GABARAP ameliorates IL-1β-induced inflammatory responses and osteogenic differentiation in bone marrow-derived stromal cells by activating autophagy

Bone mesenchymal stem cells (BMSCs) are the most commonly investigated progenitor cells in bone defect repair and osteoarthritis subchondral bone regeneration; however, these studies are limited by complex inflammatory conditions. In this study, we investigated whether pro-autophagic γ-aminobutyric acid receptor-associated protein (GABARAP) promotes BMSCs proliferation and osteogenic differentiation by modulating autophagy in the presence or absence of interleukin-1 beta (IL-1β) in vitro. The expression levels of all relevant factors were evaluated by qRT-PCR or western blotting where appropriate. BMSCs differentiation were assessed by Alizarin Red, alkaline phosphatase, safranin O, and Oil Red O staining. Furthermore, the interactions between autophagy and osteogenic differentiation were investigated by co-treatment with the autophagy inhibitor 3-methyladenine (3-MA). As the results, we found that treatment with recombinant human His6-GABARAP protein promoted cell proliferation, inhibited apoptosis, and reduced ROS generation by increasing autophagic activity, particularly when co-cultured with IL-1β. Moreover, His6-GABARAP could effectively increase the osteogenic differentiation of BMSCs. The expression levels of inflammatory factors were significantly decreased by His6-GABARAP treatment, whereas its protective effects were attenuated by 3-MA. This study demonstrates that GABARAP maintains BMSCs survival and strengthens their osteogenic differentiation in an inflammatory environment by upregulating mediators of the autophagy pathway.

Mesenchymal stem cells (MSCs), including BMSCs, display a robust proliferative capacity and multipotency that allows them to differentiate into adipocytes 1 , chondrocytes 2 , osteoblasts 3 , and other non-mesodermal lineage cells 4 in different microenvironments. Therefore, these cells are considered promising therapeutic candidates for tissue engineering and display potential for clinical application in conditions such as osteoarthritis (OA). Previous studies have strongly indicated that BMSCs can effectively promote cartilage erosion regeneration in OA by not only differentiating into cartilage-forming chondrocytes but also modulating the behavior of subchondral bone 5 . However, the abnormal microenvironment of OA characterized by stress conditions, such as inflammation 6 and oxidative stress 7,8 , reduces the survival capacity of BMSCs. Moreover, high pro-inflammatory cytokine levels inhibit BMSCs osteogenesis 9 . Therefore, approaches to promote BMSCs osteogenic differentiation and improve their cell survival under severe stress is particularly important for tissue renewal and subsequent regeneration 10 .
IL-1β is a primary initiator of inflammatory progression in OA 11 , with studies reporting that elevated IL-1β levels play a central role in inflammation-induced bone destruction 12,13 . Indeed, IL-1β expression levels are significantly higher in OA than in healthy sites and is associated with subchondral destruction 14 . Mechanistically, the inhibitory effects of IL-1β have been associated with MSCs proliferation and the activation of osteoclastogenesis 15,16 . IL-1β treatment can also dramatically induce the production of ROS, nitric oxide (NO), and proteolytic enzymes such as matrix metalloproteinase (MMP) during bone formation, thus inhibits BMSCs osteogenic differentiation and bone tissue generation in vitro and in vivo [17][18][19] . Although anti-inflammation has been studied for decades, our understanding of the mechanism underlying the effects of IL-1β on cell viability and osteoblast differentiation remains poor.
Previous studies have shown that the maintenance of bone mass stability is associated with the activation of cellular autophagy 20 , which is a highly conserved catabolic process that maintains cellular homeostasis and recycles degraded cytoplasmic materials [21][22][23] . Autophagy can also maintain MSCs stemness and protect cells against stress pathology signals, including ROS, inflammation, and metabolic precursors 24,25 ; for instance, inhibiting autophagy in osteoblast-like cells increases oxidative stress and stimulates their apoptosis 26 . During osteogenesis, autophagy is critical for decreasing osteopenia by accelerating mineralization and osteogenesis in vitro and in vivo 20,27 , indicating that increased autophagy may maintain the BMSCs phenotype after IL-1β treatment and enable the promotion of osteoblast differentiation.
GABARAP is a member of the autophagy-related protein 8 (Atg8) family 28 that displays a high degree of sequence homology with the autophagy marker light chain 3 (LC3). Kabeya et al. 29 showed that GABARAP binds to autophagic vesicles in a similar manner to LC3 and may also be involved in autophagosome formation. By modulating autophagy, GABARAP has been reported play a key role in suppressing tumor growth 30 , inhibiting inflammation progression 31 , and regulating angiogenic activity 32 via processes associated with vesicle transport, apoptotic cell death, and ROS generation. However, the role of GABARAP in regulating the differentiation fate of BMSCs via autophagy remains unclear.
Based on the studies described above, we hypothesized that the mediation of ROS generation and cellular autophagy by GABARAP may play pivotal roles in IL-1β-induced BMSCs injury. This study is the first to demonstrate the regulatory effects of GABARAP on IL-1β-induced cell apoptosis and BMSCs osteogenic differentiation in vitro, with a particular focus on the relationship of GABARAP with autophagy signaling pathways. Our longterm goal is to utilize GABARAP in bone regenerative therapy, such as improving the efficacy of BMSCs-based cellular regenerative therapies.

Materials and methods
BMSCs isolation and culture. Bone marrow was flushed from the femurs and tibias of 1-5-day-old New Zealand rabbits (Animal Resources Centre of Guangxi Medical University, Nanning, Guangxi, China) with α-modified Eagle's medium (α-MEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% (v/v) fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% (v/v) penicillin/streptomycin (Solarbio, Beijing, China) using a 27-gage syringe. The cell suspension was strained through a 70 μm mesh filter and cultured in the same medium at 37 °C under 5% CO 2 . At 80-90% confluence, the cells were trypsinized, expanded, and used for different assays after their third passage. The animal protocol was approved by the Animal Ethical Committee of the Animal Resources Centre of Guangxi Medical University (ethic cord: 201902003). All animal experiments were performed in accordance with relevant guidelines and regulations. Meanwhile, animal studies were reported in compliance with the ARRIVE guidelines 33,34 and complied with the principles of replacement, refinement and reduction (the 3Rs).
To induce osteogenesis, BMSCs were cultured in osteogenic medium in the presence of three doses of His6-GABARAP (30, 60, and 120 nM) for 21 days, after which osteogenic differentiation was determined by Alizarin Red and alkaline phosphatase (ALP) staining. Based on the levels of intracellular calcium accumulation, 60 nM His6-GABARAP were selected as the most appropriate concentrations for observing the effect of His6-GABARAP on BMSCs osteogenic differentiation. Inflammation was stimulated by treating the cells with 10 ng/mL of IL-1β, after which the cells were treated with His6-GABARAP with or without the autophagy inhibitor 3-MA (5 mM, Sigma) to clarify its relationship with the autophagy pathway in conventional culture or osteogenic differentiation.
Cell proliferation assay. BMSCs proliferation was assessed using MTT assays. One tenth of the volume of 5 mg/mL MTT solution was added to stimulated BMSCs per well and continuously incubated at 37 °C for Intracellular ROS measurement. Intracellular ROS were measured using a fluorescent 2,7-dichlorodi- Transmission electron microscopy (TEM). Harvested BMSCs were fixed for 24 h with 2.5% glutaraldehyde, incubated with 1% osmium tetroxide for 1 h in 4 °C, and stained with 2% uranyl acetate. The BMSCs were then dehydrated using an acetone gradient and embedded in araldite. Sample sections were cut, stained with toluidine blue, and observed by TEM (Hitachi, Tokyo, Japan).
Autophagy flux measurement. StubRFP-sensGFP-LC3 lentiviruses were constructed by Genechem Co. (Shanghai, China). Primary first passage BMSCs were seeded in 6-well plates, cultured overnight, and transduced with the lentivirus in serum-free medium at a multiplicity of infection of 50. After 12 h, the media was replaced with complete α-MEM and then the cells were treated with IL-1β, His6-GABARAP, and 3-MA. Autophagic flux was observed using a laser scanning confocal microscope (Nikon America Inc., Melville, NY). stubRFP and sensGFP punctae were counted manually in at least 40 cells per sample.
Total RNA isolation and quantitative RT-PCR (qRT-PCR). Total RNA was isolated from BMSCs using Trizol reagent (Invitrogen), purified using a RNeasy mini kit (QIAGEN, Valencia, CA, USA), and reverse transcribed (1 mg total RNA per sample) using a Transcriptor First-strand cDNA synthesis kit (Roche, Basel, Switzerland). qRT-PCR was performed using SYBR green master mix on an Applied Biosystems 7500 Real Time Cycler (Applied Biosystems, CA, USA) under the following cycling conditions: 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min, followed by a standard melting curve. Samples were assessed in triplicate, with gene expression assessed using the 2 -ΔΔCT method and normalized to GAPDH. The primer pairs for the target genes are listed in Table 1.
Western blotting. Protein was extracted from cell samples using RIPA Lysis Buffer (Beyotime) containing phenylmethanesulfonyl fluoride (PMSF) (Beyotime, China). Equal protein quantities (60 µg) were denatured using SDS-PAGE loading buffer, separated by 6-15% polyacrylamide gel electrophoresis, and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Prior to hybridization with antibodies, the blots were cut according to the weight of target proteins. Blots were probed at 4 °C overnight using primary antibodies against mTOR, p-mTOR, LC3, (1:1000, Cell Signaling Technology, Beverly, MA, USA), and GAPDH (1:10,000, Cell Signaling Technology). The blots were washed three times with PBST and then probed with an appropriate DyLight™ 800 4X PEG conjugated secondary antibody (1:10,000, Cell Signaling Technology) for 1 h. Protein bands were identified using an Odyssey Infrared Imaging System and protein levels were quantified relative to the GAPDH loading control using ImageJ software (NIH, Bethesda, Maryland, USA).
Statistical analysis. Data were presented as the mean ± SD and assessed using SPSS version 22.0. Parametric data were compared by one-way analysis of variance (ANOVA) with least significant difference (LSD) post-hoc tests, while non-parametric data were assessed by Mann-Whitney U tests. p values of < 0.05 were considered statistically significant.

GABARAP increases BMSCs viability and proliferation by inhibiting intracellular ROS generation.
BMSCs were treated with varying concentrations of His6-GABARAP for 3 days, with no apparent toxicity observed for doses of 0-50 nM (Fig. 1). BMSCs exposed to 40 www.nature.com/scientificreports/ displayed significantly higher proliferation, with the most dramatic increase seen for 60 nM; therefore, 30, 60, and 120 nM of His6-GABARAP were selected for the subsequent experiments. First, we carried out HE staining to evaluate changes in cell morphology after His6-GABARAP treatment for 7 days. His6-GABARAP did not affect the typical fibroblastic cell morphology of cultured BMSCs but did increase the cell density compared to the normal group (Fig. 2B), as confirmed by MTT assays. As shown in Fig. 2C, His6-GABARAP concentrations of 30, 60, and 120 nM increased cell proliferation at days 1, with 60 nM of His6-GABARAP particularly increasing proliferation on days 3 (21.8 ± 5.2% higher than the control group), 5 (8.9 ± 1.4% higher than the control group), and 7 (12.4 ± 1.6% higher than the control group).
Next, we examined the effects of His6-GABARAP on BMSCs viability by Annexin V-FITC, PI, and FDA/ PI staining. BMSCs treated with 30 and 60 nM of His6-GABARAP showed a significantly higher proportion of viable cells after 7 days of treatment (Fig. 2E), with the highest percentage of live cells observed in the 60 nM His6-GABARAP-treated group (94.4 ± 0.7%). Staining with FDA/PI dyes, which differentiate between dead and live cells, revealed that BMSCs exposed to 30 and 60 nM of His6-GABARAP showed significantly higher viability after 1, 3, 5, and 7 days of treatment ( Fig. 2A), with the optimal effects observed at 60 nM. After 7 days, 30 www.nature.com/scientificreports/ and 60 nM of His6-GABARAP also decreased intracellular ROS levels to 21.8 ± 1.9 and 9.4 ± 1.5%, respectively (Fig. 2D). Taken together, these results demonstrate that GABARAP increases BMSCs viability by blocking intracellular ROS generation.

GABARAP up-regulates cell survival by targeting autophagy mechanisms.
To clarify the role of autophagy in the protective effects of His6-GABARAP in BMSCs viability and inflammation, we treated IL-1βinduced BMSCs with His6-GABARAP with or without 3-MA. MTT assays confirmed that 3-MA negatively affected the proliferation of IL-1β-treated BMSCs and effectively blocked the promotion of cell proliferation by His6-GABARAP (Fig. 4B). By detecting the expression of apoptosis-, antioxidant-, and inflammation-related cytokines in BMSCs, we found that 3-MA prevented the His6-GABARAP-mediated inhibition of cell apoptosis, oxidative stress, and inflammation ( Fig. 5A-I), indicating that 3-MA treatment has an inhibitory effect on BMSCs viability that is promoted by His6-GABARAP in an inflammatory microenvironment.

