USP7 Attenuates Endoplasmic Reticulum Stress and NF-κB Signaling to Modulate Chondrocyte Proliferation, Apoptosis, and Inflammatory Response under Inflammation

The purpose of this research was to observe the functions and mechanisms of ubiquitin-specific peptidase 7 (USP7) on chondrocytes under tumor necrosis factor alpha- (TNF-α-) induced inflammation. Knee osteoarthritis (OA) models of mice were constructed by anterior cruciate ligament transection. The knee joint of mice was observed by histological staining, and the expression of USP7 was measured by immunohistochemistry staining. After knocking down or inhibiting USP7, chondrocyte proliferation was measured by histological staining and the CCK-8 assay; apoptosis was measured by western blot, flow cytometry, Caspase-3 activity, and TUNEL staining; and inflammatory response was measured by qRT-PCR and ELISA. The 4-phenylbutyric acid (4-PBA), siRNA of CHOP (si-CHOP), and QNZ were used to verify the signaling pathways. It was found that USP7 was reduced in the knee joint cartilage of OA mice. The knockdown of USP7 or its inhibitor decreased chondrocyte proliferation and accelerated apoptosis and inflammatory response under inflammation. The USP7 inhibitor exacerbated cartilage destruction in mice with OA. The knockdown of USP7 or its inhibitor activated the BiP-eIF2α-ATF4-CHOP signaling of endoplasmic reticulum stress (ERS) and NF-κB/p65 signaling. 4-PBA, si-CHOP, and QNZ partly reversed chondrocyte proliferation, apoptosis, and inflammatory response caused by USP7 knockdown. In conclusion, through inhibiting the BiP-eIF2α-ATF4-CHOP signaling of ERS and NF-κB/p65 signaling, USP7 promotes chondrocyte proliferation and suppresses the apoptosis and inflammatory response under TNF-α-induced inflammation.


Introduction
Deubiquitinases play essential roles in various diseases by modulating the posttranslational modification of related proteins. There are six families of deubiquitinases, and the largest one is the ubiquitin-specific peptidase (USP) family. Among the nearly 60 members of the USP family, ubiquitin-specific peptidase 7 (USP7) is researched extensively [1]. Increasing researches have confirmed that USP7 regulates multifaceted key protein deubiquitination to affect cell differentiation, tissue development, and disease occurrence [2,3].
As a chronic degenerative disease of articular cartilage, osteoarthritis (OA) is commonly found with pain and dysfunction of joints and is increasing in incidence worldwide [4]. Although OA is often considered as a noninflammatory arthropathy, increased secretion of inflammatory cytokines has been observed in many patients and animal models [5]. Increasing evidence has found that proinflammatory cytokines secreted by the synovium and chondrocytes link closely to the cartilage destruction, and inflammatory pathways are vital in the progression of OA [6,7].
Chondrocytes are the target of the inflammatory stimulation since they are the principal cells in the articular cartilage. The inflammation from OA induces chondrocyte apoptosis, which accelerates the progression of OA [8,9]. It was found that hypoxia-inducible factor-1 alpha (HIF-1α) stabilization inhibited chondrocyte apoptosis and alleviated cartilage degradation in a surgical OA model [10]. Previous research has indicated that USP7 inhibits HIF-1α ubiquitinatioin [11]. Moreover, the activation of inflammasome plays an important role during the progression of OA, and USP7 was reported to regulate the inflammasome activation in macrophages [12,13]. Therefore, it is reasonable to suggest that USP7 may regulate chondrocytes under inflammation, but how USP7 exerts its effect is still unknown. A previous study has found that USP7 modulated nuclear factor-kappa B (NF-κB) signaling [14]. The aberrant activation of NF-κB signaling was often observed in OA, and the inhibition of NF-κB signaling suppressed chondrocyte apoptosis and delayed the progression of OA [15][16][17]. Collectively, USP7 may modulate NF-κB signaling and be an important adjustor of chondrocytes under inflammation.
On the other hand, inflammation-induced endoplasmic reticulum stress (ERS) acts a key role in OA and is positively related to the cartilage degradation [18]. PERK is one of three ERS sensors involved in the BiP-PERK-eIF2α-ATF4-CHOP signaling of ERS [19]. Under pathological conditions, BiP binds to misfolded proteins and dissociates with PERK, which further promotes eIF2α phosphorylation and the subsequent high expression of ATF4 and CHOP [20]. Curcumin attenuated OA via inhibiting PERK-eIF2α-CHOP signaling in rats [21]. Cartilage-specific autophagy promoted PERK-ATF4-CHOP signaling to hinder the growth plate development in vivo and increase chondrocyte apoptosis and decrease chondrocyte proliferation in vitro [22]. In addition, Sirtuin-1 (SIRT1) has been reported to promote the chondrogenesis of growth plate by inhibiting the PERK-eIF2α-CHOP pathway, and USP7 was found to stabilize SIRT1 [23,24]. Therefore, it was hypothesized that USP7 might modulate chondrocytes under inflammation through BiP-eIF2α-ATF4-CHOP signaling.
To confirm this hypothesis, the expression of USP7 in the knee articular cartilage of OA mice, caused by the anterior cruciate ligament transection (ACLT), was measured. Functions of USP7 on chondrocyte proliferation, apoptosis, and inflammatory response both in vitro and in vivo were tested, and the underlying mechanisms were explored.

