Journal: International Journal of Biological Sciences

Article Title: SphK1/S1P signaling-mediated crosstalk between pancreatic acinar cell and macrophage M1 polarization aggravates acute pancreatitis progression

doi: 10.7150/ijbs.120627

Figure Lengend Snippet: SphK1 is overexpressed in the PACs of AP mice. (A) Venn diagram showing the overlap of differentially expressed genes associated with AP, identified from RNA-sequencing data of CCK-treated 266-6 cells and datasets from the GEO database. (B) Generation of a caerulein or L-arginine induced AP model. (C) Representative images of H&E staining in the pancreatic tissues of AP mice (n=5). Scale bars, 100 μm. (D) Pancreatic exocrine function was evaluated by measuring serum AMY and LPS levels. (E) The mRNA and protein levels of SphK1 in pancreatic tissues were evaluated by RT-qPCR and Western blot in AP mice. (F) Representative images of IHC staining for SphK1 in the pancreatic tissues of AP mice (n=6). Scale bars, 100 μm. (G) The level of S1P in mouse serum was measured by ELISA (n=4). (H) The expression of SphK1 (in green) and Amylase (in red) was detected by tissue immunofluorescence double localization. Nuclei were counterstained with DAPI (in blue). Scale bar: 50 μm. (I) The level of S1P in AP patients was measured by ELISA (n=5). (J) The mRNA and protein levels of SphK1 in CCK-treated 266-6 cells were evaluated by RT-qPCR and Western blot. (K) The S1P level in the culture media of CCK-treated 266-6 cells was measured by ELISA. *P <0.05; **P <0.01; ***P <0.001.

Article Snippet: To investigate the role of S1P in macrophages, RAW264.7 cells were treated with 1 μM S1P (Sigma) and 1 μM JTE-013 (MedChemExpress).

Techniques: RNA Sequencing, Staining, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: SphK1/S1P signaling-mediated crosstalk between pancreatic acinar cell and macrophage M1 polarization aggravates acute pancreatitis progression

doi: 10.7150/ijbs.120627

Figure Lengend Snippet: Knockdown of SphK1 mitigates the severity of AP with decreased macrophage M1 polarization. AP was induced in WT and SphK1 -/- mice by caerulein and L-arginine, respectively. (A) Representative images of the pancreatic tissues in WT and SphK1 -/- AP mice on naked eye. (B) Representative images of H&E staining in the pancreatic tissues of WT and SphK1 -/- AP mice (n=5). Scale bar: 100 μm. (C) RT-qPCR and Western blot analysis of SphK1 expression in the pancreatic tissues of WT and SphK1 -/- AP mice. (D) The S1P levels in the serum of WT and SphK1 -/- AP mice. (E) The expression of F4/80, and CD86 in the pancreatic tissues of WT and SphK1 -/- AP mice were evaluated by IHC (n=6). Scale bar: 100 μm. (F) The expression of F4/80 and CD86 in the pancreatic tissues of WT and SphK1 -/- AP mice were evaluated by IF (n=6). Scale bar: 20 μm. (G) The mRNA expression of CD86 and iNOS in the pancreatic tissues of WT and SphK1 -/- AP mice. (H) The protein expression of CD86 and iNOS in the pancreatic tissues of WT and SphK1 -/- AP mice. *P <0.05; **P <0.01; ***P <0.001.

Article Snippet: To investigate the role of S1P in macrophages, RAW264.7 cells were treated with 1 μM S1P (Sigma) and 1 μM JTE-013 (MedChemExpress).

Techniques: Knockdown, Staining, Quantitative RT-PCR, Western Blot, Expressing

Journal: International Journal of Biological Sciences

Article Title: SphK1/S1P signaling-mediated crosstalk between pancreatic acinar cell and macrophage M1 polarization aggravates acute pancreatitis progression