GABARAP maintains BMSC osteogenic differentiation in normal and inflammatory microenvironments.
Osteogenic differentiation was induced by culturing BMSCs in osteogenic media with or without 30, 60, and 120 nM of His6-GABARAP. As shown in Fig. 6C and D, ALP and Alizarin Red staining after 21 days indicated that His6-GABARAP stimulated BMSCs osteogenesis, with 60 nM of His6-GABARAP causing the most significant change in calcium deposition. We then quantified the mRNA levels of the osteoblast marker genes Col I, ALP, OCN, and OPN after 7, 14, and 21 days (Fig. 6E-H). His6-GABARAP increased Col I, OPN, ALP, and OCN transcription, particularly at a treatment concentration of 60 nM His6-GABARAP. Therefore, we used 60 nM of His6-GABARAP for subsequent experiments.
In the presence of IL-1β, 60 nM of His6-GABARAP stimulated denser deposition of Alizarin Red and ALP staining after 7, 14, and 21 days of osteogenic differentiation culture (Fig. 7A and B). Moreover, IL-1β-treated BMSCs had dramatically lower mRNA expression of the osteoblastic differentiation factors Col I, ALP, OCN, and OPN (Fig. 7C-F); however, treatment with 60 nM of His6-GABARAP yielded higher COL I, ALP, OCN, and OPN mRNA expression levels than in IL-1β treated BMSCs. Taken together, these results suggest that His6-GABARAP can maintain their BMSCs osteogenic differentiation under inflammatory conditions.

Autophagy plays a role in GABARAP-promoted BMSCs osteogenic differentiation.
To determine the mechanisms responsible for the increased osteoblastogenesis observed in His6-GABARAP-treated BMSCs under inflammatory conditions, we used 3-MA to suppress autophagy. Interestingly, inhibiting

Discussion
Previously, study reported that weekly intra-articular His6-GABARAP administration effectively promotes the therapeutic effect of BMSC-derived chondrocytes in OA lesions 38 ; however, the initiation and progression of OA involves a variety of pathological mechanisms, including the loss of bone mass in subchondral bone 39,40 and synovium lesions 41 . Meanwhile, its specific therapeutic mechanisms, particularly the associations between GABARAP and BMSCs or chondrocytes under inflammatory conditions, remain unknown. In present study, it had been demonstrated that autophagy plays an important role in promoting BMSCs osteogenic differentiation and restricts intracellular ROS generation 27 . Furthermore, it was also revealed that autophagy activation could enhance BMSCs proliferation 42 . Consistently, our pilot study also found that GABARAP could mediate BMSCs cell viability and osteogenic differentiation, which are inhibited by the pro-inflammatory factor IL-1β; thus, it may provide a potential application of GABARAP in OA repairment by promoting subchondral bone remodeling. The induction of cellular ROS generation and activation of apoptosis signaling by IL-1β has been increasingly recognized to play a vital role in cell stress 43,44 , and followed to induce senescence and apoptosis in various cell types, including BMSCs 6 , chondrocytes 45 , and osteocytes 46 , thus, IL-1β with significant pathological implications in the progression of many inflammatory diseases. Consistently, we found that IL-1β in the microenvironment may serve as an important modulator of BMSCs viability and proliferation and increase the expression of the inflammatory markers IL-6, TNF-ɑ, and MMP-13. However, His6-GABARAP treatment effectively maintained cell survival, restored the fibroblastic-like morphology of BMSCs, and downregulated pro-inflammatory cytokines and apoptosis-related markers. Furthermore, GABARAP not only suppressed inflammatory damage in BMSCs, but also enhanced BMSC proliferation and viability without IL-1β. www.nature.com/scientificreports/ Intracellular ROS generation is a major pathological driver of apoptosis in BMSCs 47 , which in turn plays a vital role in triggering inflammation 48,49 . Studies have demonstrated that autophagy plays a key role in suppressing ROS formation 50 ; therefore, we examined the effect of GABARAP on ROS generation in BMSCs. GABARAP stimulation significantly decreased ROS levels in BMSCs and the effect was maintained when co-cultured with IL-1β. In addition, His6-GABARAP treatment increased the expression of MnSOD and CuZnSOD, which are antioxidant factors in the superoxide dismutase pathways 51 .