Materials and Methods
2.1. Construction of an OA Mouse Model. The animal study was approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University (HZAUMO-2020-0014). Male C57BL/6 mice were housed in specific pathogen-free facilities and randomly divided into sham and OA groups. Knee OA models were constructed by ACLT as previously described [25]. Briefly, mice were anesthetized with pentobarbital sodium (70 mg/kg) by intraperitoneal injection. Mice in the OA group received a parapatellar skin incision at the medial side of the right knee joint, the dislocation of the patella, and the ACLT. Mice in the sham group received the joint incision without ACLT.

Haematoxylin-Eosin (HE) Staining, Safranin O-Fast
Green Staining, and Immunohistochemical Analysis. Eight weeks later, the knee cartilage tissues were fixed in 4% paraformaldehyde, decalcified in 10% EDTA for 4 weeks, and then sectioned (5 μm). The sections were stained with HE and Safranin O-Fast Green and incubated with of USP7 (1 : 250, Bethyl, AL, USA). Density was measured by Image-Pro Plus software 6.0.
2.3. Cell Culture and Induction. ATDC5 cell line was obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were firstly cultured in a growth medium and then in a chondrogenicinduced medium after 80-90% confluence as our previous study described [26]. Various concentrations of tumor necrosis factor alpha (TNF-α) (Novoprotein, Shanghai, China) were added. HBX41108 was from Toris (MN, USA). 4-Phenylbutyric acid (4-PBA) and QNZ were from MCE (NJ, USA).

Decreased Expression of USP7 in OA Mice after ACLT.
Eight weeks after ACLT, all mice in both groups were alive, although those in the OA group showed symptoms of claudication. The HE staining showed that mice in the OA group had fewer chondrocytes, while the sham group had normal morphology (Figure 1(a)). Safranin O-Fast Green staining also showed decreased cartilage thickness and chondrocytes in the OA group (Figures 1(b) and 1(c)), suggesting that OA mouse models were successfully constructed. Immunohistochemical staining showed that USP7 was mainly situated in the nuclei of the mouse knee joint chondrocytes, and USP7 was decreased in the OA group (Figures 1(d) and 1(e)).

