Journal: Frontiers in Pharmacology

Article Title: Sphingosine 1-Phosphate Induces Cyclooxygenase-2/Prostaglandin E 2 Expression via PKCα-dependent Mitogen-Activated Protein Kinases and NF-κB Cascade in Human Cardiac Fibroblasts

doi: 10.3389/fphar.2020.569802

Figure Lengend Snippet: S1P induces COX-2 expression via a G q - or PTX-sensitive G i protein-coupled S1PR1/3. (A,B) Cells were pretreated with GPA2A (G q antagonist) for 1 h or pertussis toxin (PTX, G i protein inactivator) for 24 h and then incubated with 15 μM S1P for 8 h. (C) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated with GPA2A (1 μM) or PTX (100 ng/ml) for 1 h, and then incubated with 15 μM S1P for 4 h (mRNA) or 1 h (promoter). The COX-2 mRNA and promoter activity were analyzed by real time-PCR (open bar) and promoter assay (gray bar). (D) Cells were transfected with siRNA for scrambled, G q α, or G i α for 24 h and then exposed to 15 μM S1P for 8 h (A,B,D) The levels of COX-2, GAPDH, G q α, and G i α proteins were analyzed by Western blot. Data are expressed as mean ± SEM of three individual experiments (n = 3). # p < 0.05, as compared with the cells treated with S1P alone.

Article Snippet: GPA2A, pertussis toxin (PTX), Gö6976, PD98059, SB202190, SP600125, and helenalin were from Biomol (Plymouth Meeting, PA, United States).

Techniques: Expressing, Incubation, Transfection, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Promoter Assay, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Sphingosine 1-Phosphate Induces Cyclooxygenase-2/Prostaglandin E 2 Expression via PKCα-dependent Mitogen-Activated Protein Kinases and NF-κB Cascade in Human Cardiac Fibroblasts

doi: 10.3389/fphar.2020.569802

Figure Lengend Snippet: PKCα mediates S1P-induced COX-2 expression. (A) Cells were pretreated with Gö6976 for 1 h and then incubated with 15 μM S1P for 8 h. (B) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated without or with Gö6976 (1 μM) for 1 h, and then incubated with 15 μM S1P for 4 h (mRNA) or 1 h (promoter). Western blot, real time-PCR, and promoter assay were performed to determine the levels of COX-2 protein, mRNA expression, and promoter activity, respectively. (C) Prior to exposure with S1P for 8 h, cells were transfected with siRNA of scrambled or PKCα. (D) Cells were incubated with S1P (15 μM) for the indicated time intervals. (E) Cells were incubated with S1P (15 μM) for 5 min in the absence or presence of Gö6976 (1 μM), W123 (10 μM), CAY (10 μM), GPA2A (10 μM), or PTX (100 ng/ml) for 1 h. The membrane and cytosol fractions were prepared and analyzed by Western blot. Data are expressed as mean ± SEM of three individual experiments (n = 3). # p < 0.05, as compared with the cells treated with S1P alone.

Article Snippet: GPA2A, pertussis toxin (PTX), Gö6976, PD98059, SB202190, SP600125, and helenalin were from Biomol (Plymouth Meeting, PA, United States).

Techniques: Expressing, Incubation, Transfection, Luciferase, Western Blot, Real-time Polymerase Chain Reaction, Promoter Assay, Activity Assay, Membrane

Journal: Frontiers in Pharmacology

Article Title: Sphingosine 1-Phosphate Induces Cyclooxygenase-2/Prostaglandin E 2 Expression via PKCα-dependent Mitogen-Activated Protein Kinases and NF-κB Cascade in Human Cardiac Fibroblasts

doi: 10.3389/fphar.2020.569802

Figure Lengend Snippet: S1P-induced COX-2 expression is mediated through p42/p44 MAPK phosphorylation. (A) Cells were pretreated with PD98059 for 1 h and then incubated with 15 μM S1P for 8 h. (B) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated without or with PD98059 (10 μM) for 1 h, and then incubated with 15 μM S1P for 4 h (mRNA) or 1 h (promoter). The levels of COX-2 protein, mRNA expression, and promoter activity were determined by Western blot, real time-PCR, and promoter assay, respectively. (C) Cells were transfected with siRNA of scrambled or p42 MAPK and then exposed to S1P for 8 h. (D) Cells were incubated with S1P (15 μM) for the indicated time intervals in the absence or presence of PD98059 (10 μM), W123 (10 μM), CAY (10 μM), GPA2A (10 μM), PTX (100 ng/ml), or Gö6976 (10 μM). The cell lysates were collected and analyzed by Western blot. The fold of basal was defined as normalization of the data to the respective “0,” and then compared the data of corresponding time points of control vs inhibitor with a statistic method, as described in the section of Methods . Data are expressed as mean ± SEM of three individual experiments (n = 3). # p < 0.05, as compared with the cells treated with S1P alone.

Article Snippet: GPA2A, pertussis toxin (PTX), Gö6976, PD98059, SB202190, SP600125, and helenalin were from Biomol (Plymouth Meeting, PA, United States).

Techniques: Expressing, Phospho-proteomics, Incubation, Transfection, Luciferase, Activity Assay, Western Blot, Real-time Polymerase Chain Reaction, Promoter Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Sphingosine 1-Phosphate Induces Cyclooxygenase-2/Prostaglandin E 2 Expression via PKCα-dependent Mitogen-Activated Protein Kinases and NF-κB Cascade in Human Cardiac Fibroblasts

doi: 10.3389/fphar.2020.569802

Figure Lengend Snippet: S1P-induced COX-2 expression is mediated through p38 MAPK phosphorylation. (A) Cells pretreated with SB202190 for 1 h were followed by incubation with 15 μM S1P for 8 h. (B) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated without or with SB202190 (30 μM) for 1 h, and then incubated with 15 μM S1P for 4 h (mRNA) or 1 h (promoter). The levels of COX-2 protein, mRNA expression, and promoter activity were individually examined by Western blot, real time-PCR, and promoter assay. (C) Cells transfected with p38α MAPK siRNA were incubated with S1P for 8 h. ( D ) Cells were incubated with S1P (15 μM) for the indicated time intervals in the absence or presence of SB202190 (30 μM), W123 (10 μM), CAY (10 μM), GPA2A (10 μM), or Gö6976 (10 μM) for 1 h and PTX (100 ng/ml) for 24 h. The cell lysates were collected and analyzed by Western blot. The fold of basal was defined as normalization of the data to the respective “0,” and then compared the data of corresponding time points of control vs inhibitor with a statistic method, as described in the section of Methods . Data are expressed as mean ± SEM of three individual experiments (n = 3). # p < 0.05, as compared with the cells treated with S1P alone.

Article Snippet: GPA2A, pertussis toxin (PTX), Gö6976, PD98059, SB202190, SP600125, and helenalin were from Biomol (Plymouth Meeting, PA, United States).

Techniques: Expressing, Phospho-proteomics, Incubation, Transfection, Luciferase, Activity Assay, Western Blot, Real-time Polymerase Chain Reaction, Promoter Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Sphingosine 1-Phosphate Induces Cyclooxygenase-2/Prostaglandin E 2 Expression via PKCα-dependent Mitogen-Activated Protein Kinases and NF-κB Cascade in Human Cardiac Fibroblasts

doi: 10.3389/fphar.2020.569802

Figure Lengend Snippet: JNK1/2 phosphorylation is involved in S1P-induced COX-2 expression. (A) After pretreatment with SP600125 for 1 h, cells were challenged with 15 μM S1P for 8 h. (B) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated without or with SP600125 (10 μM) for 1 h, and then incubated with 15 μM S1P for 4 h (mRNA) or 1 h (promoter). The levels of COX-2 protein, mRNA expression, and promoter activity were determined by Western blot, real time-PCR, and promoter assay, respectively. (C) Cells were transfected with siRNA of JNK1 and then exposed to S1P for 8 h. (D) Cells were incubated with S1P (15 μM) for the indicated time intervals in the absence or presence of SP600125 (10 μM), W123 (10 μM), CAY (10 μM), GPA2A (10 μM), PTX (100 ng/ml), or Gö6976 (10 μM) for 1 h. The cell lysates were collected and analyzed by Western blot. The fold of basal was defined as normalization of the data to the respective “0,” and then compared the data of corresponding time points of control vs inhibitor with a statistic method, as described in the section of Methods . Data are expressed as mean ± SEM of three individual experiments (n = 3). # p < 0.05, as compared with the cells treated with S1P alone.

Article Snippet: GPA2A, pertussis toxin (PTX), Gö6976, PD98059, SB202190, SP600125, and helenalin were from Biomol (Plymouth Meeting, PA, United States).

Techniques: Phospho-proteomics, Expressing, Transfection, Luciferase, Incubation, Activity Assay, Western Blot, Real-time Polymerase Chain Reaction, Promoter Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Sphingosine 1-Phosphate Induces Cyclooxygenase-2/Prostaglandin E 2 Expression via PKCα-dependent Mitogen-Activated Protein Kinases and NF-κB Cascade in Human Cardiac Fibroblasts

doi: 10.3389/fphar.2020.569802

Figure Lengend Snippet: NF-κB is essential for S1P-induced COX-2 expression. (A) Cells were pretreated with helenalin for 1 h and then incubated with 15 μM S1P for 8 h. (B) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated without or with helenalin (1 μM) for 1 h, and then incubated with 15 μM S1P for 4 h (mRNA) or 1 h (promoter). The levels of COX-2 protein, mRNA expression, and promoter activity were determined by Western blot, real time-PCR, and promoter assay, respectively. (C) Cells were transfected with siRNA of p65 and then exposed to S1P for 8 h. (D) Cells were incubated with S1P (15 μM) for the indicated time intervals. (E) Cells were treated with S1P (15 μM) for 15 min in the absence or presence of helenalin (1 μM). (D,E) The nuclear and cytosol fractions were prepared and analyzed by Western blot. The p65 NF-κB translocation by S1P was also determined by immunofluorescent staining as described in Materials and Methods . (F) To determine which S1PR subtypes, Gi or Gq protein, and MAPKs involved in S1P-stimulated the nuclear localization of p65 NF-κB, cells were incubated with S1P (15 μM) for 15 min in the absence or presence of Gö6976 (Gö, 10 μM), SB202190 (SB, 30 μM), SP600125 (SP, 10 μM), PD98059 (PD, 10 μM), W123 (W, 10 μM), CAY (10 μM), GPA2A (GPA, 10 μM), or PTX (100 ng/ml), and the nuclear fraction was analyzed by Western blot. To fit the construct of data layout, the data were rearranged from the same gel with the exception of non-related inhibitors and disclosed by the insertion of white spaces rearranged from the original capture. Data are expressed as mean ± SEM of three individual experiments (n = 3). # p < 0.05, as compared with the cells treated with S1P alone.

Article Snippet: GPA2A, pertussis toxin (PTX), Gö6976, PD98059, SB202190, SP600125, and helenalin were from Biomol (Plymouth Meeting, PA, United States).

Techniques: Expressing, Incubation, Transfection, Luciferase, Activity Assay, Western Blot, Real-time Polymerase Chain Reaction, Promoter Assay, Translocation Assay, Staining, Construct

Journal: Frontiers in Pharmacology

Article Title: Sphingosine 1-Phosphate Induces Cyclooxygenase-2/Prostaglandin E 2 Expression via PKCα-dependent Mitogen-Activated Protein Kinases and NF-κB Cascade in Human Cardiac Fibroblasts

doi: 10.3389/fphar.2020.569802

Figure Lengend Snippet: COX-2 promoter activity is stimulated by S1P through an NF-κB-dependent pathway. (A) Cells were incubated with S1P for the indicated time intervals (upper panel). Cells were pretreated with PD98059 (10 μM), SB202190 (30 μM), SP600125 (10 μM) for 1 h and then incubated with S1P for 30 min. The binding activity of NF-κB p65 and promoter was analyzed by ChIP assay (n = 3), as described in Methods . Left panel, to fit the construct of data layout, the data were rearranged from the same gel with the exception of non-related inhibitors and disclosed by the insertion of white spaces rearranged from the original capture. (B,C) Cells were transfected with an NF-κB-luciferase reporter gene, pretreated with W123 (10 μM), CAY (10 μM), GPA2A (10 μM), PTX (100 ng/ml), Gö6976 (10 μM), PD98059 (10 μM), SB202190 (30 μM), SP600125 (10 μM), or helenalin (1 μM) for 1 h and then incubated with S1P for 2 h. (D) The schematic picture represented two different 5′-promoter regions of the mouse COX-2 promoter constructs, both wild-type (WT) and mt-κB modified by single-point mutation of the κB binding site fused to the pGL-luciferase reporter gene. “↲ ” indicated the translational start site (+1) of the luciferase reporter gene. WT COX-2 promoter reporter gene (WT-COX-2) or NF-κB mutated COX-2 promoter reporter gene (mt-κB-COX-2) were transfected into cells, which then were incubated with or without S1P for 1 h. The promoter reporter activity was determined. (E) Cells were pretreated with W123, CAY, GPA2A, PTX, Gö6976, PD98059, SB202190, SP600125, or helenalin for 1 h and then incubated with S1P for 8 h. The PGE 2 levels were analyzed by EIA. Data are expressed as mean ± SEM of three individual experiments (n = 3). # p < 0.05, as compared with the cells treated with S1P alone.

Article Snippet: GPA2A, pertussis toxin (PTX), Gö6976, PD98059, SB202190, SP600125, and helenalin were from Biomol (Plymouth Meeting, PA, United States).

Techniques: Activity Assay, Incubation, Binding Assay, Construct, Transfection, Luciferase, Modification, Mutagenesis

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Human Salivary Histatin-1 Promotes Osteogenic Cell Spreading on Both Bio-Inert Substrates and Titanium SLA Surfaces

doi: 10.3389/fbioe.2020.584410

Figure Lengend Snippet: The effects of signaling pathway inhibitors on Hst1-induced spreading of osteogenic cells. Light micrographs depicting the spreading of osteogenic cells that were treated with (A) extracellular-signal-regulated kinase (ERK) signaling (10 μM U0126), (C) : p38 signaling (10 μM SB203580) and (E) G protein-coupled receptor (GPCR) (200 ng/mL Pertussis toxin, PTx). Bar = 50 μm. Relative spreading surface area of osteogenic cells that were treated with Hst1 with or without the pretreatment with the inhibitors of (B) extracellular-signal-regulated kinase (ERK) signaling (10 μM U0126), (D) p38 signaling (10 μM SB203580) and (F) G protein-coupled receptor (GPCR) (200 ng/mL Pertussis toxin, PTx). Data are shown as mean ± SD ( n = 6). ** p < 0.01, *** p < 0.001.

Article Snippet: To investigate the role of potential signaling pathways in the effects of Hst1 on the spreading of osteogenic cells, we applied the following signaling pathway-specific inhibitors of ERK1/2 (U0126, 10 μM; LC Laboratories, Woburn, MA, United States), GPCR (pertussis toxin (PTx), 200 ng/mL; LC Laboratories, Woburn, MA, United States), and p38 MAPK (SB203580, 10 μM; LC Laboratories, Woburn, MA, United States) using the cell spreading model on bio-inert glass.

Techniques: