WLS/wntless is essential in controlling dendritic cell homeostasis via a WNT signaling-independent mechanism

Abstract

We propose that beyond its role in WNT secretion, WLS/GPR177 (wntless, WNT ligand secretion mediator) acts as an essential regulator controlling protein glycosylation, endoplasmic reticulum (ER) homeostasis, and dendritic cell (DC)-mediated immunity. WLS deficiency in bone marrow-derived DCs (BMDCs) resulted in poor growth and an inability to mount cytokine and T-cell responses in vitro, phenotypes that were irreversible by the addition of exogenous WNTs. In fact, WLS was discovered to integrate a protein complex in N-glycan-dependent and WLS domain-selective manners, comprising ER stress sensors and lectin chaperones. WLS deficiency in BMDCs led to increased ER stress response and macroautophagy/autophagy, decreased calcium efflux from the ER, and the loss of CALR (calreticulin)-CANX (calnexin) cycle, and hence protein hypo-glycosylation. Consequently, DC-specific wls-null mice were unable to develop both Th1-, Th2- and Th17-associated responses in the respective autoimmune and allergic disease models. These results suggest that WLS is a critical chaperone in maintaining ER homeostasis, glycoprotein quality control and calcium dynamics in DCs.Abbreviations: ATF6: activating transcription factor 6; ATG5: autophagy related 5; ATG12: autophagy related 12; ATG16L1: autophagy related 16 like 1; ATP2A1/SERCA1: ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 1; BALF: bronchoalveolar lavage fluid; BFA: brefeldin A; BMDC: bone marrow-derived dendritic cell; CALR: calreticulin; CANX: calnexin; CCL2/MCP-1: C-C motif chemokine ligand 2; CNS: central nervous system; CT: C-terminal domain; DTT: dithiothreitol; DNAJB9/ERDJ4: DnaJ heat shock protein family (Hsp40) member B9; EAE: experimental autoimmune encephalomyelitis; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ERN1/IRE1: endoplasmic reticulum (ER) to nucleus signaling 1; GFP: green fluorescent protein; HSPA5/GRP78/BiP: heat shock protein A5; IFNA: interferon alpha; IFNAR1: interferon alpha and beta receptor subunit 1; IFNB: interferon beta; IFNG/INFγ: interferon gamma; IFNGR2: interferon gamma receptor 2; IL6: interleukin 6; IL10: interleukin 10; IL12A: interleukin 12A; IL23A: interleukin 23 subunit alpha; ITGAX/CD11c: integrin subunit alpha X; ITPR1/InsP3R1: inositol 1,4,5-trisphosphate receptor type 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; OVA: ovalbumin; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLF: predicted lipocalin fold; PPP1R15A/GADD34: protein phosphatase 1 regulatory subunit 15A; RYR1/RyanR1: ryanodine receptor 1, skeletal muscle; SD: signal domain; TGFB/TGF-β: transforming growth factor beta family; Th1: T helper cell type 1; Th17: T helper cell type 17; TM: tunicamycin; TNF/TNF-α: tumor necrosis factor; UPR: unfolded protein response; WLS/wntless: WNT ligand secretion mediator.

Keywords: Dendritic cells; er stress; gpr177; protein glycosylation; unfold protein response.

Figure 1.

Effect of WLS deficiency on BMDCs. (A) Colony formation of BMDCs with wild-type, heterozygous, or wls-null (wls^fx/fx) genotypes (n = 8). (B and C) Percentage of apoptotic and autophagic BMDCs with wild-type, heterozygous, or wls-null genotypes (n = 8). (D) Western blots of autophagic markers (LC3B, PIK3C3, ATG5, ATG12 and ATG16L1) in lysates of wild-type and wls-null BMDCs (n = 3; p< 0.001). (E) Percentage of apoptotic BMDCs with wild-type and wls-null genotypes by WNT1 and WNT3A treatment. (F) Percentage of autophagic BMDCs with wild-type and wls-null genotypes after WNT1 and WNT3A treatment. (G) Level of cytokine-expressing cells in wild-type or wls-null (wls^fx/fx) BMDCs. (H) Levels of IL12A, IL6, and IL10 secreted by wild-type and wls-null BMDCs, or (I) IFNG and TNF secreted by cocultured CD4⁺ T cells in the presence or absence of LPS. (J) Spleen size in wild-type and DC-specific wls-null mice with or without LPS treatment. Flow cytometric analysis of the total number of (K) splenocytes and (L) DCs in wild-type and DC-specific wls-null mice after LPS treatment (n = 8; p< 0.001)

Figure 2.

WLS deficiency results in ER stress and the loss of ER quality control. (A) Western blot analysis of UPR sensors, EIF2AK3-ElF2A signals, and calcium regulators in wild-type (+/+), heterozygous (fx/+), and wls-null (fx/fx) Itgax-Cre BMDCs. (B) TEM analysis of the ultrastructure and translational ribosome complex in wild-type and wls-null BMDCs. Red arrow, translational ribosomal complex. (C) intracellular calcium levels in wild-type and wls-null BMDCs following treatment with CCL2 (0.7 nM). (D-F) Western blotting analysis of WLS, ERN1, EIF2AK3, ATF6, HSPA5 CANX, and CALR in anti-WLS, anti-ERN1, and anti-EIF2AK3 immunoprecipitates probed with the respective antibodies in wild-type or wls-deficient BMDCs. Data were generated from three independent experiments. (G) Confocal immunofluorescent imaging of WLS, ERN1, EIF2AK3, and ATF6 in wild-type or wls-null BMDCs. (H-I), Western blot analysis of WLS, ERN1, EIF2AK3, ATF6, HSPA5, CANX, and CALR in anti-WLS immunoprecipitates of BMDCs treated with WNT1 and WNT3A, respectively

Figure 3.