doi: 10.7150/ijbs.120627

Figure Lengend Snippet: SphK1/S1P signaling is required for iPAC-induced M1 polarization of macrophages. 266-6 cells were treated with CCK to simulate the injured PACs (iPACs) in AP. (A) Schematics of the co-culture system involving CCK-treated 266-6 cells and RAW264.7 cells. (B) The mRNA level of CD86, iNOS, and TNF-α in RAW264.7 cells co-cultured with CCK-injured 266-6 cells. (C) The protein level of CD86 in RAW264.7 cells co-cultured with CCK-injured 266-6 cells. (D) The positive cells of CD86 in RAW264.7 cells following co-cultured with CCK-injured 266-6 cells was assessed by flow cytometry. (E-H) The mRNA level of CD86, iNOS, and TNF-α and the protein level of CD86 and iNOS in CM treated RAW264.7 cells when SphK1 was knockdown by siRNA or PF-543. (I) The content of S1P in RAW264.7 cells supernatant following co-cultured with CM CCK was determined by ELISA. (J) The S1P level in RAW264.7 cells co-cultured with pretreated with siSphK1 or PF-543 CM CCK . (K) The mRNA level of CD86, iNOS, and TNF-α in S1P-treated RAW264.7 cells. (L) The protein level of CD86 and iNOS in S1P-treated RAW264.7 cells. (M) The positive cells of CD86 in S1P-treated RAW264.7 cells by flow cytometry. (N-O) The mRNA and protein levels of MCP1, IL-1β, and IL-6 in S1P-treated RAW264.7 cells. (P) The level of TNF-α and IL-1β in the supernatant of S1P-treated RAW264.7 cells. (Q-R) The mRNA level of CD86, iNOS, and TNF-α and the protein level of CD86 and iNOS in S1P-treated BMDMs. *P <0.05; **P <0.01; ***P <0.001; NS, no significance.

Article Snippet: To investigate the role of S1P in macrophages, RAW264.7 cells were treated with 1 μM S1P (Sigma) and 1 μM JTE-013 (MedChemExpress).

Techniques: Co-Culture Assay, Cell Culture, Flow Cytometry, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Biological Sciences

Article Title: SphK1/S1P signaling-mediated crosstalk between pancreatic acinar cell and macrophage M1 polarization aggravates acute pancreatitis progression

doi: 10.7150/ijbs.120627

Figure Lengend Snippet: S1PR2 is necessary for S1P-induced M1 macrophages polarization. (A) RT-qPCR was performed to measure the mRNA levels of S1PRs in pancreatic tissues from AP mice. (B-C) Western blot and IHC analysis of S1PR2 expression in pancreatic tissues from AP mice. (D) IF images of AP mice pancreatic tissues stained with F4/80(in red) and S1PR2 (in green). Nuclei were counterstained with DAPI (in blue). Scale bar: 20 μm. (E) The mRNA levels of S1PRs in RAW264.7 cells following S1P treatment. (F) Western blot analysis of S1PR2 expression in S1P-treated RAW264.7 cells. (G-I) The mRNA levels of CD86, iNOS, and TNF-α and the protein level of CD86 and iNOS in RAW264.7 cells treated with CM CCK after transfection of siS1PR2 or addition of JTE-013. (J) When exogenous S1P was added, the mRNA and protein levels of CD86, iNOS, and TNF-α in RAW264.7 cells treated with CM CCK after transfection of siS1PR2 or addition of JTE-013. *P <0.05; **P <0.01; ***P <0.001; NS, no significance.

Article Snippet: To investigate the role of S1P in macrophages, RAW264.7 cells were treated with 1 μM S1P (Sigma) and 1 μM JTE-013 (MedChemExpress).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining, Transfection

Journal: International Journal of Biological Sciences

Article Title: SphK1/S1P signaling-mediated crosstalk between pancreatic acinar cell and macrophage M1 polarization aggravates acute pancreatitis progression

doi: 10.7150/ijbs.120627

Figure Lengend Snippet: S1P/S1PR2 signaling induces M1 polarization in macrophages by activating PI3K/JNK and ERK pathways. (A) Protein levels of PI3K, JNK and ERK in pancreatic tissues from AP mice were analyzed by Western blot. (B) Protein levels of PI3K, JNK and ERK in RAW264.7 cells after treatment with CM CCK or LPS were analyzed by western blot. (C) Protein levels of PI3K, JNK and ERK in S1P-treated RAW264.7 cells were analyzed by western blot. (D) Protein levels of PI3K, JNK and ERK in S1P-treated RAW264.7 cells were assessed by western blot following JTE-013 addition. *P <0.05; **P <0.01; ***P <0.001; NS, no significance.

Article Snippet: To investigate the role of S1P in macrophages, RAW264.7 cells were treated with 1 μM S1P (Sigma) and 1 μM JTE-013 (MedChemExpress).

