Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Pro-inflammatory cytokines TNF-related weak inducer of apoptosis (TWEAK) and TNFalpha induce the mitogen-activated protein kinase (MAPK)-dependent expression of sclerostin in human osteoblasts.

doi: 10.1359/jbmr.090305

Figure Lengend Snippet: FIG. 3. Effect on osteogenic gene expres- sion of a dose response of TWEAK alone (0– 100 ng/ml) or in the presence of TNF at 1 or 5 ng/ml. NHBC were cultured for 72 h under the indicated treatments and analyzed by real-time RT-PCR, as described, for the transcription factors (A) RUNX2 and (B) osterix. RUNX2 and osterix mRNA levels in response to TWEAK were inversely related, as determined by linear regression analysis (C). RT-PCR analysis was also performed for (D) OCN, (E) BSP-1, (F) SOST, (G) LRP5, and (H) LRP6. Superscripts a, b, and c indi- cate significant difference to the untreated control for TWEAK only, TWEAK ± TNF (1 ng/ml), and TWEAK ± TNF (5 ng/ml), re- spectively (p < 0.05). Data shown are nor- malized to GAPDH mRNA levels and are means of triplicate reactions ± SD. Similar results were obtained in three independent experiments.

Article Snippet: Recombinant soluble human TWEAK and the TWEAKneutralizing hamster anti-TWEAK monoclonal antibody (MAb) ABG11 were generated as previously described. (26,27) Mouse anti-TWEAK MAb P2D10 was generated as described previously. (1,10) Recombinant human TNFa, IL-1b, sclerostin, and mouse anti-human sclerostin MAb (MAB220902) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Pro-inflammatory cytokines TNF-related weak inducer of apoptosis (TWEAK) and TNFalpha induce the mitogen-activated protein kinase (MAPK)-dependent expression of sclerostin in human osteoblasts.

doi: 10.1359/jbmr.090305

Figure Lengend Snippet: FIG. 4. Effect of TWEAK and TNF, alone and in combination, on sclerostin expression. (A) Expression of sclerostin protein deter- mined by in situ immunofluorescence in ei- ther untreated, TWEAK (100 ng/ml), TNF (5 ng/ml), or TWEAK + TNF–treated NHBC, using either anti-SOST MAb or isotype- matched IgG control. Cells were counter- stained with DAPI. (B) Intracellular sclerostin protein expression in either untreated (dot- ted histogram) or TWEAK-treated cells (dashed histogram) determined by flow cy- tometry. Solid histogram represents cells stained with isotype matched IgG control. TWEAK treatment resulted in significant increase in the mean fluorescence intensity of sclerostin staining (p < 0.05). (C) Real-time RT-PCR analysis of SOST mRNA in re- sponse to TWEAK (50 ng/ml), TNF (1 ng/ ml), or TWEAK/TNF, overlaid with relative OCN mRNA expression in control cultures, under conditions otherwise permissive for in vitro mineralization. (D) Effect of protein synthesis inhibitor cycloheximide (CHX) on SOST mRNA expression with and without exposure to TWEAK (100 ng/ml) for 72 h. (E) Level of SOST mRNA (as a ratio to 18S rRNA) expressed by NHBC (three donors) by TWEAK (100 ng/ml) in the presence or absence of neutralizing anti-TWEAK Ab, ABG.11 (10 mg/ml), compared with basal SOST:18S mRNA expression in human age- and sex-matched femoral trabecular bone. (F) Expression of SOST:18S mRNA in ex vivo cultures of human bone, cultured for 24 h in the presence or absence of TWEAK (100 ng/ml), TNF (5 ng/ml), or TWEAK/TNF. All real-time RT-PCR data shown are means of triplicate reactions ± SD. Results are repre- sentative of at least three independent ex- periments. *Significant differences in gene expression compared with untreated (UT) control (p < 0.05). #Not different to TWEAK alone treatment.

Article Snippet: Recombinant soluble human TWEAK and the TWEAKneutralizing hamster anti-TWEAK monoclonal antibody (MAb) ABG11 were generated as previously described. (26,27) Mouse anti-TWEAK MAb P2D10 was generated as described previously. (1,10) Recombinant human TNFa, IL-1b, sclerostin, and mouse anti-human sclerostin MAb (MAB220902) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, In Situ, Control, Staining, Quantitative RT-PCR, In Vitro, Ex Vivo, Cell Culture, Gene Expression

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Pro-inflammatory cytokines TNF-related weak inducer of apoptosis (TWEAK) and TNFalpha induce the mitogen-activated protein kinase (MAPK)-dependent expression of sclerostin in human osteoblasts.

doi: 10.1359/jbmr.090305

Figure Lengend Snippet: FIG. 5. Sclerostin expression in response to TWEAK occurs in proliferating, Ki67-positive osteoblasts. NHBC were treated for 72 h as indicated, stained for intracellular sclerostin expression and Ki67 antigen, and analyzed by flow cytometry. Quadrants were set such that positive staining was >99% of that observed in the presence of either negative control antibody. The percentage of cells in each quadrant is shown. Similar results were obtained in two independent experiments.

Article Snippet: Recombinant soluble human TWEAK and the TWEAKneutralizing hamster anti-TWEAK monoclonal antibody (MAb) ABG11 were generated as previously described. (26,27) Mouse anti-TWEAK MAb P2D10 was generated as described previously. (1,10) Recombinant human TNFa, IL-1b, sclerostin, and mouse anti-human sclerostin MAb (MAB220902) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Staining, Cytometry, Negative Control

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Pro-inflammatory cytokines TNF-related weak inducer of apoptosis (TWEAK) and TNFalpha induce the mitogen-activated protein kinase (MAPK)-dependent expression of sclerostin in human osteoblasts.

doi: 10.1359/jbmr.090305

Figure Lengend Snippet: FIG. 6. (A) Effect of TWEAK on MAPK and NF-kB activation. Western blot analysis of NHBC treated with TWEAK (100 ng/ml), TNF (5 ng/ml), or a combination of both, for the times indicated (min). Blots were analyzed for phosphorylated and total Erk1/2, JNK and IkBa, or b-actin. Similar data were obtained on replicate blots from three independent experiments. The relative levels of phosphorylated to total Erk were quantified using ImageQuant software (Molecular Dynamics) and are expressed as mean ratios from three experiments ± SE (*significant difference compared with time 0 control, p < 0.05). (B) Effect of MAPK inhibitors on sclerostin transcription. NHBC were cultured in the presence or absence of TWEAK (100 ng/ml), TNF (5 ng/ml), or a combination of both, in the co-presence of the JNK inhibitor SP600125 or the MEK inhibitor, PD98059. SOST mRNA expression was analyzed by real-time RT-PCR. Shown are the observed fold-change in expression from the control levels for each inhibitor. Data shown are means of triplicate reactions ± SD. *Significant difference to the no inhibitor treatment for each cytokine. (C) Effect of TWEAK and TNF or the combination of both on cell proliferation in the presence or absence of SP600125 and PD90859, under identical conditions to those used in B. Cells were first labeled with CFSE, and culture was continued for 4 days. Proliferation was calculated from flow cytometry data as the relative percentage of cells that had undergone more than one cell division.

Article Snippet: Recombinant soluble human TWEAK and the TWEAKneutralizing hamster anti-TWEAK monoclonal antibody (MAb) ABG11 were generated as previously described. (26,27) Mouse anti-TWEAK MAb P2D10 was generated as described previously. (1,10) Recombinant human TNFa, IL-1b, sclerostin, and mouse anti-human sclerostin MAb (MAB220902) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Western Blot, Software, Control, Cell Culture, Expressing, Quantitative RT-PCR, Labeling, Cytometry

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Pro-inflammatory cytokines TNF-related weak inducer of apoptosis (TWEAK) and TNFalpha induce the mitogen-activated protein kinase (MAPK)-dependent expression of sclerostin in human osteoblasts.

doi: 10.1359/jbmr.090305

Figure Lengend Snippet: FIG. 7. Comparison of the effect of TWEAK (TW, 50 ng/ml), TNF (1 ng/ml), and recombinant human sclerostin (SCL, 10 ng/ ml) on (A) RUNX2 and (B) OCN mRNA levels after a 72-h treatment. Data are means of triplicate reactions ± SD and are repre- sentative of two independent experiments. *Significant differences in gene expression compared with untreated (UT) control. (C) Western blot showing the effect after 24-h exposure of recombinant TWEAK (100 and 200 ng/ml), TNF (5 ng/ml), a combination of both cytokines, and recombinant human sclerostin (100 ng/ml) on NHBC expression of phosphorylated and total GSK3b, active (dephosphorylated) and total forms of b-catenin (bCAT), and sclerostin. (D) Western blot showing activation by human recombinant sclerostin of ERK1/2 signaling. Serum-starved NHBC were treated with ei- ther TWEAK (100 ng/ml) or with human recombinant SCL at 10 and 100 ng/ml for the times indicated (min). Blots were analyzed for phosphorylated and total Erk1/2 and b-actin. Data are representative of two in- dependent experiments.

Article Snippet: Recombinant soluble human TWEAK and the TWEAKneutralizing hamster anti-TWEAK monoclonal antibody (MAb) ABG11 were generated as previously described. (26,27) Mouse anti-TWEAK MAb P2D10 was generated as described previously. (1,10) Recombinant human TNFa, IL-1b, sclerostin, and mouse anti-human sclerostin MAb (MAB220902) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Comparison, Recombinant, Gene Expression, Control, Western Blot, Expressing, Activation Assay

Journal: American journal of hypertension

Article Title: Association Between High Serum Soluble Corin and Hypertension: A Cross-Sectional Study in a General Population of China.

doi: 10.1093/ajh/hpv002

Figure Lengend Snippet: Figure 2. Scatterplot showing a significant and positive correlation of log-transformed serum corin concentration to systolic (r = 0.143, P < 0.01) and diastolic (r = 0.245, P < 0.01) blood pressure among participants not receiving any antihypertensive drugs.

Article Snippet: Soluble corin was reported to be stable in blood samples frozen at −80 °C after several cycles of freezing and thawing.17 We used a quantikine human corin immunoassay (R&D Systems, Minneapolis, MN.

Techniques: Transformation Assay, Concentration Assay

Journal: American journal of hypertension

Article Title: Association Between High Serum Soluble Corin and Hypertension: A Cross-Sectional Study in a General Population of China.

doi: 10.1093/ajh/hpv002

Figure Lengend Snippet: Figure 3. Scatterplot showing a significant and positive correlation of log-transformed serum corin concentration to systolic (r = 0.096, P < 0.01) and diastolic (r = 0.158, P < 0.01) blood pressure among nonhypertensive participants.

Article Snippet: Soluble corin was reported to be stable in blood samples frozen at −80 °C after several cycles of freezing and thawing.17 We used a quantikine human corin immunoassay (R&D Systems, Minneapolis, MN.

Techniques: Transformation Assay, Concentration Assay

Journal: American journal of hypertension

Article Title: Association Between High Serum Soluble Corin and Hypertension: A Cross-Sectional Study in a General Population of China.

doi: 10.1093/ajh/hpv002

Figure Lengend Snippet: Figure 1. Scatterplot showing a significant and positive correlation of log-transformed serum corin concentration to systolic (r = 0.153, P <0.01) and diastolic (r = 0.243, P <0.01) blood pressure among total participants.

Article Snippet: Soluble corin was reported to be stable in blood samples frozen at −80 °C after several cycles of freezing and thawing.17 We used a quantikine human corin immunoassay (R&D Systems, Minneapolis, MN.

Techniques: Transformation Assay, Concentration Assay

Journal: American journal of hypertension

Article Title: Association Between High Serum Soluble Corin and Hypertension: A Cross-Sectional Study in a General Population of China.

doi: 10.1093/ajh/hpv002

Figure Lengend Snippet: Figure 4. Median levels of serum soluble corin in different subgroups of hypertensive individuals.

Article Snippet: Soluble corin was reported to be stable in blood samples frozen at −80 °C after several cycles of freezing and thawing.17 We used a quantikine human corin immunoassay (R&D Systems, Minneapolis, MN.

Techniques: