Compressive mechanical stress enhances susceptibility to interleukin-1 by increasing interleukin-1 receptor expression in 3D-cultured ATDC5 cells

Background Mechanical overload applied on the articular cartilage may play an important role in the pathogenesis of osteoarthritis. However, the mechanism of chondrocyte mechanotransduction is not fully understood. The purpose of this study was to assess the effects of compressive mechanical stress on interleukin-1 receptor (IL-1R) and matrix-degrading enzyme expression by three-dimensional (3D) cultured ATDC5 cells. In addition, the implications of transient receptor potential vanilloid 4 (TRPV4) channel regulation in promoting effects of compressive mechanical loading were elucidated. Methods ATDC5 cells were cultured in alginate beads with the growth medium containing insulin-transferrin-selenium and BMP-2 for 6 days. The cultured cell pellet was seeded in collagen scaffolds to produce 3D-cultured constructs. Cyclic compressive loading was applied on the 3D-cultured constructs at 0.5 Hz for 3 h. The mRNA expressions of a disintegrin and metalloproteinases with thrombospondin motifs 4 (ADAMTS4) and IL-1R were determined with or without compressive loading, and effects of TRPV4 agonist/antagonist on mRNA expressions were examined. Immunoreactivities of reactive oxygen species (ROS), TRPV4 and IL-1R were assessed in 3D-cultured ATDC5 cells. Results In 3D-cultured ATDC5 cells, ROS was induced by cyclic compressive loading stress. The mRNA expression levels of ADAMTS4 and IL-1R were increased by cyclic compressive loading, which was mostly prevented by pyrollidine dithiocarbamate. Small amounts of IL-1β upregulated ADAMTS4 and IL-1R mRNA expressions only when combined with compressive loading. TRPV4 agonist suppressed ADAMTS4 and IL-1R mRNA levels induced by the compressive loading, whereas TRPV4 antagonist enhanced these levels. Immunoreactivities to TRPV4 and IL-1R significantly increased in constructs with cyclic compressive loading. Conclusion Cyclic compressive loading induced mRNA expressions of ADAMTS4 and IL-1R through reactive oxygen species. TRPV4 regulated these mRNA expressions, but excessive compressive loading may impair TRPV4 regulation. These findings suggested that TRPV4 regulates the expression level of IL-1R and subsequent IL-1 signaling induced by cyclic compressive loading and participates in cartilage homeostasis. Supplementary Information The online version contains supplementary material available at 10.1186/s12891-021-04095-x.


Background
Mechanical overload applied to articular cartilage may play an important role in the pathogenesis of osteoarthritis (OA). However, the pathomechanisms of mechanical overload-induced cartilage degradation are poorly understood. The initial process of cartilage degradation has been shown to be associated with proteoglycan loss [1] and subsequent decreases in tissue hydration. As negatively charged glucosaminoglycan chains in proteoglycan draw in and maintain water within the cartilage matrix [2], proteoglycan loss decreases the interstitial osmotic pressure. Joint loading causes deformation of the cartilage layer and associated loss of interstitial water, followed by recovery of the fluid as the load is released [3][4][5]. The membrane stretch due to joint loading as well as osmotic pressure change reportedly elicit the activation of pumps and channels such as transient receptor potential vanilloid 4 (TRPV4), which initiates intracellular signaling cascade and changes cellular volume, designated as a regulatory volume decrease (RVD) or increase [6][7][8][9][10][11]. Although compressive stress on the cartilage results in various environmental changes, such as osmotic pressure, fluid flows and tensile stress, which lead to different biological responses [12][13][14][15], most important mechanisms underlying mechanical stressinduced cartilage degradation is overproduction of matrix-degrading enzymes, such as matrix metalloproteinase (MMPs) and a disintegrin and metalloproteinases with thrombospondin motifs 4 (ADAMTS) [16]. In this context, reactive oxygen species (ROS) reportedly acts as integral factors in intracellular signaling mechanisms to produce matrix-degrading enzymes [17].
