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Original Article

Agonist-Induced Activation of Transient Receptor Potential Vanilloid 4 Promotes Autophagy and Extracellular Matrix Synthesis in the Rat Intervertebral Disc

Kohei Kuroshimaorcid, Yoshiki Takeokaorcid, Takashi Yurubeorcid, Takeru Tsujimotoorcid, Yutaro Kandaorcid, Kunihiko Miyazakiorcid, Hiroki Ohnishiorcid, Tomoya Matsuoorcid, Masao Ryuorcid, Naotoshi Kumagaiorcid, Yoshiaki Hiranakaorcid, Masahiko Furuyaorcid, Daisuke Nakagawaorcid, Yu Inoueorcid, Ryosuke Kuroda, Kenichiro Kakutaniorcid
Neurospine 2026;23(2):347-364.
Published online: April 30, 2026

Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan

Corresponding Author Yoshiki Takeoka Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Email: yoshiki_tkk@hotmail.com
• Received: November 2, 2025   • Revised: November 21, 2025   • Accepted: November 30, 2025

Copyright © 2026 by the Korean Spinal Neurosurgery Society

This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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  • Objective
    Transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel, has been implicated in intervertebral disc homeostasis; however, its role in autophagy regulation remains unclear. This study aimed to investigate whether agonist-induced TRPV4 activation promotes autophagy and extracellular matrix (ECM) synthesis in rat intervertebral discs.
  • Methods
    In vitro, rat nucleus pulposus (NP) cells were treated with the TRPV4 agonist (GSK1016790) under normal, serum-deprived, or interleukin-1β-stimulated conditions. Cell viability, intracellular Ca2+ influx, adenosine monophosphate-activated protein kinase/mammalian target of rapamycin (mTOR) (AMPK/mTOR) pathway, autophagy, ECM metabolism, apoptosis, and senescence were evaluated. In vivo, TRPV4 agonist was injected into the caudal discs subjected to temporary static compression, and disc changes were assessed by radiography, histomorphology, and immunofluorescence.
  • Results
    In vitro, agonist-induced TRPV4 activation rapidly increased intracellular Ca2+ influx and enhanced AMPK phosphorylation. A noncytotoxic concentration of the TRPV4 agonist (10 nM) was selected after dose-response testing. Under the inflammatory stress, TRPV4 agonist enhanced autophagy, promoted ECM synthesis, and suppressed apoptosis and senescence, leading to improved NP cell viability. In vivo, TRPV4 agonist treatment preserved radiographic disc height (p<0.01), reduced histomorphological degeneration (p<0.01), and increased expression of COL2A1, Brachyury, p-AMPK (phosphorylated AMPK), and autophagy markers (p<0.01) compared with controls.
  • Conclusion
    These findings demonstrated that TRPV4 activation promotes autophagy and ECM synthesis via the AMPK/mTOR pathway in rat discs and attenuates stress-induced degeneration, suggesting TRPV4 as a potential therapeutic target for disc degeneration.
  • Keywords: Transient receptor potential vanilloid 4, Autophagy, Intervertebral disc degeneration, Nucleus pulposus cell, Extracellular matrix, Spine
Low back pain affects up to 85% of people during their lifetime [1]. This condition often leads to disability, increased medical costs, and decreased workforce productivity [1]. Although the mechanism of low back pain is multifactorial, intervertebral disc degeneration is one of the independent causes [2]. Intervertebral disc is a complex, disc-shaped tissue consisting of the nucleus pulposus (NP) enclosed by the annulus fibrosus (AF) and cartilaginous endplates, forming the largest avascular organ in the human body [3]. Consequently, its nutrient reaches the disc primarily through diffusion across the endplates. With aging, however, endplate calcification and subchondral bone sclerosis hinder this nutrient transport [3], and such deprivation is suspected to accelerate disc degeneration [3]. Recent studies have suggested that disc degeneration involves a cascade of pathophysiological events in NP cells, particularly in autophagy [4]. Autophagy is a vital cellular process that preserves metabolic balance and removes misfolded proteins and damaged organelles, particularly under stress conditions such as hypoxia and nutrient deficiency [5]. The mammalian target of rapamycin (mTOR) signaling pathway acts as a key negative regulator of autophagy [6]. In parallel, degenerative changes in the disc are accompanied by biochemical degradation of the extracellular matrix (ECM) [7], which is controlled by the equilibrium between catabolic enzymes, mainly matrix metalloproteinases (MMPs), and their anticatabolic inhibitors, tissue inhibitors of metalloproteinases (TIMPs) [8]. Our previous study demonstrated that selective suppression of mTORC1 (mTOR complex 1) enhances autophagy and protects against ECM catabolism in human NP cells [9]. Moreover, autophagy mediated by autophagy‐related gene 5 was shown to maintain homeostasis in the rat intervertebral disc [10]. Collectively, these findings suggest a tight association among autophagy, tissue homeostasis, and ECM metabolism within the disc.
In addition to biochemical regulation, NP cells and their ECM respond to physiological mechanical loading through the activation of transient receptor potential (TRP) vanilloid 4 (TRPV4), as reported in bovine and human discs [11]. TRPV4 belongs to the TRP channel superfamily, a group of nonselective cation channels comprising several subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPP (polycystin), TRPML (mucolipin), TRPN (drosophila NOMPC) and TRPY (yeast) [12]. TRP channels serve essential functions in cellular physiology, acting as sensors for various stimuli, including temperature, pH, chemicals, cytokines, osmolarity and mechanical stress, and maintaining ion homeostasis [12]. Recent investigations in articular cartilage have further clarified the role of TRPV4: its activation by specific agonists enhances anabolic matrix turnover while suppressing catabolic processes, thereby promoting ECM accumulation [13]. Similarly, TRPV4 activation inhibited interleukin-1 beta (IL-1β)-induced nitric oxide release and prevented cartilage degradation and loss of mechanical properties in bovine cartilage explants [14].
Among TRP channels expressed in the disc tissue, TRPV4 shows the highest expression level [15-17]. Under hyperosmotic stress, notochordal cells exhibit autophagy induction through the Ca2+-dependent adenosine monophosphate-activated protein kinase (AMPK)/mTOR pathway, in which TRPV4-mediated Ca2+ influx is proposed as the initiating event [18]. In our previous loss-of-function study using siRNA-mediated TRPV4 knockdown in rat NP cells and disc tissues, suppression of TRPV4 resulted in reduced autophagy and ECM synthesis and ultimately led to progressive structural disc disruption [17].
Taken together, these observations led us to hypothesize that TRPV4 contributes to the maintenance of intradiscal homeostasis through the Ca2+‐dependent AMPK/mTOR pathway and autophagy (Fig. 1A). Therefore, the present study aimed to elucidate that TRPV4 activation by agonist promotes autophagy and ECM synthesis in rat intervertebral disc, both in vitro and in vivo.
