HSF1 Increases EOGT-Mediated Glycosylation of Notch1 to Promote IL-1β-Induced Inflammatory Injury of Chondrocytes

Abstract

Objective: Osteoarthritis (OA) is the most common arthritic disease in humans. Nevertheless, the pathogenic mechanism of OA remains unclear. This study aimed to explore that heat-shock transcription factor 1 (HSF1) facilitated interleukin-1 beta (IL-1β) chondrocyte injury by increasing Notch1 O-linked N-acetylglucosamine (O-GlcNAc) modification level.

Design: Human chondrocytes were incubated with 5 ng/ml interleukin-1 beta (IL-1β) for 24 h to establish OA cell model. The messenger RNA (mRNA) or protein expressions were assessed using reverse transcription-quantitative polymerase chain reaction, western blot, or immunofluorescence. Chondrocyte viability was examined by Cell Counting Kit-8 assay. Enzyme-linked immunosorbent assay was employed to detect the secretion levels of interleukin-6 (IL-6) and interleukin-8 (IL-8). Immunoprecipitation was adopted to detect Notch1 O-GlcNAc modification level. The interaction between HSF1 and epidermal growth factor-like (EGF) domain-specific O-GlcNAc transferase (EOGT) promoter was analyzed by dual-luciferase reporter gene and chromatin immunoprecipitation assays.

Results: Herein, our results demonstrated that HSF1, EOGT, Notch1, and Notch1 intracellular domain (NICD1) expressions in chondrocytes were markedly increased by IL-1β stimulation. EOGT elevated Notch1 expression in IL-1β-treated chondrocytes by increasing Notch1 O-GlcNAc modification level. EOGT silencing reduced IL-1β-induced chondrocyte inflammatory injury. In addition, HSF1 knockdown relieved IL-1β-induced chondrocyte inflammatory injury. Molecular interaction experiment proved that HSF1 transcriptionally activated EOGT expression in IL-1β-treated chondrocytes.

Conclusions: HSF1 promoted IL-1β-induced inflammatory injury in chondrocytes by increasing EOGT-mediated glycosylation of Notch1.

Keywords: EOGT; HSF1; Notch; O-glycosylation; chondrocytes; osteoarthritis.

Figure 1.

The high expression of Notch1 in IL-1β-treated chondrocytes was associated with EOGT-mediated O-GlcNAc modification. Human chondrocytes were incubated with 5 ng/ml IL-1β for 24 h. (A) IP was adopted to detect Notch1 O-GlcNAc modification level in cells. (B) EOGT protein level in cells was measured by western blot. (C-D) EOGT expression in chondrocytes following si-NC or si-EOGT transfection was measured by RT-qPCR and western blot. IL-1β-treated chondrocytes were transfected with si-NC or si-EOGT. (E) Western blot was adopted to examine EOGT, Notch1, and NICD1 protein levels in cells. (F) Notch1 O-GlcNAc modification level was measured using IP. Data were expressed as mean ± SD. All our data were obtained from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. EOGT = epidermal growth factor–like domain–specific O-GlcNAc transferase; IL-1β = interleukin-1 beta; RT-qPCR = reverse transcription-quantitative polymerase chain reaction.

Figure 2.

EOGT knockdown alleviated IL-1β-induced chondrocyte inflammatory injury. EOGT knockdown was induced in IL-1β-treated chondrocytes. (A) IL-6 and IL-8 secretion levels in chondrocytes were assessed by ELISA. (B-C) IF was adopted to detect MMP13 and ADAMTS5 fluorescence intensity in chondrocytes. Data were expressed as mean ± SD. All our data were obtained from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. EOGT = epidermal growth factor–like domain–specific O-GlcNAc transferase; IL-1β = interleukin-1 beta; ELISA = enzyme-linked immunosorbent assay.

Figure 3.

HSF1 was highly expressed in IL-1β-treated chondrocytes and was involved in Notch1-mediated inflammatory injury. (A-B) HSF1 expression level in human chondrocytes after IL-1β stimulation was measured by RT-qPCR and western blot. (C-D) RT-qPCR and western blot were adopted to examine HSF1 expression level in human chondrocytes following si-NC or si-HSF1 transfection. HSF1 knockdown was induced in IL-1β-treated chondrocytes. (E-F) HSF1 expression in chondrocytes was measured by RT-qPCR and western blot. (G) Notch1 and NICD1 protein levels in cells were examined using western blot. (H) ELISA was adopted to detect IL-6 and IL-8 secretion levels in chondrocytes. (I-J) IF was adopted to detect MMP13 and ADAMTS5 fluorescence intensity in cells. Data were expressed as mean ± SD. All our data were obtained from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. HSF1 = heat-shock transcription factor 1; EOGT = epidermal growth factor–like domain–specific O-GlcNAc transferase; IL-1β = interleukin-1 beta; RT-qPCR = reverse transcription-quantitative polymerase chain reaction; ELISA = enzyme-linked immunosorbent assay; IF = immunofluorescence.

Figure 4.

HSF1 transcriptionally activated EOGT expression in IL-1β-treated chondrocytes. (A) The potential binding sites between HSF1 and EOGT promoter was predicted using JASPAR. (B-C) IL-1β-treated chondrocytes were transfected with si-NC or si-HSF1, and EOGT expression level in cells was assessed by RT-qPCR and western blot. (D) The interaction between HSF1 and EOGT promoter was analyzed using dual-luciferase reporter gene assay. (E-F) The interaction between HSF1 and EOGT promoter was analyzed using ChIP assay. Data were expressed as mean ± SD. All our data were obtained from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. HSF1 = heat-shock transcription factor 1; EOGT = epidermal growth factor–like domain–specific O-GlcNAc transferase; IL-1β = interleukin-1 beta; RT-qPCR = reverse transcription-quantitative polymerase chain reaction; ChIP = chromatin immunoprecipitation.