FGF2 regulates melanocytes viability through the STAT3-transactivated PAX3 transcription.
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ABSTRACT:
SUBMITTER: Dong L
PROVIDER: S-EPMC3307977 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Recent studies indicate a strong correlation of zinc transporter ZIP4 and pancreatic cancer progression; however, the underlying mechanisms are unclear. We have recently found that ZIP4 is overexpressed in pancreatic cancer. In this study, we investigated the signaling pathway through which ZIP4 regulates pancreatic cancer growth.The expression of cyclin D1, interleukin 6 (IL-6), and signal transducer and activator of transcription 3 (STAT3) in pancreatic cancer xenografts and cells were examined by real-time PCR, Bio-Plex cytokine assay, and Western blot, respectively. The activity of cAMP response element-binding protein (CREB) is examined by a promoter activity assay.Cyclin D1 was significantly increased in the ZIP4 overexpressing MIA PaCa-2 cells (MIA-ZIP4)-injected orthotopic xenografts and was downregulated in the ZIP4-silenced ASPC-1 (ASPC-shZIP4) group. The phosphorylation of STAT3, an upstream activator of cyclin D1, was increased in MIA-ZIP4 cells and decreased in ASPC-shZIP4 cells. IL-6, a known upstream activator for STAT3, was also found to be significantly increased in the MIA-ZIP4 cells and xenografts and decreased in the ASPC-shZIP4 group. Overexpression of ZIP4 led to a 75% increase of IL-6 promoter activity and caused increased phosphorylation of CREB.Our study suggest that ZIP4 overexpression causes increased IL-6 transcription through CREB, which in turn activates STAT3 and leads to increased cyclin D1 expression, resulting in increased cell proliferation and tumor progression in pancreatic cancer. These results elucidated a novel pathway in ZIP4-mediated pancreatic cancer growth and suggest new therapeutic targets, including ZIP4, IL-6, and STAT3, in pancreatic cancer treatment.
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Project description:Changes in lipid metabolism and iron content are observed in the livers of patients with fatty liver disease. The expression of hepcidin, an iron-regulatory and acute phase protein synthesized by the liver, is also modulated. The potential interaction of lipid and iron metabolism is largely unknown. We investigated the role of lipid intermediate, ceramide in the regulation of human hepcidin gene, HAMP. Human hepatoma HepG2 cells were treated with cell-permeable ceramide analogs. Ceramide induced significant up-regulation of HAMP mRNA expression in HepG2 cells. The effect of ceramide on HAMP expression was mediated through transcriptional mechanisms because it was completely blocked with actinomycin D treatment. Reporter assays also confirmed the activation of 0.6 kb HAMP promoter by ceramide. HepG2 cells treated with ceramide displayed increased phosphorylation of STAT3, JNK, and NF-?B proteins. However, ceramide induced the binding of STAT3, but not NF-?B or c-Jun, to HAMP promoter, as shown by the chromatin immunoprecipitation assays. The mutation of STAT3 response element within 0.6 kb HAMP promoter region significantly inhibited the stimulatory effect of ceramide on HAMP promoter activity. Similarly, the inhibition of STAT3 with a pan-JAK kinase inhibitor and STAT3 siRNA pool also diminished the induction of both HAMP promoter activity and mRNA expression by ceramide. In conclusion, we have shown a direct role for ceramide in the activation of hepatic HAMP transcription via STAT3. Our findings suggest a crosstalk between lipid and iron metabolism in the liver, which may contribute to the pathogenesis of obesity-related fatty liver disease.
