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Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response

Pokharel, Swechha M., Shil, Niraj K., GC, Jeevan B., Colburn, Zachary T., Tsai, Su-Yu, Segovia, Jesus A., Chang, Te-Hung, Bandyopadhyay, Smarajit, Natesan, Senthil, Jones, Jonathan C. R., Bose, Santanu·2019·Nature Communications
DOI 10.1038/s41467-019-09453-xPMC PMC6443809
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Abstract

Integrins are components of cell-matrix adhesions, and function as scaffolds for various signal transduction pathways. So far no lipid ligand for integrin has been reported. Here we show that a lipid, oxysterol 25-hydroxycholesterol (25HC), directly binds to α5β1 and αvβ3 integrins to activate integrin-focal adhesion kinase (FAK) signaling. Treatment of macrophages and epithelial cells with 25HC results in an increase in activated αvβ3 integrin in podosome and focal adhesion matrix adhesion sites. Moreover, activation of pattern recognition receptor on macrophages induces secretion of 25HC, triggering integrin signaling and the production of proinflammatory cytokines such as TNF and IL-6. Thus, the lipid molecule 25HC is a physiologically relevant activator of integrins and is involved in positively regulating proinflammatory responses. Our data suggest that extracellular 25HC links innate immune inflammatory response with integrin signaling.

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Fig. 1 FAK-NFκB pathway is required for 25HC-dependent proinflammatory response. a Structure of 25-hydroxycholesterol (25HC). b–g Cells were treated with 50 µM 25HC for 8 h. FAK and NFκB activation and proinflammatory response in treated cells were evaluated. b Western blot and densitometric analyses of FAK activation (phospho-FAK, Tyr925) status in 25HC-treated RAW 264.7 macrophages. c IL-6 secretion from wild-type (WT) and FAK knockout (KO) MEFs (mouse embryo fibroblasts) treated with 25HC. d TNF secretion from NR-9456 macrophages treated with 25HC in the absence or presence of FAK inhibitor (5 µM). e TNF secretion from BMDMs treated with 25HC in the absence or presence of NFκB inhibitor Bay 11-7082 (BAY-11) (10 µM). f TNF secretion from WT and NFκB p105 KO BMDMs treated with 25HC. g Western blot and densitometric analyses of phospho-IκB status in 25HC-treated WT and FAK KO MEFs. The ELISA values (mean ± standard deviation) are representative from two or three independent experiments (n = 4). *p ≤ 0.05 using a Student’s t-test. The densitometric quantification values for phospho-FAK (p-FAK) and phospho-IκB (p-IκB) immunoblots represent the ratio of phospho-FAK:actin and phospho-IκB:actin and the fold-induction was calculated after normalizing with the control untreated (UT) or control 0 h group. The densitometric values represent the mean ± standard deviation from three independent studies. *p ≤ 0.05 using a Student’s t-test
Fig. 1 FAK-NFκB pathway is required for 25HC-dependent proinflammatory response. a Structure of 25-hydroxycholesterol (25HC). b–g Cells were treated with 50 µM 25HC for 8 h. FAK and NFκB activation and proinflammatory response in treated cells were evaluated. b Western blot and densitometric analyses of FAK activation (phospho-FAK, Tyr925) status in 25HC-treated RAW 264.7 macrophages. c IL-6 secretion from wild-type (WT) and FAK knockout (KO) MEFs (mouse embryo fibroblasts) treated with 25HC. d TNF secretion from NR-9456 macrophages treated with 25HC in the absence or presence of FAK inhibitor (5 µM). e TNF secretion from BMDMs treated with 25HC in the absence or presence of NFκB inhibitor Bay 11-7082 (BAY-11) (10 µM). f TNF secretion from WT and NFκB p105 KO BMDMs treated with 25HC. g Western blot and densitometric analyses of phospho-IκB