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

Abstract

ObjectiveOsteoarthritis (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.DesignHuman 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.ResultsHerein, 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.ConclusionsHSF1 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.