Techniques: Western Blot

Journal: International Journal of Biological Sciences

Article Title: SphK1/S1P signaling-mediated crosstalk between pancreatic acinar cell and macrophage M1 polarization aggravates acute pancreatitis progression

doi: 10.7150/ijbs.120627

Figure Lengend Snippet: M1-polarized macrophages enhance SphK1 expression in PACs through the TNF-α/NF-κB pathway. (A-B) LDH level in the culture media of 266-6 cells treated with CM S1P or CM LPS . (C) Western blot analysis of SphK1 expression in CM S1P or CM LPS -treated 266-6 cells. (D) The mRNA level of SphK1 in 266-6 cells under the treatment of TNF-α or IL-1β. (E) Schematic illustration of the NF-κB in the promoter of SphK1. (F) The protein level of NF-κB p65 and p-p65 in CM S1P or CM LPS -treated 266-6 cells. (G-H) The protein level of SphK1, NF-κB p65 and p-p65 in 266-6 cells pretreated with CM CM-CCK . (I) ChIP assays with anti-NF-κB antibody verifying the binding of NF-κB in the SphK1 promoter, and with knockdown of NF-κB. (J) 266-6 cells were transfected with pGL3 reporter vector containing WT or MUT SphK1 promoter. Luciferase activity was measured using the dual-luciferase reporter assay system. (K) The mRNA and protein levels of SphK1 were determined when NF-κB was knockdown in 266-6 cells. *P <0.05; **P <0.01; ***P <0.001; NS, no significance.

Article Snippet: To investigate the role of S1P in macrophages, RAW264.7 cells were treated with 1 μM S1P (Sigma) and 1 μM JTE-013 (MedChemExpress).

Techniques: Expressing, Western Blot, Binding Assay, Knockdown, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay

Journal: International Journal of Biological Sciences

Article Title: SphK1/S1P signaling-mediated crosstalk between pancreatic acinar cell and macrophage M1 polarization aggravates acute pancreatitis progression

doi: 10.7150/ijbs.120627

Figure Lengend Snippet: Inhibiting the SphK1/S1P/S1PR2 pathway impedes M1 macrophage polarization and alleviates inflammation in AP mice. The activity of SphK1 and S1PR2 of AP mice was inhibited by intraperitoneal injection of PF-543 and JTE-013 respectively. (A) Representative images of H&E staining in the pancreatic tissues of AP mice (n=4). Scale bar: 100 μm. (B) The expression of F4/80 and CD86 in the pancreatic tissues of AP mice were evaluated by IHC (n=6). Scale bar: 100 μm. (C) The mRNA levels of CD86, iNOS, and TNF-α in the pancreatic tissues of AP mice were determined by RT-qPCR. (D) The protein levels of CD86 and iNOS in the pancreatic tissues of AP mice were analyzed by Western blot. (E-F) The serum levels of TNF-α and IL-1β were measured by ELISA in AP mice. (G) Schematic diagram of iPACs induced M1 polarization of macrophage via SphK1/S1P signaling during AP. *P <0.05; **P <0.01; ***P <0.001; NS, no significance.

Article Snippet: To investigate the role of S1P in macrophages, RAW264.7 cells were treated with 1 μM S1P (Sigma) and 1 μM JTE-013 (MedChemExpress).

Techniques: Activity Assay, Injection, Staining, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Circulation

Article Title: Tumor necrosis factor-α-mediated downregulation of the cystic fibrosis transmembrane conductance regulator drives pathological sphingosine-1-phosphate signaling in a mouse model of heart failure.

doi: 10.1161/CIRCULATIONAHA.111.047316

Figure Lengend Snippet: Figure 1. The cystic fibrosis transmembrane conductance regulator (CFTR) mediates cellular fluorescein isothiocyanate (FITC)– sphingosine-1-phosphate (S1P) uptake. A, Western blots confirm CFTR protein expression in isolated mouse cerebral and mesenteric arteries, as well as primary vascular smooth muscle cells (VSMCs) isolated from mouse mesenteric arteries. No CFTR signal was detected from mesenteric VSMCs isolated from CFTR knockout (CFTR/) mice. The blots displayed are representative of at least 3 separate experiments. B, A standard fluorescence-activated cell sorting analysis technique reveals a shift in the median fluorescence intensity at 525 nm for VSMCs incubated with FITC-S1P compared with cells incubated with unlabeled S1P (background [Bkgd]). This median shift represents a measure of FITC-S1P uptake. C, In wild-type littermate VSMCs (CFTR/), the median fluorescence intensity shift (n6) is abolished by CFTR inhibition [100 nmol/L CFTR(inh)-172 for 30 minutes; n5]. In VSMCs isolated from CFTR knockout mice, no shift in median fluorescence intensity is observed after FITC-S1P treatment (n6), nor is there an effect of CFTR inhibition (n5). Con indicates control. D, The S1P uptake deficit in CFTR/ VSMCs was rescued by transfecting the cells with a wild-type CFTR construct (CFTRwt; n6); the rescued S1P uptake was abolished by CFTR inhibition (n5). E, In baby hamster kidney (BHK) cells, stable expression of CFTRwt significantly increased S1P uptake compared with nontransfected cells (n4). F, Maintaining BHK cells that express CFTRF508 at 27°C for 24 hours (a procedure that increases plasma membrane abundance of CFTR) also significantly increased S1P uptake (n6). *P0.05 for multiple, unpaired comparisons within each genotype in C; multiple, unpaired comparisons in D; and single, unpaired comparisons in E and F. E and F display data normalized to their respective backgrounds.

