Cellular and Molecular Life Sciences最新文献

Objective: Immunosuppressive M2 macrophages in the tumor microenvironment (TME) limit the efficacy of immune checkpoint inhibitors (ICIs), with only ∼20% of lung adenocarcinoma (LUAD) patients benefiting from ICI monotherapy. Targeting macrophage polarization represents a promising strategy to reprogram the TME and enhance antitumor immunity.

Methods: Integrated bioinformatics analysis of TCGA and GEO datasets elucidated the role of IQGAP3 in LUAD progression. A tissue microarray was utilized to compare IQGAP3 expression between tumor and adjacent normal tissues and to evaluate the infiltration relationship between IQGAP3⁺ macrophages and CD8 + T cells. Conditioned medium (CM) from IQGAP3-knockdown macrophage was applied to evaluate its impact on LUAD cell malignancy. Subcutaneous xenograft models tested the impact of IQGAP3 targeting on tumor growth and anti-PD-1 synergy.

Results: IQGAP3 was upregulated in LUAD and correlated with poor survival and reduced ICI benefit. IQGAP3⁺ macrophage infiltration inversely correlated with CD8 + T-cell abundance. CM from macrophage with IQGAP3 knockdown resulted in reduced proliferation, migration and invasion of LUAD cells. In macrophage-cancer cell and macrophage-CD8 + T cell coculture systems, IQGAP3 suppression enhanced macrophage phagocytosis and promoted T cell activation. Mechanistically, IQGAP3 regulates macrophage polarization through binding and sequestering IGF2BP2 at the cell periphery, thereby limiting IGF2BP2-mediated stabilization of FYN mRNA. This reduction in FYN expression led to decreased STAT1 phosphorylation. Targeting IQGAP3 reprogrammed TAMs toward an antitumor phenotype, suppressed tumor growth, and synergized with anti-PD-1 therapy.

Conclusion: IQGAP3 drives immunosuppressive macrophage polarization in LUAD. Its inhibition represents a novel strategy to improve immunotherapy response.

There is growing demand for improved in vitro liver models to better predict in vivo pharmacology, specifically drug disposition mediated by hepatic transporters and assessment of transporter-mediated drug interaction risk. While 2D sandwich-cultured human hepatocytes (SCHH) remain valuable, they are limited to short-term use due to hepatocyte de-differentiation and absence of non-parenchymal cells. Multicellular hepatic spheroids (MHS) offer a promising alternative, but transporter concentrations, functionality, and suitability for hepatobiliary transport studies remain unclear. We evaluated an all-human MHS model, comprised of transporter-certified™ cryopreserved primary human hepatocytes (PHH), Kupffer, stellate, and endothelial cells, for long-term hepatic transporter assessment. Over a 21-day culture period, we monitored transporter concentrations (targeted proteomics), regulation (RNA-seq), localization (immunofluorescence), bile acid profiles (LC-MS/MS), and functional transport (B-CLEAR^®). This is the first report of protein concentrations of 13 transporters in MHS over 21 days directly compared to freshly thawed PHH and SCHH from the same donor. Most transporters declined in MHS compared to PHH, while SCHH maintained or increased transporter concentrations by day 5. However, multidrug resistance-associated protein (MRP) 4 and organic solute transporter (OST)-α/β were upregulated in MHS, likely reflecting adaptation to bile acid accumulation. Bile acid profiling confirmed functional synthesis, metabolism and excretion. Functional MRP2 efflux into sealed canalicular compartments was demonstrated with the MRP2 substrate, 5(6)-carboxy-2',7'-dichlorofluorescein (CDF). Tight junction disruption of canaliculi with Ca²⁺-free buffer resulted in CDF release from canalicular compartments, with partial entrapment within MHS, likely due to the 3D architecture. These findings highlight key strengths and limitations of MHS as a model for assessing hepatobiliary transport.

