Targeting NCAPD2 as a Therapeutic Strategy for Crohn’s Disease: Implications for Autophagy and Inflammation

Abstract

Background

Our earlier studies identified that non-SMC condensin I complex subunit D2 (NCAPD2) induces inflammation through the IKK/NF-κB pathway in ulcerative colitis. However, its role in the development of Crohn’s disease (CD) and the specific molecular mechanism still need to be further studied.

Methods

NCAPD2 expression in clinical ileal CD mucosa vs normal mucosa was examined, alongside its correlation with CD patients’ clinical characteristics via their medical records. The biological function and molecular mechanism of NCAPD2 in CD were explored using a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced CD mouse model, along with immunofluorescence, western blot, quantitative real-time PCR, immunohistochemistry, hematoxylin and eosin staining, and cell functional analysis.

Results

NCAPD2 was overexpressed in CD tissues and significantly correlated with disease activity in CD patients (P = .016). In a TNBS-induced CD mouse model, NCAPD2 knockdown inhibited the development of TNBS-induced intestinal inflammation in mice. In addition, we found that NCAPD2 inhibited autophagy. Mechanistically, NCAPD2 promoted the phosphorylation of mammalian target of the rapamycin (mTOR) and its direct effector S6K and downregulated the expression of autophagy-related proteins Beclin1, LC3II, and Atg5. In addition, NCAPD2 activates the NF-κB signaling pathway, and the downstream inflammatory factors are continuously released, leading to the persistence of inflammation.

Conclusions

Our results show that NCAPD2 suppresses autophagy and worsens intestinal inflammation by modulating mTOR signaling and impacting the NF-κB pathway, suggesting a critical role in CD progression. Targeting NCAPD2 could be a promising therapeutic approach to stop CD advancement.

Introduction

Crohn’s disease (CD) is a type of inflammatory bowel disease characterized by transmural, segmental, and asymmetric inflammation that can affect any part of the digestive tract.^1,2 In Western developed countries, including North America, the incidence of CD has stabilized at a high level in recent years.^3,4 Current treatment strategies focus on achieving deep and long-term remission to prevent complications and stop the progression of the disease. However, patients still require long-term medication and even surgical treatment, and symptoms can persist throughout their lifetime, imposing significant economic and social burdens on both individuals and national healthcare systems.^5-7 Therefore, there is an urgent clinical need to explore new potential therapeutic approaches and targets.

The pathogenesis of CD is multifaceted, involving dysregulated immune responses, impaired barrier function, and aberrant autophagy processes.^8,9 Among the genetic factors associated with CD, variations in genes related to autophagy, such as ATG16L1 and IRGM, have been well documented, suggesting that autophagy plays a critical role in disease progression.¹⁰ Autophagy refers to the phagocytosis, degradation, and digestion of the cell’s substances in its lysosome. Autophagy, a cellular protection mechanism, degrades and recycles damaged organelles and proteins to maintain cell homeostasis.¹¹ Mammalian target of the rapamycin (mTOR) is a major negative regulator of autophagy, which controls cell growth by inhibiting ULK1 activity. Inhibition of mTOR or the use of mTOR inhibitors can activate autophagy.¹² Yet, the exploration of additional genetic actors and molecular pathways that contribute to the autophagy-centric inflammation characteristic of CD has been markedly limited, leaving a vast expanse of unknowns within this crucial area of research.

