American journal of physiology. Cell physiology最新文献

Radioresistance in esophageal squamous cell carcinoma (ESCC) is a critical factor leading to treatment failure and recurrence, yet its underlying molecular mechanisms remain unclear. This study aimed to investigate the role of replication factor C4 (RFC4) in ESCC radioresistance and to explore the underlying mechanisms. We utilized online bioinformatics tools to analyze the properties, functions, and prognostic significance of RFC4 in ESCC. We established cell lines with varying RFC4 expression levels and subjected them to radiation exposure. RFC4 expression was assessed using quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry, and immunoblotting. Cell proliferation was evaluated with MTT, 5-ethynyl-2'-deoxyuridine (EdU), and colony formation assays. Apoptosis and cell cycle distribution were analyzed by flow cytometry. Western blotting and immunofluorescence were used to study the impact of RFC4 on the DNA damage response in ESCC cells. A xenograft mouse model was employed to assess tumor growth in vivo. RFC4 expression was significantly upregulated in ESCC tissues and cells, particularly in radioresistant cases. Functional experiments revealed that RFC4 promotes cell proliferation, inhibits apoptosis, induces cell cycle arrest, and mitigates radiation-induced DNA damage responses. Mechanistically, RFC4-mediated radioresistance in ESCC may involve the inactivation of the p53 signaling pathway. In animal studies, RFC4 knockdown, either alone or in combination with radiation therapy, effectively suppressed the growth of xenograft tumors. These findings highlight the potential of targeting RFC4 to overcome radioresistance by modulating the DNA damage response in ESCC, offering promising therapeutic avenues for patients with ESCC.NEW & NOTEWORTHY Our research indicates that replication factor C4 (RFC4) plays a role in conferring radioresistance to esophageal squamous cell carcinoma (ESCC) by bolstering DNA damage repair, primarily through the inhibition of the p53 signaling pathway. This finding positions RFC4 as a promising therapeutic target for combating radioresistance in ESCC, although further research is required to fully comprehend its intricate role in the disease.

This study aimed to investigate the regulation of fibroblast phenotypes by mesenchymal stem cells (MSCs) delivering copper sulfide (CuS) nanoparticles (NPs) loaded with CDKN1A plasmids and their role in cartilage repair during osteoarthritis (OA). Single-cell RNA sequencing data from the GEO database were analyzed to identify subpopulations within the OA immune microenvironment. Quality control, filtering, principal component analysis (PCA) dimensionality reduction, and tSNE clustering were performed to obtain detailed cell subtypes. Pseudotime analysis was used to understand the developmental trajectory of fibroblasts, and GO/KEGG enrichment analyses highlighted biological processes related to fibroblast function. Transcriptomic data and WGCNA identified CDKN1A as a key regulatory gene. A biomimetic CuS@CDKN1A nanosystem was constructed and loaded into MSCs to create MSCs@CuS@CDKN1A. The characterization of this system confirmed its efficient cellular uptake by fibroblasts. In vitro experiments demonstrated that MSCs@CuS@CDKN1A significantly modulated fibroblast phenotypes and improved the structure, proliferation, reduced apoptosis, and enhanced migration of IL-1β-stimulated chondrocytes. In vivo, an OA mouse model was treated with intra-articular injections of MSCs@CuS@CDKN1A. Micro-CT scans revealed a significant reduction in osteophyte formation and improved joint space compared with control groups. Histological analysis, including H&E, Safranin O-Fast Green, and toluidine blue staining, confirmed improved cartilage integrity, whereas the International Osteoarthritis Research Society (OARSI) scoring indicated reduced disease severity. Immunofluorescence showed upregulated CDKN1A expression, decreased MMP13, and reduced α-SMA expression in fibroblast subtypes. Major organs exhibited no signs of toxicity, confirming the biocompatibility and safety of the treatment. These findings suggest that MSCs@CuS@CDKN1A can effectively regulate fibroblast activity and promote cartilage repair, providing a promising therapeutic strategy for OA treatment.NEW & NOTEWORTHY This study introduces MSCs@CuS@CDKN1A, a nanoengineered MSC platform that targets fibroblast phenotypes in osteoarthritis (OA). By modulating CDKN1A expression, this innovative approach not only enhances cartilage repair but also effectively mitigates fibroblast-driven inflammation, marking a significant advancement in OA therapeutics with demonstrated efficacy and biocompatibility.

