61 research outputs found

    CPT II activities and thermal instability in CPT II-deficient fibroblasts.

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    <p>Fibroblast lysates were preincubated at 37°C or 41°C. Enzymatic reactions commenced by the addition of substrates at 37°C. Data are the means of five separate experiments. The average of three independent experiments is shown ± SEM (*P<0.05).</p

    Reduction of mitochondrial membrane potential of control and variant CPT IIs in cultured fibroblasts.

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    <p>Control (A, D), V368I (homozygous) (B, E), and F352C (heterozygous) + V368I (homozygous) (C, F) fibroblasts were cultured at 37°C and 41°C. Mitochondrial depolarization was monitored by 15 min treatment with 10 μM of JC-1 in the dark and visualized under a fluorescence microscope. Scale bars, 100 μm.</p

    CPT II expression and the dominant—negative effect of <i>CPT II</i> variants on substrate-dependent kinetics.

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    <p>(A) CPT II expression was analyzed by Western Blotting with an anti-CPT II antibody. (B) Dominant—negative effect of <i>CPT II</i> variants was analyzed by substrate-dependent kinetics. (C) Relative protein expression of control and variant CPT II. (D) Real-time PCR analysis of control and variant <i>CPT II</i> expression. Lane 1, control fibroblasts; lane 2, V368I (heterozygous); lane 3, V368I (homozygous); lane 4, F352C (heterozygous) + V368I (homozygous). CPT II protein was expressed relative to β-actin. <i>V</i><sub>max</sub> and <i>K</i><sub>m</sub> values were obtained from kinetic analysis (1/V versus 1/[S] plots) by varying the concentrations of L-[methyl-<sup>3</sup>H] carnitine between 0–300 μM at a fixed 50 μM palmitoyl-CoA concentration. (●) control, (▲) V368I (Homo), (◆) F352C (Hetero) + V368I (Homo). Data are means of three separate experiments. The average of three independent experiments is shown ± SEM (*P<0.05)</p

    Pulse-chase (left) and half-lives (right) of control and variant CPT II in fibroblasts.

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    <p>Cultured fibroblasts were pulse-labeled with L-[<sup>35</sup>S] methionine for 2 h and chased for 0, 6, 12, and 18 h. CPT II from fibroblast lysates was immunoprecipitated with anti-CPT II antibodies, then subjected to SDS-PAGE followed by autoradiography. (●) control, (■) V368I (homozygous), (▲) F352C (heterozygous) + V368I (homozygous). The average of three independent experiments is shown ± SEM.</p

    Cell apoptosis analysis of <i>CPT II</i> variants in fibroblasts.

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    <p>(A) Apoptotic fibroblasts were measured by flow cytometry. Fibroblasts incubated with DMSO were used as a control. (B) LDH assay of control and <i>CPT II</i> variants in fibroblasts. (C) Apoptotic factor release was analyzed by Western Blotting with antibodies against caspase-3, caspase-8, cytochrome c, and Bid for control and variant CPT IIs in control and CPT II-deficient fibroblasts. CPT II proteins are expressed relative to β-actin. Data are means of three separate experiments. The average of three independent experiments is shown ± SEM (*P<0.05).</p

    TGF-β Negatively Regulates CXCL1 Chemokine Expression in Mammary Fibroblasts through Enhancement of Smad2/3 and Suppression of HGF/c-Met Signaling Mechanisms