Non-SMC condensin I complex subunit D2 (NCAPD2) has recently been identified as a potential player in the regulation of autophagy and inflammation,^13,14 but its role in the pathogenesis of CD remains unclear. NCAPD2 is a non-chromosomal structure maintenance protein subunit in condensin complex II, which is mainly enriched in the promoter region of actively transcribed genes, centromeres, and telomeres, and plays an important role in chromosome agglutination and chromatid separation.¹⁵ Currently, numerous studies have highlighted the abnormal overexpression of NCAPD2 in various cancer cells, demonstrating its role in promoting tumor progression by modulating cell cycle, migration, and apoptosis.^16-18 Our prior investigation unveiled the aberrant upregulation of NCAPD2 in colorectal cancer cells, where it facilitates cancer development by activating the Ca2⁺/CAMKK2/AMPK/mTORC1 signaling pathway while inhibiting autophagy.¹³ Additionally, we found that NCAPD2 induces inflammation in ulcerative colitis through the IKK/NF-κB pathway.¹⁹ Despite these advancements, there remain significant gaps in our understanding of the precise mechanisms through which NCAPD2 impacts autophagy and inflammation in CD. Most studies have explored the general role of autophagy in CD without delving into the specific contribution of components like NCAPD2. Furthermore, the therapeutic potential of targeting NCAPD2, particularly in modulating autophagy and inflammatory pathways in CD, remains largely unexplored.

In this study, our hypothesis suggested that NCAPD2 promotes the inhibition of autophagy by regulating the mTOR/NF-κB pathway and promotes the development of intestinal inflammation in CD patients. Through in vivo studies, we elucidate a novel role of NCAPD2 in regulating the development of CD, which may facilitate the development of novel therapeutic strategies to specifically regulate autophagy to control inflammation in CD, thus providing new hope for patients with limited therapeutic options.

Methods

Patient Samples

Patients diagnosed with CD were recruited from the Affiliated Hospital of Nanjing University of Chinese Medicine. CD mucosal tissue of the ileum and matched normal mucosal tissue were collected from these patients through surgery. Fresh samples were snap frozen in liquid nitrogen for subsequent experiments. Formalin-fixed tissue samples were embedded with paraffin for immunohistochemistry (IHC) analysis. In addition, the datasets involved were analyzed with Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/). The detailed experimental method can be found in Supplementary Material.

Mice, TNBS-Induced Mice Model, and Sample Collection

NCAPD2^+/− mice with a C57BL/6N background were generated by Cyagen Biosciences (https://www.cyagen.com) using CRISPR/Cas9 method. The mouse genotypes were detected using PCR. All experimental procedures received approval from the Animal Experiment Ethics Committee of Nanjing University of Chinese Medicine (license number: 2021DW-23-02).

Cell Culture and Transfection

Human colon cancer cell lines (HT-29 and Caco-2) were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco’s modified Eagle medium (DMEM) according to the instructions provided by ATCC. The siRNA oligonucleotides targeting NCAPD2 and negative control siRNA were purchased from GenePharma (Shanghai, China). The si-NCAPD2 sequence is as follows: GAAUCAGUAUGUUGUGCAA.

IHC Analysis

IHC analysis was performed on paraffin-embedded colonic tissue sections (5 μm) to assess the expression of NCAPD2 in CD tissues and normal specimens.

Quantitative Real-Time PCR

Total RNA was extracted from tissues and cells to assess the expression of inflammatory cytokines and NCAPD2 in the inflammation models of Caco-2 and HT-29 cells, and to evaluate the knockout status of the NCAPD2 gene in NCAPD2 knockout mice.

IL-6, IL-10, IL-17, and TNF-α Expression Analysis by ELISA

Cell culture supernatants and mouse serum were collected to assess the expression levels of the inflammatory cytokines IL-6, IL-10, IL-17, and TNF-α.

Western Blotting

Proteins were extracted from cells and tissues to assess the expression levels of mTOR, autophagy markers, and proteins related to the NF-κB signaling pathway.

Immunofluorescence Analysis

Tissue samples were collected from mice in different treatment groups, and immunofluorescence staining was performed to detect the localization and expression of p-p65, LC3II, and P62.

Assessment of Colonic Damage

Bodyweight, stool features, and fecal occult blood of the mice were monitored throughout the experiment on alternate days. The Disease Activity Index (DAI) was evaluated according to the previously established criteria.²⁰ The length and weight of the colons were determined. Colon Gross Morphological Damage Index (CMDI) score was calculated according to the previously established criteria.²¹ Scores of 2 gross morphology values were averaged for statistical analysis.

