Stage-dependent Expression of Autophagy-related Genes in Patients With Knee Osteoarthritis

Discussion

In this work, we carried out a study on transcriptional changes related to autophagy at different stages in patients with knee OA by using blood samples. The main result is that ULK1, TFEB, WIPI1 and NBR1 genes showed significant stage-dependent changes, concomitant with OA progression, while ATG5, ATG7, LC3B, and FOXO3 did not change significantly at the mRNA expression level with OA stage. This finding indicates that the transcriptional changes observed along OA progression might mainly be concerned with regulation and early-stage components of the autophagy pathway rather than affecting all autophagy-related genes evenly.

Autophagy is a complex, tightly controlled cellular pathway with several stages, and loss of control at different points in this pathway can result in various biological consequences (26). ULK1 is the main autophagy-initiating protein that responds to cellular stress signals, whereas TFEB is the predominant transcription factor that regulates the expression of autophagy- and lysosome-related genes (27, 28). WIPI1 functions in the initial steps of autophagosome formation and membrane remodeling, and it is identified as a component of the core early autophagy machinery (29). On the other hand, NBR1 is a selective autophagy adaptor protein that plays a role in cargo recognition and delivery of ubiquitinated proteins to autophagosomes (30). The finding that the expression of these regulatory and early-process genes varies at different stages and that their transcriptional differences are dependent on the stage supports the idea that the molecular changes associated with OA may mainly impact the upstream regulatory control of autophagy (31, 32).

Our results largely align with previous studies investigating the role of autophagy in OA (31, 32). Cartilage-based and experimental investigations have demonstrated that autophagic activity and the expression of regulatory autophagy-related genes, including ULK1-associated signaling pathways, decline with aging and OA progression (16, 31, 32). In contrast, certain core components of the autophagy machinery appear to remain relatively stable at the transcriptional level. Our findings showing no significant stage-dependent changes in ATG5, ATG7, and LC3B mRNA expression are consistent with this observation. Collectively, both previous experimental data and our results suggest that OA progression may preferentially affect upstream regulatory nodes of the autophagy pathway rather than uniformly altering downstream structural components (26, 31, 32).

TFEB, a major transcription factor that integrates cellular stress responses with metabolic and proteostatic adaptations, regulates transcriptions of genes involved in lysosomal biogenesis and autophagy (11). Through the coordinated lysosomal expression and regulation network, TFEB coordinates numerous autophagy and lysosome genes, playing a central role in the long-term maintenance of cellular homeostasis (33). In the context of OA, studies on experimental OA models and human osteoarthritic cartilage tissues showed that the expression of TFEB and/or nuclear translocation was suppressed (11, 28, 31-33). A decrease in TFEB activity has been linked to lysosomal dysfunction, insufficient autophagic flux, and an increased sensitivity of chondrocytes to oxidative stress, which ultimately leads to the degradation of the cartilage matrix and the loss of cells, thereby accelerating joint degeneration (11, 32).

Recent experimental studies have demonstrated that pharmacological or genetic activation of TFEB enhances autophagic flux, improves lysosomal function, and attenuates inflammatory signaling pathways in cellular and animal models (11, 17, 28). In OA models, TFEB activation has been associated with improved chondrocyte survival and reduced cartilage degeneration (31, 32). These findings suggest that TFEB may represent not only a downstream consequence of degenerative processes but also a potential disease-modifying therapeutic target. Within this context, our study detected stage-dependent TFEB transcriptional changes in peripheral blood, which, as OA severity increases, support systemic impairment of autophagy upper regulatory mechanisms (31). This situation is consistent with the idea that OA progression might be associated with a gradual impact on the regulatory nodal points which are responsible for the initiation and maintenance of autophagy (31-33).

However, it is not realistic to consider the transcriptional profiles obtained from peripheral blood to be a direct reflection of the molecular changes specific to joint tissue. Peripheral TFEB expression is less about intra-articular chondrocytes and more about the combined response of circulating immune cells exposed to systemic inflammatory and metabolic signals (34). Thus, while our findings are consistent with tissue-based studies showing a decrease in TFEB activity in late-stage OA (32), these results should not be considered as direct evidence of intra-articular TFEB dysfunction but rather as an indication of systemic regulatory changes associated with disease stage (24). In the future, holistic studies in which peripheral biomarkers are considered together with joint tissue expression, protein level analyses, and functional autophagy assessments will clarify the real role of TFEB in OA.

WIPI1 is a key regulator of the early stages of autophagy and is recruited to phosphatidylinositol 3-phosphate-enriched membranes during phagophore formation, where it contributes to membrane expansion (29, 35). In recent years, gene expression studies have shown that genes involved in early phases of autophagy can exhibit transcriptional changes in degenerative and aging-related diseases whereas the structural markers representing the late stages of autophagosome (e.g., LC3B) may remain more stable (20-22, 26, 31, 36). This suggests that rather than a complete loss of autophagy, regulatory and initiating mechanisms are selectively.

In this context, the stage-dependent transcriptional changes of WIPI1 identified in our study suggest that the genes involved in the early stages of autophagy are differently regulated during OA progression. However, these findings do not provide direct evidence whether autophagy is functionally upregulated or downregulated; instead of demonstrating functional autophagy activation or suppression, these results indicate that disease severity is associated with transcriptional modulation of genes regulating early autophagy.

Hence, our WIPI1 data do not reveal the presence of a selective autophagy type or a specific autophagic activation state, but rather show that early autophagy control points are transcriptionally affected in OA.

On the other hand, NBR1 is a selective autophagy adaptor protein that directly binds ubiquitinated cargo and facilitates its delivery to autophagosomes for degradation (19, 30). Experimental studies have demonstrated that NBR1 functions in cooperation with LC3 to mediate selective autophagic clearance of protein aggregates and other ubiquitinated substrates (19). NBR1 is particularly involved in protein quality control and cellular homeostasis under chronic stress conditions (37). There are only a few studies directly investigating the role of NBR1 in OA. However, the changes in expression of selective autophagy adaptor proteins have been shown to be associated with chronic inflammation, oxidative stress, and degenerative processes in various disease models (37, 38).

In this context, in our work, the changes in NBR1 mRNA levels depending on the disease stage suggest that the molecular pathways associated with selective autophagy could be involved in the adaptation process in the progression of OA (38). Nevertheless, it is not possible to say that these changes indicate a specific selective autophagy subtype (e.g., mitophagy) or a functional autophagy output directly. Instead, our data demonstrate that NBR1 might be regulated as part of the cellular stress response in OA, but the functional significance of this regulation needs to be uncovered by advanced mechanistic and tissue-based.

Remarkably, no substantial correlations were found between the expression of autophagy-related genes and the systemic inflammatory marker CRP. This suggests that the changes in gene transcription identified in this study were not simply the result of systemic inflammation as measured by CRP. On the other hand, since correlation analyses were carried out on all OA stages combined and immune cell subpopulations were not examined, more comprehensive stage-specific and cell-type-specific studies are.

At the mRNA level, the stage-dependent expression patterns of ULK1, TFEB, WIPI1, and NBR1 observed within this patient cohort suggest that regulatory autophagy-related genes may have potential as complementary molecular indicators of disease severity, particularly when interpreted alongside clinical and radiographic assessments. This interpretation is supported by the modest discriminatory performance observed in ROC analysis (Figure 4). Nevertheless, the absence of a healthy control group, the cross-sectional study design, and the lack of protein-level or functional validation preclude classification of these genes as diagnostic biomarkers. Rather, they should be considered exploratory severity-associated molecular markers requiring further validation.

One of the major features of the present work is the within-cohort design. Since there was no healthy control group, the transcriptional differences found should be seen as severity-associated changes in K-L stages rather than dysregulation compared to a normal baseline. Therefore, this method focused on determining if the gradual development of the disease was related to changes in autophagy gene expression in the OA population. By focusing exclusively on intra-cohort comparisons, this approach minimizes inter-group variability and potential confounding effects that may occur when external control groups are included.

Several limitations should be acknowledged. Firstly, the cross-sectional design of this study allows assessment of gene expression at a single time point and does not provide information about longitudinal molecular changes during OA progression. Secondly, only blood mRNA was analyzed without autophagy estimations in tissues or protein level or functional studies. Further work including repeated sampling over time, analysis of joint tissues, and use of functional tests will shed more light on autophagy regulation in knee OA.

In conclusion, this study has revealed that the genes primarily responsible for the initiation, regulation, and formation of the autophagosome early stages are differentially expressed depending on the stage of the disease in knee OA. At the same time, the genes essential in structural autophagy hardly changed at the mRNA level in peripheral blood. Our findings are consistent with previous reports indicating that dysregulation of upstream regulatory components of the autophagy pathway is associated with OA progression and cartilage degeneration (16, 31, 32). Experimental and mechanistic studies have further emphasized that impairment of key regulatory nodes, including ULK1- and TFEB-mediated pathways, may contribute to disrupted autophagic homeostasis in degenerative conditions (11, 28, 33). Together, these data support the concept that disease severity in OA may be linked to alterations at the regulatory level of the autophagy network rather than to uniform disruption of core structural machinery (26, 31, 32). This offers a platform for future integrative and mechanistic.