Propofol is a commonly used anesthetic for sedation during surgery. This drug is reported to exhibit nonanaesthetic immunomodulatory and anti-inflammatory effects. Herein, we investigated the impact of topical propofol delivery with the aim of mitigating psoriatic inflammation. The antipsoriatic potency of propofol was evaluated in a cell-based study in which keratinocytes, macrophages, and neutrophils were used as models. A significant reduction in the proinflammatory effectors interleukin (IL)-6, IL-8, and CXC motif chemokine ligand (CXCL)1 was found in activated keratinocytes (HaCaT) treated with propofol. This reduction could enable baseline control. Immunoblotting suggested that the antioxidant enzymes nuclear factor erythroid 2-related factor (Nrf)2 and heme oxygenase (HO)-1 were involved in the protective effect of propofol on keratinocyte stimulation. The increase in Nrf2 and HO-1 was mediated by kelch-like ECH-associated protein (KEAP)1 downregulation. Propofol presented scavenging activity and decreased 2,2-diphenyl-1-picrylhydrazyl (DPPH) by 47%. The downregulation of cytokines/chemokines in activated macrophages (differentiated THP-1) and mouse neutrophils was also found after propofol treatment. Macrophage migration triggered by the conditioned medium of activated keratinocytes could be blocked with the intervention of propofol. The absorption level of propofol (3 mM) into intact pig skin was 1.2 nmol/mg. Skin deposition was increased to 3.7 nmol/mg after SC lipid removal to mimic psoriasiform skin. In silico molecular docking demonstrated the facile interaction of propofol with ceramides in the stratum corneum (SC). The treatment of imiquimod (IMQ)-sensitized mice with topical propofol suppressed erythema, acanthosis, and macrophage/neutrophil infiltration. Propofol also dramatically decreased cytokine/chemokine levels and epidermal thickness in the lesion. In summary, propofol exhibits anti-inflammatory and antioxidant properties to treat psoriasiform lesions. Topical propofol delivery is useful as an ideal route to accomplish antipsoriatic therapy and avoid systemic effects.

UFMylation, similar to ubiquitination, is a unique post-translational modification which is indispensable in hematopoiesis, neurogenesis and chondrogenesis. However, its role in intervertebral disc development remains unclear. In this study, we focused on DDRGK domain containing protein 1 (DDRGK1), a pivotal component involved in UFMylation, and generated Ddrgk1^fl/fl; Acan-CreER^T2 (Ddrgk1^cKO) mice to explore DDRGK1's regulatory function in the nucleus pulposus and cartilage endplate. We found that Ddrgk1 conditional knockout led to severe retardation of spinal growth, disruption of disc cellularity and initiation of disc degeneration during early postnatal phase. Furthermore, Ddrgk1 conditional knockout in late postnatal phase resulted in profound degeneration of mouse discs, mainly characterized by substantially reduced thickness of cartilage endplate. In addition, Ddrgk1^cKO mice exhibited exacerbated disc degeneration compared to the WT mice after the lumbar spine instability surgery. RNA sequencing of disc cells from Ddrgk1^cKO mice showed upregulation of genes related to apoptosis, matrix metalloproteinase activation, extracellular matrix (ECM) degradation and endoplasmic reticulum (ER) unfolded protein response after Ddrgk1 conditional knockout. Immunohistochemical analysis further verified increased apoptosis, ECM disruption and ER stress in both nucleus pulposus and cartilage endplate after Ddrgk1 conditional knockout. In summary, this study demonstrated that DDRGK1 preserves the normal cellularity and structure of intervertebral discs by regulating the cell fate of nucleus pulposus and cartilage endplate cells, maintaining the ER homeostasis and regulating the metabolic balance of ECM.

Background: Sepsis-induced acute lung injury (ALI) is characterized by excessive inflammation and pyroptosis of lung epithelial cells. AU-rich element binding factor 1 (AUF1) is a key RNA-binding protein involved in mRNA decay and regulation of inflammatory responses. Understanding AUF1's role in ALI could reveal novel therapeutic targets.

Methods: We employed both in vivo (cecal ligation and puncture, CLP) and in vitro (lipopolysaccharide, LPS-treated cells) models of sepsis-induced ALI. Survival rates, lung histopathology, and expression levels of ZDHHC21, STING palmitoylation, and pyroptosis markers (c-Caspase-1, GSDMD-N, IL-18, IL-1β) were assessed. Knockdown and overexpression experiments for ZDHHC21, ETS2, and AUF1 were conducted. RNA immunoprecipitation (RIP) and RNA pulldown assays evaluated AUF1 binding to ETS2 mRNA, while mRNA decay was analyzed using actinomycin D.

