Stimuli Bypassing LAT Deficiency in J.CaM2 Reduce Elevated <i>RAG-1</i> Expression

<div><p>(A) LAT-deficient J.CaM2 cells were stimulated with PMA or PDBu and analyzed for <i>RAG-1</i> mRNA expression by Northern blot analysis. In (A), (B), and (C), the same blot was hybridized with a fragment encoding the constant region of <i>TCRα</i> and with <i>β-actin</i> to control for stimulation and loading.</p> <p>(B) Analysis of PMA-induced reduction of <i>RAG-1</i> expression levels in absence of protein synthesis blocked by cycloheximide treatment of J.CaM2 cells. Control hybridizations were carried out by hybridization with the constant region of <i>TCRα</i> and with <i>β-actin</i>.</p> <p>(C) Analysis of <i>RAG-1</i> transcripts in J.CaM2-HM1-1.1 cells stimulated for various time intervals with carbachol to trigger the human muscarinic (HM) receptor subtype I on the surface of these cells.</p></div

Model of the Constitutive Signaling Pathway That Provides a Basal Repression of <i>RAG</i> Gene Transcription

<p>Constitutive signaling in resting thymoytes and our model Jurkat T cell line represses <i>RAG</i> gene expression. Chemical inhibition of signaling molecules, genetic modifications, or induced expression of genes that resulted in elevated <i>RAG</i> expression are summarized in the gray-shaded boxes. The tonic signal and therefore normal expression of <i>RAG</i> genes rely strongly on signaling through the adapter LAT. The effects of basal phosphorylation of LAT are twofold: (1) recruitment and activation of PLCγ generates low levels of second messengers that signal through calcium and Ras pathways, culminating in Erk kinase activity; and (2) phosphorylation of tyrosine residue 6 of LAT, likely establishing a LAT–PLCγ–Abl complex, is required for low-level kinase activity of Abl. Abl and Erk kinase activities deliver unique repressive signals to control <i>RAG</i> gene expression.</p

Erk and Abl Synergize to Repress <i>RAG-1</i> Expression and Require Tyrosine 132 in LAT

<div><p>(A) Jurkat T cells were stimulated with 5 ng/ml PMA for 24 h to mimic a low, constitutive MAPK signal. Intracellular FACS staining for phospho-Erk, phospho-Abl, and isotype control was performed on the indicated samples.</p> <p>(B) The combined effect of blocking both Abl and MEK-1 and MEK-2 on <i>RAG-1</i> expression was determined by Northern blot analysis. Cells were treated for 24 h.</p> <p>(C) <i>RAG-1</i> expression levels in LAT-deficient J.CaM2 cells treated with the designated inhibitors compared to vehicle-treated and Jurkat T cells as control.</p> <p>(D) Intracellular FACS analysis using phospho-Erk, phospho-Abl, and isotype control antibodies on Jurkat, J.CaM2, and J.CaM2 cells stably reconstituted with cDNA expression vectors carrying the indicated mutations in LAT.</p> <p>(E) Consequences of a signaling defective LAT molecule on <i>RAG-1</i> mRNA levels in the specified resting cell lines. LAT_Y→F6,7,8 reflects mutations in tyrosine residues 132, 171, and 191, whereas LAT_Y→F6 harbors only a single mutation at tyrosine position 132.</p></div

