Mesenchymal stem cell attenuates spinal cord injury by inhibiting mitochondrial quality control-associated neuronal ferroptosis.

Ferroptosis is a newly discovered form of iron-dependent oxidative cell death and drives the loss of neurons in spinal cord injury (SCI). Mitochondrial damage is a critical contributor to neuronal death, while mitochondrial quality control (MQC) is an essential process for maintaining mitochondrial homeostasis to promote neuronal survival. However, the role of MQC in neuronal ferroptosis has not been clearly elucidated. Here, we further demonstrate that neurons primarily suffer from ferroptosis in SCI at the single-cell RNA sequencing level. Mechanistically, disordered MQC aggravates ferroptosis through excessive mitochondrial fission and mitophagy. Furthermore, mesenchymal stem cells (MSCs)-mediated mitochondrial transfer restores neuronal mitochondria pool and inhibits ferroptosis through mitochondrial fusion by intercellular tunneling nanotubes. Collectively, these results not only suggest that neuronal ferroptosis is regulated in an MQC-dependent manner, but also fulfill the molecular mechanism by which MSCs attenuate neuronal ferroptosis at the subcellular organelle level. More importantly, it provides a promising clinical translation strategy based on stem cell-mediated mitochondrial therapy for mitochondria-related central nervous system disorders.

Combined inhibition of surface CD51 and γ-secretase-mediated CD51 cleavage improves therapeutic efficacy in experimental metastatic hepatocellular carcinoma.

BACKGROUND & AIMS: Integrin αv (ITGAV, CD51) is regarded as a key component in multiple stages of tumor progression. However, the clinical failure of cilengitide, a specific inhibitor targeting surface CD51, suggests the importance of yet-unknown mechanisms by which CD51 promotes tumor progression. METHODS: In this study, we used several hepatocellular carcinoma (HCC) cell lines and murine hepatoma cell lines. To investigate the role of CD51 on HCC progression, we used a 3D invasion assay and in vivo bioluminescence imaging. We used periostin-knockout transgenic mice to uncover the role of the tumor microenvironment on CD51 cleavage. Moreover, we used several clinically relevant HCC models, including patient-derived organoids and patient-derived xenografts, to evaluate the therapeutic efficacy of cilengitide in combination with the γ-secretase inhibitor LY3039478. RESULTS: We found that CD51 could undergo transmembrane cleavage by γ-secretase to produce a functional intracellular domain (CD51-ICD). The cleaved CD51-ICD facilitated HCC invasion and metastasis by promoting the transcription of oxidative phosphorylation-related genes. Furthermore, we identified cancer-associated fibroblast-derived periostin as the major driver of CD51 cleavage. Lastly, we showed that cilengitide-based therapy led to a dramatic therapeutic effect when supplemented with LY3039478 in both patient-derived organoid and xenograft models. CONCLUSIONS: In summary, we revealed previously unrecognized mechanisms by which CD51 is involved in HCC progression and uncovered the underlying cause of cilengitide treatment failure, as well as providing evidence supporting the translational prospects of combined CD51-targeted therapy in the clinic. IMPACT AND IMPLICATIONS: Integrin αv (CD51) is a widely recognized pro-tumoral molecule that plays a crucial role in various stages of tumor progression, making it a promising therapeutic target. However, despite early promising results, cilengitide, a specific antagonist of CD51, failed in a phase III clinical trial. This prompted further investigation into the underlying mechanisms of CD51's effects. This study reveals that the γ-secretase complex directly cleaves CD51 to produce an intracellular domain (CD51-ICD), which functions as a pro-tumoral transcriptional regulator and can bypass the inhibitory effects of cilengitide by entering the nucleus. Furthermore, the localization of CD51 in the nucleus is significantly associated with the prognosis of patients with HCC. These findings provide a theoretical basis for re-evaluating cilengitide in clinical settings and highlight the importance of identifying a more precise patient subpopulation for future clinical trials targeting CD51.

Nestin-dependent mitochondria-ER contacts define stem Leydig cell differentiation to attenuate male reproductive ageing.

