45 affects the interaction of PP2A 289 with p65. Intriguingly, we observed that TC45 enhanced the interaction of PP2A with p65 290 ( Figure 6B). On the other hand, the interaction of PP2A with p65 was dramatically 291 impaired when TC45 was knocked out ( Figure 6C) or depleted by siRNA ( Figure   292 6-figure supplement 1A). Moreover, we observed that over-expression of PP2A failed to 293 mediate the dephosphorylation of p65 in TC45-deficient MEFs ( Figure 6D, lanes 11-12), 294 but strongly decreased the phosphorylation level in the WT cells ( Figure 6D, lanes 5-6).

295
These results suggest that TC45 is required for PP2A-mediated dephosphorylation of 296 p-p65.

297
Further results revealed that TC45, PP2A and p65 forms a hetero-trimer complex  Figure E), suggesting that Y100 is critical for p65 to interact with 300 both PP2A and TC45. Molecular docking analyses provided the structural base for the 301 hetero-trimer complex, where TC45 binds to the N-terminus, and PP2A binds to the 302 C-terminus separately (Figure 6Fc). In phosphoylated p65, the C-terminus is covered 303 by the N-terminus (Figure 6Fa). When the C-terminus of p65 is released by TC45 ( Figure   304 6Fb), PP2A associates with it and initiates the dephosphorylation of S536 (Figure 6Fc), 305 leading to p65 dephosphorylation ( Figure 6Fd).

306
Next, we determined whether GdX affects the interaction of PP2A with p65.

307
Interestingly, IP experiments demonstrated that over-expression of Myc-GdX inhibited 308 the interaction of HA-PP2A with Flag-p65 ( Figure 6G). Consistently, we observed that 309 the interaction of PP2A and p65 was dramatically increased when GdX was deleted in

311
To valid that the inhibitory role of GdX on the interaction of PP2A and p65 is due to its 312 interaction with TC45, we used mutant GdX(L29P), which lost the ability to associate     Immunohistochemical analyses demonstrated that p-p65 staining was much stronger in 377 the nucleus in the colon section from WT mice than that from GdX -/Y mice treated with suggested that deletion of GdX impaired the activation of NF-κB in colon tissue.

381
Consistently, we observed that specific deletion of GdX in DCs and Mφ significantly 382 decreased the p-p65 levels during acute colitis ( Figure 7T). Taken together, these data 383 suggested that GdX deficiency alleviates the colon inflammation through regulation of 384 NF-κB activity in DCs and Mφ.

387
In this study, we revealed a previously unrecognized regulatory mechanism of 388 NF-κB signaling in innate immune cells. GdX forms a complex with tyrosine 389 phosphatase TC45, traps PP2A by blocking its termination to warranty a sufficient 390 activation of NF-κB ( Figure 6I). GdX deletion impaired the production of  Although NF-κB signaling is tightly associated with IBDs, the cell-type specific 396 mechanisms of NF-κB is poorly understood. GdX deficiency led to reduced 397 phosphorylation of p65 in colon sections during mucosal inflammation, which resulted in 398 a less severe colitis. Consistent with our studies, the level of p65 has been reported 399 increased in the nuclear extracts of intestinal lamina propria biopsy from IBD patients 400 20 (Ardite et al., 1998). Moreover, activated p65 was found in either Mφ or epithelial cells 401 from inflamed mucosa but was almost absent in normal mucosa (Roesky et al., 2000). We 402 previously observed that IECs from GdX -Y mice had a greater proliferative ability which 403 endowed an enhanced ability to maintain mucosal integrity (Wang et al., 2014). In this 404 study, we found that deletion of GdX in IECs had no effect on DSS-induced colitis, but  p65 phosphorylation is critical for activation of NF-κB-dependent transcription 420 (Sakurai et al., 1999;Sizemore et al., 2002;Zhong et al., 1998). Our data showed that 421 21 GdX prolonged LPS-induced phosphorylation of p65 by trapping TC45 and PP2A.

422
Consequently, p65 phosphorylation is sustained by GdX. Mechanistically, we observed 423 that TC45 was critical for GdX-maintained p65 phosphorylation on serine 536. As  The WT and GdX-deficient mice were injected (i.p.) with 20 mg/kg LPS, and then the 455 splenocytes were collected after 1.5 or 6 h. RNAs were purified and reverted into cDNA 456 libraries. High-throughput sequencing was performed by BGISEQ-500 (Beijing

457
Genomics Institute, BGI). The RNA-seq was carried out with two biological replicates.

458
The RNA-seq reads were mapped to mm10 genome by HISAT2 v2.    None of the authors have conflicting financial interests with any findings in this paper.  presented as mean ± SD from three repeats. **, p < 0.01; ***, p < 0.001.       The endogenous interaction of PP2A and p65 was decreased in TC45-deficient cells. (D) PP2A failed to dephosphorylate p-p65 without TC45. p-p65 levels were examined in MEF TC45+/+ and MEF TC45-/cells infected with Ad-GdX or Ad-PP2A and treated with TNF-α (10 ng/mL) for 15 min, followed by TNF-α withdrawal for 30 min before harvesting. (E) Y100 is critical for the interaction of PP2A and p65. IP assays were performed after HEK-293T cells were transfected with the Flag-tagged p65, p65(Y100F), or p65(Y152F), as well as HA-PP2A for 24 h. (F) A molecular docking model showed TC45/p65/PP2A complex formation and disassociation. (a) The C-terminus of p65 is hided at the N-terminus of p65 to maintain an active form of p65. Without association of TC45 with p65 at its N-terminus, p65 remains a coated confirmation where the C-terminus, in particular S538, is hided. (b) TC45 starts to bind to the C-terminus of p65 and release the N-terminus of p65 from hiding. (c) PP2A gets a chance to associate with the released C-terminus and the heter-trimer complex of TC45/p65/PP2A is formed. In this complex, PP2A dephosphorylates S536 and finally maintains p65 unphosphorylated status (d). (G) GdX inhibited the interaction of p65 and PP2A. (H) GdX(L29P) mutant failed to decrease the interaction of PP2A and p65. HEK293T cells were transfected with the indicated plasmids for the IP experiment. (I) A model of the competition of GdX with TC45 to interact with p65. When TC45 interacts with the N-terminus of p65, the C-terminus of p65 is exposed to interact with PP2A. GdX interacts with TC45 and blocks its interaction with p65. In this way p65 maintains at its active form without interaction of PP2A. All of the in vitro experiments were repeated three times, and the results shown were representative.   GdX maintained the level of p-p65 by blocking the complex formation of TC45 and PP2A with p65, leading to abrogated dephosphorylation of p65. Without GdX, TC45 brings PP2A to associate with p-p65 and mediates dephosphorylation of p-p65 for the termination of NF-kB signaling. In the presence of GdX, GdX interacts with TC45 and competes TC45 from interaction of PP2A and p-p65, leading to impaired dephosphorylation of p-p65. In such a way, GdX guards TC45 and PP2A from interaction with p65 to maintain DCs and Mφs alert to pathogen attacks.