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Acta Neuropathologica Communications Jun 2024Neurofibromatosis Type 1 (NF1) is caused by loss of function variants in the NF1 gene. Most patients with NF1 develop skin lesions called cutaneous neurofibromas (cNFs)....
snRNA-seq of human cutaneous neurofibromas before and after selumetinib treatment implicates role of altered Schwann cell states, inter-cellular signaling, and extracellular matrix in treatment response.
Neurofibromatosis Type 1 (NF1) is caused by loss of function variants in the NF1 gene. Most patients with NF1 develop skin lesions called cutaneous neurofibromas (cNFs). Currently the only approved therapeutic for NF1 is selumetinib, a mitogen -activated protein kinase (MEK) inhibitor. The purpose of this study was to analyze the transcriptome of cNF tumors before and on selumetinib treatment to understand both tumor composition and response. We obtained biopsy sets of tumors both pre- and on- selumetinib treatment from the same individuals and were able to collect sets from four separate individuals. We sequenced mRNA from 5844 nuclei and identified 30,442 genes in the untreated group and sequenced 5701 nuclei and identified 30,127 genes in the selumetinib treated group. We identified and quantified distinct populations of cells (Schwann cells, fibroblasts, pericytes, myeloid cells, melanocytes, keratinocytes, and two populations of endothelial cells). While we anticipated that cell proportions might change with treatment, we did not identify any one cell population that changed significantly, likely due to an inherent level of variability between tumors. We also evaluated differential gene expression based on drug treatment in each cell type. Ingenuity pathway analysis (IPA) was also used to identify pathways that differ on treatment. As anticipated, we identified a significant decrease in ERK/MAPK signaling in cells including Schwann cells but most specifically in myeloid cells. Interestingly, there is a significant decrease in opioid signaling in myeloid and endothelial cells; this downward trend is also observed in Schwann cells and fibroblasts. Cell communication was assessed by RNA velocity, Scriabin, and CellChat analyses which indicated that Schwann cells and fibroblasts have dramatically altered cell states defined by specific gene expression signatures following treatment (RNA velocity). There are dramatic changes in receptor-ligand pairs following treatment (Scriabin), and robust intercellular signaling between virtually all cell types associated with extracellular matrix (ECM) pathways (Collagen, Laminin, Fibronectin, and Nectin) is downregulated after treatment. These response specific gene signatures and interaction pathways could provide clues for understanding treatment outcomes or inform future therapies.
Topics: Humans; Schwann Cells; Skin Neoplasms; Benzimidazoles; Extracellular Matrix; Signal Transduction; Neurofibroma; Female; Male; RNA-Seq; Middle Aged; Adult; Neurofibromatosis 1; Protein Kinase Inhibitors; Transcriptome
PubMed: 38907342
DOI: 10.1186/s40478-024-01821-z -
Scientific Reports Jun 2024Glioblastoma (GBM) is a highly aggressive and deadly brain cancer. Temozolomide (TMZ) is the standard chemotherapeutic agent for GBM, but the majority of patients...
Glioblastoma (GBM) is a highly aggressive and deadly brain cancer. Temozolomide (TMZ) is the standard chemotherapeutic agent for GBM, but the majority of patients experience recurrence and invasion of tumor cells. We investigated whether TMZ treatment of GBM cells regulates matrix metalloproteinases (MMPs), which have the main function to promote tumor cell invasion. TMZ effectively killed GL261, U343, and U87MG cells at a concentration of 500 µM, and surviving cells upregulated MMP9 expression and its activity but not those of MMP2. TMZ also elevated levels of MMP9 mRNA and MMP9 promoter activity. Subcutaneous graft tumors survived from TMZ treatment also exhibited increased expression of MMP9 and enhanced gelatinolytic activity. TMZ-mediated MMP9 upregulation was specifically mediated through the phosphorylation of p38 and JNK. This then stimulates AP-1 activity through the upregulation of c-Fos and c-Jun. Inhibition of the p38, JNK, or both pathways counteracted the TMZ-induced upregulation of MMP9 and AP-1. This study proposes a potential adverse effect of TMZ treatment for GBM: upregulation of MMP9 expression potentially associated with increased invasion and poor prognosis. This study also provides valuable insights into the molecular mechanisms by which TMZ treatment leads to increased MMP9 expression in GBM cells.