To date, numerous studies have reported a close pathological relationship between interleukin-1β (IL-1β) and OA. Whether the chondrocyte itself possesses the capacity to produce IL-1β remains highly controversial, but high levels of caspase-1 and the active form of IL-1β have been detected in OA cartilage [18]. IL-1β has been shown to elicit transient changes in Ca 2+ levels in chondrocytes and to affect responses to hypoosmotic stress by preventing decreases in regulatory volume [19]. Moreover, a previous study showed that expression levels of IL-1 receptor (IL-1R) doubled in OA chondrocytes as compared to those in normal chondrocytes, suggesting that susceptibility to IL-1 is increased in OA cartilage [20].
The present study used 3D cartilage constructs in a Type I collagen scaffold, since cellular phenotypes and culture conditions such as monolayer culture, threedimensional (3D) culture, and cartilage explants influenced experimental results [21][22][23][24]. In fact, responses to mechanical loading by cells having the mature chondrocyte phenotype remain to be elucidated in the 3D environment. We developed a cyclic load bioreactor that can apply compressive deformation of the 3D constructs, mimicking the in vivo environment of human articular cartilage [25]. The bioreactor can be adopted to examine chondrocyte responses to the cyclic compressive stress. We hypothesized that IL-1 susceptibility and matrixdegrading enzyme expression would be increased by compressive mechanical stress and subsequent ROS induction, and this would be controlled by TRPV4, as a calcium channel mechanosensor that reportedly plays an important role in the regulation of cellular volume changes [26]. The purpose of this study was to assess compressive mechanical stress-induced IL-1R expression in 3D-cultured ATDC5 cells, and the effects of TRPV4 modification on IL-1R expression.
Cell differentiation in alginate beads culture ATDC5 cells were suspended at a density of 2 × 10 6 cells/ml in a 1.2% solution of sterile alginate (Kelco, Chicago, IL) in 0.15 M NaCl. The cell suspension was slowly expressed through a 22-gauge needle dropwise into a 100 mM calcium chloride solution. Beads were allowed to polymerize in this solution for 10 min before two consecutive washes with 0.15 M NaCl followed by two washes in DMEM/F12. After washing, beads containing ATDC5 cells were then placed into 12-well culture plates (10 beads/well) in 2 ml of DMEM/F12 containing 5% FBS, penicillin (100 μg/mL)-streptomycin (50 μg/ mL), insulin-transferrin-selenium, recombinant human bone morphogenetic protein (BMP)-2 (0.25 μg/mL, R&D Systems, Minneapolis MN), and 50 μg/ml ascorbic acid. ATDC5 cells were cultured in alginate beads with the growth medium containing insulintransferrin-selenium and BMP-2 for 6 days. Medium was changed once in three days. Medium was then removed from individual wells, and the beads were dissolved by incubation for 10-15 min at 4°C in dissolving solution (55 mM sodium citrate, 30 mM Na 2 EDTA, 0.15 M NaCl, pH 6.8). The resulting suspension was centrifuged at 900 rpm for 10 min at 4°C to separate the ATDC5 cells.
Cell seeding in collagen scaffold and production of 3Dcultured constructs The cultured cell pellet was seeded in collagen scaffolds to produce 3D-cultured constructs. In brief, cultured cells (5 × 10 5 /scaffold) were suspended in 1% collagen gel (Nitta gelatin, Osaka, Japan) on ice. The cell suspension was incorporated into collagen scaffolds (5-mm diameter and 3-mm thick, Atelocollagen Sponge Mighty; Koken, Tokyo, Japan;) using a 27gauge needle syringe. The collagen scaffold had an interconnected pore size of 30 nm to 200 nm. The scaffolds were produced via the process of freezedrying of 10% collagen gel and cross-linking to reinforce the mechanical property, which is similar to those of articular cartilage [25]. Cyclic compressive load of 40 kPa can apply a 10% compressive deformation of the constructs, which is stronger than physiological loading, since articular cartilage deformation under physiological loading is reportedly < 10% in human knees [27,28].