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Project description:In this study, three-dimensional (3-D) poly(ethylene glycol) (PEG) hydrogel arrays were used to screen for the effects of fibroblast growth factor-2 (FGF2), combined with multiple hydrogel matrix parameters, on human mesenchymal stem cell (hMSC) viability and spreading. In particular, we examined the effects of FGF2 while co-varying hydrogel matrix degradability, cell adhesion ligand type, and cell adhesion ligand density. FGF2 significantly improved viability of hMSCs in a dose-dependent manner in both nondegrading and degrading PEG hydrogels in the absence of extracellular matrix-derived cell adhesion ligands. The presence of a small molecule that inhibits autophosphorylation of the FGF2 receptor blocked the effects of FGF2 on hMSC viability in PEG hydrogels, both in the presence and absence of the Arg-Gly-Asp-Ser-Pro (RGDSP) ligand. FGF2 effects on hMSC viability were less pronounced when FGF2 was presented in combination with the RGDSP cell adhesion ligand or the IKVAV cell adhesion ligand in nondegrading PEG hydrogels. Importantly, spread hMSC morphologies were observed and quantified in a select subset of hydrogel networks, which were degradable and included both FGF2 and RGDSP. These results indicate that the hydrogel arrays described here can be used to efficiently study the influence of soluble and insoluble hydrogel matrix parameters on stem cell behavior, and to identify synthetic, 3-D environments that promote specific hMSC behaviors.
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Project description:Circular RNAs (circRNAs) are a novel class of noncoding RNAs that play an important role in cancer. However, the mechanisms by which circRNAs regulate gene expression in pancreatic ductal adenocarcinoma (PDAC) remain unclear. This study seeks to elucidate the role that circRNAs play in the proliferation of PDAC cells. On the basis of previous studies of circRNA expression profiles in PDAC, we found that the circRNA_100782 was markedly upregulated in PDAC tissue. Functional experiments revealed that circRNA_100782 down-regulation inhibited BxPC3 cell proliferation and colony formation. Loss-of-function studies showed that knockdown of circRNA_100782 inhibited cell proliferation by downregulating the microRNA-124 (miR-124) target genes interleukin-6 receptor (IL6R) and signal transducer and activator of transcription 3 (STAT3). Overexpression of miR-124 also inhibited BxPC3 cell proliferation by reducing the expression of IL6R and STAT3, which was consistent with the result of silencing circRNA_100782. In addition, luciferase assay revealed that miR-124 was a direct target of circRNA_100782. Silencing STAT3 inhibited BxPC3 cell proliferation and colony formation. Cell viability was reduced in BxPC3 cells treated with si-circRNA_100782 and miR-124 mimic, and this effect could be attenuated by activating STAT3. In vivo study validated that circRNA_100782 knockdown suppressed BxPC3 xenografts in nude mice. Taken together, these results suggest that circRNA_100782 regulates BxPC3 cell proliferation by acting as miR-124 sponge through the IL6-STAT3 pathway.
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Project description:Melanoma is a highly aggressive and drug resistant form of skin cancer. It arises from melanocytes, the pigment producing cells of the skin. The formation of these melanocytes is driven by the transcription factor PAX3 early during embryonic development. As a result of alternative splicing, the PAX3 gene gives rise to eight different transcripts which encode isoforms that have different structures and activate different downstream target genes involved in pathways of cell proliferation, migration, differentiation and survival. Furthermore, post-translational modifications have also been shown to alter the functions of PAX3. We previously identified PAX3 downstream target genes in melanocytes and melanoma cells. Here we assessed the effects of PAX3 down-regulation on this panel of target genes in primary melanocytes versus melanoma cells. We show that PAX3 differentially regulates various downstream target genes involved in cell proliferation in melanoma cells compared to melanocytes. To determine mechanisms behind this differential downstream target gene regulation, we performed immunoprecipitation to assess post-translational modifications of the PAX3 protein as well as RNAseq to determine PAX3 transcript expression profiles in melanocytes compared to melanoma cells. Although PAX3 was found to be post-translationally modified, there was no qualitative difference in phosphorylation and ubiquitination between melanocytes and melanoma cells, while acetylation of PAX3 was reduced in melanoma cells. Additionally, there were differences in PAX3 transcript expression profiles between melanocytes and melanoma cells. In particular the PAX3E transcript, responsible for reducing melanocyte proliferation and increasing apoptosis, was found to be down-regulated in melanoma cells compared to melanocytes. These results suggest that alternate transcript expression profiles activate different downstream target genes leading to the melanoma phenotype.
