14-3-3 epsilon is an intracellular component of TNFR2 receptor complex and its activation protects against osteoarthritis

Abstract

Objectives: Osteoarthritis (OA) is the most common joint disease; however, the indeterminate nature of mechanisms by which OA develops has restrained advancement of therapeutic targets. TNF signalling has been implicated in the pathogenesis of OA. TNFR1 primarily mediates inflammation, whereas emerging evidences demonstrate that TNFR2 plays an anti-inflammatory and protective role in several diseases and conditions. This study aims to decipher TNFR2 signalling in chondrocytes and OA.

Methods: Biochemical copurification and proteomics screen were performed to isolate the intracellular cofactors of TNFR2 complex. Bulk and single cell RNA-seq were employed to determine 14-3-3 epsilon (14-3-3ε) expression in human normal and OA cartilage. Transcription factor activity screen was used to isolate the transcription factors downstream of TNFR2/14-3-3ε. Various cell-based assays and genetically modified mice with naturally occurring and surgically induced OA were performed to examine the importance of this pathway in chondrocytes and OA.

Results: Signalling molecule 14-3-3ε was identified as an intracellular component of TNFR2 complexes in chondrocytes in response to progranulin (PGRN), a growth factor known to protect against OA primarily through activating TNFR2. 14-3-3ε was downregulated in OA and its deficiency deteriorated OA. 14-3-3ε was required for PGRN regulation of chondrocyte metabolism. In addition, both global and chondrocyte-specific deletion of 14-3-3ε largely abolished PGRN's therapeutic effects against OA. Furthermore, PGRN/TNFR2/14-3-3ε signalled through activating extracellular signal-regulated kinase (ERK)-dependent Elk-1 while suppressing nuclear factor kappa B (NF-κB) in chondrocytes.

Conclusions: This study identifies 14-3-3ε as an inducible component of TNFR2 receptor complex in response to PGRN in chondrocytes and presents a previously unrecognised TNFR2 pathway in the pathogenesis of OA.

Keywords: Chondrocytes; Inflammation; Osteoarthritis.

Fig. 1.. 14-3-3ε is an intracellular component of TNFR2 complex in chondrocyte and down-regulated in OA cartilage.

(a) Schematic of the experimental design to identify potential molecules recruited to TNFR2 intracellular domain (ICD) in response to PGRN stimulation. Summary of the hits that were specifically recruited to activated TNFR2 complexes in human C28I2 chondrocytes. (b) Chondrocytes isolated from WT or 14-3-3ε^Agc1 mice were treated with 10ng/ml TNFα or/and 200ng/ml PGRN for 30 min, then immunoprecipitated with 14-3-3ε or TNFR2 antibodies, and detection of TNFR2 and 14-3-3ε by immunoblotting. Results shown are representative of 3 biological replicates. (c) Volcano plots for gene expression of human OA (n = 4) versus normal (n=3) cartilage. Genes in red (up-regulated in OA) and blue (down-regulated in OA) have Benjamini–Hochberg adjusted P < 0.05. (d, e) Relative mRNA expressions of GRN (gene encoding PGRN) (d) and YWHAE (gene encoding 14-3-3ε) (e) in human OA versus normal cartilage by RNA-seq. (f) Unbiased clustering of scRNA-seq data from human nonarthritic (n = 3) and OA (n = 4) revealed 7 distinct cell clusters. (g-j) Expression of COMP (g), GRN (h), YWHAE (i) and TNFRSF1B (gene encoding TNFR2) (j) across the cell clusters. Each dot represents a single cell and colors correspond to the expression level of a gene in each cell. (k) qRT-PCR analysis of 14-3-3ε in human OA (n = 22) and normal (n = 21) cartilage. (l, m) Immunohistochemical staining of 14-3-3ε and quantification of 14-3-3ε positive cells in joint section collected from WT mice subjected to sham or DMM surgery (n = 8 mice per group). Scale bar, 50μm. (n) Relative 14-3-3ε mRNA level in cartilage isolated from sham or DMM operated mice (n = 8 mice per group). Data are mean ± SD.

Fig. 2.. 14-3-3ε deletion exaggerates naturally occurring phenotype with age.

(a) Immunohistochemical staining for 14-3-3ε, Safranin O, Movat pentachrome, and H&E staining in knee joint section collected from 14-3-3ε^f/f (n = 3) and 14-3-3ε−/− (n = 4) mice at age 18 months. Scale bar, 100μm. Representative image is shown. (b, c) Scoring of proteoglycan loss and cartilage thickness in 14-3-3ε^f/f and 14-3-3ε−/− mice at age 18 months, respectively. (d) Quantitation of the composition of the articular cartilage in 14-3-3ε^f/f and 14-3-3ε−/− mice at 18 months based on Movat pentachrome staining (yellow: bone; blue: cartilage). (e) Micro-CT scan and three-dimensional reconstruction of the knee joint from 18 months old 14-3-3ε^f/f and 14-3-3ε−/− mice, the region marked in red is osteophyte. (f, g) Three-dimensional microCT images and quantification of thickness for the medial compartment of the tibial subchondral bone of 18 months old 14-3-3ε^f/f and 14-3-3ε−/− mice. (h) Representative image of immunohistochemical staining for Aggrecan neoepitope, COMP fragment, ColX and MMP13 in WT and 14-3-3ε−/− knee section at age 18 months. Scale bar, 50μm. (i-l) Mmp13, Adamts5, Cox2 and Nos2 mRNA levels in cartilage from WT (n = 3) and 14-3-3ε−/− (n = 4) at age 18 months. Data are mean ± SD, * P < 0.05 or ** P < 0.01.

Fig. 3.. 14-3-3ε is required for PGRN regulation of chondrocyte metabolism.

(a) Schematic for generating 14-3-3ε−/− human C28I2 chondrocytes using CRISPR/Cas9 technology. (b) Western blotting to confirm the loss of 14-3-3ε in 14-3-3ε knockout C28I2 cells. Cell lysates were examined by immunoblotting with 14-3-3ε antibody. (c) mRNA levels of Col2, Acan and COMP in control and 14-3-3ε knockout C28I2 cells treated with or without 200ng/ml PGRN for 24hrs, assayed by qRT-PCR analysis. (d) mRNA levels of Mmp13, Adamts5, Cox2 and Nos2 in control and 14-3-3ε knockout C28I2 cells treated with 10ng/ml TNFα in the absence or presence of 200ng/ml PGRN for 24 hrs, assayed by qRT-PCR analysis. (e) Expression of Flag-14-3-3ε in control and 14-3-3ε knockout C28I2 cells, assayed by western blot. (f) mRNA levels of Col2 and Acan in PGRN (200ng/ml) treated control or 14-3-3ε knockout C28I2 cells with or without re-expression of 14-3-3ε, assayed by qRT-PCR analysis. (g) Control and 14-3-3ε knockout C28I2 cells with or without re-expression of 14-3-3ε were treated with 10ng/ml TNFα in the absence or presence of 200ng/ml PGRN for 24 hrs. mRNA levels of Mmp13, Adamts5, Cox2 and Nos2 were measured by qRT-PCR. Data are mean ± SD; n = 4 biological replicates; * P < 0.05 or ** P < 0.01.

Fig. 4.. Global deletion of 14-3-3ε regulates OA pathogenesis and largely abrogates PGRN’s therapeutic effects against OA.

(a) Schematic of the experimental outline. 14-3-3ε^f/f and Rosa26-ERT2;14-3-3ε^f/f (i.e. 14-3-3ε−/−) mice are injected with tamoxifen at 10-weeks old, and DMM operation is performed on 3months old mice. n = 8 mice per group. (b) Representative images of Safranin O/Fast green stained sections of knee joints from 14-3-3ε^f/f and 14-3-3ε−/− mice treated with or without PGRN for 8 weeks. Scale bar, 50μm. (c) Quantitative analysis of OARSI score, osteophyte development, and subchondral bone plate (SBP) thickness in different group of mice. (d) Representative images of immunohistochemical staining for MMP13, Aggrecan neoepitope, COMP fragment, and ColX in knee joint sections of 14-3-3ε^f/f and 14-3-3ε−/− mice treated with or without PGRN for 8 weeks. Scale bar, 50μm. (e) Serum COMP fragment levels in 14-3-3ε^f/f and 14-3-3ε−/− mice treated with or without PGRN for 8 weeks. (f) 2min travel distance and von Frey pain assay in DMM-operated WT and 14-3-3ε−/− mice treated with or without PGRN at the indicated time after surgery. Data are mean ± SD; ** P < 0.01.

Fig. 5.. Chondrocyte specific deletion of 14-3-3ε attenuates PGRN mediated protection against experimental OA.

(a) Schematic of the experimental outline. 14-3-3ε^f/f and Agc1-ERT2; 14-3-3ε^f/f (14-3-3ε^Agc1) mice are injected with tamoxifen at 10 weeks old, and DMM operation is performed on 3 months old mice. n = 8 mice per group. (b) Representative images of Safranin O/Fast green stained sections of knee joints from 14-3-3ε^f/f and 14-3-3ε^Agc1 mice treated with or without PGRN for 8 weeks. Scale bar, 50μm. (c) Quantitative analysis of OARSI score, osteophyte development, and subchondral bone plate (SBP) thickness in different group of mice. (d) Representative images of immunohistochemical staining for MMP13, Aggrecan neoepitope, COMP fragment, and ColX in knee joint sections of 14-3-3ε^f/f and 14-3-3ε^Agc1 mice treated with or without PGRN for 8 weeks. Scale bar, 50μm. (e) Serum COMP fragment levels in 14-3-3ε^f/f and 14-3-3ε^Agc1 mice treated with or without PGRN for 8 weeks. (f) 2min travel distance and von Frey pain assay in DMM-operated 14-3-3ε^f/f and 14-3-3ε^Agc1 mice treated with or without PGRN at the indicated time after surgery. Data are mean ± SD; ** P < 0.01.

Fig. 6.. Transcription factor Elk-1 is indispensable for the regulation of chondrocyte anabolism by PGRN/TNFR2/14-3-3ε.

(a) Transcriptional activities of 45 transcription factors are scanned using the transcription factor array. Primary articular chondrocytes isolated from WT, TNFR2−/−, and 14-3-3ε mice are transfected with the construct included in the kit for 48hrs, followed by treatment with 200ng/ml PGRN for another 24 hrs. (b) Elk-1 transcription activity in primary articular chondrocytes isolated from WT, TNFR2−/−, and 14-3-3ε−/− mice treated with PGRN for 24hrs. n = 6 for each group. (c) Immunoblotting of indicated protein in WT, TNFR2−/− and 14-3-3ε−/− primary articular chondrocytes treated with PGRN for different time points, as indicated. n = 4 for each group. (d) Densitometry analysis of immunoblotting results shown in (c). (e) Immunoblotting of indicated protein in control and 14-3-3ε knockout human C28I2 cells transfected with Flag-14-3-3ε construct prior to treatment with PGRN for indicated time. n = 4 for each group. (f) Densitometry analysis of immunoblotting results shown in (e). (g) Elk-1 transcription activity analysis in human chondrocytes treated with 10μM U0126 for 1h prior to treatment with 200ng/ml PGRN for 24hrs. n =6 for each group. (h) Immunoblotting of indicated protein in human chondrocytes treated with 10μM U0126 for 1h prior to treatment with 200ng/ml PGRN for different time points, as indicated. n = 4 for each group. (i) Densitometry analysis of immunoblotting results shown in (h). (j) mRNA levels of indicated molecules in human chondrocytes treated with 10μM U0126 for 1h prior to treatment with 200ng/ml PGRN for 24hrs. n = 4 for each group. (k) A proposed model depicting the signaling pathway by which PGRN (its derivative Atsttrin as well) binds to TNFR2 and recruits 14-3-3ε to the receptor complex, leading to the activation of chondrocyte anabolism and protection against OA. Data are mean ± SD; ** P < 0.01.