Male reproductive system ageing is closely associated with deficiency in testosterone production due to loss of functional Leydig cells, which are differentiated from stem Leydig cells (SLCs). However, the relationship between SLC differentiation and ageing remains unknown. In addition, active lipid metabolism during SLC differentiation in the reproductive system requires transportation and processing of substrates among multiple organelles, e.g., mitochondria and endoplasmic reticulum (ER), highlighting the importance of interorganelle contact. Here, we show that SLC differentiation potential declines with disordered intracellular homeostasis during SLC senescence. Mechanistically, loss of the intermediate filament Nestin results in lower differentiation capacity by separating mitochondria-ER contacts (MERCs) during SLC senescence. Furthermore, pharmacological intervention by melatonin restores Nestin-dependent MERCs, reverses SLC differentiation capacity and alleviates male reproductive system ageing. These findings not only explain SLC senescence from a cytoskeleton-dependent MERCs regulation mechanism, but also suggest a promising therapy targeting SLC differentiation for age-related reproductive system diseases.

CBX4 contributes to HIV-1 latency by forming phase-separated nuclear bodies and SUMOylating EZH2.

The retrovirus HIV-1 integrates into the host genome and establishes a latent viral reservoir that escapes immune surveillance. Molecular mechanisms of HIV-1 latency have been studied extensively to achieve a cure for the acquired immunodeficiency syndrome (AIDS). Latency-reversing agents (LRAs) have been developed to reactivate and eliminate the latent reservoir by the immune system. To develop more promising LRAs, it is essential to evaluate new therapeutic targets. Here, we find that CBX4, a component of the Polycomb Repressive Complex 1 (PRC1), contributes to HIV-1 latency in seven latency models and primary CD4+ T cells. CBX4 forms nuclear bodies with liquid-liquid phase separation (LLPS) properties on the HIV-1 long terminal repeat (LTR) and recruits EZH2, the catalytic subunit of PRC2. CBX4 SUMOylates EZH2 utilizing its SUMO E3 ligase activity, thereby enhancing the H3K27 methyltransferase activity of EZH2. Our results indicate that CBX4 acts as a bridge between the repressor complexes PRC1 and PRC2 that act synergistically to maintain HIV-1 latency. Dissolution of phase-separated CBX4 bodies could be a potential intervention to reactivate latent HIV-1.

Nestin promotes pulmonary fibrosis via facilitating recycling of TGF-ß receptor I.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease that is characterised by aberrant proliferation of activated myofibroblasts and pathological remodelling of the extracellular matrix. Previous studies have revealed that the intermediate filament protein nestin plays key roles in tissue regeneration and wound healing in different organs. Whether nestin plays a critical role in the pathogenesis of IPF needs to be clarified. METHODS: Nestin expression in lung tissues from bleomycin-treated mice and IPF patients was determined. Transfection with nestin short hairpin RNA vectors in vitro that regulated transcription growth factor (TGF)-ß/Smad signalling was conducted. Biotinylation assays to observe plasma membrane TßRI, TßRI endocytosis and TßRI recycling after nestin knockdown were performed. Adeno-associated virus serotype (AAV)6-mediated nestin knockdown was assessed in vivo. RESULTS: We found that nestin expression was increased in a murine pulmonary fibrosis model and IPF patients, and that the upregulated protein primarily localised in lung α-smooth muscle actin-positive myofibroblasts. Mechanistically, we determined that nestin knockdown inhibited TGF-ß signalling by suppressing recycling of TßRI to the cell surface and that Rab11 was required for the ability of nestin to promote TßRI recycling. In vivo, we found that intratracheal administration of AAV6-mediated nestin knockdown significantly alleviated pulmonary fibrosis in multiple experimental mice models. CONCLUSION: Our findings reveal a pro-fibrotic function of nestin partially through facilitating Rab11-dependent recycling of TßRI and shed new light on pulmonary fibrosis treatment.

Histone chaperone CAF-1 promotes HIV-1 latency by leading the formation of phase-separated suppressive nuclear bodies.

HIV-1 latency is a major obstacle to achieving a functional cure for AIDS. Reactivation of HIV-1-infected cells followed by their elimination via immune surveillance is one proposed strategy for eradicating the viral reservoir. However, current latency-reversing agents (LRAs) show high toxicity and low efficiency, and new targets are needed to develop more promising LRAs. Here, we found that the histone chaperone CAF-1 (chromatin assembly factor 1) is enriched on the HIV-1 long terminal repeat (LTR) and forms nuclear bodies with liquid-liquid phase separation (LLPS) properties. CAF-1 recruits epigenetic modifiers and histone chaperones to the nuclear bodies to establish and maintain HIV-1 latency in different latency models and primary CD4+ T cells. Three disordered regions of the CHAF1A subunit are important for phase-separated CAF-1 nuclear body formation and play a key role in maintaining HIV-1 latency. Disruption of phase-separated CAF-1 bodies could be a potential strategy to reactivate latent HIV-1.

Inhibition of TGFß improves hematopoietic stem cell niche and ameliorates cancer-related anemia.

BACKGROUND: Cancer cachexia is a wasting syndrome that is quite common in terminal-stage cancer patients. Cancer-related anemia is one of the main features of cancer cachexia and mostly results in a poor prognosis. The disadvantages of the current therapies are obvious, but few new treatments have been developed because the pathological mechanism remains unclear. METHODS: C57BL/6 mice were subcutaneously injected with Lewis lung carcinoma cells to generate a cancer-related anemia model. The treated group received daily intraperitoneal injections of SB505124. Blood parameters were determined with a routine blood counting analyzer. Erythroid cells and hematopoietic stem/progenitor cells were analyzed by flow cytometry. The microarchitecture changes of the femurs were determined by micro-computed tomography scans. Smad2/3 phosphorylation was analyzed by immunofluorescence and Western blotting. The changes in the hematopoietic stem cell niche were revealed by qPCR analysis of both fibrosis-related genes and hematopoietic genes, fibroblastic colony-forming unit assays, and lineage differentiation of mesenchymal stromal cells. RESULTS: The mouse model exhibited hematopoietic suppression, marked by a decrease of erythrocytes in the peripheral blood, as well as an increase of immature erythroblasts and reduced differentiation of multipotent progenitors in the bone marrow. The ratio of bone volume/total volume, trabecular number, and cortical wall thickness all appeared to decrease, and the increased osteoclast number has led to the release of latent TGFß and TGFß signaling over-activation. Excessive TGFß deteriorated the hematopoietic stem cell niche, inducing fibrosis of the bone marrow as well as the transition of mesenchymal stromal cells. Treatment with SB505124, a small-molecule inhibitor of TGFß signaling, significantly attenuated the symptoms of cancer-related anemia in this model, as evidenced by the increase of erythrocytes in the peripheral blood and the normalized proportion of erythroblast cell clusters. Meanwhile, hindered hematopoiesis and deteriorated hematopoietic stem cell niche were also shown to be restored with SB505124 treatment. CONCLUSION: This study investigated the role of TGFß released by bone remodeling in the progression of cancer-related anemia and revealed a potential therapeutic approach for relieving defects in hematopoiesis.

Targeting Nestin+ hepatic stellate cells ameliorates liver fibrosis by facilitating TßRI degradation.

