Leptin induces IL-6 expression through OBRl receptor signaling pathway in human synovial fibroblasts

Abstract

Background: Leptin, an adipocyte-secreted hormone that centrally regulates weight control, may exert proinflammatory effects in the joint, depending on the immune response. Leptin is abundantly expressed in osteoarthritis (OA) cartilage and synovium. However, the relationship between leptin and interleukin-6 (IL-6) in OA synovial fibroblasts (OASFs) remains obscure.

Methodology/principal findings: Stimulation of OASFs with leptin induced IL-6 expression in a concentration- and time-dependent manner. OASFs expressed the long (OBRl) and short (OBRs) isoforms of the leptin receptor. However, OBRl, but not OBRs, antisense oligonucleotide (AS-ODN) abolished the leptin-mediated increase of IL-6 expression. Transfection with insulin receptor substrate (IRS)-1 siRNA decreased leptin-induced IL-6 production. In addition, pretreatment of cells with PI3K, Akt, or AP-1 inhibitor also inhibited the potentiating action of leptin. Leptin-induced AP-1 activation was inhibited by OBRl, IRS-1, PI3K, or Akt inhibitors and siRNAs.

Conclusions/significance: Our results showed that leptin activates the OBRl receptor, which in turn activates IRS-1, PI3K, Akt, and AP-1 pathway, leading to up-regulation of IL-6 expression.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Leptin induces IL-6 expression in human synovial fibroblasts through OBRl receptor.

(A and B) OASFs were incubated with various concentrations of leptin for 24 h. Media and total RNA were collected, and the expression of IL-6 was examined by qPCR and ELISA assay (n=4). (C and D) OASFs were incubated with leptin (3 µM) for 6, 12, or 24 h. Media and total RNA were collected, and the expression of IL-6 was examined by qPCR and ELISA assay (n=4). (E) OASFs were incubated with leptin (3 µM) for 24 h. OBRl and OBRs mRNA expression was examined by qPCR (n=4). (F) OASF cells were transfected with OBRl and OBRs AS-ODN or OBRl and OBRs MM-ODN, and mRNA level of OBRl and OBRs was analyzed by qPCR (n=4). (G and H) OASFs were transfected with OBRl and OBRs AS-ODN or OBRl and OBRs MM-ODN for 24 h and then stimulated with leptin (3 µM) for 24 h; IL-6 expression was examined by qPCR and ELISA assay (n=4). Results are expressed as mean ± S.E.M. of four independent experiments. *: p < 0.05 as compared with basal level. #: p < 0.05 as compared with the leptin-treated group.

Figure 2. The IRS-1 signaling pathway is involved in leptin-mediated increase of IL-6 expression.

(A and B) OASFs were transfected with IRS-1 siRNA for 24 h. Media and total RNA were collected, and the expression of IL-6 was examined by qPCR and ELISA assay (n=4). (C) OASFs were incubated with leptin for the indicated time intervals. Total protein was collected and the phosphorylation of IRS-1 was examined by western blotting assay (n=4). (D) OASFs were transfected with OBRl AS-ODN and MM-ODN for 24 h and then stimulated with leptin (3 µM) for 30 min. Total protein was collected and the phosphorylation of IRS-1 was examined by western blotting (n=4). Results are expressed as mean ± S.E.M. of four independent experiments. *: p < 0.05 as compared with basal level. #: p < 0.05 as compared with the leptin-treated group.

Figure 3. The PI3K signaling pathway is involved in leptin-mediated increase of IL-6 expression.

(A and B) OASFs were pretreated for 30 min with Ly294002 and wortmannin or transfected with p110 siRNA for 24 h, followed by stimulation with leptin for 24 h. Media and total RNA were collected, and the expression of IL-6 was examined by qPCR and ELISA assay (n=4). (C) OASFs were incubated with leptin for the indicated time intervals. Total protein was collected and the phosphorylation of p110 was examined by western blotting (n=4). (D) OASFs were transfected with IRS-1 siRNA for 24 h and then incubated with leptin (3 µM) for 60 min; Total protein was collected and the phosphorylation of p110 was examined by western blotting (n=4). Results are expressed as mean ±S.E.M. of four independent experiments. *: p < 0.05 as compared with basal level. #: p < 0.05 as compared with the leptin-treated group.

Figure 4. The Akt signaling pathway is involved in the potentiating action of leptin

(A and B) OASFs were pretreated for 30 min with Akt inhibitor or transfected with Akt siRNA for 24 h, followed by stimulation with leptin for 24 h. Media and total RNA were collected, and the expression of IL-6 was examined by qPCR and ELISA assay (n=4). (C) OASFs were incubated with leptin for the indicated time intervals. Total protein was collected and the phosphorylation of AKT was examined by western blotting (n=4). (D) OASFs were pretreated for 30 min with Ly294002 or transfected with IRS-1 siRNA for 24 h and then incubated with leptin (3 µM) for 60 min; Total protein was collected and the phosphorylation of AKT was examined by western blotting (n=4). Results are expressed as mean ±S.E.M. of four independent experiments. *: p < 0.05 as compared with basal level. #: p < 0.05 as compared with the leptin-treated group.

Figure 5. Involvement of AP-1 in leptin induced IL-6 production.

(A) OASFs were transfected with indicated IL-6 promoter luciferase plasmids for 24 h. Cells were then incubated with leptin (3 µM) for 24 h. IL-6 luciferase activity was measured, and the results were normalized to β-galactosidase activity and expressed as the mean ± SE for 3 independent experiments performed in triplicate (n=4). (B and C) OASFs were pretreated for 30 min with curcumin or transfected for 24 h with c-Jun siRNA, followed by stimulation with leptin for 24 h. Media and total RNA were collected, and the expression of IL-6 was examined by qPCR and ELISA assay (n=4). (D) OASFs were incubated with leptin for the indicated time intervals. Total protein was collected and the phosphorylation of c-Jun was examined by western blotting (n=4). (E) OASFs were pretreated with Ly294002 and Akt inhibitor for 30 min or transfected with IRS-1 siRNA for 24 h and then incubated with leptin for 120 min. Total protein was collected and the phosphorylation of c-Jun was examined by western blotting (n=4). Results are expressed as mean ±S.E.M. of four independent experiments. *: p < 0.05 as compared with basal level. #: p < 0.05 as compared with the leptin-treated group.

Figure 6. Leptin induces AP-1 activation through OBRl receptor/IRS-1/PI3K/Akt pathway.

(A) OASFs were pretreated with Ly294002 and Akt inhibitor for 30 min or transfected with OBRl AS-ODN and IRS-1 siRNA for 24 h and then stimulated with leptin for 120 min, and chromatin immunoprecipitation assay was performed. Chromatin was immunoprecipitated with anti-c-Jun. One percent of the precipitated chromatin was assayed to verify equal loading (input) (n=4). (B and C) OASFs were transfected with AP-1-luciferase expression vector and then pretreated with Ly294002, Akt inhibitor, and curcumin or cotransfected with OBRl AS-ODN, IRS-1 siRNA, p110 siRNA, Akt siRNA, and c-Jun siRNA before incubation with leptin for 24 h. Luciferase activity was then assayed. Results are expressed as mean ±S.E.M. of four independent experiments. *: p < 0.05 as compared with basal level. #: p < 0.05 as compared with the leptin-treated group.