Results: In ALI models, ZDHHC21 expression, STING palmitoylation, and pyroptosis markers were significantly increased. Inhibition of NLRP3/AIM2 improved survival and reduced lung injury without affecting ZDHHC21 levels. Knockdown of ZDHHC21 reduced STING palmitoylation and pyroptosis. ETS2 was upregulated in ALI and directly activated ZDHHC21 transcription. AUF1 expression was downregulated in ALI, leading to decreased decay of ETS2 mRNA and elevated ETS2/ZDHHC21/STING axis activity. Overexpression of AUF1 reversed these changes, reducing STING palmitoylation and pyroptosis. AUF1 directly bound and promoted the decay of ETS2 mRNA.

Conclusion: AUF1 mitigates sepsis-induced ALI by promoting ETS2 mRNA decay via the ETS2/ZDHHC21 axis, thereby reducing STING palmitoylation and pyroptosis in lung epithelial cells. Targeting the AUF1/ETS2/ZDHHC21/STING pathway offers a promising therapeutic strategy for improving outcomes in sepsis-induced ALI. Future studies should validate these findings in human samples and explore upstream regulators of AUF1.

Since the first report in 1972, several studies have documented an association between Parkinson's disease (PD) and melanoma. Up to 20-fold increased risk of melanoma was reported in PD patients, while a personal/family history of melanoma was linked to a 1.85-fold PD risk. Neurons and melanocytes, which both derive from the neuroectodermal crest, share biological pathways that may be dysregulated in both diseases. In particular, accumulation of the alpha-synuclein (α-syn, SNCA) protein, a pathological hallmark of PD, is also observed in melanoma. Indeed, dysregulated α-syn proteostasis is known to disrupt several biological pathways which can co-incidentally, albeit paradoxically contribute to both neurodegeneration and hyper-proliferative cell growth. These include abnormalities in dopamine (DA), melanin, and iron metabolism, oxidative stress, DNA damage/repair response, inflammation, as well as alterations in mitochondrial function, and cell-clearing machinery. Although α-syn depletion was shown to attenuate melanoma cell proliferation and neurodegeneration, it remains unclear whether α-syn accumulation is a mere culprit of disease, if it represents a common outcome from shared upstream mechanisms, or, finally, a compensatory response to cellular stress. In an effort to elucidate how α-syn bridges melanomagenesis and the neurodegenerative events of PD, this review discusses specific cellular and molecular pathways related to α-syn proteostasis, including environmental factors implicated in melanocytic transformation, such as UV radiation. Addressing open questions and establishing novel experimental models remain essential for developing effective therapeutic approaches to target melanoma and PD without overlooking their comorbidity.

Background: Ischemia/reperfusion injury (I/RI) impedes the progress of flap and allograft transplantation. Among various strategies to address oxidative stress (OS) and mitochondrial dysfunction associated with I/RI, exosomes derived from adipose-derived stem cells (ADSCs) subjected to hypoxia pretreatment show significant therapeutic potential.

Methods and results: This study assessed the effects of ADSC-derived exosomes (ADSC-Ex) from normoxic and hypoxic conditions on reactive oxygen species (ROS), mitochondrial calcium ion (Ca²⁺) influx, mitochondrial potential, and cell apoptosis in an ischemia/reperfusion (I/R) model. Mass spectrometry (MS) was utilized to analyze differentially expressed proteins in hypoxic ADSC-Ex compared to normoxic controls. The functions of significantly upregulated proteins were investigated through knockdown experiments in hypoxic ADSC-Ex on alleviating I/R injury (I/RI) in HUVECs. Hypoxic ADSC-Ex significantly mitigated I/RI in vascular endothelial cells both in vitro and in vivo. This effect was associated with reduced ROS and mitochondrial Ca²⁺ influx, and protection of mitochondrial potential. MS identified several proteins that were significantly upregulated in hypoxic ADSC-Ex, with Vesicle-associated membrane protein 4 (VAMP4) emerging as a pivotal molecule involved in alleviating I/RI in vascular endothelial cells.