Plasma membrane repair (PMR) restores membrane integrity of cells, preventing cell death in vital organs, and has been studied extensively in skeletal muscle. Dysferlin, a sarcolemmal Ca²⁺-binding protein, plays a crucial role in PMR in skeletal muscle. Previous studies have suggested that PMR uses membrane trafficking and membrane fusion, similar to neurotransmission. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate membrane fusion in neurotransmission with the help of synaptotagmin, a crucial Ca²⁺-binding protein. Interestingly, dysferlin shares structural similarity with synaptotagmin and was shown to promote SNARE-mediated membrane fusion in a liposome-based assay. However, whether dysferlin facilitates SNARE-mediated membrane fusion in PMR in muscle cells remains unclear. In this study, we aimed to test if SNARE-mediated PMR requires dysferlin in muscle cells with pharmacological and genetic approaches. TAT-NSF700, which disrupts the disassembly of SNARE complexes, was used to disrupt functions of SNAREs in muscle cells. We found that human-induced pluripotent stem cells-derived cardiomyocytes (hiPS-CMs) treated with TAT-NSF700 showed a higher loss of membrane integrity after repetitive mechanical strains. Moreover, laser-wounded mouse flexor digitorum brevis (FDB) fibers treated with TAT-NSF700 showed an increased Ca²⁺ influx, but a decreased FM1-43 uptake, which depends on dynamin-regulated endocytosis as we previously showed in FDB fibers. Importantly, overexpression of STX4-mCitrine or eGFP-SNAP23 decreased Ca²⁺ influx in laser-wounded FDB fibers. Furthermore, overexpression of STX4-mCitrine also decreased Ca²⁺ influx in laser-wounded dysferlin-deficient FDB fibers. Overall, these results suggest that disassembly of SNARE complexes is required for efficient PMR and STX4-enhanced PMR does not require dysferlin in skeletal muscle.NEW & NOTEWORTHY Dysferlin, a crucial Ca²⁺-binding protein in plasma membrane repair (PMR), shares homology with synaptotagmin, which binds Ca²⁺ and regulates SNARE-mediated vesicle fusion in neurons. Dysferlin was thus hypothesized to function as synaptotagmin in PMR. We demonstrate here that the activity of SNAREs is important for PMR, and overexpression of STX4 enhances PMR in both intact and dysferlin-deficient skeletal muscle. These data suggest that SNARE-mediated PMR may be independent of dysferlin in skeletal muscle.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder leading to end-stage renal disease. ADPKD arises from mutations in the PKD1 and PKD2 genes, which encode polycystin 1 (PC1) and polycystin 2 (PC2), respectively. PC2 is a nonselective cation channel, and disease-linked mutations disrupt normal cellular processes, including signaling and fluid secretion. In this study, we investigate whether disease-causing missense mutations compromise PC2 folding, an event that can lead to endoplasmic reticulum-associated degradation (ERAD). To this end, we first developed a new yeast PC2 expression system. We show that the yeast system provides a tractable model to investigate PC2 biogenesis and that a disease-associated PC2 mutant, D511V, exhibits increased polyubiquitination and accelerated proteasome-dependent degradation compared with wild-type PC2. In contrast to wild-type PC2, the PC2 D511V variant also failed to improve the growth of yeast strains that lack endogenous potassium transporters, highlighting a loss of channel function at the cell surface and a new assay for loss-of-function PKD2 variants. In HEK293 cells, both D511V along with another disease-associated mutant, R322Q, were targeted for ERAD. Consistent with defects in protein folding, the surface localization of these PC2 variants was increased by incubation at low-temperature in HEK293 cells, underscoring the potential to pharmacologically rescue these and perhaps other misfolded PC2 alleles. Together, our study supports the hypothesis that select PC2 missense variants are degraded by ERAD, the potential for screening PKD2 alleles in a new genetic system, and the possibility that chemical chaperone-based therapeutic interventions might be used to treat ADPKD.NEW & NOTEWORTHY This study indicates that select missense mutations in PC2, a protein that when mutated leads to ADPKD, result in protein misfolding and degradation via the ERAD pathway. Our work leveraged a new yeast model and an HEK293 cell model to discover the mechanism underlying PC2 instability and demonstrates the potential for pharmacological rescue. We also suggest that targeting the protein misfolding phenotype with chemical chaperones may offer new therapeutic strategies to manage ADPKD-related protein dysfunction.

CD147 has the potential to serve as a specific target with therapeutic characteristics in several respiratory diseases. Studies have demonstrated that CD147 regulates levels of oxidative phosphorylation (OXPHOS) through the process of mitochondrial translocations. However, there is still limited insight in the distinct mechanism of CD147 in asthmatic macrophages. Here, we found that CD147 expression levels increased significantly both in vivo and in vitro. CD147 undergoes mitochondrial translocation in M2 macrophages. Reducing the expression of CD147 resulted in a decline in M2 polarization levels within macrophages, as well as a decrease in the levels of mitochondrial respiratory chain complexes I, II, and IV proteins. This effect may be attained by interacting with adenine nucleotide translocase 1 (ANT1), subsequently impacting the levels of mitophagy. We also discovered that CD147 knockdown significantly reduced airway remodeling and inflammation in addition to lowering the polarization level of M2 in the lung tissues of chronic asthmatic model mice. The findings represent the first evidence of the distinct function of CD147 in the process of airway remodeling in asthma.NEW & NOTEWORTHY The interaction between CD147 and ANT1 in M2 macrophages occurs via mitochondrial translocation, resulting in alterations in ANT1 expression levels. This, in turn, triggers the activation of the mitophagy pathway, leading to modifications in OXPHOS levels. Ultimately, these changes contribute to the enhancement of M2 polarization, thereby exacerbating airway remodeling in asthma.

Cellular senescence has been implicated in the aging-related dysfunction of satellite cells, the resident muscle stem cell population primarily responsible for the repair of muscle fibers. Despite being in a state of permanent cell cycle arrest, these cells remain metabolically active and release an abundance of factors that can have detrimental effects on the cellular microenvironment. This phenomenon is known as the senescence-associated secretory phenotype (SASP), and its metabolic profile is poorly characterized in senescent muscle. In the present investigation, we examined the intracellular and extracellular metabolome of C₂C₁₂ myoblasts using a bleomycin (BLEO)-mediated model of DNA damage-induced senescence. We also evaluated the relationship between the senescent metabolic phenotype and SASP signaling through molecular and network-based analyses. Senescent myoblasts exhibited a significantly altered extracellular metabolome (i.e., exometabolome), including increased secretion of several aging-associated metabolites. Four of these metabolites-trimethylamine-N-oxide (TMAO), xanthine, choline, and oleic acid-were selected for individual dose-response experiments to determine whether they could drive the senescence phenotype. Although most of the tested metabolites did not independently alter senescence markers, oleic acid treatment of healthy myoblasts significantly upregulated the SASP genes Ccl2, Cxcl12, and Il33 (p < 0.05). A gene-metabolite interaction network further revealed that oleic acid was one of the most interconnected metabolites to key senescence-associated genes. Notably, oleic acid interacted with several prominent SASP genes, suggesting a potential epigenetic effect between this monounsaturated fatty acid and SASP regulation. In summary, the exometabolome, particularly oleic acid, is implicated in SASP signaling within senescent myoblasts.NEW & NOTEWORTHY Cellular senescence and its accompanying secretory phenotype [i.e., the senescence-associated secretory phenotype (SASP)] have been linked to the aging-associated dysfunction of skeletal muscle, yet little is known about this phenomenon in satellite cells. We report that senescent myoblasts experience a significantly altered extracellular metabolome primarily characterized by the substantial release of nonesterified fatty acids. Targeted evaluation of several extracellular senescence-associated metabolites reveals a potential epigenetic role for long-chain fatty acids, particularly oleic acid, in regulating SASP-related gene expression.

It has been proposed that bone marrow contributes to the pathogenesis of arteriosclerosis. Nerve growth factor receptor (NGFR) is expressed in bone marrow stromal cells; it is also present in peripheral blood and ischemic coronary arteries. We hypothesized that bone marrow-derived NGFR-positive (NGFR⁺) cells regulate arterial remodeling. We found that human NGFR⁺ mononuclear cells (MNCs) in peripheral blood expressed markers for plasmacytoid dendritic cells (DCs) and were susceptible to apoptosis in response to proNGF secreted by activated arterial smooth muscle cells (SMCs). Bone marrow-specific depletion of NGFR⁺ cells increased neointimal formation following arterial ligation in mice. Bone marrow-derived NGFR⁺ cells accumulated in the neointima and underwent apoptosis. In contrast, in a bone marrow-specific NGFR-knockout model, SMCs occupied the neointima with augmented proliferation. NGFR⁺ cells in the neointima promoted mannose receptor C-type 1-positive anti-inflammatory macrophage accumulation and secreted anti-inflammatory IL-10, thereby inhibiting SMC proliferation in the neointima. In patients with acute coronary syndrome (ACS), NGFR⁺ peripheral MNCs increased after ACS onset. Multiple linear regression analysis showed that an insufficient increase in NGFR⁺ peripheral MNCs in ACS was an adjusted independent risk factor for 9-mo intimal progression of a nontargeted lesion. Taken together, these observations imply that bone marrow-derived NGFR⁺ DCs are suppressors of arteriosclerosis.NEW & NOTEWORTHY We propose a new concept of arterial remodeling after injury in which bone marrow-derived NGFR⁺ dendritic cells inhibit neointimal progression mediated by apoptosis. NGFR⁺ dendritic cells promote anti-inflammatory MRC1⁺ M2 macrophage accumulation and production of interleukin-10, inhibiting smooth muscle cell proliferation within the neointima. In a clinical study, insufficient mobilization of NGFR⁺ peripheral mononuclear cells in acute coronary syndrome was an independent risk factor for 9-mo nontargeted coronary intimal progression.