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    <div><p>Fibroblasts are major cellular components of the breast cancer stroma, and influence the growth, survival and invasion of epithelial cells. Compared to normal tissue fibroblasts, carcinoma associated fibroblasts (CAFs) show increased expression of numerous soluble factors including growth factors and cytokines. However, the mechanisms regulating expression of these factors remain poorly understood. Recent studies have shown that breast CAFs overexpress the chemokine CXCL1, a key regulator of tumor invasion and chemo-resistance. Increased expression of CXCL1 in CAFs correlated with poor patient prognosis, and was associated with decreased expression of TGF-β signaling components. The goal of these studies was to understand the role of TGF-β in regulating CXCL1 expression in CAFs, using cell culture and biochemical approaches. We found that TGF-β treatment decreased CXCL1 expression in CAFs, through Smad2/3 dependent mechanisms. Chromatin immunoprecipitation and site-directed mutagenesis assays revealed two new binding sites in the CXCL1 promoter important for Smad2/3 modulation of CXCL1 expression. Smad2/3 proteins also negatively regulated expression of Hepatocyte Growth Factor (HGF), which was found to positively regulate CXCL1 expression in CAFs through c-Met receptor dependent mechanisms. HGF/c-Met signaling in CAFs was required for activity of NF-κB, a transcriptional activator of CXCL1 expression. These studies indicate that TGF-β negatively regulates CXCL1 expression in CAFs through Smad2/3 binding to the promoter, and through suppression of HGF/c-Met autocrine signaling. These studies reveal novel insight into how TGF-β and HGF, key tumor promoting factors modulate CXCL1 chemokine expression in CAFs.</p></div

    HGF positively regulates CXCL1 expression in mammary fibroblasts.

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    <p>(A) The indicated cell lines were analyzed for HGF expression in conditioned media by ELISA. (B-C) 41CAFs expressing control siRNA (Con) or siRNAs to HGF were analyzed for expression of HGF (B) or CXCL1 (C) by ELISA. (D) 41CAFs were treated with increasing concentrations of HGF for 48 hours and analyzed for CXCL1 expression by ELISA. Statistical analysis was performed using Two Tailed T-Test (B,C), or One Way ANOVA followed by Bonferonni post-hoc comparisons (A,D). Statistical significance was determined by p<0.05; *p<0.05, **p<0.01, ***p<0.001. Values are expressed as Mean ± SEM.</p

    TGF-β enhances binding of Smad2 and Smad3 to the CXCL1 promoter.

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    <p>Genomic DNA was isolated from 41CAFs treated with 5 ng/ml of TGF-β for 6 hours, and immunoprecipated with: control IgG, or antibodies to Smad2 or Smad3. Samples were analyzed by real-time PCR analysis for (A) Smad2 or (B) Smad3 binding to SBE1 and SBE2 or SBE2 alone on the CXCL1 promoter. Smad2/3 binding to the PAI1 promoter was evaluated as a positive control for TGF-β responsiveness. Background from IgG control was subtracted from samples. Left panels show fraction of Smad2 or 3 binding to DNA relative to input control. Right panel shows Smad2 or Smad3 binding normalized to (-) TGF-β group for each promoter region. Statistical analysis was performed using Two Tailed T-test. Statistical significance was determined by p<0.05; *p<0.05, **p<0.01. Values are expressed as Mean ± SEM.</p

    c-Met receptor tyrosine kinases positively regulate CXCL1 expression in mammary CAFs.

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    <p>(A) Normal breast (n = 3) or breast tumor tissues (n = 4) were immunostained for c-Met expression. Magnified inset shows representative staining in fibroblasts. E = epithelium, BV = blood vessel. Scale bar = 50 microns. Secondary antibody control = anti-rabbit-biotinylated. (B) 311NAFs and 41CAFs were analyzed for c-Met expression by immunoblot analysis. c-Met expression was normalized to actin by densitometry analysis. 4T1 mammary carcinoma cells are shown as a positive control. (C) 41CAFs and 311 NAFs were treated with 200 nM c-Met kinase inhibitor type II (CKII) for 1 hour, and analyzed for expression of phospho-c-Met (Tyr-1234/1235) by immunoblot. (D) 41CAFs or 311NAFs treated with CKII for 48 hours were analyzed for CXCL1 expression by ELISA. Statistical analysis was performed using Two Tailed T-Test. Statistical significance was determined by p<0.05; *p<0.05, n.s; not significant. Values are expressed as Mean ± SEM.</p
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