Cellular and Molecular Life Sciences最新文献

Adult neurogenesis in the hippocampus, involving the generation and integration of new neurons, is essential for behavioral pattern separation, which supports accurate memory recall and cognitive plasticity. Here, we explore the role of the DNA repair protein NEIL3 in adult hippocampal neurogenesis and behavioral pattern separation. NEIL3 is required for efficient proliferation and neuronal differentiation of neonatal NSPCs and adult-born NPCs in the hippocampus following a behavioral pattern separation task. NEIL3-depleted mice exhibited a reduced preference for the novel object location, indicating a deficit in pattern separation. NEIL3-deficient adult-born neurons exhibited a significant reduction in mature-like membrane properties, indicating impaired functional maturation. Interestingly, these impairments were not associated with the decreased genomic integrity but with the altered transcriptional regulation of the Wnt signaling pathway. Given the importance of adult neurogenesis in cognitive function, targeting NEIL3 could offer therapeutic potential for addressing age-related hippocampal dysfunction and cognitive decline.

Background: Nuclear-cytoplasmic transport proteins (NCTPs) impact the transport of proteins and RNA molecules between the nucleus and cytoplasm in tumor cells, making them promising targets for cancer therapy. Currently, the molecular mechanism and function of Nuclear RNA export factor 3 (NXF3) in gastric cancer (GC) remains unclear.

Methods: We used Univariate Cox regression analysis and LASSO regression analysis, Receiver Operating Characteristic (ROC) curves to construct and evaluate a NCTP prognosis risk scoring model (NCTP model). Moreover, we identified the key NCTP (NXF3) affecting GC through differential expression and prognosis analysis. Subsequently, we introduced NXF3 shRNA into GC cells to investigate the impact of NXF3 on the cell proliferation, cell migration, invasion, and cell cycle and apoptosis and tumor growth by CCK-8 assay, transwell, wound healing assay, Flow cytometry, and nude mice subcutaneous tumor in vitro and in vivo. Furthermore, we investigated the key molecules influenced by NXF3 through piRNA-Seq, RNA-Seq, RIP-Seq, IP-MS, and Nuclear-cytoplasmic transcriptomics.

Results: We constructed a prognostic risk model related to 3 NCTPs, including NXF3, GLE1 and RANGAP. The NCTP model effectively predicts the prognosis of GC patients. The low-risk group exhibited a significantly higher overall survival rate than that of the high-risk group. Notably, NXF3 is identified as a crucial NCTP in GC, and its high expression is associated with poor prognosis of GC patients. Knocking down of NXF3 significantly inhibited the proliferation, invasion, migration, cell cycle, tumor growth and induced cell apoptosis of GC cells in vitro and in vivo. Mechanistically, NXF3 modulates the cell cycle, cellular senescence related oncogenic pathways via piRNA-target network. Specifically, our findings highlighted several piRNA-related signaling pathways in GC, such as piRNA_3457319-CCND1/CDKN1A-p53, piRNA_2847077-TGFB3/TGFBR2-Cellular senescence, piRNA_448895-IGF1/PDGFRA/ACTB/MAP2K6-Rap1. Moreover, NXF3 was shown to facilitate the nuclear export of CDK5RAP3 mRNA, thereby promoting cell cycle progression and increasing cancer cell proliferation in gastric cancer.

Conclusion: Our study demonstrates that NXF3 modulates cell cycle progression and promotes gastric cancer development through piRNA-related pathways and the nuclear export of CDK5RAP3 mRNA. Targeting NXF3 represents a promising strategy for developing novel therapeutic approaches for gastric cancer.

Campomelic Dysplasia (CD) is a rare congenital disease caused by haploinsufficiency (HI) in SOX9. Patients with CD typically present with skeletal abnormalities and 75% of them have sex reversal. In this study, we use CRISPR/Cas9 to generate a human induced pluripotent stem cell (hiPSC) model from a heathy male donor, based on a previously reported SOX9 splice site mutation in a CD patients. This hiPSCs-derived chondrocytes from heterozygotes (HT) and homozygotes (HM) SOX9 mutation carriers showed significant defects in chondrogenesis. Bulk RNA profiling revealed that the BMP-SMAD signaling pathway, ribosome-related, and chromosome segregation-related gene sets were altered in the HT chondrocytes. The profile also showed significant noggin upregulation in CD chondrocytes, with ChIP-qPCR confirming that SOX9 binds to the distal regulatory element of noggin. This suggests SOX9 plays a feedback role in the BMP signaling pathway by modulating noggin expression rather than acting solely as a downstream regulator. This provides further insights into its dosage sensitivity in chondrogenesis. Overexpression of SOX9 showed promising results with improved sulfated glycosaminoglycans (GAGs) aggregation and COL2A1 expression following differentiation. We hope this finding could provide a better understanding of the dosage-dependent role of SOX9 in chondrogenesis and contribute to the development of improved therapeutic targets for CD patients.

Tyrosine kinase 2 (TYK2) deficiency and loss or inhibition of kinase activity in men and mice leads to similar immune compromised phenotypes, predominantly through impairment of interferon (IFN) and interleukin 12 family responses. Here we relate the transcriptome changes to phenotypical changes observed in TYK2-deficient (Tyk2^-/-) and TYK2 kinase-inactive (Tyk2^K923E) mice in naïve splenic immune cells and upon ex vivo IFN treatment or in vivo tumor transplant infiltration. The TYK2 activities under homeostatic and both challenged conditions are highly cell-type-specific with respect to quantity and quality of transcriptionally dependent genes. The major impact of loss of TYK2 protein or kinase activity in splenic homeostatic macrophages, NK and CD8⁺ T cells and tumor-derived cytolytic cells is on IFN responses. While reportedly TYK2 deficiency leads to partial impairment of IFN-I responses, we identified cell-type-specific IFN-I-repressed gene sets completely dependent on TYK2 kinase activity. Reported kinase-inactive functions of TYK2 relate to signaling crosstalk, metabolic functions and cell differentiation or maturation. None of these phenotypes relates to respective enriched gene sets in the TYK2 kinase-inactive cell types. Nonetheless, the scaffolding functions of TYK2 are capable to change transcriptional activities at single gene levels and chromatin accessibility at promoter-distal regions upon cytokine treatment most prominently in CD8⁺ T cells. The cell-type-specific transcriptomic and epigenetic effects of TYK2 shed new light on the biology of this JAK family member and are relevant for current and future treatment of autoimmune and inflammatory diseases with TYK2 inhibitors.

PPP1R13L is a conserved inhibitor of p53, selectively regulating a subset of p53 target genes. Previous studies have reported that PPP1R13L promotes cervical cancer progression, yet its precise mechanism remains unclear and warrants further investigation. In this study, we utilized public databases to reveal the correlation between PPP1R13L and tumor progression pathways. Subsequently, we performed functional assays both in vitro and in xenograft models to assess the impact of PPP1R13L on cervical cancer. Our results demonstrate that PPP1R13L promotes cervical cancer cell proliferation, epithelial-mesenchymal transition, cycle progression, and glycolysis via the PTEN/AKT/mTOR pathway. Mechanistically, PPP1R13L regulates the transcription of PTEN through its Ank-SH3 domain interaction with p53 family, p53 and p63. In 293T cells, p53 originally exhibits significantly higher transcriptional activity than p63. However, in cervical cancer-where E6 continuously degrades p53 and p63 is highly expressed-p63 demonstrates a transcriptional activity for PTEN that is comparable to, or even surpasses, that of p53, depending on E6 expression levels. Additionally, in C33A, an HPV-negative cervical cancer cell line, the p53 R273C mutation causes PPP1R13L to exert an opposite effect, and p63 is shown to be inhibited by PPP1R13L independently of p53. Finally, the response elements of PPP1R13L-regulated p53 family target genes were experimentally validated on p63 for the first time. This provides a sequence basis for the selective regulation of p53 family target genes by PPP1R13L. In summary, we underscore the specificity of the PPP1R13L/p63/PTEN axis in cervical cancer and propose that PPP1R13L holds potential as a therapeutic target for cervical cancer treatment.

Lymphangiogenesis has gained considerable interest due to its established role in cancer progression and dissemination of metastatic cells through lymph nodes. Deciphering the molecular mechanisms that govern lymphangiogenesis within lymph nodes holds promise for revealing novel targetable molecules and pathways to inhibit metastasis. In this study, we revealed a previously unrecognized role of AXL, a tyrosine kinase receptor, in the lymphatic vessel formation. We first validated the expression of AXL in lymphatic endothelial cells (LECs), followed by functional studies using RNA interference and pharmacological inhibition with R428/Bemcentinib. These approaches provided compelling evidence that AXL promotes LEC migration in both 2D and 3D culture systems. Our findings demonstrated that AXL activation was induced by VEGF-C (Vascular Endothelial Growth Factor C) and further amplified downstream signaling via the AKT pathway. In vivo, the role of AXL in lymphatic vessel sprouting was demonstrated using R428 in a model of VEGF-C-induced lymphangiogenesis in lymph nodes. Interestingly, we discovered that AXL was predominantly expressed in MARCO⁺ LECs. Strikingly, under metastatic conditions, there was a notable increase in the density and penetration extent of these AXL-expressing LECs into the lymph node parenchyma. Collectively, our findings pinpoint AXL as a potent enhancer of lymphangiogenesis operating through the VEGF-C/AKT pathway. Furthermore, the identification of AXL expression within a distinct LEC subpopulation, particularly in the context of metastasis, underscores the intricate interplay between AXL signaling and lymphatic dynamics within the lymph node microenvironment.

