Journal: Arthritis and rheumatism

Article Title: Down-regulation of collagen and connective tissue growth factor expression with hepatocyte growth factor in lung fibroblasts from white scleroderma patients via two signaling pathways.

doi: 10.1002/art.22874

Figure Lengend Snippet: Figure 1. Effect of hepatocyte growth factor (HGF) and selected inhibitors on expression of connective tissue growth factor (CTGF) and type I collagen in lung fibroblasts from systemic sclerosis patients. A, Confluent cultures of lung fibroblasts were serum-starved for 24 hours, then incubated for 72 hours with or without HGF (50 ng/ml), MEK-1/2 inhibitor U0126 (1 M), matrix metalloproteinase (MMP) inhibitor GM1489 (GM; 10 nM), or NF-B inhibitor BAY 11-7082 (BAY; 10 M). In some experiments, cells were transfected with Grb2 small interfering RNA (siRNA), MMP-1 siRNA, or control siRNA, as described in Materials and Methods. The cells were collected and then analyzed by Western blotting using anti-CTGF and anti–type I collagen antibodies. Anti–-actin was used as a sample loading control; anti-Grb2 was used to validate the knockdown effect of Grb2 siRNA. Cell culture supernatant was collected and subjected to enzyme-linked immunosorbent assay to measure the depletion of MMP-1 siRNA. Findings with control siRNA treatment were identical to findings with serum-free medium (lane 1). Immunoblots are representative of 3 independent experiments. B, The results of immunoblot analysis were quantified. This analysis represents bands corresponding to the 1 and 2 chains of type I collagen; numbers below the columns correspond to the lanes in A. Values are the mean and SD from 3 independent experiments. P 0.05 versus unstimulated cells.

Article Snippet: Recombinant human HGF and the Quantikine human proMMP-1 immunoassay kit were purchased from R&D Systems (Minneapolis, MN); an EnzoLytePlus 520 MMP-1 assay kit was obtained from AnaSpec (San Jose, CA); a NoShift transcription factor assay kit, NoShift NF- B (p65) reagents, and a NucBuster protein extraction kit were purchased from Novagen (Madison, WI); MEK-1/2 inhibitor U0126, MMP inhibitor GM1489, and NF- B inhibitor BAY 11-7082 were purchased from EMD Biosciences (San Diego, CA); Grb2 siRNA, MMP-1 siRNA, control siRNA-A, anti- CTGF, and fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti–type I collagen was obtained from Southern Biotechnology (Birmingham, AL); and anti– p44/p42 MAPK (anti–ERK-1/2), anti–phospho–p44/p42 MAPK, and anti-I B were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Incubation, Transfection, Small Interfering RNA, Control, Western Blot, Knockdown, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Arthritis and rheumatism

Article Title: Down-regulation of collagen and connective tissue growth factor expression with hepatocyte growth factor in lung fibroblasts from white scleroderma patients via two signaling pathways.

doi: 10.1002/art.22874

Figure Lengend Snippet: Figure 2. Effect of HGF on p44/p42 MAPK (ERK-1/2) phosphorylation in lung fibroblasts from systemic sclerosis (SSc) patients. A, HGF-induced ERK-1/2 phosphorylation was determined in cells treated for various time periods with HGF (50 ng/ml). Cell extracts were immunoblotted with anti–phospho–ERK-1/2 (ph-ERK-1/2) or anti–ERK-1/2 (as a control) (see Materials and Methods). B, Dose-dependence of ERK-1/2 phosphorylation was determined by adding HGF at varying doses (5–500 ng/ml) to SSc lung fibroblasts for 10 minutes. C, Lung fibroblasts were transfected with or without Grb2 siRNA, MMP-1 siRNA, and control siRNA or pretreated for 40 minutes with or without MEK-1/2 inhibitor U0126 (1 M), MMP inhibitor GM1489 (10 nM), or NF-B inhibitor BAY 11-7082 (10 M), then incubated with HGF (50 ng/ml) for 10 minutes. Anti-Grb2 was used to validate the knockdown effect of Grb2 siRNA. Cell culture supernatant was collected and subjected to enzyme-linked immunosorbent assay to measure the depletion of MMP-1 siRNA. Findings with control siRNA treatment were identical to findings with serum-free medium (lane 1). Quantitative results of densitometric analysis of immunoblots are also shown; values are the mean and SD from 3 independent experiments. P 0.05 versus unstimulated cells. See Figure 1 for other definitions.

