SP1/CTR1 mediated oxidative stress-induced cuproptosis in intervertebral disk degeneration

doi:10.21203/rs.3.rs-2585336/v1

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SP1/CTR1 mediated oxidative stress-induced cuproptosis in intervertebral disk degeneration

https://doi.org/10.21203/rs.3.rs-2585336/v1

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Intervertebral disk degeneration (IDD) is the primary pathology responsible for lower back pain. Oxidative stress-induced cell death plays a fundamental role in IDD pathogenesis. Cuproptosis is a recently discovered form of programmed cell death dependent on copper availability. Whether cuproptosis is involved in IDD progression remains unknown. Herein, we established in vitro and in vivo models to investigate the presence of cuproptosis in IDD and the mechanisms by which oxidative stress interacts with copper sensitivity in nucleus pulposus cells (NPCs). We found that expression of the key cuproptosis regulator ferrodoxin-1 (FDX1) increased with disk degeneration in both rat and human disks. Sublethal oxidative stress on NPCs led to increased FDX1 expression and cell death in the presence of Cu²⁺ at physiological concentrations, whereas knockdown of FDX1 inhibited cell death. Since copper homeostasis is involved in copper-induced cytotoxicity, we investigated the role of copper transport-related proteins, including importers (transporter copper transporter 1 [CTR1] ) and efflux pumps (ATPase transporter, ATP7A, and ATP7B). We found that expression of CTR1 and ATP7A increased under oxidative stress, and blocking CTR1 significantly reduced oxidative stress/copper-induced cell death. Moreover, we found that oxidative stress promoted the expression of specific protein 1 (SP1) and SP1-mediated CTR1 transcription. SP1 inhibition improved cell viability and decreased cell death rates, which was reversed by the administration of elesclomol, a copper ionophore. Importantly, SP1 inhibition both significantly preserved disk hydration and alleviated tissue degeneration. This suggest that oxidative stress mediates cuproptosis by increasing copper flux through the promotion of SP1-mediated CTR1 transcription. This study highlights the importance of cuproptosis in IDD progression and provides a promising therapeutic target for IDD treatment.

Biological sciences/Cell biology/Cell death

Biological sciences/Cell biology/Cell signalling

Intervertebral disc degeneration

Cuproptosis

oxidative stress

CTR1

SP1

Lower back pain (LBP) remains a leading cause of disability and its related financial burden¹. Approximately 40% of LBP cases are attributed to intervertebral disk degeneration (IDD)^2,3. Although surgery and medical treatment have progressed in recent years, IDD remains prevalent and difficult to reverse or alleviate^4,5. Therefore, the mechanisms underlying its development and progression should be clarified to unveil novel targets for therapy.

Cell death has been proven to be critical in the pathogenesis of IDD. Oxidative stress, proinflammatory mediators, and mechanical stress are fundamental components of IDD microenvironments, which lead to progressive loss of the extracellular matrix (ECM), increased cell senescence, altered healthy disk cell phenotypes, and, ultimately, increased cell death^6,7. Apoptosis, including the intrinsic and extrinsic apoptotic pathways, is the most widely studied programmed cell death involved in IDD⁸. Cell shrinkage, plasma membrane bubbling, and nuclear condensation and fragmentation are typical morphological changes in apoptotic cells⁹. Necroptosis, a defined form of regulated necrosis, is also involved in IDD-related cell death via the receptor-interacting protein kinase 1 (RIPK1)/receptor-interacting protein kinase 3 (RIPK3)/mixed-lineage kinase domain-like protein (MLKL) pathway¹⁰. Pyroptosis, a newly discovered inflammasome-mediated cell death pathway, has also been shown to participate in IDD as a cellular response to chronic inflammation¹¹. Although many types of programmed cell death have been identified in IDD, inhibition of these pathways does not fully prevent chemical or mechanical stress from leading to cell death, indicating that there may be other forms of apoptosis involved in the pathogenesis of IDD^12–14.

Cuproptosis, a newly discovered form of programmed cell death, is dependent on Cu availability, and is linked to ferrodoxin-1 (FDX1) levels¹⁵. After cupric ions are imported into cells by copper ion carriers, FDX1 reduces the cupric ions to cuprous ions with stronger toxicity. At the same time, FDX1 also promotes the lipoacylation and aggregation of tricarboxylic acid (TCA) cycle-related proteins in the mitochondrial matrix, such as DLAT and DLST, increasing the binding of cuprous ions to these lipoylation proteins, causing intracellular protein toxicity and leading to cell death¹⁶. Chemometric analysis of trace elements in the disk tissue showed that the concentration of Cu in the IDD tissue ranged from 0.97–6.089 mg/kg¹⁷. However, the biochemical properties of copper that may have relevance in IDD and the involvement of cuproptosis in IDD-related cell death remain unclear.