Topics: Temozolomide; Glioblastoma; Matrix Metalloproteinase 9; Humans; p38 Mitogen-Activated Protein Kinases; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; MAP Kinase Signaling System; Antineoplastic Agents, Alkylating; Animals; Brain Neoplasms; Transcription Factor AP-1; Up-Regulation; Mice
PubMed: 38906916
DOI: 10.1038/s41598-024-65398-2 -
Cell Death & Disease Jun 2024Colorectal cancer (CRC) remains a major global cause of cancer-related mortality, lacking effective biomarkers and therapeutic targets. Revealing the critical pathogenic...
Colorectal cancer (CRC) remains a major global cause of cancer-related mortality, lacking effective biomarkers and therapeutic targets. Revealing the critical pathogenic factors of CRC and the underlying mechanisms would offer potential therapeutic strategies for clinical application. G protein signaling (RGS) protein family modulators play essential role within regulating downstream signaling of GPCR receptors, with function in cancers unclear. Our study focused on the expression patterns of RGS proteins in CRC, identifying Regulator of G protein signaling 16 (RGS16) as a prospective diagnostic and therapeutic target. Analyzing 899 CRC tissues revealed elevated RGS16 levels, correlating with clinicopathological features and CRC prognosis by immunohistochemistry (IHC) combined with microarray. We confirmed the elevated RGS16 protein level in CRC, and found that patients with RGS16-high tumors exhibited decreased disease-specific survival (DSS) and disease-free survival (DFS) compared to those with low RGS16 expression. Functional assays demonstrated that RGS16 promoted the CRC progression, knockdown of RGS16 led to significantly increased apoptosis rates of CRC in vitro and in vivo. Notably, we also confirmed these phenotypes of RGS16 in organoids originated from resected primary human CRC tissues. Mechanistically, RGS16 restrained JNK/P38-mediated apoptosis in CRC cells through disrupting the recruitment of TAB2/TAK1 to TRAF6. This study provides insights into addressing the challenges posed by CRC, offering avenues for clinical translation.
Topics: Humans; Colorectal Neoplasms; Apoptosis; RGS Proteins; MAP Kinase Kinase Kinases; Female; Animals; Male; Adaptor Proteins, Signal Transducing; p38 Mitogen-Activated Protein Kinases; TNF Receptor-Associated Factor 6; Mice; Cell Line, Tumor; MAP Kinase Signaling System; Signal Transduction; Mice, Nude; Middle Aged; Intracellular Signaling Peptides and Proteins
PubMed: 38906869
DOI: 10.1038/s41419-024-06803-6 -
International Journal of Biological... 2024Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and the production of autoantibodies. Previous studies have...
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and the production of autoantibodies. Previous studies have indicated an association between high-salt diets (HSD) and an increased risk of RA, yet the underlying mechanisms remain unclear. Macrophage pyroptosis, a pro-inflammatory form of cell death, plays a pivotal role in RA. In this study, we demonstrate that HSD exacerbates the severity of arthritis in collagen-induced arthritis (CIA) mice, correlating with macrophage infiltration and inflammatory lesions. Given the significant alterations observed in macrophages from CIA mice subjected to HSD, we specifically investigate the impact of HSD on macrophage responses in the inflammatory milieu of RA. In our experiments, pretreatment with NaCl enhances LPS-induced pyroptosis in RAW.264.7 and THP-1 cells through the p38 MAPK/NF-κB signaling pathway. Subsequent experiments reveal that Slc6a12 inhibitors and SGK1 silencing inhibit sodium-induced activation of macrophage pyroptosis and the p38 MAPK/NF-κB signaling pathway, whereas overexpression of the SGK1 gene counteracts the effect of sodium on macrophages. In conclusion, our findings verified that high salt intake promotes the progression of RA and provided a detailed elucidation of the activation of macrophage pyroptosis induced by sodium transportation through the Slc6a12 channel.
Topics: Animals; Mice; Arthritis, Rheumatoid; Macrophages; Pyroptosis; Protein Serine-Threonine Kinases; Sodium Chloride; RAW 264.7 Cells; Humans; Male; Immediate-Early Proteins; Arthritis, Experimental; Signal Transduction; p38 Mitogen-Activated Protein Kinases; Mice, Inbred DBA
PubMed: 38904021
DOI: 10.7150/ijbs.93242 -
Frontiers in Cell and Developmental... 2024Extrachromosomal DNAs (eccDNAs) frequently carry amplified oncogenes. This investigation aimed to examine the occurrence and role of eccDNAs in individuals diagnosed...
