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STING Negatively Regulates Double-Stranded DNA-Activated JAK1-STAT1 Signaling via SHP-1/2 in B Cells.

Dong G, You M, Ding L, Fan H, Liu F, Ren D, Hou Y - Mol. Cells (2015)

Bottom Line: In the present study, we found that dsDNA directly triggered the JAK1-STAT1 signaling by inducing phosphorylation of the Lyn kinase.Moreover, this response is not dependent on type I IFN receptors.In addition, compared with normal B cells, the expression of STING was significantly lower and the phosphorylation level of JAK1 was significantly higher in B cells from MRL/lpr lupus-prone mice, highlighting the close association between STING low-expression and JAK1-STAT1 signaling activation in B cells in autoimmune diseases.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University, Nanjing 210093, China.

ABSTRACT
Recognition of cytosolic DNA initiates a series of innate immune responses by inducing IFN-I production and subsequent triggering JAK1-STAT1 signaling which plays critical roles in the pathogenesis of infection, inflammation and autoimmune diseases through promoting B cell activation and antibody responses. The stimulator of interferon genes protein (STING) has been demonstrated to be a critical hub of type I IFN induction in cytosolic DNA-sensing pathways. However, it still remains unknown whether cytosolic DNA can directly activate the JAK1-STAT1 signaling or not. And the role of STING is also unclear in this response. In the present study, we found that dsDNA directly triggered the JAK1-STAT1 signaling by inducing phosphorylation of the Lyn kinase. Moreover, this response is not dependent on type I IFN receptors. Interestingly, STING could inhibit dsDNA-triggered activation of JAK1-STAT1 signaling by inducing SHP-1 and SHP-2 phosphorylation. In addition, compared with normal B cells, the expression of STING was significantly lower and the phosphorylation level of JAK1 was significantly higher in B cells from MRL/lpr lupus-prone mice, highlighting the close association between STING low-expression and JAK1-STAT1 signaling activation in B cells in autoimmune diseases. Our data provide a molecular insight into the novel role of STING in dsDNA-mediated inflammatory disorders.

No MeSH data available.


Related in: MedlinePlus

dsDNA-induced activation of the JAK1-STAT1 signaling requires Lyn and is independent of IFNAR. (A, B) BJAB cells were treated with the neutralizing antibody for the IFN-α/β receptor (Merck & Millipore) for 30 min prior to transfection with poly(dA:dT) or stimulation of IFN-α for 30 min or 6 h. The expression levels of IFIT1, MX1, OAS1 and IFI44 were detected at 6 h by qPCR (A). The phosphorylation of JAK1 was detected at 30 min by Western blot (B). (C, D) BJAB cells were treated with Lyn inhibitor Saracatinib for 1 h prior to tansfection with poly(dA:dT) for 30 min or 6 h. The expression levels of IFIT1, IFI44, MX1 and OAS1 were detected at 6 h by qPCR (C). The phosphorylation of JAK1 was detected at 30 min by Western blot (D). (E, F) BJAB cells were tansfected with si-Lyn or si-NC for 36 h and then tansfected with poly(dA:dT) for 30 min or 6 h. The expression levels of IFIT1, MX1, OAS1 and IFI44 were detected at 6 h by qPCR (E). The phosphorylation of JAK1 and STAT1 were detected at 30 min by Western blot (F). The data shown represent the means of three independent experiments, and the error bars represent the s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001; ns denotes p > 0.05.
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f2-molce-38-5-441: dsDNA-induced activation of the JAK1-STAT1 signaling requires Lyn and is independent of IFNAR. (A, B) BJAB cells were treated with the neutralizing antibody for the IFN-α/β receptor (Merck & Millipore) for 30 min prior to transfection with poly(dA:dT) or stimulation of IFN-α for 30 min or 6 h. The expression levels of IFIT1, MX1, OAS1 and IFI44 were detected at 6 h by qPCR (A). The phosphorylation of JAK1 was detected at 30 min by Western blot (B). (C, D) BJAB cells were treated with Lyn inhibitor Saracatinib for 1 h prior to tansfection with poly(dA:dT) for 30 min or 6 h. The expression levels of IFIT1, IFI44, MX1 and OAS1 were detected at 6 h by qPCR (C). The phosphorylation of JAK1 was detected at 30 min by Western blot (D). (E, F) BJAB cells were tansfected with si-Lyn or si-NC for 36 h and then tansfected with poly(dA:dT) for 30 min or 6 h. The expression levels of IFIT1, MX1, OAS1 and IFI44 were detected at 6 h by qPCR (E). The phosphorylation of JAK1 and STAT1 were detected at 30 min by Western blot (F). The data shown represent the means of three independent experiments, and the error bars represent the s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001; ns denotes p > 0.05.

Mentions: Considering that poly(dA:dT) shows a better effect on activating the JAK1-STAT1 signaling compared with poly(dG:dC) and ISD, we focused on exploring the mechanism of poly(dA:dT)-activated JAK1-STAT1 signaling in this study. As is known, type I IFN binds to the interferon α/β receptors (IFNAR) and stimulates JAK1 and STAT1 tyrosine phosphorylation (Darnell, 1997; Platanias, 2005). To evaluate the effect of transactivation by IFNAR on poly(dA:dT)-activated JAK1-STAT1 signaling, BJAB cells were pre-incubated with the anti-IFNAR neutralizing antibody for 30 min prior to transfected with poly(dA:dT). As shown in Supplementary Fig. S2, the neutralizing antibody could significantly inhibit IFN-α-induced expression of IFIT1, MX1, OAS1 and IFI44, while it didn’t affect poly(dA:dT)-induced expression of IFIT1, MX1, OAS1 and IFI44 (Fig. 2A). Moreover, IFN-α-induced phosphorylation of JAK1 was abrogated in cells treated with the neutralizing antibody, whereas there was no inhibitory effect of the antibody on poly(dA:dT)-induced phosphorylation of JAK1 (Fig. 2B). These data indicate that poly(dA:dT)-triggered activation of JAK1-STAT1 signaling is independent of IFNAR.


STING Negatively Regulates Double-Stranded DNA-Activated JAK1-STAT1 Signaling via SHP-1/2 in B Cells.

Dong G, You M, Ding L, Fan H, Liu F, Ren D, Hou Y - Mol. Cells (2015)

dsDNA-induced activation of the JAK1-STAT1 signaling requires Lyn and is independent of IFNAR. (A, B) BJAB cells were treated with the neutralizing antibody for the IFN-α/β receptor (Merck & Millipore) for 30 min prior to transfection with poly(dA:dT) or stimulation of IFN-α for 30 min or 6 h. The expression levels of IFIT1, MX1, OAS1 and IFI44 were detected at 6 h by qPCR (A). The phosphorylation of JAK1 was detected at 30 min by Western blot (B). (C, D) BJAB cells were treated with Lyn inhibitor Saracatinib for 1 h prior to tansfection with poly(dA:dT) for 30 min or 6 h. The expression levels of IFIT1, IFI44, MX1 and OAS1 were detected at 6 h by qPCR (C). The phosphorylation of JAK1 was detected at 30 min by Western blot (D). (E, F) BJAB cells were tansfected with si-Lyn or si-NC for 36 h and then tansfected with poly(dA:dT) for 30 min or 6 h. The expression levels of IFIT1, MX1, OAS1 and IFI44 were detected at 6 h by qPCR (E). The phosphorylation of JAK1 and STAT1 were detected at 30 min by Western blot (F). The data shown represent the means of three independent experiments, and the error bars represent the s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001; ns denotes p > 0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4443286&req=5