Conclusion: This study demonstrated that hypoxic ADSC-Ex reduced ROS and mitochondrial dysfunction in vascular endothelial cells through VAMP4, thereby attenuating I/RI. This finding might provide a new approach for treating post-transplant I/RI.

Advances in assisted reproductive technology (ART) have expanded treatment options for many infertile families. However, outcomes in women of advanced maternal age remain suboptimal, primarily due to fewer retrievable oocytes and diminished embryonic developmental competence. In this study, we show that adding an appropriate concentration of amphiregulin (AREG) to the embryo culture medium significantly enhances the developmental competence of embryos from aged mice. Mechanistically, AREG binds to the epidermal growth factor receptor and induces receptor phosphorylation, thereby upregulating Ras like protein REM2. REM2 suppresses voltage gated calcium channel-mediated Ca²⁺ influx, reducing the elevated Ca²⁺ levels observed in aged embryos relative to young embryos and maintaining ATP supply. The improvement in ATP levels, in turn, restrains the excessive expression of LDHA in aged embryos, thereby decreasing pyruvate to lactate conversion and global protein lactylation, ultimately shifting embryonic physiology toward a more youthful state. These findings support supplementation with AREG during embryo culture as a rational strategy to improve ART outcomes in women of advanced maternal age.

Epitranscriptomics modifications play an important role in sex-dependent biological phenomena. N6-adenosine methylation (m6A), the most prevalent epitranscriptomics modification in eukaryotic mRNA, participates in regulating various sex-specific physiological processes. Here, we generated METTL4 knockout mice lacking methyltransferase-like 4, which mediates m6A. Behavioral analyses revealed that only female METTL4^-/- mice exhibited pain hypersensitivity, with subsequent experiments showing the involvement of METTL4-mediated m6A in this sex-differentiated biological phenotype. Further exploration demonstrated that this sex-specific pain hypersensitivity is closely associated with sex-dependent expression of uncoupling protein 2 (UCP2) in synapses. Specifically, elevated UCP2 expression in METTL4^-/- female mice enhances the efficiency of synaptic transmission by modulating mitochondrial energy metabolism at synapses. Collectively, this study identifies a distinct pathway mediated by METTL4-driven m6A modification, providing critical insights into the molecular basis of sex-specific differences in pain transmission. These findings also highlight the potential of targeting METTL4 for sex-differentiated pain management strategies in clinical settings.

The accumulation of HIFs regulated by the PHD-pVHL pathway represents the classical mechanism that transcriptionally mediates cellular adaptation to hypoxia. Extensive hypoxic stress activates cell death. Comprehensive understanding the mechanisms of hypoxia-induced cell death is essential for treatment of several diseases. Here, we revealed that β-TrCP1 degradation is essential for hypoxia-induced cell death and tissue injury. Hypoxia promotes β-TrCP1 degradation via proteasome pathway in HIFs-independent manner, and SMURF2 is identified as the corresponding E3 ligase. Additionally, acetylation of β-TrCP1 decreases after hypoxia, which is required for β-TrCP1 degradation. Tip60 establishes the acetylation of β-TrCP1 under normoxic conditions and is prolyl-hydroxylated by PHD2. Prolyl Hydroxylation stabilizes Tip60 under normoxic conditions, while hypoxia promotes the degradation of Tip60 by decreasing its prolyl hydroxylation. HDAC8 catalyses the deacetylation of β-TrCP1, which is enhanced after hypoxia. Loss of β-TrCP1 acetylation after hypoxia promotes the binding of SMURF2 to β-TrCP1 and its degradation. p53 is a substrate of β-TrCP1, and loss of β-TrCP1 upon hypoxia results in the accumulation of p53, which is responsible for hypoxia-induced cell death and tissue injury. Thus, this study illustrates a previously unappreciated posttranscriptional hypoxia-responsive mechanism constituted by PHD2-Tip60-HDAC8-SMURF2-β-TrCP1 degradation axis to promote p53 accumulation to mediate cell death and tissue injury.