Calumenin prevents fibroblast senescence and lung aging by promoting vimentin proteostasis

Significance

Progressive lung fibrosis is closely linked to aging-related dysfunction in fibroblasts, yet our understanding of this relationship is limited. Therefore, exploring the molecular mechanisms through which fibroblast senescence impacts lung fibrosis is essential. This study identifies the critical role of calumenin in maintaining vimentin proteostasis and regulating fibroblast senescence. These findings offer valuable insights into the molecular mechanisms of aging-related lung fibrosis and position calumenin as a promising therapeutic target for managing fibrosis.

Abstract

Progressive lung fibrosis is linked to aging-related dysfunction in fibroblasts, which remains poorly understood. To investigate the alterations in fibroblasts, particularly the molecular programs driving this profibrotic evolution in the aging lung, we isolated senescent lung fibroblasts from aged mice. We observed aberrant vimentin aggregates, which correlate with accelerated fibroblast senescence. CRISPR-based screening identified calumenin as a chaperone protein essential for vimentin proteostasis. A fibroblast-specific knockout of calumenin promotes the accumulation of vimentin aggregates and profibrotic factors migracytosis, exacerbating fibroblast senescence and lung aging. Mechanistically, calumenin collaborates with the TRiC complex to facilitate proper vimentin folding and recruits the chaperonin subunit Chaperonin Containing TCP1 Subunit 2 (CCT2) to degrade misfolded vimentin aggregates. Pathologically, external profibrotic stimuli trigger calcium transients and induce calumenin degradation, resulting in fibroblast senescence and the initiation of fibrosis. The natural product 9-85, derived from high-content screening, specifically targets and disrupts vimentin aggregates upon stimulation, alleviating aging-related lung fibrosis. Our findings reveal that calumenin coordinates vimentin quality control to shape cell structure and suppress the secretome of senescent fibroblasts, providing a promising therapeutic strategy for aging-related organ fibrosis.

Accelerated aging and cellular senescence drive various fibrotic diseases (1), directly contributing to pathological changes, such as idiopathic pulmonary fibrosis (IPF) and renal fibrosis (2–4). A key feature of senescence is that senescent cells release damaging senescence-associated secretory phenotype (SASP) factors, particularly those that promote inflammation and to remodel the extracellular matrix, leading to excessive scar formation and exacerbating fibrosis development (5). Researchers are rapidly developing therapeutic strategies for the targeted clearance of senescent cells, with some already demonstrating success in combating lung fibrosis. Schafer et al. observed pathological senescent cell accumulation in IPF lung tissue, and the deletion of these cells rejuvenates pulmonary health in aged mice (6). Hashimoto et al. eliminated p19^ARF-expressing cells, reversing the aging-associated gene expression profile and enhancing pulmonary function in mice (7). Rehan et al. revealed that restoring the SIRT3–FoxO3a pathway induces senescent myofibroblast depletion and facilitates lung fibrosis resolution (8). In addition to these genetic strategies, both senolytic interventions (which eliminate a portion of senescent cells) and senomorphic interventions (which suppress their proinflammatory and damaging secretory phenotype) restore lung function in aged mice (9, 10). Despite strong evidence supporting senescent cells’ role in the physiological remodeling of aged lungs, further investigation into the underlying mechanisms will undoubtedly provide more clinical opportunities.

A characteristic of many aging-related diseases is disrupted protein homeostasis (proteostasis), which leads to the accumulation of protein aggregates. Misfolded proteins of Parkinson’s disease (α-synuclein) and Alzheimer’s disease (tau and amyloid-β (Aβ)) are well-known contributors to disease pathogenesis (11, 12). However, the connection between protein aggregates, fibroblast function, and fibrosis pathology remains unclear. Vimentin, a type III intermediate filament in fibroblasts, has gained attention as a key player in the proliferative and remodeling phases of wound healing (13). It forms a flexible cytoplasmic network that supports cellular architecture and function (14). Additionally, vimentin may play a protective role in protein quality control by sequestering misfolded proteins into cage-like aggregates (15). Evidence also suggests that vimentin aggregates regulate mitosis, stem cell activation, and the cellular stress response (16). Interestingly, inhibiting vimentin assembly with withaferin A (WFA) reduces fibroblast invasiveness in IPF and offers protection against fibrosis (17). Although the precise mechanism remains unknown, it is clear that vimentin aggregates play a crucial role in tissue aging and organ fibrosis.

Chaperones serve as the primary defense against various stressors, preventing misfolded protein aggregation and regulating these proteins through refolding or degradation pathways. For example, heat shock protein 70 (HSP70) interacts with nascent polypeptides to ensure correct folding or initiate degradation via the proteasome (18). Calumenin, a low-affinity calcium-binding protein, also acts as a chaperone, likely regulating F508del-CFTR folding and CFTR maturation in cystic fibrosis-related epithelial cells (19). Additionally, calumenin has emerged as a marker of epithelial–mesenchymal transition (EMT) in various cancers and organ fibrosis (20). However, its chaperone activity remains poorly understood due to uncertainty about its specific substrate proteins.

In this study, we identified a functional link between aging-related lung fibrosis and disrupted vimentin proteostasis. Our findings reveal that calumenin acts as a chaperone, responsible for ensuring proper vimentin folding and clearing disordered aggregates in response to pathological stimuli. The fibroblast-specific knockout of calumenin accelerated both cellular senescence and organ fibrosis. Under profibrotic conditions, calcium transients trigger calumenin degradation, and we identified two phosphorylation sites crucial for calumenin stability. Considering that bisbibenzyl compounds exhibit various bioactivities such as antibiotic, antioxidative, and antitumor activities (21), we conducted targeted screening of our laboratory’s existing bisbibenzyl compound library and identified a natural compound, 9-85, which targets vimentin aggregates and alleviates aging-related lung fibrosis in vivo. Collectively, our data uncover a role for calumenin in regulating vimentin proteostasis under stress conditions. By coordinating the secretion of profibrotic factors through migracytosis, calumenin influences fibroblast senescence, positioning it as a potential therapeutic target for aging-related fibrotic diseases.

