The UBA5-GABARAP-PERK axis promotes cartilage degeneration in osteoarthritis by inhibiting autophagy and promoting endoplasmic reticulum stress

doi:10.21203/rs.3.rs-2131841/v1

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Research article

The UBA5-GABARAP-PERK axis promotes cartilage degeneration in osteoarthritis by inhibiting autophagy and promoting endoplasmic reticulum stress

https://doi.org/10.21203/rs.3.rs-2131841/v1

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purpose

Osteoarthritis (OA) is one of the most common causes of disability in the elderly. Ubiquitin-like modifier-activated enzyme 5 (UBA5) is a critical factor in preventing cellular autophagy and causing endoplasmic reticulum stress but has not been studied in OA. We aimed and explored the involvement of the UBA5-GABARAP-PERK axis in regulating cartilage matrix metabolism and apoptosis in osteoarthritis.

Methods

Oxidative stress was induced using IL-1β, which disrupted the homeostatic balance of cartilage. In in vivo and in vitro experiments, Western blot, qt-QPCR, scanning electron microscopy, immunofluorescence, and mCherry-GFP-LC3 plasmid were applied to observe OA-associated cartilage degeneration, ROS production, mitochondrial function, autophagic flux, endoplasmic reticulum stress and matrix after application of UBA5 selective inhibitor DKM2-93, knockdown or overexpression of UBA5 changes in metabolic indicators. UBA5 adeno-associated virus was injected into the cavity of mice, and a mouse OA model was induced by DMM surgery. Histological analysis of cartilage degeneration was performed using immunohistochemistry, Safranin-O staining, HE staining, Micro-CT, OARSI, and synovitis score.

Results

Expression of UBA5 was increased in chondrocytes receiving IL-1β intervention. Knockdown of UBA5 in vivo and in vitro inhibited OA-related chondrogenic degeneration, alleviated mitochondrial dysfunction, stimulated autophagy, inhibited endoplasmic reticulum stress, reduced catabolism, and increased anabolism. Overexpression of UBA5 also promotes oxidative stress and disrupts the molecular signature of healthy chondrocytes. Mechanistically, the destructive function of UBA5 may be attributed to the activation of the PERK/ATF4 signaling pathway. Through immunoprecipitation experiments, UBA5 was shown to inhibit autophagy by interacting with GABARAP to activate the PERK signaling pathway. Inhibition of PERK attenuated UBA5-induced osteoarthritis. Our findings suggest that Jun-B and C-Jun transcription factors may promote UBA5 expression and inhibition of UBA5 expression by in vivo application of adeno-associated virus, reduce chondrocyte death, attenuate cartilage degeneration, and promote subchondral bone remodeling.

Conclusions

This study revealed that UBA5 might regulate chondrocyte matrix catabolism and anabolism through the UBA5-GABARAP-PERK axis, suggesting a potential role for UBA5 in OA cartilage injury.

osteoarthritis

chondrocyte

autophagy

UBA5

GABARAP

Endoplasmic Reticulum

There are approximately 10% of men and 18% of women in the developed world suffer from osteoarthritis (OA) over the age of 60. Direct and indirect socio-economic burdens resulting from the disease in developed countries are between 1.0% and 2.5% of GDP. Population aging is deepening its impact as time goes on(1). For patients with advanced osteoarthritis, total joint replacement is the primary treatment option because there is no intervention to halt or reverse disease progression. It has been a commitment to prevent and treat early osteoarthritis due to the short lifespan of prosthetics and the side effects of joint replacement surgery(2, 3). In osteoarthritis, cartilage is gradually destroyed, extracellular matrix synthesis is inadequate, and matrix-degrading enzymes are accumulated, resulting in progressive joint damage(4). Despite the identification of numerous risk factors, including genetics, mechanical stress, and metabolism, the pathogenesis of osteoarthritis remains poorly understood, necessitating the search for effective preventive and pharmacological targets(5, 6).

In eukaryotic cells, autophagy is a mechanism of self-protection that maintains metabolic homeostasis within the cells. Chondrocyte autophagy increases during the early stages of OA and decreases during the late stages(7).

