American journal of physiology. Cell physiology最新文献

Cancer cachexia is the involuntary loss of body and skeletal muscle mass, which negatively impacts physical function, quality of life, treatment tolerance, and survival. Skeletal muscles of cachectic people and mice with pancreatic tumors also exhibit skeletal muscle damage, non-resolute immune cell infiltration, and impaired regeneration. These phenotypes may be influenced by the accumulation of senescent cells, which secrete factors detrimental to skeletal muscle health. However, there is currently no comprehensive research on senescent cell accumulation in skeletal muscle of tumor-bearing hosts, with or without chemotherapy. To address this gap, we cross-referenced the SenMayo panel of 125 senescence-related genes with our RNA-seq dataset in mouse skeletal muscle during the initiation and progression of cancer cachexia, which revealed a differential expression of 39 genes at pre-cachexia, 64 genes at cachexia onset, and 72 genes when cachexia is severe. Since p16 is a canonical marker of senescence, we subsequently orthotopically injected p16-tdTomato reporter mice with murine KPC pancreatic cancer cells and treated a subset of mice with chemotherapy. At experimental endpoint, when KPC treatment-naïve mice were cachectic, we observed an increased accumulation of p16+ cells, along with increased mRNA levels of hallmark senescence markers (Cdkn1a/p21, Cdkn2a/p16, Glb1/senescent-associated-β-galactosidase), which were exacerbated by chemotherapy. Lastly, we demonstrate an increase in CDKN1A/p21 in the muscle of cachectic patients with pancreatic cancer, which associated with cachexia severity. These findings suggest that senescent cells accumulate in skeletal muscle of cachectic pancreatic tumor-bearing hosts and that chemotherapy can exacerbate this accumulation.

The Calcium Homeostasis Modulator (CALHM) proteins comprise a family of six genes, some of which have been demonstrated to function as ion channels. CALHM1, the founding member, is an extracellular Ca²⁺- and voltage-gated large-pore non-selective ion channel. The mechanisms by which Ca²⁺ and voltage regulate CALHM1 channel gating are unknown. Cryo-electron microscopic structures of CALHM1 and its paralogs have provided little insights into these features, although they have suggested that the amino-termini, including an amino-terminal helix (NTH) and the first transmembrane helix (TM1) may possess significant flexibility. Here we investigated the role of the amino terminus in gating regulation of human CALHM1 channels expressed in Xenopus oocytes. Deletion of the NTH and the proximal end of TM1 markedly reduced the voltage-dependence of channel gating, whereas extracellular Ca²⁺ retained ability to close the channel, indicating that the amino-terminus is not the Ca²⁺-regulated gate. Furthermore, inhibition of channel currents by ruthenium red was independent of the presence of the amino terminus and was mediated by effects on channel gating rather than pore block. Introduction of a cysteine residue into the proximal end of TM1 enabled complete inhibition of the channel by a crosslinking reagent under conditions in which the channel was in a closed state. Our findings indicate that while the NTH plays a role in voltage-dependent gating, it does not act as the gate itself. Instead, our results suggest that the gate in CALHM1 is formed by proximal regions of the first transmembrane domain.

Enhanced endothelial sodium channel (EnNaC) functioning causes an increase in vessel stiffness. Here, we investigated the regulation of EnNaC in mouse aortic endothelial cells (mAoEC) by the actin cytoskeleton and lipid raft association protein myristoylated alanine-rich C-kinase substrate like protein 1 (MLP1). We hypothesized that mutation of specific amino acid residues within the effector domain of MLP1 or loss of association between MLP1 and the anionic phospholipid phosphate PIP2 would significantly alter membrane association and EnNaC activity in mAoEC. mAoEC transiently transfected with a mutant MLP1 construct (three serine residues in the effector domain replaced with aspartate residues) showed a significant decrease in EnNaC activity compared to cells transfected with wildtype MLP1. Compared to vehicle treatment, mAoEC treated with the PIP2 synthesis blocker wortmannin showed less colocalization of EnNaC and MLP1. In other experiments, Western blot and densitometric analysis showed a significant decrease in MLP1 and caveloin-1 protein expression in mAoEC treated with wortmannin compared to vehicle. Finally, wortmannin treatment decreased sphingomyelin content and increased membrane fluidity in mAoEC. Taken together, these results suggest that constitutive phosphorylation of MLP1 attenuates the function of EnNaC in aortic endothelial cells by a mechanism involving a decrease in association with MLP1 and EnNaC at the membrane, while deletion of PIP2 decreases MLP1 expression and overall membrane fluidity.