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Project description:BackgroundPax3 is a developmental transcription factor that is required for neural tube and neural crest development. We previously showed that inactivating the p53 tumor suppressor protein prevents neural tube and cardiac neural crest defects in Pax3-mutant mouse embryos. This demonstrates that Pax3 regulates these processes by blocking p53 function. Here we investigated the mechanism by which Pax3 blocks p53 function.Methodology/principal findingsWe employed murine embryonic stem cell (ESC)-derived neuronal precursors as a cell culture model of embryonic neuroepithelium or neural crest. Pax3 reduced p53 protein stability, but had no effect on p53 mRNA levels or the rate of p53 synthesis. Full length Pax3 as well as fragments that contained either the DNA-binding paired box or the homeodomain, expressed as GST or FLAG fusion proteins, physically associated with p53 and Mdm2 both in vitro and in vivo. In contrast, Splotch Pax3, which causes neural tube and neural crest defects in homozygous embryos, bound weakly, or not at all, to p53 or Mdm2. The paired domain and homeodomain each stimulated Mdm2-mediated ubiquitination of p53 and p53 degradation in the absence of the Pax3 transcription regulatory domains, whereas Splotch Pax3 did not stimulate p53 ubiquitination or degradation.Conclusions/significancePax3 inactivates p53 function by stimulating its ubiquitination and degradation. This process utilizes the Pax3 paired domain and homeodomain but is independent of DNA-binding and transcription regulation. Because inactivating p53 is the only required Pax3 function during neural tube closure and cardiac neural crest development, and inactivating p53 does not require Pax3-dependent transcription regulation, this indicates that Pax3 is not required to function as a transcription factor during neural tube closure and cardiac neural crest development. These findings further suggest novel explanations for PAX3 functions in human diseases, such as in neural crest-derived cancers and Waardenburg syndrome types 1 and 3.
| S-EPMC3247257 | biostudies-literature
Project description:BackgroundThe study of the immune microenvironment in prostate cancer (PRAD) has brought new opportunities for the current traditional treatment regimens. Therefore, our goal is to develop a universal immunodiagnostic marker to improve patient survival.MethodsBioinformatics analysis: We collected 591 samples from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) cohorts and evaluated the abundance and distribution of immune cell members in the PRAD expression profile matrix in the mixed cell population by CIBERSORT, ESTIMATE, single-sample gene set enrichment analysis (ssGSEA), and other methods. The target genes related to PRAD immune microenvironment and tumor mutation load were obtained by overlap analysis and verified by pan-cancer analysis. Cell experiment: The cell transfection scheme was designed, and the experiment was divided into three groups: overexpressing lactoferrin (LTF) group, empty plasmid group, and control group. After obtaining cells in each group, the gene and protein expression levels of LTF and signal transduction of signal transducer and activator of transcription 3 (STAT3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the above three groups were detected by real-time PCR and Western blot, respectively. Finally, the level of GM-CSF secretion in the three groups was detected by ELISA.ResultsMacrophages, resting mast cells, and plasma cells play an important role in PRAD immune microenvironment. In addition, high tumor mutation load [tumor mutational burden (TMB)] was positively correlated with lymph node metastasis in patients with PRAD. As the core gene of the PRAD immune microenvironment, the low expression of LTF in PRAD promotes the occurrence of immunodeficiency, PRAD, and the enrichment of the Janus kinase (JAK)/STAT3 signal pathway. Through cell experiments, it was found that the content of LTF mRNA and protein increased significantly, while the content of STAT3 and GM-CSF mRNA and protein decreased significantly in the overexpressed LTF group. The level of GM-CSF in the supernatant of cell culture was significantly decreased in the overexpression group of LTF.ConclusionThe core gene we proposed is one of the most promising biomarkers to improve the overall survival rate of PRAD and provides an important theoretical basis for the study of the mechanism of the LTF-mediated JAK/STAT3 pathway in PRAD.
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