Autophagy is a catabolic process wherein damaged proteins and organelles are phagocytosed and degraded to maintain energy levels and support organelle renewal 52 . Under stress conditions such as hypoxia and starvation, activation of autophagy can prevent apoptosis and maintain cellular homeostasis [53][54][55] . Indeed, we found that autophagic flux was significantly suppressed in IL-1β-induced BMSCs and restored by His6-GABARAP treatment. The phosphoinositide 3-kinase (PI3K) blocker 3-MA, which is routinely used to inhibit autophagy 56 , markedly abrogated the effects of GABARAP on apoptosis, the inflammatory response, and ROS generation. (G) Western blot was used to analyze the protein expression of LC3, mTOR, and p-mTOR. OS (with osteogenic differentiation); OS + IL-1β (with osteogenic differentiation and 10 ng/mL IL-1β); OS + IL-1β + 3-MA (with osteogenic differentiation, 10 ng/mL IL-1β, and 3-MA); OS + IL-1β + GA (with osteogenic differentiation, 10 ng/ mL IL-1β, and 60 nM His6-GABARAP); OS + IL-1β + GA + 3-MA (with osteogenic differentiation, 10 ng/ mL IL-1β, 60 nM His6-GABARAP, and 3-MA). Values are presented as means ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 relative to the OS group; #p < 0.05, ##p < 0.01, ###p < 0.001 relative to the OS + IL-1β group. Scale bar, 1000 µm. www.nature.com/scientificreports/ Therefore, our findings suggest that GABARAP promotes BMSCs proliferation and reduces inflammationinduced apoptosis by activating autophagy; however, more studies are required to clarify the mechanism further. The capacity to differentiate into several cell lines is a crucial characteristic of BMSCs, and it has been reported that the stimulation of intracellular ROS degradation by autophagy activation plays a vital role in regulating BMSCs osteogenic differentiation 27 . In this study, we observed that the pharmacological induction of GABARAP was closely associated with BMSCs osteogenesis and calcium deposition. Due to the importance of IL-1β in MSCs differentiation, multiple studies have demonstrated that IL-1β plays a crucial role in suppressing osteogenesis 57 and upregulating metalloproteinase expression to degrade the extracellular matrix of bone tissue 18 . Conversely, studies have also suggested that IL-1β stimulation promotes calcium deposition and endochondral ossification 58,59 ; however, recent studies have shown that the effects of IL-1β on stem cells is dependent upon the target cell line and the IL-1β concentration 36 . Notably, in our study BMSCs displayed a lower osteoblast capacity when incubated with 10 ng/mL of IL-1β, with His6-GABARAP treatment improving osteogenic differentiation in the presence of IL-1β via a mechanism closely related to the up-regulation of autophagy markers. As hypothesized, 3-MA stimulation effectively inhibited the effect of GABARAP, suggesting that GABARAP stimulates the osteogenic differentiation of IL-1β-induced BMSCs by activating autophagy. This stimulatory effect of GABARAP may be due to the role of autophagy maintenance in ROS generation, since autophagy has been shown to be a good candidate for maintaining the redox homeostasis of BMSCs 27 . Moreover, elevated ROS levels in BMSCs have been reported to modulate osteogenesis, with ROS scavenging able to effectively restore their osteogenic capacity 60 . However, the specific mechanisms during osteogenic differentiation, particularly in the presence of GABARAP, require further exploration. Taken together, we showed that GABARAP promotes osteogenic differentiation in IL-1β-induced BMSCs, at least partly by activating the autophagy pathway.
In conclusion, GABARAP improves BMSCs proliferation and partially protects cell against IL-1β-induced inflammation and intracellular ROS generation. In addition, our findings suggest that GABARAP promotes the osteogenic differentiation of BMSCs exposed to IL-1β and that autophagy activation is involved in its effect on BMSCs viability and osteogenic differentiation. Further studies will be conducted focusing on the mechanisms underlying the effects of GABARAP under more complicated inflammatory conditions. However, GABARAP may represent a novel therapeutic option for treating inflammation in OA and an effective method for promoting bone tissue regeneration.