Journal: The Journal of Biological Chemistry

Article Title: Protein kinase a regulates cyclooxygenase-2 expression through the RNA-binding proteins HuR and TTP

doi: 10.1016/j.jbc.2025.111064

Figure Lengend Snippet: PKA activation is required for COX-2 induction and stabilization in macrophages . A , THP-1 macrophages (Mϕ) were serum-starved for 12 h and stimulated with 1 μM PGE 2 , 5 ng/ml IL-1β, or a combination of both for the indicated times. Cell lysates were analyzed by immunoblotting for COX-2 and GAPDH. Representative blots from three independent experiments are shown. B , THP-1 Mϕ were serum-starved for 12 h and stimulated with 1 μM PGE 2 , 1 μM forskolin/50 μM IBMX, 5 ng/ml IL-1β, or combinations for 6 h. Cell lysates were analyzed by immunoblotting for COX-2 and GAPDH. Representative blots from three independent experiments are shown. C , THP-1 Mϕ were serum-starved for 12 h and stimulated as in B for 6 h. Total RNA was extracted, and COX-2 and GAPDH mRNA levels were determined by RT-qPCR. Graph shows ΔΔCt values (mean ± SD) from four independent experiments (n = 4). Statistical significance was determined by one-way ANOVA followed by Sidak's multiple comparisons test; p values are indicated. D , THP-1 Mϕ were serum-starved for 12 h, preincubated with 1 μM ONO-AE3-208 (EP4 inhibitor) for 1 h, and then stimulated with 1 μM PGE 2 , 5 ng/ml IL-1β, or a combination for 6 h. Cell lysates were analyzed by immunoblotting for COX-2 and GAPDH. Representative blots from three independent experiments are shown. E , THP-1 Mϕ were serum-starved for 12 h, preincubated with 1 μM BLU0588 (PKA inhibitor) for 12 h, and then stimulated with 1 μM PGE 2 , 5 ng/ml IL-1β, or a combination for 6 h. Cell lysates were analyzed by immunoblotting for COX-2 and GAPDH. Representative blots from three independent experiments are shown. F , THP-1 Mϕ were serum-starved for 12 h, preincubated with 1 μM BLU0588 for 12 h, and then stimulated as in E for 6 h. Total RNA was extracted, and COX-2 and GAPDH mRNA levels were determined by RT-qPCR. Graph shows ΔΔCt values (mean ± SD) from three independent experiments (n = 3). Statistical significance was determined by one-way ANOVA followed by Sidak's multiple comparisons test; p values are indicated. G , schematic representation of COX-2 regulation at transcriptional, posttranscriptional (RNA stability), and protein stability levels. H , THP-1 Mϕ were stimulated with 1 μM PGE 2 and 5 ng/ml IL-1β for the indicated short time points. Total RNA was extracted, and COX-2 mRNA levels were determined by RT-qPCR. Graph shows ΔΔCt values (mean ± SD) from three independent experiments (n = 3). Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparisons test; p values are indicated. I , THP-1 Mϕ were serum-starved for 12 h, stimulated with 1 μM PGE 2 and 5 ng/ml IL-1β for the indicated times followed by the addition of 5 μg/ml actinomycin D to inhibit transcription for the indicated time points, in the presence or absence of 1 μM PKA inhibitor BLU0588. Total RNA was extracted, and COX-2 mRNA levels were determined by RT-qPCR. Graph shows the percentage of COX-2 mRNA remaining at each time point, normalized to time 0, from three independent experiments. COX-2, cyclooxygenase-2; IL-1β, interleukin 1β; Mϕ, macrophages; PKA, protein kinase A; RT-qPCR, reverse transcription quantitative PCR.