status in 25HC-treated WT and FAK KO MEFs. The ELISA values (mean ± standard deviation) are representative from two or three independent experiments (n = 4). *p ≤ 0.05 using a Student’s t-test. The densitometric quantification values for phospho-FAK (p-FAK) and phospho-IκB (p-IκB) immunoblots represent the ratio of phospho-FAK:actin and phospho-IκB:actin and the fold-induction was calculated after normalizing with the control untreated (UT) or control 0 h group. The densitometric values represent the mean ± standard deviation from three independent studies. *p ≤ 0.05 using a Student’s t-testCC BY · PubMed Central
Fig. 2 25HC interacts with α5β1 and αvβ3 integrins. a, b Biotinylation reactions were performed in the presence of either 25HC or DMSO (control). Cell lysates collected from BMDMs (a) or RAW 264.7 macrophages (b) incubated with biotinylated 25HC (biotin-25HC) or DMSO at 4 °C were precipitated (PPT) with avidin–agarose and subsequently immuno-blotted (IB) with either α5 integrin (a) or αv integrin (b) antibody. Total lysates were also blotted with α5 integrin (a), αv integrin (b), and actin (a, b) antibodies. c, d Biotin-25HC and DMSO (control) were incubated (PPT; precipitated) with avidin–agarose beads. 25HC conjugated beads were then incubated either with purified α5β1 integrin (c) or αvβ3 integrin (d). The avidin–agarose bound complex was subsequently immuno-blotted with either α5 integrin (c) or αv integrin (d) antibody. e, f Protein G-agarose beads were conjugated with either control IgG or antibody (Ab) specific for α5 integrin (e) and αv integrin (f). The beads were then incubated with either purified α5β1 integrin (e) or αvβ3 integrin (f) protein. Control beads (control IgG + purified integrin protein) and integrin protein bound beads (integrin-specific antibody + purified integrin protein) was incubated with tritiated 3H-25HC. Bead-bound radioactivity (count per minute; CPM) was measured by scintillation counter. g Cell lysate collected from RAW 264.7 macrophages incubated with biotin-25HC, biotinylated 27-hydroxycholesterol (biotin-27HC), biotinylated 4β-hydroxycholesterol (biotin-4βHC) or DMSO (control) at 4 °C was precipitated (PPT) with avidin–agarose and subsequently immuno-blotted with α5 integrin antibody. Total cell lysate was also blotted with α5 integrin and β-actin antibodies. h Surface plasmon resonance (SPR) analyses were performed by injecting increasing concentrations of 25HC (16 nM, 40 nM, 160 nM, 640 nM, 1.6 µM) into both the αvβ3 integrin-immobilized and control surface of the CM5 chips. Each background-corrected sensorgram is a representative of replicate runs. i Binding of radioactive 3H-25HC to immobilized αvβ3 integrin protein in the presence or absence of purified chemokine domain of fractalkine protein was assessed as described in the “methods” section. Immunoblot images are representative from three independent experiments. The radioactive values (mean ± standard deviation) are representative from two independent experiments. *p ≤ 0.05 using a Student’s t-test
Fig. 2 25HC interacts with α5β1 and αvβ3 integrins. a, b Biotinylation reactions were performed in the presence of either 25HC or DMSO (control). Cell lysates collected from BMDMs (a) or RAW 264.7 macrophages (b) incubated with biotinylated 25HC (biotin-25HC) or DMSO at 4 °C were precipitated (PPT) with avidin–agarose and subsequently immuno-blotted (IB) with either α5 integrin (a) or αv integrin (b) antibody. Total lysates were also blotted with α5 integrin (a), αv integrin (b), and actin (a, b) antibodies. c, d Biotin-25HC and DMSO (control) were incubated (PPT; precipitated) with avidin–agarose beads. 