Suppressor of cytokine signaling-1 selectively inhibits LPS-induced IL-6 production by regulating JAK-STAT

Abstract

Suppressor of cytokine signaling-1 (SOCS-1) is one of the negative-feedback regulators of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. We previously showed that SOCS-1 participates in LPS signaling, but it is not entirely clear yet how SOCS-1 suppresses LPS signaling. In this study, we demonstrate that SOCS-1 selectively inhibits LPS-induced IL-6 production through regulation of JAK-STAT but not production of TNF-alpha, granulocyte colony-stimulating factor, IFN-beta, and other cytokines. We found that LPS directly activated Jak2 and Stat5, whereas SOCS-1 inhibited LPS-induced Jak2 and Stat5 activation. Furthermore, AG490, a Jak-specific inhibitor, and dominant negative Stat5 only reduced LPS-induced IL-6 production. Additionally, Stat5 interacted with p50, resulting in recruitment of Stat5 to the IL-6 promoter together with p50 in response to LPS stimulation. These findings suggest that the JAK-STAT pathway participates in LPS-induced IL-6 production and that SOCS-1 suppresses LPS signaling by regulating JAK-STAT.

Fig. 1.

SOCS-1 selectively inhibits IL-6 production by LPS. (A) Raw/Neo and Raw/SOCS-1 cells were stimulated by LPS at the indicated time points. Expression of LPS-induced cytokines genes was examined by RT-PCR. (B) Raw/Neo and Raw/SOCS-1 cells were stimulated by LPS as indicated. IL-6 and TNF-α production was measured by using ELISA. (C) SOCS-1 He mice and WT mice were injected i.p. with 1 mg of LPS. Serum IL-6 levels were measured by ELISA at 2 h. Data show means ± SE of three independent experiments.

Fig. 2.

Jak2 participates directly in LPS-induced IL-6 production. (A) Raw/Neo and Raw/SOCS-1 cells were stimulated by LPS at the indicated time points. Tyrosine phosphorylation of Jak2 was analyzed by immunoprecipitation and Western blotting. (B) COS-7 cells were cotransfected with MyD88-Flag, TLR4-Flag, and Jak2. After 2 days, the cells were lysed and immunoprecipitated with anti-Flag Ab, followed by detection of Jak2 by means of Western blotting. Raw cells were stimulated by LPS with or without AG490. (C) Cytokine induction by LPS was examined by using RT-PCR. (D) LPS-induced IL-6 production was measured by means of ELISA. Data show means ± SE of three independent experiments.

Fig. 3.

Stat5 participates directly in LPS-induced IL-6 production. (A) Raw/Neo and Raw/SOCS-1 cells were incubated with LPS at the indicated time points. Whole-cell lysates were used for immunoblotting (IB) analysis with anti-phospho-tyrosine Stat5 Ab. (B) Peritoneal macrophages were isolated from BALB/c mice and stimulated by LPS at the indicated time points. Tyrosine phosphorylation of Stat5 was examined by means of immunoprecipitation (IP) and Western blotting. (C) The interaction of Stat5 with TLR4 was examined by immunoprecipitation and Western blotting in COS-7 cells, which were inducted with Stat5, TLR4, and Raw cells. Raw/Neo, Raw/Stat5 1^*6, and Raw/Stat5 DN were stimulated by LPS at the indicated time points, followed by an examination of IL-6 and TNF-α induction by means of RT-PCR (D) and ELISA (E and F). Data show means ± SE of three independent experiments.

Fig. 4.

Stat5 associates with p50 and mediates LPS-induced IL-6 production. (A) COS-7 cells were cotransfected with Stat5 WT, Stat5 1^*6, or Stat5 DN and p50. Whole-cell lysates were immunoprecipitated with anti-p50 Ab after which Stat5 was detected with Western blotting. (B) Raw/Stat5 1^*6 cells were stimulated by LPS followed by examination of the association of Stat5 with endogenous p50 by means of immunoprecipitation (IP) and Western blotting. (C) Raw cells were stimulated with LPS for 2 h, and the ChIP assay was performed by using anti-Stat5a, anti-Stat5, and anti-p50 Abs. Purified DNA fragments were amplified by using primers specific for the IL-6 promoter, as described in Materials and Methods. IB, immunoblotting.