Extrachromosomal circular DNA (eccDNA) characteristics in the bile and plasma of advanced perihilar cholangiocarcinoma patients and the construction of an eccDNA-related gene prognosis model.
Extrachromosomal DNAs (eccDNAs) frequently carry amplified oncogenes. This investigation aimed to examine the occurrence and role of eccDNAs in individuals diagnosed with advanced perihilar cholangiocarcinoma (pCCA) who exhibited distinct prognostic outcomes. Five patients with poor survival outcomes and five with better outcomes were selected among patients who received first-line hepatic arterial infusion chemotherapy from June 2021 to June 2022. The extracted eccDNAs were amplified for high-throughput sequencing. Genes associated with the differentially expressed eccDNAs were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The differentially expressed bile eccDNA-related genes were used to construct a prognostic model. Across all 10 patients, a total of 19,024 and 3,048 eccDNAs were identified in bile and plasma, respectively. The concentration of eccDNA detected in the bile was 9-fold higher than that in plasma. The chromosome distribution of the eccDNAs were similar between bile and matched plasma. GO and KEGG pathway analyses showed enrichment in the mitogen-activated protein kinase (MAPK) and Wnt/β-catenin pathways in patients with poor survival outcomes. According to the prognostic model constructed by eccDNA-related genes, the high-risk group of cholangiocarcinoma patients displayed significantly shorter overall survival ( < 0.001). Moreover, the degree of infiltration of immunosuppressive cells was higher in patients in the high-risk group. In conclusion, EccDNA could be detected in bile and plasma of pCCA patients, with a higher concentration. A prognostic model based on eccDNA-related genes showed the potential to predict the survival and immune microenvironment of patients with cholangiocarcinoma.
PubMed: 38903532
DOI: 10.3389/fcell.2024.1379435 -
Frontiers in Plant Science 2024Sugarcane aphid has emerged as a major pest of sorghum recently, and a few sorghum accessions were identified for resistance to this aphid so far. However, the molecular...
Sugarcane aphid has emerged as a major pest of sorghum recently, and a few sorghum accessions were identified for resistance to this aphid so far. However, the molecular and genetic mechanisms underlying this resistance are still unclear. To understand these mechanisms, transcriptomics was conducted in resistant Tx2783 and susceptible BTx623 sorghum genotypes infested with sugarcane aphids. A principal component analysis revealed differences in the transcriptomic profiles of the two genotypes. The pathway analysis of the differentially expressed genes (DEGs) indicated the upregulation of a set of genes related to signal perception (nucleotide-binding, leucine-rich repeat proteins), signal transduction [mitogen-activated protein kinases signaling, salicylic acid (SA), and jasmonic acid (JA)], and plant defense (transcription factors, flavonoids, and terpenoids). The upregulation of the selected DEGs was verified by real-time quantitative PCR data analysis, performed on the resistant and susceptible genotypes. A phytohormone bioassay experiment showed a decrease in aphid population, plant mortality, and damage in the susceptible genotype when treated with JA and SA. Together, the results indicate that the set of genes, pathways, and defense compounds is involved in host plant resistance to aphids. These findings shed light on the specific role of each DEG, thus advancing our understanding of the genetic and molecular mechanisms of host plant resistance to aphids.
PubMed: 38903420
DOI: 10.3389/fpls.2024.1324085 -
Stem Cell Research & Therapy Jun 2024Adult skeletal muscle contains resident muscle stem cells (MuSC) with high myogenic and engraftment potentials, making them suitable for cell therapy and regenerative...
BACKGROUND
Adult skeletal muscle contains resident muscle stem cells (MuSC) with high myogenic and engraftment potentials, making them suitable for cell therapy and regenerative medicine approaches. However, purification process of MuSC remains a major hurdle to their use in the clinic. Indeed, muscle tissue enzymatic dissociation triggers a massive activation of stress signaling pathways, among which P38 and JNK MAPK, associated with a premature loss of MuSC quiescence. While the role of these pathways in the myogenic progression of MuSC is well established, the extent to which their dissociation-induced activation affects the functionality of these cells remains unexplored.