Reactive oxygen species (ROS) are widely associated with signal transduction, cell senescence, apoptosis, and phenotypic transformation, and play a critical role in the development of IDD^18,19. An imbalance between ROS production and the protective mechanism of antioxidants leads to the accumulation of ROS in IDD and causes oxidative stress²⁰. Although oxidative stress has been reported to be associated with a variety of cell death pathways, including apoptosis, necroptosis, and pyroptosis, a direct relationship between oxidative stress and cuproptosis has not been fully clarified^21–23.

Copper ion, a cofactor critical for the growth and development of all organisms, is involved in oxidative stress responses²⁴. Cu ions act as cofactors for enzymes, including Cu/Zn-superoxide dismutase, cytochrome C oxidase, and ascorbate oxidase, which mediate redox cycling between Cu²⁺ and Cu⁺ and catalyze the production of highly toxic hydroxyl radicals^24,25. Excessive copper exposure has also been shown to induce cellular oxidative stress and lead to over-activation of autophagy and eventual cell death, suggesting that oxidative stress does mediate copper toxicity^26–28. To our knowledge, most of the existing reports have focused on the regulatory effects of copper on oxidative stress responses, but whether oxidative stress mediates the metabolism of copper ions, such as intracellular transportation and cuproptosis, remains a research gap in the literature.

Herein, we investigated cuproptosis in IDD and determined its role in IDD-related cell death. More importantly, our team, for the first time, clarified the relationship between oxidative stress and cuproptosis, and identified that oxidative stress increases copper import and induces cuproptosis by upregulating the transcription of the high-affinity copper transporter (CTR1) protein by promoting the expression of its transcription factor, specific protein 1 (SP1).

Isolation and culture of primary rat nucleus pulposus cells (NPCs)

Sprague-Dawley rats (male, three months old) were used in this study. Rat NPCs were obtained as previously described²⁹. Briefly, NP tissues were isolated from rat lumbar disks after sacrifice. The tissues were shredded and digested in 0.25% type II collagenase (Sigma, St. Louis, MO, USA) at 37°C for 15 min. The digested mixture was centrifuged at 300 g for 5 min and suspended in DMEM/F-12 (Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, USA) at 37°C and 5% CO₂. The medium was changed every 2 days, and when the confluence reached 80–90%, the cells were passaged with trypsin-EDTA solution (Gibco, USA). The third passage was used for subsequent experiments.

Experimental design

To explore the effects of oxidative stress on copper sensitivity, NPCs were cultured with nonlethal tert-butyl hydroperoxide (TBHP, 0, 15, 30 µM) (Sigma-Aldrich, Missouri, USA) and/or CuCl₂ (0, 2, 4 µM, Sigma-Aldrich, USA) for 24 h. To determine whether SP1 mediated oxidative stress-induced cuproptosis, NPCs were incubated with an SP1 DNA-binding inhibitor, Mithramycin A (MA, 2 µM) (HY-A0122, MedChemExpress, New Jersey, USA), TBHP (30 µM), and CuCl₂ (4 µM) for 24 h. To ensure that SP1 mediated cuproptosis by affecting copper transport, after NPCs were treated with MA (2 µM), TBHP (30 µM) or/and CuCl₂ (4 µM) for 24 h, Elesclomol (0.1 µM) (HY-12040 MedChemExpress, USA) was administered for another 24 h.

IDD model establishment and injection

Experiments were approved by the Animal Experimentation Committee of the Huazhong University of Science and Technology ([2021] IACUC Number 3077). Male Sprague-Dawley rats (200–300 g) were acquired from the Laboratory Animal Center of the Huazhong University of Science and Technology (Wuhan, China). Rats were randomly divided into four groups: intact, IDD, IDD treated with normal saline (NS), and IDD treated with MA. Rats were anesthetized with 0.6% (w/v) pentobarbital sodium, and coccygeal vertebrae Co5/6 were located via manual palpation and counting, which was confirmed by trial radiography. The rat IDD model was established using a 22-gauge sterile needle puncture at a depth of 5 mm. The needle was rotated 360° and held for 30 s. Surgery was not performed in the intact group. Three days later, 2 µL of NS or MA (100 µg/ml) was injected into the operated NP disks via a 29-gauge Hamilton needle, and the needle was held for 1 min for solution diffusion. The IDD group underwent the same procedure without liquid injection. NS and MA injections were repeated every two weeks. Four weeks after the first dose, the rats were sacrificed, and disks were harvested for further radiography and histological analysis.