BACKGROUND & AIMS: Liver fibrosis is a wound healing response that arises from various aetiologies. The intermediate filament protein Nestin has been reported to participate in maintaining tissue homeostasis during wound healing responses. However, little is known about the role Nestin plays in liver fibrosis. This study investigated the function and precise regulatory network of Nestin during liver fibrosis. METHODS: Nestin expression was assessed via immunostaining and quantitative real-time PCR (qPCR) in fibrotic/cirrhotic samples. The induction of Nestin expression by transforming growth factor beta (TGFß)-Smad2/3 signalling was investigated through luciferase reporter assays. The functional role of Nestin in hepatic stellate cells (HSCs) was investigated by examining the pathway activity of profibrogenic TGFß-Smad2/3 signalling and degradation of TGFß receptor I (TßRI) after interfering with Nestin. The in vivo effects of knocking down Nestin were examined with an adeno-associated virus vector (serotype 6, AAV6) carrying short-hairpin RNA targeting Nestin in fibrotic mouse models. RESULTS: Nestin was mainly expressed in activated HSCs and increased with the progression of liver fibrosis. The profibrogenic pathway TGFß-Smad2/3 induced Nestin expression directly. Knocking down Nestin promoted caveolin 1-mediated TßRI degradation, resulting in TGFß-Smad2/3 pathway impairment and reduced fibrosis marker expression in HSCs. In AAV6-treated murine fibrotic models, knocking down Nestin resulted in decreased levels of inflammatory infiltration, hepatocellular damage, and a reduced degree of fibrosis. CONCLUSION: The expression of Nestin in HSCs was induced by TGFß and positively correlated with the degree of liver fibrosis. Knockdown of Nestin decreased activation of the TGFß pathway and alleviated liver fibrosis both in vitro and in vivo. Our data demonstrate a novel role of Nestin in controlling HSC activation in liver fibrosis. LAY SUMMARY: Liver fibrosis has various aetiologies but represents a common process in chronic liver diseases that is associated with high morbidity and mortality. Herein, we demonstrate that the intermediate filament protein Nestin plays an essential profibrogenic role in liver fibrosis by forming a positive feedback loop with the TGFß-Smad2/3 pathway, providing a potential therapeutic target for the treatment of liver fibrosis.

Dact1 involvement in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation by regulating cx43

Abstract Objective: To determine the role of the dishevelled binding antagonist of beta catenin 1 (DACT1) in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation (AF). Methods: The DACT1 expression and its associations with the degree of fibrosis and β-catenin in valvular disease patients were analyzed by immunohistochemistry and Masson's staining. DACT1 was overexpressed in the atrial myocyte cell line (HL-1) and the cardiac cell line (H9C2) by adenoviral vectors. Alterations in the fibrous actin (F-actin) content and organization and the expression of β-catenin were detected by flow cytometry, immunofluorescence, and Western blotting. Additionally, the association of DACT1 with gap junctions connexin 43 (Cx43) was detected by immunohistochemistry, immunofluorescence, and Western blotting. Results: Decreased cytoplasmic DACT1 expression in the myocardium was associated with AF (P=0.037) and a high degree of fibrosis (weak vs. strong, P=0.028; weak vs. very strong, P=0.029). A positive association was observed between DACT1 and β-catenin expression in clinical samples (P=0.028, Spearman's rho=0.408). Furthermore, overexpression of DACT1 in HL-1 and H9C2 cells induced an increase in β-catenin and subsequent partial colocalization of DACT1 and β-catenin. In addition, F-actin content and organization were enhanced. Interestingly, DACT1 was positively correlated with the Cx43 expression in clinical samples (P=0.048, Spearman's rho=0.370) and changed the Cx43 distribution in cardiac cell lines. Conclusion: DACT1 proved to be a novel AF-related gene by regulating Cx43 via cytoskeletal organization induced by β-catenin accumulation in cardiomyocytes. DACT1 could thus serve as a potential therapeutic marker for AF.

Nestin regulates cellular redox homeostasis in lung cancer through the Keap1-Nrf2 feedback loop.

Abnormal cancer antioxidant capacity is considered as a potential mechanism of tumor malignancy. Modulation of oxidative stress status is emerging as an anti-cancer treatment. Our previous studies have found that Nestin-knockdown cells were more sensitive to oxidative stress in non-small cell lung cancer (NSCLC). However, the molecular mechanism by which Nestin protects cells from oxidative damage remains unclear. Here, we identify a feedback loop between Nestin and Nrf2 maintaining the redox homeostasis. Mechanistically, the ESGE motif of Nestin interacts with the Kelch domain of Keap1 and competes with Nrf2 for Keap1 binding, leading to Nrf2 escaping from Keap1-mediated degradation, subsequently promoting antioxidant enzyme generation. Interestingly, we also map that the antioxidant response elements (AREs) in the Nestin promoter are responsible for its induction via Nrf2. Taken together, our results indicate that the Nestin-Keap1-Nrf2 axis regulates cellular redox homeostasis and confers oxidative stress resistance in NSCLC.

DACT1 Involvement in the Cytoskeletal Arrangement of Cardiomyocytes in Atrial Fibrillation by Regulating Cx43.

OBJECTIVE: To determine the role of the dishevelled binding antagonist of beta catenin 1 (DACT1) in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation (AF). METHODS: The DACT1 expression and its associations with the degree of fibrosis and ß-catenin in valvular disease patients were analyzed by immunohistochemistry and Masson's staining. DACT1 was overexpressed in the atrial myocyte cell line (HL-1) and the cardiac cell line (H9C2) by adenoviral vectors. Alterations in the fibrous actin (F-actin) content and organization and the expression of ß-catenin were detected by flow cytometry, immunofluorescence, and Western blotting. Additionally, the association of DACT1 with gap junctions connexin 43 (Cx43) was detected by immunohistochemistry, immunofluorescence, and Western blotting. RESULTS: Decreased cytoplasmic DACT1 expression in the myocardium was associated with AF (P=0.037) and a high degree of fibrosis (weak vs. strong, P=0.028; weak vs. very strong, P=0.029). A positive association was observed between DACT1 and ß-catenin expression in clinical samples (P=0.028, Spearman's rho=0.408). Furthermore, overexpression of DACT1 in HL-1 and H9C2 cells induced an increase in ß-catenin and subsequent partial colocalization of DACT1 and ß-catenin. In addition, F-actin content and organization were enhanced. Interestingly, DACT1 was positively correlated with the Cx43 expression in clinical samples (P=0.048, Spearman's rho=0.370) and changed the Cx43 distribution in cardiac cell lines. CONCLUSION: DACT1 proved to be a novel AF-related gene by regulating Cx43 via cytoskeletal organization induced by ß-catenin accumulation in cardiomyocytes. DACT1 could thus serve as a potential therapeutic marker for AF.

TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb.

Comprehensively elucidating the molecular mechanisms of human immunodeficiency virus type 1 (HIV-1) latency is a priority to achieve a functional cure. As current 'shock' agents failed to efficiently reactivate the latent reservoir, it is important to discover new targets for developing more efficient latency-reversing agents (LRAs). Here, we found that TRIM28 potently suppresses HIV-1 expression by utilizing both SUMO E3 ligase activity and epigenetic adaptor function. Through global site-specific SUMO-MS study and serial SUMOylation assays, we identified that P-TEFb catalytic subunit CDK9 is significantly SUMOylated by TRIM28 with SUMO4. The Lys44, Lys56 and Lys68 residues on CDK9 are SUMOylated by TRIM28, which inhibits CDK9 kinase activity or prevents P-TEFb assembly by directly blocking the interaction between CDK9 and Cyclin T1, subsequently inhibits viral transcription and contributes to HIV-1 latency. The manipulation of TRIM28 and its consequent SUMOylation pathway could be the target for developing LRAs.

Nuclear Nestin deficiency drives tumor senescence via lamin A/C-dependent nuclear deformation.

Emerging evidence has revealed that Nestin not only serves as a biomarker for multipotent stem cells, but also regulates cell proliferation and invasion in various tumors. However, the mechanistic contributions of Nestin to cancer pathogenesis are still unknown. In the present study, previously thought to reside exclusively in the cytoplasm, Nestin can also be found in the nucleus and participate in protecting tumor cells against cellular senescence. Specifically, we reveal that Nestin has a nuclear localization signal (aa318-aa347) at the downstream of rod domain. We then find nuclear Nestin could interact with lamin A/C. Mechanistic investigations demonstrate that Nestin depletion results in the activation of cyclin-dependent kinase 5 (Cdk5), which causes the phosphorylation of lamin A/C (mainly at S392 site) and its subsequent translocation to the cytoplasm for degradation. The findings establish a role for nuclear Nestin in tumor senescence, which involves its nucleus-localized form and interaction with lamin A/C.