25HC conjugated beads were then incubated either with purified α5β1 integrin (c) or αvβ3 integrin (d). The avidin–agarose bound complex was subsequently immuno-blotted with either α5 integrin (c) or αv integrin (d) antibody. e, f Protein G-agarose beads were conjugated with either control IgG or antibody (Ab) specific for α5 integrin (e) and αv integrin (f). The beads were then incubated with either purified α5β1 integrin (e) or αvβ3 integrin (f) protein. Control beads (control IgG + purified integrin protein) and integrin protein bound beads (integrin-specific antibody + purified integrin protein) was incubated with tritiated 3H-25HC. Bead-bound radioactivity (count per minute; CPM) was measured by scintillation counter. g Cell lysate collected from RAW 264.7 macrophages incubated with biotin-25HC, biotinylated 27-hydroxycholesterol (biotin-27HC), biotinylated 4β-hydroxycholesterol (biotin-4βHC) or DMSO (control) at 4 °C was precipitated (PPT) with avidin–agarose and subsequently immuno-blotted with α5 integrin antibody. Total cell lysate was also blotted with α5 integrin and β-actin antibodies. h Surface plasmon resonance (SPR) analyses were performed by injecting increasing concentrations of 25HC (16 nM, 40 nM, 160 nM, 640 nM, 1.6 µM) into both the αvβ3 integrin-immobilized and control surface of the CM5 chips. Each background-corrected sensorgram is a representative of replicate runs. i Binding of radioactive 3H-25HC to immobilized αvβ3 integrin protein in the presence or absence of purified chemokine domain of fractalkine protein was assessed as described in the “methods” section. Immunoblot images are representative from three independent experiments. The radioactive values (mean ± standard deviation) are representative from two independent experiments. *p ≤ 0.05 using a Student’s t-testCC BY · PubMed Central
Fig. 3 Modeling identifies specific interactions between αvβ3 integrin and 25HC. a Ectodomain structure of αvβ3 integrin containing ‘RGD’’-binding site (site I) (blue) and site-II (red) residues highlighted by a layer of solvent molecule of radius 1.4 Å. The site-II-bound 25HC molecule is presented as a surface model in green and Mn2+ ions from metal-ion-dependent ligand-binding site (MIDAS) and adjacent to MIDAS (ADMIDAS) sites of βI domain, β-propeller domain and genu knee region are presented in cyan. b Potential interacting residues for molecular recognition of αvβ3 integrin by 25HC were identified in a docking simulation. H-bonds are highlighted in dashed lines in red. c, d Ser399 of β-propeller (red), Ser162 (blue) and Ala263 (black) of βI domain make stable H-bond interactions with 3- and 25-hydroxyl groups of 25HC, respectively. The H-bond distances (c) and H-bond angles (d) are plotted against MD simulation time (200 ns). e The root-mean-square deviations (RMSD) measured for specificity-determining loop (SDL), α1- and α7 helices during the entire 200 ns long simulation. The SDL region undergoes significant conformational change (RMSD of ~6 Å) upon 25HC binding. f The conformational change in the SDL is accompanied by disruption of H-bond interaction between Tyr122-Thr182 residues, as well as change in the β-propeller blade that interacts with SDL. Movement of the regions from the beginning 0 ns (blue) to the end 200 ns (red) is indicated by arrows. g Correlation matrices (as heat maps) indicating correlated (red) or anti-correlated motions (blue) of α- and β-subunits of αvβ3 integrin in unbound and 25HC-bound states. h, i The distance between the amide NH of Q120 of the β-propeller domain and backbone carbonyl oxygen of P169 of the SDL. Breakage of this electrostatic interaction in the 25HC-bound protein (blue) during the simulation, around ~30 ns simulation causes significant increase in the distance, which remain intact in the unbound protein (magenta) during the entire 200 ns simulation time
