SCFFBXO17 E3 ligase modulates inflammation by regulating proteasomal degradation of glycogen synthase kinase-3β in lung epithelia

Abstract

Glycogen synthase kinase-3β (GSK3β) has diverse biological roles including effects on cellular differentiation, migration, and inflammation. GSK3β phosphorylates proteins to generate phosphodegrons necessary for recognition by Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligases leading to subsequent proteasomal degradation of these substrates. However, little is known regarding how GSK3β protein stability itself is regulated and how its stability may influence inflammation. Here we show that GSK3β is degraded by the ubiquitin-proteasome pathway in murine lung epithelial cells through lysine 183 as an acceptor site for K48 polyubiquitination. We have identified FBXO17 as an F-box protein subunit that recognizes and mediates GSK3β polyubiquitination. Both endogenous and ectopically expressed FBXO17 associate with GSK3β, and its overexpression leads to decreased protein levels of GSK3β. Silencing FBXO17 gene expression increased the half-life of GSK3β in cells. Furthermore, overexpression of FBXO17 inhibits agonist-induced release of keratinocyte-derived cytokine (KC) and interleukin-6 (IL-6) production by cells. Thus, the SCF^FBXO17 E3 ubiquitin ligase complex negatively regulates inflammation by targeting GSK3β in lung epithelia.

Keywords: E3 ubiquitin ligase; inflammation; lung; lung injury; ubiquitin.

Figure 1.

GSK3β degradation occurs through the ubiquitin-proteasome pathway. A, MLE-12 cells were treated with CHX alone (40 μg/ml) or in combination with MG132 (20 μm) or leupeptin (20 μg/ml) for 0, 2, 4, and 8 h. Immunoblots of lysates for endogenous GSK3β and β-actin as a loading control were performed. B, shown are the relative densitometries of GSK3β protein over time for each immunoblot. The data represent mean ± S.E. of n = 4 independent experiments. *, p value <0.05 by a nonparametric test for trend. C, MLE-12 cells were transfected with plasmid encoding HA-tagged ubiquitin (HA-Ub) using 0, 1, 2, and 4 μg of DNA. Cells were cultured for 48 h. Immunoblots for GSK3β and β-actin as a loading control are shown. Bar graph depicts relative densitometry representative of the immunoblot shown. D, MLE-12 cells were treated with MG132 for 3 and 6 h prior to harvesting lysates. Immunoprecipitation (IP) of endogenous GSK3β was performed and samples were immunoblotted (IB) with antibodies against K48-ubiquitin or K63-ubiquitin.

Figure 2.

Lysine 183 is an acceptor site for K48 polyubiquitination in GSK3β. A, plasmids expressing HA-tagged wild-type, K183R, or K205R mutant GSK3β were transfected into MLE-12 cells. Cells were cultured for 48 h and then treated with CHX for 0, 2, 4, and 8 h. Lysates were prepared and immunoblotted for HA and β-actin as a loading control. B, the relative densitometries of GSK3β protein plotted over time for each immunoblot are shown. The data represent mean ± S.E. of n = 4 independent experiments. *, p value <0.05 by a nonparametric test for trend. C, MLE-12 cells were transfected with HA-tagged wild-type, or K183R GSK3β plasmids and cultured for 48 h. Cells were then treated with MG132 for 6 h and harvested. Immunoprecipitation (IP) was performed with ubiquitin antibody and samples were probed with HA antibody. D and E, cells were transfected with HA-tagged wild-type, K183R, or K205R mutant GSK3β plasmids and subjected to MG132 treatment prior to HA-antibody pulldown and ubiquitin (D) or K48 chain specific immunoblotting (E). Far right panel: input samples were immunoblotted (IB) with HA-antibody and β-actin as a loading control.

Figure 3.

FBXO17 targets GSK3β for degradation in lung epithelial cells. A, MLE-12 cells were transfected with 0, 1, 2, and 4 μg of FBXO17-V5 expression plasmids and cultured for 48 h. Endogenous GSK3β, FBXO17-V5, and β-actin protein levels were analyzed by immunoblotting (IB). B, MLE12 cells were transfected with 2 μg of FBXO17-V5 plasmid for 48 h. RNA was isolated and analyzed by RT-PCR using primers against GSK3β and GAPDH as an internal control. Inset, FBXO17-V5 protein expression was confirmed by immunoblotting using V5 antibody. C and D, FBXO17-V5 expression plasmid (2 μg) was transfected into MLE-12 cells and cells were cultured for 48 h. Immunoprecipitation (IP) of lysates was performed using Skp1 antibody. Samples were immunoblotted with Skp1, V5, and β-actin (loading control) antibodies. The data in each panel are representative of at least n = 3 independent experiments. D, MLE-12 cells were co-transfected with HA-tagged wild-type, K183R, or K205R mutant GSK3β plasmids with or without FBXO17-V5 plasmid. Samples were immunoblotted with HA, V5, and β-actin (loading control) antibodies.