MRI Analysis

T2-weighted midsagittal sections of the coccygeal vertebra were obtained using an MRI scanner (Skyra 3.0T, Siemens, Germany). Three radiologists independently participated in the evaluation of disk grading based on the Pfirrmann MRI grade system³⁰. The T2-weighted signal intensity was measured using ImageJ software 1.52i (National Institutes of Health, Bethesda, MD, USA). The relative water content, representing the hydration of disk tissues, was calculated as: relative water content = \(\frac{{D}_{s}}{{D}_{o}}\) (D_s, T2-weighted intensity of disks after surgery; D_o, T2-weighted intensity of intact disks).

Histopathological and immunohistochemical evaluation

The rat disk tissues were fixed in paraformaldehyde (4%, w/v). After embedding in paraffin, 4.0 µm sections were cut using a rotary microtome (Thermo Scientific, Massachusetts, USA). Histological evaluation of the slides was performed using hematoxylin and eosin (H&E) and Safranin O-fast green (S&O) staining, following the manufacturer’s protocols. Histological grading was performed independently by three pathologists based on the histological grading scale of Han et al.³¹.

Immunohistochemical staining, with primary antibodies against SP1 (A14662, Abclonal, China), FDX1 (12592-1-AP, proteintech, Chicago, USA), CTR1 (A0773, Abclonal, China), ATP7A (A8399, Abclonal, China), and ATP7B (A5676, Abclonal, China), were performed to observe the expression levels of these proteins in the tissues. Under high magnification using an optical microscope (Olympus, Tokyo, Japan), five visual fields for each slide were randomly chosen for quantitative analysis and the proportion of positively stained cells was calculated.

The primary antibodies mentioned above were also used for immunofluorescence analysis. After dewaxing and dehydration, the slides were incubated with the corresponding primary antibody working solution overnight at 4°C. Fluorescence-conjugated secondary antibodies (AS0743 and AS074; Abclonal, China) were added to detect fluorescent signals before adding DAPI staining solution (Sigma, USA). An anti-fluorescence quenching sealer (Thermo Fisher Scientific, USA) was used for sealing. A fluorescence microscope (Olympus, Tokyo, Japan) was used to photograph sample fluorescence signals.

Western blot analysis

The culture medium was removed, and the cells were washed with PBS and lysed in lysis buffer (Beyotime, China) supplemented with the protease inhibitor phenylmethanesulfonylfluoride (PMSF, Beyotime) and phosphatase inhibitor cocktail I (Sigma, USA). The cell lysates were sonicated and centrifuged at 13,000 g for 15 min at 4°C. A BCA protein assay kit (Boster, China) was used to determine protein concentration, and the supernatants were denatured with SDS-PAGE denaturation buffer (Servicebio, China). 10 micrograms of protein were loaded into 10% or 15% SDS-PAGE gels, then transferred onto polyvinylidene fluoride (PVDF) membranes and blocked. The antibodies used were SP1 (A14662, Abclonal), FDX1 (12592-1-AP, Proteintech), CTR1 (A0773, Abclonal), ATP7A (A8399, Abclonal), ATP7B (A5676, Abclonal), and GAPDH (10494-1-AP, Proteintech). Membranes were immunoblotted overnight at 4°C. and were then incubated with secondary antibodies for 1 h at room temperature. Finally, the protein bands were visualized using enhanced chemiluminescence following the manufacturer’s instructions (Amersham Biosciences, Piscataway, NJ, USA). Data were processed using ImageJ and GraphPad Prism 8.