Results

Vimentin Aggregates Accumulate in Senescent Fibroblasts.

Cellular senescence in fibroblasts contributes to the progression of fibrotic pulmonary disease (22). To uncover the molecular mechanisms driving fibroblast senescence and organ fibrosis, we isolated lung fibroblasts from aged mice and observed an increased senescence demonstrated by transcriptional activation of p16 and the SASP (SI Appendix, Fig. S1A). Consistently, similar results were observed in lung tissues from both young and aged mice (SI Appendix, Fig. S1A). Monocyte chemotactic protein 1 (Mcp1), plasminogen activator inhibitor 1 (Pai1), and tumor necrosis factor-α (Tnfα) were the SASP factors that were primarily proinflammatory, while Matrix Metallopeptidase 12 (Mmp12), Collagen Type I Alpha 1 Chain (Col1a1), Matrix Metallopeptidase 10 (Mmp10), and transforming growth factor-β (Tgfβ) belong to the SASP factors that were predominantly involved in fibrogenesis (23). To further assess the profibrotic effects in vivo, we engrafted lung fibroblasts from aged mice into the lungs of C57BL/6 mice, which led to aging-related lung fibrosis (Fig. 1 A–C and SI Appendix, Fig. S1 B and C). In the experimental group transplanted with aged mouse lung fibroblasts, significant weight loss, progressive decline in pulmonary function (reduced both FVC and lung compliance), and decreased grip strength were observed compared to control groups (SI Appendix, Fig. S1 D–G). In contrast, lung fibroblasts from young mice did not exhibit similar effects (Fig. 1 A–C and SI Appendix, Fig. S1 B and C). RNA-seq analysis of lung tissue from the mice injected with aged lung fibroblasts revealed an increase in cellular senescence and activation of profibrotic pathways, along with alterations in cytoskeleton-associated genes (SI Appendix, Fig. S1H and Table S3). Changes in the cellular cytoskeleton primarily affect mechanical properties and are closely linked to accelerated organ aging and fibrosis (14, 17).

Fig. 1.

Vimentin aggregates accumulate in senescent fibroblasts. (A–C) 3-mo-old C57BL/6 mice were randomized to receive intratracheal instillation of lung fibroblasts isolated from young (3-mo-old) or aged (18-mo-old) mice. They were then killed after 3 mo, and lungs were harvested for analysis. (A) Schematic diagram of mouse models. (B) Masson trichrome staining of lung tissues from various groups. (Scale bar: 500 µm.) (C) Western blot results and quantification of ECM and p16 in lung tissues from the indicated mice groups. (D) Immunofluorescence staining of vimentin aggregates in lung fibroblasts. Vimentin aggregates were quantified as outlined. (Scale bar:5 µm.) (E) Representative electron microscopy (EM) photos of lung fibroblasts. The area outlined by red shows the enlarged details of the cell. [Scale bars: 5 µm (Left) and 1 µm (Right).] (F) Membrane fractionation scheme for the isolation of autophagosomes. Western blot was performed on aged-mice lung fibroblasts to examine the distribution of vimentin in different gradient fractions. (G) Schematic representation of experimental models. Lung fibroblasts were subjected to mechanical stress for 48 h and subsequently analyzed. (H) Immunofluorescence of vimentin aggregates in lung fibroblasts. Young murine lung fibroblasts were subjected to mechanical stress for 48 h. (Scale bar: 5 µm.) (I) Schematic diagram of mouse models. 3-mo-old C57BL/6 mice were randomized to receive intratracheal instillation of lung fibroblasts from young mice. Cells were subjected to mechanical stress for 48 h prior to instillation. Mice were killed after 3 mo, and lungs were harvested for further analysis. (J) Masson trichrome staining of lung tissues from the indicated mice groups. (Scale bar: 500 µm.) (K) Western blot analysis and quantification of ECM proteins and p16 in lung tissues from the indicated mice groups.

Vimentin, a predominant cytoplasmic intermediate filament protein, plays a crucial role in stabilizing intracellular structures, mechanosensing, and fibroblast functions (24, 25). While young murine lung fibroblasts display robust vimentin filaments stretching from the nuclear edge to the cell membrane, aged murine lung fibroblasts exhibit a cage-like vimentin structure that colocalizes with protein aggregates (Fig. 1D and SI Appendix, Fig. S1I). Ultrastructural analysis using TEM further revealed the accumulation of large autophagic vesicles in aged murine lung fibroblasts (Fig. 1E). CCT2 has been shown to facilitate the autophagy-mediated clearance of solid protein aggregates (26). We found that LC3 inclusion bodies coincided with CCT2 and vimentin, suggesting that vimentin aggregates form a cage-like structure around CCT2 (Fig. 1F). Together, these findings suggest that the cage-like structures formed in senescent fibroblasts primarily consist of vimentin aggregates.

We next explored the relationship between vimentin aggregate formation and fibroblast aging. Since mechanical stress influences the mechanotransduction and metabolism of fibroblasts in fibrotic diseases (27, 28), we assessed the impact of mechanical stress on vimentin proteostasis in murine lung fibroblasts. Sustained mechanical stress significantly increased vimentin aggregates, resembling the behavior observed in aged fibroblasts (Fig. 1 G and H and SI Appendix, Fig. S1J). Furthermore, fibroblasts exposed to mechanical stress displayed an accelerated senescence phenotype, evidenced by a higher proportion of SA-β-gal-positive cells, as well as increased expression of p16 and SASP markers (SI Appendix, Fig. S1 K–N). These findings suggest a strong correlation between vimentin aggregate formation and fibroblast senescence. To confirm these results in vivo, we isolated young murine fibroblasts and subjected them to mechanical stress for 48 h. We then injected the stressed fibroblasts into the lungs of C57BL/6 mice via intratracheal administration, allowing lung fibrosis to develop over 3 mo (Fig. 1I). Via Masson’s trichrome staining and the hydroxyproline assay, it was observed that mice injected with mechanically stressed fibroblasts showed significantly higher histopathological fibrosis scores compared to those injected with unstressed fibroblasts (Fig. 1 J–K and SI Appendix, Fig. S1 O and P). The pulmonary protein level of the senescence marker p16 was elevated, suggesting that mechanical stress disrupts vimentin proteostasis and may contribute to cellular senescence (Fig. 1K). Additionally, mice that received injections of mechanically stressed fibroblasts exhibited significant weight loss, a progressive decline in pulmonary function (decreased both FVC and lung compliance), and reduced grip strength (SI Appendix, Fig. S1 Q–T).

