O-GlcNAc transferase regulates intervertebral disc degeneration by targeting FAM134B-mediated ER-phagy

Both O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) and endoplasmic reticulum-phagy (ER-phagy) are well-characterized conserved adaptive regulatory mechanisms that maintain cellular homeostasis and function in response to various stress conditions. Abnormalities in O-GlcNAcylation and ER-phagy have been documented in a wide variety of human pathologies. However, whether O-GlcNAcylation or ER-phagy is involved in the pathogenesis of intervertebral disc degeneration (IDD) is largely unknown. In this study, we investigated the function of O-GlcNAcylation and ER-phagy and the related underlying mechanisms in IDD. We found that the expression profiles of O-GlcNAcylation and O-GlcNAc transferase (OGT) were notably increased in degenerated NP tissues and nutrient-deprived nucleus pulposus (NP) cells. By modulating the O-GlcNAc level through genetic manipulation and specific pharmacological intervention, we revealed that increasing O-GlcNAcylation abundance substantially enhanced cell function and facilitated cell survival under nutrient deprivation (ND) conditions. Moreover, FAM134B-mediated ER-phagy activation was regulated by O-GlcNAcylation, and suppression of ER-phagy by FAM134B knockdown considerably counteracted the protective effects of amplified O-GlcNAcylation. Mechanistically, FAM134B was determined to be a potential target of OGT, and O-GlcNAcylation of FAM134B notably reduced FAM134B ubiquitination-mediated degradation. Correspondingly, the protection conferred by modulating O-GlcNAcylation homeostasis was verified in a rat IDD model. Our data demonstrated that OGT directly associates with and stabilizes FAM134B and subsequently enhances FAM134B-mediated ER-phagy to enhance the adaptive capability of cells in response to nutrient deficiency. These findings may provide a new option for O-GlcNAcylation-based therapeutics in IDD prevention.


Western blot analysis
The corresponding protein extraction kits (Beyotime, Nantong, China) were used to lyse cells and extract protein samples according to the kit instructions. 40 μg protein of each sample were loaded onto appropriate 8%-12% sodium dodecyl sulfatepolyacrylamide gels to separate protein bands based on the molecular weight of the target protein and transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). Then, the membranes were incubated with primary antibodies at the appropriate dilutions overnight at 4 °C prior to incubation with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature, detection with an enhanced chemiluminescence imaging system (Bio-Rad) and quantification of band densities with ImageJ software. The antibody dilutions were as follows: anti-OGT
Fluorescence signals were visualized by fluorescence microscopy (Olympus, BX53, Melville, NY, USA) and analyzed using ImageJ software.

Apoptosis assay
An Annexin V-FITC/PI Apoptosis Detection Kit (KeyGEN, Nanjing, China) was used to evaluate apoptosis according to the manufacturer's instructions. In brief, after the indicated treatment, NP cells were harvested with 0.25% trypsin (containing no EDTA).
After washed three times with PBS, the cells were stained with Annexin V-FITC (annexin V) and propidium iodide (PI) and analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA). the labeled cells of Annexin V+/PI-and Annexin V+/PI+ were considered as the apoptotic cells.

Transmission electron microscopy
Transmission electron microscopy (TEM) was used to detect the formation of autophagosomes and autolysosomes. In brief, the cells were collected and fixed with 2.5% glutaraldehyde for 4 h, rinsed in 0.1 M phosphate buffer (pH 7.4) and subsequently fixed with 1% osmium tetroxide (OsO4) at room temperature for 2 h. After fixation, the samples were dehydrated twice through an ethanol gradient (30%-50%-70%-80%-85%-90%-95%-100%) and embedded in epoxy resin. After ultrathin (70 nm) sectioning, the sections were double stained with 2% uranyl acetate and lead citrate for 20 min at room temperature and examined using transmission electron microscopy (Tecnai, FEI, USA).

Coimmunoprecipitation
Cell samples were lysed with NP-40 lysis buffer (1% NP-40, 30 mM Tris-HCl (pH 7.4), 10 μg/mL aprotinin, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 10 μg/mL leupeptin) containing protease inhibitor cocktail (MCE) for 20 min on ice, the lysates were then centrifuged at 12,000 × g at 4 °C for 10 min, the supernatants were collected. Then, a bicinchoninic acid (BCA) kit (Beyotime) was used to measure the total protein concentrations. After preclearing with 20 µL of protein A/G magnetic beads for 1 h at 4 °C, equal amounts of supernatants were incubated with IgG, anti-OGT, and anti-FAM134B antibodies plus protein A/G or anti-HA magnetic beads and anti-Flag magnetic bead conjugates overnight at 4 °C. Then, the beads were washed three times with NP-40 lysis buffer and boiled in 2 × SDS sample loading buffer for subsequent western blot analysis. :   Supplementary Fig. 1 The protein expression profile of OGA in human NP tissues.

Supplemental Figures
The protein expression level of OGA in human NP tissues was detected using IHC staining, representative images were shown (a) and relative OGA immunopositve staining cellratio were calculated (b), scale bar: 250 μm and 100 μm. The data are presented as the mean ± S.D. values. * P < 0.05. Supplementary Fig. 2 Effect of ND treatment, TMG, and OSMI-1 on cell viability in human NP cells. Atter exposed to ND treatment (a), TMG (b), and OSMI-1 (c) for the indicated time points (0, 6, 12, 24, and 36 hours), the viability of the human NP cells was measured by CCK-8 assay. The data are presented as the mean ± S.D. values. * * P < 0.01, * P < 0.05.

Supplementary Fig.3 Validation of OGT overexpression and knockdown in human NP cells.
(a, b) 72 hours post lentiviral transfection, the human NP cells were harvested and OGT expression were analyzed using western blot analysis, and the relative band densities were quantified. (c, d) 48 hours post siRNA transfection, the human NP cells were harvested and OGT expression were analyzed using western blot analysis, and the relative band densities were quantified. The data are presented as the mean ± S.D.
values. * * P < 0.01, * P < 0.05. Supplementary Fig. 4 Effects of OGA knockdown on ND-induced senescence and apoptosis in human NP cells. (a, b) after transfected with OGA siRNA for 48 hours, the OGA knockdown efficacy in NP cells was validated by western blot analysis, and the relative band densities were quantified. (c-f) NP cells were transduced with the OGA siRNA for 48 h and then subjected to ND treatment for 36 h. The senescence-and apoptosis-associated proteins p53, p21, p16, Bcl-2, Bax, and cleaved caspase 3 were evaluated using western blot analysis, and the relative band densities were quantified.
The data are presented as the mean ± S.D. values. * * P < 0.01, * P < 0.05. (a, b) 48 hours post siRNA transfection, the human NP cells were harvested and FAM134B expression were analyzed using western blot analysis, and the relative band densities were quantified. The data are presented as the mean ± S.D. values. * * P < 0.01, * P < 0.05.