1. Ethics Statement
All animal experiments were carried out in compliance with the guidelines by the Institutional Animal Care and Use Committee of the authors’ institution. The animal study protocol was approved by the Institutional Review Board of Kobe University Graduate School of Medicine (P210801, 23 August 2021).
2. Antibodies and Reagents
All antibodies and chemical reagents employed in this study are listed in Supplementary Table 1.
3. Cells
A total of forty‐four 12‐week‐old male Sprague Dawley rats (mean±standard deviation [SD], 464.7±8.7 g) obtained from CLEA Japan (Japan) were randomly assigned to in vitro and in vivo experiments. Coccygeal (C) intervertebral discs from 20 rats (mean±SD, 463.8±9.0 g) were harvested posteuthanasia and separated into NP and AF regions. NP tissues were enzymatically digested for 1 hour at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 0.114% collagenase type II. Isolated NP cells were cultured as a monolayer under 2% O2 at 37°C in DMEM containing 10% FBS until reaching approximately 80% confluence. Only first‐passage cells were used to preserve phenotypic characteristics [19-21].
Cells were seeded at specific densities depending on the experiment: 5.0×103 cells/well in 96‐well plates for viability assays, 1.5×104 cells/well in 96‐well plates for Ca2+ imaging, 1.5×105 cells/well in 6‐well plates for protein extraction, and 1.2×104 cells/well in 8‐well chamber slides for staining. For each assay, 5 replicates from 5 individual animals were used (each n=5) in accordance with previous studies [19-21].
To assess cytotoxicity, cell viability was first examined following treatment with various concentrations (0–100 nM) of the TRPV4 agonist (GSK1016790A; Sigma-Aldrich, USA) for 24 hours in DMEM containing 10% FBS. Viability was calculated using the cell counting kit-8 (CCK-8) and microscopic cell counting. In parallel, intracellular Ca2+ influx was evaluated using Fluo-4 AM to assess TRPV4-dependent activation of the AMPK/mTOR pathway.
To mimic pathological conditions such as nutrient deprivation and/or inflammation, NP cells were treated for 24 hours with TRPV4 agonist or dimethyl sulfoxide (DMSO, negative control) in DMEM with either 10% FBS, 0% FBS, or 0% FBS supplemented with 10-ng/mL IL-1β, a proinflammatory cytokine implicated in disc degeneration [22,23]. For all in vitro experiments, TRPV4 agonist was dissolved in DMSO and further diluted in culture medium to a final DMSO concentration of 0.1% (v/v). The negative control contained the same final concentration of DMSO (0.1% v/v) without TRPV4 agonist.
After treatment, CCK‐8 and microscopic cell counting were performed to evaluate viability. Western blotting (WB) was conducted to assess markers of autophagy, ECM metabolism, apoptosis, and senescence. Apoptotic cells were identified using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and cellular senescence was evaluated by senescence-associated beta-galactosidase (SA-β-gal) staining (Fig. 1B).
4. Animals and Surgical Procedures
Twenty-four rats (465.5±8.0 g) were used for in vivo experiments. TRPV4 expression in rat NP cells was confirmed by immunofluorescence prior to the in vivo procedures. To reproduce mechanical stress‐induced disc degeneration, a tail static compression model was utilized [19,24-26]. Under general anesthesia, an Ilizarov‐type external fixator equipped with springs was applied between the C8 and C10 vertebrae (n=24). Using a 33‐gauge needle inserted through a 5‐mm longitudinal skin incision, 2‐μL of TRPV4 agonist or control DMSO was injected into the C8–9 and C11–12 discs or the C9–10 and C12–13 discs, respectively [23,27]. A 1.3‐MPa axial force, comparable to that experienced in the human lumbar spine during moderate lifting [1,28], was applied to C8–9 and C9–10 discs for 24 hours and subsequently released. At various time points (0–56 days), disc tissues (C8–9, C9–10, C11–12, and C12–13) were harvested following radiographic imaging and analyzed by histomorphological safranin‐O staining and immunofluorescence (Fig. 1C).
Sample size was determined based on prior studies (n=5 in vitro and 6 in vivo/time point).19,23-27 Animals were maintained in specific pathogen‐free conditions with ad libitum access to food and water under a 12‐hour light/dark cycle, temperature of 23°C±2°C, and humidity of 55%±5%. Humane endpoints included ≥20% weight loss or significant behavioral changes. For intradiscal injections in vivo, TRPV4 agonist and control solutions were prepared in 0.1% (v/v) DMSO, similar to in vitro experiments.
5. Cell Viability Assay
Cell viability was quantified using CCK‐8 assay based on dehydrogenase activity, with absorbance measured at 450 nm on the Model 680 microplate reader. Microscopic images were obtained using the BZ‐X700 microscope, and adherent cells were counted in duplicate across 4 randomly selected low‐power fields (×100) (LPFs) using the ImageJ software (https://imagej.nih.gov/ij/).
6. Ca2+ Imaging
Cells were incubated with loading buffer containing Fluo-4 AM (342-90961, Dojindo Laboratories, Japan) at 37°C for 1 hour, and subsequently treated with recording medium containing 1.25 mmol/L of Probenecid. Following administration of TRPV4 agonist (0–20 nM), fluorescence images were captured using BZ‐X700 microscope, and fluorescent intensity was measured every 7 seconds for 3 minutes using a plate reader (EnSpire, PerkinElmer, USA).
7. Protein Extraction, Sodium Dodecyl Sulfate‐Polyacrylamide Gel Electrophoresis, and WB
Cells were lysed on ice using 3‐(N‐morpholino) propanesulfonic acid buffer supplemented with protease and phosphatase inhibitors. Tissue samples were homogenized at 4°C using the MS‐100R bead‐beating disrupter (2 cycles of 30 seconds) in tissue protein extraction reagent containing protease and phosphatase inhibitors. After centrifugation (20,000×g for 15 minutes at 4°C), the supernatant was collected, and protein concentration was measured by bicinchoninic acid assay.
Equal amounts of protein (30 μg) were mixed with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis buffer, denatured at 95°C for 5 minutes, separated on 7.5%–15% polyacrylamide gels, and transferred to polyvinylidene difluoride membranes. Membranes were incubated overnight (4°C) with primary antibodies (1:200–1:1,000 dilution) and then for 1 hour with secondary antibodies (1:400 dilution) at room temperature. Protein bands were visualized using enhanced chemiluminescence and imaged with the LAS‐3000 mini analyzer. Band intensity was quantified using ImageJ software.
WB was designed to analyze the intracellular expression of notochordal Brachyury and CD24 [22], anabolic Aggrecan, COL2A1 [29], anticatabolic TIMPs, catabolic MMPs, TRPV4, AMPK, mTOR, p70S6K, autophagy‐related p62/SQSTM1 and LC3 [30], apoptosis‐related poly (ADP‐ribose) polymerase (PARP) and cleaved caspase‐9 [31], and senescence‐related p53, p21/CIP1, and p16/INK4a [32] in vitro. Aggrecan samples were deglycosylated using chondroitinase ABC and endo‐β‐galactosidase before analysis [33]. Protein expression was normalized to tubulin and presented as relative percentage of control.
8. Paraffin‐Embedded Tissue Preparation