Figure 4.

FBXO17 overexpression increases polyubiquitination and proteasomal degradation of GSK3β. A, MLE-12 cells were transfected with 2 μg of empty pcDNA 3.1 TOPO vector or ΔFboxFBXO17-V5-expressing plasmids and cultured for 48 h. Cells were then treated with cycloheximide (40 μg/ml) and lysates were collected at 0, 2, 4, and 8 h. Samples were immunoblotted with GSK3β, V5, and β-actin (loading control) antibodies. B, knockdown experiments were performed by co-transfecting GSK3β-V5 plasmids combined with FBXO17 siRNA (100 nm) or control scrambled RNA into BEAS-2B cells. Cells were cultured for 72 h and then treated with CHX (40 μg/ml). Samples were collected at 0, 2, 4, and 8 h. The relative densitometries of GSK3β protein plotted over time for each immunoblot are shown. The data represent mean ± S.E. of n = 3 independent experiments. *, p value <0.05 by a nonparametric test for trend. C, MLE-12 cells were all transfected with plasmids expressing HA-ubiquitin combined with empty vector, FBXO17-V5, or ΔFboxFBXO17-V5. Cells were cultured for 48 h and then treated with MG132 (20 μm) for 6 h prior to preparing lysates. Immunoprecipitation (IP) was done using GSK3β antibody. Samples were immunoblotted (IB) with antibodies against HA, V5, GSK3β, and β-actin as a loading control. D, BEAS-2B cells were transfected with 100 nm scrambled RNA (negative control) or FBXO17 siRNA. Cells were cultured for 72 h and then treated with MG132 (20 μm) for 6 h. Lysates were prepared and immunoprecipitation was done using a GSK3β antibody. Samples were immunoblotted with antibodies for ubiquitin, FBXO17, and β-actin (loading control). The data in each panel are representative of at least n = 3 independent experiments.

Figure 5.

FBXO17 associates with GSK3β through a docking motif. A, MLE cells were transfected with FBXO17-V5 plasmid and cultured for 48 h. Samples were collected and V5 antibody was used for immunoprecipitation (IP). Cells were collected and assayed for GSK3β, V5, Skp1, and β-actin (loading control) by immunoblotting (IB). B, MLE-12 cells were transfected with FBXO17-V5 and GSK3β-HA plasmids and cultured on glass bottom dishes for 48 h. Cells were fixed and immunostained with antibodies to V5 (green) and HA (red). Nuclei were stained with DAPI (blue). Colocalization is demonstrated by yellow on the merged image. Representative images from three independent experiments are shown. Scale bars = 10 μm. C, in vitro transcription and translation of wild-type and deletion mutants of V5-tagged GSK3β and FBXO17 (no tag) was performed. V5-GSK3β samples included full-length protein (FL) and deletion mutants expressing amino acids 1–200, 1–250, 1–360, 51–420, 101–420, 151–420, and 201–420. Samples were immunoprecipitated using the FBXO17 antibody and immunoblotted with V5 antibody. Data represent mean ± S.D. of duplicate measurements. The data in each panel are representative of at least n = 3 independent experiments.

Figure 6.

FBXO17 abrogates inflammatory cytokine production in lung epithelial cells. A, MLE-12 cells were stimulated with LPS (10 μg/ml) and lysates were collected at 0, 2, 4, and 8 h. Samples were immunoblotted (IB) with antibodies against GSK3β, FBXO17, and β-actin (loading control). A control (C) untreated sample is shown collected at 8 h. B, MLE-12 cells were transfected with FBXO17-V5 plasmid and cultured for 48 h. Cells were treated with TNFα (10 ng/ml) for 16 h. Samples were collected and analyzed by ELISA for IL-6 or KC. C, MLE-12 cells were transfected with FBXO17-V5 plasmid and cultured for 48 h. Cells were then treated with LPS (10 μg/ml) for 16 h. Samples were collected and analyzed by ELISA for IL-6 or KC. Data represent mean ± S.D. of duplicate measurements and were analyzed by Student's t test. The data in each panel are representative of at least n = 3 independent experiments with p values <0.05 (*) and <0.01 (**). D, MLE-12 cells were transfected with FBXO17-V5 with or without GSK3β-HA plasmid and cultured for 48 h. Cells were then treated with LPS (10 μg/ml) for 6 h. Samples were collected and analyzed by ELISA for KC. Data represent mean ± S.D. of duplicate measurements and were analyzed by Student's t test. The data in panel D are representative of at least n = 3 independent experiments with p values <0.01 (*) and <0.001 (**).