crfr1 agonist stressin-1  (Tocris)

Journal: Pain

Article Title: Mast cell corticotropin-releasing factor receptor 1 contributes to pancreatic cancer pain via mitogen-activated protein kinase/sphingosine kinases type 1 signaling

doi: 10.1097/j.pain.0000000000003909

Figure Lengend Snippet: Mast cell CRFR1 expression is elevated in patients with PDAC and mouse models. (A and B) CRF levels increased in both patients with PDAC and mouse models with abdominal hyperalgesia based on ELISA assay. (C) UMAP plot of reanalyzed scRNA-seq data from dataset GSE155698 showing distinct pancreatic cell populations. (D) The percentage of mast cell cluster increased in PDAC tissues compared to normal pancreatic tissues. (E) Average CRFR1 expression per mast cell was significantly elevated in PDAC tissues, whereas CRFR2 expression remained unchanged. (F and G) qRT-PCR and western blotting confirmed the upregulation of CRFR1 in orthotopic PDAC mouse models. (H and I) Flow cytometry demonstrated an increased proportion of CD117 + FcεR + mast cells (gated within CD45 + leukocytes) and elevated CRFR1 expression in CD117 + FcεR + CD45 + mast cells in PDAC mice. (J–M) Immunofluorescence staining further confirmed elevated CRFR1 expression in tryptase + mast cells in PDAC experimental mice and painful human patients with PDAC. n = 5 mice per group. Data with error bars represented as mean ± SD and analyzed using Mann–Whitney U test (A, M) or Student t test (B, F, I, K). * P < 0.05, ** P < 0.01, *** P < 0.001. CRF, corticotropin-releasing factor; CRFR1, corticotropin-releasing factor receptor 1; ELISA, enzyme-linked immunosorbent assay; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; UMAP, uniform manifold approximation and projection.

Article Snippet: To assess the role of CRFR1, 1 μM CRF, a CRFR1 agonist (HY-P1533A, MCE), was added to MC/9 cells for 48 hours.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Flow Cytometry, Immunofluorescence, Staining, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: Pain

Article Title: Mast cell corticotropin-releasing factor receptor 1 contributes to pancreatic cancer pain via mitogen-activated protein kinase/sphingosine kinases type 1 signaling

doi: 10.1097/j.pain.0000000000003909

Figure Lengend Snippet: CRFR1 promotes the activation of mast cell and enhances abdominal pain sensitivity. (A) qRT-PCR verified that CRF (1 μM) significantly upregulated mast cell activation markers, including TPSAB1, IL-4, TNF-α, and CCL2. (B) Western blotting confirmed the increased protein expression of TPSAB1 after CRF stimulation. (C) ELISA assays demonstrated elevated levels of secreted TPS, His, IL-4, TNF-α, and CCL2 in the supernatant of CRF-treated cells. (D) Administration of ANT (a CRFR1 antagonist) significantly reduced abdominal withdrawal response frequency in PDAC mice. (E and F) qRT-PCR and western blotting revealed reduced expression of TPSAB1, IL-4, TNF-α, and CCL2 after ANT treatment. (G) ELISA results further confirmed the suppression of TPS, His, IL-4, TNF-α, and CCL2 secretion in the ANT-treated group. n = 5 mice per group. Data with error bars represented as mean ± SD and analyzed using 2-tailed Student t test. * P < 0.05, ** P < 0.01, *** P < 0.001. ANT, Antisauvagine; CCL2, C-C motif chemokine ligand 2; CRF, corticotropin-releasing factor; CRFR1, corticotropin-releasing factor receptor 1; ELISA, enzyme-linked immunosorbent assay; IL-4, interleukin-4; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; TNF-α, tumor necrosis factor-alpha; TPS, tryptase; TPSAB1, tryptase alpha/beta 1.

Article Snippet: To assess the role of CRFR1, 1 μM CRF, a CRFR1 agonist (HY-P1533A, MCE), was added to MC/9 cells for 48 hours.