After euthanasia, rat caudal spinal units (vertebral body-discvertebral body) were excised, fixed in 4% paraformaldehyde for 24 hours, decalcified in 10% ethylenediaminetetraacetic acid for 7 days, embedded in paraffin, and sectioned midsagittally at 7 μm for histomorphological staining and immunofluorescence.
9. TUNEL Staining
Cells were fixed with 4% paraformaldehyde for 10 minutes and subjected to fluorescein‐labeled TUNEL staining to detect apoptotic DNA fragmentation, with 4´,6‐diamidino‐2‐phenylindole (DAPI) used for nuclear counterstaining [29,34]. The percentage of TUNEL‐positive cells was calculated relative to the total number of DAPI‐positive cells in 4 random LPFs using the BZX700 microscope and ImageJ software.
10. SA‐β‐Gal Staining
Cellular senescence was assessed using cytochemical SA‐β‐gal staining at pH 6.0.35 The percentage of positively stained cells was determined in duplicate across 4 random LPFs.
11. Immunofluorescence
For in vivo analysis, multicolor immunofluorescence was conducted to evaluate TRPV4, ECM, AMPK, and autophagy-related markers. Following antigen retrieval, permeabilization, and blocking, sections were incubated with primary antibodies against TRPV4, COL2A1, Brachyury, p‐AMPK, p62/SQSTM1, and LC3‐II (1:100 dilution) overnight at 4°C, followed by Alexa Fluor 488/568/647 secondary antibodies (1:200 dilution) and DAPI for 1 hour at room temperature. The percentage of positive cells was quantified relative to DAPI‐positive cells in 4 random LPFs.
12. Radiography
Lateral tail radiographs were obtained using a VPX‐30E system with IXFR film (exposure: 40 seconds, distance: 40 cm, current: 3 mA, voltage: 35 kV). Disc height was assessed using ImageJ twice at a 1‐week interval by each of 2 investigators, normalized to adjacent vertebral body heights as the disc height index (DHI), shown as the percent of preoperative DHI (%DHI=[postoperative DHI/preoperative DHI]×100), and further normalized to the intact disc as the normalized %DHI (normalized %DHI=[experimental %DHI/intact %DHI]×100) [36].
13. Histomorphology
Safranin‐O, fast green, and hematoxylin staining were performed to assess structural integrity of disc tissues. Degeneration grade was scored from 0 (normal) to 16 (severely degenerated) [37]. Each specimen was evaluated twice by 2 independent investigators, and median values were used for analysis.
14. Statistical Analysis
All quantitative data are presented as the mean±95% confidence interval in text and as box and dot plots in figures. Normality was tested before analysis. Multiway repeated measures analysis of variance followed by Tukey-Kramer post hoc test was performed to evaluate the effects of “treatment,” “experimental condition,” and “time” on in vitro cell viability, cell count, Ca2+ imaging, WB, TUNEL staining, and SA‐β‐Gal staining, on in vivo radiography, histomorphology, and immunofluorescence, respectively. Statistical analysis was conducted using IBM SPSS Statistics 23.0 (IBM, USA).
1. Confirmation of TRPV4 Expression in the Rat Intervertebral Disc
Immunofluorescence verified TRPV4 expression in the rat intervertebral disc tissue (Fig. 1D).
2. Effect of TRPV4 Agonist on Cell Viability
The viability of rat disc NP cells was analyzed using CCK-8 and microscopic cell counting. Agonist-induced cytotoxicity in the rat disc NP cells increased in a dose-dependent manner (Fig. 2A). TRPV4 agonist at 20 nM concentration significantly decreased cell viability (CCK-8, versus control, 89.2%±7.5%, p=0.02; cell number, 134.8±10.8/LPF vs. 106.4±13.7/LPF, p<0.01). Based on these findings, 10 nM was selected as an effective but nontoxic concentration for subsequent experiments.
3. Increase in Intracellular Ca2+ Concentration by TRPV4 Agonist
The intracellular Ca2+ influx, a trigger for AMPK/mTOR pathway, was assessed. The Ca2+ imaging revealed that TRPV4 agonist immediately increased intracellular Ca2+ level in the rat disc NP cells. Moreover, TRPV4 agonist induced the dose-dependent accumulation of Ca2+ (0 nM, 1,632.2±152.8 relative fluorescence units [RFU]; 5 nM, 2,472.0±555.7 RFU; 10 nM, 3,056.2±423.7 RFU). Significant differences were observed among 0 nM, 5 nM, and 10 nM (p<0.01); however, there was no significant difference in fluorescence intensity between 10 nM and 20 nM (3,056.2±423.7 RFU vs. 3,126.6±473.3 RFU, p=0.97) (Fig. 2B).
4. In Vitro Agonist-Induced Activation of TRPV4 Promotes Autophagy and ECM Synthesis in Rat Disc NP Cells
First, we assessed cell viability by CCK‐8 and conducted microscopic cell counting to understand the effects of TRPV4 agonist on disc cellular physiology. They showed an increasing tendency by treatment of TRPV4 agonist in serum-free DMEM (CCK‐8, 71.6%±11.6% vs. 79.4%±11.1%; cell number, 73.0±12.2/LPF vs. 86.4±15.2/LPF) and a significant increase under IL-1β stimulation (CCK‐8, 63.0%±12.7% vs. 74.7%±9.9%, p=0.04; cell number, 48.6±13.9/LPF vs. 71.8±11.6/LPF, p<0.01). These results indicated that TRPV4 agonist increased the cell viability and cell number under the inflammatory condition (Fig. 2C).
Second, we assessed the effects of TRPV4 agonist on disc ECM metabolism. Notochordal marker expression was evaluated to validate the rat disc NP phenotype. In WB, protein extracts from tested samples all showed positive expression of notochord‐related Brachyury and CD24. The WB demonstrated that treatment of TRPV4 agonist significantly increased p-AMPK/AMPK (144.5%±56.4%, p=0.04) protein expression without proinflammatory IL‐1β stimulation, while the other markers did not change significantly. Proinflammatory IL‐1β stimulation resulted in significant downregulation of the AMPK/mTOR pathway (p‐AMPK/AMPK, 49.7%±28.8%, p=0.02; mTOR, 265.7%±66.8%, p<0.01; RAPTOR, 187.7%±68.8%, p<0.01; p70S6K, 208.4%±113.4%, p=0.01), autophagy (LC3‐II, 40.7%±22.3%, p<0.01; p62/SQSTM1, 160.5%±54.3%, p<0.01), and ECM metabolism (COL2A1, 37.0%±23.9%, p<0.01; Aggrecan, 39.2%±22.3%, p<0.01; MMP3, 236.3%±46.7%, p<0.01; MMP13, 185.7%±68.3%, p<0.01; TIMP1, 47.6%±24.1%, p<0.01) compared to the control. In the presence of IL‐1β, treatment of TRPV4 agonist significantly increased COL2A1 (70.4%±33.1%, p=0.04) and Aggrecan (84.2%±17.0%, p<0.01), decreased MMP13 (124.3%±44.7%, p=0.02) protein expression, and increased TIMP1 (80.5%±34.4%, p=0.04) expression compared to the DMSO control by autophagy promotion via the AMPK/mTOR pathway (p‐AMPK/AMPK, 109.6%±55.9%, p<0.01; mTOR, 173.1%±30.8%, p<0.01; RAPTOR, 123.6%±33.9%, p<0.01; p70S6K, 127.4%±49.9%, p=0.01; LC3‐II, 68.6%±22.8%, p<0.01; p62/SQSTM1, 106.1%±32.2%, p<0.01) (Fig. 3).
5. In Vitro Agonist-Induced Activation of TRPV4 Suppresses the Incidence of Apoptosis and Senescence in Rat Disc NP Cells
In the absence of IL‐1β, TRPV4 agonist did not significantly change apoptosis and senescence markers. The IL‐1β stimulation induced significant downregulation of apoptosis‐related PARP (36.7%±25.0%, p<0.01) and upregulation of apoptotic cleaved PARP (151.3%±108.2%, p<0.01), cleaved caspase‐9 (210.1%±131.5%, p<0.01), senescent p53 (185.1%±121.8%, p<0.01), p21/CIP1 (166.5%±147.4%, p<0.01) and p16/INK4a (218.5%±154.5%, p<0.01) expression. Under IL-1β stimulation, TRPV4 agonist decreased these apoptotic and senescent markers (PARP, 69.0%±23.2%, p<0.01; cleaved PARP, 108.2%±31.1%, p<0.01; cleaved caspase‐9, 131.5%±52.6%, p<0.01; p53, 121.8%±32.4%, p<0.01; p16/INK4a, 154.5%±66.4%, p=0.04) (Fig. 4A). These results suggest that TRPV4 agonist decelerates apoptosis and senescence under the stressful serum deprivation and inflammation.
In vitro effects of TRPV4 agonist on disc cellular apoptosis and senescence were further evaluated by TUNEL and SA‐β‐gal staining. The percentage of TUNEL‐positive cells increased following IL‐1β stimulation (42.7%±12.1% vs. 3.6%±5.3%, p<0.01), significantly suppressed by TRPV4 agonist (26.8%±9.9% vs. 42.7%±12.1%, p<0.01) (Fig. 4B). Similarly, the percentage of SA‐β‐gal‐positive cells increased following IL‐1β stimulation (43.7%±8.4% vs. 14.1%±8.7%, p<0.01), significantly suppressed by TRPV4 agonist (33.2%±8.1% vs. 43.7%±8.4%, p=0.02) (Fig. 4C).
6. In Vivo Agonist-Induced Activation of TRPV4 Suppresses Radiographic and Histomorphological Disc Disruption in the Rat Tail Temporary Static Compression Model
Based on the in vitro cytoprotective effects of TRPV4 activation, in vivo intradiscal injection experiments with TRPV4 agonist were conducted. All rats underwent surgery well and gained body weight throughout the experiment period (mean 575.9±9.8 g at 56 days). All springs maintained their compressive length and fully recovered after release, indicating sustained axial loading. There were no surgery‐related complications. Successful administration was confirmed by radiography upon injecting a contrast agent (Fig. 5A).
In vivo effects of TRPV4 agonist on disc tissue protection were evaluated under the temporary static compression model. In control DMSO-injected loaded discs, %DHI was significantly lower than control DMSO-injected unloaded discs at 28 days (67.1%±12.9% vs. 86.5%±10.8%, p<0.01) and 56 days (51.8%± 11.4% vs. 82.7%±9.6%, p<0.01). Similarly, in TRPV4 agonist-injected loaded discs, %DHI was significantly lower than TRPV4 agonist-injected unloaded discs at 28 days (71.1%±81.1% vs. 85.8%±11.1%, p<0.01) and 56 days (62.9%±9.4% vs. 82.7%±9.2%, p<0.01). Meanwhile, in TRPV4 agonist‐injected loaded discs, %DHI was significantly larger than control DMSO-injected loaded discs at 56 days (62.9%±9.4% vs. 51.8%±11.4%, p=0.04) (Fig. 5B). These results showed that TRPV4 agonist slowed mechanically induced radiological disc degeneration, suggesting that TRPV4 agonist has the effect of maintaining disc height against mechanical stress.
Similarly, in safranin‐O staining, DMSO-injected loaded discs showed significantly higher degenerative scores than DMSO-injected unloaded discs at 28 days (4.8±1.3 vs. 1.0±1.6, p<0.01) and 56 days (7.3±1.5 vs. 1.5±1.0, p<0.01), and TRPV4-injected loaded discs showed significantly higher degenerative scores than TRPV4-injected unloaded discs at 28 days (3.8±1.3 vs. 0.8±1.3, p<0.01) and 56 days (5.8±2.1 vs. 1.2±1.3, p<0.01). In TRPV4 agonist‐injected loaded discs, histomorphological changes were significantly lower than control DMSO-injected loaded discs at 56 days (5.8±2.1 vs. 7.3±1.5, p=0.03) (Fig. 5C). These results implied that TRPV4 agonist suppresses disc disruption under the mechanical load.
7. In Vivo Agonist-Induced Activation of TRPV4 Promotes ECM Synthesis, AMPK Phosphorylation, and Autophagy Activity in the Rat Tail Temporary Static Compression Model
We finally performed multicolor immunofluorescence for TRPV4, ECM metabolism, AMPK phosphorylation, and autophagy activity in the rat tail temporary static compression model. TRPV4 immunopositivity under loaded conditions declined significantly over time in both DMSO control-injected and TRPV4 agonist-injected discs (DMSO control: 0 day, 96.1%±3.3%; 7 days, 79.4%±5.3%; 28 days, 52.5%±11.8%; 56 days, 35.8%±13.5%, TRPV4 agonist: 0 day, 94.9%±2.9%; 7 days, 81.8%±7.1%; 28 days, 67.0%±11.1%; 56 days, 52.3%±16.3%, all p<0.01). In addition, immunopositivity for TRPV4 under loaded conditions was significantly lower than that under unloaded conditions at all time points (DMSO control: 7 days, 79.4%±5.3% vs. 94.8%±2.3%; 28 days, 52.5%±11.8% vs. 90.9%±3.8%; 56 days, 35.8%±13.5% vs. 90.7%±4.3%, TRPV4 agonist: 7 days, 81.8%±7.1% vs. 93.7%±4.5%; 28 days, 67.0%±11.1% vs. 91.9%±5.2%; 56 days, 52.3%±16.3% vs. 87.8%±2.6%, all p<0.01). In TRPV4 agonist‐injected loaded discs, moreover, the percentage of TRPV4‐positive cells was significantly higher than control DMSO-injected loaded discs at 28 days (67.0%±11.1% vs. 52.5%±11.8%, p<0.01) and 56 days (52.3%±16.3% vs. 35.8%±13.5%, p<0.01).
The percentage of COL2A1‐positive cells under loaded conditions decreased significantly over time in both DMSO control-injected and TRPV4 agonist- injected discs (DMSO control: 0 day, 96.2%±4.0%; 7 days, 79.9%±6.5%; 28 days, 47.1%±9.4%; 56 days, 25.8%±13.1%, TRPV4 agonist: 0 day, 97.4%±3.9%; 7 days, 81.8%±8.0%; 28 days, 60.2%±12.6%; 56 days, 41.4%±10.2%, all p<0.01), and was significantly lower than that under unloaded conditions at all time points (DMSO control: 7 days, 79.9%±6.5% vs. 91.3%±3.7%; 28 days, 47.1%±9.4% vs. 91.7%±4.8%; 56 days, 25.8%±13.1% vs. 91.7%±3.7%, TRPV4 agonist: 7 days, 81.8%±8.0% vs. 93.5%±2.3%; 28 days, 60.2%±12.6% vs. 92.3%±6.1%; 56 days, 41.4%±10.2% vs. 89.1%±2.8%, all p<0.01).
The percentage of Brachyury‐positive cells under loaded conditions decreased significantly over time in both DMSO control-injected and TRPV4 agonist- injected discs (DMSO control: 0 day, 96.5%±2.7%; 7 days, 85.6%±6.8%; 28 days, 65.1%±8.9%; 56 days, 49.0%±9.6%, TRPV4 agonist: 0 day, 95.7%±3.7%; 7 days, 88.5%±5.3%; 28 days, 74.0%±7.2%; 56 days, 61.4%±9.4%, all p<0.01), and was significantly lower than that under unloaded conditions at all time points (DMSO control: 7 days, 85.6%±6.8% vs. 93.2%±5.0%, p=0.04; 28 days, 65.1%±8.9% vs. 90.1%±3.5%, p<0.01; 56 days, 49.0%±9.6% vs. 89.4%±3.7%, p<0.01, TRPV4 agonist: 28 days, 74.0%±7.2% vs. 90.9%±6.2%, p<0.01; 56 days, 61.4%±9.4% vs. 89.9%±5.2%, p<0.01). In TRPV4 agonist‐injected loaded discs, the percentage of COL2A1‐positive cells did not significantly increase compared with control DMSO‐injected loaded discs at 7 days (81.8%±8.0% vs. 79.9%±6.5%, p>0.99), however, significantly increased at 28 days (60.2%±12.6% vs. 47.1%±9.4%, p<0.01) and 56 days (41.4%±10.2% vs. 25.8%±13.1%, p<0.01) with an increase in Brachyury expression (28 days, 74.0%±7.2% vs. 65.1%±8.9%, p<0.01; 56 days, 61.4%±9.4% vs. 49.0%±9.6%, p<0.01) (Fig. 6).
In control DMSO‐injected loaded discs, the immunopositivity of p‐AMPK significantly decreased compared with control DMSO‐injected unloaded discs at all time points (7 days, 68.4%±11.4% vs. 82.7%±11.3%; 28 days, 55.6%±6.5% vs. 79.5%±7.6%; 56 days, 39.2%±13.8% vs. 76.1%±8.9%, all p<0.01). In TRPV4 agonist‐injected loaded discs, the percentage of p‐AMPK‐positive cells was significantly higher than in control DMSO‐injected loaded discs at 28 days (67.2%±8.2% vs. 55.6%±6.5%, p=0.04) and 56 days (58.0%±10.1% vs. 39.2%±13.8%, p<0.01) (Fig. 7).
In addition, the percentage of p62/SQSTM1‐positive and LC3‐II‐negative cells under loaded condition increased significantly over time in both TRPV4 agonist-injected and control DMSO-injected discs (DMSO control: 0 day, 3.1%±3.3%; 7 days, 18.7%±6.3%; 28 days, 42.6%±12.6%; 56 days, 74.3%±18.9%, TRPV4 agonist: 0 day, 2.6%±3.3%; 7 days, 14.0%±6.4%; 28 days, 30.4%±12.9%; 56 days, 51.1%±8.8%, all p<0.01), and was significantly higher than that under unloaded condition at all time points (DMSO control: 7 days, 18.7%±6.3% vs. 3.3%±2.4%; 28 days, 42.6%±12.6% vs. 3.4%±2.0%; 56 days, 74.3%±18.9% vs. 3.6%±4.4%, TRPV4 agonist: 7 days, 14.0%±6.4% vs. 2.3%±3.4%; 28 days, 30.4%±12.9% vs. 2.4%±2.5%; 56 days, 51.1%±8.8% vs. 2.6%±3.1%, all p<0.01). In TRPV4 agonist‐injected loaded discs, the percentage of p62/SQSTM1‐positive and LC3‐II‐negative cells was significantly lower than control DMSO-injected loaded discs at 28 days (30.4%±12.9% vs. 42.6%±12.6%, p<0.01) and 56 days (51.1%±8.8% vs. 74.3%±18.9%, p<0.01) (Fig. 8).
Collectively, the observed findings support that the TRPV4 activation by agonist promoted autophagy and ECM synthesis via AMPK/mTOR pathway, which contributes to the maintenance of the disc homeostasis, particularly under mechanical stress.
This study is the first to demonstrate both in vitro and in vivo evidence that pharmacological activation of TRPV4 enhances autophagy and ECM synthesis, ultimately mitigating mechanically induced intervertebral disc degeneration [7,26]. TRPV4 is known to mediate cell-ECM interactions in disc NP cells and articular chondrocytes, functioning as a mechanosensitive and osmosensitive ion channel that regulates cellular responses to microenvironmental changes in both healthy and degenerated tissues [38]. Previous studies have explored the involvement of TRPV4 in disc inflammation. In bovine NP cells, reduced osmolarity upregulated TRPV4 expression and triggered TRPV4-mediated Ca2+ signaling, which consequently increased the expression of proinflammatory cytokines [15]. Elevated TRPV4 levels were also detected in aggrecan-depleted regions of degenerated human discs, suggesting that TRPV4 may contribute to swelling-related inflammation and ECM degradation [15]. Conversely, repeated activation of TRPV4 produced anabolic effects on the ECM, improving overall disc stiffness and enhancing glycosaminoglycan content in the NP [39]. Furthermore, transient TRPV4 activation elicited an acute proregenerative inflammatory response in NP cells, supporting its potential role in preserving the immuneprivileged status of the healthy disc [40]. Also, TRPV4 may be involved in mechanical signal transduction, that is hydrostatic pressure in disc NP cells and disc tissues, as well as osmotic sensing [11]. In notochordal cells, hyperosmotic stress induced autophagy via AMPK/mTOR pathway, with TRPV4-dependent Ca2+ influx acting as a key initiating signal [18]. Based on these previous studies, it is suggested that under osmotic or mechanical stress, TRPV4 is involved in maintaining intervertebral disc homeostasis, and that one of the mechanisms mediating this effect is autophagy induction via the AMPK/mTOR pathway. However, there have been no reports on the relationship among TRPV4 activation, homeostasis, and autophagy in the intervertebral disc.
In the present study, we examined this hypothesis using both in vitro inflammatory stimulation and an in vivo mechanical compression model. In vitro, we first confirmed that administration of TRPV4 agonist-induced intracellular Ca2+ influx. WB subsequently revealed that TRPV4 activation under proinflammatory conditions enhanced autophagy and ECM synthesis via the AMPK/mTOR pathway. We also observed that TRPV4 activation significantly suppressed apoptosis and senescence, leading to increased cell viability. These results collectively suggest that TRPV4 exerts anabolic, anti-apoptotic, and antisenescent effects that support disc homeostasis through autophagy modulation. Our in vivo findings were consistent with these in vitro observations. Activation of TRPV4 attenuated radiographic, histomorphological, and immunofluorescent signs of mechanically induced disc degeneration, accompanied by increased AMPK expression and autophagy activity. Compared with DMSO control‐injected loaded discs, TRPV4 agonist‐injected loaded discs maintained larger radiographical disc height, exhibited diminished histomorphological degenerative changes, and showed increased expression of AMPK, autophagy markers, and COL2A1. In this study, Brachyury and CD24 protein levels were preserved after 24-hour IL-1β stimulation in vitro, whereas Brachyury-positive cells progressively decreased over 56 days under loaded conditions in vivo. This apparent discrepancy is likely attributable to differences in both the type and duration of stress. Compared to acute inflammatory stimulation for 24 hours in monolayer culture in vitro, the in vivo temporary static compression model showed more sensitive and accurate results possibly due to the more physiological conditions.
Previous studies have separately explored the relationship between mechanical stress and TRPV4 signaling [11] and the role of autophagy in ECM metabolism [21]. Moreover, loss‐of‐function experiments using TRPV4 siRNA demonstrated that TRPV4 suppression led to reduced autophagy and ECM synthesis, ultimately resulting in progressive structural disc deterioration [17]. Together with our current findings as a gain-of-function study, these results indicate that TRPV4 activation contributes to maintaining intradiscal homeostasis against physical loading by promoting autophagy.
This study has several limitations. First, this study adopted a single concentration and administration of TRPV4 agonist in in vivo experiments. Moderate TRPV4 activation appears to be beneficial for disc maintenance, whereas excessive or prolonged stimulation could induce inflammatory or degenerative responses [39]. Second, although our in vivo model reproduced mechanical loading by temporary static compression, the in vitro experiments were performed under inflammatory and nutrient-deprived conditions without direct application of hydrostatic pressure. Such biochemical stimuli are widely used to mimic the degenerative disc microenvironment; however, they do not fully recapitulate the complex mechanical cues, including dynamic hydrostatic pressure, that NP cells experience in vivo. Third, the expression pattern and functional role of TRPV4 in degenerating human discs remain insufficiently characterized. Further studies are required to determine whether TRPV4 behaves similarly in human pathological conditions. Finally, our rat tail compression model cannot fully replicate the human aging process, as rats retain notochordal cells throughout their lifespan, unlike humans. We also did not quantify viable cell numbers nor evaluate apoptosis/senescence-related markers in vivo due to limited rat samples.
In summary, our in vitro and in vivo findings indicate that agonist-induced activation of TRPV4 promotes autophagy and ECM synthesis in the rat intervertebral disc. These results suggest that targeting TRPV4 may offer a novel therapeutic strategy for the treatment of disc degenerative diseases.
Supplementary Table 1 are available at https://doi.org/10.14245/ns.2551582.791.
Supplementary Table 1.
List of the antibodies, reagents, and instruments used
ns-2551582-791-Supplementary-Table-1.pdf

