Targeted regulation of FoxO1 in chondrocytes prevents age‐related osteoarthritis via autophagy mechanism

Abstract Autophagy is designated as a biological recycling process to maintain cellular homeostasis by the sequestration of damaged proteins and organelles in plasma and cargo delivery to lysosomes for degradation and reclamation. This organelle recycling process promotes chondrocyte homeostasis and has been previously implicated in osteoarthritis (OA). Autophagy is widely involved in regulating chondrocyte degeneration markers such as MMPs, ADAMSTs and Col10 in chondrocytes. The critical autophagy‐related (ATG) proteins have now been considered the protective factor against late‐onset OA. The current research field proposes that the autophagic pathway is closely related to chondrocyte activity. However, the mechanism is complex yet needs precise elaboration. This review concluded that FoxO1, a forkhead O family protein, which is a decisive mediator of autophagy, facilitates the pathological process of osteoarthritis. Diverse mechanisms regulate the activity of FoxO1 and promote the initiation of autophagy, including the prominent AMPK and Sirt‐2 cellular pathways. FoxO1 transactive is regulated by phosphorylation and acetylation processes, which modulates the downstream ATGs expression. Furthermore, FoxO1 induces autophagy by directly interacting with ATGs proteins, which control the formation of autophagosomes and lysosomes fusion. This review will discuss cutting‐edge evidence that the FoxO–autophagy pathway plays an essential regulator in the pathogenesis of osteoarthritis.

the decline in quality of life, the economic burden in medical rehabilitation and unemployment, are even more magnificent. 2 The WHO data reveal that, by 2015, the years lived with disability (YLD) caused by OA accounted for approximately 3.9% of all diseases globally. By 2020, OA will become the fourth-largest cause of YLD loss. 5 Therefore, research in the pathogenesis and progression mechanism of OA disease and striving to explore feasible solutions and targets for treating or delaying the progression of the disease has significant clinical value and social significance.

| AG EING AND S ENE SCEN CE-REL ATED CHONDROC Y TE AP OP TOS IS IS THE CRITI C AL DE TERMINANT IN THE PATHOG ENE S IS OF OS TEOARTHRITIS
The aetiology of OA is complex and needs elaboration. Evidence shows a significant correlation between increased susceptibility to OA and ageing. 4 In the early stage of OA progression, ageing promotes articular cartilage degeneration, elastic cartilage gradually degrades into fibrocartilage, and its cushioning and stabilizing effect on As an essential factor in stabilizing articular cartilage homeostasis, chondrocytes undergo a series of alterations in the OA progression. Disequilibrate of chondrocyte homeostasis by impairment causes the chondrocytes hypertrophy and further promotes osteogenic differentiation, eventually inducing cartilage degradation and apoptosis of chondrocytes. 6 In the pathogenesis of degeneration, the expression of matrix metalloproteinases (MMP9 and MMP13) and various cartilage matrix proteases, such as ADAMTS5 and ADAMTS7, can enzymolysis the type II collagen (Col2α1) and proteoglycan (aggrecan, Acan) in the cartilage matrix.
It produces decomposition, which further triggers the degeneration of the cartilage matrix and the transformation into a bone matrix. 7 In the early stage of OA, autophagy-mediator LC3-II, beclin-1 and ATG5 in articular chondrocytes increased as the feedback reaction to homeostasis impairment. The expression of matrix proteases, such as MMP9, MMP13 and ADAMTS5, ADAMTS7 is inhibited, the metabolic decomposition of Acan, Col2α1, and other multiple helix collagen protects cartilage is prevented. However, with the progress of OA disease, oxidative stress damage continues to accumulate, and the level of autophagy-mediated by LC3-II, beclin-1 and ATG5 in chondrocytes gradually decreases, and it promotes the hypertrophy marker of chondrocytes such as Runx2, Col10α1 and MMP9. The increase in osteogenic differentiation markers leads to hypertrophy of chondrocytes and further differentiation into osteoblasts, which eventually triggers the activation of caspase3 and ccaspase9 and initiates the apoptosis program 8 (Figure 1). In addition, as the content of elastic fibres in cartilage decreases, the increase in mechanical stress will also cause the reduction in the expression of ULK1, LC3-II The ATG5 knockout mouse model indicated that the gene and protein levels of Col2α1 and Acan decreased significantly, while the expression of matrix protease MMP13 and ADAMTS5 increased. 13 This study also reflects that autophagy in chondrocytes functions as the protector of cartilage degradation. 13 The sirolimus or rapamycin inhibits the mammalian target of rapamycin (mTOR) signalling pathway and significantly increases the expression of Col2α1, Acan and Sox9 in ATDC5 chondrocytes. The catabolism markers Col10α1, MMP9, MMP13 and ADAMTS5 and the apoptosis markers caspase3, caspase9, beclin-1 and beclin-2 significantly reduced. 11 The above-mentioned evidence further confirms that autophagy is important in maintaining chondrocytes and cartilage matrix homeostasis and inhibiting chondrocyte apoptosis. The histological study of clinical cases also found similar results, and mTOR expression increased in joint samples of patients with OA. The specific ligand rapamycin can bind to the intracellular receptor FKBP-12 to form a complex and act directly on mTOR. The FRB domain (FKBP-12rapamycin binding site) can effectively inhibit its protein activity and increase the level of autophagy. Therefore, regulating the mTOR signalling pathway of autophagy upstream can effectively reduce the destruction of the cartilage matrix. 14,15 Similarly, the mTOR knockout mouse model reveals that autophagy was significantly activated in chondrocytes, effectively reducing chondrocyte apoptosis and maintaining the homeostasis of the articular cartilage microenvironment. Articular cartilage degeneration decelerated in the surgicalinduced OA mouse model. 16 On the contrary, chondrocyte-targeted ATG5 and ATG7 knocked out mouse model reveals the accumulation in caspase-mediated apoptosis and acceleration of OA disease progression. 17

| FoxO1 IS THE DOMINANT TR ANSCRIP TION FAC TOR REG UL ATING THE OA PROG RE SS I ON
The FoxO (Forkhead-box class O) transcription factor family for mammals mainly includes FoxO1, FoxO3, FoxO4 and FoxO6, which perform distinct and overlapping functions. The domain of its specific DNA binding site comprises three alpha-helices and two characteristic outer loops, 18 as the shape of a fork. FoxO is mainly involved in the physiological regulation of biological development and ageing, which is closely related to the lifespan of vertebrates.
In addition, FoxO transcription factors are also involved in critical roles such as preventing oxidative stress damage and maintaining homeostasis of the intracellular environment. 19 Even though FoxO1, FoxO3 and FoxO4 are expressed ubiquitously in multiple organs, the gene regulation of each FoxO conforms to a tissue-specific pattern, while FoxO6 expression largely correlates to neurological function. 20,21 FoxO1 is primarily expressed in bone and cartilage and functions as an essential factor in regulating bone tissue development and maintaining chondrocyte homeostasis. 22 FoxO1 interacts with Runx2 23,24 and Wnt/β-catenin cellular pathway in osseous tissue to regulate osteoblast differentiation and further modulates energy metabolism to influence bone development. 22,25 The longevity of homosapien is associated with the polymorphisms of FoxO3. 26 FoxOs are the downstream target of phosphoinositide-3 kinase (PI3K)/Akt signalling, which modulate cell proliferation, growth, apoptosis, and the expression of antioxidant and autophagy proteins. 27 FoxO controls the cellular homeostasis and the subsistence of stem cells in development to maintain the biological function of the tissue. 27 FoxOs also regulate the self-renewal of stem cell populations 28 and are involved in the regulation of cell differentiation. 29 Oxidative stress induces the FoxOs expression and transcription. The downstream antioxidant enzymes, including catalase and manganese superoxide dismutase, are activated to prevent impairment of cellular homeostasis. 30 Besides, autophagy and ubiquitinproteasome system are the two primary intracellular clearance mechanisms regulated by the transcriptional function of FoxOs.
Recycling and eliminating damaged organelle and dysfunctioned protein are significant for cellular homeostasis.
FoxO-meditated autophagy has been implicated as the essential regulator for chondrocytes homeostasis preventing the F I G U R E 1 In the progression of OA, chondrocytes undergo a series of pathological alterations of homeostasis impairment-hypertrophyosteogenic differentiation-apoptosis and finally trigger the OA phenomenon. In the early stage of OA, autophagy activation can effectively prevent chondrocyte hypertrophy (MMP9, Col10 increase) and chondrocytes marker (Prg4, Col2α1) decrease. With the deterioration of OA, the level of autophagy continues to decrease, leading to the osteogenesis markers (ADAMTS, MMP13 and Runx2) and apoptosis markers (Caspase3, Caspase8 and Beclin1) increase and eventually resulting in the cartilage matrix calcification. Ageing and chronic damage are the main initiating factors of OA and directly or indirectly activate inflammatory factors inducing stress damage to chondrocytes. Inflammatory factors also aggravate the joints by synovial hyperplasia and inflammation intensification intervertebral disc and articular cartilage from degradation. 19,31 FoxO1 expression decreases in the cartilage of elderly OA patients, especially in the superficial layer of the cartilage in the weightbearing area. Similarly, in the fibrosis area and osteophytes hyperplasia area, FoxO1 presents a significantly lower expression, which reveals the phenomenal correlation between OA progression and FoxO1 expression. 22 Animal experiments reveal that FoxO1 expression gradually decreased with increased mouse age and the chondrocytes with lower FoxO1 expression aggregated in the cartilage not covered by the meniscus. The accumulation of microdamage induced by biomechanical stress on the cartilage regulates the FoxO1 expression and OA progression. 31 Apart from cartilage degradation, the meniscus damage accounted for the primary factor for knee OA. Ageing is the leading risk factor for meniscus degeneration. 32 The histological autopsy analysis of human knee joints reveals that meniscus from donors with normal articular cartilage presented minimal reduced FoxO1 or FoxO3 expression. 33 On the contrary, mice showed ageing-related meniscus damage and lowered FoxO1 and  The structural alteration of LC3-I to LC3-II is the fundamental of autophagosome maturation. The conversion of LC3-I to LC3-II is an essential process in the maturation of autophagosomes, which facilitates the amalgamation of lysosomes for substance degradation and recycling. 58 In the autophagy formation process, the beclin-1 complex is regulated by phosphatidylinositol 3-kinase (PI3K) and binds to the inner membrane structure in the cell (endoplasmic reticulum, mitochondria or nuclear membrane), promoting the ULK1 complex to forms the outer membrane structure of the autophagosomes. The colonization of ATG14L facilitates the transport and maturation of autophagosomes. On the contrary, ULK1 can also negatively regulate AMPK and deactivate autophagy. The mTOR can phosphorylate ATG13 to inhibit the outer membrane formation for the ULK1 complex. 11 The impacts of FoxO1 phosphorylation on autophagy are significantly different in various pathways and cells. Phosphorylation induced by AKT, 59 ERK, 60 MEK 61 and AMPK is the essential PTMs of Foxo1 that trigger the alteration of subcellular localization. 62 Phosphorylated-Foxo1(p-FoxO1) is restricted in the cytoplasm and interacts with ubiquitin E3 ligases, evoking the degeneration of Foxo1. Furthermore, p-FoxO1 binding to 14-3-3 protein extends the physical distance of FoxO1 and downstream target, preventing the binding and activation of targeted DNA from transcription. 63,64 That ERK cellular pathway is one of the critical signals of FoxO1 phosphorylation. The inhibition of ERK and MEK attenuate phosphorylation resulting in the activation of Foxo1 in rat hepatocytes. 61 Phosphorylation of FoxO1 is invalid in binding to ATG7 for autophagy initiation in mouse ovarian granulosa cells. The FSH or melatonin induces both Akt-mediated phosphorylation and Sirt1-mediated deacetylation of FoxO1. However, neither FoxO1-ATG7 interaction nor autophagy upregulation was verified. 42,45 Interestingly, a recent study revealed a specific result that phosphorylated cytosolic FoxO1 associates with Atg7 to induce autophagy in iNKs, which is essential for NK cell development. Modified FoxO1 promotes the interaction with ATG7 and stabilizes FoxO1 in iNKs cytoplasm, leading to the autophagy initiation. 43 A comparable study has suggested that Akt-induced FoxO1 phosphorylation excludes FoxO1 from the nucleus to cytoplasm, resulting in the enhancement of FoxO1-Atg7 interaction and promoting autophagy activation in the cytoplasm. 43,65 The AMPK-FoxO-autophagy pathway contributes to cell viability with the essential adaptation mechanism, maintaining cellu-  69 and activates AMPK via SESN to promote autophagy activation. 50,51 Apart from binding to the promotors to upregulate the expression of autophagy-related genes, FoxOs independently activate autophagy without transactivation functions. 45 FoxOs translocate from the nucleus to the cytosolic compartment and combine directly to the E1-like enzyme, ATG7, to elevate autophagy activity. The acetylation (e.g. Lys262, Lys265 and Lys274 in Homo sapiens FoxO1) 45,70 and Akt-induced phosphorylation (e.g. Thr24, Ser256 and Ser319 for Mus musculus Foxo1, and Thr32, Ser253 and Ser315 for H. sapiens FoxO1) 43  by ATG7 is indispensable for inducing ATG12-ATG5 conjugates and membrane-bound LC3-II in mammalian cells. 77 The acetylation of FoxO1 is essential for autophagy initiation. However, enhancing ATG7 activity after the interaction with acetylated FoxO1 still needs detailed elaboration. The physiological mechanism of the FoxO1 signalling pathway in chondrocytes requires specific exposition. This review discussed, at least in part, how the FoxO1 correlated with autophagy initiation and the protective ability in cartilage and bone homeostasis.

CO N FLI C T O F I NTE R E S T S
All authors declare that they have no conflict of interest.

AUTH O R CO NTR I B UTI O N
Jiaji Yue:Funding acquisition (lead); Writing -original draft (lead).

E TH I C A L A PPROVA L
The ethical approved was exempted by the institutional review board of Shenzhen Second People's Hospital.

Jiaji Yue
https://orcid.org/0000-0001-8113-4577 F I G U R E 3 Activation process of autophagy regulated by FoxO1. In response to the stimulation of various harmful factors, AMPK activates the FoxO1 phosphorylation and formation of the ULK1 complex, which initiates autophagy with Beclin-1 complex, ATG14L and other molecules. In the elongation phase of the autophagosome formation, the transformation of LC3-I to LC3-II is essential for continuing the autophagy process. Acetylated FoxO1 can regulate the expression of ATG7. Meanwhile, phosphorylated FoxO1 form a FoxO1-ATG7 complex, activate the ubiquitinase activity of ATG7 and perform ubiquitination on the ATG5-ATG12 protein complex