Fig. 3 Modeling identifies specific interactions between αvβ3 integrin and 25HC. a Ectodomain structure of αvβ3 integrin containing ‘RGD’’-binding site (site I) (blue) and site-II (red) residues highlighted by a layer of solvent molecule of radius 1.4 Å. The site-II-bound 25HC molecule is presented as a surface model in green and Mn2+ ions from metal-ion-dependent ligand-binding site (MIDAS) and adjacent to MIDAS (ADMIDAS) sites of βI domain, β-propeller domain and genu knee region are presented in cyan. b Potential interacting residues for molecular recognition of αvβ3 integrin by 25HC were identified in a docking simulation. H-bonds are highlighted in dashed lines in red. c, d Ser399 of β-propeller (red), Ser162 (blue) and Ala263 (black) of βI domain make stable H-bond interactions with 3- and 25-hydroxyl groups of 25HC, respectively. The H-bond distances (c) and H-bond angles (d) are plotted against MD simulation time (200 ns). e The root-mean-square deviations (RMSD) measured for specificity-determining loop (SDL), α1- and α7 helices during the entire 200 ns long simulation. The SDL region undergoes significant conformational change (RMSD of ~6 Å) upon 25HC binding. f The conformational change in the SDL is accompanied by disruption of H-bond interaction between Tyr122-Thr182 residues, as well as change in the β-propeller blade that interacts with SDL. Movement of the regions from the beginning 0 ns (blue) to the end 200 ns (red) is indicated by arrows. g Correlation matrices (as heat maps) indicating correlated (red) or anti-correlated motions (blue) of α- and β-subunits of αvβ3 integrin in unbound and 25HC-bound states. h, i The distance between the amide NH of Q120 of the β-propeller domain and backbone carbonyl oxygen of P169 of the SDL. Breakage of this electrostatic interaction in the 25HC-bound protein (blue) during the simulation, around ~30 ns simulation causes significant increase in the distance, which remain intact in the unbound protein (magenta) during the entire 200 ns simulation timeCC BY · PubMed Central
Fig. 4 25HC activates αvβ3 integrin in epithelial cell focal adhesions and macrophage podosomes. a Human lung epithelial cells (A549 cells) or differentiated THP-1 macrophages were subjected to various treatments and immunostained as indicated. In untreated (UT) A549 and THP-1 cells (first row), αvβ3 integrin, immunostained using the antibody LM609, was present in paxillin-positive focal adhesions and podosomes, respectively. In contrast, active αvβ3 integrin staining, indicated using the antibody AP5 (second and third row), was found almost exclusively in cells treated for 2 h with 0.5 µM 25HC compared with DMSO-treated controls. Merged images show the co-localization of αvβ3 integrin (total or activated) with focal adhesions/podosomes. Bar, 10 µm. b Quantification of the number of focal adhesions (FAs) and AP5+ FAs in A549 cells (three experiments, >12 cells per experiment; n ≥ 30). c Quantification of the number of podosomes and AP5+ podosomes in THP-1 cells (three experiments, 25 cells per experiment; n ≥ 70). Two- and One-tailed Wilcoxon rank-sum tests were performed to evaluate differences between total and AP5+ FA/podosome counts, respectively. Graphs depict mean ± SEM. *p ≤ 0.05
Fig. 4 25HC activates αvβ3 integrin in epithelial cell focal adhesions and macrophage podosomes. a Human lung epithelial cells (A549 cells) or differentiated THP-1 macrophages were subjected to various treatments and immunostained as indicated. In untreated (UT) A549 and THP-1 cells (first row), αvβ3 integrin, immunostained using the antibody LM609, was present in paxillin-positive focal adhesions and podosomes, respectively. In contrast, active αvβ3 integrin staining, indicated using the antibody AP5 (second and third row), was found almost exclusively in cells treated for 2 h with 0.5 µM 25HC compared with DMSO-treated controls. Merged images show the co-localization of αvβ3 integrin (total or activated) with focal adhesions/podosomes. Bar, 10 µm. b Quantification of the number of focal adhesions (FAs) and AP5+ FAs in A549 cells (three experiments, >12 cells per experiment; n ≥ 30). c Quantification of the number of podosomes and AP5+ podosomes in THP-1 cells (three experiments, 25 cells per experiment; n ≥ 70). Two- and One-tailed Wilcoxon rank-sum tests were performed to evaluate differences between total and AP5+ FA/podosome counts, respectively. Graphs depict mean ± SEM. *p ≤ 0.05CC BY · PubMed Central
Fig. 5 α5β1 and αvβ3 integrins regulate 25HC-mediated proinflammatory response. a, c–g α5β1 and αvβ3 integrin-deficient cells were treated with 50 µM 25HC for 8 h to evaluate NFκB activation and proinflammatory response. a TNF secretion from 25HC-treated NR-9456 macrophages in the presence of either control IgG or α5β1 integrin blocking antibody (Ab). b Western blot analyses of α5 integrin protein expression in THP-1 cells transfected with either control siRNA (con siRNA) or α5 integrin-specific siRNA (α5 siRNA). c RT-PCR analyses of TNF expression in 25HC-treated THP-1 cells transfected with either control siRNA (con siRNA) or α5 integrin-specific siRNA (α5 siRNA). d Western blot and densitometric analyses of phospho-IκB status in 25HC-treated wild-type (WT) and α5 integrin knockout (KO) HAP1 cells. e TNF secretion from 25HC-treated WT and β3 integrin-deficient (β3+/− cells) BMDMs. f, g TNF (f) and IL-6 (g) secretion from 25HC-treated THP-1 macrophages in the presence of either control IgG or αvβ3 integrin blocking antibody (Ab). RT-PCR images are representative from two independent experiments. The ELISA values (mean ± standard deviation) are representative from two or three independent experiments (n = 4). *p ≤ 0.05 using a Student’s t-test. The densitometric quantification values for phospho-IκB (p-IκB) immunoblot represent the ratio of phospho-IκB:actin and the fold-induction was calculated after normalizing with the control 0 min group. The densitometric values represent the mean ± standard deviation from three independent studies. *p ≤ 0.05 using a Student’s t-test
Fig. 5 α5β1 and αvβ3 integrins regulate 25HC-mediated proinflammatory response. a, c–g α5β1 and αvβ3 integrin-deficient cells were treated with 50 µM 25HC for 8 h to evaluate NFκB activation and proinflammatory response. a TNF secretion from 25HC-treated NR-9456 macrophages in the presence of either control IgG or α5β1 integrin blocking antibody (Ab). b Western blot analyses of α5 integrin protein expression in THP-1 cells transfected with either control siRNA (con siRNA) or α5 integrin-specific siRNA (α5 siRNA). c RT-PCR analyses of TNF expression in 25HC-treated THP-1 cells transfected with either control siRNA (con siRNA) or α5 integrin-specific siRNA (α5 siRNA). d Western blot and densitometric analyses of phospho-IκB status in 25HC-treated wild-type (WT) and α5 integrin knockout (KO) HAP1 cells. e TNF secretion from 25HC-treated WT and β3 integrin-deficient (β3+/− cells) BMDMs. f, g TNF (f) and IL-6 (g) secretion from 25HC-treated THP-1 macrophages in the presence of either control IgG or αvβ3 integrin blocking antibody (Ab). RT-PCR images are representative from two independent experiments. The ELISA values (mean ± standard deviation) are representative from two or three independent experiments (n = 4). *p ≤ 0.05 using a Student’s t-test. The densitometric quantification values for phospho-IκB (p-IκB) immunoblot represent the ratio of phospho-IκB:actin and the fold-induction was calculated after normalizing with the control 0 min group. The densitometric values represent the mean ± standard deviation from three independent studies. *p ≤ 0.05 using a Student’s t-testCC BY · PubMed Central