The ubiquitin-like modifier activator enzyme 5 (UBA5) belongs to the E1-like ubiquitin activator enzyme family and regulates autophagy specificity by activating ubiquitin fold modifier 1(8). There are five components in the ubiquitin-proteasome system: ubiquitin, E1 ubiquitin-activating enzyme, E2 ubiquitin-binding enzyme, E3 ubiquitin ligase, 26S proteasome, and DUB. UFMylation is initiated by UBA5, which affects DNA damage response, transcriptional regulation, cell cycle control, dynamic homeostasis of ER proteins, and autophagy(9) (10–13). Our previous study found that UBA5 is involved in ER-phagy, which increases autophagic flux in cancer cells(14, 15); in addition to autophagy, ER-phagy is considered an effective method of removing damaged organelles(8). Endoplasmic reticulum stress pathways may be involved in the pathogenesis of osteoarthritis. Reducing endoplasmic reticulum stress attenuates cartilage degeneration and chondrocyte apoptosis(16, 17). By studying UBA5, we suggest that it disrupts cartilage homeostatic balance and promotes osteoarthritis progression and that the previous role of UBA5 in osteoarthritis remains unclear. In addition, UBA5 promotes endoplasmic reticulum stress by enhancing PERK/ATF4 signaling and inhibits GABARAP expression leading to reduced autophagic flux, thereby promoting osteoarthritis. We found that the UBA5-GABARAP-PERK axis is involved in the regulation of chondrocyte matrix metabolism, apoptosis, inhibition of autophagy, and promotion of endoplasmic reticulum stress. The biological role of UBA5 in OA is explored in our study for the first time. As a result, our data demonstrate the importance of UBA5 in regulating cartilage health as an osteoarthritis treatment target.

Reagent

MCE Chemicals (State of New Jersey, USA) supplied DKM2-93 (98.87%), 3-Methyladenine (3-MA), Thapsigargin (TG), E64d, and pepstatin A. Sigma Chemical provided chloroquine (CQ) dissolved in 0.1% dimethyl sulfoxide (DMSO). Recombinant mouse IL-1β was obtained from the R&D system (Minneapolis Research and Development Center, Minneapolis, Minnesota, USA). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM/F12) were provided by Gibco (New York, NY, USA). Anti-COL2A1 and Anti-UBA5 were purchased from Proteintech Group (Wuhan, China); Anti-MMP13 was purchased from Abcam (Cambridge, UK). Anti-β-Actin, anti-MMP3, FITC-labeled goat anti-rabbit secondary antibodies, cy3-labeled goat anti-mouse secondary antibodies, trypsin, and type II collagenase were purchased from Boster (Wuhan, China). Cell Signaling Technology (Beverly, USA) provided SQSTM-1, LC3 II/I, and Ubiquitin. Abclonal (Wuhan, China) provided PERK, P-PERK, ATF4, BiP/GRP78, and IRE1.

Isolation and culture of mouse chondrocytes

Chondrocytes were obtained from knee cartilage of 5-day-old C57BL/6J mice (from Tongji medical college, Wuhan, China). Chondrocytes from isolated knee cartilage were incubated with 0.25% trypsin-EDTA for 30 minutes in a 37℃ incubator; after trypsin removal and incubation with 0.2% collagenase for 6–8 h, chondrocytes were collected and cultured in DMEM/F12 medium containing 10% fetal bovine serum. The medium was changed every two days. Cultured first and second-generation chondrocytes were used for experiments after 80% of the cells reached 80% in the flasks.

Cellular activity and drug toxicity assays

A cell counting kit (CCK8, Boster, China) was used to measure DKM2-93's effects on chondrocyte activity. Inoculation of chondrocyte suspensions (100 µL/well) in 96-well plates was carried out for 24 hours. DKM2-93, a selective inhibitor of UBA5, was applied to chondrocytes for 6 h, 12 h, 18 h, and 24 h. After washing the cells with PBS, CCK8 solution was added and incubated for one hour at 37°C. An enzyme meter (Molecular facility in Sunnyvale, California) measured absorbance at 450 mm wavelength.

Histopathological evaluation

The knee joints of mice were collected after they were injected with 1% sodium pentobarbital intraperitoneally. After the knee muscles and skin were removed, the knee joint was fixed in 4% paraformaldehyde for 24 hours, decalcified in decalcifying solution for 25 days, embedded in paraffin, and the medial joint space of the knee joint was cut using a microscopic cutter (LeicaBios systems, USA). The sections were stained with safranin-O, toluidine blue, and HE. According to the recommendations of the Osteoarthritis Research Society International (OARSI), cartilage degeneration in mice was histologically graded according to the severity of cartilage damage. A grading system was used to determine the severity of synovitis. A scoring system was developed by two independent investigators who did not know the grouping; immunohistochemistry was performed as previously described, and sections were incubated with antibodies against MMP3, MMP13, COL2A1, and UBA5(18).

siRNA

Designed and synthesized siRNAs for mouse UBA5, GABARAP genes (RiboBio, Guangzhou, China), UBA5 sequence is sense strand 5'-GTCCACAACTACAATATCA-3'. Sequence of GABARAP is: sense strand 5' GTACCAGGAACACCATGAA-3'. After inoculation into six-well plates, chondroblasts were transfected for 48 hours with lipofectamine 3000 siRNA transfection reagent from Thermo Fisher.

Adeno-associated viruses and overexpression plasmids

Mouse UBA5 gene of AAV9-UBA5 and AAV9-GFP were purchased from Jikai Gene, Shanghai, China. A plasmid overexpressing the mouse UBA5 gene was designed and synthesized by Jiman Gene, Shanghai, China, and the sequence was senses trand5'-GTCCACAACTACAATATCA-3'. We constructed the UBA5 overexpression plasmid according to the instructions, and we used Lipo3000 and P3000 to transfect chondrocytes for 48h using the liposome transduction method.

Western blot

The cells were washed three times with phosphate-buffered saline (PBS), placed on ice, and lysed for 30 minutes in RIPA lysis buffer containing a 1% protease inhibitor phosphatase inhibitor mixture. The protein samples were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Billerica, MA), which were blocked in 5% BSA for 60 min, incubated overnight in specific primary antibody. The membranes were incubated in a 1:5000 dilution of membranes incubated in 1:5000 dilution of horseradish peroxidase-conjugated secondary antibody (BA2913, Boster, Wuhan, China) for 60 min, washed three times with TBST, and then incubated with Western ECL Substrate Kit. The protein bands were visualized by Bio-Rad scanner (Hercules, CA) and subsequently analyzed by ImageJ.

Immunofluorescence

We inoculated chondrocytes into 24-well plates, fixed them with 4% paraformaldehyde at room temperature for 10 minutes, washed them three times with PBS, permeabilized them with 0.1% TritonX-100 for 10 minutes, and closed the wells with 5% BSA at room temperature for 1 hour. In the dark room, we incubated chondrocytes for 1h with FITC goat anti-rabbit secondary antibodies, washed them three times with PBS, stained them with DAPI for 10 minutes, and blocked them for 10 minutes. The images were taken with a fluorescence microscope (Evosfl AUTO, Life Technologies, USA).

Reactive oxygen (ROS)testing

A Reactive Oxygen Species Assay Kit (S0033, Beyotime, Wuhan, China) was used to measure cellular ROS levels. By detecting DCF fluorescence through fluorescence microscopy, the researchers confirmed the level of intracellular reactive oxygen species by oxidizing non-fluorescent DCFH to generate fluorescent DCF.

Mitochondrial membrane potential detection

A mitochondrial membrane potential assay kit with JC-1 probe was used to detect mitochondrial transmembrane potential (MMP) changes. A decrease in cell membrane potential can be easily detected by the change in the color of JC-1 from red to green, and the change in color of JC-1 can also be used as an indicator of early apoptosis.

Co-immunoprecipitation (Co-IP)

After removing insoluble precipitate from chondrocytes, 1–2 µg of UBA5 or GABARAP antibody or rabbit IgG389 (B900610, Proteintech, china) was added and incubated for 16 h at 4°C. Protein A/G magnetic beads (HY-K0202, MCE) were added and incubated at 4°C for 4 hours. We washed the proteins four times in 1XPBS, precipitated them at 95°C for 10 min, separated the proteins using SDS-PAGE gels, and analyzed them by protein blotting.

TUNEL staining

The level of apoptosis was detected using TUNEL staining. We stained sections with the TUNEL Apoptosis Detection Kit (Alexa Fluor 488, Yeeasen, China), stained cell nuclear with DAPI, and observed images under a fluorescence microscope (Evosfl AUTO, Life Technologies, USA).

Transmission electron microscope observation of mitochondrial morphology

The chondrocytes were fixed in 2.5% glutaraldehyde fixative at room temperature, protected from light for 5 minutes, scraped off with a cell scraper, and centrifuged for 2 minutes at 2500 rpm. Use new electron microscope fixative, fix for 30 minutes at room temperature, avoid light, store at 4 ℃, and observe using Hitachi TEM.

Animal experiments procedure

This study used forty-eight 7-week-old (23g ± 2g) male pathogen-free C57BL/6 mice, which were approved by the ethical committee of Tongji Hospital, Tongji Medical College, and Huazhong University of Science and Technology, China. C57BL/6J mice were randomly divided into 6 groups (n = 8): Sham group, sham + AAV9-GFP group, AAV9-GFP + DMM group, DMM group, AAV9-UBA5 + Sham group, AAV9-UBA5 + DMM group. The adeno-associated virus was injected into the joint cavity of mice requiring injections of AAV9-GFP and AAV9-UBA5. For the expression of the relevant genes, all mice were kept for two weeks. Sterilization, anesthesia with 1% sodium pentobarbital intraperitoneally, and supine immobilization were performed routinely before surgery. As part of the sham-operated group, the skin was performed, but DMMs were not performed. According to the published protocol, a transection of the medial meniscal ligament and medial collateral ligament (DMM) caused joint instability in mice without damaging other ligaments(18, 19). animals were euthanized 8 weeks after surgery.

Statistical Analysis

In order to conduct statistical analysis, GraphPad Prism 7.0 was used, and the results are presented as mean ± standard deviation (SD). ANOVA and Student tests were used to compare two or more groups of data. p < 0.05 was considered to be a statistically significant difference.

Increased expression of UBA5 in knee cartilage of mice with surgically induced OA

By screening the GEO database, the investigators found that UBA5 was highly expressed in osteoarthritic cartilage (Fig. 1A, 1B, Support table1); to further explore the relationship between UBA5 activity and cartilage degeneration in osteoarthritis, we constructed a DMM-induced OA mouse model and performed immunohistochemical staining (IHC) to examine the expression of UBA5 in articular cartilage of normal and OA mice. We found that UBA5 expression was low in normal chondrocytes and increased in the cartilage of OA mice 8 weeks after DMM surgery (Fig. 1C). It seems that the high level of UBA5 protein expression is associated with cartilage damage (Fig. 1D). According to Safranin-O staining, articular cartilage thickness was impaired in severely degraded articular cartilage samples. Expression of UBA5 was significantly increased in the superficial region of cartilage, indicating that UBA5 may be associated with cartilage damage. This finding could be confirmed by RT-qPCR (Fig. 1E). There is evidence that UBA5 may contribute to cartilage erosion and the development of OA.

UBA5 is involved in regulating the anabolism and catabolism of chondrocytes

UBA5 is involved in regulating chondrocyte anabolism and catabolism. With the selection of the small molecule UBA5-specific inhibitor DKM2-93 (DKM), we explored the molecular mechanisms of UBA5 in regulating the extracellular matrix (ECM) of chondrocytes (Fig. 2A). DKM did not affect cell viability at less than 50 µM and 24 hours of intervention time compared to the normal group (Fig. 2B, 2C). The intervention of chondrocytes with IL-1β to construct an in vitro OA cell model revealed that DKM alleviated the IL-1β-induced decrease in COL2A1 and increased in MMP3 and MMP13 and that this therapeutic effect was dose-dependent, indicating that 25 µM is the optimal DKM2-93 concentration for treating chondrocytes with an optimal intervention time of 24 h (Fig. 2D, 2G, 2E, 2H). In addition, siRNA-UBA5 was applied to assess the blocking effect on UBA5. UBA5 knockdown resulted in reduced MMP13 expression and upregulated COL2A1 expression, and the above data indicated that blocking UBA5 expression could promote cartilage anabolism and inhibit ECM degradation (Fig. 2F, 2I). Meanwhile, the investigators searched for upstream transcription factors regulating UBA5 using the cistromeDB database and initially found that transcription factors Jun-B and C-Jun were involved in regulating UBA5 expression by rt-qPCR validation (Fig. 2J)(20). Previous studies have shown that C-Jun and JUN-B are involved in the regulation of MMP3, ADAMTS-5, and COL2A1 expression in chondrocytes, which are involved in a variety of cellular pathological processes in joint tissue(21). Therefore, we suggest that UBA5 is involved in chondrocyte homeostatic homeostasis.

Inhibition of UBA5 reduces reactive oxygen species production with mitochondrial dysfunction to inhibit oxidative stress-induced chondrocyte apoptosis

Aging, oxidative stress, and mitochondrial dysfunction are the pathogenesis of OA. atg8 autophagy is heavily dependent on mitochondria, which is associated with mitochondrial autophagy. Excessive ROS production leads to DNA and mitochondrial damage, and the damaged outer membrane slowly accumulates in the nucleus and cytoplasm. Apoptosis and accelerated senescence are caused by excessive reactive oxygen species (ROS) production and antioxidant deficiency. DKM treatment reduced ROS levels in chondrocytes and IL-1β-induced oxidative stress in chondrocytes (Fig. 2K). Images observed by transmission electron microscopy showed that IL-1β intervention resulted in chondrocytes exhibiting reduced mitochondrial volume, increased membrane density, and loss of intermediate cristae. To some extent, silencing UBA5 alleviated these mitochondrial dysfunctions (Fig. 2L). The early stages of apoptosis are characterized by a decrease in mitochondrial membrane potential (MMP). To investigate whether UBA5 is involved in IL-1β-induced collapse of the mitochondrial membrane potential, chondrocytes were treated with IL-1β or IL-1β in combination with DKM. As a control, starvation reduced the level of mitochondrial membrane potential and disrupted mitochondrial autophagy. When the mitochondrial membrane potential was high, the JC-1 fluorescent probe produced red fluorescence and formed J-aggregates. Mitochondrial depolarization was assessed by measuring the ratio of red to green light when mitochondrial membrane potential was low because JC-1 is a monomer (monomer) and cannot aggregate in the matrix. The results showed that IL-1β increased the green fluorescence intensity of the JC-1 monomer, which led to a decrease in MMP and impaired mitochondrial function. Inhibition of UBA5 may alleviate mitochondrial autophagy and rescue the collapse of mitochondrial membrane potential in the experimental group under DKM intervention (Fig. 3A). The above results suggest that inhibition of UBA5 may alleviate the negative effects of oxidative stress, protect chondrocytes from ROS-induced collapse of mitochondrial membrane potential and altered mitochondrial morphology, and reduce chondrocyte apoptosis.

UBA5 inhibits autophagic flux in chondrocytes and the extension process of autophagosomes

Autophagy is thought to target and remove misfolded, aggregated, or damaged proteins, dysfunctional organelles, and intracellular pathogens(22). Previous studies have shown that tumor cell autophagy is increased in the absence of UBA5(15, 23). The presence of a bispecific LIR/UFM pattern at the C-terminus of UBA5 binds to Atg8 proteins, specifically GABARAP, and exerts a variety of biological effects, including endoplasmic reticulum stress; also, GABARAP is involved in the localization of UBA5 at the endoplasmic reticulum membrane (24–27). Based on the above studies, we set out to explore how UBA5 affects autophagy in chondrocytes and the relationship between the roles of UBA5 and GABARAP. First, the subcellular localization of UBA5 was confirmed (28). EMBL-EBI database(29) search suggested GST pull-down experiments to confirm the interaction between UBA5 and GABARAP(30). Immunofluorescence results showed that DKM decreased the IL-1β-induced expression level of UBA5 (Fig. 3B). Autophagy is widely believed to be promoted by starvation and the mTOR inhibitor rapamycin (Rapa). Under starvation conditions, chondrocytes expressed less UBA5, and the results suggest that UBA5 may not affect autophagy via the trophic pathway. The degree of inhibition of UBA5 expression by DKM was comparable to Rapa levels, suggesting that UBA5 affects autophagy (Fig. 3A and 3C). the LC3II/I and Beclin-1 expression. GFP-LC3 spots were used to confirm changes in chondrocyte autophagy. When cells are not autophagic, LC3 shows a diffuse state in the cytoplasm. When GFP-LC3 was activated by external stimuli, a speckled state was observed on autophagosomal membranes. The appearance of LC3 spots on the autophagosome membrane after silencing UBA5 indicated that silencing UBA5 promoted autophagy, and a similar phenomenon was observed in chondrocytes under IL-1β intervention, suggesting that silencing UBA5 promotes the formation of chondrocyte autophagosomes and autophagic lysosomes (Fig. 3D). Gain-of-function and loss-of-function interventions with UBA5 revealed that silencing UBA5 in chondrocytes reduced the expression of SQSTM-1, MMP3, and MMP13, increased GABARAP, LC3B-II/I, and Beclin-1, and restored autophagy levels. Overexpression of UBA5 stimulated the expression of MMP13, SQSTM-1, inhibited LC3B-II/I, GABARAP, and Beclin-1 expression, and inhibited autophagy, while silencing of GABARAP reduced SQSTM-1, LC3II/I and Beclin-1 expression. As a result of silencing/overexpressing UBA5, the expression levels of SQSTM-1 decreased and increased, respectively, as well as that of autophagy-related proteins (LC3 II/I, GABARAP, and Beclin-1) increased and decreased, indicating that UBA5 is related to SQSTM-1, LC3B-II/I, and Beclin-1. It is possible that UBA5 affects autophagic flux by relocating SQSTM-1 (Fig. 3E-G, 3I-K). At the same time, we verified the interaction between UAB5 and GABARAP by CO-IP experiment, which further proved that UBA5 regulates the autophagy process (Fig. 3H).

There are two reasons for the increase in LC3 expression. In the autophagy-activated state, LC3 expression increases to promote autophagosome formation, or in the late stage of autophagy, LC3 cannot be degraded, resulting in accumulation. In order to investigate the effect of UBA5 on autophagy, we used E64d + pepstatin A, CQ, and 3-MA to block autophagy. A combination of e64d and pepstatin A inhibited lysosomal acidic protease activity, resulting in significant inhibition of lysosomal degradation. However, autophagosome formation was not affected. In comparison with other experimental groups, E64d + pepstatin A with DKM increased the expression of UBA5 and SQSTM-1 in chondrocytes. UBA5 protein expression was found to increase after inhibition of lysosomal degradation and not interfere with autophagosome formation, indicating that autophagy inhibition by UBA5 occurs before autophagosome lysosomal cleavage (Fig. 4A, 4D). By preventing autophagosome formation and nucleation, 3-MA inhibits early autophagy. Compared with DKM alone for intervention in chondrocytes, 3-MA inhibits autophagy and increases UBA5 expression (Fig. 4B, 4G). In comparison with other experimental groups, 3-MA intervention inhibited autophagic flux by suppressing ubiquitination levels, promoting SQSTM-1 expression, and partially reversing the extent of autophagy rescue by DKM (Fig. 4E, 4H); CQ and DKM combined did not affect ubiquitination levels (Fig. 4F, 4I). The above results show that LC3 expression is suppressed by UBA5 by inhibiting the extension of autophagosomes, but not the cleavage phase of autophagy-lysosomes.

UBA5 promotes endoplasmic reticulum stress

Sixteen genes were obtained from the Genecards database (31) and GEO database (including UBA5, MAP1LC3A, RFC1, NOP56, RBMX, EDEM1, LMO7, MAGED1, NOLC1, WDFY3, DNAJC3, DNAJC10, SEC62, CCPG1, GET4, and TEX264) are involved in biological processes such as endoplasmic reticulum stress, endoplasmic reticulum autophagy and mitochondrial dysfunction (Fig. 5A). Autophagy can be inhibited to some extent by sustained endoplasmic reticulum stress. Protein blotting experiments showed that Silencing of UBA5 reduced the expression of endoplasmic reticulum stress markers PERK, P-PERK, ATF4, and BiP/GRP78 in IL-1β-intervened chondrocytes (Fig. 5B, 5E). Silencing PERK using siRNA-PERK decreased the expression of MMP13, PERK, and BiP/GRP78 and increased the expression of COL2A1, indicating that the PERK pathway was activated by IL-1β intervention. The above results demonstrated that silencing UBA5 could inhibit the activation of the PERK signaling pathway by IL-1β, inhibit chondrocyte catabolism and promote anabolism (Fig. 5C, 5F). UBA5 could exert biological functions through the PERK signaling pathway.

Inhibition of UBA5 alleviated OA progression

We tested whether AAV9-shRNA-UBA5 adeno-associated viruses could mitigate OA progression by injecting specific AAV9-shRNA-UBA5 adeno-associated viruses into mice's joint cavities. Two weeks after AAV9-UBA5 injection, DMM surgery was performed on the joint cavity, and the mice were killed four weeks later. UBA5 immunofluorescence was used to detect infection and knockdown efficiency after adeno-associated virus injection (Fig. 6A). After four weeks of treatment, the remaining mice were executed. A safranin O stain, toluidine blue stain, and the OARSI scoring system were used to assess the histomorphological features, and immunofluorescence staining was used to determine the expression levels of UBA5, MMP3, MMP13, and COLL2. Comparatively to the DMM group, UBA5 injection reduced cartilage surface calcification to some extent (Fig. 6B).

Conversely, cartilage damage and proteoglycan loss were reduced in the group injected with AAV9-UBA5 intervention compared to the DMM group. In the DMM group, the OARSI score was significantly higher than that in the sham-operated group. In the AAV9-UBA5 group, the OARSI score was lower than that in the DMM group, with significant differences between the groups (Fig. 6C, 6E). According to HE staining, the DMM group could cause synovial cell proliferation and synovial hypertrophy, whereas the AAV9-UBA5 intervention group showed some improvement in the severity of synovial inflammation (Fig. 6D, 6E). AAV9-UBA5 injection restored COL2A1 expressions in cartilage, while MMP3 and MMP13 expression decreased (Fig. 6F, 6H); TUNEL staining results indicated that silencing UBA5 inhibited apoptosis of chondrocytes (Fig. 6G).