Conflict of Interest

YT and KKakutani are the faculty members of the endowed course by Surgical Spine Inc. and SMI Japan Inc. The other authors have nothing to disclose.

Funding/Support

This research was funded by JSPS KAKENHI, Grant Numbers JP22K16743 and JP25K19972, and Grant of Japan Orthopaedics and Traumatology Research Foundation No. 554.

Acknowledgments

The authors thank Kyoko Tanaka, Maya Yasuda, and Minako Nagata (Department of Orthopedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan) for their technical assistance.

Author Contribution

Conceptualization: TY; Data curation: NK, TY, MR; Formal analysis: NK, TY, MR, YT, YK, KKuroshima, YH, MF, DN, YI, RK, KKakutani; Funding acquisition: YT; Methodology: NK, TY, MR; Project administration: TY, MR; Visualization: NK, TY, MR; Writing – original draft: NK, TY, MR; Writing – review & editing: TY, YT, YK, KKuroshima, YH, MF, DN, YI, RK, KKakutani.

Fig. 1.
Overview of disc cell autophagy and extracellular matrix (ECM) turnover under mechanical and chemical stimuli, experimental setups in vitro and in vivo, and confirmation of transient receptor potential vanilloid 4 (TRPV4) expression in intervertebral disc. (A) Conceptual diagram illustrating the protective effects of autophagy and ECM metabolism mediated through the Ca2+‐dependent AMPK/mTOR pathway under mechanical or chemical stress. By TRPV4 agonist, Ca2+ influx increases and AMPK is activated. Then, mTORC1 and p70S6K are suppressed, resulting in promotion of autophagy and ECM metabolism. (B) Schematic illustration of the in vitro experiments and sample allocation. First passage, approximately 80% monolayer disc nucleus pulposus (NP) cells from 12‐week‐old male rats were cultured as a monolayer (n=20). Cells were treated with various concentrations (0–100 nM) of TRPV4 agonist for 24 hours in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), followed by cytotoxicity testing via cell count and CCK‐8 (n=5). Then, cells were treated for 24 hours with TRPV4 agonist (10 nM) or control in DMEM with 10% FBS, 0% FBS, or 0% FBS and 10‐ng/mL interleukin (IL)‐1β, and analyzed via cell counting and CCK‐8 for cell viability (n=5). Cells were also treated for 24 hours with TRPV4 agonist (10 nM) or control in DMEM with 0% FBS or 0% FBS and 10‐ng/mL IL‐1β, and analyzed by Western blotting for the phenotype, ECM metabolism, apoptosis, and senescence markers, as well as by apoptosis and senescence staining (each n=5). (C) Diagram of the in vivo temporary static compression model and sample distribution. Rat tails (n=18) were fitted with a spring-loaded Ilizarov‐type device spanning C8–10. TRPV4 agonist was injected into C8–9 and C11–12, while control DMSO was injected into C9–10 and C12–13 using a 33‐gauge needle. A compressive load of 1.3‐MPa axial force was applied to C8–9 and C9–10 for 24 hours and subsequently released. At 0–56 days, loaded and unloaded discs with TRPV4 agonist or sample were collected after radiographic height assessment and examined histomorphologically with safranin‐O and immunofluorescence for ECM, AMPK, and autophagy (n=6/time point). (D) Confirmation of TRPV4 expression in NP cells by immunofluorescence. AMPK, adenosine monophosphate- activated protein kinase; mTOR, mammalian target of rapamycin (mTOR); mTORC1, mTOR complex 1; p70S6K, p70 S6 kinase; CCK‐8, cell counting kit-8; DMSO, dimethyl sulfoxide.
ns-2551582-791f1.jpg
Fig. 2.
Transient receptor potential vanilloid 4 (TRPV4) agonist has dose-dependent cytotoxicity, increases intracellular Ca2+ concentration, and increases cell viability under inflammatory conditions in rat nucleus pulposus (NP) cells. (A) Cell morphology, viability (CCK-8), and counting after 24-hour treatment of TRPV4 agonist (0–100 nM) in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). Cell counting was performed across 4 random low‐power fields (LPFs) per duplicate. (B) Intracellular Ca2+ levels visualized by Fluo-4 AM staining and fluorescence intensity following TRPV4 agonist treatment. (C) Cell morphology, viability, and counting after 24-hour treatment of TRPV4 agonist or control in DMEM with 10% FBS, with 0% FBS, or with 0% FBS and 10‐ng/mL interleukin (IL)‐1β. Cell counting was performed across 4 random LPFs per duplicate. Data in panels A and C are shown as dot and box plots (n=5); in panel B, data are represented by the means with error bars indicating the 95% confidence intervals at each time point (n=5). One‐way repeated measures analysis of variance with Tukey-Kramer post hoc test was applied. Representative cellular images are shown. CCK‐8, cell counting kit-8; RFU, relative fluorescence units.
ns-2551582-791f2.jpg
Fig. 3.
In vitro agonist-induced activation of transient receptor potential vanilloid 4 (TRPV4) promotes autophagy and extracellular matrix (ECM) synthesis under interleukin (IL)-1β stimulation in rat nucleus pulposus (NP) cells. Western blotting analysis of TRPV4, phenotypic markers (Brachyury and CD24), AMPK/mTOR pathway proteins (AMPK, mTOR, RAPTOR, and p70S6K), autophagy markers (LC3‐II and p62/SQSTM1), and ECM metabolism markers (COL2A1, Aggrecan, catabolic MMP3 and MMP13, and anticatabolic TIMP1 and TIMP2) in supernatant protein extracts from rat NP cells treated for 24 hours with TRPV4 agonist or control in serum‐free DMEM containing 10‐ng/mL IL‐1β. Relative protein levels normalized to tubulin are shown as dot and box plots (n=5). One‐way repeated measures analysis of variance with Tukey-Kramer post hoc test was applied. Representative immunoblots are shown. AMPK, adenosine monophosphate-activated protein kinase; mTOR, mammalian target of rapamycin (mTOR); RAPTOR, regulatory-associated protein of mTOR; p70S6K, p70 S6 kinase; LC3‐II, microtubuleassociated protein 1 light chain 3-II; p62/SQSTM1, sequestosome 1; COL2A1, collagen type II alpha 1 chain; MMP3, matrix metalloproteinase-3; TIMP, tissue inhibitor of metalloproteinases; DMEM, Dulbecco’s modified Eagle’s medium.
ns-2551582-791f3.jpg
Fig. 4.
In vitro agonist-induced activation of transient receptor potential vanilloid 4 (TRPV4) suppresses apoptosis and senescence under interleukin (IL)-1β stimulation in rat nucleus pulposus (NP) cells. (A) Western blotting (WB) for apoptosis markers (PARP, cleaved PARP, and cleaved caspase‐9), and senescence markers (p53, p21/CIP1, and p16/INK4a) in total protein extracts from rat NP cells treated for 24 hours with TRPV4 agonist or control in serum‐free DMEM with 10‐ng/mL IL‐1β. Relative protein levels normalized to tubulin are shown as dot and box plots. (B) Immunofluorescence for TUNEL (green), nuclear DAPI (blue), and merged images after 24-hour treatment of TRPV4 agonist. Quantification of TUNEL‐positive cells relative to DAPIpositive cells is presented. (C) SA‐β‐gal staining after 24-hour treatment of TRPV4 agonist. Quantification of SA‐β‐gal‐positive cells relative to total cells is presented. Data are presented with dot and box plots (n=5). In (B, C), cell counting was performed across 4 random low‐power fields (LPFs) per duplicates. One‐way repeated measures analysis of variance with Tukey-Kramer post hoc test was applied. Representative immunoblots are shown. PARP, apoptosis‐related poly (ADP‐ribose) polymerase; DMEM, Dulbecco’s modified Eagle’s medium; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4´,6‐diamidino‐2‐phenylindole; FBS, fetal bovine serum.
ns-2551582-791f4.jpg
Fig. 5.