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Pain

Article Title: Mast cell corticotropin-releasing factor receptor 1 contributes to pancreatic cancer pain via mitogen-activated protein kinase/sphingosine kinases type 1 signaling

doi: 10.1097/j.pain.0000000000003909

Figure Lengend Snippet: Mast cell–specific CRFR1 knockout reduces mast cell degranulation and alleviates cancer-associated pain. (A and B) Flow cytometry analysis of CRFR1 expression in CD117 + FcεR + mast cells from para-tumor tissues confirmed efficient CRFR1 knockout in PC-CKO mice. (C and D) Immunofluorescence staining further validated decreased CRFR1 expression in tryptase + mast cells from mouse para-tumor tissues. White arrows indicated the colocalization of CRFR1 and tryptase. Quantification of CRFR1 + tryptase + mast cells was performed using ImageJ. Scale bar = 400 µm. (E) Mast cell–specific CRFR1 knockout (PC-CKO) mice exhibited reduced abdominal mechanical hyperalgesia compared with PC-WT controls, as measured by the von Frey test. No significant difference was observed between WT and CKO groups in the absence of tumor transplantation. (F) PC-CKO mice showed lower hunching scores compared to PC-WT mice, indicating reduced spontaneous abdominal pain. (G and H) qRT-PCR and western blotting demonstrated decreased expression of mast cell activation markers in CKO mice. (I) ELISA assays showed reduced secretion of TPS, His, IL-4, TNF-α, and CCL2, indicating suppressed mast cell degranulation after CRFR1 deletion. n = 5 mice per group. Data with error bars represented as mean ± SD and analyzed using Student t test (B and D) and 2-way ANOVA (E, F, H, I). * P < 0.05, ** P < 0.01, *** P < 0.001. CCL2, C-C motif chemokine ligand 2; CRFR1, corticotropin-releasing factor receptor 1; CKO, conditional knockout; ELISA, enzyme-linked immunosorbent assay; IL-4, interleukin-4; qRT-PCR, quantitative real-time polymerase chain reaction; TNF-α, tumor necrosis factor-alpha; TPS, tryptase; WT, wild type; PC, orthotopic pancreatic cancer model mice; TPSAB1, tryptase alpha/beta 1.

Article Snippet: To assess the role of CRFR1, 1 μM CRF, a CRFR1 agonist (HY-P1533A, MCE), was added to MC/9 cells for 48 hours.

Techniques: Knock-Out, Flow Cytometry, Expressing, Immunofluorescence, Staining, Transplantation Assay, Quantitative RT-PCR, Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Pain

Article Title: Mast cell corticotropin-releasing factor receptor 1 contributes to pancreatic cancer pain via mitogen-activated protein kinase/sphingosine kinases type 1 signaling

doi: 10.1097/j.pain.0000000000003909

Figure Lengend Snippet: MAPK/SPHK1 axis is a potential downstream pathway of CRFR1 in mast cell activation. (A) Volcano plot showed 134 upregulated and 394 downregulated genes in para-tumor tissues from CRFR1-CKO mice compared to WT controls. (B) Heatmap of the top 100 differentially expressed genes. (C) GO enrichment analysis revealed key altered pathways, including MAPK signaling. (D) GSEA indicated that CRFR1 knockout suppressed the phospholipid biosynthetic process. (E and F) qRT-PCR and western blotting confirmed the downregulation of SPHK1 after CRFR1 inhibition. (G) ELISA results showing reduced S1P levels in CRFR1-CKO mice. (H) CRF stimulation increased SPHK1 expression and activated MAPK signaling in MC/9 mast cells in vitro. (I) CRFR1 knockout reduced MAPK pathway activation in vivo. (J) MAPK inhibitor SB203580 blocked CRF-induced SPHK1 upregulation. Data with error bars represented as mean ± SD and analyzed using 2-tailed Student t test. * P < 0.05, ** P < 0.01, *** P < 0.001. CKO, conditional knockout; CRF, corticotropin-releasing factor; CRFR1, corticotropin-releasing factor receptor 1; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; GO, gene ontology; GSEA, gene set enrichment analysis; MAPK, mitogen-activated protein kinase; PC, orthotopic pancreatic cancer model mice; qRT-PCR, quantitative real-time polymerase chain reaction; S1P, sphingosine-1-phosphate; SPHK1, sphingosine kinases type 1; WT, wild type.

Article Snippet: To assess the role of CRFR1, 1 μM CRF, a CRFR1 agonist (HY-P1533A, MCE), was added to MC/9 cells for 48 hours.