Gene Expression Analysis of Resting Jurkat T Cells and Derived Signaling Mutants

<div><p>(A) Basal gene expression of wild-type Jurkat T cells compared to Lck-deficient J.CaM1, CD45 (PTPRC)-deficient J45, LAT-deficient J.CaM2, ZAP-70-deficient P116, and SLP-76 (LCP2)-deficient J14 signaling mutants. High-quality data were analyzed using the Statistical Analysis of Microarrays software package (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000053#pbio.0000053-Tusher1" target="_blank">Tusher et al. 2001</a>). After selecting high-quality data, array elements that were 2.5-fold above or below the median on at least two microarrays were included (337 cDNA elements). These elements are displayed in hierarchical cluster format where rows represent genes and columns represent experimental samples. Colored pixels capture the magnitude of the response for any gene. Shades of red and green represent fold above and fold below the median, respectively. Black pixels reflect the median and gray pixels represent missing data. Data in 337 rows correspond to 321 unique cDNA clones, representing 269 unique genes. Jurkat T cells were analyzed in six individual samples and each mutant cell line in triplicate, and the sample dendrogram was generated by hierarchically clustering the arrays using the elements shown. To interactively explore the data, go to <a href="http://microarray-pubs.stanford.edu/cgi-bin/tonicsignal/fig1a/gx?n=fig1a" target="_blank">http://microarray-pubs.stanford.edu/cgi-bin/tonicsignal/fig1a/gx?n=fig1a</a>. Abbreviations: FYB, Fyn-binding protein; SELL, CD62L. </p> <p>(B) Basal gene expression differences between unstimulated LAT deficient (J.CaM2) and wild-type Jurkat T cells. Data presented in the 273 rows correspond to 251 unique cDNA clones and represent the 193 unique genes most differentially expressed between the two lines that were selected (false discovery rate, <1%). Elements were further filtered for being at least 2-fold different between the lines and for technically adequate measurements in at least five of six samples analyzed. These elements are displayed as in (A). Abbreviations: MAGEA-8, melanoma antigen, family A, 8; MAGEA-11, melanoma antigen, family A, 11. To interactively explore the data, go to <a href="http://microarray-pubs.stanford.edu/cgi-bin/tonicsignal/fig1b/gx?n=fig1b" target="_blank">http://microarray-pubs.stanford.edu/cgi-bin/tonicsignal/fig1b/gx?n=fig1b</a>.</p> <p>(C) Northern blot analysis of <i>RAG-1</i> gene expression in Jurkat, J.CaM2, J14, and cDNA-reconstituted cell lines J.CaM2-LAT and J14-76-11. RNA levels are indicated by <i>β-actin</i> hybridization. All Northern blots presented in this study are representative examples of three independent experiments, unless mentioned otherwise.</p> <p>(D) Resting Jurkat, TCRα-deficient (J.RT-T3.1), TCRβ-deficient (J.RT3-T3.5), and LAT-deficient (J.CaM2) cell lines were analyzed for <i>RAG-1</i> and <i>β-actin</i> mRNA expression by Northern blot analysis.</p></div

Erk and Abl Kinases Transduce Repressive Signals That Control <i>RAG</i> Gene Expression

<div><p>(A) Northern blot analysis for <i>RAG-1</i> gene expression in STI-571-treated Jurkat T cells and comparison to PP2- or Ro-318220-treated samples.</p> <p>(B) Wild-type thymocytes were treated with the indicated inhibitors for 20 h and analyzed for <i>Rag-1</i> expression.</p> <p>(C) Northern blot analysis of <i>Rag-1</i> expression in inhibitor-treated TCR transgenic OT-I thymocytes or wild-type thymocytes of littermate controls. The relative expression level of <i>Rag-1</i> was calculated from two independent experiments, and the mean expression and standard deviation (SD) are indicated.</p> <p>(D) Western blot analysis using RIPA lysates of the indicated cell lines. Protein levels of phosphorylated Erk-1 and Erk-2, Erk-1 and Erk-2, and c-Abl were determined in 4 × 10⁶ resting cells per sample.</p> <p>(E) Analysis of phospho-Erk-1 and Erk-2 levels in Jurkat T cells treated for 24 h with the indicated inhibitors prior to RIPA lysis. Equal loading is indicated by Erk-1 and Erk-2 levels determined by stripping and reprobing the same blot.</p></div

Proper Expression of <i>RAG-1</i> and <i>RAG-2</i> Requires a Signaling-Competent LAT Molecule

<div><p>(A) Graphic representation of human LAT. Conserved tyrosine residues in mammals are numbered 1 through 9, with the corresponding amino acid numbering below. Cysteine residues 26 and 29 are required for palmitoylation. Adapted from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000053#pbio.0000053-Lin1" target="_blank">Lin and Weiss (2001</a>).</p> <p>(B) <i>RAG-1</i> expression by Northern blotting in Jurkat T cells induced to express the phosphatase CD148 for increasing periods of time.</p> <p>(C) <i>RAG-1</i> and <i>RAG-2</i> expression by DNA array analysis. Expression of the two genes was set at 1 in wild-type Jurkat cells and compared to expression in J.CaM2 cells, in J.CaM2 reconstituted with wild-type LAT (J.CaM2-LAT), or with signaling-mutant LAT molecules (J.CaM2-LATallF and J.CaM2-LATCtoS). In addition, expression profiles were compared to J.CaM2 treated with DMSO, J.CaM2 stimulated with PMA (25 ng/ml for 24 h), and Jurkat cells incubated with PP2 (20μM for 24 h).</p> <p>(D) Expression profiles of genes with increased expression in J.CaM2 and a similar expression behavior as <i>RAG-1</i> and <i>RAG-2</i>. Abbreviations: ABCB10, ATP-binding cassette, subfamily B, member 10; DTR, diphteria toxin receptor; 1309069, similar to Rattus norvegicus nuclear-encoded mitochandrial elongation factor G; SCN2A2, sodium channel, voltage-gated, type II, α2 polypeptide; 2108230, unknown human expressed sequence tag.</p></div

LAT Is Required for Two Largely Separate Pathways Marked by Constitutive Phosphorylation of Erk and Abl Kinases

<div><p>(A) Intracellular FACS staining for phospho-Erk (I and IV), phospho-Abl (II and V), and isotype control (III and VI) in the indicated cell lines. Histograms are an example of a representative experiment. The accompanying bar graphs are depicted in the same color-coding and represent the mean levels of fluorescence and standard deviation of three independent assays for all experiments. Mean levels of phosphoproteins measured by fluorescence in a resting Jurkat population were arbitrarily set at 100, and the mean fluorescence of the isotype control samples is indicated by the dotted line as a point of reference. Specifics are mentioned in the text.</p> <p>(B) Analysis of tyrosine phosphorylation levels of c-Abl in the indicated cell samples. c-Abl or control immunopercipitations were immunoblotted for total tyrosine phosphorylation by 4G10. The same blot was stripped and reprobed for c-Abl. Doxycyclin was administered for 7 d to induce CD148 expression.</p> <p>(C) Resting Jurkat T cells were treated with the indicated inhibitors for 24 h, and intracellular FACS staining for phospho-Abl and isotype control was compared to that detected in vehicle-treated Jurkat cells and the J.CaM2 line. Bar graphs (representing three experiments) display the four conditions depicted in the histograms as well as three additional samples.</p></div

Identification and Annotation of a Common Serum Response in Fibroblasts

<div><p>(A) The fibroblast common serum response. Genes with expression changes that demonstrate coordinate induction or repression by serum in fibroblasts from ten anatomic sites are shown. Each row represents a gene; each column represents a sample. The level of expression of each gene in each sample, relative to the mean level of expression of that gene across all the samples, is represented using a red–green color scale as shown in the key; gray indicates missing data. Representative genes with probable function in cell cycle progression (orange), matrix remodeling (blue), cytoskeletal rearrangement (red), and cell–cell signaling (black) are highlighted by colored text on the right. Three fetal lung fibroblast samples, cultured in low serum, which showed the most divergent expression patterns among these samples (in part due to altered regulation of lipid biosynthetic genes [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020007#pbio-0020007-Chang1" target="_blank">Chang et al. 2002</a>]), are indicated by blue branches.</p> <p>(B) Identification of cell cycle-regulated genes in the common serum response signature. The expression pattern of each of the genes in (A) during HeLa cell cycle over 46 h after synchronization by double thymidine block is shown (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020007#pbio-0020007-Whitfield1" target="_blank">Whitfield et al. 2002</a>). Transit of cells through S and M phases during the timecourse, verified by flow cytometry, is indicated below. Approximately one-quarter of genes demonstrate a periodic expression patterns and are therefore operationally annotated as cell cycle genes; the remainder of the genes are used in further analyses to define the CSR.</p> <p>(C) Validation of annotation by temporal expression profiles. Timecourse of gene expression changes in a foreskin fibroblast culture after shifting from 0.1% to 10% FBS is shown. Global gene expression patterns were determined using cDNA microarrays containing 36,000 genes; genes whose transcript levels changed by at least 3-fold during the timecourse and those in (A) are displayed. The cell cycle genes identified in the analysis illustrated in (B) were found to have a distinct temporal expression pattern with coordinate upregulation at 12 h.</p></div

Histological Architecture of CSR Gene Expression in Breast Cancer

<p>Representative ISH of <i>LOXL2</i> and <i>SDFR1</i> and IHC of PLOD2, PLAUR, and ESDN are shown (magnification, 200×). Panels for <i>LOXL2</i>, PLAUR, PLOD2, and ESDN represent cores of normal and invasive ductal breast carcinoma from different patients on the same tissue microarray. Panels for <i>SDFR1</i> demonstrate staining in adjacent normal and carcinoma cells on the same tissue section. Arrows highlight spindle-shaped stromal cells that stain positive for <i>SDFR1</i> and PLOD2. No signal was detected for the sense probe for ISH or for control IHC without the primary antibody.</p

Survey of Fibroblast CSR Gene Expression in Human Cancers

<p>Expression patterns of available CSR genes in over 500 tumors and corresponding normal tissues were extracted, filtered as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020007#s4" target="_blank">Materials and Methods</a>, and organized by hierarchical clustering. The response of each gene in the fibroblast serum response is shown on the right bar (red shows activated; green shows repressed by serum). The strong clustering of the genes induced or repressed, respectively, in fibroblasts in response to serum exposure, based solely on their expression patterns in the tumor samples, highlights their coordinate regulation in tumors. The dendrograms at the top of each data display represent the similarities among the samples in their expression of the fibroblast CSR genes; tumors are indicated by black branches, normal tissue by green branches.</p