Article Snippet: Recombinant human HGF and the Quantikine human proMMP-1 immunoassay kit were purchased from R&D Systems (Minneapolis, MN); an EnzoLytePlus 520 MMP-1 assay kit was obtained from AnaSpec (San Jose, CA); a NoShift transcription factor assay kit, NoShift NF- B (p65) reagents, and a NucBuster protein extraction kit were purchased from Novagen (Madison, WI); MEK-1/2 inhibitor U0126, MMP inhibitor GM1489, and NF- B inhibitor BAY 11-7082 were purchased from EMD Biosciences (San Diego, CA); Grb2 siRNA, MMP-1 siRNA, control siRNA-A, anti- CTGF, and fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti–type I collagen was obtained from Southern Biotechnology (Birmingham, AL); and anti– p44/p42 MAPK (anti–ERK-1/2), anti–phospho–p44/p42 MAPK, and anti-I B were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Control, Transfection, Incubation, Knockdown, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Arthritis and rheumatism

Article Title: Down-regulation of collagen and connective tissue growth factor expression with hepatocyte growth factor in lung fibroblasts from white scleroderma patients via two signaling pathways.

doi: 10.1002/art.22874

Figure Lengend Snippet: Figure 3. Effect of HGF on proMMP-1 expression in lung fibroblasts from systemic sclerosis patients. A, HGF increased proMMP-1 levels in lung fibroblast culture medium, in a dose-dependent manner, within 72 hours. B, HGF, administered for 72 hours at a dose of 50 ng/ml, induced significant increases in proMMP-1 expression after 24–72 hours of treatment. C, Grb2 siRNA and MEK-1/2 inhibitor U0126 prevented HGF-induced expression of proMMP-1, while MMP inhib- itor GM1489 and NF-B inhibitor BAY 11-7082 had no effect on HGF-induced proMMP-1 expression. MMP-1 siRNA reduced both basal and HGF-induced proMMP-1 protein expression. Findings with control siRNA treatment were identical to findings with serum-free medium (SFM). Each bar represents the mean and SD of duplicate determinations in 4 independent experiments. Note that the proteo- lytic activity of GM1489 did not affect MMP-1 expression. P 0.05 versus unstimulated cells. See Figure 1 for other definitions.

Article Snippet: Recombinant human HGF and the Quantikine human proMMP-1 immunoassay kit were purchased from R&D Systems (Minneapolis, MN); an EnzoLytePlus 520 MMP-1 assay kit was obtained from AnaSpec (San Jose, CA); a NoShift transcription factor assay kit, NoShift NF- B (p65) reagents, and a NucBuster protein extraction kit were purchased from Novagen (Madison, WI); MEK-1/2 inhibitor U0126, MMP inhibitor GM1489, and NF- B inhibitor BAY 11-7082 were purchased from EMD Biosciences (San Diego, CA); Grb2 siRNA, MMP-1 siRNA, control siRNA-A, anti- CTGF, and fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti–type I collagen was obtained from Southern Biotechnology (Birmingham, AL); and anti– p44/p42 MAPK (anti–ERK-1/2), anti–phospho–p44/p42 MAPK, and anti-I B were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Inhibition, Control, Activity Assay

Journal: Arthritis and rheumatism

Article Title: Down-regulation of collagen and connective tissue growth factor expression with hepatocyte growth factor in lung fibroblasts from white scleroderma patients via two signaling pathways.

doi: 10.1002/art.22874

Figure Lengend Snippet: Figure 4. Effect of HGF on proteolytic activity of MMP-1 in lung fibroblasts from systemic sclerosis (SSc) patients. Proteolytic activity of MMP-1 was studied by fluorescence analysis, as described in Materials and Methods. Values are presented in relative fluorescence units (RFUs), recorded with a fluorescence microplate reader with excitation set at 485 nm and emission set at 520 nm. A, HGF increased the proteolytic activity of MMP-1 in lung fibroblast culture medium in a dose-dependent manner within 72 hours. B, HGF, at a dose of 50 ng/ml, induced proteolytic activity of MMP-1 in SSc lung fibroblasts in a time-dependent manner. C, Grb2 siRNA and MEK-1/2 inhibitor U0126 prevented HGF-induced proteolytic activity of MMP-1, and MMP inhibitor GM1489 and MMP-1 siRNA significantly reduced the proteolytic activity of MMP-1. NF-B inhibitor BAY 11-7082 had no effect on the proteolytic activity of MMP-1. Each bar represents the mean and SD of duplicate determinations in 4 independent experiments. P 0.05 versus serum-free medium (SFM) from unstimulated cells. See Figure 1 for other definitions.

Article Snippet: Recombinant human HGF and the Quantikine human proMMP-1 immunoassay kit were purchased from R&D Systems (Minneapolis, MN); an EnzoLytePlus 520 MMP-1 assay kit was obtained from AnaSpec (San Jose, CA); a NoShift transcription factor assay kit, NoShift NF- B (p65) reagents, and a NucBuster protein extraction kit were purchased from Novagen (Madison, WI); MEK-1/2 inhibitor U0126, MMP inhibitor GM1489, and NF- B inhibitor BAY 11-7082 were purchased from EMD Biosciences (San Diego, CA); Grb2 siRNA, MMP-1 siRNA, control siRNA-A, anti- CTGF, and fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti–type I collagen was obtained from Southern Biotechnology (Birmingham, AL); and anti– p44/p42 MAPK (anti–ERK-1/2), anti–phospho–p44/p42 MAPK, and anti-I B were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Activity Assay, Fluorescence

Journal: Arthritis and rheumatism

Article Title: Down-regulation of collagen and connective tissue growth factor expression with hepatocyte growth factor in lung fibroblasts from white scleroderma patients via two signaling pathways.