We aimed to explore novel pathogenesis in young AR children, and thus finding novel nasal spray reagents for them, especially under 4 years old. In this study, non-targeted metabolomics analyses were employed to explore the differential metabolites in NALF of AR children. CCK-8 and flow cytometry were employed to assess cell proliferation and apoptosis in HNEpCs. HNEpCs were co-cultured with CD4⁺T cells, and flow cytometry was employed to detect Th17/Treg cells. RNA sequencing was employed to assess the key pathways in xanthine-treated Jurkat T cells. Finally, both the in vitro and in vivo experiments were employed to assess the effect of DPCPX (Adora1 inhibitor) on ATF4 expression and Th17/Treg cells. Xanthine and uric acid levels were increased in NALF of AR children. XDH, PNP, xanthine/hypoxanthine, and uric acid levels were elevated in Derp1-treated HNEpCs, and si-XDH reversed the reduced cell viability and increased cell apoptosis in Derp1-treated HNEpCs. Both xanthine and Derp1-treated HNEpCs increased Th17/Treg ratio. ERS pathway was affected in xanthine-treated Jurkat T cells, and ATF4 was markedly reduced in xanthine-treated Jurkat T cells. Xanthine exhibited no effect on Adora1 expression, while DPCPX elevated ATF4 expression and reduced Th17/Treg ratio in xanthine-treated Jurkat T cells. The in vitro experiments revealed that DPCPX reduced inflammatory infiltration, Th17/Treg ratio, IL-17, TNF-α, and IL-6 in AR mice. These results demonstrated that xanthine inhibited ATF4 expression via Adora1 to elevate Th17/Treg ratio in nasal cavity, thus participating in AR progression. These findings may provide novel therapeutic interventions for young AR children.

Heparan sulfate is a highly sulfated and structurally heterogeneous polysaccharide that plays key roles in numerous biological processes. Due to its complex structure and variable sulfation patterns, accurately characterizing and quantifying HS in biological samples poses significant analytical challenges. This review presents an advanced LC-MS/MS methodology that utilizes isotope-labeled internal standards for the precise quantification of HS disaccharides and rare 3-O-sulfated tetrasaccharides, alongside monitoring 6-O-endosulfatase enzyme activity and the metabolism of synthetic HS oligosaccharides in biological systems. The combination of isotope-labeled standards with LC-MS/MS technology provides a powerful and sensitive approach for comprehensive analysis of HS modifications, offering valuable insights into HS metabolism and its alterations across various biological contexts.

Arterial stiffening is a hallmark of chronic kidney disease (CKD)-related cardiovascular events and is primarily attributed to the elevated matrix stiffness. Stiffened arteries are accompanied by low-grade inflammation, but the causal effects of matrix stiffness on inflammation remain unknown. For analysis of the relationship between arterial stiffness and vascular inflammation, pulse-wave velocity (PWV) and aortic inflammatory markers were analyzed in an adenine-induced mouse model of CKD in chronological order. Compared with their control littermates, mice with CKD showed elevated arterial stiffness at the early stage of disease progression, which preceded the onset of vascular inflammation. Correspondingly, the increase of matrix stiffness induced vascular smooth muscle cells (VSMCs) to transdifferentiate into an inflammatory phenotype, as indicated by the increased expression and secretion of MCP-1, IL-6, IL-1β, and IL-18. RNA-sequencing analysis of stiff matrix-cultured VSMCs and bioinformatics analysis with the ChIP-Atlas database revealed the potential involvement of the transcription factor Runx2. The expression and the nuclear localization of Runx2 were significantly increased in stiff matrix-cultured VSMCs. High-throughput ChIP-sequencing and promoter luciferase assays further revealed that NLRP3 was directly transcriptionally regulated by Runx2. The inhibition of Runx2 or NLRP3 inflammasome abrogated the proinflammatory effect of matrix stiffening on VSMCs. Together, these data revealed that arterial stiffness precedes vascular inflammatory responses in CKD mice and that the Runx2-NLRP3 axis orchestrates matrix stiffness and the VSMC inflammatory phenotype, which may contribute to the pathogenic role in arterial stiffness-related vascular inflammation and CKD-related cardiovascular complications.NEW & NOTEWORTHY As a hallmark of chronic kidney disease (CKD), arterial stiffening is related to increased vascular inflammation and cardiovascular morbidity, whereas the underlying mechanism is unclear. The study demonstrates that increased arterial stiffness precedes the onset of vascular inflammation, and matrix stiffness stimulates the transdifferentiation of vascular smooth muscle cells (VSMCs) to an inflammatory phenotype via activating Runx2-NLRP3 signaling, which provides novel insights into CKD-related cardiovascular disorder treatment.

Histone lactylation is crucial in a variety of physiopathological processes; however, the function and mechanism of histone lactylation in endometriosis remain poorly understood. Therefore, the objective of this investigation was to illuminate the function and mechanism of histone lactylation in endometriosis. Immunohistochemistry was used to investigate the expression of histone lactylation. Cell Counting Kit-8 assay (CCK8), Transwell assay, and endometriosis mouse models were used to investigate the effects of histone lactylation in vitro and in vivo. Transcriptomics and immunoprecipitation-mass spectrometry (IP-MS), Western blot, co-immunoprecipitation (Co-IP), quantitative reverse transcription polymerase chain reaction (qRT-PCR), and chromatin immunoprecipitation-qPCR (ChIP-qPCR) were used to explore the intrinsic mechanisms. In this study, we found that histone lactylation was upregulated in endometriosis and could promote endometriosis progression both in vivo and in vitro. Mechanistically, histone lactylation H3K18la promoted the transcription of Ras homolog enriched in striatum (RASD2), and RASD2, in turn, increased the stability of CTP synthase 1 (CTPS1) by promoting the SUMOylation and inhibiting the ubiquitination of CTPS1, thereby promoting endometriosis progression. Overall, our findings indicated that histone lactylation could promote the progression of endometriosis through the RASD2/CTPS1 axis. This investigation uncovered a novel mechanism and identified prospective targets for endometriosis diagnosis and therapy.NEW & NOTEWORTHY Our finding reveals a novel mechanism that promotes the progression of endometriosis, namely the histone lactylation/RASD2/CTPS1 axis. This finding suggests that inhibiting histone lactylation or inhibiting RASD2 and CTPS1 might be a potential therapeutic strategy to inhibit endometriosis lesion growth.

