Unknown,Transcriptomics,Genomics,Proteomics

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In TCR-stimulated T-cells, N-ras regulates specific genes and signal transduction pathways

ABSTRACT: It has been recently shown that N-ras plays a preferential role in immune cell development and function; specifically: N-ras, but not H-ras or K-ras, could be activated at and signal from the Golgi membrane of immune cells following a low level TCR stimulus. The goal of our studies was to test the hypothesis that N-ras and H-ras played distinct roles in immune cells at the level of the transcriptome. First, we showed via mRNA expression profiling that there were over four hundred genes that were uniquely differentially regulated either by N-ras or H-ras, which provided strong evidence in favor of the hypothesis that N-ras and H-ras have distinct functions in immune cells. We next characterized the genes that were differentially regulated by N-ras in T cells following a low-level TCR stimulus. Of the large pool of candidate genes that were differentially regulated by N-ras downstream of TCR ligation, four genes were verified in qRT-PCR-based validation experiments as being differentially regulated by N-ras (Dntt, Slc9a6, Chst1, and Lars2). Finally, although there was little overlap between individual genes that were regulated by N-ras in unstimulated thymocytes and stimulated CD4+ T-cells, there was a nearly complete correspondence between the signaling pathways that were regulated by N-ras in these two immune cell types. Since we were interested primarily in genes that were differentially regulated by N-ras following a low-level TCR stimulus, our microarray data comparison was between data from TCR-stimulated, WT CD4+ T-cells and from TCR-stimulated, N-ras KO CD4+ T-cells. Genes that were differentially regulated in the comparison between stimulated N-ras KO CD4+ T-cells and unstimulated N-ras KO CD4+ T-cells, as well as those genes that were differentially regulated in the comparison between stimulated WT CD4+ T-cells and unstimulated WT CD4+ T-cells were excluded from this analysis. To determine if N-ras and H-ras regulate different sets of genes in thymocytes, a comparison was made between the set of genes that were differentially regulated by N-ras in the [WT] vs. [N-ras KO] comparison and the set of genes that were differentially regulated by H-ras in the [WT] vs. [H-ras KO] comparison. RNA was extracted from CD4+ T cell splenocytes isolated from 6-20 week old N-Ras KO and WT mice following growth in T cell growth media either with or without 1 microgram/milliliter ant-CD3 and anti-CD28 antibodies. RNA was extracted from thymocytes isolated directly from 6-20 week old N-Ras KO, H-Ras KO and WT mice.

ORGANISM(S): Mus musculus

SUBMITTER: stephen Lynch

PROVIDER: E-GEOD-45739 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:It has been recently shown that N-ras plays a preferential role in immune cell development and function; specifically: N-ras, but not H-ras or K-ras, could be activated at and signal from the Golgi membrane of immune cells following a low level TCR stimulus. The goal of our studies was to test the hypothesis that N-ras and H-ras played distinct roles in immune cells at the level of the transcriptome. First, we showed via mRNA expression profiling that there were over four hundred genes that were uniquely differentially regulated either by N-ras or H-ras, which provided strong evidence in favor of the hypothesis that N-ras and H-ras have distinct functions in immune cells. We next characterized the genes that were differentially regulated by N-ras in T cells following a low-level TCR stimulus. Of the large pool of candidate genes that were differentially regulated by N-ras downstream of TCR ligation, four genes were verified in qRT-PCR-based validation experiments as being differentially regulated by N-ras (Dntt, Slc9a6, Chst1, and Lars2). Finally, although there was little overlap between individual genes that were regulated by N-ras in unstimulated thymocytes and stimulated CD4+ T-cells, there was a nearly complete correspondence between the signaling pathways that were regulated by N-ras in these two immune cell types. Since we were interested primarily in genes that were differentially regulated by N-ras following a low-level TCR stimulus, our microarray data comparison was between data from TCR-stimulated, WT CD4+ T-cells and from TCR-stimulated, N-ras KO CD4+ T-cells. Genes that were differentially regulated in the comparison between stimulated N-ras KO CD4+ T-cells and unstimulated N-ras KO CD4+ T-cells, as well as those genes that were differentially regulated in the comparison between stimulated WT CD4+ T-cells and unstimulated WT CD4+ T-cells were excluded from this analysis. To determine if N-ras and H-ras regulate different sets of genes in thymocytes, a comparison was made between the set of genes that were differentially regulated by N-ras in the [WT] vs. [N-ras KO] comparison and the set of genes that were differentially regulated by H-ras in the [WT] vs. [H-ras KO] comparison.

