Journal: Scientific Reports

Article Title: TLR4 counteracts BVRA signaling in human leukocytes via differential regulation of AMPK, mTORC1 and mTORC2

doi: 10.1038/s41598-019-43347-8

Figure Lengend Snippet: Our working model for biliverdin/BVRA and TLR4 signaling in leukocytes. Biliverdin induced responses are shown in black. Biliverdin acts on BVRA. Our data suggest that biliverdin facilitates formation of BVRA signaling complex(s) that include mTORC2. mTORC2 induces phosphorylation of two targets: PKCζ at Thr410 and Akt at Ser473. PKCζ is involved in LKB1 Ser431 and AMPKα Thr172 phosphorylation. AMPK phosphorylates TSC1/2 at Ser1387 and Raptor at Ser792. Therefore, BVRA activation may contribute to mTORC1 inhibition via two SI: phosphorylated Raptor, which inhibits mTORC1 complex formation, and TSC1/2 mediated inhibition of mTORC1 activity. Because mTORC1 is central to TLR4 signaling in leukocytes , BVRA inhibition of mTORC1 suppresses TLR4. Pharmacological inhibitors are shown in blue. Torin and PP242, but not rapamycin, inhibited BVRA-mediated protein-protein interactions. TLR4 induced response are shown in red. Once activated, TLR4 down-regulates BVRA and PKCζ expression (dashed lines). TLR4 upregulates expression of MMP9 , which then triggers AMPKα cleavage (dashed line), leading to Raptor Ser792 dephosphorylation . This enables mTORC1 activation, which then triggers an increase in HIF-1α expression and S6K1 phosphorylation at Thr389. TLR4 also triggers an increase in haptoglobin expression, thus limiting heme availability. Using these parallel mechanisms TLR4 ensures biliverdin/BVRA and mTORC2 signaling inhibition, and on the other hand, mTORC1 activation.

Article Snippet: HIF-1α (sc-10790; 1:250), AMPKα (sc-25792; 1:1000), MMP9 (sc-10737; 1:1000), TLR4 (sc-10741; 1:500), BVRA (sc-393385; 1:1000), Akt1 (sc-5928; 1:500), p-LKB1 Ser431 (sc-271924; 1:500), LKB1 (sc-32245; 1:500), p-CaMKKβ Thr286 (sc-32289; 1:750), CaMKKβ (sc-100364; 1:1000), p-mTOR Ser2481 (sc-293132; 1:1000), mTOR (sc-517464; 1:1000), p-PKCζ Thr410 (sc-271962; 1:500), PKCζ (sc-393218; 1:500), heme oxygenase 1 (HO-1) (sc-136960; 1:1000), heme oxygenase 2 (HO-2) (sc-17786; 1:1000), tuberin (TSC1/2)(sc-271314; 1:500), haptoglobin β (sc-390962; 1:500), p-NOS3 Ser1177 (sc-81510; 1:500) and NOS3 (sc-376751; 1:500) were all from Santa Cruz Biotechnology. p-Raptor Ser792 (#2083; 1:1000), Raptor (#2280; 1:200), p-p70 S6 kinase Thr389 (#9205; 1:1000), p-AMPKα Thr172 (#2535; 1:1000), p-Akt Ser473 (#4051; 1:1000), p-Akt Thr308 (#9275; 1:1000), tuberin/TSC2 (#4308; 1:1000) and p-tuberin/TSC2 Ser1387 (#5584; 1:1000) were all from Cell Signaling Technology.

Techniques: Phospho-proteomics, Activation Assay, Inhibition, Activity Assay, Protein-Protein interactions, Expressing, De-Phosphorylation Assay

Journal: Molecules and cells

Article Title: Regulations of Reversal of Senescence by PKC Isozymes in Response to 12-O-Tetradecanoylphorbol-13-Acetate via Nuclear Translocation of pErk1/2.

doi: 10.14348/molcells.2016.2362

Figure Lengend Snippet: Fig. 4. TPA rapidly degrades PKCα in nuclei of senescent cells, whereas its regeneration is so delayed. To examine the fate of PKCα after TPA stimulation in nuclei of senescent cells, ICC and IP-IB analyses were performed in the pres- ence and absence of proteasome inhibi- tor, MG132. (A) TPA treatment signifi- cantly increased fluorescence of PKCα in the nuclei of senescent HDF cells in 3 h, indicating nuclear translocation of PKCα in the cells. In addition, the fluo- rescence derived from ubiquitin and PKCα was co-localized in the nuclei of the cells. (B) IP with anti-PKCα antibody and IB with anti-ubiquitin antibody showed polyubiquitination of PKCα after TPA + MG132 treatment as compared with that of the DMSO + MG132 treat- ment (upper panel) along with the slight accumulation of PKCα protein in the TPA + MG132 treated cells than the control (middle panel). The 10% input shows equal loading of the proteins for IP analysis (lower panels). (C) Confocal microscope findings revealing the sepa- rate location of PKCα and ubiquitin molecules in the nuclei of HDF cells in 3 h of DMSO treatment. However, TPA treatment induced co-translocation of PKCα and ubiquitin molecules in nuclei of the cells treated with TPA for 3 h (D), when scrutinized by Z-section. Note the red (Ub) and the green (PKCα) dots in the cells treated with DMSO, whereas the dots were co-localized upon treat- ment of HDF cells with TPA. (E) To in- vestigate the kinetic changes of PKCα degradation by either TPA or siRNAs, old HDF cells were harvested at the indicated times and then the degree of PKCα expression was measured by IB analyses. Expressions of PKCα relative

Article Snippet: Antibodies to pErk1/2, Erk1/2, PEA15pS104 and PEA-15 were from Cell Signaling (USA); against PKCβ1, Lamin B1, HA, ubiquitin (Ub) and α-tubulin were from Santa Cruz Biotechnology (USA); against PKCα from Novus Biologicals (Littleton, USA).

Techniques: Fluorescence, Translocation Assay, Derivative Assay, Ubiquitin Proteomics, Control, Microscopy, Expressing

Journal: PLoS ONE

Article Title: The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

doi: 10.1371/journal.pone.0049512

Figure Lengend Snippet: Penh responses to aerosolized methacholine in control mice (square) and OVA-sensitized/challenged mice (circle) treated with vehicle (black) or H89 (10 mg/kg) (grey) in the acute ( A ) and moderate ( B ) asthma models. (B = baseline). Data represent mean values ± SEM (bars) from n = 6−9 mice for control groups and n = 12 mice for OVA sensitized/challenged groups (OVA). * P <0.05 and *** P <0.001 vs corresponding controls; ### P <0.001 vs group indicated; NS: not significant.

Article Snippet: H89 ( N -[2-( p -Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt) (10 mg/kg) (LC Laboratories, PKC Pharmaceuticals Inc., Woburn, MA, USA) suspended in 5% DMSO in saline was administered i.p. two hours before each OVA challenge (or two hours before the last OVA challenge only for ).

Techniques: Control

Journal: PLoS ONE

Article Title: The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

doi: 10.1371/journal.pone.0049512

Figure Lengend Snippet: Effect of H89 (10 mg/kg) (grey blocks) or vehicle (black blocks) on total leukocyte (Total), eosinophil (Eos) and macrophage (Mac) numbers in BAL fluid 24 hours after the last challenge in control (Ctr) and OVA-sensitized/challenged mice (OVA) in the acute ( A ) and moderate ( B ) asthma models. C–D. Effect of H89 (10 mg/kg) (grey blocks) or vehicle (black blocks) on neutrophils (Neu) and lymphocytes (Lym) numbers in BAL fluid 24 hours after the last challenge in control (Ctr) and OVA-sensitized/challenged mice (OVA) in the acute ( C ) and moderate ( D ) asthma models. Data represent mean values (blocks) ± SEM (bars) from n = 6−9 mice per group (saline) and n = 9−12 mice per group (OVA). * P <0.05, ** P <0.01 and *** P <0.001 vs corresponding controls; ## P <0.01 and ### P <0.001 vs group indicated; NS: not significant.

Article Snippet: H89 ( N -[2-( p -Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt) (10 mg/kg) (LC Laboratories, PKC Pharmaceuticals Inc., Woburn, MA, USA) suspended in 5% DMSO in saline was administered i.p. two hours before each OVA challenge (or two hours before the last OVA challenge only for ).

Techniques: Control, Saline

Journal: PLoS ONE

Article Title: The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

doi: 10.1371/journal.pone.0049512

Figure Lengend Snippet: H&E-stained lung sections demonstrating peribronchial inflammatory infiltrates 24 hours after the last OVA challenge (magnification × 200). ( B–C ) Inflammation score in lung sections from control (Ctr) and OVA sensitized/challenged (OVA) mice treated with vehicle (black blocks) or H89 (grey blocks) in the acute ( B ) and moderate ( C ) asthma models. Data represent mean values ± SEM (bars) from n = 6 mice per group. *** P <0.001 vs corresponding controls; ### P <0.001 vs group indicated.

Article Snippet: H89 ( N -[2-( p -Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt) (10 mg/kg) (LC Laboratories, PKC Pharmaceuticals Inc., Woburn, MA, USA) suspended in 5% DMSO in saline was administered i.p. two hours before each OVA challenge (or two hours before the last OVA challenge only for ).

Techniques: Staining, Control

Journal: PLoS ONE

Article Title: The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

doi: 10.1371/journal.pone.0049512

Figure Lengend Snippet: Periodic acid Schiff (PAS)-stained lung sections demonstrating hyperplasia of mucus-producing goblet cells 24 hours after the last OVA challenge (magnification × 200). ( B–C ) Mucus score in lung sections from control (Ctr) and OVA sensitized/challenged (OVA) mice treated with vehicle (black blocks) or H89 (grey blocks) in the acute ( B ) and moderate ( C ) asthma models. Data represent mean values ± SEM (bars) from n = 6 mice per group. ** P <0.01 and *** P <0.001 vs corresponding controls; ## P <0.01 and ### P <0.001 vs group indicated.

Article Snippet: H89 ( N -[2-( p -Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt) (10 mg/kg) (LC Laboratories, PKC Pharmaceuticals Inc., Woburn, MA, USA) suspended in 5% DMSO in saline was administered i.p. two hours before each OVA challenge (or two hours before the last OVA challenge only for ).

Techniques: Staining, Control

Journal: PLoS ONE

Article Title: The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

doi: 10.1371/journal.pone.0049512

Figure Lengend Snippet: Acidic toluidine blue-stained lung sections 24 hours after the last OVA challenge (magnification × 200). Black arrows indicate toluidine blue-positive mast cells ( B–C ) Quantification of mast cell numbers in lung sections from control (Ctr) and OVA sensitized/challenged (OVA) mice treated with vehicle (black blocks) or H89 (grey blocks) in the acute ( B ) and moderate ( C ) asthma models. Data represent mean values ± SEM (bars) from n = 6 mice per group. ** P <0.01 and *** P <0.001 vs corresponding controls; ## P <0.01 vs group indicated.

Article Snippet: H89 ( N -[2-( p -Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt) (10 mg/kg) (LC Laboratories, PKC Pharmaceuticals Inc., Woburn, MA, USA) suspended in 5% DMSO in saline was administered i.p. two hours before each OVA challenge (or two hours before the last OVA challenge only for ).

Techniques: Staining, Control

Journal: PLoS ONE

Article Title: The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

doi: 10.1371/journal.pone.0049512

Figure Lengend Snippet: BAL fluid was collected 24 hours after the last OVA challenge. The levels of IL-4 ( A–B ) and IL-5 ( C–D ) were determined using ELISA in the acute ( A & C ) and moderate ( B & D ) asthma models in control (Ctr) and OVA-sensitized/challenged (OVA) mice treated with vehicle (black blocks) or H89 (grey blocks). Data represent mean values ± SEM (bars) from n = 6−8 mice per group. * P <0.05, ** P <0.01 and *** P <0.001 vs corresponding controls; # P <0.05 and ## P <0.01 vs group indicated.

Article Snippet: H89 ( N -[2-( p -Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt) (10 mg/kg) (LC Laboratories, PKC Pharmaceuticals Inc., Woburn, MA, USA) suspended in 5% DMSO in saline was administered i.p. two hours before each OVA challenge (or two hours before the last OVA challenge only for ).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: PLoS ONE

Article Title: The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

doi: 10.1371/journal.pone.0049512

Figure Lengend Snippet: Serum was collected 24 hours after the last OVA challenge. The levels of OVA-specific IgE ( A–B ), OVA-specific IgG1 ( C–D ), OVA-specific IgG2a ( E ) and OVA-specific IgG2c ( F ) were determined using ELISA in the acute ( A, C & E ) and moderate ( B, D & F ) asthma models in control (squares) and OVA sensitized/challenged (circles) mice treated with vehicle (black blocks) or H89 (grey blocks). Data represent mean values ± SEM (bars) from n = 4−6 mice per group (saline) and n = 6−12 mice per group (OVA). * P <0.05, ** P <0.01 and *** P <0.001 vs corresponding controls; # P <0.05 and ## P <0.01 vs group indicated.

Article Snippet: H89 ( N -[2-( p -Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt) (10 mg/kg) (LC Laboratories, PKC Pharmaceuticals Inc., Woburn, MA, USA) suspended in 5% DMSO in saline was administered i.p. two hours before each OVA challenge (or two hours before the last OVA challenge only for ).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Saline