Extended anti-inflammatory action of a degradation-resistant mutant of cell-penetrating suppressor of cytokine signaling 3

Abstract

Suppressor of cytokine signaling 3 (SOCS3) regulates the proinflammatory cytokine signaling mediated by the JAK/STAT signaling pathway. SOCS3 is rapidly induced and then targeted to the ubiquitin-proteasome pathway via a mechanism that requires the C-terminal SOCS box. Due to its rapid turnover, the intracellular stores of SOCS3 seem insufficient to control acute or protracted inflammatory diseases. Previously, we developed an intracellular protein therapy that uses a recombinant cell-penetrating form of SOCS3 (CP-SOCS3) to inhibit the JAK/STAT pathway and prevent cytokine-mediated lethal inflammation and apoptosis of the liver (Jo, D., Liu, D., Yao, S., Collins, R. D., and Hawiger, J. (2005) Nat. Med. 11, 892-898). The potent anti-inflammatory and cytoprotective activity of CP-SOCS3 prompted us to analyze its intracellular turnover, as compared with that of endogenous SOCS3 protein induced in macrophages by the proinflammatory agonists, interferon-gamma and lipopolysaccharide. We found that the half-life (t(1/2)) of endogenous SOCS3 is 0.7 h in activated macrophages, compared with a t(1/2) of 6.2 h for recombinant CP-SOCS3. Deletion of the SOCS box in CP-SOCS3 renders it more resistant to proteasomal degradation, extending its t(1/2) to 29 h. Consequently, this SOCS box-deleted form of CP-SOCS3 displays persistent inhibitory activity for 24 h toward interferon-gamma- and lipopolysaccharide-induced cytokine and chemokine production. Compared with the wild-type suppressor, this gain-of-function CP-SOCS3 mutant provides a longer acting inhibitor of cytokine signaling, a feature that offers a clear advantage for the intracellular delivery of proteins to treat acute or protracted inflammatory diseases.

FIGURE 1.

Design of recombinant CP-SOCS3 proteins for bacterial expression and affinity purification. A, schematic representation of full-length wild-type SOCS3, showing the different functional domains of the protein: KIR (kinase inhibitory region), SH2 domain, PEST motif, and SOCS box; non-CP-SOCS3, the non-cell penetrating SOCS3 that lacks the MTM, but contains an N-terminal His₆ tag (white); CP-SOCS3, cell-penetrating full-length SOCS3 with a 12-amino acid MTM (red) at the NH₂-terminal, and His₆ tag (white); CP-SOCS3ΔSB, cell-penetrating SOCS3 deletion mutant lacking the C-terminal SOCS box, but possessing the MTM (red) and His₆ tag (white) at the N terminus. B, immunoblot displaying expressed and purified non-CP-SOCS3 (26.6 kDa), CP-SOCS3 (27.9 kDa), and CP-SOCS3ΔSB (23.4 kDa).

FIGURE 2.

Endogenous SOCS3 turnover in RAW macrophages stimulated with proinflammatory agonists. RAW 264.7 cells were stimulated with 100 units/ml of IFN-γ and 250 ng/ml of LPS for 4 h to induce SOCS3 expression (t = 0). After the treatments, samples were collected at 0, 0.5, 1, 1.5, 2, 4, and 6 h. SOCS3 protein levels were quantified by immunoblotting (IB) using the infrared Odyssey Li-Cor system software. A, RAW cells were incubated without (squares) or with (circles) 15 μg/ml of cycloheximide. B, RAW cells were incubated without (squares) or with (diamonds) 15 μg/ml of cycloheximide and 1 μm epoxomicin. C, RAW cells incubated without (squares) or with (triangles) 15 μg/ml of cycloheximide plus 40 μg/ml of calpeptin. D, RAW cells were incubated without (squares) or with (inverted triangle) all three inhibitors: 15 μg/ml of cycloheximide, 40 μg/ml of calpeptin, and 1 μm epoxomicin. The bars represent the mean ± S.E. of n = 3 (A and B) or n = 4 (C and D) independent experiments.

FIGURE 3.

Intracellular delivery of recombinant SOCS3 proteins. Fluorescence confocal laser scanning microscopy of proteinase K-treated and non-fixed RAW macrophages shows intracellular localization of FITC-labeled CP-SOCS3 proteins (green). a–l, images presented represent a 13-μm midcell cross-section. a–c, confocal images of RAW cells incubated with FITC alone. a, FITC image: no fluorescent signal observed. b, differential interference contrast (DIC) image of the RAW cells depicted. c, merged view of DIC and FITC images. d–f, confocal images of RAW cells incubated with FITC-labeled non-CP-SOCS3. d, FITC image, no fluorescent signal detected. e, DIC image of the RAW cells depicted. f, merged view of DIC and FITC images. g–i, confocal images of RAW cells incubated with FITC-labeled CP-SOCS3. g, FITC image, strong fluorescence throughout the cytoplasm. h, DIC image of RAW cells depicted. i, merged view of DIC and FITC images showing localization of FITC-labeled CP-SOCS3 throughout the cytoplasm of the RAW cells. j–l, confocal images of RAW cells incubated with FITC-labeled CP-SOCS3ΔSB. j, FITC image, strong fluorescence throughout the cytoplasm. k, DIC image of RAW cells depicted. l, merged view of DIC and FITC images showing localization of FITC-labeled CPSOCS3ΔSB throughout the cytoplasm. All images are representative of multiple unfixed cells from three independent experiments.