Hematoxylin and Eosin Staining and Masson’s Staining

Colon tissues were fixed, paraffin-embedded, and then sectioned. Hematoxylin and eosin (H&E) staining was routinely performed to observe the inflammatory state and pathological alterations in the colonic tissues. The extent of colonic fibrosis in mice was observed using conventional Masson’s staining and subsequently analyzed with ImageJ software (National Institutes of Health, Bethesda, MD).

Data Acquisition and Analysis

All data analysis was conducted using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA). The results were expressed as mean ± SD. The 1-way analysis of variance (ANOVA) test was used as a statistical method to evaluate the statistical differences between groups. Each experiment was repeated at least 3 times. A difference was considered to be significant when P <.05.

Statistical methods for Supplementary Table S1 are as follows: Quantitative data that follow a normal distribution are expressed as mean ± SD and analyzed using an independent t test. Data that do not follow a normal distribution are represented by median (interquartile range) and analyzed using the Kruskal-Wallis test. Qualitative data are presented as percentages (n, %) and intergroup comparisons are performed using chi-squared test or Fisher’s exact test. All tests are 2-tailed, and a P-value <.05 is considered statistically significant.

Statistical methods for Supplementary Table S2 are as follows: Spearman and Pearson analyses were used to assess the correlation between NCAPD2 expression and clinical features in CD patients.

Results

NCAPD2 Expression Is Elevated in CD and Is Significantly Correlated With Disease Activity

NCAPD2 mRNA expression was analyzed in CD using data from the GEO database, showing high levels in CD (Figure 1A). IHC further confirmed elevated NCAPD2 levels in CD patients, with normal specimens exhibiting weak NCAPD2 signals (Figure 1B).

Subsequently, we analyzed the comprehensive medical records of the enrolled patients and investigated the correlation between NCAPD2 expression and the clinical characteristics of CD. Our results revealed a significant correlation between NCAPD2 expression and disease activity in CD (Crohn’s Disease Activity Index [CDAI] > 150, P = .016) (Supplementary Table S1). However, no significant associations with biomarkers were observed (Supplementary Table S2).

NCAPD2 Is Overexpressed in a Cellular Inflammatory Model

To validate the expression pattern of NCAPD2 in a range of human colorectal cell lines under inflammatory conditions and elucidate the relationship between NCAPD2 and inflammation, we established an in vitro inflammatory cell model.

Based on the results of enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR (qRT-PCR), which indicated elevated expression of NCAPD2 and IL-8, Caco-2 cells were cultured with 50 ng/mL IL-1β for 48 hours, while HT-29 cells were incubated with 1 μg/mL lipopolysaccharide (LPS) for 24 hours (Figure 1C and D). Transfection of HT-29 and Caco-2 cells with OE-NCAPD2 and Si-NCAPD2 showed that inflammatory factors influence NCAPD2 expression, with NCAPD2 showing a corresponding change with the increase or decrease in inflammatory markers (Figure 1E and F). However, ELISA analysis of cell supernatants indicated no significant changes in inflammatory markers TNF-α, IL-6, and IL-17 regardless of NCAPD2 expression levels (Figure 1G). This underscores a crucial finding of our study: while inflammatory factors influence the expression of NCAPD2, changes in NCAPD2 levels under basal conditions do not induce alterations in inflammatory cytokine expression.

NCAPD2 Activated mTOR/Autophagy in a Cellular Inflammatory Model

To delve deeper, we sought to ascertain whether NCAPD2 affects autophagy via mTOR in an in vitro inflammatory environment. Treatment of Caco-2 cells with IL-1β resulted in increased levels of p-mTOR, accompanied by a decrease in autophagy-related proteins such as Beclin1, LC3II, and ATG5 (Figure 2B and D). These changes were further exacerbated by IL-1β stimulation and NCAPD2 overexpression. Conversely, the knockdown of NCAPD2 mitigated the IL-1β-induced increase in p-mTOR and decrease in autophagy proteins. Similar patterns were observed in HT-29 cells. Immunofluorescence analysis showed that LPS stimulation led to decreased LC3II expression and increased p62 expression in HT-29 cells, with co-overexpression of NCAPD2 further suppressing LC3II expression and enhancing p62 expression (Figure 2A and C).

