Among known interacting partners, heat shock protein family A (Hsp70) member 5 (HSPA5, also known as 78 kDa glucose-regulated protein, GRP78; BiP) has the highest number of edges (10). HSPA5 is a chaperone in the endoplasmic reticulum (ER) lumen, which is known to regulate clusterin stability under ER stress (33) and is involved in the molecular mechanisms of ER stress induced during chondrogenesis (34). HSP90B1 (Grp94) also has a very high number of edges (7). HSP90B1 is involved in ER stress triggered by excessive mechanical load and hypoxia in chondrocytes (35).

Amyloid-β A4 protein (APP) is one of the top entities in the clusterin connectome (36). The clusterin–amyloid β-peptide complex interacts with low-density lipoprotein (LDL) receptor-related protein 2 (LRP2; megalin, known to act as a clusterin receptor), which offers a mechanism to clear the pathological accumulation of aggregates (37). Autoantibodies against LRP2 have been detected in 87% of patients with rheumatoid arthritis (RA) and 15% of patients with OA, indicating that these anti-LRP2 autoantibodies may play pathological roles by inhibiting the protein reabsorbing function of LRP2 in the proximal tubule (38). α-synuclein (SNCA) also forms a major component of amyloid plaques in AD and PD, and this process is blocked by clusterin (39). Clusterin interacts with extracellular α-synuclein fibrils and limits their uptake by astrocytes (40). Transthyretin (TTR) is an amyloidogenic protein. Clusterin is known to interact with aggregated forms of TTR, and serum clusterin levels in patients with transthyretin amyloid cardiomyopathy are significantly lower than those in healthy controls (41). TTR deposition in articular cartilage has been reported to increase disease severity in a murine model of OA (42). Moreover, both clusterin and TTR levels were higher in synovial fluid samples of patients with knee OA than in those with hand OA, indicating that they are involved in similar molecular pathways during OA pathogenesis (43). Clusterin interacts with amyloidogenic variants of lysozyme (LYZ) (44). Lysozyme has long been known to be present in the cartilage ECM (45), and cartilage degradation leads to increased serum and synovial fluid lysozyme levels in patients with OA (46). Clusterin is also involved in the folding/unfolding pathway of the extracellular protein α-lactalbumin (LALBA) (47).

Protein disulfide isomerase family A member 3 (PDIA3, ERp57), an oxidoreductase involved in native disulfide bond formation, is required for efficient clusterin oxidative folding (48). In the case of protein misfolding in the ER of chondrocytes, ECM proteins aggregate, resulting in ER stress, and the unfolded protein response (UPR) is initiated. Persistent ER stress is a pathogenic mechanism underlying OA (49). Hypoxia upregulated 1 (HYOU1), a marker of protein misfolding under cellular stress, is involved in the chondrocyte response to IL-1α (50).

Clusterin has a well-known anti-apoptotic role, partly because it reduces the activity of the pro-apoptotic protein BCL2 associated X apoptosis regulator (BAX) (51, 52). However, the regulatory mechanisms of BAX underlying chondrocyte apoptosis in OA remain largely unknown (53), and clusterin involvement in this pathway has not been implicated in OA chondrocytes. In contrast, using a text-mining-based approach, anti-apoptotic BCL2L1 (BCL-XL) was recently identified as a gene that could be exploited as a potential drug target in OA (54) and is known to regulate apoptosis through the BCL-XL protein in kidney cells (55).

ATPase copper-transporting β (ATP7B) is an important regulator of intracellular Cu homeostasis (15). COMMD1 (Copper metabolism Murr1 domain 1) is expressed in most tissues and plays a role in controlling protein degradation and stability (56). Clusterin and COMMD1 interact with ATP7B independently. As a consequence of these interactions, degradation of misfolded Cu-ATPase molecules is facilitated, which is an important factor in the quality control of ATP7B required for the maintenance of normal copper homeostasis (15). Literature on the function of copper transporters in chondrocytes is sparse (57), although a genetic predisposition to physiologically higher circulating copper and zinc status may increase the risk of OA (58). Significantly higher Cu concentrations have been detected in the synovial fluid of patients with OA than in healthy subjects (59). Therefore, Cu levels and transporter status in combination with clusterin should be further investigated in the context of OA. In contrast, COMMD1 is an important mediator of NF-κB signalling, a key player in inflammatory pathways, and clusterin has been linked to COMMD1 protein levels (60). Clusterin also has a complex regulatory interaction with NF-κB signalling (61).

