Time course of gene expression signatures in mPTECs during ex vivo culture
ABSTRACT: To further examine the gene expression of isolated primary mouse proximal tubular epithelial cells (mPTECs) during ex vivo culture, we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to distinguish the key regulatory transcription factor which was significantly altered. Primary culuture of mPTECs were cultured on culture dishes for 1 and 3 days. As compared to freshly isolated mPTECs, a 1801-gene consensus signature was identified that distinguished between day 1 and and day 3 samples. Within these genes, 78 transcriptal factors were dramatically altered. Expression of three genes (KLF5, KLF4, and CCND1) from this signature was quantified in the same RNA samples by RT-PCR. The proliferation of mouse proximal tubular epithelial cells in ex vivo culture depends on matrix stiffness. Combined analysis of the microarray and experimental data revealed that Krüppel-like factor 5 (Klf5) was the most upregulated transcription factor accompanied by Krüppel-like factor 4 (Klf4) downregulation when cells on stiff matrix. These changes were reversed by soft matrix via ERK inactivation. Knockdown of Klf5 or forced-expression of Klf4 inhibited stiff matrix-induced cell spreading and proliferation, suggesting that Klf5/Klf4 act as positive/negative regulators, respectively. Moreover, stiff matrix-activated ERK increased the protein level and nuclear translocation of mechanosensitive Yes-associated protein 1 (YAP1), which is reported to prevent Klf5 degradation. Finally, in vivo model of unilateral ureteral obstruction (UUO) revealed that matrix stiffness-regulated Klf5/Klf4 is related to the pathogenesis of renal fibrosis. In the dilated tubules of obstructed kidney, ERK/YAP1/Klf5/Cyclin D1 axis were upregulated and Klf4 was downregulated. Inhibition of collagen crosslinking by lysyl oxidase inhibitor alleviated UUO-induced tubular dilatation and proliferation with preserving Klf4 and suppressing the ERK/YAP1/Klf5/Cyclin D1 axis. This study unravels a novel mechanism how matrix stiffness regulates cellular proliferation and highlights the importance of matrix stiffness-modulated Klf5/Klf4 in the regulation of renal physiological functions and fibrosis progression. Gene expression in cultured mPTECs was measured at 0, 1 and 3 days after culturing on culture dishes.
Project description:The behavior of cells is strongly determined by the stiffness of extracellular matrix. In lung fibrosis, stiff lung tissue promotes the transformation of myofibroblasts, which secrect an excess of extracellular matrix proteins, and play an central role in the progression of fibrosis. To screen which transcripts were involved in stiff matrix induced myofibroblasts transformation, mouse fibroblasts (NIH3T3) cultured on different stiffness (1 KPa and 4 KPa) hydrogels coated with rat tail type I collagen for 48h, then the whole -genome transcriptional profiles of fibroblasts were analyzed. Of the 26,423 detected transcripts, 170 were significantly upregulated and 98 were significantly downregulated (p<0.05) in fibroblasts cultured on stiff matrix. KEGG pathway enrichment analyses found that ECM-receptor interaction and Focal adhesion were the first two significantly enriched pathway. Overall design: Fibroblasts were cultured on soft and stiff matrix for 48h, and then cells were collected for microarray experiments.
Project description:Hippo signaling pathway is pivotally involved in human cancer. Among the Hippo components, YAP1 is highly active while function of MST1,2 and SAV1 was lost in liver cancer. Based on systematic analysis, we identified KLF5 as YAP1 binding partner in silico. To investigate KLF5 in liver cancer, we performed the gene expression microarray after knocked down YAP1, TEAD1 and KLF5 in SK-Hep1 cell line. To identify the role of YAP1, TEAD1 and KLF5 in hepatocellular carcinoma cell line, we performed microarray after knocking down YAP1, TEAD1 and KLF5 in hepatocellular carcinoma cell line (3 siLuc, 3 siYAP1, 3 siTEAD1, 3 siKLF5)
Project description:Vascular extracellular matrix (ECM) stiffening is a risk factor for aortic and coronary artery disease. How matrix stiffening regulates the transcriptome profile of human aortic (Ao) and coronary (Co) vascular smooth muscle cells (VSMCs) is not well understood. Furthermore, the role of long non-coding RNAs (lncRNAs) in the cellular response to stiffening has never been explored. This study characterizes the stiffness-sensitive transcriptome of human Ao and Co VSMCs and identify potentially key lncRNA regulators of stiffness-dependent VSMC functions. Ao and Co VSMCs were cultured on hydrogel substrates mimicking physiologic and pathologic ECM stiffness. Total RNA-seq was performed to compare the stiffness-sensitive transcriptome profiles of Ao and Co VSMCs. Overall design: 4 unique sample types with 4 replicates (16 total samples). Donor-matched Aortic and Coronary VSMCs were serum starved for 48 hours, then cultured in serum containing media for 24 hours on both soft and stiff fibronectin coated hydrogel matrices.
Project description:Renal tubular atrophy and interstitial fibrosis are common hallmarks of etiologically different progressive chronic kidney diseases (CKD) that eventually result in organ failure. We identify Dickkopf-3 (Dkk3) as a stress-induced, tubular epithelia-derived mediator of kidney fibrosis. Genetic as well as antibody-mediated abrogation of Dkk3 led to reduced tubular atrophy and decreased interstitial matrix accumulation in two mouse models of renal fibrosis. This was accompanied by an amplified, anti-fibrogenic, inflammatory response within the injured kidney. Mechanistically, Dkk3 deficiency led to diminished canonical Wnt/β-catenin signaling in stressed tubular epithelial cells. To identify global changes in gene expression due to the lack of Dkk3, whole-transcriptome sequencing (mRNA-seq) was performed on RNA isolated from kidneys of Wt and Dkk3-/- mice 7 days after UUO. Overall design: Kidneys of Dkk3-/- and C57Bl/6 mice were harvested 7 days after UUO. Total RNA was isolated and each Sample was prepared as a 10-plex, which was sequenced on three lanes. HiSeq 2000 50bp SR run was used.
Project description:Transcriptional profiling of mouse chondrocytes cells comparing control untreated chondrocytes cells with chondrocytes cells plated in stiff and soft ECM. Goal was to determine the effects of ECM stiffness on gene expression. Overall design: Three-condition experiment: normal, stiff ECM, soft ECM. Biological replicates: 3 normal replicates, 3 stiff ECM replicates, 3 soft ECM replicates.
Project description:Primary human hepatic stellate cells (HSCs) isolated from healthy patients were purchased from Sciencell. They were seeded on either 400 Pa or 25000 Pa polyacrylamide hydrogels for 2 days and collected for RNA isolation. The goal of this study is to determine genes transcriptionally regulated by a stiff matrix. 2 RNA samples of HSCs on 400 Pa and 3 RNA samples of HSCs on 25600 Pa were sent to the University of Minnesota Genomics Center for RNA sequencing. Overall design: 2 RNA samples isolated from HSC on 400 Pa stiffness and 3 from HSCs on 25600 Pa stiffness. They were converted to Illumina sequencing libraries using Illumina’s Truseq Stranded mRNA Sample Preparation Kit. Truseq libraries were then subjected to cluster using Illumina cBot instrument and sequencing using HiSeq2500
Project description:Global transcriptome analysis showed that human lymphatic endothelial cells (LECs) grown on a soft matrix exhibit increased GATA2 expression, concomitant with a GATA2-dependent upregulation of genes involved in cell migration and lymphangiogenesis, including the key lymphangiogenic growth factor receptor VEGFR3. Affymetrix GeneChip analysis revealed regulation of 2771 transcripts above or below a 1.4-fold change (log2 fold change >0.5 or <-0.5) threshold on soft versus stiff matrices. Moreover, 406 (27%) of the 1485 transcripts that were increased and 207 (16 %) of the 1286 transcripts that were decreased on soft matrix were regulated in a GATA2 dependent manner. Overall design: In total 18 samples were analyzed. To identify differentially expressed genes between the ctrl stiff and ctrl soft groups, a stepwise analysis with 6 biological replicates was performed. First, exon set ID’s with an average expression lower than 5 were considered as not significantly expressed and excluded from the analysis. A threshold of 40% increase (>0.5 log2 fold change) or decrease (<-0.5 log2 fold change) of gene expression on the soft matrix (vs. stiff matrix) was considered for further analysis. For all genes with 3 or more exon probe set ID’s regulated above the defined thresholds, the average log2 fold change of the regulated exon probe ID’s was calculated and used to generate the final list of genes regulated by matrix stiffness. To determine which of the genes are regulated in a GATA2 dependent manner, a stepwise analysis with 2 biological replicates ctrl siRNA soft vs GATA2 siRNA soft groups was performed. The previously identified exon probe ID’s for genes differentially regulated between the ctrl stiff and ctrl soft groups were extracted and their average log2 fold change was calculated for the new data set. A threshold of 40% increase (>0.5 log2 fold change) or decrease (<-0.5 log2 fold change) of gene expression in the absence of the GATA2 was used to generate the final list of genes.
Project description:Performing Chromatin IP of Klf2, Klf4, Klf5 and p53 in mouse embryonic stem cells with NimbleGen custom genomic tiling arrays, we sought to decipher Klf2, Klf4, Klf5-regulated genes. Keywords: genomic tiling arrays, ES cells Overall design: 12 samples: Chromatin IP of Klf2, Klf4, Klf5 and p53 in mouse embryonic stem cells with NimbleGen custom genomic tiling arrays; three independent experimental replicates for each experimental condition were performed.
Project description:Performing Chromatin IP of Klf2, Klf4, Klf5 and p53 in mouse embryonic stem cells with NimbleGen custom genomic tiling arrays, we sought to decipher Klf2, Klf4, Klf5-regulated genes. 12 samples: Chromatin IP of Klf2, Klf4, Klf5 and p53 in mouse embryonic stem cells with NimbleGen custom genomic tiling arrays; three independent experimental replicates for each experimental condition were performed.
Project description:We tested the hypothesis that increasing matrix stiffness on which normal human lung fibroblasts are grown promotes the expression of a fibrogenic cellular transcriptomic program. Keywords: Human lung fibroblast, matrix stiffness, PTGS2, COX-2, Prostaglandin E2 Total RNA extracted from normal human lung fibroblasts from 3 human subjects and separately grown on 5 discrete matrix stiffness conditions: 100, 400, 1600, 6400, 25600 Pascals.