USP7 Inhibitor HBX41108 Inhibits ATDC5 Cell Proliferation and Enhances Apoptosis and Inflammatory
Response under TNF-α-Induced Inflammation. HBX41108 was a small molecular inhibitor of USP7. The alcian blue and toluidine blue staining intensities decreased gradually with increasing HBX41108 under TNF-α (Fig. S4A). HBX41108 also inhibited ATDC5 cell proliferation at equal or greater than 1 μM in a dose-independent manner (Fig.  S4B). Flow cytometry suggested that HBX41108 accelerated chondrocyte apoptosis at a dose-independent manner, except 0.5 μM (Figs. S4C and S4D). Considering that 0.5 μM HBX41108 had no obvious effect on chondrocyte proliferation and apoptosis, 1 μM and 2 μM HBX41108 were selected for use in the following experiment.
HBX41108 inhibited the expression of Col2a1 and Sox9 (Figs. S4E-S4G), upregulated ATDC5 cell apoptosis, and improved the expression of proinflammatory cytokines (Figs. S4F-S4J). These results were consistent with USP7 knockdown. Collectively, USP7 inhibitor HBX41108 reduced chondrocyte proliferation and aggravated apoptosis and the inflammatory response under TNF-α-induced inflammation.     Oxidative Medicine and Cellular Longevity 3.5. USP7 Inhibitor HBX41108 Aggravated Cartilage Destruction of OA Mice. To determine the functions of USP7 in vivo four weeks after ACLT, the USP7 inhibitor HBX41108 was injected intraperitoneally into the OA mice twice a week for four weeks (Figure 3(a)). The HE staining showed that the OA group with HBX41108 had fewer chondrocytes than the OA group without HBX41108 (Figure 3(b)). Safranin O-Fast Green staining also showed decreased cartilage thickness and structural breakage of cartilage tissue in the OA group with HBX41108 (Figures 3(c)  and 3(d)). This indicated that the USP7 inhibitor HBX41108 aggravated cartilage destruction in OA mice.

Discussion
Posttraumatic OA is caused by joint trauma like anterior cruciate ligament (ACL) injury and meniscus tears [27]. Acute ACL injury caused serious local inflammation in the joint, inducing the proteolysis of aggrecan and Col2a1. Although joint trauma leads to OA in only 12% of cases, it mainly troubles young patients and causes long-term implications [28]. Hence, research on reducing cartilage destruction after ACT injury for OA patients has significant implications.
Increasing evidence suggests that deubiquitinases are essential in bone metabolism; however, their roles in cartilage and related diseases are poorly understood. Our previous study demonstrated that USP7 stimulated chondrocyte proliferation and chondrogenic differentiation in vitro and in vivo [26]. In this study, the knee OA model of mice was successfully constructed by ACLT, and USP7 was found to reduce in the knee joint of OA mice. Continuous local TNF-α levels were observed in the injured joint and

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Oxidative Medicine and Cellular Longevity provided continued stimulation to the surrounding cells [29]. To further study the influence of USP7 on chondrocytes under inflammation and the underlying mechanisms, TNF-α was supplemented to mimic OA in vitro. The results revealed that under TNF-α-induced inflammation in vitro, inactivation of USP7 inhibited chondrocyte proliferation and accelerated chondrocyte apoptosis and inflammatory response through activation of ERS and NF-κB signaling. Recent studies confirm that USP7 and its inhibitors regulate cell proliferation and apoptosis, especially in cancers. Inhibition of USP7 reduced cell growth of esophageal squamous cell carcinoma and activated ERS to induce NOXAmediated apoptosis [30]. In this study, USP7 knockdown activated chondrocyte apoptosis and suppressed chondrocyte proliferation under TNF-α-induced inflammation. Further, the USP7 inhibitor HBX41108 showed the same trend in vitro. However, this result differs from a study that found USP7 inhibition alleviated hydrogen peroxide-induced injury, like reduced proliferation and increased inflammation, by regulating NOX4 ubiquitination in rat chondrocytes [31]. Supplementation of hydrogen peroxide or TNF-α can mimic OA in vitro, but it led to different expressions and functions of USP7, possibly because USP7, as a deubiquitinase, modulates deubiquitination of diverse proteins to exert its functions under various stimulations. Moreover, the simulated conditions in vitro are not the same as those in vivo, given that the USP7 inhibitor HBX41108 was injected intraperitoneally in vivo and resulted in HBX41108 exacerbated cartilage destruction in OA mice. Hence, these results strongly indicate that USP7 protects cartilage and reduces its destruction in posttraumatic OA.
ERS is regarded as necessary in cartilage development. Chondrocytes, the only cell type in cartilage, showed impaired growth and upregulated apoptosis under ERSinduced conditions [32]. In human OA, ERS contributed to enhanced chondrocyte apoptosis and increased CHOP expression in chondrocytes [33]. Our previous study also found that CHOP suppressed ATDC5 cell proliferation and differentiation after chondrogenic induction [34]. However, the current study found that USP7 promoted ATDC5 cell proliferation. Considering that CHOP is the later event of BiP-PERK-eIF2α-ATF4 axis of ERS [35], it is suggested that USP7 may exert its effects through inhibiting BiP-eIF2α-ATF4-CHOP signaling.
The results of this study confirmed the supposition. Firstly, it was found that USP7 knockdown and its inhibitor activated BiP-eIF2α-ATF4-CHOP signaling of ERS. And then, reduction of ERS with its inhibitor, 4-PBA, could partially rescue the inhibition of chondrocyte proliferation, the promotion of chondrocyte apoptosis, and inflammatory response caused by USP7 knockdown. A recent study also found that administration of 4-PBA alleviated ERS, decreasing the chondrocyte apoptosis in mouse knee joints with obesity-linked OA [36]. According to these results, 4-PBA may be used to treat human OA, but this needs to be investigated further. Furthermore, the current study found that si-CHOP had the same effect as 4-PBA. This was consistent with the finding of Uehara et al. that CHOP knockout restrained the chondrocyte apoptosis and cartilage destruction in mice with OA [37]. Such results might be explained by that CHOP directly decreased antiapoptotic Bcl-2 protein but increased proapoptotic Bax protein to induce cell apoptosis [38]. However, since there were three ERS signalings and our study only demonstrated that CHOP knockdown decreased the BiP-eIF2α-ATF4-CHOP signaling, a study of relationship between CHOP knockdown and other ERS On the other hand, inflammation also leads to ERS in the pathogenesis of inflammatory diseases [39,40]. It was found that ERS could increase proinflammatory cytokines via NF-κB signaling [41]. NF-κB signaling is crucial in OA, and its activation causes cartilage destruction to aggravate the progression of OA [15,42]. The current study found that USP7 knockdown or its inhibitor activated NF-κB signaling. Moreover, supplementation of this signaling inhibitor QNZ rescued the chondrocyte proliferation, apoptosis, and inflammatory response caused by USP7 knockdown. Other research also reported that QNZ accelerated chondrocyte proliferation and decreased chondrocyte degeneration by promoting glucose uptake [43]. Taken together, this study demonstrates that USP7 suppresses NF-κB/p65 signaling to regulate chondrocyte proliferation, apoptosis, and inflammatory response under TNF-α-induced inflammation.
Additional, small molecule inhibitors of USP7 were considered as potential therapies to delay cancer progression by many researchers [3,44]. However, the results of this study found that the inhibitor of USP7 aggravated cartilage destruction. Hence, clinical doctors should pay more attention to cartilage if the USP7 inhibitor is used for cancers patients with OA. Besides, 4-PBA and QNZ may be potential drugs to slow cartilage destruction of OA and deserve further investigation.
Anyway, this study was based on the function loss of USP7 since the overexpressed lentivirus plasmid was too large to be successfully transfected into ATDC5 cells. The USP7-overexpressed adenovirus vector might be an alternative to be studied in the future. Another limitation was the lack of in vivo studies to verify the underlying mechanisms of USP7 on OA.

Conclusions
In conclusion, this study found that USP7 was reduced at the knee articular cartilage of mice with OA. The knockdown and inhibitor HBX41108 of USP7 hindered chondrocyte proliferation and accelerated chondrocyte apoptosis and inflammatory response under inflammation. USP7 may exert these effects through inhibiting BiP-eIF2α-ATF4-CHOP signaling of ERS and NF-κB signaling (Figure 8). This study shows new sights into the effects of USP7 on chondrocyte functions under inflammation.

Data Availability
This research article data used to support the finding of this study are included within the article.