Techniques: Activation Assay, Knock-Out, Quantitative RT-PCR, Western Blot, Inhibition, Enzyme-linked Immunosorbent Assay, Expressing, In Vitro, In Vivo, Real-time Polymerase Chain Reaction

Journal: Pain

Article Title: Mast cell corticotropin-releasing factor receptor 1 contributes to pancreatic cancer pain via mitogen-activated protein kinase/sphingosine kinases type 1 signaling

doi: 10.1097/j.pain.0000000000003909

Figure Lengend Snippet: SPHK1 mediates the roles of CRFR1 on mast cell activation. (A and B) qRT-PCR and western blotting confirmed the upregulation of SPHK1 in the PDAC mice model. (C) Schematic of the experimental design. PF543, a selective SPHK1 antagonist, or vehicle was administered intraperitoneally to PDAC-bearing mice or mast cell–deficient (C-Kit W-sh ) mice. n = 5 mice per group. (D and E) Von Frey testing and hunching scores demonstrated alleviated cancer-associated abdominal pain after PF543 treatment or mast cell knockout, whereas the analgesic effect of PF543 was abrogated in mast cell deficient mice. (F and G) Inhibition of SPHK1 reduced mRNA and protein expression levels of TPSAB1, IL-4, TNF-α, and CCL2. (H) ELISA showed decreased secretion of mast cell degranulation factors after PF543 treatment, an effect blocked by mast cell knockout. (I and J) SiRNA-1 exhibited the highest knockdown efficiency of SPHK1 at both mRNA and protein levels. (K–M) Knockdown of SPHK1 attenuated CRF-induced expression of mast cell degranulation markers. Data with error bars represented as mean ± SD and analyzed using Student t test (A), 2-way ANOVA (D, E, G, H, K, M), and 1-way ANOVA (I). * P < 0.05, ** P < 0.01, *** P < 0.001. CCL2, C-C motif chemokine ligand 2; CRF, corticotropin-releasing factor; CRFR1, corticotropin-releasing factor receptor 1; ELISA, enzyme-linked immunosorbent assay; IL-4, interleukin-4; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; SPHK1, sphingosine kinases type 1; TNF-α, tumor necrosis factor-alpha; TPSAB1, tryptase alpha/beta 1.

Article Snippet: To assess the role of CRFR1, 1 μM CRF, a CRFR1 agonist (HY-P1533A, MCE), was added to MC/9 cells for 48 hours.

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, Knock-Out, Inhibition, Expressing, Enzyme-linked Immunosorbent Assay, Knockdown, Real-time Polymerase Chain Reaction

Journal: Current biology : CB

Article Title: Role of Corticotropin-Releasing Factor in Cerebellar Motor Control and Ataxia.

doi: 10.1016/j.cub.2017.07.035

Figure Lengend Snippet: Figure 2. CRFergic Neurons in the IO Directly Project to the Cerebellar IN, and CRFR1 and CRFR2 Are Co-expressed in the Cerebellar IN Glutamatergic Neurons (A) Retrograde tracer Fluoro-Gold and CRF were co-localized in neurons in the contralateral IO. Bottom: magnification of dotted squares (top). IOD, inferior olive dorsal nucleus; IOM, inferior olive medial nucleus; IOPr, inferior olive principal nucleus; py, pyramidal tract. (B) A cerebellar slice showing the injection site of Fluoro-Gold. IN, interpositus nucleus; 4V, 4th ventricle. (C) Fluoro-Gold-labeled fibers were observed in the inferior cerebellar peduncle. icp, inferior cerebellar peduncle. (D) Bar graphs showing the relative expression of Crfr1 and Crfr2 mRNAs to gapdh mRNA in the cerebellar IN (n = 6). Data represent mean ± SEM. (E) Double-immunostaining results showing that two CRF receptors, CRFR1 and CRFR2, were not only present in the cerebellar IN but also co-localized in the same IN neurons. DN, dentate nucleus; FN, fastigial nucleus; IN, interpositus nucleus. Bottom: magnification of dotted squares (top). (F and G) CRFR2 was co-localized with glutamate (F) rather than GAD67 (G), a key enzyme in GABA biosynthesis, in IN neurons, indicating a selective expression of CRF receptors in glutamatergic rather than GABAergic neurons in the cerebellar IN. White arrowheads indicate a GAD67-positive neuron that did not express CRFR2.

Article Snippet: To explore the underlying receptor and ionicmechanisms, selective CRFR1 antagonist antalarmin (300 nM), selective CRFR2 antagonist antisauvagine-30 (100 nM), selective CRFR1 agonist stressin-I (300 nM; Tocris, Bristol, UK), selective CRFR2 agonist urocortin-II (UCN-II, 300 nM; Phoenix, Burlingame, CA), selective inward-rectifier K+ channel blocker tertiapin-Q (100 nM; Tocris, Bristol, UK), and selective HCN channel blocker ZD7288 (50 mM, Tocris, Bristol, UK) were used.

Techniques: Injection, Labeling, Expressing, Double Immunostaining