GRK2 inhibits Flt-1+ macrophage infiltration and its proangiogenic properties in rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease with a complex etiology. Monocyte-derived macrophages (MDMs) infiltration are associated with RA severity. We have reported the deletion of G-protein-coupled receptor kinase 2 (GRK2) reprograms macrophages toward an anti-inflammatory phenotype by recovering G-protein-coupled receptor signaling. However, as more GRK2-interacting proteins were discovered, the GRK2 interactome mechanisms in RA have been understudied. Thus, in the collagen-induced arthritis mouse model, we performed genetic GRK2 deletion using GRK2^f/fLyz2-Cre^+/- mice. Synovial inflammation and M1 polarization were improved in GRK2^f/fLyz2-Cre^+/- mice. Supporting experiments with RNA-seq and dual-luciferase reporter assays identified peroxisome proliferator-activated receptor γ (PPARγ) as a new GRK2-interacting protein. We further confirmed that fms-related tyrosine kinase 1 (Flt-1), which promoted macrophage migration to induce angiogenesis, was inhibited by GRK2-PPARγ signaling. Mechanistically, excess GRK2 membrane recruitment in CIA MDMs reduced the activation of PPARγ ligand-binding domain and enhanced Flt-1 transcription. Furthermore, the treatment of mice with GRK2 activity inhibitor resulted in significantly diminished CIA pathology, Flt-1⁺ macrophages induced-synovial inflammation, and angiogenesis. Altogether, we anticipate to facilitate the elucidation of previously unappreciated details of GRK2-specific intracellular signaling. Targeting GRK2 activity is a viable strategy to inhibit MDMs infiltration, affording a distinct way to control joint inflammation and angiogenesis of RA.

Keywords: Flt-1; GRK2; Monocyte-derived macrophages; PPARγ; Rheumatoid arthritis.

Graphical abstract

Figure 1

Increased GRK2 expression is associated with the activation of infiltrated synovial MDMs in RA. (a) Flow cytometric analysis of CD14⁺CCR2⁺ PBMCs obtained from HCs (n = 10) and RA (n = 22). ∗∗P < 0.01 versus HC. (b) Flow cytometric analysis of GRK2 MFI in PBMCs obtained from HCs (n = 10) and RA (n = 22). ∗∗P < 0.01 versus HC. (c) Heat map summarizing statistically significant correlations between GRK2 and CD14⁺CCR2⁺, RA-related clinical manifestation (DAS28, ESR and VAS) (n = 22). ∗P < 0.05. (d–f) Representative GRK2, CD68 and CCR2 immunohistochemical staining collected from HCs (n = 3), OA (n = 3) and RA (n = 5) synovium. ∗∗P < 0.01 versus HC. ^##P < 0.01 versus OA. Scale bar: 500 μm. (g) Ctrl: PBMC obtained from HCs and induced by M-CSF (n = 3); RA: SM obtained from RA synovial tissue (n = 3). (h) GRK2 membraned expression of MCSF-induced PBMC. ∗∗P < 0.01 versus Ctrl. (i) iNOS, Arg1 and CCR2 expression of GSK180736A-treated SM (n = 3). ∗∗P < 0.01 versus RA.

Figure 2

GRK2 deficiency attenuates the development of CIA. (a) Timeline of the experimental sequence of the CIA mouse model. (b) IF staining of MHC II in CD68⁺ SMs (n = 3). LL: Lining layer. SL: Sublining layer. Scale bar: 200 μm. (c) The proportion of CD11b⁺Ly6c⁺ SM in CIA (n = 5), ns indicates no significant difference, ∗∗P < 0.01 versus sham; ^##P < 0.01 versus GRK2^f/f. (d) GRK2 expression in BMDMs and SMs of C57/BL6 mice (n = 10), ∗∗P < 0.01 versus BMDMs. (e) Correlations between SM-GRK2 and BMDM-GRK2 were assessed by linear regression analyses (n = 10). (f) Radiant efficiency of AIA synovium from Day 0 to Day 7 (n = 5). ∗∗P < 0.01 versus Day 0. (g) The ratio of CD86/CD206 in CIA mice (n = 4), ∗∗P < 0.01 versus sham; ^##P < 0.01 versus GRK2^f/f. (h) The BMDMs migration ability in CIA mice (n = 3), ∗P < 0.05, ∗∗P < 0.01 versus sham; ^#P < 0.05, ^##P < 0.01 versus GRK2^f/f. Scale bar: 100 μm.

Figure 3

GRK2 deficiency alters PPARγ signaling in BMDMs. (a) RNA-seq expression analysis of BMDMs obtained from GRK2^f/f (n = 6) and GRK2^f/fCre^+/− mice (n = 6). (b) Heatmap showing differential genes between BMDMs from GRK2^f/f and GRK2^f/fCre^+/− mice (blue = down-regulated, red = upregulated). Fold change >2, adjusted P < 0.05. (c) Top-12 significantly enriched GO annotations associated with upregulated and down-regulated genes. (d) Top-10 significantly enriched KEGG pathway associated with upregulated and downregulated genes. (e) PPARγ mRNA expression of GRK2^−/− BMDMs (n = 3). ns indicates no significant difference. (f) PPARγ expression of GRK2^−/− BMDMs (n = 6). ∗∗P < 0.01 versus Ctrl. (g) PPARγ nucleus expression of GRK2^−/− BMDMs (n = 5). ∗∗P < 0.01 versus Ctrl.

Figure 4

GRK2 activates PPARγ by targeting Tyr473. (a) PPI network of GRK2 and PPARα/β/γ. (b) FRET assay with mCherry-GRK2 and EGFP-PPARγ in HEK293 cells. Graph depicts fluorescence ratio per cell (n = 3). ∗P < 0.05, ∗∗P < 0.01 versus Ctrl. Scale bar: 3 μm. (c) The interaction between GRK2 and PPARγ in BMDMs by CO-IP, IP: GRK2 (n = 3). (d) The interaction between GRK2 and PPARγ in BMDMs by CO-IP, IP: PPARγ (n = 3). (e) The interaction of PPARγ and GRK2 in HEK293 cells by his pull-down (n = 6). (f) The backbone of protein was rendered in tube and colored in green. (g) GRK2 (left) and PPARγ (right) protein is rendered by the surface. The detail binding mode of GRK2 with PPARγ. Yellow dash represents hydrogen bond or salt bridge. (h) The point mutation strategy used in GRK2. (i) Luciferase reporter assay (n = 3), ∗P < 0.05, ∗∗P < 0.01 versus Gal4 promotor-NC, ns indicates no significant difference, ^##P < 0.01 versus GRK2-NC. (j) The point mutation strategy used in PPARγ. (k) The interaction between GRK2 and PPARγ in HEK293 cells. HEK293 cells were co-transfected with GRK2 plasmid and PPARγ-LBD plasmid or PPARγ-Y473A plasmid (n = 3). (l) The activity of PPRE is measured by luciferase reporter system. HEK293T cells are co-transfected with PPARγ-LBD plasmid or PPARγ-Y473A plasmid and PPARγ reporter for 24 h ns indicates no significant difference, ∗P < 0.05 versus Ctrl (n = 3). (m) GRK2 interacts with PPARγ via targeting Tyr473, leading to the nuclear translocation of PPARγ.

Figure 5

GRK2-PPARγ inhibited Flt-1 transcription. (a) RNA-seq expression analysis of BMDMs obtained from WT (n = 4) and PPARG knockout mice (n = 4). (b) Heatmap showing differential genes between BMDMs from WT and PPARG knockout mice (blue = down-regulated, red = upregulated). Fold change >2, adjusted P < 0.05. (c) Venn diagram indicating overlapping DEGs between GRK2^−/−, PPARγ^−/− BMDMs and RA-SM. (d) KEGG pathway enrichment chord diagram of 17 overlapping DEGs. (e) Top 10 TFs enriched by up- and downregulated DEGs in GRK2^−/− BMDMs. (f) Flt-1 mRNA expression in GRK2^−/− BMDMs (n = 3), ∗∗P < 0.01 versus Ctrl. (g) Flt-1 mRNA expression in PPARγ^−/− BMDMs (n = 3), ∗∗P < 0.01 versus Ctrl. (h) GRK2 activated PPARγ-Tyr473 may inhibit Flt-1 transcription.

Figure 6

PPARγ inhibits the transcription of Flt-1 and results in angiogenesis. (a) The strategy for constructing GRK2^−/− Raw264.7 cells. (b) The Flt-1 expression in GRK2^−/− Raw264.7 cells (n = 6). (c) The cell migration ability of GRK2^−/− Raw264.7 cells (n = 5), ∗∗P < 0.01 versus NC. Scale bar: 100 μm. (d) PPARγ binding motif. (e) Luciferase reporter assay (n = 3), ∗∗P < 0.01 versus PPARγ-NC, ns indicates no significant difference, ^##P < 0.01 versus ctrl. (f) Flt-1 expression in RA SM (n = 3), ∗∗P < 0.01 versus Ctrl. (g) Flt-1 mRNA expression in RA SM (n = 3), ∗∗P < 0.01 versus Ctrl. (h) The cell migration assay of Flt-1 knockdown BMDMs exposed to hypoxia (n = 3), ∗∗P < 0.01 versus Ctrl. Scale bar: 100 μm. (i) The expression of iNOS, Arg1 and CCR2 in VEGF-stimulated BMDMs (n = 3). (j) IF staining for CD31 and CD11b in RA synovium (n = 3). Scale bar: 200 μm. (k) Tubulogenesis assays on HUVECs that were coclutred with Raw264.7 (control) or with VEGF-pretreated Raw264.7 (n = 5), ∗∗P < 0.01 versus Ctrl. Scale bar: 100 μm. (l) Tubulogenesis assays on Raw264.7 and HUVECs mixed with Raw264.7 (n = 5). Scale bar: 100 μm.

Figure 7

The role and mechanisms of GRK2 activity inhibitor on RA macrophages. (a) The structure of CP-25. (b) Timeline of the experimental sequence of the CIA mouse model (n = 8). (c) Paw swelling of CIA mice from Day 28 to Day 55 (n = 8), ∗P < 0.05, ∗∗P < 0.01 versus Model. (d) Representative images of ankle joint histology, H&E, toluidine blue, and safranin o–fast green staining from mice harvested at Day 55 of CIA (n = 8). Scale bar: 100 μm. (e) The proportion of CD11b⁺Ly6c⁺ SM in CIA (n = 5), ∗P < 0.05, ∗∗P < 0.01 versus Normal; ns indicates no significant difference, ^##P < 0.01 versus Model. (f) Correlations between SM-Ly6c and BMDM-Ly6c were assessed by linear regression analyses (n = 5). (g) Immunofluorescent staining of CD11b and Flt-1 were performed on paraffin embedded ankle joint slices (n = 5). Scale bar: 100 μm. (h) Membrane GRK2, cytoplasm GRK2, PPARγ and Flt-1expressionin CIA BMDMs (n = 5), ∗P < 0.05, ∗∗P < 0.01 versus Normal; ^##P < 0.01 versus Model. (i) The interactions between GRK2 and PPARγ in CIA BMDMs by CO-IP (n = 5), ∗P < 0.05, ∗∗P < 0.01 versus Normal; ^##P < 0.01 versus Model. (j) The interactions between GRK2 and PPARγ in CP-25/Paxil-treated BMDMs by CO-IP (n = 3), ns indicates no significant difference.