Cyclic compressive loading on 3D-cultured constructs Cyclic compressive loading was applied to the 3Dcultured constructs using a cyclic load stimulator (Technoview, Osaka, Japan), as previously reported [29,30]. The loading stimulator was formed with metal plates and 96-well culture dishes where the 3D-cultured construct was incubated with DMEM/F12 and 10% FBS and maintained at a temperature of 37°C in 5% CO 2 (Fig. 1a). The cylindrical loading pistons connected to weights and moving stage that raises and drops the loading pistons on to the constructs can apply compressive load (Fig. 1b). In this study, cyclic compressive loading of 0 kPa, 20 kPa or 40 kPa were applied at 0.5 Hz for 3 h. The constructs at 0, 1, 3, 6 and 12 h after cyclic compressive loading of 40 kPa for 3 h were analyzed for mRNA expressions of ADAMTS4, MMP-3 and IL-1R using a realtime polymerase chain reaction (RT-PCR). The mRNA expressions of ADAMTS4, MMP-3, IL-6, IL-1β and IL-1R were quantitatively measured at 6 h after cyclic compressive loading of 0 kPa, 20 kPa and 40 kPa for 3 h (Fig. 1c). Effects of antioxidant, IL-1β and TRPV4 agonist/ antagonist on IL-1R and ADAMTS4 mRNA expressions by 3D-cultured constructs under cyclic compressive loading Mechanical stress induces ROS accumulation in chondrocytes in vivo [31]. ROS signaling is a risk factor for osteoarthritis [32]. To assess effects of the 3D-cultured constructs with or without mechanical loading, we evaluated ROS accumulation by staining with 8-hydroxy-2′ -deoxyguanosine (8OHdG). In nuclear and mitochondrial DNA, 8-OHdG is one of the predominant forms of free radical-induced oxidative lesions, and has therefore been widely used as a biomarker for oxidative stress and carcinogenesis [33,34]. 3D-cultured constructs without loading were served as a control. Just before cyclic compressive loading was applied for 3 h, 50 μM of pyrollidine dithiocarbamate (PDTC) (Sigma Aldrich), an antioxidant, was added to the 3D-cultured constructs. PDTC, an inhibitor of NF-κB, affects induction of G1 phase cell cycle arrest, and prevent induction of ROS [35,36]. The mRNA expression of ADAMTS4 and IL-1R were measured 6 h after the cyclic loading. IL-1β was purchased from R&D Systems and TRPV4 agonist (GSK1016790A) and antagonist (HC-067047) were purchased from WAKO (Osaka, Japan). IL-1β (1 pg/ml, 10 ng/ml), GSK1016790A (1 mM), or HC-067047 (1 mM) were added to the 3D-cultured construct with or without cyclic compressive loading. The mRNA expression of ADAMTS4 and IL-1R were measured 6 h after the cyclic loading.

Real-time PCR analysis
Total RNAs were isolated from each sample by RNAiso plus (Takara Bio, Kyoto, Japan), and cDNAs were synthesized from total RNAs using a Prime Script RT H reagent kit (Takara Bio). Real-time PCR was performed using SYBR Premix ExTaq IIH (Takara Bio) with a DICE thermal cyclerH (Takara Bio), according to the instructions from the manufacturer. Results were normalized to glyceraldhyde-3-phosphate dehydrogenase (GAPDH) as the fold change compared with samples.

Statistical analysis
Data are presented as mean ± standard deviation (SD). Analysis of variance (one or two factor ANOVA) was used to test for differences among more than two groups. Post hoc test was performed by a Bonferroni (See figure on previous page.) Fig. 2 Alginate beads culture and production of 3D-cultured constructs. a) Alginate bead culture. ATDC5 cells were cultured in alginate beads with growth medium containing insulin-transferrin-selenium and BMP-2 for 6 days. Toluidine blue staining showed increased cartilage matrix synthesis, especially as proteoglycan in the alginate beads. b) After dissolving with sodium citrate and Na 2 EDTA, cells were seeded within collagen gels and incorporated into type-I collagen scaffold (Koken, Osaka, Japan) using a 27-gauge needle syringe. ATDC5 cells were distributed within the scaffold. c) Collagen type-II mRNA expression in the scaffold was significantly increased by the addition of recombinant BMP-2. d) In toluidine blue staining of 3D-cultured constructs, extracellular matrix was stained violet. e) In immunostaining, positive cells for the cartilage matrix components chondroitin 4-sulfate and keratan sulfate were present a) b) Fig. 3 Effects of cyclic compressive loading on 3D-cultured constructs. a) Temporal changes in mRNA expression levels of MMP-3, ADAMTS4 and IL-1R were analyzed for 12 h (n = 6 each). All mRNA expression levels were upregulated at 6 h after compressive loading for 3 h. b) The mRNA expression levels of ADAMTS4, MMP3, IL-6, and IL-1R were all increased by 40 kPa compressive loading at 6 h, whereas these mRNA expressions were not stimulated by 20 kPa loading. IL-1β expression level was unchanged after compressive loading. Values are given as mean ± SD (n = 6 each). *P < 0.05 method. The comparisons of two groups were performed by unpaired two-tailed Student's t-test. Each experiment was performed more than three times. P < 0.05 was considered to indicate statistical significance.

Results
Chondrogenic ATDC5 cells seeded in the type-I collagen scaffold ATDC5 cells were cultured in alginate beads with the growth medium containing insulin-transferrin-selenium and BMP-2 for 6 days. Toluidine blue staining revealed increased proteoglycan synthesis (Fig. 2a). After dissolving with sodium citrate and Na 2 EDTA, cells were successfully incorporated into type-I collagen scaffold (Fig. 2b). The collagen type-II mRNA expression in the scaffold was significantly increased with the addition of recombinant BMP-2 (Fig. 2c). In toluidine blue staining of 3D-cultured constructs, extracellular matrix was stained violet (Fig. 2d). In immunostaining, positive cells for the cartilage matrix components chondroitin 4-sulfate and keratan sulfate were present in 3D-cultured constructs (Fig. 2e).

Effects of cyclic compressive loading on 3D-cultured constructs
Time courses of expression levels of MMP-3, ADAM TS4 and IL-1R mRNAs after cyclic compressive loading of 40 kPa for 3 h were examined (Fig. 3A). MMP-3 and ADAMTS4 mRNAs were gradually increased over the first 6 h and thereafter decreased to the control level by 12 h. The IL-1R mRNA expression level was increased by 1 h after loading, maintained until 6 h, and then decreased to the control level by 12 h. The mRNA expression levels of ADAMTS4, MMP3, IL-6, and IL-1R were all increased by 40 kPa of compressive loading at 6 h, whereas such increase mRNA expressions were not observed by 20 kPa of loading (Fig. 3b). IL-1β mRNA expression was unchanged, regardless of the amount of compressive loading. The 3D-cultured constructs with cyclic compressive loading exhibited ROS accumulation, whereas those without cyclic compressive loading did a) b) Fig. 4 ROS accumulation in 3D-cultured constructs following cyclic compressive loading and effects of antioxidant. 3D-cultured constructs with or without cyclic loading were fixed. Cells were then stained with mouse-anti-8OHdG antibody and observed under a microscope. Bar, 20 μm. b) PDTC, an antioxidant, markedly prevented both ADAMTS4 and IL-1R mRNA expressions induced by compressive mechanical stress. Values are given as mean ± SD (n = 6 each). *P < 0.05 not (Fig. 4a). PDTC, an antioxidant, markedly prevented mechanically induced mRNA expressions of ADAMTS4 and IL-1R (Fig. 4b).
Increased IL-1 susceptibility of 3D-cultured constructs by cyclic compressive loading Increased expressions of ADAMTS4 and IL-1R mRNA were observed when 3D-cultured constructs were treated with a sufficient amount of IL-1β (10 ng/ml) in the absence of compressive loading (Fig. 5). Although a slight amount of IL-1β (1 pg/ml) alone failed to upregulate expression levels of ADAMTS4 and IL-1R mRNA, a slight amount of IL-1β (1 pg/ml) in combination with 40-kPa compressive loading dramatically increased both ADAMTS4 and IL-1R mRNA expressions by 5-and 8fold, respectively.

Effects of TRPV4 channel modification on ADAMTS4 and IL-1R mRNA expression levels in 3D-cultured constructs
Neither TRPV4 agonist nor antagonist affected expression levels of ADAMTS4 and IL-1R mRNA in the absence of cyclic compressive loading. However, TRPV4 agonist suppressed upregulation of ADAMTS4 and IL-1R mRNA levels induced by 40 kPa of cyclic compressive loading. TRPV4 antagonist conversely accelerated both ADAMTS4 and IL-1R mRNA expressions. Increased expressions of the both mRNAs were not observed by 20 kPa of cyclic compressive loading (Fig. 6).

Immunoreactivities of TRPV4 and IL-1R increase with cyclic compressive loading
The 3D-cultured constructs in the type-I collagen scaffold stained with toluidine blue exhibited a round morphology after applying cyclic compressive loading, as compared to those without cyclic compressive loading serving as controls (Fig. 7a, b). The Cells extended their cell protrusions at 0 kPa and the cell protrusions disappeared at 40 kPa in 3D-cultured constructs. The cells tended to exhibit a round shape, which is a morphology resistant to compressive mechanical stress ( Supple Fig). Immunoreactivities to TRPV4 (Fig. 7d, e) and IL-1R (Fig. 7g, h) significantly increased in constructs with cyclic compressive loading (Fig. 7e, h), but not in constructs without cyclic compressive loading (Fig. 7d, g).

Discussion
Development of anti-OA therapy has been focused on the mechanisms of interaction between physical factors (i.e., mechanical or osmotic) and biochemical factors, including inflammatory cytokines and cartilage matrixdegrading enzymes, such as IL-1, ADAMTS, and MMPs. Hyper-osmotic stress has been implicated as a potential surrogate for static compressive loading in cartilage [37], and the osmotic environment plays an important role in controlling the synthesis and breakdown of components of the extracellular matrix [38]. Previous studies have suggested that the effects of static compression on cartilage was attributed to IL-1 action [39] and osmotic pressure acted synergistically with IL-1 to stimulate Fig. 5 Effects of a slight amount of IL-1β with cyclic compressive loading. The mRNA levels of ADAMTS4 and IL-1R were substantially increased by the cyclic compressive stress. Increased expressions of both mRNAs were observed in the 3D-cultured constructs treated with a sufficient amount of IL-1β (10 ng/ml) as a positive control. Compressive stress plus 1 pg/ml IL-1β upregulated the levels of ADAMTS4 and IL-1R expression, but 1 pg/ml IL-1β alone failed to do so. Values are given as mean ± SD (n = 6 each). *P < 0.05 cyclo-oxygenase-2 and prostaglandin E2 productions in cartilage [40].
In the present study, cyclic compressive loading induced expression of IL-1R and ADAMTS4 genes through ROS induction by 3D-cultured ATDC5 cells, and that process was suppressed by TRPV4 agonist and enhanced by TRPV4 antagonist. TRPV4 has been known as a calcium channel receptor, transporting Ca 2+ intracellularly and regulating cell volume. In steady state, physiological mechanical loading may activate TRPV4 and stimulate the chondrocyte to elicit a Ca 2+ influx, causing regular RVD response [41]. Compression loading lead to mobilization of chondrocyte intracellular Ca 2+ and to changes of cartilage synthesis [42,43]. Lewis et al. showed that loss of chondrocyte volume regulation is initially a consequence of cartilage degeneration [44]. In our study, 20 kPa of loading condition, in which TRPV4 may promote normal RVD response, did not stimulate production of IL-1R or ADAMTS4, whereas a 40 kPa loading increased IL-1R production and this may result in compromising cell volume regulation by TRPV4. In fact, TRPV4 is highly expressed in articular chondrocytes, and loss of TRPV4 function is reportedly associated with joint arthropathy and osteoarthritis [45,46]. The biological activity of IL-1 reportedly stabilizes intracellular F-actin [47] and increases cellular stiffness and viscosity [48]. TRPV4-induced Ca2+ influx counteracts the F-actin stabilization evoked by IL-1 receptor signaling [49]. Mechanical overload-induced IL-1R thus carries a risk of obstructing the normal RVD response by F-actin-induced increments in cellular stiffness, as increased cellular stiffness was accompanied by impairment of channel receptors at the cell membrane [50,51]. Collectively, increased IL-1 susceptibility caused by mechanical overload may further impair cell volume regulation of TRPV4 and increase the risk of OA development, compatible with our results for 3D-cultured ATDC5 cells in the type-I collagen scaffold. Mechanical signal transduction appears to be triggered by a diverse array of biophysical conditions, such as cell deformation [52], alternation of hydrostatic pressure [53], fluid flow [54], and changes in extra-or intracellular osmotic pressure [55]. Numerous studies dealing with mechanotransduction in cartilage have introduced cyclic tensile stress in the monolayer culture and hydrostatic pressure in 3D culture [56,57]. The former experimental condition of tensile mechanical stress applied on a monolayer culture potentially causes dedifferentiation of chondrocytes. The present study utilized cyclic compressive stress of 40 kPa at 0.5 Hz, aiming at 10% deformity using the specific bioreactor, which might physiologically mimic the overloaded condition of the human knee joint. Moreover, ATDC5 cells gained the mature chondrocyte phenotype using quick alginate Fig. 7 Immunohistochemistry of TRPV4 and IL-1R. a, b) In 3D-cultured constructs in type-I collagen scaffold stained with toluidine blue, cells with cyclic compressive loading showed a round morphology as compared with cells without cyclic compressive loading. c-h) Immunofluorescent staining of TRPV4 (d and e) and IL-1R (g and h) revealed significant increases in TRPV4 (e) and IL-1R (h) immunoreactivities after cyclic compressive load was applied on the 3D-cultured constructs bead embedding followed by culture in collagen gel and type-I collagen scaffold. Such a culture environment may provide optimal experimental conditions for assessing the effects of compressive mechanical loading on mature chondrocytes.
Although whether chondrocytes possess the ability to produce IL-1β in OA cartilage remains disputable, several reports in the literature have provided positive evidence for slight amounts of IL-1β (< 1 pg/ml) in the synovial fluid of OA joints [58][59][60]. Indeed, subtle amount of IL-1β is assumed to be produced by surrounding synoviocytes and to gain access to and bind IL-1R on the surface of chondrocytes, and to exert biological activities as a proinflammatory cytokine. In our experiments, 1 pg/ml of IL-1β stimulated production of IL-1R and ADAMTS4 in combination with 40 kPa of cyclic compressive loading, because excessive mechanical stress substantially induced IL-1R in 3D-cultured ATDC5 cells. Another in vivo study in mice supported a critical role of IL-1R in the development of arthritis. Kagari et al. showed that arthritis induced by anti-type II collagen antibody was unable to be reproduced in IL-1R-KO mice [61]. In addition, a recent study demonstrated that IL-1R gene polymorphisms were associated with susceptibility to knee OA in the Chinese population [62]. Taken together with this evidence, IL-1R expression may make the cells more susceptible to IL-1β signaling, potentially leading to increased cartilage degeneration and OA. Finally, there are several limitations to this study. First, ATDC5 cells rather than actual chondrocytes were differentiated and embedded in a Type I collagen scaffold to create a cartilage tissue. However, responsiveness to the mechanical stress might be different from the actual articular cartilage. Second, the effects of fluid flow by piston moving of the load stimulator was not taken into account in the unloading samples. Third, protein production levels of IL-1R, ADAMTS4 and other molecules were not evaluated in this study. We assumed that compression stress causes the cell volume change, and TRPV4 may act as a cell volume regulator. If so, Ca 2+ influx would occur, however, we have not directly analyzed Ca 2+ influx and cell volume change. Last, the direct relationship between ROS production and TRPV4 function in response to compressive mechanical stress remains unclear. It is undoubtedly a subject of our further studies.
In summary, cyclic compressive stress is capable of stimulating expression of IL-1R and matrix-degrading enzymes such as ADAMTS4 potentially through reactive oxygen species in 3D-cultured ATDC5 cells. When the degree of compressive stress is physiological, cell volume regulation by TRPV4 may function and abrogate further downstream events, but excessive compressive stress may impair TRPV4 function and subsequently increase production of IL-1R and ADAMTS4. In the articular cartilage at the initial OA stage, proteoglycan loss begins and interstitial osmotic pressure starts decreasing. In this context, even physiological mechanical stress carries a risk of causing dysfunction of cell volume control and further progression of OA. From the perspective of mechanical loading, calcium channel receptors such as TRPV4 is a key mechanosensor and osmosensor and plays an important role not only in the regulation of cell deformation and osmotic pressure, but also in suppression of the subsequent catabolic reaction of chondrocytes. Understanding chondrocyte responses to excessive mechanical stress may provide new insights into the pathogenesis of and future therapeutic strategies for OA.

Conclusions
Cyclic compressive loading induced mRNA expressions of ADAMTS4 and IL-1R through ROS in 3D-cultured ATDC5 cells, which was regulated by TRPV4. Excessive compressive loading may impair TRPV4 regulation. These findings suggested that TRPV4 regulates the expression level of IL-1R and subsequent IL-1 signaling induced by cyclic compressive loading and participates in cartilage homeostasis.