The complement system is involved in host defense mechanisms that aim to eliminate potentially harmful structures from the body. Clusterin potently inhibits terminal complement assembly by blocking complement protein (C9), thereby reducing the rate of complement-mediated cytolysis and providing higher levels of protection (62). Complement protein C9 has been described in the hypertrophic zone of the epiphyseal growth plate (63). C9 appears to be predominantly present in SC5b-9 complexes in synovial membrane samples from patients with OA (64). In cases of acute arthritis, such as OA flare-up, marked C9 deposits were detected in the synovium; however, C9 deposits were not found in chronic conditions associated with degenerative diseases, such as OA (65).

Clusterin is also implicated in DNA repair. Given the often fatal consequences of DNA breaks, several pathways exist for the recognition and repair of these lesions. One such pathway involves the DNA-dependent protein kinase (DNA-PK) complex, which consists of a catalytic subunit and heterodimeric Ku autoantigen comprising Ku70 (XRCC6) and Ku80 proteins (66). Clusterin was identified as an interacting partner of Ku70, likely initiating complex signalling mechanisms leading to cell death (66). In colon cancer, interleukin 6 (IL-6) affects pro-survival pathways by modulating the expression and molecular interactions between the pro-apoptotic factor BAX, DNA repair proteins Ku70/86, and clusterin (67). However, no experimental data are available on the role of clusterin in mediating repair pathways involving Ku70 in OA.

Selenoprotein R (SelR, also known as methionine sulfoxide reductase B1, MSRB1) plays an important role in maintaining intracellular redox balance by reducing the R-form of methionine sulfoxide. Given that selenium is an essential trace element, selenoproteins that mediate its metabolism are involved in key cellular functions such as redox homeostasis (68). SelR interacts with clusterin (69). Co-overexpression of SelR and clusterin in an AD model significantly decreased intracellular ROS levels. Furthermore, the interaction between clusterin and β-amyloid peptide was confirmed, suggesting a putative effect of SelR and β-amyloid peptide via clusterin (69). Appropriate selenium levels are required to maintain cartilage development and homeostasis (68), and experimental evidence suggests that selenoproteins are expressed in chondrocyte cell lines in vitro (70). Selenium deficiency is linked to the development of Kashin–Beck disease (KBD), which is an endemic osteoarthropathy (prevalent in low-selenium areas of China, North Korea, and Siberia in Russia) caused by disturbances in the closure of the epiphyseal plate and manifests as skeletal deformities and movement disorders (71). Certain polymorphisms in selenoprotein genes are associated with a higher risk of KBD (72). Furthermore, a cross-sectional analysis of dietary selenium intake revealed that high dietary selenium consumption may be associated with an increased risk of OA (73). However, SelR has not yet been directly discussed in the context of OA development.

Plexins are receptors of the semaphorin family of signalling proteins (74). Plexin A4 (PLXNA4) acts as a clusterin receptor in the central nervous system and is an emerging therapeutic target for AD (68). Although plexins in chondrocytes have only been partially mapped, semaphorin-3A (Sema3A) has been implicated in OA chondrocyte physiology because excessive Sema3A signalling stimulated by the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) promotes apoptosis (75). Sema4D has recently been shown to be involved in chondrocyte apoptosis triggered by lipopolysaccharide (LPS) (76). Therefore, elucidating aberrant semaphorin signalling in the context of plexins and clusterin may lead to the identification of new targets in OA.

We then analyzed the overall clusterin interactome in the IPA knowledge base and identified additional pathways and interacting partners. In the overall molecular network of clusterin, the top canonical pathways were “colorectal cancer metastasis signaling,” “regulation of the epithelial mesenchymal transition by growth factors pathway,” “pancreatic adenocarcinoma signaling,” “IL-12 signaling and production in macrophages,” and “glucocorticoid receptor signaling” ( Table 5A ). These results are in line with the well-established role of clusterin in tumor biology.

The top upstream regulators were IL-6, CLU, TGFB1, TP53, and EZH2 ( Table 6A ). Interleukin-6 (IL-6) has been reported to influence pro-survival pathways in colon cancer progression via Bax, Ku70/86, and clusterin (67). IL-6 is especially relevant in the context of OA; an increase in IL-6 serum levels has been associated with decreased physical function and increased risk of knee OA progression (77). Transforming growth factor β1 (TGFB1) regulates clusterin expression (78–80). TGFB1 is an essential factor in chondrogenesis and cartilage maintenance, and a recent study confirmed that a SNP associated with OA susceptibility affects TGFB1 expression by influencing its enhancer (81). The tumor suppressor protein p53 (TP53) represses clusterin expression, which may be important for p53-mediated cell death (82). p53 has a well-established role in OA (82). EZH2, a histone methyltransferase involved in polycomb repressor complex 2 (PRC2), represses clusterin expression; therefore, aberrant upregulation of EZH2 may contribute to the progression of various tumors (83). EZH2 is upregulated in OA (84); however, the link between EZH2, clusterin, and OA progression has not yet been established.

The entities in the causal network of clusterin were PPARG, UCH1, TXNIP, PIAS4, and DICER1 ( Table 6A ). Clusterin overexpression upregulates the adipogenic marker peroxisome proliferator-activated receptor γ (PPARG) during adipocyte differentiation (85). PPARG signalling is involved in skeletal muscle regeneration via growth/differentiation factor 3 (GDF3) (86). PPARG expression is upregulated in synovitis, indicating its role in mediating tissue recovery (87). Although ubiquitin carboxyl-terminal hydrolase (UCL1), thioredoxin-interacting protein (TXNIP), and DICER1 are among the top members of the causal network, we did not find a direct association between these factors and clusterin. Nevertheless, TXNIP, an inhibitor of antioxidant activity, is downregulated by sirtuin 6 (SIRT6) in chondrocytes (88). Furthermore, TXNIP forms a complex with DDIT/REDD1, an endogenous inhibitor of mTOR that regulates cellular stress responses; the TXNIP/REDD1 complex is required for the activation of autophagy in chondrocytes, but its expression is reduced in OA (89). A protein inhibitor of activated STAT (PIAS4/PIASY) interferes with the binding of NF-κB, an important regulator of inflammation, to its target genes (60). As previously discussed, clusterin may mediate COMMD1, which induces NF-κB destabilization and proteasomal degradation (60). DICER-dependent pathways play critical roles in chondrocyte proliferation and differentiation during skeletal development (90).

The top five networks with the involvement of clusterin were as follows: “cell death and survival, cellular assembly and organization, cancer,” “cancer, organismal injury and abnormalities, cellular development,” “cellular development, connective tissue development and function, tissue development,” “cell death and survival, lipid metabolism, molecular transport,” and “cellular assembly and organization, DNA replication, recombination, and repair, cellular compromise” ( Table 7A ).

Given that one of the top networks above was “cellular development, connective tissue development and function, and tissue development,” we repeated the IPA knowledgebase analysis focusing on connective tissue development and function. In this analysis, the top five canonical pathways were as follows: “pancreatic adenocarcinoma signaling,” “hepatic fibrosis signaling pathway,” “regulation of the epithelial mesenchymal transition by growth factors pathway,” “colorectal cancer metastasis signaling,” and “chronic myeloid leukemia signaling” ( Table 5B ).

The top upstream regulators were TGFB1, PD98059, IGF-1, HRAS, and EGF ( Table 6B ). TGFB1, an upstream regulator of clusterin, has been discussed previously. PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK1/MAPKK), abrogated clusterin-stimulated proliferation, indicating that clusterin may activate the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway (91). As discussed previously, clusterin stimulates MMP-9 expression via ERK1/2 and NF-κB pathways (92). In an in vitro model of OA, ADAMTS and MMP upregulation correlated with the activation of ERK1/2 signalling, and PD98059 reversed the overexpression of matrix metalloproteinases (93). Insulin-like growth factor-1 (IGF-1) is also known to induce clusterin expression (94) and is involved in protecting cells from premature senescence (95). IGF-1 plays key roles in cartilage by promoting chondrocyte proliferation, enhancing ECM production, and inhibiting chondrocyte apoptosis, and is therefore highly relevant in OA therapy (96). Induction of the HRAS proto-oncogene represses clusterin expression in a MEK/ERK and methylation-dependent manner, indicating that DNA hypermethylation of the clusterin promoter is controlled by oncogenic signalling pathways (97). HRAS is involved in modulating chondrocyte apoptosis, senescence, and ECM degradation via MAPK signalling in OA (98). Epidermal growth factor (EGF) regulates clusterin expression via the Ras/ERK/AP-1 signalling pathway (99). While EGF signalling plays an important role in endochondral bone formation and joint homeostasis, conflicting results on its role in OA have been reported, which is likely attributable to the activation of specific downstream molecules as well as crosstalk with other signalling pathways (100).

The entities in the causal network were ADAM12, HTATIP2, EPHA4, Zibotentan, and BMS-387032 ( Table 6B ). We discussed the inclusion of ADAMs in an extended clusterin connectome (see Supplementary Material ). HTATIP2 is an oxidoreductase required for tumor suppression in gliomas (101). Ephrin type-A receptor 4 (EPHA4) plays an emerging role in OA. Activation of EPHA4 signalling attenuates pro-inflammatory cytokine and MMP production in synoviocytes and augments the expression of chondrogenic genes in chondrocytes (102). Zibotentan is an endothelin A receptor antagonist (103). In vascular smooth muscle cells, endothelin was found to be significantly differentially expressed in response to clusterin (104). Endothelin-1 signalling plays an emerging role in OA pathogenesis by stimulating the expression of MMP-1 and MMP-13 (105). BMS-387032 is a potent inhibitor of cyclin-dependent kinases (CDK) 2, 7, and 9 (106). CDK inhibitors reduce the injury response after joint trauma, indicating that this pathway can be exploited for the prevention and/or treatment of early OA (107).

The top five networks in the clusterin interactome in the context of connective tissue development and function were as follows: “cellular development, cellular growth and proliferation, connective tissue development and function,” “cellular development, cellular growth and proliferation, lymphoid tissue structure and development,” “cell-to-cell signaling and interaction, carbohydrate metabolism, cellular development,” “cellular development, connective tissue development and function, skeletal and muscular system,” and “cellular development, cellular growth and proliferation, cancer” ( Table 7B ).

We extracted these molecules from the IPA knowledgebase, and their expression levels were modulated by clusterin, as shown in the published literature ( Figure 3 ). In OA, clusterin-dependent regulation of several molecules is particularly relevant.

Clusterin increases the production of TNF-α and MMP-9 in macrophages (108), and both proteins are critical mediators of the OA pathophysiology (109). In cultured human fibroblast-like synoviocytes, clusterin knockdown by siRNA increased the production of pro-inflammatory cytokines IL-6 and IL-8 (CXCL8), indicating that clusterin plays a negative regulatory role in NF-κB-regulated cytokine production (24). Clusterin inhibits NF-κB signalling by stabilizing IκBs in neuroblastoma cells (110). Clusterin decreases the production of PGE₂ (prostaglandin E₂) (111), a principal mediator of inflammation, in RA and OA (112). Clusterin gene silencing in human OA chondrocytes shifted the cell phenotype towards hypertrophy and increased apoptosis, downregulated NF-κB-regulated genes, and increased MMP13 and TNF-α levels, suggesting a protective role of clusterin in these cells (113). Clusterin is involved in upregulating MMP-2 and downregulating E-cadherin expression in tumor cells (114). As discussed previously, clusterin and COMMD1 interact to downregulate ATP7A and ATP7B copper-transporting ATPases, thereby mediating Cu homeostasis (15). Overexpression of clusterin blocks TNF-α-mediated induction of p21 (CDKN1A) and abrogates proteolytic activation of the apoptosis regulator BAX, rendering clusterin-overexpressing breast cancer cells significantly more resistant to cytokines (115). Clusterin regulates the expression of proteins in mitochondrial apoptosis pathways, such as Bcl‐2, BAX, Bcl‐xL and caspase‐9, and phosphorylation of Akt (116). Clusterin blocks hepatic lipid accumulation by inhibiting SREBP-1c expression, suggesting that it may be a suitable target for preventing hepatic fat accumulation in insulin-resistant patients (117).

Clusterin is also an emerging modulator of TGF-β signalling that regulates SMAD2/3 proteins (118). These proteins are essential for the formation and maintenance of healthy cartilage and SMAD3 mutations are associated with OA (119). Clusterin overexpression increased SMAD2/3 protein levels via enhancing TGF-β-induced transcriptional activity (118). Clusterin is also involved in stabilising SMAD2/3. In tumor cells, clusterin plays a protective role against ER stress-induced apoptosis by interacting with glucose-regulated protein 78 (GRP78; also known as HSPA5), a central regulator of the unfolded protein response (120). GRP78 is upregulated in advanced OA, suggesting that chondrocytes experience ER stress during its pathogenesis (121). Clusterin may also be involved in regulating cellular cholesterol homeostasis under both normal and pathological conditions (122). Cholesterol homeostasis plays a key role in skeletal development, the dysregulation of which contributes to the development of cartilage diseases, including OA (123).