Article Snippet: Sphingosine-1-phosphate (S1P) was purchased from Biomol International (Plymouth Landing, USA), fluorescein-labeled S1P (FITC-S1P) from Echelon Biosciences (Salt Lake City, USA), JTE013 from Tocris Bioscience (Ellisville, USA) and etanercept from Amgen (Thousand Oaks, USA).

Techniques: Western Blot, Expressing, Isolation, Knock-Out, FACS, Incubation, Inhibition, Control, Construct, Clinical Proteomics, Membrane

Journal: Circulation

Article Title: Tumor necrosis factor-α-mediated downregulation of the cystic fibrosis transmembrane conductance regulator drives pathological sphingosine-1-phosphate signaling in a mouse model of heart failure.

doi: 10.1161/CIRCULATIONAHA.111.047316

Figure Lengend Snippet: Figure 2. The cystic fibrosis transmembrane conductance regulator (CFTR) modulates sphingosine-1-phosphate (S1P)–dependent responses in cultured vascular smooth muscle cells and isolated arteries. A, S1P (100 nmol/L) significantly inhibits wild-type (CFTR/) vascular smooth muscle cell proliferation, an effect that is enhanced by CFTR inhibition [100 nmol/L CFTR(inh)-172; n5 for all groups]. B, CFTR knockout (CFTR/) vascular smooth muscle cell proliferation is lower under control (con) conditions than CFTR/ vascular smooth muscle cells (P0.05 comparing white bars in A and B; n5). S1P inhibits cell proliferation in CFTR/ vascular smooth muscle cells; however, CFTR inhibition has no further effect (n5). C, S1P-stimulated vasoconstriction is stronger in posterior cerebral arteries iso- lated from CFTR/ compared with CFTR/ littermate controls (n6 for both groups). Posterior cerebral artery responses to phenylephrine (D) and acetylcholine (E) are similar in the 2 genotypes (n4 for CFTR/; n3 for CFTR/). F, Posterior cerebral artery myogenic tone is stronger at all transmural pressures 20 mm Hg (ie, 40–100 mm Hg); a similar pattern is observed for mesenteric arteries (G) (for both cere- bral and mesenteric arteries: n6 for CFTR/; n8 for CFTR/). The enhanced myogenic tone in CFTR/ cerebral (F) and mesenteric (G) arteries is abolished by S1P2 receptor inhibition (1 mol/L JTE013; 30 minutes; n4 for both artery types). Maximal vessel diameters at 45 mm Hg (diamax) were as follows: posterior cerebral artery CFTR/ 1668 m (n8), posterior cerebral artery CFTR/ 1625 m (n6), PNS; and mesenteric CFTR/ 24011 m (n8), mesenteric CFTR/ 30134 m (n6), PNS. In A and B, *P0.05 relative to control; P0.05 relative to S1P for multiple, unpaired comparisons; in C through E, *P0.05 after 2-way repeated-measures ANOVA; in F and G, *P0.05 relative to the CFTR/ genotype after 2-way ANOVA.

Article Snippet: Sphingosine-1-phosphate (S1P) was purchased from Biomol International (Plymouth Landing, USA), fluorescein-labeled S1P (FITC-S1P) from Echelon Biosciences (Salt Lake City, USA), JTE013 from Tocris Bioscience (Ellisville, USA) and etanercept from Amgen (Thousand Oaks, USA).