Fig. 6 The 25HC-integrin-FAK signaling network is required for optimal Nod2 response. a IL-6 in serum of mice injected (via intraperitoneal route) with 25HC (50 mg/kg; 4 h) (n = 5). b, c, e, g–m Cells were treated with 25 µg/ml of MDP for 8 h and d, f mice were injected intraperitoneally with 20 mg/kg of MDP for 4 h. C25H expression, 25HC production, FAK activation, and proinflammatory response were assessed. b RT-PCR analyses of C25H expression in MDP-treated wild-type (WT) and C25H knockout (KO) BMDMs. c 25HC levels in the medium supernatant of MDP-treated BMDMs. d 25HC levels in the serum of WT mice treated with MDP (n = 4). e IL-6 secretion from WT and C25H KO BMDMs treated with MDP. f IL-6 levels in the serum of WT and C25H KO mice treated with MDP (n = 4). g Western blot and densitometric analyses of FAK activation (phospho-FAK, Tyr397) status in MDP-treated THP-1 macrophages. h IL-6 production from MDP-treated WT and FAK KO MEFs (mouse embryo fibroblasts). i IL-6 secretion from MDP-treated NR-9456 macrophages in the presence of either control IgG or α5β1 integrin blocking antibody (Ab). j RT-PCR analyses of TNF expression in MDP-treated THP-1 cells transfected with either control siRNA (con siRNA) or α5 integrin-specific siRNA (α5 siRNA). k TNF secretion from MDP-treated WT and β3 integrin-deficient (β3+/− cells) BMDMs. l TNF secretion from MDP-treated THP-1 macrophages in the presence of either control IgG or αvβ3 integrin blocking antibody. m Western blot and densitometric analyses of FAK activation (phospho-FAK, Tyr925) status in MDP-treated WT and C25H KO BMDM. RT-PCR images are representative from two independent experiments. The ELISA values (mean ± standard deviation) are representative from two or three independent experiments (n = 4). *p ≤ 0.05 using a Student’s t-test. The densitometric quantification values for phospho-FAK (p-FAK) immunoblot represent the ratio of phospho-FAK:actin and the fold-induction was calculated after normalizing with the control untreated (UT) group. The densitometric values represent the mean ± standard deviation from three independent studies. *p ≤ 0.05 using a Student’s t-test
Fig. 6 The 25HC-integrin-FAK signaling network is required for optimal Nod2 response. a IL-6 in serum of mice injected (via intraperitoneal route) with 25HC (50 mg/kg; 4 h) (n = 5). b, c, e, g–m Cells were treated with 25 µg/ml of MDP for 8 h and d, f mice were injected intraperitoneally with 20 mg/kg of MDP for 4 h. C25H expression, 25HC production, FAK activation, and proinflammatory response were assessed. b RT-PCR analyses of C25H expression in MDP-treated wild-type (WT) and C25H knockout (KO) BMDMs. c 25HC levels in the medium supernatant of MDP-treated BMDMs. d 25HC levels in the serum of WT mice treated with MDP (n = 4). e IL-6 secretion from WT and C25H KO BMDMs treated with MDP. f IL-6 levels in the serum of WT and C25H KO mice treated with MDP (n = 4). g Western blot and densitometric analyses of FAK activation (phospho-FAK, Tyr397) status in MDP-treated THP-1 macrophages. h IL-6 production from MDP-treated WT and FAK KO MEFs (mouse embryo fibroblasts). i IL-6 secretion from MDP-treated NR-9456 macrophages in the presence of either control IgG or α5β1 integrin blocking antibody (Ab). j RT-PCR analyses of TNF expression in MDP-treated THP-1 cells transfected with either control siRNA (con siRNA) or α5 integrin-specific siRNA (α5 siRNA). k TNF secretion from MDP-treated WT and β3 integrin-deficient (β3+/− cells) BMDMs. l TNF secretion from MDP-treated THP-1 macrophages in the presence