doi: 10.1002/art.22874

Figure Lengend Snippet: Figure 5. Effect of HGF on DNA binding activity of NF-B in lung fibroblasts from systemic sclerosis (SSc) patients. A competitive analysis of NF-B DNA binding activity in SSc lung fibroblasts and HeLa cell nuclear extracts was performed, as described in Materials and Methods. A, HGF added to the cells for 72 hours inhibited NF-B DNA binding activity in a dose- dependent manner. B, Nuclear extract from SSc lung fibroblasts treated with HGF significantly reduced NF-B DNA binding activity in a time-dependent manner. C, Grb2 siRNA prevented HGF-induced inhibition of NF-B, whereas MEK-1/2 inhibitor U0126, MMP inhibitor GM1489, and MMP-1 siRNA had no effect on NF-B DNA binding activity. Lightly shaded bars binding activity assessed with biotinylated NF-B wild-type (WT) double-stranded DNA (dsDNA); darkly shaded bars binding activity assessed with WT dsDNA plus nonspecific, nonbiotinylated dsDNA with a mutant NF-B consensus-binding motif; solid bars binding activity assessed with WT dsDNA plus specific NF-B competitor dsDNA without biotin end labels. Each bar represents the mean and SD of duplicate determinations in 3 independent experiments. P 0.05 versus nuclear extracts from unstimulated cells. Neg negative control; SFM serum-free medium (see Figure 1 for other definitions).

Article Snippet: Recombinant human HGF and the Quantikine human proMMP-1 immunoassay kit were purchased from R&D Systems (Minneapolis, MN); an EnzoLytePlus 520 MMP-1 assay kit was obtained from AnaSpec (San Jose, CA); a NoShift transcription factor assay kit, NoShift NF- B (p65) reagents, and a NucBuster protein extraction kit were purchased from Novagen (Madison, WI); MEK-1/2 inhibitor U0126, MMP inhibitor GM1489, and NF- B inhibitor BAY 11-7082 were purchased from EMD Biosciences (San Diego, CA); Grb2 siRNA, MMP-1 siRNA, control siRNA-A, anti- CTGF, and fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti–type I collagen was obtained from Southern Biotechnology (Birmingham, AL); and anti– p44/p42 MAPK (anti–ERK-1/2), anti–phospho–p44/p42 MAPK, and anti-I B were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Binding Assay, Activity Assay, Inhibition, Mutagenesis, Negative Control

Journal: Arthritis and rheumatism

Article Title: Down-regulation of collagen and connective tissue growth factor expression with hepatocyte growth factor in lung fibroblasts from white scleroderma patients via two signaling pathways.

doi: 10.1002/art.22874

Figure Lengend Snippet: Figure 6. HGF-induced accumulation of IB in lung fibroblasts from systemic sclerosis (SSc) patients. A, SSc lung fibroblasts were incubated for 72 hours with various doses of HGF (5–500 ng/ml) to determine the dose-dependence of IB expression. B, HGF-induced IB accumu- lation was determined in cells treated for various time periods with HGF (50 ng/ml). C, Lung fibroblasts were transfected with or without Grb2 siRNA, MMP-1 siRNA, and control siRNA, or with or without 40-minute pretreatment with MEK-1/2 inhibitor U0126 (1 M), MMP inhibitor GM1489 (10 nM), or NF-B inhibitor BAY 11-7082 (10 M), then incubated with HGF (50 ng/ml) for 72 hours. Anti-Grb2 was used to validate the knockdown effect of Grb2 siRNA. Cell culture supernatant was collected and subjected to enzyme-linked immunosorbent assay to measure the depletion of MMP-1 siRNA. Findings with control siRNA treatment were identical to findings with serum-free medium (lane 1). Anti–-actin was used as a loading control. Immunoblots are representative of 3 independent experiments. Of note, BAY 11-7082 did not affect HGF-induced IB accumulation. Quantitative results of densitometric analysis of immunoblots are also shown; values are the mean and SD from 3 independent experiments. P 0.05 versus unstimulated cells. See Figure 1 for other definitions.

Article Snippet: Recombinant human HGF and the Quantikine human proMMP-1 immunoassay kit were purchased from R&D Systems (Minneapolis, MN); an EnzoLytePlus 520 MMP-1 assay kit was obtained from AnaSpec (San Jose, CA); a NoShift transcription factor assay kit, NoShift NF- B (p65) reagents, and a NucBuster protein extraction kit were purchased from Novagen (Madison, WI); MEK-1/2 inhibitor U0126, MMP inhibitor GM1489, and NF- B inhibitor BAY 11-7082 were purchased from EMD Biosciences (San Diego, CA); Grb2 siRNA, MMP-1 siRNA, control siRNA-A, anti- CTGF, and fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti–type I collagen was obtained from Southern Biotechnology (Birmingham, AL); and anti– p44/p42 MAPK (anti–ERK-1/2), anti–phospho–p44/p42 MAPK, and anti-I B were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Incubation, Expressing, Transfection, Control, Knockdown, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot