While there is mounting evidence that interleukin (IL)-23-IL-17 axis plays a critical role in the pathogenesis of various autoimmune diseases, much remains to be elucidated on how IL-23 is induced in the pathologica...While there is mounting evidence that interleukin (IL)-23-IL-17 axis plays a critical role in the pathogenesis of various autoimmune diseases, much remains to be elucidated on how IL-23 is induced in the pathological processes. IL-23 is a heterodimer composed of p19 and p40, the latter being shared with IL-12. We previously reported that prostaglandin (PG) E2 promotes CD40-mediated induction of 1123a (p19) expression through its E receptor subtype 4 (EP4) receptor in splenic dendritic cells (DCs). Here, we have analyzed signaling pathways regulating 1123a induction in the cross talk between EP4 and CD40 in bone marrow-derived DCs. We found that PGE2 synergistically induced 1123a transcription with CD40 signaling. An EP4 agonist, but not agonists of EP1, EP2, or EP3, reproduced this action. Stimulation of CD40 with an agonist antibody evoked biphasic induction of 1123a expression, with the early phase peaking at 1 h and the late phase peaking at 12 h and lasting up to 36 h after stimulation, whereas induction by lipopolysaccharide or tumor necrosis factor-α was transient. The early phase induction by CD40 stimulation was absent in DCs derived from Nfkbl-deficient mice, and the late phase induction was eliminated by RNA interference of nuclear factor-kappa B (NF-κB) p100 subunit. Further, cAMP response element-binding protein (CREB) depletion completely eliminated the induction of 1123a by CD40 stimulation. The addition of the EP4 agonist amplified the induction in both phases through the cAMP-protein kinase A (PKA) pathway. These results suggest that 1123a expression in DCs is synergistically triggered by the PG E2-EP4-cAMP-PKA pathway and canonical/non-canonical NF-KB pathways and CREB activated by CD40 stimulation.展开更多
Rheumatoid arthritis(RA)is an autoimmune disease and is mainly characterized by abnormal proliferation of fibroblast-like synoviocytes(FLS).The up-regulated cellular membrane expression of G protein coupled receptor k...Rheumatoid arthritis(RA)is an autoimmune disease and is mainly characterized by abnormal proliferation of fibroblast-like synoviocytes(FLS).The up-regulated cellular membrane expression of G protein coupled receptor kinase 2(GRK2)of FLS plays a critical role in RA progression,the increase of GRK2 translocation activity promotes dysfunctional prostaglandin E4 receptor(EP4)signaling and FLS abnormal proliferation.Recently,although our group found that paeoniflorin-6’-O-benzene sulfonate(CP-25),a novel compound,could reverse FLS dysfunction via GRK2,little is known as to how GRK2 translocation activity is suppressed.Our findings revealed that GRK2 expression up-regulated and EP4 expression down-regulated in synovial tissues of RA patients and collagen-induced arthritis(CIA)rats,and prostaglandin E2(PGE2)level increased in arthritis.CP-25 could down-regulate GRK2 expression,up-regulate EP4 expression,and improve synovitis of CIA rats.CP-25 and GRK2 inhibitors(paroxetine or GSK180736 A)inhibited the abnormal proliferation of FLS in RA patients and CIA rats by down-regulating GRK2 translocation to EP4 receptor.The results of microscale thermophoresis(MST),cellular thermal shift assay,and inhibition of kinase activity assay indicated that CP-25 could directly target GRK2,increase the protein stability of GRK2 in cells,and inhibit GRK2 kinase activity.The docking of CP-25 and GRK2 suggested that the kinase domain of GRK2 might be an important active pocket for CP-25.G201,K220,K230,A321,and D335 in kinase domain of GRK2 might form hydrogen bonds with CP-25.Site-directed mutagenesis and co-immunoprecipitation assay further revealed that CP-25 down-regulated the interaction of GRK2 and EP4 via controlling the key amino acid residue of Ala321 of GRK2.Our data demonstrate that FLS proliferation is regulated by GRK2 translocation to EP4.Targeted inhibition of GRK2 kinase domain by CP-25 improves FLS function and represents an innovative drug for the treatment of RA by targeting GRK2.展开更多
文摘While there is mounting evidence that interleukin (IL)-23-IL-17 axis plays a critical role in the pathogenesis of various autoimmune diseases, much remains to be elucidated on how IL-23 is induced in the pathological processes. IL-23 is a heterodimer composed of p19 and p40, the latter being shared with IL-12. We previously reported that prostaglandin (PG) E2 promotes CD40-mediated induction of 1123a (p19) expression through its E receptor subtype 4 (EP4) receptor in splenic dendritic cells (DCs). Here, we have analyzed signaling pathways regulating 1123a induction in the cross talk between EP4 and CD40 in bone marrow-derived DCs. We found that PGE2 synergistically induced 1123a transcription with CD40 signaling. An EP4 agonist, but not agonists of EP1, EP2, or EP3, reproduced this action. Stimulation of CD40 with an agonist antibody evoked biphasic induction of 1123a expression, with the early phase peaking at 1 h and the late phase peaking at 12 h and lasting up to 36 h after stimulation, whereas induction by lipopolysaccharide or tumor necrosis factor-α was transient. The early phase induction by CD40 stimulation was absent in DCs derived from Nfkbl-deficient mice, and the late phase induction was eliminated by RNA interference of nuclear factor-kappa B (NF-κB) p100 subunit. Further, cAMP response element-binding protein (CREB) depletion completely eliminated the induction of 1123a by CD40 stimulation. The addition of the EP4 agonist amplified the induction in both phases through the cAMP-protein kinase A (PKA) pathway. These results suggest that 1123a expression in DCs is synergistically triggered by the PG E2-EP4-cAMP-PKA pathway and canonical/non-canonical NF-KB pathways and CREB activated by CD40 stimulation.
基金supported by the Key Project of National Natural Science Foundation of China(No.81330081)Surface Project of National Natural Science Foundation of China(No.81673444)Youth Science Fund Project of National Natural Science Foundation of China(No.81502123)
文摘Rheumatoid arthritis(RA)is an autoimmune disease and is mainly characterized by abnormal proliferation of fibroblast-like synoviocytes(FLS).The up-regulated cellular membrane expression of G protein coupled receptor kinase 2(GRK2)of FLS plays a critical role in RA progression,the increase of GRK2 translocation activity promotes dysfunctional prostaglandin E4 receptor(EP4)signaling and FLS abnormal proliferation.Recently,although our group found that paeoniflorin-6’-O-benzene sulfonate(CP-25),a novel compound,could reverse FLS dysfunction via GRK2,little is known as to how GRK2 translocation activity is suppressed.Our findings revealed that GRK2 expression up-regulated and EP4 expression down-regulated in synovial tissues of RA patients and collagen-induced arthritis(CIA)rats,and prostaglandin E2(PGE2)level increased in arthritis.CP-25 could down-regulate GRK2 expression,up-regulate EP4 expression,and improve synovitis of CIA rats.CP-25 and GRK2 inhibitors(paroxetine or GSK180736 A)inhibited the abnormal proliferation of FLS in RA patients and CIA rats by down-regulating GRK2 translocation to EP4 receptor.The results of microscale thermophoresis(MST),cellular thermal shift assay,and inhibition of kinase activity assay indicated that CP-25 could directly target GRK2,increase the protein stability of GRK2 in cells,and inhibit GRK2 kinase activity.The docking of CP-25 and GRK2 suggested that the kinase domain of GRK2 might be an important active pocket for CP-25.G201,K220,K230,A321,and D335 in kinase domain of GRK2 might form hydrogen bonds with CP-25.Site-directed mutagenesis and co-immunoprecipitation assay further revealed that CP-25 down-regulated the interaction of GRK2 and EP4 via controlling the key amino acid residue of Ala321 of GRK2.Our data demonstrate that FLS proliferation is regulated by GRK2 translocation to EP4.Targeted inhibition of GRK2 kinase domain by CP-25 improves FLS function and represents an innovative drug for the treatment of RA by targeting GRK2.