In vivo agonist-induced activation of transient receptor potential vanilloid 4 (TRPV4) mitigates radiographic and histomorphological disc degeneration in the rat tail temporary static compression model. (A) Confirmation of intradiscal 33‐gauge needle and 2‐μL contrast under fluoroscopy. (B) Lateral radiographs of rat tail C8–9 TRPV4 agonist‐injected loaded, C9–10 DMSO control‐injected loaded, C11–12 TRPV4 agonist‐injected unloaded, and C12–13 DMSO control‐injected unloaded discs at 0, 7, 28, and 56 days after 24-hour temporary static compression (1.3 MPa). Disc height changes are shown. (C) Safranin‐O histology of the same groups across time points with grading of degeneration. Data are presented as dot and box plots (n=6). Two‐way analysis of variance with Tukey-Kramer post hoc test was applied. Representative radiographs and histological sections are shown. DMSO, dimethyl sulfoxide.
ns-2551582-791f5.jpg
Fig. 6.
In vivo agonist-induced activation of transient receptor potential vanilloid 4 (TRPV4) promotes extracellular matrix (ECM) synthesis in the rat tail temporary static compression model. Immunofluorescence for TRPV4 (red), COL2A1 (green), phenotypic Brachyury (purple), nuclear DAPI (blue), and merged signals of rat tail C12–13 DMSO control‐injected unloaded, C11–12 TRPV4 agonist‐injected unloaded, C9–10 DMSO control‐injected loaded, and C8–9 TRPV4 agonist‐injected loaded discs at 0, 7, 28, and 56 days after 24-hour temporary static compression (1.3 MPa). Percentages of TRPV4‐, COL2A1‐, and Brachyury‐positive cells relative to DAPI are shown. Cell counting was performed across 4 random low‐power fields per duplicate. Data are shown as dot and box plots (n=6). Two‐way analysis of variance with Tukey-Kramer post hoc test was applied. Representative immunofluorescent images are shown. DAPI, 4´,6‐diamidino‐2‐phenylindole; COL2A1, collagen type II alpha 1 chain; DMSO, dimethyl sulfoxide.
ns-2551582-791f6.jpg
Fig. 7.
In vivo agonist-induced activation of transient receptor potential vanilloid 4 (TRPV4) promotes AMPK phosphorylation in the rat tail temporary static compression model. Immunofluorescence for p‐AMPK (red), nuclear DAPI (blue), and merged signals of rat tail C12–13 DMSO control‐injected unloaded, C11–12 TRPV4 agonist‐injected unloaded, C9–10 DMSO controlinjected loaded, and C8–9 TRPV4 agonist‐injected loaded discs at 0, 7, 28, and 56 days after 24-hour temporary static compression (1.3 MPa). Percentage of p‐AMPK‐positive cells relative to DAPI are shown. Cell counting was performed across 4 random low‐power fields per duplicate. Data are shown as dot and box plots (n=6). Two‐way analysis of variance with Tukey-Kramer post hoc test was applied. Representative immunofluorescent images are shown. AMPK, adenosine monophosphate-activated protein kinase; p‐AMPK, phosphorylated AMPK; DAPI, 4´,6‐diamidino‐2‐phenylindole; DMSO, dimethyl sulfoxide.
ns-2551582-791f7.jpg
Fig. 8.
In vivo agonist-induced activation of transient receptor potential vanilloid 4 (TRPV4) promotes autophagy activity in the rat tail temporary static compression model. Immunofluorescence for LC3‐II (red), p62/SQSTM1 (green), nuclear DAPI (blue), and merged signals of rat tail C12–13 DMSO control‐injected unloaded, C11–12 TRPV4 agonist‐injected unloaded, C9–10 DMSO control‐injected loaded, and C8–9 TRPV4 agonist‐injected loaded discs at 0, 7, 28, and 56 days after 24-hour temporary static compression (1.3 MPa). Percentages of p62/SQSTM1‐positive and LC3‐II‐negative cells relative to DAPI‐positive cells are shown. Cell counting was performed across 4 random low‐power fields per duplicate. Data are shown as dot and box plots (n=6). Two‐way analysis of variance with Tukey-Kramer post hoc test was applied. Representative immunofluorescent images are shown. LC3‐II, microtubule-associated protein 1 light chain 3-II; p62/SQSTM1, sequestosome 1; DAPI, 4´,6‐diamidino‐2‐ phenylindole; DMSO, dimethyl sulfoxide.
ns-2551582-791f8.jpg
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Agonist-Induced Activation of Transient Receptor Potential Vanilloid 4 Promotes Autophagy and Extracellular Matrix Synthesis in the Rat Intervertebral Disc
Neurospine. 2026;23(2):347-364.   Published online April 30, 2026
DOI: https://doi.org/10.14245/ns.2551582.791
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Agonist-Induced Activation of Transient Receptor Potential Vanilloid 4 Promotes Autophagy and Extracellular Matrix Synthesis in the Rat Intervertebral Disc
Neurospine. 2026;23(2):347-364.   Published online April 30, 2026
DOI: https://doi.org/10.14245/ns.2551582.791
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Agonist-Induced Activation of Transient Receptor Potential Vanilloid 4 Promotes Autophagy and Extracellular Matrix Synthesis in the Rat Intervertebral Disc
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Fig. 1. Overview of disc cell autophagy and extracellular matrix (ECM) turnover under mechanical and chemical stimuli, experimental setups in vitro and in vivo, and confirmation of transient receptor potential vanilloid 4 (TRPV4) expression in intervertebral disc. (A) Conceptual diagram illustrating the protective effects of autophagy and ECM metabolism mediated through the Ca2+‐dependent AMPK/mTOR pathway under mechanical or chemical stress. By TRPV4 agonist, Ca2+ influx increases and AMPK is activated. Then, mTORC1 and p70S6K are suppressed, resulting in promotion of autophagy and ECM metabolism. (B) Schematic illustration of the in vitro experiments and sample allocation. First passage, approximately 80% monolayer disc nucleus pulposus (NP) cells from 12‐week‐old male rats were cultured as a monolayer (n=20). Cells were treated with various concentrations (0–100 nM) of TRPV4 agonist for 24 hours in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), followed by cytotoxicity testing via cell count and CCK‐8 (n=5). Then, cells were treated for 24 hours with TRPV4 agonist (10 nM) or control in DMEM with 10% FBS, 0% FBS, or 0% FBS and 10‐ng/mL interleukin (IL)‐1β, and analyzed via cell counting and CCK‐8 for cell viability (n=5). Cells were also treated for 24 hours with TRPV4 agonist (10 nM) or control in DMEM with 0% FBS or 0% FBS and 10‐ng/mL IL‐1β, and analyzed by Western blotting for the phenotype, ECM metabolism, apoptosis, and senescence markers, as well as by apoptosis and senescence staining (each n=5). (C) Diagram of the in vivo temporary static compression model and sample distribution. Rat tails (n=18) were fitted with a spring-loaded Ilizarov‐type device spanning C8–10. TRPV4 agonist was injected into C8–9 and C11–12, while control DMSO was injected into C9–10 and C12–13 using a 33‐gauge needle. A compressive load of 1.3‐MPa axial force was applied to C8–9 and C9–10 for 24 hours and subsequently released. At 0–56 days, loaded and unloaded discs with TRPV4 agonist or sample were collected after radiographic height assessment and examined histomorphologically with safranin‐O and immunofluorescence for ECM, AMPK, and autophagy (n=6/time point). (D) Confirmation of TRPV4 expression in NP cells by immunofluorescence. AMPK, adenosine monophosphate- activated protein kinase; mTOR, mammalian target of rapamycin (mTOR); mTORC1, mTOR complex 1; p70S6K, p70 S6 kinase; CCK‐8, cell counting kit-8; DMSO, dimethyl sulfoxide.