Genome-Wide CRISPR Screen of Fibroblasts Reveals Calumenin as a Negative Regulator of Vimentin Aggregate Formation.

To identify genes that modulate fibroblast senescence by regulating vimentin aggregates, we conducted a genome-wide CRISPR-Cas9 knockout screen using an engineered human fibroblast HFL1 cell line stably expressing RFP-tagged α-SMA and GFP-tagged p16, which are upregulated upon mechanical stress stimulation (SI Appendix, Fig. S2 A and B). We transduced these engineered HFL1 cells with a lentiviral CRISPR knockout library. After 7 d of culture, we sorted and sequenced cells that simultaneously expressed high levels of RFP and GFP. Preliminary analysis revealed that sgRNAs targeting Calumenin were the top-ranked candidates, positively enriched in RFP⁺ GFP⁺ cells (Fig. 2 A and B). Next, we selected the top nine candidates from the initial screen to evaluate their roles in vimentin aggregate formation (SI Appendix, Fig. S2C). We transduced HFL1 cells with sgRNA lentivirus targeting these candidate genes, followed by 48 h of mechanical stress stimulation. HFL1 cells transduced with Calumenin sgRNAs displayed significant vimentin aggregates and high enrichment in autophagosome fractions (Fig. 2 C–E and SI Appendix, Fig. S2 D–F). Similar results were observed in Calumenin knockdown HFL1 cells and Calumenin knockout L929 cells (SI Appendix, Fig. S2 G–K). Consistently, TEM analysis showed that calumenin deficiency increased vimentin accumulation in autophagic vesicles in both HFL1 and L929 cells (Fig. 2 F and G and SI Appendix, Fig. S2 L and M). Together, these results suggest that calumenin negatively regulates vimentin aggregate formation in response to cellular stress.

Fig. 2.

Genome-wide CRISPR screening of fibroblasts reveals calumenin as a negative regulator of vimentin aggregate formation (A) Score of the genes exhibiting enriched sgRNAs. (B) Normalized read counts of Calu sgRNA between the two groups. (C) Western blot validation of Calu knockout cell lines. (D and E) HFL1 cells were subjected to mechanical stress for 48 h. (D) Immunofluorescence and calculation of vimentin aggregate proportions in HFL1 cells. The single-channel images are shown in SI Appendix, Fig. S2F. (Scale bar: 5 µm.) (E) Western blot of vimentin aggregates in autophagosome fractions of HFL1 cells. (F and G) Transmission Electron Microscope in HFL1 (F) and L929 (G) cells. (Scale bar: 5 µm and 1 µm.)

Fibroblast-Specific Knockout of Calumenin Exacerbates Cellular Senescence and Aging-Related Fibrosis.

Given calumenin’s critical role in regulating vimentin proteostasis, we aimed to assess the impact of calumenin deficiency on fibroblast senescence and lung fibrosis. RNA-seq analysis identified 595 differentially expressed genes between Calumenin knockdown and control mouse primary lung fibroblasts (p_adj < 0.05, |log₂Fold Change| > 1). The upregulated genes were primarily associated with aging-related fibrosis (Fig. 3A and SI Appendix, Fig. S3A and Table S4). Similar findings were observed in RNA-seq analysis of HFL1 cells (SI Appendix, Fig. S3 B and C and Table S5). Additionally, quantitative proteomic analysis revealed 773 proteins had increased expression and 250 proteins had decreased expression in Calumenin knockdown mouse primary lung fibroblasts compared to controls (Fig. 3B). The clustering heatmap of differential proteins showed that upregulated proteins in Calumenin knockdown cells were largely related to aging-associated fibrosis, consistent with RNA-seq data (Fig. 3C and SI Appendix, Table S6). Increased fibrosis induction was also confirmed by the substantial enrichment of fibrosis-associated ECM proteins indicated by integrated transcriptomic and proteomic analysis, supporting the critical role of calumenin in fibrosis (SI Appendix, Fig. S3D). In contrast, ubiquitin-related genes were significantly downregulated in Calumenin knockdown fibroblasts, suggesting a reduction in intracellular ubiquitin-dependent degradation (SI Appendix, Fig. S3A). This observation implies that CCT2-mediated aggrephagy plays a role in maintaining vimentin proteostasis (26). Validation studies in Calumenin knockout HFL1 cells and Calumenin knockdown mouse primary lung fibroblasts further demonstrated increased vimentin aggregates, elevated ECM-associated proteins, and higher levels of senescence biomarkers (Fig. 3 D and E and SI Appendix, Fig. S3 E and F). Together, these findings underscore the pivotal role of calumenin in regulating fibroblast senescence and fibrogenesis.

Fig. 3.

Calumenin deficiency exacerbates age-related lung fibrosis (A–C) Volcano plot and heatmap (Related to SI Appendix, Table S6) of differentially expressed genes (A) and proteins (B and C) in Calu knockdown mouse primary lung fibroblasts compared to control cells. (D) Western blot of ECM proteins in total lysates and vimentin aggregates in autophagosomes of HFL1 cells and mice primary lung fibroblasts. (E) SA-β-gal staining of the indicated HFL1 cells. (F) RT-qPCR analysis of calumenin in different tissues of 8-wk-old mice. (G) Western blot analysis of calumenin in lung endothelial cells and fibroblasts isolated from adult Calu^flox/flox; Acta2-CreERT2 mice. (H and I) Analysis of FVC (H) and lung compliance (I) of young (3-mo-old) and aged (18-mo-old) Calu^flox/flox and Calu^−/− mice (mean ± SEM, n = 5). (J) Hydroxyproline levels in the lungs from Calu^flox/flox and Calu^−/− mice (mean ± SEM, n = 5). (K) Masson trichrome staining of lung tissues from Calu^flox/flox and Calu^−/− mice. (Scale bar: 500 µm.) (L) RT-qPCR data of p16 and SASP components in lung fibroblasts isolated from Calu^flox/flox and Calu^−/− mice. (M) Western blot of ECM proteins and calumenin in lung, ovarian, and heart tissues from C57BL/6 mice of four different ages (3, 9, 12, and 24 mo).

