Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

SOX9 regulated matrix proteins are increased in patients serum and correlate with severity of liver fibrosis

ABSTRACT: Extracellular matrix (ECM) deposition and resultant scar play a major role in the pathogenesis and progression of liver fibrosis. Identifying core regulators of ECM deposition may lead to urgently needed diagnostic and therapetic strategies for the disease. The transcription factor Sex determining region Y box 9 (SOX9) is actively involved in scar formation and its prevalence in patients with liver fibrosis predicts progression. In this study, transcriptomic approaches of Sox9-abrogated myofibroblasts identified >30% of genes regulated by SOX9 relate to the ECM.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Leo Zeef

PROVIDER: E-MTAB-7298 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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SOX9 regulated matrix proteins are increased in patients serum and correlate with severity of liver fibrosis.

Athwal Varinder S VS Pritchett James J Martin Katherine K Llewellyn Jessica J Scott Jennifer J Harvey Emma E Zaitoun Abed M AM Mullan Aoibheann F AF Zeef Leo A H LAH Friedman Scott L SL Irving William L WL Hanley Neil A NA Guha Indra N IN Piper Hanley Karen K

Scientific reports 20181217 1

Extracellular matrix (ECM) deposition and resultant scar play a major role in the pathogenesis and progression of liver fibrosis. Identifying core regulators of ECM deposition may lead to urgently needed diagnostic and therapetic strategies for the disease. The transcription factor Sex determining region Y box 9 (SOX9) is actively involved in scar formation and its prevalence in patients with liver fibrosis predicts progression. In this study, transcriptomic approaches of Sox9-abrogated myofibro ...[more]

PMID: 30559459

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Project description:Vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular ageing. Sox9 (SRY-Box Transcription Factor 9) is a master regulator of chondrogenesis, also expressed in the vasculature, that has been implicated in vascular smooth muscle cell (VSMC) osteo-chondrogenic conversion. Here, we investigated the relationship between vascular ageing, calcification and Sox9-driven ECM regulation in VSMCs. Immunohistochemistry in human aortic samples showed that Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16. Analysis of Sox9 expression in vitro showed it was mechanosensitive with increased expression and nuclear translocation in senescent cells and on stiff matrices. Manipulation of Sox9 via overexpression and depletion, combined with atomic force microscopy (AFM) and proteomics, revealed that Sox9 regulates ECM stiffness and organisation by orchestrating changes in collagen expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs whereby senescent cells plated onto ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (LH3) was identified as a Sox9 target, and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles (EVs) and Sox9 promoted EV secretion, leading to increased LH3 deposition within the ECM. These findings identify cellular senescence and Sox9 as a key regulators of ECM stiffness during VSMC ageing and highlight a crucial role for ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle whereby cellular senescence and increased ECM stiffening promote Sox9 expression which drives further ECM modifications that act to accelerate vascular stiffening and cellular senescence.

Project description:Introduction: In addition to the well-known cartilage extracellular matrix-related expression of Sox9, we demonstrated that chondrogenic differentiation of progenitor cells is driven by a sharply defined bi-phasic expression of Sox9: an immediate early and a late (extracellular matrix associated) phase expression. In this study we aimed to determine what biological processes are driven by Sox9 during this early phase of chondrogenic differentiation. Materials: Sox9 expression in ATDC5 cells was knocked-down by siRNA transfection at the day before chondrogenic differentiation or at day 6 of differentiation. Samples were harvested at 2 hours, and 7 days of differentiation. The mRNA sequencing library was generated using TruSeq mRNA sample preparation kit (Illumina, Eindhoven, the Netherlands). In short, mRNA was enriched using magnetic beads coated with poly-dT, followed by fragmentation. The fragmented mRNA enriched samples were subjected to cDNA synthesis by reverse transcriptase, followed by dA-tailing and ligation of specific double-stranded bar-coded adapters. Next, the library was amplified and following cleanup the sizes of the libraries were determined on an Agilent 2100 Bioanalyzer via a DNA 1000 chip according manufacturer’s protocol. Pooled libraries consisting of equal molar samples were sequenced on a high-output 75bp single read on the NextSeq500 (Illumina). For each sample, the number of reads covering one or more exons of a given transcript were extracted. Triplicates of samples that were treated with either Scrambled or Sox9 siRNAs, at two different time points, were grouped separately. A transcript was defined as expressed when all replicates of a group had at least 5 reads extracted within the transcript's region. The grouped data were then compared to one another. The fold-change difference and the p-value were calculated using R-package edgeR, after which the p-value was corrected for multiple testing (false discovery rate (FDR)-corrected). The transcriptomes using a RNA-seq approach was analyzed using pathway and network analyses. Results: Early Sox9 knockdown severely inhibited chondrogenic differentiation weeks later. Sox9 expression during the immediate early phase of ATDC5 chondrogenic differentiation regulated the expression of ribosome biogenesis factors and ribosomal protein subunits. This was accompanied by decreased translational capacity following Sox9 knockdown, and this correlated to lower amounts of active mono- and polysomes. Moreover, cap- versus IRES-mediated translation was altered by Sox9 knockdown. Sox9 overexpression was able to induce reciprocal effects to the Sox9 knockdown.

Dataset Information

SOX9 regulated matrix proteins are increased in patients serum and correlate with severity of liver fibrosis

Publications

SOX9 regulated matrix proteins are increased in patients serum and correlate with severity of liver fibrosis.

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