FIGURE 4.

Intracellular delivery of CP-SOCS3 and CP-SOCS3ΔSB is mostly independent of endosomal membrane compartment. Fluorescence confocal laser scanning microscopy of RAW macrophages incubated with FITC-labeled recombinant proteins (green), and the endosomal marker FM-595 (red). a–i, images presented represent a 10-μm midcell cross-section. a–c, RAW cells incubated with FITC-labeled non-CP-SOCS3 (green) and FM-595 (red). a, FITC image, no fluorescent signal detected. b, FM-595 only, endosomes detected throughout the cell. c, merged view of FITC and FM-595 images. d–f, confocal images of RAW cells incubated with FITC-labeled CP-SOCS3 and FM-595. d, FITC image, fluorescent signal throughout the cytoplasm. e, FM-595 only, endosomes detected throughout the cell. f, merged view of FITC and FM-595 images, no overlapping green and red fluorescent signals. g–i, confocal images of RAW cells incubated with FITC-labeled CP-SOCS3ΔSB and FM-595. g, FITC images, fluorescent signal throughout the cytoplasm. h, FM-595 only, endosomes detected throughout the cell. i, merged view of FITC and FM-595 images, no overlapping green and red fluorescent signals. All images are representative of multiple unfixed cells from three independent experiments.

FIGURE 5.

Proteasomal inhibitor extends the half-life of CP-SOCS3 and deletion of the SOCS box dramatically improves the intracellular stability of CP-SOCS3. A, stimulated RAW macrophages were treated for 1 h with CP-SOCS3 in the presence (inverted triangle) or absence (squares) of 1 μm epoxomicin. Half-life was determined by immunoblot analysis of samples collected at 0, 0.5, 2, 4, 6, 12, and 24 h. B, stimulated RAW macrophages were treated for 1 h with CP-SOCS3ΔSB in the absence (open squares) or presence (solid circles) of 1 μm epoxomicin. Half-life was determined by immunoblot analysis of samples collected at 0, 0.5, 2, 4, 6, 12, and 24 h. The bars represent the mean ± S.E. of three independent experiments (n = 3).

FIGURE 6.

CP-SOCS3ΔSB inhibits STAT1 phosphorylation and displays prolonged anti-inflammatory activity associated with intracellular persistence as compared with full-length CP-SOCS3 in AMJ2.C8 macrophage cell line. A and B, after a 1-h pre-treatment of macrophages with cell-penetrating proteins, cells were stimulated with 100 units/ml of IFN-γ and 0.2 μg/ml of LPS for 15 min. Cells were harvested with 1× CBA lysis buffer and analyzed for phosphorylated STAT1 levels by CBA. A, pSTAT1 (units/ml). B, immunoblotting results of pSTAT1 in AMJ2.C8 macrophages treated with CP-SOCS3 or CP-SOCS3ΔSB for 1 h and stimulated for 15 min. C and D, the cells were treated for 1 h with CP-SOCS3 or CP-SOCS3ΔSB. Six or 24 h following protein treatment, cells were stimulated with 100 units/ml of IFN-γ and 0.5 μg/ml of LPS for 6 h. Supernatants were collected before treatment (t = 0 h) and after the 6-h stimulation, at 12 and 30 h, respectively. Samples analyzed for inflammatory cytokine/chemokine production by CBA. C, TNF-α (pg/ml). D, MCP-1 (pg/ml). E, immunoblotting results of CP-SOCS3 or CP-SOCS3ΔSB protein levels in cells after 6 h stimulation, at 12 and 30 h. The bars represent the mean ± S.E. of n = 3 (A and B) or n = 4 (C–E) independent experiments.

FIGURE 7.

CP-SOCS3ΔSB inhibits STAT1 phosphorylation and displays prolonged anti-inflammatory activity associated with intracellular persistence in primary macrophages. BMDM obtained from C3H/HeJ mice were treated for 1 h with 10 μm CP-SOCS3 or 10 μm CP-SOCS3ΔSB. A and B, after a 1-h pre-treatment of macrophages with cell-penetrating proteins, cells were stimulated with 100 units/ml of IFN-γ and 0.2 μg/ml of LPS for 15 min. Cells were harvested with 1× CBA lysis buffer and analyzed for phosphorylated STAT1 by CBA. A, pSTAT1 (units/ml). B, immunoblots of pSTAT1 levels in BMDM treated with CP-SOCS3 or CP-SOCS3ΔSB for 1 h and stimulated for 15 min. C and D, 6 or 24 h following protein treatment, cells were stimulated with 100 units/ml of IFN-γ and 0.5 μg/ml of LPS for 6 h. Supernatants were collected before treatment (t = 0 h) and after the 6-h stimulation, at 12 and 30 h, respectively. Samples were analyzed for inflammatory cytokine/chemokine production by CBA. C, TNF-α (pg/ml). D, MCP-1 (pg/ml). E, immunoblots of CP-SOCS3 or CP-SOCS3ΔSB protein levels in cells after a 6-h stimulation, at 12 and 30 h. The bars represent the mean ± S.E. of n = 3 (A and B) or n = 4 (C–E) independent experiments.