Project description:Serum-dependent transcriptional networks identify distinct functional roles for H-ras and N-ras during initial stages of the cell cycle; Using oligonucleotide microarrays, we compared gene expression transcriptional profiles corresponding to the initial cell cycle stages of mouse fibroblasts lacking H-Ras and/or NRas with those of matching, WT controls. The similarity of transcription profiles among serum-starved fibroblasts of all different WT and ras knockout genotypes tested, indicated that H-Ras and N-Ras do not play significant roles in control of transcriptional responses to serum deprivation stress. In contrast, genomic disruption of H-ras or N-ras, individually or in combination, determined highly specific, differential gene expression profiles in response to post-starvation stimulation with serum for 1 hour (G0/G1 transition) or for 8 hours (mid-G1 progression). The absence of N-Ras caused significantly higher changes than the absence of H-Ras on the wave of transcriptional activation linked to G0/G1 transition. In contrast, the absence of H-Ras affected more potently the profile of the transcriptional wave detected during mid-G1 progression. Functional analysis demonstrated a predominant functional association of H-Ras with growth and proliferation, whereas N-Ras exhibited a closer functional link to development or cell cycle regulation as well as immunomodulation and apoptosis. Mechanistic analysis indicated that ERK-dependent activation of Stat1 mediates the regulatory effect of N-Ras on defense and immunity, whereas the pro-apoptotic effects of N-Ras are mediated through ERK and p38 signaling. Our observations support previous reports of an absolute requirement for different peaks of Ras activity during the initial stages of the cell cycle and confirm the notion of functional specificity for the H-Ras and N-Ras isoforms. Experiment Overall Design: Mus musculus cell lines from the appropriate ras genotype were harvested on Dulbeccoâs modified Eagleâs medium (DMEM) supplemented with fetal bovine serum (10% FBS), glutamine (2mM), penicillin (100 U/ml) and streptomycin (100 mg/ml). Cultures were grown in a humidified CO2 (5%) atmosphere at 37Â°C and when subconfluent cells were starved for 24 hours. After starvation cells were either used for RNA isolation, or induced for 1 hour or 8 hours with 20% fetal bovine serum and then RNA extraction and isolation was carried out for hybridization on Affymetrix microarrays.

Project description:We have compared the response to TGFbeta1 in normal and v-rasHa transduced primary mouse keratinocytes using NCI cDNA microarrays. This analysis reveals that Ha-ras alters global TGFbeta1 mediated gene expression in a gene specific manner. The expression pattern of TGFbeta1 immediate early response genes and down regulation of cell cycle control genes is not altered by Ha-ras but the induction of most extracellular matrix genes is blocked. Using Smad3 null keratinocytes, we find that the majority of TGFbeta1 responsive genes in primary keratinocytes are Smad3 dependent, but patterns of transcriptional responses suggest that the ability of Ha-ras to block TGFbeta1 mediated gene expression is not dependent on Smad3. However, the combination of oncogenic ras and loss of Smad3 prevents the TGFbeta1 dependent suppression of genes associated with cell cycle progression and apoptosis observed with either genotype alone. In addition several extracellular matrix genes are super-repressed by TGFbeta1 in the Ha-ras Smad3 null keratinocytes. These data provide a genomic framework for understanding how disruptions of TGFbeta signaling and oncogenic ras cooperate to promote premalignant progression of primary keratinocytes. Keywords: time series design RNA from Balb/c primary and ras keratinocytes treated for 1, 6, 24, and 48 hours with TGFbeta1 at 1ng/ml was compared to RNA from the corresponding untreated controls. For each timepoint sample from primary and ras cells, multiple arrays (including dye swaps) were analyzed. There are 6, 7, 6, and 5 arrays analyzed for RNA from primary keratinocytes treated for 1, 6, 24, and 48 hours as compared to RNA from untreated primary cells, respectively. There are 6, 8, 7, and 5 arrays analyzed for RNA from ras keratinocytes treated for 1, 6, 24, and 48 hours as compared to RNA from untreated ras cells, respectively. There are 50 arrays analyzed for the Balb/c time-course dataset. RNA from Smad3 WT and KO primary and ras keratinocytes treated 48 hours with TGFbeta1 were compared to RNA from its untreated corresponding controls. There are 6 arrays analyzed for the Smad3 WT/KO data set. There are 56 arrays in this series.

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In TCR-stimulated T-cells, N-ras regulates specific genes and signal transduction pathways

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