f2-molce-38-5-441: dsDNA-induced activation of the JAK1-STAT1 signaling requires Lyn and is independent of IFNAR. (A, B) BJAB cells were treated with the neutralizing antibody for the IFN-α/β receptor (Merck & Millipore) for 30 min prior to transfection with poly(dA:dT) or stimulation of IFN-α for 30 min or 6 h. The expression levels of IFIT1, MX1, OAS1 and IFI44 were detected at 6 h by qPCR (A). The phosphorylation of JAK1 was detected at 30 min by Western blot (B). (C, D) BJAB cells were treated with Lyn inhibitor Saracatinib for 1 h prior to tansfection with poly(dA:dT) for 30 min or 6 h. The expression levels of IFIT1, IFI44, MX1 and OAS1 were detected at 6 h by qPCR (C). The phosphorylation of JAK1 was detected at 30 min by Western blot (D). (E, F) BJAB cells were tansfected with si-Lyn or si-NC for 36 h and then tansfected with poly(dA:dT) for 30 min or 6 h. The expression levels of IFIT1, MX1, OAS1 and IFI44 were detected at 6 h by qPCR (E). The phosphorylation of JAK1 and STAT1 were detected at 30 min by Western blot (F). The data shown represent the means of three independent experiments, and the error bars represent the s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001; ns denotes p > 0.05.
Mentions: Considering that poly(dA:dT) shows a better effect on activating the JAK1-STAT1 signaling compared with poly(dG:dC) and ISD, we focused on exploring the mechanism of poly(dA:dT)-activated JAK1-STAT1 signaling in this study. As is known, type I IFN binds to the interferon α/β receptors (IFNAR) and stimulates JAK1 and STAT1 tyrosine phosphorylation (Darnell, 1997; Platanias, 2005). To evaluate the effect of transactivation by IFNAR on poly(dA:dT)-activated JAK1-STAT1 signaling, BJAB cells were pre-incubated with the anti-IFNAR neutralizing antibody for 30 min prior to transfected with poly(dA:dT). As shown in Supplementary Fig. S2, the neutralizing antibody could significantly inhibit IFN-α-induced expression of IFIT1, MX1, OAS1 and IFI44, while it didn’t affect poly(dA:dT)-induced expression of IFIT1, MX1, OAS1 and IFI44 (Fig. 2A). Moreover, IFN-α-induced phosphorylation of JAK1 was abrogated in cells treated with the neutralizing antibody, whereas there was no inhibitory effect of the antibody on poly(dA:dT)-induced phosphorylation of JAK1 (Fig. 2B). These data indicate that poly(dA:dT)-triggered activation of JAK1-STAT1 signaling is independent of IFNAR.

Bottom Line: In the present study, we found that dsDNA directly triggered the JAK1-STAT1 signaling by inducing phosphorylation of the Lyn kinase.Moreover, this response is not dependent on type I IFN receptors.In addition, compared with normal B cells, the expression of STING was significantly lower and the phosphorylation level of JAK1 was significantly higher in B cells from MRL/lpr lupus-prone mice, highlighting the close association between STING low-expression and JAK1-STAT1 signaling activation in B cells in autoimmune diseases.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University, Nanjing 210093, China.

ABSTRACT
Recognition of cytosolic DNA initiates a series of innate immune responses by inducing IFN-I production and subsequent triggering JAK1-STAT1 signaling which plays critical roles in the pathogenesis of infection, inflammation and autoimmune diseases through promoting B cell activation and antibody responses. The stimulator of interferon genes protein (STING) has been demonstrated to be a critical hub of type I IFN induction in cytosolic DNA-sensing pathways. However, it still remains unknown whether cytosolic DNA can directly activate the JAK1-STAT1 signaling or not. And the role of STING is also unclear in this response. In the present study, we found that dsDNA directly triggered the JAK1-STAT1 signaling by inducing phosphorylation of the Lyn kinase. Moreover, this response is not dependent on type I IFN receptors. Interestingly, STING could inhibit dsDNA-triggered activation of JAK1-STAT1 signaling by inducing SHP-1 and SHP-2 phosphorylation. In addition, compared with normal B cells, the expression of STING was significantly lower and the phosphorylation level of JAK1 was significantly higher in B cells from MRL/lpr lupus-prone mice, highlighting the close association between STING low-expression and JAK1-STAT1 signaling activation in B cells in autoimmune diseases. Our data provide a molecular insight into the novel role of STING in dsDNA-mediated inflammatory disorders.

No MeSH data available.


Related in: MedlinePlus