METHODS
We assessed the effect of P38 and JNK MAPK induction on stemness marker expression and MuSC activation state during isolation by pharmacological approaches. MuSC functionality was evaluated by in vitro assays and in vivo transplantation experiments. We performed a comparative analysis of the transcriptome of human MuSC purified with pharmacological inhibitors of P38 and JNK MAPK (SB202190 and SP600125, respectively) versus available RNAseq resources.
RESULTS
We monitored PAX7 protein levels in murine MuSC during muscle dissociation and demonstrated a two-step decline partly dependent on P38 and JNK MAPK activities. We showed that simultaneous inhibition of these pathways throughout the MuSC isolation process preserves the expression of stemness markers and limits their premature activation, leading to improved survival and amplification in vitro as well as increased engraftment in vivo. Through a comparative RNAseq analysis of freshly isolated human MuSC, we provide evidence that our findings in murine MuSC could be relevant to human MuSC. Based on these findings, we implemented a purification strategy, significantly improving the recovery yields of human MuSC.
CONCLUSION
Our study highlights the pharmacological limitation of P38 and JNK MAPK activities as a suitable strategy to qualitatively and quantitatively ameliorate human MuSC purification process, which could be of great interest for cell-based therapies.
Topics: p38 Mitogen-Activated Protein Kinases; Animals; Humans; Mice; MAP Kinase Signaling System; Muscle, Skeletal; Stem Cells; Cell Differentiation; Mice, Inbred C57BL; Male; Anthracenes; JNK Mitogen-Activated Protein Kinases
PubMed: 38902774
DOI: 10.1186/s13287-024-03795-0 -
BMC Genomics Jun 2024The genotype-by-environment interaction (GxE) in beef cattle can be investigated using reaction norm models to assess environmental sensitivity and, combined with...
BACKGROUND
The genotype-by-environment interaction (GxE) in beef cattle can be investigated using reaction norm models to assess environmental sensitivity and, combined with genome-wide association studies (GWAS), to map genomic regions related to animal adaptation. Including genetic markers from whole-genome sequencing in reaction norm (RN) models allows us to identify high-resolution candidate genes across environmental gradients through GWAS. Hence, we performed a GWAS via the RN approach using whole-genome sequencing data, focusing on mapping candidate genes associated with the expression of reproductive and growth traits in Nellore cattle. For this purpose, we used phenotypic data for age at first calving (AFC), scrotal circumference (SC), post-weaning weight gain (PWG), and yearling weight (YW). A total of 20,000 males and 7,159 females genotyped with 770k were imputed to the whole sequence (29 M). After quality control and linkage disequilibrium (LD) pruning, there remained ∼ 2.41 M SNPs for SC, PWG, and YW and ∼ 5.06 M SNPs for AFC.
RESULTS
Significant SNPs were identified on Bos taurus autosomes (BTA) 10, 11, 14, 18, 19, 20, 21, 24, 25 and 27 for AFC and on BTA 4, 5 and 8 for SC. For growth traits, significant SNP markers were identified on BTA 3, 5 and 20 for YW and PWG. A total of 56 positional candidate genes were identified for AFC, 9 for SC, 3 for PWG, and 24 for YW. The significant SNPs detected for the reaction norm coefficients in Nellore cattle were found to be associated with growth, adaptative, and reproductive traits. These candidate genes are involved in biological mechanisms related to lipid metabolism, immune response, mitogen-activated protein kinase (MAPK) signaling pathway, and energy and phosphate metabolism.
CONCLUSIONS
GWAS results highlighted differences in the physiological processes linked to lipid metabolism, immune response, MAPK signaling pathway, and energy and phosphate metabolism, providing insights into how different environmental conditions interact with specific genes affecting animal adaptation, productivity, and reproductive performance. The shared genomic regions between the intercept and slope are directly implicated in the regulation of growth and reproductive traits in Nellore cattle raised under different environmental conditions.
Topics: Animals; Cattle; Genome-Wide Association Study; Reproduction; Polymorphism, Single Nucleotide; Female; Whole Genome Sequencing; Gene-Environment Interaction; Male; Genotype; Phenotype; Quantitative Trait Loci; Linkage Disequilibrium
PubMed: 38902640
DOI: 10.1186/s12864-024-10520-x -
Molecular Biomedicine Jun 2024Hepatic ischemia-reperfusion injury (HIRI) is a critical pathophysiological process during liver transplantation (LT). Multiple genes and signal pathways are...
Hepatic ischemia-reperfusion injury (HIRI) is a critical pathophysiological process during liver transplantation (LT). Multiple genes and signal pathways are dysregulated during HIRI. This study aims to identify genes as potential therapeutic targets for ameliorating HIRI. Datasets containing samples from the human donor liver (GSE151648) and mouse HIRI model (GSE117066) were analyzed to determine differentially expressed genes (DEGs). The selected DEGs were confirmed by real-time PCR and western blot in the hepatocyte hypoxia-reoxygenation (HR) model, mouse HIRI model, and human liver samples after transplantation. Genetic inhibition was used to further clarify the underlying mechanism of the gene in vitro and in vivo. Among the DEGs, CSRNP1 was significantly upregulated (|log FC|= 2.08, P < 0.001), and was positively correlated with the MAPK signal pathway (R = 0.67, P < 0.001). CSRNP1 inhibition by siRNA significantly suppressed apoptosis in the AML-12 cell line after HR (mean Annexin ratio = 60.62% vs 42.47%, P = 0.0019), but the protective effect was eliminated with an additional MAPK activator. Knocking down CSRNP1 gene expression by intravenous injection of AAV-shRNA markedly reduced liver injury in mouse HIRI model (ALT: AAV-NC vs AAV-shCsrnp1 = 26,673.5 ± 2761.2 vs 3839.7 ± 1432.8, P < 0.001; AST: AAV-NC vs AAV-shCsrnp1 = 8640.5 ± 1450.3 vs 1786.8 ± 518.3, P < 0.001). Liver-targeted delivery of siRNA by nanoparticles effectively inhibited intra-hepatic genetic expression of Csrnp1 and alleviated IRI by reducing tissue inflammation and hepatocyte apoptosis. Furthermore, CSRNP1 inhibition was associated with reduced activation of the MAPK pathway both in vitro and in vivo. In conclusion, our results demonstrated that CSRNP1 could be a potential therapeutic target to ameliorate HIRI in an MAPK-dependent manner.
Topics: Reperfusion Injury; Animals; Liver Transplantation; Humans; Mice; Apoptosis; MAP Kinase Signaling System; Male; Cell Line; Liver; Hepatocytes; Disease Models, Animal; Mice, Inbred C57BL
PubMed: 38902590
DOI: 10.1186/s43556-024-00185-z -
Molecular & Cellular Proteomics : MCP Jun 2024Osmotic stress significantly hampers plant growth and crop yields, emphasizing the need for a thorough comprehension of the underlying molecular responses. Previous...
Osmotic stress significantly hampers plant growth and crop yields, emphasizing the need for a thorough comprehension of the underlying molecular responses. Previous research has demonstrated that osmotic stress rapidly induces calcium influx and signaling, along with the activation of a specific subset of protein kinases, notably the Raf-SnRK2 kinase cascades within minutes. However, the intricate interplay between calcium signaling and the activation of RAF-SnRK2 kinase cascades remains elusive. Here in this study, we discovered that Raf-like protein (RAF) kinases undergo hyperphosphorylation in response to osmotic shocks. Intriguingly, treatment with the calcium chelator EGTA robustly activates RAF-SnRK2 cascades, mirroring the effects of osmotic treatment. Utilizing high-throughput DIA-based phosphoproteomics, we unveiled the global impact of EGTA on protein phosphorylation. Beyond the activation of RAFs and sucrose non-fermenting-1-related protein kinase 2s (SnRK2s), EGTA treatment also activates mitogen-activated protein kinase (MAPKs) cascades, Calcium-dependent protein kinases (CDPKs), and receptor-like protein kinases, etc. Through overlapping assays, we identified potential roles of mitogen-activated protein kinase kinase kinase kinases (MAP4Ks) and receptor-like protein kinases in the osmotic-stress-induced activation of RAF-SnRK2 cascades. Our findings illuminate the regulation of phosphorylation and cellular events by Ca signaling, offering insights into the (exocellular) Ca deprivation during early hyperosmolality sensing and signaling.
PubMed: 38901673
DOI: 10.1016/j.mcpro.2024.100804