To examine the role of calumenin in vivo, we first assessed its expression levels in various organs, finding significant expression in the lung, ovary, and heart (Fig. 3F and SI Appendix, Fig. S3G). We then generated tamoxifen-inducible, fibroblast-specific Calumenin knockout mice by mating Acta2-CreERT2 mice with Calu^flox/flox animals (SI Appendix, Fig. S3H). Tamoxifen treatment resulted in a specific reduction of calumenin expression in lung fibroblasts, while lung endothelial cells in Calu^−/− mice remained unaffected (Fig. 3G). Eighteen-month-old Calu^−/− mice exhibited a significant decline in body weight, grip strength, and lung function compared to control (Calu^flox/flox) mice (Fig. 3 H and I and SI Appendix, Fig. S3 I and J). Analyzing hydroxyproline levels and conducting Masson’s trichrome staining revealed severe fibrosis in aged Calu^−/− mice (Fig. 3 J and K). Furthermore, qPCR and IHC analyses of senescence markers indicated signs of accelerated lung tissue aging in Calu^−/− mice (Fig. 3L and SI Appendix, Fig. S3K).

To further investigate calumenin’s protective role in organ fibrosis under both physiological and pathological conditions, we harvested various tissues and observed a decrease in calumenin expression in an age-dependent manner (Fig. 3M). Accordingly, we established acute organ fibrosis models (lung, heart, and ovary) by treating mice with bleomycin, AGII combined with PE, and 3-MCPD, respectively. Western blot analysis indicated significant downregulation of calumenin levels in the acute fibrotic mouse group compared to the control groups (SI Appendix, Fig. S3L). Collectively, these findings demonstrate that the loss of calumenin in fibroblasts, induced by aging or chemical stimuli, contributes to the development of organ fibrosis.

Calumenin Interacts with the Chaperonin TRiC and Vimentin to Support Chaperone-Mediated Proteostasis of Vimentin.

To elucidate the mechanism underlying calumenin-mediated vimentin proteostasis, we aimed to identify potential interaction partners of calumenin in HEK293T cells overexpressing Flag-tagged calumenin. We identified calumenin-associated protein complexes using immunoprecipitation coupled with mass spectrometry (IP-MS). The IP-MS analysis revealed that proteins associated with calumenin primarily fell into three categories: 12 components of the cytoskeletal protein complex, 10 members of the TRiC/CCT complex, and 13 chaperone proteins (SI Appendix, Fig. S4A). These findings suggest that calumenin may interact with cytoskeletal proteins and molecular chaperones. Consistent with these results, silver staining analysis demonstrated that vimentin and the TRiC complex, particularly CCT2, are the main proteins interacting with calumenin (Fig. 4A). To validate the interaction, we cotransfected Flag-tagged calumenin, HA-tagged CCT2, and Myc-tagged vimentin into HEK293T cells. A coimmunoprecipitation (co-IP) assay confirmed the formation of a calumenin–CCT2–vimentin protein complex (Fig. 4B). Furthermore, endogenous immunoprecipitation in both HFL1 and L929 cells revealed a more robust interaction complex under mechanical stress (SI Appendix, Fig. S4B). Immunofluorescence analysis further confirmed a more pronounced colocalization of this complex in stressed fibroblasts compared to unstressed cells (Fig. 4C). Thus, calumenin interacts with vimentin and the TRiC complex, and these interactions are enhanced in response to external stimuli.

Fig. 4.

Calumenin interacts with the chaperonin TRiC and with vimentin, supporting chaperone-mediated vimentin proteostasis (A) Flag pull-down assay used to identify calumenin-binding proteins in 293T cells. (B) Co-IP of calumenin, vimentin, and CCT2 in HEK 293T cells. (C) Immunofluorescence showing calumenin (blue), CCT2 (red), and vimentin (green) in human primary lung fibroblasts. Cells were subjected to mechanical stress. (Scale bars: 3 µm and 1 µm.) (D) Vimentin–calumenin–TRiC complex structure predicted by ZDOCK. (E) Representative autoradiograms of folding, at the 2 min and 16 min time points, respectively. (F and G) HFLl cells were subjected to mechanical stress for 48 h. (F) Western blot analysis of vimentin aggregates and CCT2 in autophagosome gradient fractions 1-8. (G) Western blot of vimentin aggregates in proteinase-K and Triton X-100-treated autophagosomes. (H and I) Immunofluorescence and calculation of vimentin aggregates in HFL1 cells transfected with HA-CCT2^WT or HA-CCT2^T400P. Cells were subjected to mechanical stress. Single-channel images are shown in SI Appendix, Fig. S4K. (Scale bar: 5 µm.) (J and K) Analysis of vimentin aggregates in WT or CCT2-knockdown HFL1 cells with or without HA-CCT2^WT (J) and HA-CCT2^T400P (K) reexpression. Cells were subjected to mechanical stress. (L) Analysis of vimentin aggregates in WT or ATG5-knockdown HFL1 cells with or without HA-CCT2 overexpression. (M) Quantification of the data in Fig. 4 J–L. (N) Schematic depiction of calumenin-mediated vimentin quality control.

Next, we aimed to identify the downstream signaling pathways and functions associated with the TRiC/calumenin/vimentin complex. KEGG and GO pathway analyses of calumenin interactors revealed a strong association between calumenin and stress-sensing as well as proteostasis pathways (SI Appendix, Fig. S4 C and D). Similarly, we conducted IP-MS of vimentin and CCT2, and in the CCT2 immunoprecipitation, we observed enrichment of tubulin and other TRiC-associated proteins, aligning with previous reports (SI Appendix, Fig. S4 E and F) (29). The GO pathway analyses derived from the vimentin and CCT2 IP-MS results showed similarities to those from the calumenin interactors (SI Appendix, Fig. S4 G and H), highlighting pathways related to cytoskeletal filament bundles, chaperone-mediated protein folding, and degradation. These analyses suggest that TRiC and calumenin play crucial roles in chaperone-assisted vimentin quality control.

The TRiC/CCT complex is known for its role in ATP-dependent actin folding (29). To determine whether the TRiC/CCT–calumenin complex functions similarly in vimentin folding, we predicted the structure and arrangement of the vimentin–calumenin–TRiC complex using ZDOCK. Vimentin can insert into the internal cavity of the TRiC protein through structural folding, while calumenin binds to both ends of the TRiC (Fig. 4D). To test this hypothesis, we used human TRiC protein and calumenin proteins to evaluate their roles in vimentin folding (SI Appendix, Fig. S4I). We incubated denatured ^[35S]vimentin with the TRiC complex, with or without calumenin, and initiated the folding process by increasing the temperature to 37 °C and adding ATP. Upon ATP treatment, the TRiC complex facilitated vimentin folding, as indicated by a marked increase in folded vimentin (Fig. 4E). In the absence of calumenin, approximately 23% of vimentin achieved proper folding, while about 15% formed aggregates. When calumenin was added to the ^[35S]vimentin–TRiC complex, the yield of folded vimentin increased nearly threefold, and vimentin aggregation was reduced by half (Fig. 4E). Thus, our results suggest that calumenin assists in TRiC-mediated vimentin folding during the later phase, particularly after the formation of the vimentin–TRiC complex.

Given that CCT2 serves as an aggrephagy receptor for solid protein clearance, we next tested whether the TRiC/calumenin complex facilitates the degradation of misfolded vimentin. We performed membrane fractionation and LC3 lipidation assays in stress-stimulated cells to identify compartments within the autophagosomes. We found that CCT2 coeluted with vimentin in the autophagosome fraction (F-AG), and this synchronization decreased upon calumenin depletion (Fig. 4F and SI Appendix, Fig. S4J). Additionally, both vimentin and CCT2 resisted digestion by proteinase K, meaning they were enclosed within the autophagosomes (Fig. 4G). Therefore, CCT2 promotes the entry of vimentin aggregates into the autophagosome in a calumenin-dependent manner. In the immunofluorescence assays and cycloheximide (CHX) chase, we rescued this degradation by restoring the expression of wild-type CCT2, whereas the CCT2^T400P mutant failed to promote recruitment of autophagic membranes to the aggresome for degradation (Fig. 4 H–K and M and SI Appendix, Fig. S4K). Furthermore, we found that the degradation of vimentin in autophagosomes was impeded by the knockdown of ATG5, a major autophagy regulator, confirming the clearance of vimentin aggregates via autophagy (Fig. 4 L and M). Additionally, overexpressing RAB5 or RAB7 in Calu KO cells, crucial GTPases involved in regulating endocytic trafficking and degradation, reversed the increase in vimentin aggregate formation (SI Appendix, Fig. S4 L–N). Together, these data confirm that calumenin and CCT2 regulate vimentin proteostasis by coordinating its correct folding and aggrephagy (Fig. 4N).

Calumenin Deficiency Induces Fibroblast Senescence by Aggravating Profibrotic Secretome via Migracytosis.

Calumenin is required in mediating vimentin proteostasis, which is necessary for cell migration. Notably, migrasomes are newly identified vesicular organelles that transfer cellular contents and release unwanted materials (30, 31). Previous studies have shown that migrasome formation is caused by shaping retraction fibers during cell migration, with vimentin acting as one of the key regulators (32). We hypothesized that calumenin regulates migrasome-supported extracellular matrix (ECM) secretion by mediating vimentin proteostasis. To verify this hypothesis, we assessed the colocalization of migrasomes with vimentin and ECM proteins. We expressed exogenous TSPAN4-GFP in young human lung and ovarian fibroblasts to label the migrasomes and conducted endogenous immunofluorescent staining for vimentin and ECM proteins. We found minimal colocalization of α-SMA or fibronectin with TSPAN4 in young human lung and ovarian fibroblasts, indicating limited ECM protein accumulation in the migrasomes of these cells (Fig. 5 A and B and SI Appendix, Fig. S5 A and B). In contrast, we observed increased vimentin aggregate formation and ECM protein accumulation in the migrasomes of young human lung and ovarian fibroblasts exposed to mechanical stress or calumenin deprivation (Fig. 5 A and B and SI Appendix, Fig. S5 A and B). Furthermore, we corroborated these findings by isolating migrasomes and autophagosomes from young human lung and ovarian fibroblasts.

Fig. 5.

Calumenin-mediated vimentin dynamics promote fibroblast activation by aggravating ECM secretion from migrasomes. (A–C) Fibroblasts isolated from human lung (A) or ovarian donors (B) were stably expressing TSPAN4-GFP for migrasome labeling. Cells were subsequently exposed to mechanical stress or underwent calumenin knockout. (A and B) Immunofluorescence of vimentin (blue), α-SMA (red), and TSPAN4 (green) in fibroblasts from young human lung or ovarian donors. (Scale bar: 10 µm.) (C) Western blot for ECM in cell lysates and released by migrasomes from young human fibroblasts. The quantification of protein levels is shown in SI Appendix, Fig. S5E. (D) Immunofluorescence of vimentin (blue), α-SMA (red), and TSPAN4 (green) in the indicated cells. (Scale bar: 10 µm.) (E) Western blot analysis of ECM protein levels in cell lysates and released by migrasomes in the indicated cells. The quantification of protein levels is shown in SI Appendix, Fig. S5K.

We detected an increase in vimentin aggregates in autophagosomes, along with elevated levels of ECM proteins in migrasomes and those secreted into the supernatant, in both stress-stimulated and Calu KO young human lung and ovarian fibroblasts (Fig. 5C and SI Appendix, Fig. S5 C–F). Similarly, we observed that ECM accumulation in migrasomes was increased in Calu KO HFL1 cells (Fig. 5 D and E and SI Appendix, Fig. S5 G–K). Tailless vimentin (Vimentin^1-411) disrupts the cytoskeletal network and exhibits a cage-like structure (33). We introduced either the vimentin truncated mutant (Vimentin^1-411) or full-length (Vimentin^FL) into vimentin KO HFL1 cells. We found that reexpression of Vimentin^1-411, but not Vimentin^FL, promoted vimentin aggregation, ECM migracytosis, and secretion (Fig. 5 D and E and SI Appendix, Fig. S5 G–K). These results indicate that stressed or Calu KO fibroblasts deliver a greater quantity of ECM proteins to migrasomes and secrete them into the microenvironment, demonstrating a more severe senescence secretory phenotype. Previous results showed that CCT2 promotes vimentin proteostasis by removing vimentin aggregates. Overexpression of CCT2 in HFL1 cells reduced ECM protein levels in cell lysates and migrasomes under stress stimulation (SI Appendix, Fig. S5 L–P). In contrast, cells transfected with the control vector or the CCT2^T400P mutant failed to restore disrupted vimentin proteostasis and retained a high degree of ECM protein accumulation (SI Appendix, Fig. S5 L and M). Together, these results demonstrate that calumenin deficiency disrupts vimentin proteostasis and increased ECM migracytosis.

Loss of Calumenin Phosphorylation Prevents Calumenin Degradation Triggered by Profibrotic Stimuli and Inhibits Fibroblast Senescence.

After establishing that vimentin proteostasis alters in response to profibrotic stimuli, we aimed to elucidate how calumenin senses these signals and modulates vimentin proteostasis. Calumenin, a low-affinity calcium-binding protein in the secretory pathway, has seven EF-hand domains that change conformation with calcium (34, 35). Typically, physiological stimuli induce cytoplasmic calcium influx from the ER, triggering cell stress responses (36). IP3R mediates calcium ion release through IP3 binding and plays a critical role in regulating calcium transients and maintaining ER calcium homeostasis (37, 38). In L929 cells, we demonstrate that Ip3r knockdown effectively prevents mechanical stress-induced vimentin aggregation (SI Appendix, Fig. S6 A and B). This finding suggests that calcium transients are essential for the formation of vimentin aggregates. To determine how calcium affects calumenin stability, we overexpressed Flag-tagged WT calumenin or an EF-hand mutant calumenin (E56/57/174/175/211/212/252/288/289Q) and incubated the cells with calcium before lysis. Calcium protected the WT calumenin protein from proteasome-mediated ubiquitination and degradation in a dose-dependent manner. However, this protection diminished in cells overexpressing the EF-hand mutant calumenin, indicating that calumenin’s binding to Ca²⁺ through the EF-hand domain is critical for maintaining protein stability (Fig. 6A and SI Appendix, Fig. S6C).

Fig. 6.

Loss of calumenin phosphorylation prevents calumenin degradation triggered by profibrotic stimuli and inhibits fibroblast senescence. (A–C) L929 cells expressing Flag-calumenin treated with MG132 (10 µM, 2 h) and harvested with IP lysis buffer. (A) Western blot showing calumenin ubiquitination in L929 cells exposed to various concentrations of Ca²⁺. (B) Schematic diagram showing the phosphorylation and calcium-binding sites of calumenin. (C) Identification of calumenin phosphorylation sites via mass spectrometry. (D–G) Generation of the CRISPR/Cas9 system mediated by Y275D, S277D, Y275A, and S277A mutant stable cell lines from Calu KO L929 cells. (D) Western blot showing calumenin ubiquitination and phosphorylation with anti-Ub and anti-phospho-(Ser/Tyr) antibodies in the indicated mutant cells. (E) Schematic diagram showing the mutations of the phosphorylation sites of calumenin. (F) Western blot analysis of vimentin aggregates in autophagosome fractions, ECM proteins, and senescence markers in total lysates of the indicated mutant cells. The quantification of protein levels is shown in SI Appendix, Fig. S6F. (G) Western blot analysis of ECM protein levels released by migrasomes in the indicated mutant L929 cells. The quantification of protein levels is shown in SI Appendix, Fig. S6G.

Phosphorylation serves as an upstream signaling step that triggers proteasome-mediated calumenin degradation. Mass spectrometry (MS) analysis of purified calumenin identified four phosphorylation sites (S125, Y275, S277, and Y294) that were enriched in cells stimulated by mechanical stress (Fig. 6 B and C and SI Appendix, Fig. S6 D and E). Mutating two of these phosphorylation sites to alanine, Y275A and S277A, reduced the protein’s ability to bind Ser/Tyr kinases and inhibited ubiquitination, thereby protecting calumenin from degradation (Fig. 6D). In line with this, the resuspension of wild-type calumenin and Calu^Y275A/S277A mutants in Calu KO fibroblasts effectively downregulated vimentin aggregates in the autophagosome fraction, as well as ECM proteins in total lysates and migrasomes (Fig. 6 E–G and SI Appendix, Fig. S6 F and G). This effect corresponded with improved senescence phenotypes, given lower SA-β-gal-positive cells (SI Appendix, Fig. S6H). Overall, these results demonstrate that the two phosphorylation sites and EF binding sites are crucial for calumenin stability. Mutations in these sites mimic the effects of Calu KO and promote fibroblast senescence.

9-85 Ameliorates Lung Aging by Targeting Vimentin Aggregates.

After establishing the crucial role of vimentin aggregates in fibroblast senescence, we aimed to identify novel compounds that target the elimination of these aggregates through high-throughput phenotypic screening. Notably, treatment with the bibenzyl compound 9-85 effectively reduced the accumulation of vimentin aggregates in mechanical-stress-stimulated cells dose-dependently (Fig. 7A and SI Appendix, Fig. S7 A–D). Our data suggest that 9-85 could be a potent compound for reducing vimentin aggregate levels.

Fig. 7.

9-85 ameliorated organ fibrosis by targeting vimentin aggregates. (A) Chemical structures of 9-85. (B) Silver staining and western blot analysis of SDS-PAGE-separated protein samples immunoprecipitated with the 9-85-biotin probe. (C) Surface plasmon resonance (SPR) assessing the affinity between 9-85 and vimentin proteins. (D) Molecular docking of vimentin (red) with the 9-85–PAL probe (yellow). (E) Photo crosslinking coupled with MS indicating the binding sites of 9-85–PAL complexes with vimentin. (F) The ECM levels in senescent HFL1 cells (Passage 8) treated with different concentrations of 9-85 for 24 h. (G) Representative image of pulmospheres from young and old IPF patients treated with 9-85 (1 µM and 5 µM). (H) Masson trichrome staining of lung sections from each group of the treated mice. (I) Levels of hydroxyproline in the lungs from 3-mo-old (young) and 18-mo-old (aged) mice administered with the vehicle or 9-85 (mean ± SEM, n = 5). (J) Ashcroft score of the Masson stains in Fig. 7H; n = 5.

To explore the mechanism of 9-85, we designed and synthesized three novel chemical probes based on structure–activity relationship studies: biotin, a photoaffinity label (PAL), and rhodamine (SI Appendix, Fig. S7E and Dataset S1). We treated mouse primary lung fibroblasts with either 9-85-biotin or free biotin, and then, they were precipitated with streptavidin-coated agarose beads and subjected to gel electrophoresis before mass spectrometry analysis. Notably, only one band with a molecular mass of approximately 55 kD precipitated with 9-85-biotin but not with free biotin (Fig. 7B). Mass spectrometry analysis confirmed that the protein bound to 9-85 is vimentin. Furthermore, 9-85-biotin effectively bound to recombinant vimentin via competitive inhibition due to higher concentrations of unlabeled 9-85 (SI Appendix, Fig. S7F). Additionally, immunofluorescence staining demonstrated the colocalization of 9-85-rhodamine and vimentin in L929 cells (SI Appendix, Fig. S7G). Together, these results support the conclusion that 9-85 specifically binds to vimentin.

We confirmed the direct interaction between 9-85 and vimentin using a surface plasmon resonance (SPR) binding assay. The binding response curve indicates that vimentin binds directly to 9-85 in a dose-dependent manner, demonstrating rapid and strong association (K_D = 28.1 nM) (Fig. 7C). Additionally, residues N283 and Y291 play a crucial role in the hydrophobic interactions between vimentin and 9-85 (Fig. 7D). We validated the predicted binding orientations through tandem mass spectrometry analysis of vimentin and 9-85–PAL. This analysis revealed a tryptic peptide (SVAAKN₂₈₃ (+538.27) LQEAE EWYKS) resulting from the covalent attachment of photoactivated 9-85 (Fig. 7E). Furthermore, vimentin with mutated N283 and Y291 sites (Vim^{N283A, Y291A}) did not interact with 9-85, as shown by SPR analysis and a pull-down assay (SI Appendix, Fig. S7 H and I). These results suggest that the N283 and Y291 residues in vimentin are essential for its interaction with 9-85.

To further validate the antiaging effect of 9-85, we treated senescent HFL1 cells (Passage 8) with 9-85. Western blot analysis confirmed that 9-85 treatment reduced key ECM protein expression and senescence biomarkers in senescent HFL1 cells (Passage 8) (Fig. 7F and SI Appendix, Fig. S7J), along with decreased SA-β-gal activity (SI Appendix, Fig. S7K). Invasiveness in pulmospheres from IPF patients with 9-85 treatment showed a decreased zone of invasion (Fig. 7G and SI Appendix, Fig. S7L). Additionally, we treated 12-mo-old mice with various concentrations of 9-85 through intravenous injections four times a month for a total of 6 mo to investigate 9-85’s role in aging-related fibrosis. We then killed the mice at 18 mo to assess lung function and histology. 9-85 treatment significantly alleviated weight loss, restored pulmonary function (as evidenced by improved FVC and increased lung compliance), and enhanced grip strength (SI Appendix, Fig. S7 M–P). A hydroxyproline assay, along with Masson staining, revealed reduced fibrosis in the lungs treated with 9-85 (Fig. 7 H–J). Additionally, vimentin aggregates in autophagosome fractions isolated from lung fibroblasts of 9-85-treated mice were downregulated (SI Appendix, Fig. S7 Q and R). Given that vimentin is an intermediate filament protein critical for fundamental cellular processes, we employed the same dosing regimen to evaluate potential organ toxicity of 9-85 in 8-wk-old mice. Following chronic administration, no signs of toxicity were detected in liver or kidney tissues, and no adverse immune effects were observed (SI Appendix, Fig. S7 S–Y). Collectively, our findings underscore the therapeutic potential of 9-85 for treating lung fibrosis by suppressing vimentin aggregates and fibroblast senescence (SI Appendix, Fig. S7Z).

Discussion

Fibrosis presents a significant challenge in healthcare, often resulting in severe organ dysfunction and failure across various systems, contributing to up to 45% of global mortality (39). Cellular senescence is closely linked with tissue fibrosis, particularly pulmonary fibrosis (PF), which shows increased markers of cellular senescence in epithelial cells and fibroblasts (39). In IPF, senescent fibroblasts are abnormally activated, exhibiting telomere shortening and SASP that promote the onset and progression of IPF (40). Although lung fibrosis and overall health can be improved by targeting senescent cells in preclinical IPF animal models, the precise signaling network in fibroblasts that drives the senescent profibrotic phenotype remains incompletely understood. Therefore, we isolated senescent fibroblasts from aged mice and confirmed their profibrotic effects in vivo. RNA sequencing analysis revealed that the senescent fibroblasts displayed abnormal alterations in the cytoskeleton, which are associated with aging-related symptoms and diseases (41). Further investigation indicated that senescent lung fibroblasts contained more vimentin aggregates than healthy cells.

Vimentin, an abundant cytoplasmic intermediate filament protein, serves multiple functions, including matrix remodeling and signal transduction (42). While most studies have focused on its role in ECM remodeling, understanding how vimentin orchestrates fibroblast activation and regulates mechanical homeostasis is crucial for uncovering novel therapies. In this study, we isolated lung fibroblasts from aged mice and observed cellular senescence along with the formation of cage-like structures. In contrast, fibroblasts from young mice exhibited robust filaments from the nuclear periphery extending to the cell membrane. Morrow et al. reported similar cage-like aggresome formation during neural stem cell activation, elucidating the vital role of vimentin in proteasome recruitment (15). However, the characteristics of disordered vimentin within the aggresome remain unexplored. Notably, Duarte et al. demonstrated the essential intrinsically disordered tail domain of vimentin for cage-like redistribution (33), confirming the presence of disordered vimentin within aggresomes. Additionally, disassembling vimentin aggregates using WFA suppressed fibroblast activation and pulmonary fibrosis in mice (17), suggesting that the aberrant proteostasis of vimentin significantly contributes to the pathogenesis of fibrosis.

To elucidate vimentin proteostasis and fibroblast senescence, we performed genome-wide CRISPR screens and identified calumenin as a key regulator. Calumenin has been implicated as a putative cystic fibrosis transmembrane conductance regulator (CFTR) chaperone (43). Our immunoprecipitation-coupled proteomic analysis revealed calumenin-interacting partners, including vimentin and the TRiC/CCT complex. The TRiC complex is responsible for the folding of over 10% of cellular proteins and consists of eight paralogous subunits, each contributing uniquely to complex assembly, allosteric regulation, and substrate folding (29, 44). Our findings show that CCT2 plays a critical role in vimentin proteostasis, while the functional roles of the other subunits require further investigation. Our results support this hypothesis; both in silico and experimental data demonstrate that calumenin binds to vimentin, facilitating TRiC-mediated vimentin folding. Additionally, we showed that CCT2 mediates the autophagic clearance of misfolded vimentin, corroborating a previous study that identified an aggrephagy receptor (CCT2) for solid protein aggregate clearance (26).

We next investigated the role of calumenin-mediated vimentin proteostasis in age-associated fibrosis. We found that fibroblast-specific knockout of calumenin exacerbates cellular senescence and mice lung fibrosis. Vimentin plays a critical role in mediating the growth, maturation, and integrin-dependent adhesive strength, enabling adjustment of cells to their collagen attachment, regulation of their formation of cell extensions, and control of connective tissue cell migration (45). The migrasome, a newly discovered organelle, emerges from migrating fibroblasts and requires integrin α5-fibronectin pairing, which is essential for mediating cell adhesion to the ECM (46). Our findings suggest that aberrant vimentin proteostasis orients fibroblasts, resulting in increased migracytosis of ECM proteins such as collagen, fibronectin, and α-smooth muscle actin (α-SMA). These data further reveal the potential functions of migrasomes in fibroblasts.

Finally, we investigated the pathological significance of calumenin in various disease contexts. We found that profibrotic stimuli disrupt ER calcium homeostasis, leading to proteasome-mediated degradation of calumenin and subsequent fibroblast activation. Accordingly, calumenin expression decreased dramatically in mouse models of both aged and chemically induced acute organ fibrosis. Notably, young Calu^−/− mice did not exhibit accelerated aging phenomenon compared to young normal mice, suggesting that calumenin primarily contributes to aging-related lung fibrosis. Targeting vimentin aggregates with small molecules effectively mitigated lung fibrosis in mice, providing promising strategies to intervene in the fibrotic process and restore mechanical homeostasis.

In summary, our findings identify calumenin as a chaperone that facilitates vimentin folding, thereby reducing its aggregation in fibroblasts. Paradoxically, while vimentin supports migration (47), we show that its abnormal aggregation does not enhance motility. Instead, it drives morphological remodeling that promotes migrasome formation and profibrotic factor secretion, ultimately inducing cellular senescence and consequently contributing to fibrogenesis in multiple organs. Importantly, our data offer insights for coordinated actions of distinct chaperone proteins in preventing misfolded protein aggregation and maintaining cellular homeostasis.

Limitations of the Study.

The current study highlights a TRiC/calumenin chaperone system that orchestrates vimentin proteostasis. While our evidence primarily relies on in silico predictions and indirect validations, the crystal structure of the calumenin–TRiC–vimentin complex remains to be elucidated. Additional evidence is necessary to support our proposed model. Furthermore, we primarily focused on the involvement of the TRiC–Calumenin complex in regulating vimentin homeostasis during fibroblast senescence. However, whether this protein complex similarly regulates other intermediate filament proteins, such as desmin and keratin, and whether the homeostatic balance of these proteins participates in fibroblast senescence and dysfunction, was not explored in greater depth in this section. Most fibrosis models utilize mice that do not capture the complexity of human organ fibrosis, potentially limiting the translational impact of our research on clinical applications.

Materials and Methods

Ethical Statement.

All experiments involving human lung and ovary tissue samples were approved by the Institutional Review Board of Qilu Hospital of Shandong University (No. 202304), Yanan University Affiliated Hospital (No. S-S20220001), and the Center for Reproductive Medicine, Shandong University (No. 202236). We obtained lung and ovary tissues from routine surgical and biopsy procedures, with patient consent and ethics board approval.

PROTEOSTAT^® Aggresome Detection.

We detected PROTEOSTAT^® aggresomes in the indicated cells using a PROTEOSTAT^® Aggresome Detection Kit (ENZ-51035) according to the manufacturer’s protocol. For fluorescence experiments on fresh human fibroblasts, we precoated the culture dishes with fibronectin (1 µg/mL) for 2 h before cell seeding.

Synthetic Method of 9-85.

Compound 9-85 was synthesized following a specific route. Synthetic pathways are provided in Dataset S1, with detailed procedures available in the Synthetic Methods section of the SI Appendix.

Additional methods are described in SI Appendix.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

RNA-seq and Proteomic data are publicly accessible from the GEO database (GSE304185 (48), GSE262803 (49), and GSE304187 (50)) and the iProX database (IPX0008498000) (51), respectively. Source data, unique materials, and reagents included in this study can be supplied by contacting the corresponding author. Other data are included in the article and/or SI Appendix.