Effect of Combinatorial Extracellular Matrix and Substrate Stiffness on Gene Expression of Activated Hepatic Stellate Cells
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ABSTRACT:
PROVIDER: PRJNA868328 | ENA |
REPOSITORIES: ENA
Dataset's files
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| SRR21006700.fastq.gz | Fastqsanger.gz | |||
| SRR21006701.fastq.gz | Fastqsanger.gz | |||
| SRR21006702.fastq.gz | Fastqsanger.gz | |||
| SRR21006703.fastq.gz | Fastqsanger.gz | |||
| SRR21006704.fastq.gz | Fastqsanger.gz |
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Project description:Effect of Combinatorial Extracellular Matrix and Substrate Stiffness on Chromatin Accessibility of Activated Hepatic Stellate Cells
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Project description:Extracellular matrix proteomic profiling Mass spectrometry characterization illustrated a reprogrammed proteome in response to matrix stiffness, including a core subset of 84 ECM-specific proteins, including various ECM regulators and tissue-associated ECM-related proteins. Further, we observe remodeling of ECM proteome with softer substrates associated with metabolic/mitochondrial network (178 proteins), while with 40 kPa substrate (92 proteins enriched) resulting in networks associated with enrichment of collagen biosynthesis, integrin cell surface interactions, and extracellular matrix organization networks.
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Project description:The extracellular matrix (ECM) stiffness influences many physiological and pathological processes. Cells can sense mechanical changes within the ECM through mechanosensors, most notably through the focal adhesion kinase (FAK). Using human-derived data and 2D/3D engineered models, we identified a ubiquitous and uncharacterized FAK isoform lacking exon 4 (FAKΔe4). We demonstrated that FAKΔe4 splicing is regulated by substrate stiffness in a biphasic manner, in which an optimal stiffness is required for a maximal FAKΔe4 expression. We further showed that FAKΔe4 dictates at which stiffness optimal migration speed and invasion occurs, impairs focal adhesion dynamics and maturation, and shifts its autophosphorylation and downstream YAP nuclear translocation toward lower stiffness compared to the canonical FAK isoform. Moreover, the FAKΔe4 expression levels to canonical FAK determine at which stiffness cells will converge during durotaxis. Our results reveal how FAKΔe4 acts as a fine-tuning mechanosensing switch and reframe our understanding of mechanotransduction.
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Project description:Matrix stiffness has significant effects on cell behavior, however, less is known regarding the epigenomic and transcriptional regulation underling the effect of matrix stiffness on cells. In this study, we use an in vitro system to assess the phenotypic shifts of hepatic stellate cells (HSCs) following changes in matrix stiffness, and integrate multi-omics with imaging and biochemical assays to investigate the molecular mechanisms. We show that cells cultured on a stiff matrix display more accessible chromatin sites, which consist of primed chromatin regions that become more accessible prior to the upregulation of nearby genes. These regions are enriched in fibrosis-associated genes that function in cytoskeletal organization and response to mechanical stimuli. We also identify activation of p-JUN in response to the stiff matrix and promoting phenotypic shifts. The identified chromatin accessibility-dependent effect of matrix stiffness may be responsible for various fibrotic diseases and provide insight into
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Project description:Elevated intraocular pressure, a major risk factor of glaucoma, is caused by the abnormal function of trabecular outflow pathways. Human trabecular meshwork (HTM) tissue plays an important role in the outflow pathways. However, the molecular mechanisms that how TM cells respond to the elevated IOP are largely unknown. We cultured primary HTM cells on polyacrylamide gels with tunable stiffness corresponding to Young's moduli ranging from 1.1 to 50 kPa. Then next‐generation RNA sequencing (RNA‐seq) was performed to obtain the transcriptomic profiles of HTM cells. Bioinformatics analysis revealed that genes related to glaucoma including DCN, SPARC, and CTGF, were significantly increased with elevated substrate stiffness, as well as the global alteration of HTM transcriptome. Extracellular matrix (ECM)‐related genes were selectively activated in response to the elevated substrate stiffness, consistent with the known molecular alteration in glaucoma. Human normal and glaucomatous TM tissues were also obtained to perform RNA‐seq experiments and supported the substrate stiffness‐altered transcriptome profiles from the in vitro cell model. The current study profiled the transcriptomic changes in human TM cells upon increasing substrate stiffness. Global change of ECM‐related genes indicates that the in vitro substrate stiffness could greatly affect the biological processes of HTM cells. The in vitro HTM cell model could efficiently capture the main pathogenetic process in glaucoma patients, and provide a powerful method to investigate the underlying molecular mechanisms.
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