The epidermal growth factor receptor (EGFR) signaling pathway is an evolutionary conserved mechanism to control cell behavior during tissue development and homeostasis. Deregulation of this pathway has been associated with abnormal cell behavior, including hyperproliferation, senescence, and an inflammatory cell phenotype, thereby contributing to pathologies across a variety of organs, including the kidneys, skin, and lungs. To date, there are seven distinct EGFR ligands described. Although binding of these ligands to the receptor is cell type-specific and spatio-temporally controlled with distinct affinities and kinetics, epiregulin (EREG) stands out as a long-acting EGFR ligand that emerges under pathological conditions, particularly in tissue fibrosis. Although EREG has been extensively studied in cancer, its contribution to the maladaptive remodeling of tissue is elusive. The aim of this review is to highlight the role of EREG in skin, kidney, and lung fibrosis and to discuss opportunities for therapeutic intervention.

Intestinal microbiota are pathophysiologically involved in diabetic nephropathy (DN). Dapagliflozin, recognized for its blood glucose-lowering effect, has demonstrated efficacy in improving DN. However, the mechanisms beyond glycemic control that mediate the impact of dapagliflozin on DN remain unclear. Here, we investigated the effects of dapagliflozin on DN and gut microbiota, elucidating how it mitigates DN via the gut-kidney axis. Low-dose dapagliflozin markedly ameliorated renal inflammation and fibrosis and improved gut barrier function in high-fat diet (HFD)/streptozotocin (STZ)-induced DN mice and db/db mice without affecting blood glucose levels. These effects were associated with altered gut microbial composition and function. Eradication of the resident microbiota abolished the protective effects of dapagliflozin against kidney injury in DN mice. Moreover, dapagliflozin significantly altered microbial metabolites in DN mice, decreasing argininosuccinic acid (ASA) and palmitic acid (PA), while increasing S-allylcysteine (SAC) levels. ASA and PA increased the expression of renal inflammation- and fibrosis-related markers in HK-2 cells, whereas SAC ameliorated renal damage and altered the microbial composition in a manner similar to dapagliflozin in DN mice. Notably, Muribaculaceae and Desulfovibrionaceae were correlated with the alleviation of DN-associated renal dysfunction by low- and high-dose dapagliflozin treatments in DN mice. These findings demonstrate a potential application of dapagliflozin in managing DN by targeting the gut microbiota.NEW & NOTEWORTHY We demonstrated that dapagliflozin administration alleviated renal inflammation and fibrosis in vivo and in vitro, along with reshaping the gut microbiota composition and altering levels of key microbial metabolites, including argininosuccinic acid (ASA) and palmitic acid (PA), while increasing S-allylcysteine (SAC). Importantly, the genera Muribaculaceae and Desulfovibrionaceae emerged as pivotal microbial genera mediating the protective effects of dapagliflozin against diabetic nephropathy.

Thoracic aortic aneurysm (TAA) is a vascular disease associated with high mortality rates. Ferroptosis has been shown to mediate the transformation of vascular smooth muscle cells (VSMCs). However, the regulatory mechanisms by which ferroptosis influences TAA remain unclear. In this study, we induced TAA mouse models using angiotensin II (Ang II) and evaluated the impact of ferroptosis on the pathological changes observed in TAA mice, employing liproxstatin-1 as a treatment. In addition, we assessed the regulatory effect of METTL14 on the ferroptosis of VSMCs after treating them with a ferroptosis activator (imidazole ketone erastin, IKE). RNA binding protein immunoprecipitation (RIP) and RNA pull-down assays were conducted to investigate the interaction between acyl-CoA synthase long-chain family member 4 (ACSL4) mRNA and the proteins METTL14 or IGF2BP2. The results indicated that the level of ferroptosis was elevated in the thoracic aorta of TAA mice, and METTL14 was upregulated in TAA models and IKE-induced VSMCs. Knockdown of METTL14 was found to inhibit the progression of TAA by reducing the ferroptosis of VSMCs. Furthermore, IGF2BP2 recognized METTL14-modified ACSL4 mRNA and regulated its stability, thereby mediating the ferroptosis of VSMCs. Collectively, the effects of METTL14 on VSMC ferroptosis present therapeutic potential for the treatment of TAA.NEW & NOTEWORTHY Our study confirmed that METTL14 can induce ferroptosis in vascular smooth muscle cells during the progression of thoracic aortic aneurysm by mediating the m⁶A modification of ACSL4 mRNA.