Flow cytometry of PI

The culture medium was removed, and the cells were washed with PBS. After digestion, cells were centrifuged at 300 g for 5 min, and then harvested for suspension with 100 µl of 1x binding buffer with addition of 2 µl propidium iodide (PI) (HYC2019, HYCEZMBIO, Wuhan, China). Cells were incubated in the dark at room temperature for 10 min and detected by flow cytometry (BD LSRII, Becton Dickinson) within 30 min. The proportion of PI + cells was calculated as the rate of cell death.

siRNA transfection

CTR1 and FDX1 siRNAs were purchased from GenePharma Co., Ltd. (Suzhou, China). Rat NPCs were transfected with CTR1 or FDX1 siRNAs (10 nM) using Lipofectamine 3000 (Thermo Fisher Scientific), according to the manufacturer’s instructions. The culture medium was then replaced with DMEM/F-12 medium after 24 h transfection, and incubated for a further 48 h for later experiments. The siRNA sequences used are listed in Supplementary Table 1.

Real-time PCR

Total RNA was harvested using the RNA-easy™ Isolation Reagent (Vazyme Biotech Co., Ltd., Nanjing, China), following the manufacturer’s instructions. NanoDrop 2000 analysis software was used to calculate the concentration of the extracted RNA. A reverse transcription kit (Vazyme Biotech Co., Ltd., Nanjing, China) was used for cDNA synthesis. The cycle threshold (CT) was determined using the SYBR PrimeScript RT-PCR Kit (Vazyme Biotech Co., Ltd, Nanjing, China) on a Step One Plus Real-Time PCR system (Bio-Rad, Hercules, CA, USA). The results were quantified using the 2 -△△CT method, with GAPDH levels used for normalization. The primer sequences used are listed in Supplementary Table 2.

Chip-qpcr

An Enzymatic Chromatin IP Kit (9003, CST, USA) was used to extract chromatin and DNA according to the manufacturer's protocol. A ChIP-grade anti-SP1 antibody (1 ug/6 ug chromatin, ab231778, Abcam, USA) was used to precipitate chromatin bound to SP1. Subsequent qPCR followed the above procedure, and the result was expressed as a percentage of the input. Primers were designed using SP1-related ChIP-seq datasets from the Cistrome Data Browser. Primers were designed in the promoter proximal region with the highest ChIP-seq peaks (Supplementary Table 3).

Preprocessing of bulk Transcriptome data

The bulk RNA-seq dataset consists of three control and six degenerative NP tissues obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo; accession number: GSE176205). We applied Salmon ver1.9.0 based on the Galaxy platform (https://usegalaxy.org/) in quasi-mapping mode with the Human Transcript sequences reference file from Genecode (https://www.gencodegenes.org/human/) to quantify the FASTA files of the GSE176205 dataset.

RNA-seq data analysis

All analyses were performed using R (ver 4.2.1) and Rstudio (https://posit.co/ver 2022.12.0 + 353). We aggregated Salmon-quant transcript level expression data at the gene level and imported the results into DESeq2 with Tximport (ver. 1.24.0). Differential analysis was performed using DESeq2 (ver 1.36.0)³². The GSVA package (version 1.44.5) was used to calculate the ssGSEA score as the cutoff value.

Single-cell RNA-seq data analysis

R packet Seurat ver4.2.0 was used to analyze the scRNA-seq data³³. After creating Seurat objects from GSE165722, we filtered out cells with less than 200 genes and more than 10% mitochondrial genes. Then, the gene expression of each cell was standardized using the SCTransform method, and the standardized samples were integrated using the CCA algorithm. After the best dimension was determined by the PCA method, the integrated Seurat object was reduced and visualized by t-distributed stochastic neighbor embedding (t-SNE)³⁴. Based on the Human Primary Cell Atlas (HPCA), SingleR was used to automatically annotate the reduced-dimensional dataset³⁵. The “FeaturePlot()” command was used to map the expression of a single gene in different cell populations.

Statistical analysis

Each experiment was performed independently with at least three biological replicates. All data are presented as the mean ± standard deviation (SD), and t-tests were conducted to analyze differences between the two groups. Multiple datasets were analyzed using one-way repeated-measures analysis of variance (ANOVA). Non-parametric data (Pfirrmann grade and histological score) were analyzed using the Kruskal-Wallis H test. Statistical charts were created using GraphPad Prism 8. p < 0.05 was considered statistically significant.

Cuproptosis may present in degenerated disks

To investigate the role of cuproptosis in disk degeneration, we first explored the expression of cuproptosis-related genes (CRGs) (LIAS, LIPT1, MTF1, ATP7A, ATP7B, CTR1, CDKN2A, DLAT, DLD, FDX1, GLS, PDHA1, and PDHB) in NP tissue, based on GSE176205, and found that more than half of the CRGs were significantly upregulated in degenerative NP tissue (Fig. 1A). We then used the ssGSEA score based on CRGs as an index to describe the degree of cuproptosis in NP tissues. The results showed that the cuproptosis index was significantly higher in degenerated NP tissues (Fig. 1B). We then enrolled tissue samples from patients who underwent lumbar diskectomy surgery (Supplementary Table 4), and categorized them into degenerated (Pfrimann Grade > II) and control (Pfrimann Grade I-II) groups. Disk tissues with different degrees of degeneration exhibited varied signal intensities on a T2-weighted MRI, indicating a decrease in the water content of NP tissues with greater IDD progression (Fig. 1C). S&O and toluidine blue–fast green staining displayed a reduction in proteoglycan content in the degenerated disks (Fig. 1D). Additionally, the expression of FDX1 and ATP7A increased with the progression of intervertebral disk degeneration, suggesting an increased potential of cuproptosis in degenerated disks (Fig. 1D, E).

Oxidative Stress Promotes Cuproptosis In Npcs In The Presence Of Copper

To examine the sensitivity of NPCs to copper, physiological concentrations of Cu^2 + 16 were administrated to NPCs with or without TBHP. PI flow cytometry showed no significant alteration in the cell death rate occurred when treated with Cu²⁺ at physiological concentrations, while the cell death rate correlated with the copper concentration gradient in the presence of TBHP at nonlethal doses (Fig. 2A). The CCK8 assay also revealed that cell viability was not impaired by Cu²⁺ treatment alone, but decreased when treated with TBHP, indicating that oxidative stress increased the sensitivity of cells to copper (Fig. 2B). FDX1 is the key regulator of cuproptosis ¹⁶. Western blotting and immunofluorescence staining showed that FDX1 expression gradually increased, correlating with the TBHP concentration gradient (Fig. 2C-D, and S1). FDX1 knockdown significantly reduced cell death induced by TBHP + Cu²⁺, and reversed the impaired cell viability (Fig. 2E). Therefore, oxidative stress increases the sensitivity of NPCs to copper and promotes cuproptosis.

Oxidative Stress Enlarged Copper Flux To Induce Cuproptosis

Copper ionophores (such as elesclomol) are small molecular transporters of copper ions in cells that increase the cytotoxicity of copper ions³⁶. Copper homeostasis plays a critical role in preventing the adverse effects of copper on cells³⁷. Therefore, we examined the copper importer CTR1, and the Cu-efflux pumps ATP7A and ATP7B, based on GSE165722. We found that CTR1, ATP7A, and ATP7B were mainly expressed in chondrocyte-like cells, among which the average expression and percentage of expressed CTR1 cells were the most distinct (Figure A, B, S2). Western blot analysis revealed a 5.76-fold increase in CTR1 expression and a 4.02-fold increase in ATP7A expression when treated with TBHP (30 µM), while no significant increase was observed in ATP7B (Fig. 3C)³⁸. Immunofluorescence staining also supported an increase in CTR1 and ATP7A, but not ATP7B (Fig. 3D, S3). SiCtr1-307 and siCtr1-428 were identified as were effective at knocking down the gene expression of Ctr1, were denoted as siCtr1-1 and siCtr1-2, respectively (Fig. 3E and 3F). We found that silencing of CTR1 improved cell viability and reduced the cell death rate (Fig. 3G and 3H). Therefore, oxidative stress elevates the expression of CTR1 and ATP7A to increase copper flux in cells, and a higher fold increase in CTR1 may lead to copper accumulation in cells, which leads to cuproptosis.

The Transcription Factor Of Ctr1, Sp1, Is Upregulated By Oxidative Stress

SP1 promotes CTR1 transcription during copper deficiency, and plays a critical role in copper homeostasis³⁹. We found that SP1 was highly expressed in degenerated human disks and in the NP tissues of rat IDD models (Fig. 4A and 4B). Immunofluorescence staining and western blotting showed that SP1 expression increased gradually with increasing TBHP concentration (Fig. 4C-E). Therefore, SP1 responds to oxidative stress and may serve as a stress sensor to mediate copper homeostasis.