Techniques: Cell Culture, Isolation, Inhibition, Knock-Out, Control

Journal: Circulation

Article Title: Tumor necrosis factor-α-mediated downregulation of the cystic fibrosis transmembrane conductance regulator drives pathological sphingosine-1-phosphate signaling in a mouse model of heart failure.

doi: 10.1161/CIRCULATIONAHA.111.047316

Figure Lengend Snippet: Figure 3. Heart failure (HF) downregulates the cystic fibrosis transmembrane conductance regulator (CFTR) but not sphingosine-1- phosphate (S1P) signaling components. HF (4–6 weeks after myocardial infarction) is associated with the downregulation of posterior cerebral artery CFTR protein expression (n4) (A), which negatively correlated with the extent of the myocardial infarction (B). C through E, HF does not affect the mRNA expression of mesenteric artery sphingosine kinase 1 (SphK1; n12), S1P phosphohydrolase 1 (SPP1; n7), or the S1P2 receptor subtype (S1P2R; n6). *P0.05 for single, unpaired comparisons.

Article Snippet: Sphingosine-1-phosphate (S1P) was purchased from Biomol International (Plymouth Landing, USA), fluorescein-labeled S1P (FITC-S1P) from Echelon Biosciences (Salt Lake City, USA), JTE013 from Tocris Bioscience (Ellisville, USA) and etanercept from Amgen (Thousand Oaks, USA).

Techniques: Expressing

Journal: Circulation

Article Title: Tumor necrosis factor-α-mediated downregulation of the cystic fibrosis transmembrane conductance regulator drives pathological sphingosine-1-phosphate signaling in a mouse model of heart failure.

doi: 10.1161/CIRCULATIONAHA.111.047316

Figure Lengend Snippet: Figure 6. Tumor necrosis factor- (TNF-) downregulates cystic fibrosis transmembrane conductance regulator (CFTR) expression and stimulates a reduction in plasma membrane–associated CFTR. A, TNF- (10 ng/mL; 24 hours) stimulates the downregulation of mesen- teric vascular smooth muscle cell CFTR protein expression (n6), with (B) a concomitant reduction in fluorescein isothiocyanate– sphingosine-1-phosphate (S1P) uptake (n6). Both the reduced CFTR expression (n4) (C) and attenuated fluorescein isothiocyanate– S1P uptake (n4–10) (D) were completely reversed 24 hours after TNF- washout. E, TNF- treatment (10 ng/mL) stimulates a steady decline in CFTR protein expression (n4). Analysis of the 165-kDa (immature, nonglycosylated CFTR; band B) and 170-kDa (mature, glyco- sylated CFTR; band C) bands indicated that only the mature form of CFTR declined. F, Analysis of plasma membrane and intracellular frac- tions (n6) indicated that membrane-associate CFTR was reduced without a concomitant accumulation of intracellular-associated CFTR. In A and B, *P0.05 for unpaired comparisons with control (con)/background (Bkgd); in C through F, *P0.05 for multiple, unpaired compari- sons with the control value at time0 hours; P0.05 for difference between pre–TNF- removal and TNF- removal.

Article Snippet: Sphingosine-1-phosphate (S1P) was purchased from Biomol International (Plymouth Landing, USA), fluorescein-labeled S1P (FITC-S1P) from Echelon Biosciences (Salt Lake City, USA), JTE013 from Tocris Bioscience (Ellisville, USA) and etanercept from Amgen (Thousand Oaks, USA).

Techniques: Expressing, Clinical Proteomics, Membrane, Control

Journal: Circulation

Article Title: Tumor necrosis factor-α-mediated downregulation of the cystic fibrosis transmembrane conductance regulator drives pathological sphingosine-1-phosphate signaling in a mouse model of heart failure.

doi: 10.1161/CIRCULATIONAHA.111.047316

Figure Lengend Snippet: Figure 8. Schematic representation of the proposed relationship between cystic fibrosis transmembrane conductance regu- lator (CFTR) and sphingosine-1-phosphate (S1P) signaling. S1P produced by sphin- gosine kinase 1 (Sphk1) is released to the extracellular compartment, where it (1) ac- tivates S1P receptor (S1PR)–dependent signaling pathways or (2) is transported by CFTR across the plasma membrane for degradation by S1P phosphohydrolase 1 (SPP1). During heart failure, tumor necro- sis factor- (TNF-) stimulates the down- regulation of CFTR (3), thus inhibiting S1P degradation and concomitantly enhancing S1P receptor signaling. sER indicates smooth endoplasmic reticulum.

Article Snippet: Sphingosine-1-phosphate (S1P) was purchased from Biomol International (Plymouth Landing, USA), fluorescein-labeled S1P (FITC-S1P) from Echelon Biosciences (Salt Lake City, USA), JTE013 from Tocris Bioscience (Ellisville, USA) and etanercept from Amgen (Thousand Oaks, USA).

Techniques: Produced, Protein-Protein interactions, Clinical Proteomics, Membrane