NCAPD2 Activated NF-κB in a Cellular Inflammatory Model

We next sought to investigate whether NCAPD2 influences NF-κB in a cellular inflammatory model. Immunofluorescence analysis of p-p65 revealed that both LPS and IL-1β upregulated NF-κB expression in HT-29 and Caco-2 cells, respectively (Figure 3A). Notably, this upregulation was augmented by NCAPD2 overexpression and mitigated by its knockdown. Further insights into the pathway dynamics were gained through western blot analysis (Figure 3B). In HT-29 cells, LPS stimulation led to an increase in phosphorylated p65 levels. NCAPD2 overexpression accentuated this elevation, while its knockdown attenuated it. Similarly, in Caco-2 cells, IL-1β induced phosphorylation of p65, which was further enhanced by NCAPD2 overexpression and reduced by its silencing. Additionally, ELISA results showed that overexpression of NCAPD2 significantly increased the levels of proinflammatory cytokines TNF-α, IL-6, and IL-17 in the supernatant of HT-29 and Caco-2 cells under LPS and IL-1β stimulation, while reducing the expression of the anti-inflammatory cytokine IL-10. Conversely, knocking down NCAPD2 resulted in the opposite effects (Figure 3C). As NF-κB acts as a transcription factor activating downstream proinflammatory genes, our findings collectively suggest that NCAPD2 enhances NF-κB signaling activation and the expression of the inflammatory factors in an in vitro inflammatory milieu.

NCAPD2 Regulates Inflammation Through the mTOR/NF-κB Signaling Pathway

We have established that NCAPD2 can activate mTOR and its downstream effectors to suppress autophagy, while also enhancing the activation of NF-κB signaling. Therefore, it is imperative to further elucidate the interplay between NCAPD2, mTOR, and NF-κB.

Western blot analysis was employed to assess the levels of mTOR, NF-κB, and autophagy-related proteins. Following the overexpression of NCAPD2 in HT-29 cells, there was a marked increase in the levels of p-mTOR (S2448), p-p70S6K, p-p65, and P62, accompanied by a decrease in the expression of Beclin1, ATG5, and LC3II (Figure 4A). Similarly, overexpression of NCAPD2 in Caco-2 cells yielded comparable results (Figure 4B). To delve deeper, we utilized rapamycin (RAPA), an mTOR inhibitor. Intriguingly, RAPA not only inhibited mTOR pathway activation but also attenuated NF-κB activation, while increasing the expression of autophagy-related proteins. Importantly, RAPA reversed the overactivation of the mTOR pathway and NF-κB induced by NCAPD2 overexpression. In essence, RAPA significantly counteracted NCAPD2-induced mTOR/NF-κB activation. These findings suggest that NCAPD2 regulates inflammation through the mTOR/NF-κB signaling pathway and plays a crucial role in CD.

Knockout of NCAPD2 Gene Impaired the Development of LPS-Induced CD In Vivo

To further identify the key role of NCAPD2 in CD, we generated the NCAPD2 knockout (NCAPD2 KO; NCAPD2^(+/−)) mouse model by deleting exons 3-13 of the mouse NCAPD2 genomic DNA. The genomic DNA of wild-type (WT) and NCAPD^(+/−) mice was analyzed by PCR (Figure 5A). In comparison to WT mice, NCAPD2^(+/−) mice exhibited notable improvements in the context of experimental colitis. These enhancements included a higher survival rate, an increase in body weight, a reduction in the DAI score, and mitigation of intestinal inflammatory injuries (Figure 5B, E, and G). Similar results were obtained for the expression of inflammatory factors measured by ELISA (Figure 5H). Additionally, histological analysis using Masson’s staining, in line with H&E staining (Figure 5C), revealed that WT mice displayed extensive epithelial architecture disruption, near-total crypt loss, compromised epithelial integrity, and significant colonic tissue fibrosis when juxtaposed with NCAPD2^+/− mice.

Western blot analysis further substantiated these findings by indicating a decline in the expression of phosphorylated forms of mTOR, p4E-BP1, and p65 in NCAPD2 KO mice. Furthermore, the expressions of autophagy markers, ATG5, Beclin1, and the LC3II/I ratio, in tissues were notably elevated compared to those in the model group (Figure 5F). Immunofluorescence analysis of LC3, P62, and p-p65 also indicated the similar results (Figure 5D). Electron microscopy of autophagosomes revealed acute colonic inflammation in WT mice treated with 2,4,6-trinitrobenzene sulfonic acid (TNBS), showing inhibited autophagy and a diminished presence of autophagosomes (Figure 5I). Importantly, the suppression of autophagy observed due to NCAPD2 knockdown seemed to counteract the autophagy inhibition imposed by the model group. These findings demonstrate that NCAPD2 deficiency curtails CD progression by regulating the mTOR signaling pathway and affecting the NF-κB signaling pathway.

Discussion

CD and other inflammatory bowel diseases represent severe, chronic conditions that disrupt patients’ lives significantly. The exact etiology of CD remains elusive. Current understanding suggests that it arises from a complex interplay of genetic, environmental, and immune factors.^21,22 Our study reveals NCAPD2 as a crucial mediator in the balance of autophagy and inflammation in the gastrointestinal tract. We found that NCAPD2 inhibits autophagy via the mTOR pathway, disrupting cellular homeostasis and debris clearance, while simultaneously activating the NF-κB pathway, amplifying proinflammatory responses (Figure 6). The significance of these findings lies not only in their contribution to our understanding of the molecular underpinnings of intestinal inflammation but also in highlighting the potential of NCAPD2 as a novel therapeutic target for conditions characterized by dysregulated autophagy and excessive inflammatory responses.

The mTOR pathway is a central regulator of cell growth and metabolism and is known for inhibiting autophagy in response to nutrient abundance. In the context of CD, NCAPD2 inhibits autophagy via the mTOR pathway, disrupting a critical cellular process responsible for the degradation and recycling of cellular components. This disruption is particularly detrimental because efficient autophagy is essential for controlling inflammation and maintaining intestinal homeostasis. Under normal conditions, mTOR is activated by signals such as growth factors, amino acids, and energy status, which reflect the availability of nutrients.²³ When mTOR is active, it suppresses autophagy by phosphorylating and inhibiting key autophagy-initiating complexes, such as the ULK1 complex.²⁴ This suppression is beneficial in nutrient-rich conditions, allowing cells to prioritize growth and proliferation over the catabolic process of autophagy. However, in disease states like CD, where chronic inflammation and cellular stress are prevalent, the role of autophagy becomes critically important.²⁵ Therefore, efficient autophagy is essential for mitigating inflammation and preserving the integrity of intestinal epithelial cells, which form the first line of defense in the gut.

Inflammation is one of the hallmarks of CD, which is characterized by several indicators such as cytokine production, immune cell infiltration, and tissue damage.²⁶ Our study found that NCAPD2 exacerbates these inflammatory markers. By inhibiting autophagy through the mTOR pathway, NCAPD2 disrupts the cellular defense against stress and damaged organelles, essential for maintaining intestinal epithelial integrity.²⁷ This disruption may lead to a pathological state prone to inflammation. Moreover, the NF-κB pathway activated by NCAPD2 not only acts as a beacon of inflammatory response but also enhances the expression of proinflammatory cytokines, thus creating a feedback loop that exacerbates intestinal inflammation. Under normal conditions, NF-κB activity is tightly controlled and is activated only in response to stress or pathogen infection.²⁸ However, in CD, aberrant NF-κB activation driven by dysregulated mTOR signaling. By focusing on the specific interactions between NCAPD2, mTOR, and NF-κB, we can identify novel points of intervention. For example, targeting the interface at which NCAPD2 affects mTOR signaling may provide a means of restoring autophagic function and inhibiting NF-κB-driven inflammation. Similarly, exploring how the effects of NCAPD2 on NF-κB activation aggravate inflammatory pathways in CD may reveal novel strategies to modulate immune responses in affected individuals. In conclusion, understanding these interactions could elucidate CD pathogenesis and discover targeted therapeutic approaches, paving the way for precision medicine strategies.

This intricate interplay between autophagy inhibition and inflammation promotion provides us with a clearer understanding of the molecular mechanisms underlying gut pathology.^29,30 Our study advances this idea by integrating the regulatory role of NCAPD2 and providing a new perspective that bridges these 2 critical pathways. This integration not only enriches our understanding of the complex network of interactions that control inflammation and autophagy but also emphasizes the novelty and importance of our findings. Through this perspective, NCAPD2 emerges as a potential master regulator, providing new avenues for therapeutic intervention in inflammatory bowel disease and potentially other diseases driven by similar pathological mechanisms.

However, our study acknowledges several limitations. The complexity of the mTOR and NF-κB pathways, along with CD’s multifactorial nature, means our findings are just a small piece of the puzzle. The interplay between genetics, environmental factors, and patient variability challenges the translation of these results into therapies. While in vitro and animal models offer insights into NCAPD2’s role, they do not fully replicate human gut pathology, and compensatory mechanisms in humans remain unexplored. Thus, further clinical validation is needed. Despite confirming NCAPD2’s regulation of this pathway, translating these findings into effective therapies has proven challenging. Future research should explore NCAPD2’s role in other inflammatory diseases, develop specific inhibitors for testing in animal models, and investigate its interactions with other signaling pathways and cellular processes.

In summary, our study elucidated the elevated expression of NCAPD2 in both clinical samples and inflammatory cell models derived from patients with CD. We discovered that NCAPD2 inhibits autophagy by modulating the mTOR signaling pathway and influences the NF-κB pathway, exacerbating intestinal inflammation. Additionally, the data obtained from CD-induced NCAPD2^(+/−) mice further support the notion that NCAPD2 may play a crucial role in the progression of CD. Hence, targeting NCAPD2 emerges as a promising therapeutic strategy to impede the advancement of CD.

Supplementary Data

Supplementary data are available at Inflammatory Bowel Diseases online.

Acknowledgments

We thank the patients who participated in the survey. Special thanks to Professor Liu Ping for the valuable feedback on the manuscript draft. We also thank the Laboratory at the College of Life Sciences, Nanjing Normal University, for technical support.

Author Contributions

H.G. analyzed the data and wrote the manuscript. C.W., H.Z., and H.C. performed the experiments. Y.G., L.Q., and Y.Z. performed the literature search and data extraction. P.L. revised the manuscript. B.Y. was responsible for the design of the study and funding acquisition. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (NNSFC) (81673973, 82074431), the Jiangsu Province Hospital of Chinese Medicine Peak Talent Program (y2021rc27), and the Phase III Project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (ZYX03KF034). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflicts of Interest

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability

Some data used in this study were obtained from the publicly accessible websites (https://www.ncbi.nlm.nih.gov/geo/) described in the “Methods” section. Other data are available from the corresponding author; please contact [email protected].

Ethical Statement

This study was approved (or granted exemption) by the ethics committee of the Affiliated Hospital of Nanjing University of Chinese Medicine (approval no. 2021NL-091-02). We certify that the study was performed in accordance with the 1964 declaration of HELSINKI and later amendments. This study and included experimental procedures were approved by the institutional animal care and use committee of the Animal Experiment Ethics Committee of Nanjing University of Chinese Medicine (approval no. 2021DW-23-02). All animal housing and experiments were conducted in strict accordance with the institutional guidelines for care and use of laboratory animals. Written informed consent was obtained from all the participants prior to the enrollment (or for the publication) of this study (or case report). Ethics approval and consent to participate: The experimental protocol was approved by the Ethics Board of Jiangsu Province Hospital of Chinese Medicine, Affiliated Hospital of Nanjing University of Chinese Medicine.

References

Author notes