Oxidative Stress Promoted Cuproptosis Via Increasing Sp1-mediated Ctr1 Transcription

Next, a CHIP-qPCR assay was performed to evaluate the effects of SP1 on CTR1 transcription in the presence of oxidative stress. Primers flanking CTR1 specifically amplified the DNA sequence immunoprecipitated by SP1 in TBHP-treated NPCs (Fig. 5A). The occupancy of SP1 near the CTR1 promoter increased significantly when the cells were treated with TBHP (Fig. 5A), indicating that oxidative stress promoted SP1-mediated CTR1 transcription. Mithramycin A (MA, 2 µM), an inhibitor of SP1 DNA binding, was used to block the effects of SP1⁴⁰. The expression of CTR1 was significantly reduced, whereas that the expressions of FDX1, ATP7A, and ATP7B were not significantly affected by MA (Fig. 5B).

Moreover, SP1 inhibition reduced cell death induced by TBHP and Cu²⁺ (Fig. 5C). Cell viability was significantly impaired in the TBHP + CuCl₂ group, and MA significantly reversed these effects (Fig. 5D). Elesclomol (0.1 µM) was used to promote the uptake of copper ions, which resulted in a reversal of the MA-mediated reduction in cell death (Fig. 5E), and further impairment of cell viability (Fig. 5F). These results suggest that oxidative stress promotes the influx of copper ions by increasing SP1-mediated CTR1 transcription, thus leading to cuproptosis.

Inhibiting Sp1 Alleviated Disk Degeneration In Vivo

We further investigated the role of SP1 in the rat IDD model. T2-weighted MRI images showed that needle puncture of Co 5/6 significantly reduced NP signaling, while supplementary treatment with MA preserved NP intensity (Fig. 6A). A modified Pfrimann grading was performed to analyze the degenerative grade of each group, which showed that the severity of IDD was partly diminished by MA (Fig. 6B). Quantitative analysis of the water content based on MRI images indicated that MA preserved NP hydration (Fig. 6C). H&E and S&O staining showed a sparse number of surviving cells, little proteoglycan content, and irregular distribution of extracellular matrix in the NP tissues after needle puncture, whereas the application of MA partially reversed the disruption of NP tissues (Fig. 6D). Furthermore, histological grading showed that the impaired morphology of the disks could be rescued by exenatide application (Fig. 6E). IHC staining of cuproptosis-related proteins demonstrated that FDX1 and CTR1 expression significantly increased in the rat IDD model, whereas MA application reduced CTR1 expression (Fig. 6F). In addition, ATP7A and ATP7B levels were not obviously altered after needle puncture and MA application (Fig. 6F). Therefore, blocking SP1 to reduce copper flux is an effective approach for inhibiting cuproptosis, thus alleviating intervertebral disk degeneration.

Cuproptosis is a newly reported programmed cell death that relies on FDX1-induced Cu²⁺ to Cu⁺ reduction and TCA protein lipoylation. Since cell death plays a critical role in the pathogenesis of IDD, we were curious about whether cuproptosis was involved³⁶. Based on RNA-seq data, we found a significant association between cuproptosis and disk degeneration, which was verified by the clinical data that FDX1 expression was increased in IDD samples of higher grades compared to lower grades. Knockdown of FDX1 alleviated cell death, suggesting the presence of cuproptosis in IDD. Trace element analysis revealed that a higher concentration of copper was found in healthy disks than in IDD disks⁴¹. The upregulation of cuproptosis proteins coupled with the simultaneous reduction in detectable dry weight of copper in IDD suggests that the regulation of copper homeostasis may play a critical role in this phenomenon.

For Cu uptake, Cu²⁺ is reduced to Cu⁺ by cell membrane-associated reductases for CTR1, which has a pseudo-cone-shaped pore through the lipid bilayer, with a narrow extracellular Cu⁺ entry site⁴². After Cu⁺ is transported through the pores formed by CTR1 monomers, it is delivered from the C-terminal end of CTR1 at the cytoplasmic membrane to antioxidant protein 1 (Atox1), a Cu chaperone³⁸. The Cu-Atxo1 complex delivers Cu⁺ to the Cu efflux pumps ATP7A and ATP7B³⁸. Previous studies have reported that the distribution of ATP7A and 7B is tissue-specific. ATP7A is mainly expressed in the intestinal epithelium, whereas ATP7B is mainly expressed in the liver and brain^43,44. We first identified a significant increase in the expressions of CTR1 and ATP7A in disks with higher degeneration grades, suggesting a relatively higher Cu flux in IDD. Overexpression of CTR1 has been reported to increase cell death by inducing higher cellular sensitivity to copper ions at physiological concentrations (0.5-4 µM) ³⁶. Therefore, upregulation of CTR1 in IDD may also contribute to increased copper sensitivity and lead to increased cuproptosis. However, the regulation of CTR1 in IDD has not yet been reported.