WLS coordinates a multi-protein complex and regulates ER stress response in BMDCs

Figure 4.

WLS-mediated molecular chaperone supercomplex modulates protein glycosylation, cytokine secretion, and cell fate in BMDCS. (A) Total glycosylation levels as measured by ELISA in BMDCs with wild-type and wls-null genotypes after TM, BFA, or DTT treatment. (B) Glycosylation levels of different kinds of glycan modification of total proteins determined by phenol-sulfuric acid method. (C) Heatmap of 507 biomarkers in wild-type and wls-null (wls^fx/fx) BMDCs; (D) Comparison of the number of hypoglycosylated proteins between wild-type and wls-null cells. #, hypo-glycosylation, no significance (*, p< 0.05); (E) GO biological process over-representation analysis with 15 signal pathways between wild-type and wls-null cells; (F) Glycosylation levels of CDH5/VE-Cadherin, BMPR1B, TGFBR1, LRP6, FZD1/Frizzled-1, FZD3, FZD4, FZD5, FZD6, and CTNNB1/β-catenin in wild-type and wls-null cells. (G) Glycosylation levels as measured by ELISA in anti-WNT1, WNT3A, and WNT5A immunoprecipitates of wild-type and wls-null BMDC. (H) Levels of WNT1, WNT3A, and WNT5A as measured by ELISA in wild-type and wls-null BMDCs. (I) Glycosylation levels of INS (insulin), IGF1, GRB2, IGF2R, INSR, GCG (glucagon), SLC2A1/GLUT1, SLC2A2, SLC2A3, and SLC2A5 in wild-type and wls-null cells. (J-K) Levels of GCG, glucose, and ATP as measured by ELISA in wild-type and wls-null BMDCs. (L) Glycosylation levels of CD14, TLR1, TLR2, TLR3, TLR4, IFNB, IL6, and TNF in wild-type and wls-null cells. (M) Glycosylation levels of IFNA, IFNG, IFNAR1, IFNGR2, IL10, IL12A, IL23A and TGFA in wild-type and wls-null cells

Figure 5.

WLS coordinates a multi-protein complex and regulates ER stress response in A549 cells. (A) Wild-type and various deletion mutants of WLS as indicated. (B) Western blot analysis of UPR sensors and CANX-CALR complex expression in anti-GFP immunoprecipitates from wild-type and mutant WLS-transfected A549 cells. Arrow, HSPA5. (C) Western blot analysis of UPR sensors and calcium regulators (ITPR1, RYR1, and ATP2A1) in A549 cells transfected with wild-type or mutant WLS (Arrow, ITPR1). (D) Confocal microscopy imaging of the interaction between WLS and EIF2AK3 in A549 cells transfected with wild-type or WLSΔCT mutants. WLS, green; EIF2AK3, red; and nuclei, blue. (E) Percentage of autophagic A549 cells transfected with wild-type or WLS mutants. (F) Intracellular calcium levels in A549 cells transfected with wild-type or WLS mutants. (G) glycosylation levels as measured by ELISA in A549 cells transfected with wild-type or WLS mutants. Data were generated from three independent experiments for each panel (***, p< 0.001)

Figure 6.

In vivo mouse EAE model. (A) Clinical score (means ± SEM) of MOG_35–55-induced EAE in wild-type or DC-specific, wls-null (wls^fx/fx) mice (N = 8 mice/group; ***p < 0.0001). (B-E) Number of DCs, pDCs, cDCs, and CNS-infiltrating Th1, Th17, or Th1/Th17 (CD4⁺ IFNG⁺ IL17A⁺) CD4⁺ T cells from MOG_35–55-induced wild-type and DC-specific wls-null mice on day 21 (N = 8 mice/group; p < 0.001 in two-tailed unpaired t-test; n.s., not significant n ≥ 12). (F) Histological analysis of spinal cords of wild-type and wls-null mice with EAE using hematoxylin & eosin (H&E) staining. (G-K) Total number of inflammatory cells and cell differentials in BALFs after OVA challenge (p < 0.001; n.s., not significant). (L) H&E staining of lung tissues in wild-type and wls-null mice (N = 12)

Figure 7.

A schematic diagram illustrates a hypothetical model of WLS as an essential chaperone docking ER stress sensors and CANX-CALR complex and controlling ER homeostasis. According to our results, WLS integrates the CANX-CALR cycle, UPR sensors (ERN1, EIF2AK3, ATF6, HSPA5) and calcium regulators (not depicted in the scheme) via different interacting domains. Functionally, the WLS-associated complex controls glycoprotein quality, ER homeostasis and, at least in part, cellular survival, growth and response, whereas WLS deficiency releases UPR sensors and dissociates the CANX-CALR complex, resulting in global hypo-glycosylation and activation of UPR signaling, leading to autophagy, apoptosis and cell death