Article Snippet: Antibodies for COX-2 (#12282), GAPDH (#2118), pVASP S157 (#84519), VASP (#3132), pP38 T180/182 (#4511), P38 (#8690), pNFκB S536 (#3033), NFκB (#8042), TTP (#71632), RIα/β (#3927), Cα (#5842), GFP (#2956), GST (#2622), phospho-CREB S133 (#9198), CREB (#4820), and PKA substrates (#9624) were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Protein kinase a regulates cyclooxygenase-2 expression through the RNA-binding proteins HuR and TTP

doi: 10.1016/j.jbc.2025.111064

Figure Lengend Snippet: PKA interacts with HuR, a regulator of COX-2 mRNA stability . A , interaction of endogenous PKA with HuR in THP-1 macrophages (Mϕ). THP-1 Mϕ were serum-starved for 12 h and stimulated with 100 ng/ml LPS for 2 h. Cell lysates were subjected to pull-down using 8-AHA-cAMP (RIα), Rp-8-AHA-cAMPS (holoenzyme), or HaloLink resin (control), followed by immunoblot analysis for HuR, TTP, PKA-Cα, and RIα. B , interaction of HuR with the PKA catalytic subunit. HEK293T cells expressing EGFP-HuR, Flag-Cα, Flag-RIα, and Flag-HaloTag (negative control) were serum-starved for 12 h. Cell lysates were immunoprecipitated using DYKDDDDK Fab-Trap agarose and analyzed by immunoblotting for EGFP and Flag. C , purification of HuR and PKA-Cα for in vitro interaction assays. Coomassie blue-stained gels showing purified HaloTag-TEV-HuR (soluble) and DYKDDDDK Fab-Trap agarose-immobilized PKA-Cα and HaloTag (negative control). D , in vitro interaction between HuR and PKA-Cα. Purified PKA-Cα or HaloTag (immobilized on beads) was incubated with purified HuR (soluble). Bound HuR was detected by immunoblotting. E , schematic representation of HuR's RNA recognition motifs (RRMs) used to generate GST-tagged constructs for interaction studies. F , interaction of PKA-Cα with HuR RRM domains. HEK293T cells coexpressing GST-HuR RRM domains, GFP-PKA-Cα, and PKA-RIα were subjected to GST pull-down and analyzed by immunoblotting for GFP, Flag, and GST. G , enhancement of HuR-PKA-Cα interaction by forskolin/IBMX stimulation. HEK293T cells expressing GST-HuR, GFP-PKA-Cα, and PKA-RIα were serum-starved for 12 h and stimulated with 1 μM forskolin and 50 μM IBMX for the indicated times. Cell lysates were subjected to GST pull-down and analyzed by immunoblotting for GFP, Flag, and GST. COX-2, cyclooxygenase-2; HEK293T, human embryonic kidney 293 cell line; HuR, Hu antigen R; LPS, lipopolysaccharide; PKA, protein kinase A; TTP, tristetraprolin.

Article Snippet: Antibodies for COX-2 (#12282), GAPDH (#2118), pVASP S157 (#84519), VASP (#3132), pP38 T180/182 (#4511), P38 (#8690), pNFκB S536 (#3033), NFκB (#8042), TTP (#71632), RIα/β (#3927), Cα (#5842), GFP (#2956), GST (#2622), phospho-CREB S133 (#9198), CREB (#4820), and PKA substrates (#9624) were purchased from Cell Signaling Technology.

Techniques: Control, Western Blot, Expressing, Negative Control, Immunoprecipitation, Purification, In Vitro, Staining, Incubation, Construct

Journal: The Journal of Biological Chemistry

Article Title: Protein kinase a regulates cyclooxygenase-2 expression through the RNA-binding proteins HuR and TTP