Fig. 2. Transient receptor potential vanilloid 4 (TRPV4) agonist has dose-dependent cytotoxicity, increases intracellular Ca2+ concentration, and increases cell viability under inflammatory conditions in rat nucleus pulposus (NP) cells. (A) Cell morphology, viability (CCK-8), and counting after 24-hour treatment of TRPV4 agonist (0–100 nM) in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). Cell counting was performed across 4 random low‐power fields (LPFs) per duplicate. (B) Intracellular Ca2+ levels visualized by Fluo-4 AM staining and fluorescence intensity following TRPV4 agonist treatment. (C) Cell morphology, viability, and counting after 24-hour treatment of TRPV4 agonist or control in DMEM with 10% FBS, with 0% FBS, or with 0% FBS and 10‐ng/mL interleukin (IL)‐1β. Cell counting was performed across 4 random LPFs per duplicate. Data in panels A and C are shown as dot and box plots (n=5); in panel B, data are represented by the means with error bars indicating the 95% confidence intervals at each time point (n=5). One‐way repeated measures analysis of variance with Tukey-Kramer post hoc test was applied. Representative cellular images are shown. CCK‐8, cell counting kit-8; RFU, relative fluorescence units.
Fig. 3. In vitro agonist-induced activation of transient receptor potential vanilloid 4 (TRPV4) promotes autophagy and extracellular matrix (ECM) synthesis under interleukin (IL)-1β stimulation in rat nucleus pulposus (NP) cells. Western blotting analysis of TRPV4, phenotypic markers (Brachyury and CD24), AMPK/mTOR pathway proteins (AMPK, mTOR, RAPTOR, and p70S6K), autophagy markers (LC3‐II and p62/SQSTM1), and ECM metabolism markers (COL2A1, Aggrecan, catabolic MMP3 and MMP13, and anticatabolic TIMP1 and TIMP2) in supernatant protein extracts from rat NP cells treated for 24 hours with TRPV4 agonist or control in serum‐free DMEM containing 10‐ng/mL IL‐1β. Relative protein levels normalized to tubulin are shown as dot and box plots (n=5). One‐way repeated measures analysis of variance with Tukey-Kramer post hoc test was applied. Representative immunoblots are shown. AMPK, adenosine monophosphate-activated protein kinase; mTOR, mammalian target of rapamycin (mTOR); RAPTOR, regulatory-associated protein of mTOR; p70S6K, p70 S6 kinase; LC3‐II, microtubuleassociated protein 1 light chain 3-II; p62/SQSTM1, sequestosome 1; COL2A1, collagen type II alpha 1 chain; MMP3, matrix metalloproteinase-3; TIMP, tissue inhibitor of metalloproteinases; DMEM, Dulbecco’s modified Eagle’s medium.
Fig. 4. In vitro agonist-induced activation of transient receptor potential vanilloid 4 (TRPV4) suppresses apoptosis and senescence under interleukin (IL)-1β stimulation in rat nucleus pulposus (NP) cells. (A) Western blotting (WB) for apoptosis markers (PARP, cleaved PARP, and cleaved caspase‐9), and senescence markers (p53, p21/CIP1, and p16/INK4a) in total protein extracts from rat NP cells treated for 24 hours with TRPV4 agonist or control in serum‐free DMEM with 10‐ng/mL IL‐1β. Relative protein levels normalized to tubulin are shown as dot and box plots. (B) Immunofluorescence for TUNEL (green), nuclear DAPI (blue), and merged images after 24-hour treatment of TRPV4 agonist. Quantification of TUNEL‐positive cells relative to DAPIpositive cells is presented. (C) SA‐β‐gal staining after 24-hour treatment of TRPV4 agonist. Quantification of SA‐β‐gal‐positive cells relative to total cells is presented. Data are presented with dot and box plots (n=5). In (B, C), cell counting was performed across 4 random low‐power fields (LPFs) per duplicates. One‐way repeated measures analysis of variance with Tukey-Kramer post hoc test was applied. Representative immunoblots are shown. PARP, apoptosis‐related poly (ADP‐ribose) polymerase; DMEM, Dulbecco’s modified Eagle’s medium; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4´,6‐diamidino‐2‐phenylindole; FBS, fetal bovine serum.
Fig. 5. In vivo agonist-induced activation of transient receptor potential vanilloid 4 (TRPV4) mitigates radiographic and histomorphological disc degeneration in the rat tail temporary static compression model. (A) Confirmation of intradiscal 33‐gauge needle and 2‐μL contrast under fluoroscopy. (B) Lateral radiographs of rat tail C8–9 TRPV4 agonist‐injected loaded, C9–10 DMSO control‐injected loaded, C11–12 TRPV4 agonist‐injected unloaded, and C12–13 DMSO control‐injected unloaded discs at 0, 7, 28, and 56 days after 24-hour temporary static compression (1.3 MPa). Disc height changes are shown. (C) Safranin‐O histology of the same groups across time points with grading of degeneration. Data are presented as dot and box plots (n=6). Two‐way analysis of variance with Tukey-Kramer post hoc test was applied. Representative radiographs and histological sections are shown. DMSO, dimethyl sulfoxide.
Fig. 6. In vivo agonist-induced activation of transient receptor potential vanilloid 4 (TRPV4) promotes extracellular matrix (ECM) synthesis in the rat tail temporary static compression model. Immunofluorescence for TRPV4 (red), COL2A1 (green), phenotypic Brachyury (purple), nuclear DAPI (blue), and merged signals of rat tail C12–13 DMSO control‐injected unloaded, C11–12 TRPV4 agonist‐injected unloaded, C9–10 DMSO control‐injected loaded, and C8–9 TRPV4 agonist‐injected loaded discs at 0, 7, 28, and 56 days after 24-hour temporary static compression (1.3 MPa). Percentages of TRPV4‐, COL2A1‐, and Brachyury‐positive cells relative to DAPI are shown. Cell counting was performed across 4 random low‐power fields per duplicate. Data are shown as dot and box plots (n=6). Two‐way analysis of variance with Tukey-Kramer post hoc test was applied. Representative immunofluorescent images are shown. DAPI, 4´,6‐diamidino‐2‐phenylindole; COL2A1, collagen type II alpha 1 chain; DMSO, dimethyl sulfoxide.
Fig. 7. In vivo agonist-induced activation of transient receptor potential vanilloid 4 (TRPV4) promotes AMPK phosphorylation in the rat tail temporary static compression model. Immunofluorescence for p‐AMPK (red), nuclear DAPI (blue), and merged signals of rat tail C12–13 DMSO control‐injected unloaded, C11–12 TRPV4 agonist‐injected unloaded, C9–10 DMSO controlinjected loaded, and C8–9 TRPV4 agonist‐injected loaded discs at 0, 7, 28, and 56 days after 24-hour temporary static compression (1.3 MPa). Percentage of p‐AMPK‐positive cells relative to DAPI are shown. Cell counting was performed across 4 random low‐power fields per duplicate. Data are shown as dot and box plots (n=6). Two‐way analysis of variance with Tukey-Kramer post hoc test was applied. Representative immunofluorescent images are shown. AMPK, adenosine monophosphate-activated protein kinase; p‐AMPK, phosphorylated AMPK; DAPI, 4´,6‐diamidino‐2‐phenylindole; DMSO, dimethyl sulfoxide.
Fig. 8. In vivo agonist-induced activation of transient receptor potential vanilloid 4 (TRPV4) promotes autophagy activity in the rat tail temporary static compression model. Immunofluorescence for LC3‐II (red), p62/SQSTM1 (green), nuclear DAPI (blue), and merged signals of rat tail C12–13 DMSO control‐injected unloaded, C11–12 TRPV4 agonist‐injected unloaded, C9–10 DMSO control‐injected loaded, and C8–9 TRPV4 agonist‐injected loaded discs at 0, 7, 28, and 56 days after 24-hour temporary static compression (1.3 MPa). Percentages of p62/SQSTM1‐positive and LC3‐II‐negative cells relative to DAPI‐positive cells are shown. Cell counting was performed across 4 random low‐power fields per duplicate. Data are shown as dot and box plots (n=6). Two‐way analysis of variance with Tukey-Kramer post hoc test was applied. Representative immunofluorescent images are shown. LC3‐II, microtubule-associated protein 1 light chain 3-II; p62/SQSTM1, sequestosome 1; DAPI, 4´,6‐diamidino‐2‐ phenylindole; DMSO, dimethyl sulfoxide.

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Agonist-Induced Activation of Transient Receptor Potential Vanilloid 4 Promotes Autophagy and Extracellular Matrix Synthesis in the Rat Intervertebral Disc
NS : Neurospine
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