RACK1 mediates the advanced glycation end product‐induced degradation of HIF‐1α in nucleus pulposus cells via competing with HSP90 for HIF‐1α binding

Abstract

Hyperglycemia can drive advanced glycation end product (AGE) accumulation and associated nucleus pulposus cell (NPC) dysfunction, but the basis for this activity has not been elucidated. Hypoxia‐inducible factor‐1α (HIF‐1α) is subject to cell‐type‐specific AGE‐mediated regulation. In the current study, we assessed the mechanistic relationship between AGE accumulation and HIF‐1α degradation in NPCs. Immunohistochemical staining of degenerated nucleus pulposus (NP) samples was used to assess AGE levels. AGE impact on NPC survival and glycolysis‐related gene expression was assessed via 3‐(4,5)‐dimethylthiazol(‐z‐y1)‐3,5‐di‐phenyltetrazolium bromide assay and quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR), while HIF‐1α expression in NPCs following AGE treatment was monitored via Western blot analysis and qRT‐PCR. Additionally, a luciferase reporter assay was used to monitor HIF‐1α transcriptional activity. The importance of the receptor for activated C‐kinase 1 (RACK1) as a mediator of HIF‐1α degradation was evaluated through gain‐ and loss‐of‐function experiments. Competitive binding of RACK1 and HSP90 to HIF‐1α was evaluated via immunoprecipitation. Increased AGE accumulation was evident in NP samples from diabetic patients, and AGE treatment resulted in reduced HIF‐1α protein levels in NPCs that coincided with reduced HIF‐1α transcriptional activity. AGE treatment impaired the stability of HIF‐1α, leading to its RACK1‐mediated proteasomal degradation in a manner independent of the canonical PHD‐mediated degradation pathway. Additionally, RACK1 competed with HSP90 for HIF‐1α binding following AGE treatment. AGE treatment of NPCs leads to HIF‐1α protein degradation. RACK1 competes with HSP90 for HIF‐1α binding following AGE treatment, resulting in posttranslational HIF‐1α degradation. These results suggest that AGE is an intervertebral disc degeneration risk factor, and highlight potential avenues for the treatment or prevention of this disease.

Melatonin alleviates the heat stress‐induced impairment of Sertoli cells by reprogramming glucose metabolism

“…In studies on nucleus pulposus cell dysfunction, HSP90 was shown to help stabilize HIF-1α in normoxia. 55 In our study, after blocking the binding of HSP90 to HIF-1α, glycolysis became weaker, and ATP, NADPH, and GSH levels decreased, while ROS levels increased markedly. These results demonstrate that the MTNR1B-HSP90-HIF-1α axis is essential to melatonin reprogramming in glucose metabolism and to attenuate heat stress-induced injury in SCs (Figure 8).…”

“…However, under normoxic conditions, HIF‐1α is commonly degraded. In studies on nucleus pulposus cell dysfunction, HSP90 was shown to help stabilize HIF‐1α in normoxia 55 . In our study, after blocking the binding of HSP90 to HIF‐1α, glycolysis became weaker, and ATP, NADPH, and GSH levels decreased, while ROS levels increased markedly.…”

Stimuli-responsive therapeutic systems for the treatment of diabetic infected wounds

“…31 The high concentrations of blood glucose in the physiological environment not only reduce the bioactivity of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α (HIF-1α) but also induce the non-enzymatic glycosylation reaction of biological macromolecules, further resulting in advanced glycation end products. 32 The resulting products will decrease the hydrophilicity of the extracellular matrix, further making a diabetic wound chronically remained inflamed. 33…”

Role of Advanced Glycation End Products in Intervertebral Disc Degeneration: Mechanism and Therapeutic Potential

“…Following AGEs treatment, the receptor for activated C-kinase 1 (RACK1) competes with heat shock protein 90 (HSP90) for binding to HIF-1 α , resulting in posttranslational HIF-1 α degradation and RACK1-mediated proteasomal degradation, independently of the canonical iron-dependent prolyl-hydroxylase domain- (PHD-) mediated degradation pathway. Under normoxic conditions, PHD proteins promote HIF-1 α degradation by the 26S proteasome [ 76 , 77 ]. The degradation of HIF-1 α disrupts the biological function of NP cells and promotes IDD.…”