People with type 2 diabetes (T2D) have a greater risk of developing neurodegenerative diseases, like Alzheimer's disease, in later life. Exogenous ketone supplements containing the ketone body β-hydroxybutyrate (β-OHB) may be a strategy to protect the brain as β-OHB can support cerebral metabolism and promote neuronal plasticity via expression of brain-derived neurotrophic factor (BDNF). Parallel human (ClinicalTrials.gov ID NCT04194450, ClinicalTrials.gov ID NCT05155410) and rodent trials were conducted to characterize the effect of acute and short-term exogenous ketone supplementation on indices of brain health. First, we aimed to investigate the effect of acute and short-term supplementation of exogenous ketone monoester on circulating BDNF and cognition in adults with T2D. There were no effects of ketone supplementation on plasma BDNF or cognition. Second, we aimed to investigate the mechanistic effects of acute and chronic β-OHB supplementation on cortical BDNF content and recognition memory in C57BL/6J mice with and without insulin resistance. Acutely, β-OHB did not alter recognition memory or BDNF content. Similarly, chronic β-OHB supplementation did not alter recognition memory or BDNF content. Collectively, our data demonstrates that ketone supplementation does not elevate BDNF content in humans or mice. Furthermore, our data does not support the involvement of BDNF in the potential cognitive benefits of β-OHB supplementation.NEW & NOTEWORTHY Ketone supplementation does not alter circulating BDNF levels or cognition in humans with T2D. Acute and chronic ketone supplementation in C57BL/6J mice did not change BDNF protein content or improve recognition memory. Ketone supplementation in C57BL/6J mice positively modulated β-site amyloid precursor protein cleaving enzyme 1 (BACE1) activity, providing a potential future therapeutic strategy.

Ubiquitin-specific protease 35 (USP35) was found to be involved in various tumor progression, but its role in breast cancer remains largely unknown. USP35 mRNA and protein expression in breast cancer tissues and cells were evaluated by quantitative real-time PCR and Western blot, respectively. Subsequently, flow cytometry and 5-ethynyl-2'-deoxyuridine labeling were used to evaluate breast cancer cell apoptosis and proliferation. Cellular glycolytic function was analyzed using the Seahorse assay and various kits. Furthermore, co-immunoprecipitation (Co-IP) and immunoprecipitation assays were utilized to validate the deubiquitylation mechanism of USP35. Finally, a subcutaneous human xenograft tumor model was established in nude mice to verify the effect of USP35 in vivo. By examining the clinical samples and cell lines, we found that USP35 expression was significantly upregulated in breast cancer. Further functional studies showed that knockdown USP35 expression inhibited cell proliferation and promoted apoptosis. In addition, knockdown of USP35 decreased phosphofructokinase1 (PFK-1) expression and was associated with lower extracellular acidification rate and oxygen consumption rate compared with sh-Control. Co-IP assays identified PFK-1 as a direct deubiquitiation target of USP35. Importantly, we demonstrated that PFK-1 is an essential mediator for USP35-induced cell proliferation and glycolysis in vitro and in vivo. This study identified that USP35 regulates the proliferation and glycolysis of breast cancer cells by mediating the ubiquitination level of PFK-1. The USP35/PFK-1 axis offers novel insight for the treatment of breast cancer.NEW & NOTEWORTHY This study identified that USP35 regulates the proliferation and glycolysis of breast cancer cells by mediating the ubiquitination level of PFK-1. The USP35/PFK-1 axis offers novel insight for the treatment of breast cancer.