of either control IgG or αvβ3 integrin blocking antibody. m Western blot and densitometric analyses of FAK activation (phospho-FAK, Tyr925) status in MDP-treated WT and C25H KO BMDM. RT-PCR images are representative from two independent experiments. The ELISA values (mean ± standard deviation) are representative from two or three independent experiments (n = 4). *p ≤ 0.05 using a Student’s t-test. The densitometric quantification values for phospho-FAK (p-FAK) immunoblot represent the ratio of phospho-FAK:actin and the fold-induction was calculated after normalizing with the control untreated (UT) group. The densitometric values represent the mean ± standard deviation from three independent studies. *p ≤ 0.05 using a Student’s t-testCC BY · PubMed Central
Fig. 7 The 25HC-integrin-FAK signaling network regulates proinflammatory response during virus infection. a 25HC levels in the medium supernatant of BMDMs infected with human respiratory syncytial virus (RSV). b TNF production from wild-type (WT) and C25H knockout (KO) BMDMs infected with RSV. c Western blot and densitometric analyses of FAK activation (phospho-FAK, Tyr925) status in RSV-infected BMDMs. d Western blot and densitometric analyses of FAK activation (phospho-FAK, Tyr925) status in influenza A virus (IAV)-infected BMDMs. e IL-6 production from RSV-infected WT and FAK KO MEFs. f IL-6 production from IAV-infected WT and FAK KO MEFs. g TNF production from RSV-infected BMDMs in the presence of either control IgG or α5β1 integrin blocking antibody (Ab). h IL-6 production from IAV-infected BMDMs in the presence of either control IgG or α5β1 integrin blocking antibody. i TNF secretion from IAV-infected WT and β3 integrin-deficient (β3+/− cells) BMDMs. j IL-6 secretion from IAV-infected THP-1 cells in the presence of either control IgG or αvβ3 integrin blocking antibody. k IL-6 in the lungs of mice injected (via intratracheal or I.T route) with 25HC (5 mg/kg; 6 h) in the presence of either IgG or β1 integrin blocking antibody (Ab) administered to the mice via I.T route (n = 4). l IL-6 in the lungs of mice injected (via I.T route) with 25HC (5 mg/kg; 6 h) in the presence of either vehicle (control) or FAK inhibitor (PND-1186) administered to the mice via I.T route (n = 4). The ELISA values (mean ± standard deviation) are representative from two or three independent experiments (n = 4). *p ≤ 0.05 using a Student’s t-test. The densitometric quantification values for phospho-FAK (p-FAK) immunoblot represent the ratio of phospho-FAK:actin and the fold-induction was calculated after normalizing with the control mock-infected group. The densitometric values represent the mean ± standard deviation from three independent studies. *p ≤ 0.05 using a Student’s t-test
Fig. 7 The 25HC-integrin-FAK signaling network regulates proinflammatory response during virus infection. a 25HC levels in the medium supernatant of BMDMs infected with human respiratory syncytial virus (RSV). b TNF production from wild-type (WT) and C25H knockout (KO) BMDMs infected with RSV. c Western blot and densitometric analyses of FAK activation (phospho-FAK, Tyr925) status in RSV-infected BMDMs. d Western blot and densitometric analyses of FAK activation (phospho-FAK, Tyr925) status in influenza A virus (IAV)-infected BMDMs. e IL-6 production from RSV-infected WT and FAK KO MEFs. f IL-6 production from IAV-infected WT and FAK KO MEFs. g TNF production from RSV-infected BMDMs in the presence of either control IgG or α5β1 integrin blocking antibody (Ab). h IL-6 production from IAV-infected BMDMs in the presence of either control IgG or α5β1 integrin blocking antibody. i TNF secretion from IAV-infected WT and β3 integrin-deficient (β3+/− cells) BMDMs. j IL-6 secretion from IAV-infected