Triple-negative breast cancer (TNBC) cells are rich in glycocalyx (GCX) that is closely correlated with the reorganization of cytoskeletal filaments. Most studies have focused on cell membrane glycoproteins in this context, but rarely on the significance of glycosaminoglycans, particularly the hyaluronan (HA)-associated GCX. Here, we reported that removal of GCX HA could significantly increase breast cancer cells (BCCs) stiffness, leading to impaired cell growth and decreased stem-like properties. Furthermore, we found that the delay of TNBC cells progression could be restored after the cells were re-softened. Meanwhile, in vivo studies revealed that hyaluronidase (HAase)-pretreated BCCs displayed reduced tumor growth and migration. Intriguingly, we identified that ZC3H12A, a zinc-finger RNA binding protein encoded gene, was significantly upregulated after the GCX HA impairment. Of note, knockdown of ZC3H12A could soften the HAase-treated TNBC cells, implying a GCX HA-ZC3H12A regulation on cell stiffening. Taken together, our findings suggested that the breakdown of pericellular HA coat could influence TNBC cells mechanical properties which might be helpful to the future breast cancer research.

Over the last decades, organ transplantation has made rapid progress as a curative therapy for organ failure. However, the adaptive immune system-alloreactive T cells and antibodies targeting human leukocyte antigens (HLA)-is the leading cause of graft rejection. The presence of anti-donor HLA antibodies is considered a risk factor that disqualifies a particular donor-recipient pair. However, alloantibodies are found in some long-term graft survivors, suggesting a protective blocking function of some alloantibodies. Therefore, whether alloantibodies can have a positive as well as a negative effect in transplantation remains unclear. Here, HLA-A*11:01-specific monoclonal antibodies were generated from a human non-immune antibody library, and the effect of these antibodies was investigated on activation of A*11:01- specific T cells. We identified an A*11:01-specific monoclonal antibody with the capacity to block TCR recognition, TCR recruitment to the immune synapse, and T cell activation. The antibody reduced translocation of the transcription factor NFAT1 and phosphorylation of the MAP kinase ERK, which are both required for T cell effector function and TCR signal transduction. Cross-linking mass spectrometry was used to identify the epitope, demonstrating that this alloantibody can inhibit TCR from binding to the HLA molecule. These findings indicate that some HLA-specific alloantibodies can reduce T cell responses to the allograft. This has significant implications for interpretation of the existence of donor-specific antibodies, since some of them can protect the graft. Moreover, such antibodies may have therapeutic potential as specific treatments targeting mismatched donor HLA molecules.

Alzheimer's disease (AD) is a common and increasing societal problem due to the extending human lifespan. In males, loss of chromosome Y (LOY) in leukocytes is strongly associated with AD. We studied here DNA methylation and RNA expression in sorted monocytes and granulocytes with and without LOY from male AD patients. Through multi-omic analysis, we identified new candidate genes along with those previously associated with AD. Global analyses of DNA methylation in samples with LOY vs. normal state showed that hypomethylation dominated both in granulocytes and monocytes. Our findings highlight LOY-related differences in DNA methylation that occur in gene regulatory regions. Specifically, we observed alterations in key genes involved in leukocyte differentiation: FLI1, involved in early hematopoiesis; RUNX1, essential for blood cell development; RARA, regulating gene expression in response to retinoic acid; CANX, crucial for protein folding; CEBPB, a transcription factor important for immune responses; and MYADM, implicated in cell adhesion and migration. Moreover, protein-protein interaction analysis in granulocytes identified that products of two of these genes, CANX and CEBPB, are key hub proteins. This research underscores the potential of multi-omic approach in pure hematopoietic cell populations to uncover the molecular underpinnings of AD. Finally, our results link previous analysis showing impact of LOY on leukocyte differentiation, LOY-associated transcriptional dysregulation and GWAS studies of LOY.

Bcl-2 associated athanogene-5 (BAG5) represents a unique BAG cochaperone family member, regulating chaperone activity. We first demonstrated significant differences in Bag5 expression by RNA seq analysis of teratozoospermia and healthy male sperm samples, but the genetic and molecular mechanisms governing this process remain elusive. We further found that BAG5 has highest expression in human and mouse testes. BAG5 expression is elevated in late stage pachytene spermatocytes and spermatids. Targeted Bag5 inactivation in mice induces massive apoptosis in male germ cells and abrogates male infertility. The ordered loading of sperm basic nuclear proteins on chromatin is altered, with lost TNPs and PRMs, resulting in severe sperm head deformity and partial 9 + 2 microtubule structure disorder. In terms of mechanism, immunoprecipitation (IP)-mass spectroscopy (MS) revealed BAG5 interacts with HSPA2, a testis-specific HSP70 family member regulating the transcription of the transition protein TNPs as well as spermatogenesis. RNA-sequencing assessment of Bag5 deficient testis confirmed Bag5 participation in transcriptional repression and revealed significant changes in Hspa2 expression. Bag5 deficiency resulted in decreased levels of HSPA2, germ cell apoptosis and subsequent inappropriate nuclear protein deposition and chromatin condensation. Decreased BAG5 expression levels in patients with non-obstructive azoospermia and oligoasthenospermia were also detected. These results uncovered an intriguing HSPA2-mediated key function of BAG5, which may constitute a potential prognostic biomarker of male infertility.