Effect of Ras/Raf-1 modulation on transforming growth factor-beta-1 (TGF-β1)-induced expression of the gene

<p><b>Copyright information:</b></p><p>Taken from "Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes"</p><p>http://arthritis-research.com/content/9/6/R122</p><p>Arthritis Research & Therapy 2007;9(6):R122-R122.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2246241.</p><p></p> Rat chondrocytes were electroporated with empty vector, wild-type, constitutively active, or dominant-negative plasmids for Ras or Raf-1 (2 μg/well of six-well plate) before stimulation with 10 ng/mL of TGF-β1 for 12 hours. Total RNA was extracted and subjected to real-time polymerase chain reaction analysis. The mRNA level of Ank was normalized to that of S29 mRNA and is expressed as mean percentages (± standard deviation) over control values from three independent experiments. Statistically significant differences from the control are indicated as *< 0.05 and from TGF-β1-treated cells as #< 0.05. Effect of TGF-β1 on extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in electroporated cells with wild-type, constitutively active, or dominant-negative plasmids for Ras (2 μg/well of six-well plate). Total proteins were extracted from rat chondrocytes exposed to 10 ng/mL of TGF-β1 for 15 minutes and subjected to Western blotting using anti-phospho- and anti-total-ERK 1/2 antibodies. The relative abundance of these proteins was normalized to that of β-actin protein

Respective contributions of and to transforming growth factor-beta-1 (TGF-β1)-induced increase in extracellular inorganic pyrophosphate (ePPi) production

<p><b>Copyright information:</b></p><p>Taken from "Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes"</p><p>http://arthritis-research.com/content/9/6/R122</p><p>Arthritis Research & Therapy 2007;9(6):R122-R122.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2246241.</p><p></p> Effect of small interfering RNA (siRNA) on Ank and PC-1 mRNA levels. Rat chondrocytes were transfected with siRNA 24 hours before TGF-β1 stimulation. Total RNA was extracted from rat chondrocytes exposed to 10 ng/mL of TGF-β1 for 12 hours (Ank) or 24 hours (PC-1) and then subjected to real-time polymerase chain reaction analysis. The level of Ank, PC-1, and L27 mRNAs was normalized to that of S29 mRNA and expressed as mean percentages (± SD) over control values. Effect of Ank or PC-1 siRNA on ePPi levels. Shown are levels of ePPi in culture supernatant of rat chondrocytes transfected with siRNA and then stimulated for 12 or 24 hours with 10 ng/mL of TGF-β1. ePPi levels were normalized to the amount of total cell proteins (= 6) and are expressed as mean (± SD) in picomoles per microgram of protein. Statistically significant differences from the control are indicated as *< 0.05 and from TGF-β1-treated cells as #< 0.05

Effect of transforming growth factor-beta-1 (TGF-β1) on proteins regulating inorganic pyrophosphate metabolism

<p><b>Copyright information:</b></p><p>Taken from "Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes"</p><p>http://arthritis-research.com/content/9/6/R122</p><p>Arthritis Research & Therapy 2007;9(6):R122-R122.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2246241.</p><p></p> Phenotypic characterization of chondrocytes. Total RNA was extracted from untreated rat chondrocytes and subjected to real-time polymerase chain reaction (PCR) analysis. The relative abundance of gene mRNAs was normalized to that of S29 mRNA. Results are presented in histograms as mean percentages (± standard deviation [SD]) over S29 value. Effect of TGF-β1 on Ank, PC-1, and TNAP mRNA levels. Total RNA was extracted from rat chondrocytes exposed to 10 ng/mL of TGF-β1 from 1 to 48 hours and subjected to real-time PCR analysis. The relative abundance of gene mRNAs was normalized to that of S29 mRNA. Results are expressed as mean percentages (± SD) over control values. Statistically significant differences from the control are indicated as *< 0.05. Effect of TGF-β1 on ANK or PC-1 protein levels. Total proteins were extracted from rat chondrocytes exposed to 10 ng/mL of TGF-β1 from 6 to 48 hours and subjected to Western blotting using polyclonal anti-ANK and anti-PC-1 antibody. The protein band intensities were quantified by densitometry from enhanced chemiluminescence immunoblots. The relative abundance of these proteins was normalized to that of β-actin protein and expressed as induction folds over control value. N.D., not detected; TNAP, tissue-nonspecific alkaline phosphatase

4-MU4-deoxy-xyloside counteracts TGF-β1-induced fibroblast-to-myofibroblast trans-differentiation.

<p>Lung fibroblasts were treated with 500 μM of 4-MU4-deoxy-xyloside and/or TGF-β1 (5 ng/ml) for 24 h and the expression of αSMA was analyzed by qPCR (<b>A</b>) and by Western Blot (<b>B</b>). The relative expression was normalized with ribosomal protein S29. Data are expressed as mean ± SD of three separate experiments; *P<0.05 versus control. For Western blot, whole cell lysates were analysed αSMA content. β-actin was used as a loading control. <b>(C)</b> Bar graph shows the expression of αSMA relative to β-actin level and expressed relative to control lysate. Data are expressed as mean ± SD of three separate experiments; *P<0.05 versus control.</p

4-MU4-deoxy-xyloside antagonized TGF-β1-induced PG synthesis in lung fibroblasts.

<p>Cells were treated with 500 μM or 1 mM of 4-MU4-deoxy-xyloside or DMSO (control) and/or TGF-β1 (5 ng/ml) for 24 h and (<b>A</b>) the level of PG synthesis was measured by the ³⁵Sulfate incorporation for the last 6 h of the treatment. The radioactivity associated with GAGs was evaluated by liquid scintillation counting after CPC precipitation and DNA normalization. Data are expressed as mean ± SD of three separate experiments; *P<0.05 versus control. (<b>B</b>) GAG chains were radiolabelled by ³⁵Sulfate incorporation and isolated by CPC method at 24 h after the treatment and analyzed by SDS-PAGE then visualized by autoradiography. Bar graph shows the amount of ³⁵Sulfate GAGs normalized to DNA and relative to control. Data are expressed as mean ± SD of three separate experiments; *P<0.05 versus control.</p

4-MU4-deoxy-xyloside blocks TGF-β1 signaling.

<p>(<b>A</b>) Lung fibroblasts were co-transfected with (CAGA)₁₂-Lux reporter and pRL-TK (Renilla reporter) constructs and were incubated with DMSO (control) or with various concentrations of 4-MU4-deoxy-xyloside in the presence and absence TGF-β1 (5 ng/ml) for 24 h. The induction of the (CAGA)₁₂-Lux reporter was measured by luciferase assay. Luciferase activities were normalized to pRL-TK vector activity. Data are expressed as mean ± SD of three separate experiments; *P<0.05 versus control. <b>(B)</b> Cells were incubated with 500 μM of 4-MU4-deoxy-xyloside or <b>(C)</b> with 500 μM of 4-MU-xyloside for 24 h then stimulated or not with TGF-β1 (5 ng/ml) for 3 h and assayed by Western blot to assess the levels of phosphorylation of Smad2 and Smad3. Bar graphs show the expression of phospho-Smad2 and phospho-Smad3 relative to total Smad2/3 level normalized to β-actin and expressed relative to control lysates. Data are expressed as mean ± SD of three separate experiments; *P<0.05 versus control.</p

4-MU4-deoxy-xyloside inhibits PG synthesis.

<p>(<b>A</b>) Primary rat lung fibroblasts were treated with various concentrations of 4-MU4-deoxy-xyloside for 24 h and the level of the PG synthesis was measured by ³⁵Sulfate incorporation for the last 6 h of the treatment. The control was treated with vehicle (DMSO). PG synthesis level was normalized to the amount of DNA. (<b>B</b>) Lung primary fibroblasts were incubated with or without 100 μM of 4-MU-xyloside in the presence and absence of 4-MU4-deoxy-xyloside (500 μM) and GAGs were metabolically labelled for 24 h with ³⁵Sulfate incorporation. Radiolabeled GAGs were isolated from the medium by CPC precipitation and analyzed by SDS-PAGE then visualized by autoradiography. Bar graph shows total ³⁵Sulfated GAGs normalized to DNA and relative to control (untreated). Data are expressed as mean ± SD of three separate experiments. *P<0.05 versus control.</p

4-MU4-deoxy-xyloside reduces cell viability and inhibits cell proliferation.

<p>Lung fibroblasts were treated with DMSO (Control) or with 500 μM of 4-MU4-deoxy-xyloside and/or TGF-β1 (5 ng/ml) for 24 h. <b>(A)</b> Cell viability was measured by MTT assay. The relative cell viability (%) was expressed as a percentage relative to control cells. <b>(B)</b> Cell proliferation was analyzed by CSFC labelling. <b>(C)</b> Bar graph shows cell proliferation expressed as percentage relative to control cells (DMSO). Data are expressed as mean ± SD of three separate experiments. *P<0.05 versus control.</p

Effect of Smad 7 overexpression on transforming growth factor-beta-1 (TGF-β1)-induced responses in rat chondrocytes

<p><b>Copyright information:</b></p><p>Taken from "Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes"</p><p>http://arthritis-research.com/content/9/6/R122</p><p>Arthritis Research & Therapy 2007;9(6):R122-R122.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2246241.</p><p></p> Rat chondrocytes were electroporated with either empty vector or wild-type Smad 7 overexpressing plasmid (2 μg/well of six-well plate) and then treated for 12 hours with 10 ng/mL of TGF-β1. Total RNA was extracted and subjected to real-time polymerase chain reaction analysis. The mRNA level of aggrecan and Ank was normalized to that of S29 mRNA and is expressed as mean percentages (± standard deviation) over control values from three independent experiments. Statistically significant differences from the control are indicated as *< 0.05 and from TGF-β1-treated cells as #< 0.05

4-MU4-deoxy-xyloside reduced TGF-β1-induced increased expression of collagen type 1.

<p>Lung fibroblasts were treated with 500 μM of 4-MU4-deoxy-xyloside and/or TGF-β1 (5 ng/ml) for 6 h and the expression of endogenous TGF-β1was analyzed by qPCR. The relative expression was normalized with ribosomal protein S29. Data are expressed as mean ± SD of three separate experiments, (*P<0.05).</p