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FGF23 Induction of O-Linked N-Acetylglucosamine Regulates IL-6 Secretion in Human Bronchial Epithelial Cells

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FGF23 Induction of O-Linked N-Acetylglucosamine Regulates IL-6 Secretion in Human Bronchial Epithelial Cells

Stefanie Krick et al. Front Endocrinol (Lausanne).

Abstract

The hexosamine biosynthetic pathway (HBP) generates the substrate for the O-linked β-N-acetylglucosamine (O-GlcNAc) modification of proteins. The HBP also serves as a stress sensor and has been reported to be involved with nuclear factor of activated T-cells (NFAT) activation, which can contribute to multiple cellular processes including cell metabolism, proliferation, and inflammation. In our previously published report, Fibroblast Growth Factor (FGF) 23, an important endocrine pro-inflammatory mediator, was shown to activate the FGFR4/phospholipase Cγ (PLCγ)/nuclear factor of activated T-cells (NFAT) signaling in chronic inflammatory airway diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). Here, we demonstrate that FGF23 increased the O-GlcNAc modification of proteins in HBECs. Furthermore, the increase in O-GlcNAc levels by FGF23 stimulation resulted in the downstream activation of NFAT and secretion of interleukin-6 (IL-6). Conversely, inhibition of FGF23 signaling and/or O-GlcNAc transferase (OGT)/O-GlcNAc reversed these effects. Collectively, these data suggest that FGF23 induced IL-6 upregulation and secretion is, at least, partially mediated via the activation of the HBP and O-GlcNAc levels in HBECs. These findings identify a novel link whereby FGF23 and the augmentation of O-GlcNAc levels regulate airway inflammation through NFAT activation and IL-6 upregulation in HBECs. The crosstalk between these signaling pathways may contribute to the pathogenesis of chronic inflammatory airway diseases such as COPD and CF as well as metabolic syndromes, including diabetes.

Keywords: FGF23 = fibroblast growth factor 23; IL-6 (Interleukin 6); NFAT (nuclear factor expression of activated T cell); O-GlcNAc; inflammation.

Figures

Figure 1
Figure 1
FGF23 stimulates the HBP/O-GlcNAc modification of proteins via the PLCγ signaling pathway in human bronchial epithelial cells. (A) Representative Immunoblots showing global O-GlcNAc, OGT, OGA, and β-Actin from HBECs treated as described. (B–D) Densitrometric quantitation of O-GlcNAc, OGT, and OGA from (A). (E) Representative Immunoblots showing phosphorylation of PLCγ and ERK, total PLCγ and ERK, and β-Actin from HBECs treated as described. (F,G) Densitrometric quantitation of Immunoblots from (A). (H) Representative Immunoblots of global O-GlcNAc, OGT, OGA, and β-Actin from HBECs treated with a PLCγ inhibitor (U73122 at 0, 0.1, and 1.0 μM) and FGF23 (20 ng/ml) for 24 h. (I–K) Densitrometric quantitation of O-GlcNAc, OGT, and OGA from (H). Western blots were performed as triplicates of the same experiment. Statistical analysis was done using ANOVA or Student's t-test showing means ± S.E.M. with *p < 0.05, **p < 0.01, and ***p < 0.001. Ctrl, Control; FGF23, fibroblast growth factor 23; TG, thiamet G (OGA inhibitor); OSMI-1, OGT inhibitor.
Figure 2
Figure 2
Knockdown of OGT reduces the O-GlcNAc modification of proteins. (A) Representative Immunoblots showing global O-GlcNAc, OGT, OGA, and β-Actin from HBECs treated as described or (B) HBECs were transfected with siRNA against OGT in the presence and absence of FGF23. Western blots were performed as triplicates of the same experiment. Ctrl, Control; FGF23, fibroblast growth factor 23; si ctrl, small interfering RNA control; and si OGT, small interfering RNA against OGT. Statistical analysis was done using ANOVA or Student's t-test showing means ± S.E.M. with *p < 0.05, **p < 0.01, and ***p < 0.001.
Figure 3
Figure 3
FGF23 and OGA inhibition both increase IL-6 secretion in human bronchial epithelial cells. (A) Bar graphs showing transcript analysis and fold change of IL-6 and IL-8 levels in HBECs treated in the presence or absence of FGF23, TG, or OSMI-1 for 24h. (B) IL-6 and IL-8 protein amounts determined by ELISA from conditioned media of HBECs. All experiments were done in triplicate and bar graphs shown respresent the mean ± S.E.M.(*p < 0.05; **p < 0.01, and ***p < 0.001). Ctrl, Control; FGF23, fibroblast growth factor 23; TG, thiamet G (OGA inhibitor); OSMI-1, OGT inhibitor.
Figure 4
Figure 4
The NFAT activation is regulated by FGF23 and OGA inhibition (TG) and is downstream of O-GlcNAc regulation. (A) FGF23 and TG activate NFAT as assessed by a luciferase-based reporter gene assay in HBECs and is reduced to normal levels by an FGFR4 inhibitor (R4) or blocked by OSMI-1 similar to the effects of the NFAT activation inhibitor, cyclosporine (CsA). (B) Western blots of O-GlcNAc, OGT, and OGA following knockdown of NFAT2c and NFAT3c. Experiments were performed in triplicate. Statistical analysis was done using ANOVA or Student's t-test showing means ± S.E.M. with *p < 0.05, and ***p < 0.001. Ctrl, Control; FGF23, fibroblast growth factor 23; TG, thiamet G (OGA inhibitor); R4, FGFR4 inhibitor; OSMI-1, OGT inhibitor; CsA, cyclosporine; and siNFAT, small interfering RNA against NFAT2c or 3c.
Figure 5
Figure 5
Gene silencing of NFAT2c leads to downregulation of IL-6 expression, while knockdown of both NFAT2c/3c results in reduced IL-6 secretion in HBECs. (A) Bar graphs showing transcript analysis and fold change of IL-6 levels following NFAT2/3C knockdown with siRNA in HBECs. (B) IL-6 protein level from conditioned media of HBECs as determined by ELISA. All experiments were done in triplicate and statistical analyses was done using ANOVA or Student's t-test showing means ± S.E.M. with **p < 0.01 and ***p < 0.001. si control, small interfering RNA control; siNFAT, small interfering RNA against NFAT2c or 3c; small interfering RNA against OGT; and CsA, cyclosporin.
Figure 6
Figure 6
Model. Upon binding to FGFR4 in human bronchial epithelial cells, FGF23 stimulates the phosphorylation of PLCγ, which increases the O-GlcNAc modification of proteins. The increase in O-GlcNAc results in NFAT activation and translocation from the cytoplasm to the nucleus where it drives the expression of IL-6 and subsequent secretion out of the cell.

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