doi: 10.1016/j.jbc.2025.111064

Figure Lengend Snippet: HuR inhibition prevents COX-2 expression induced by PGE 2 and IL-1β in macrophages . A , effect of HuR inhibition on COX-2 protein expression in THP-1 macrophages (Mϕ) stimulated with PGE 2 and IL-1β. THP-1 Mϕ were serum-starved for 12 h, preincubated with 1 μM MS-444 (HuR inhibitor) for 12 h, and then stimulated with 1 μM PGE 2 , 5 ng/ml IL-1β, or a combination for 6 h. Cell lysates were analyzed by immunoblotting for COX-2 and GAPDH. Representative blots from three independent experiments are shown. B , effect of HuR inhibition on COX-2 mRNA expression in THP-1 Mϕ stimulated with PGE 2 and IL-1β. THP-1 MΦ were treated as in A . Total RNA was extracted, and COX-2 and GAPDH mRNA levels were determined by RT-qPCR. Graph shows ΔΔCt values (mean ± SD) from three independent experiments (n = 3). Statistical significance was determined by one-way ANOVA followed by Sidak's multiple comparisons test; p values are indicated. C , effect of HuR inhibition on COX-2 protein expression in murine bone marrow-derived macrophages (mBMDMs) stimulated with PGE 2 and IL-1β. mBMDMs were treated as in A . Cell lysates were analyzed by immunoblotting for mCOX-2 and mGAPDH. Representative blots from three independent experiments are shown. D , effect of HuR inhibition on COX-2 mRNA expression in mBMDMs stimulated with PGE 2 and IL-1β. mBMDMs were treated as in A . Total RNA was extracted, and COX-2 and GAPDH mRNA levels were determined by RT-qPCR. Graph shows ΔΔCt values (mean ± SD) from three independent experiments (n = 3). Statistical significance was determined by one-way ANOVA followed by Sidak's multiple comparisons test; p values are indicated. E , effect of HuR inhibition on COX-2 protein expression in THP-1 Mϕ stimulated with forskolin, IBMX, and IL-1β. THP-1 Mϕ were serum-starved for 12 h, preincubated with 1 μM MS-444 for 12 h, and then stimulated with 1 μM forskolin, 50 μM IBMX, and 5 ng/ml IL-1β, or a combination for 6 h. Cell lysates were analyzed by immunoblotting for COX-2 and GAPDH. Representative blots from three independent experiments are shown. F , effect of HuR inhibition on COX-2 mRNA expression in THP-1 Mϕ stimulated with forskolin, IBMX, and IL-1β. THP-1 Mϕ were treated as in E . Total RNA was extracted, and COX-2 and GAPDH mRNA levels were determined by RT-qPCR. Graph shows ΔΔCt values (mean ± SD) from three independent experiments (n = 3). Statistical significance was determined by one-way ANOVA followed by Sidak's multiple comparisons test; p values are indicated. G , effect of PKA-Cα knockout (KO) on COX-2 mRNA expression in HEK293T cells stimulated with forskolin and IBMX. HEK293T cells (Cas9 control or PKA-Cα KO) were serum-starved for 12 h and stimulated with 1 μM forskolin and 50 μM IBMX for 1 h. Total RNA was extracted, and COX-2 and GAPDH mRNA levels were determined by RT-qPCR. Graph shows ΔΔCt values (mean ± SD) from four independent experiments (n = 4). Statistical significance was determined by one-way ANOVA followed by Sidak's multiple comparisons test; p values are indicated. H , effect of HuR knockout (KO) on COX-2 mRNA expression in HEK293T cells stimulated with forskolin and IBMX. HEK293T cells (Cas9 control or HuR KO) were treated as in G . Graph shows ΔΔCt values (mean ± SD) from four independent experiments (n = 4). Statistical significance was determined by one-way ANOVA followed by Sidak's multiple comparisons test; p values are indicated. I , effect of PKA and HuR KO on COX-2 protein expression in HEK293T cells stimulated with forskolin and IBMX. HEK293T cells (Cas9 control, PKA-Cα KO, or HuR KO) were serum-starved for 12 h and stimulated with 1 μM forskolin and 50 μM IBMX for 1 h. Cell lysates were analyzed by immunoblotting for COX-2 and GAPDH. Representative blots from three independent experiments are shown. COX-2, cyclooxygenase-2; HuR, Hu antigen R; IL-1β, interleukin 1β; PKA, protein kinase A; RT-qPCR, reverse transcription quantitative PCR.

Article Snippet: Antibodies for COX-2 (#12282), GAPDH (#2118), pVASP S157 (#84519), VASP (#3132), pP38 T180/182 (#4511), P38 (#8690), pNFκB S536 (#3033), NFκB (#8042), TTP (#71632), RIα/β (#3927), Cα (#5842), GFP (#2956), GST (#2622), phospho-CREB S133 (#9198), CREB (#4820), and PKA substrates (#9624) were purchased from Cell Signaling Technology.

Techniques: Inhibition, Expressing, Western Blot, Quantitative RT-PCR, Derivative Assay, Knock-Out, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Protein kinase a regulates cyclooxygenase-2 expression through the RNA-binding proteins HuR and TTP

doi: 10.1016/j.jbc.2025.111064

Figure Lengend Snippet: PKA promotes phosphorylation of TTP at serine 273 and decreases its binding to the COX-2 3′UTR . A , interaction of PKA with HuR or TTP. HEK293T cells coexpressing EGFP-PKA-Cα and GST-tagged HuR or TTP were lysed and subjected to GST pulldown. Pull-down and input samples were analyzed by immunoblotting for PKAS, GST, and GFP. B , effect of PKA inhibition on TTP interaction. HEK293T cells coexpressing EGFP-PKA-Cα and GST-tagged TTP were incubated with 1 μM BLU0588 (PKA inhibitor) for 12 h, lysed, and subjected to GST pulldown. Pulldown and input samples were analyzed by immunoblotting for PKAs and GST. C , effect of forskolin/IBMX stimulation on TTP interaction. HEK293T cells expressing GST-TTP were serum-starved for 12 h, stimulated with 1 μM forskolin and 50 μM IBMX for the indicated times, lysed, and subjected to GST pull-down. Pull-down and input samples were analyzed by immunoblotting for PKAs and GST. D , schematic representation of TTP modular architecture and potential PKA phosphorylation sites based on Scansite 4.0 and PhosphoSitePlus. E , identification of PKA phosphorylation site on TTP. HEK293T cells coexpressing EGFP-PKA-Cα and GST-tagged TTP wild-type or Ser/Thr mutants were lysed and subjected to GST pulldown. Pulldown and input samples were analyzed by immunoblotting for PKAs, GST, and GFP. Representative blots from three independent experiments are shown. F , effect of PKA activation on TTP interaction with COX-2 3′UTR. HEK293T cells coexpressing GST, GST-TTP wild-type or S273A, and COX-2 3′UTR-luciferase were serum-starved, treated with 1 μM forskolin and 50 μM IBMX for 1 h, lysed, and subjected to RNA immunoprecipitation (RIP). COX-2 and GAPDH mRNA levels were determined by RT-qPCR on RIP and input samples. Graph shows relative COX-2 mRNA binding (normalized to GST-TTP wild-type control) as ΔΔCt values (mean ± SD) from four independent experiments (n = 4). Statistical significance was determined by one-way ANOVA followed by Sidak's multiple comparisons test; p values are indicated. COX-2, cyclooxygenase-2; HuR, Hu antigen R; PKA, protein kinase A; TTP, tristetraprolin.

Article Snippet: Antibodies for COX-2 (#12282), GAPDH (#2118), pVASP S157 (#84519), VASP (#3132), pP38 T180/182 (#4511), P38 (#8690), pNFκB S536 (#3033), NFκB (#8042), TTP (#71632), RIα/β (#3927), Cα (#5842), GFP (#2956), GST (#2622), phospho-CREB S133 (#9198), CREB (#4820), and PKA substrates (#9624) were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, Binding Assay, Western Blot, Inhibition, Incubation, Expressing, Activation Assay, Luciferase, RNA Immunoprecipitation, Quantitative RT-PCR, Control

Journal: The Journal of Biological Chemistry

Article Title: Protein kinase a regulates cyclooxygenase-2 expression through the RNA-binding proteins HuR and TTP

doi: 10.1016/j.jbc.2025.111064

Figure Lengend Snippet: Proposed model for PKA-dependent posttranscriptional regulation of COX-2 expression via HuR and TTP . Active Gs-coupled EP4 receptors stimulate adenylate cyclase, generating cAMP that binds to PKA regulatory subunits and promotes holoenzyme dissociation. Free PKA catalytic subunits (PKA-C) bind to HuR and phosphorylate TTP at serine 273, inhibiting TTP binding to AU-rich elements in the COX-2 3′UTR. This favors HuR binding and stabilizes COX-2 mRNA. PKA-C binding to HuR provides proximity to COX-2 mRNA, efficiently protecting it from TTP-mediated degradation. Stabilized COX-2 mRNA enhances translation, and the resulting COX-2 protein increases PGE 2 production. Secreted PGE 2 can bind autocrinally to EP4 receptors, establishing a potential positive feedback loop. COX-2, cyclooxygenase-2; PKA, protein kinase A; TTP, tristetraprolin.

Article Snippet: Antibodies for COX-2 (#12282), GAPDH (#2118), pVASP S157 (#84519), VASP (#3132), pP38 T180/182 (#4511), P38 (#8690), pNFκB S536 (#3033), NFκB (#8042), TTP (#71632), RIα/β (#3927), Cα (#5842), GFP (#2956), GST (#2622), phospho-CREB S133 (#9198), CREB (#4820), and PKA substrates (#9624) were purchased from Cell Signaling Technology.

Techniques: Expressing, Binding Assay

Journal: Biomedicines

Article Title: Effect of Serping1 siRNA Injection on Dopaminergic Cell Reduction in an MPTP-Induced Parkinson’s Disease Mouse Model

doi: 10.3390/biomedicines14030569

Figure Lengend Snippet: Changes in cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS) levels in the striatum by injecting Serping1 siRNA. ( A ) Immunoblot results of COX2 and iNOS in the striatum of C, NC, SER1, and NAC groups. ( B ) The immunoblot of COX2 and iNOS is shown in a graph. COX2 and iNOS levels increased significantly in the NC group. However, COX2 levels decreased significantly in the SER1 and NAC groups, and iNOS levels also decreased in the SER1 and NAC groups; (COX2, n = 3, F(3,8) = 14.270, ANOVA = 0.001; iNOS, n = 3, F(3,8) = 4.366, ANOVA = 0.042) means ± standard errors, * p < 0.05, ** p < 0.005, # p = 0.067.

Article Snippet: Then, the membranes were incubated with primary antibodies: anti-TH (1:2000; Santa Cruz Biotechnology, Dallas, TX, USA), anti- Serping1 (1:2000; Cloud clone Corp., Katy, TX, USA), anti-β-actin (1:5000; Santa Cruz Biotechnology), anti-pSer129-α-syn (1:1000; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), anti-cyclooxygenase-2 (COX2) (1:2000; Proteintech group, Rosemont, IL, USA), and anti-nitric oxide synthase (iNOS) (1:2000; Calbiochem, Darmstadt, Germany).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: TREM-1 Is Induced in Tumor Associated Macrophages by Cyclo-Oxygenase Pathway in Human Non-Small Cell Lung Cancer

doi: 10.1371/journal.pone.0094241

Figure Lengend Snippet: A) Western blot analysis from human macrophages with COX-2 siRNA confirmed knock down of COX-2 protein in cells with siCOX-2 compared to the mock (control) siRNA. B) FACS images demonstrating TREM-1 expression from human macrophages expressing control siRNA or COX-2 siRNA co-cultured with NL-20 cells or cancer cells (A549, H23 or H 838 cells). The expression of TREM-1 was increased in macrophages expressing control siRNA co-cultured with cancer cells whereas macrophages with siCOX-2 showed an attenuated expression of TREM-1. C) Percentage of cells that stained positive for TREM-1 staining n = 3, p<0.05). D) Representative RT-PCR from macrophages with siCOX-2 show a decreased TREM-1 message compared to macrophages with control siRNA.

Article Snippet: Cells were transfected with siRNA oligonucleotides targeting human COX-2 mRNA, and a non-related control siRNA (Santa Cruz) through specific LONZA transfection reagents (LONZA) according to the manufacturer’s instructions.

Techniques: Western Blot, Knockdown, Control, Expressing, Cell Culture, Staining, Reverse Transcription Polymerase Chain Reaction