Effect of UBA5 on subchondral bone remodeling in mice with OA

Using micro-CT, we confirmed the effect of intra-articular injection of AAV9-UBA5 adeno-associated virus on tibial articular surface flatness and subchondral bone remodeling in mice. DMM-injected groups had a disruption of the tibial articular surface; the tibial articular surface was flatter in the AAV9-UBA5 intervention group than in the DMM group (Fig. 7A). There was a comparison of the bone volume ratio, the number of trabeculae, and the thickness and separation of trabeculae in the medial subchondral bone of the tibial plateau between the experimental groups. Comparing the DMM group with the sham-operated group, there were no significant differences in BV/TV, Tb.N, Tb.Th, and Tb.Sp. BV/TV index increased in the AAV9-UBA5 group compared with the DMM group and Tb.Th index increased compared with the sham + AAV9-GFP group, indicating that UBA5 is involved in subchondral bone remodeling (Fig. 7B).

For the development of new treatments for osteoarthritis, it is essential to identify the genes that play a role in the synthesis and metabolism of the cartilage matrix(5, 32, 33). UBA5 is an enzyme that plays a key role in the UFM1 pathway, which can be found in the nucleus of the cell as well as in the membrane of the cell, initiating the binding of UFM1 to form the UFMylation pathway(34, 35).

Our study shows that UBA5 plays a significant role in OA pathogenesis, which is the first evidence that UBA5 may play a role in the pathogenesis of OA. We applied IL-1β treatment to chondrocytes to mimic OA in vitro, and as reported in this study, DKM2-93, a selective UBA5 inhibitor, significantly inhibited MMP3 and MMP13, promoted COL2A1, and reversed IL-1β-mediated collapse of mitochondrial membrane potential and mitochondrial morphological dysfunction to alleviate chondrocyte oxidative stress. A key role that C-Jun and Jun-B play in regulating UBA5 is that of transcriptional activation. In vitro, we suggest that inhibitors of UBA5 could reduce the degeneration of cartilage associated with OA as well as maintain mitochondrial function and cartilage integrity. There is evidence that ER surface proteins can recruit the ER-autophagy machinery through the LC3/GABARAP interaction region (LIR/GIM), which serves as a receptor to send endoplasmic reticulum to lysosomes for degradation and promote autophagosome formation(36).

In the maturation of autophagosomes, GABARAP is involved in membrane fusion and transport, which affect the localization and function of UBA5 on the endoplasmic reticulum membrane during the maturation process. It is important to note that UBA5 interacts with GABARAP through a bispecific LIR/UFM pattern, and this process is accompanied by critical proximity to the structural rearrangement of the gated K46 residue on the GABABRAP side chain(8, 37, 38). It has been shown in this study that UBA5 interacts with GABARAP, disrupting cartilage homeostasis, and promoting OA progression by inhibiting GABARAP expression. Using serum starvation and rapamycin as positive controls for autophagy induction, we observed that promoting autophagy decreased UBA5 expression, whereas silencing UBA5 enhanced autophagic flux. Atg12 binding and LC3 modification determine the expansion of autophagosomes in mammals. ATG12 binding requires ubiquitin-activating enzyme E1 (including UBA5) and ubiquitin-activating enzyme E2(38). 3-MA and CQ inhibit autophagy. UBA5 negatively regulates autophagy and participates in autophagosome extension but not in the fusion or degradation of autophagic lysosomes, according to the results.

Unfolded protein response (UPR) occurs when the endoplasmic reticulum is stressed by external factors or intracellular damage, resulting in protein synthesis, lipid metabolism, and calcium storage(39). Direct contact between mitochondria and ER or indirect processing through intermediate metabolites can affect mitochondrial membrane potential, mitochondrial biosynthesis, and reactive oxygen species production(32, 40). Pro-apoptotic or pro-survival signaling is determined by the degree of activation of the UPR or ER stress. As the degree of injury increases or UPR signaling is prolonged, mitochondrial damage and outer mitochondrial membrane permeabilization induce caspase-9 activation and initiate apoptotic mechanisms. Endoplasmic reticulum stress increases the level of autophagy, promotes the clearance of unfolded proteins and damaged organelles, and restores the stability of the internal environment. It has been suggested that the IRE1 and PERK-eIF2α signaling pathways are activated during autophagy activation by the UPR(41–44). Components of the UFMylation system can regulate ER stress directly or indirectly by establishing regulatory feedback loops. In the event that the UFM1 cascade fails to control ER stress, it leads to metabolic disorders (12, 45–48). In addition, the ubiquitin E3 ligase FBXO21, a component of the UFMylation system, is thought to be involved in promoting OA progression(49, 50).

With age, oxidative stress and mitochondrial dysfunction lead to abnormal protein homeostasis in OA and aging chondrocytes. Compared to young articular cartilage, OA or senescent cartilage exhibits a high expression of ER stress markers (including ATF4, CHOP), activation of JNK signaling pathway, and increased apoptosis. Blocking endoplasmic reticulum stress can alleviate the imbalance between chondrocyte apoptosis and cartilage matrix homeostasis(17, 51). ER stress-related signaling pathways are now considered to be key targets widely involved in the maintenance of cartilage homeostasis, and given the close relationship between UBA5 and the endoplasmic reticulum, we discussed the possibility that UBA5 is involved in ER stress-related signaling pathways as a component of the UFM system to affect OA. We found that UBA5 may disrupt articular cartilage homeostasis through the PERK-ATF4 signaling pathway. With the progression of OA, cartilage surface degeneration with synovitis occurs. In a DMM mouse model, silencing UBA5 with adeno-associated virus significantly reduced OARSI and synovitis scores. In the DMM mouse model, silencing UBA5 also reduced apoptosis and affected subchondral bone remodeling. According to our findings, UBA5 interferes with autophagic flux and mitochondrial function by inhibiting IL-1β, a process associated with the PERK pathway.

However, we demonstrated that cellular experiments could use more diverse stimuli to induce oxidative stress in chondrocytes. It is expected that these issues will be addressed in the future.

In summary, based on our in vivo and in vitro experiments, we found that UBA5 may be involved in the pathogenesis of osteoarthritis by reducing the expression of essential autophagy proteins, leading to mitochondrial dysfunction and increasing endoplasmic reticulum stress. As an inhibitor of osteoarthritis, DKM2-93 maintains the dynamic homeostasis of chondrocytes. UBA5 may regulate chondrocyte matrix catabolism and anabolism through the UBA5-GABARAP-PERK axis, suggesting a potential regulatory role for UBA5 in OA cartilage injury. The use of DKM2-93 to target UBA5 is a potential therapeutic strategy for the treatment of osteoarthritis.

OA, Osteoarthritis; UBA5, Ubiquitin Like Modifier Activating Enzyme 5; UFM1, Ubiquitin Fold Modifier 1; ECM, Extracellular Matrix; MMP3, Matrix Metallopeptidase 3; MMP13, Matrix Metallopeptidase 13; COL2A1, Collagen Type II Alpha 1 Chain; GABARAP, GABA Type A Receptor-Associated Protein; Rapa, Rapamycin; 3-MA, 3-Methyladenine; CQ, Chloroquine; TG, Thapsigargin; E64d, Aloxistatin; UPR, Unfolded Protein Response; PERK, Eukaryotic Translation Initiation Factor 2 Alpha Kinase 3; P-PERK, Phospho-PERK; ATF4, Activating Transcription Factor 4; BiP/GRP78, Glucose-regulated Protein 78; IRE1, Endoplasmic Reticulum To Nucleus Signaling 1; ER-phagy, Autophagy of the ER;

Ethical approval and consent to participate

All animal experiments were approved by the Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IACUC number: TJH-202201013).

Consent for publication

Not applicable.

Availability of data and materials

The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.

Competing interests

The authors declared no conflict of interest.

Funding

This work was supported by the National Natural Science Foundation of China (No: 82172498, 82172435);

Authors' contributions

Fengjing Guo: Conception, design, and obtaining of funding. Genchun Wang: data collection, analysis, and article drafting; Kai sun: data analysis and technical support; Zhou Guo: data collection; Liang Cai Hou: data collection. Zehang Zheng: technical support; jingTing Xu: data acquisition; Xiong zhang: technical support; yaPing Ye: technical support and funding acquisition. All authors gave final approval for the version to be submitted.

Acknowledgments

No applicable.

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The UBA5-GABARAP-PERK axis promotes cartilage degeneration in osteoarthritis by inhibiting autophagy and promoting endoplasmic reticulum stress

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Abstract

purpose

Methods

Results

Conclusions

Figures

Introduction

Material And Methods

Reagent

Isolation and culture of mouse chondrocytes

Cellular activity and drug toxicity assays

Histopathological evaluation

siRNA

Adeno-associated viruses and overexpression plasmids

Western blot

Immunofluorescence

Reactive oxygen (ROS)testing

Mitochondrial membrane potential detection

Co-immunoprecipitation (Co-IP)

TUNEL staining

Transmission electron microscope observation of mitochondrial morphology

Animal experiments procedure

Statistical Analysis

Results

Increased expression of UBA5 in knee cartilage of mice with surgically induced OA

UBA5 is involved in regulating the anabolism and catabolism of chondrocytes

UBA5 inhibits autophagic flux in chondrocytes and the extension process of autophagosomes

UBA5 promotes endoplasmic reticulum stress

Inhibition of UBA5 alleviated OA progression

Effect of UBA5 on subchondral bone remodeling in mice with OA

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

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