ROS are ubiquitous second messengers that fine-tune a variety of physiological and pathological processes within the cell. ROS can indirectly induce gating of TRPM2, a Ca²⁺ transport protein, by promoting the second messenger adenosine diphosphate⁴⁵, while directly activating many other transporters via modification of cysteine residues at the pore-forming regions located within the plasma membrane⁴⁶. However, the role of ROS in copper transport has not been clarified. We found that oxidative stress increased the protein expression of CTR1 and ATP7A, as well as the key regulator of cuproptosis, FDX1, but not ATP7B. Moreover, Cu²⁺ at physiological concentrations caused cell death in a dose-dependent manner in the presence of TBHP, whereas the effect was diminished its absence. Knocking down CTR1 and FDX1 reversed TBHP-induced cellular sensitivity to Cu²⁺, suggesting that oxidative stress caused cuproptosis by promoting copper flux in NPCs.

We previously investigated the effects of specificity protein 1 (SP1), a ubiquitously expressed transcription regulator, in response to compression stress on NPCs and found that SP1 regulates ROS accumulation by activating TP53-induced glycolysis and apoptosis regulator (TIGAR)⁴⁷. SP1 has also been reported to regulate proinflammatory cytokine-induced catabolic enzyme gene expression in NPCs⁴⁸. Notably, SP1 is oxidative stress-inducible and mediates many cellular processes, including inflammation, oxidative stress, and apoptosis ^49,50. Therefore, SP1 might play a critical role in regulating the pathophysiological processes of NPCs under oxidative stress. More importantly, Sp1 is a key regulator of copper homeostasis³⁹. Under copper deficiency, SP1 binds to its own promoter to upregulate its own expression, and further binds to the GC box sequences at the promoters of CTR1 to promote its expression and increase copper import^51,52. Thus, we were curious whether SP1 regulates CTR1 expression under oxidative stress with copper ions at physiological concentrations. We identified an upregulation of SP1 in IDD and in response to oxidative stress and found that oxidative stress promoted SP1-mediated CTR1 transcription. Moreover, Sp1 inhibition reversed the oxidative stress-induced upregulation of CTR1 and protected NPCs against cuproptosis. Therefore, the alteration of copper flux by oxidative stress is regulated by SP1 and its DNA binding to CTR1 promoters.

In summary, our research is the first to identify that cuproptosis participates in IDD-related cell death and revealed that oxidative stress mediated cuproptosis by increasing copper flux by promoting SP1-mediated CTR1 transcription (Fig. 7). This study highlights the importance of cuproptosis in IDD progression and provides a promising therapeutic target for IDD treatment.

Data availability

The data used to support the findings of this study have been included in this article.

Declaration of competing interest

The authors declare no conflicts of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China [82202764] [81974352] [82202765] [82202763], China Postdoctoral Science Foundation [2021M701331], Natural Science Foundation of Hubei Province [2020CFB778], and Scientific Research Training Program for Young Talents from Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.

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SP1/CTR1 mediated oxidative stress-induced cuproptosis in intervertebral disk degeneration

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Abstract

Figures

Introduction

Methods

Isolation and culture of primary rat nucleus pulposus cells (NPCs)

Experimental design

IDD model establishment and injection

MRI Analysis

Histopathological and immunohistochemical evaluation

Western blot analysis

Flow cytometry of PI

siRNA transfection

Real-time PCR

Chip-qpcr

Preprocessing of bulk Transcriptome data

RNA-seq data analysis

Single-cell RNA-seq data analysis

Statistical analysis

Results

Cuproptosis may present in degenerated disks

Oxidative Stress Promotes Cuproptosis In Npcs In The Presence Of Copper

Oxidative Stress Enlarged Copper Flux To Induce Cuproptosis

The Transcription Factor Of Ctr1, Sp1, Is Upregulated By Oxidative Stress

Oxidative Stress Promoted Cuproptosis Via Increasing Sp1-mediated Ctr1 Transcription

Inhibiting Sp1 Alleviated Disk Degeneration In Vivo

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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