THP-1 cells in the presence of either control IgG or αvβ3 integrin blocking antibody. k IL-6 in the lungs of mice injected (via intratracheal or I.T route) with 25HC (5 mg/kg; 6 h) in the presence of either IgG or β1 integrin blocking antibody (Ab) administered to the mice via I.T route (n = 4). l IL-6 in the lungs of mice injected (via I.T route) with 25HC (5 mg/kg; 6 h) in the presence of either vehicle (control) or FAK inhibitor (PND-1186) administered to the mice via I.T route (n = 4). The ELISA values (mean ± standard deviation) are representative from two or three independent experiments (n = 4). *p ≤ 0.05 using a Student’s t-test. The densitometric quantification values for phospho-FAK (p-FAK) immunoblot represent the ratio of phospho-FAK:actin and the fold-induction was calculated after normalizing with the control mock-infected group. The densitometric values represent the mean ± standard deviation from three independent studies. *p ≤ 0.05 using a Student’s t-testCC BY · PubMed Central
Fig. 8 A schematic model showing regulation of proinflammatory response by 25HC-integrin-FAK signaling network. Nod2 activation and virus (RSV and IAV) infection triggers expression of C25H, which results in production of 25HC. Extracellular 25HC activates integrin-FAK-NFκB signaling. Thus, 25HC links PRR (Nod2) pathway with integrin-FAK-NFκB signaling to confer optimal proinflammatory response following Nod2 activation and virus infection. INTG, integrin; FAK, focal adhesion kinase
Fig. 8 A schematic model showing regulation of proinflammatory response by 25HC-integrin-FAK signaling network. Nod2 activation and virus (RSV and IAV) infection triggers expression of C25H, which results in production of 25HC. Extracellular 25HC activates integrin-FAK-NFκB signaling. Thus, 25HC links PRR (Nod2) pathway with integrin-FAK-NFκB signaling to confer optimal proinflammatory response following Nod2 activation and virus infection. INTG, integrin; FAK, focal adhesion kinaseCC BY · PubMed Central

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  1. Guo, W. & Giancotti, F. G. Integrin signalling during tumour progression. Nat. Rev. Mol. Cell Biol. 5, 816–826 (2004). · DOI
  2. Hynes, R. O. Integrins: bidirectional, allosteric signaling machines. Cell 110, 673–687 (2002). · DOI
  3. Shattil, S. J., Kim, C. & Ginsberg, M. H. The final steps of integrin activation: the end game. Nat. Rev. Mol. Cell Biol. 11, 288–300 (2010). · DOI · Paper page
  4. Lelievre, S., Weaver, V. M. & Bissell, M. J. Extracellular matrix signaling from the cellular membrane skeleton to the nuclear skeleton: a model of gene regulation. Recent Prog. Horm. Res. 51, 417–432 (1996).
  5. Eke, I. et al. beta(1)Integrin/FAK/cortactin signaling is essential for human head and neck cancer resistance to radiotherapy. J. Clin. Invest. 122, 1529–1540 (2012). · DOI
  6. Campbell, I. D. & Humphries, M. J. Integrin structure, activation, and interactions. Cold Spring Harb. Perspect. Biol. 3, a004994 (2011). · DOI
  7. Zhu, J., Zhu, J. & Springer, T. A. Complete integrin headpiece opening in eight steps. J. Cell Biol. 201, 1053–1068 (2013). · DOI
  8. Müller, M. A. et al. The glycophorin A transmembrane sequence within integrin αvβ3 creates a non-signaling integrin with low basal affinity that is strongly adhesive under force. J. Mol. Biol. 425, 2988–3006 (2013). · DOI
  9. Ye, F., Snider, A. K. & Ginsberg, M. H. Talin and kindlin: the one-two punch in integrin activation. Front. Med. 8, 6–16 (2014). · DOI
  10. Calderwood, D. A., Campbell, I. D. & Critchley, D. R. Talins and kindlins: partners in integrin-mediated adhesion. Nat. Rev. Mol. Cell Biol. 14, 503–517 (2013). · DOI
  11. Murphy, D. A. & Courtneidge, S. A. The’ins’ and’outs’ of podosomes and invadopodia: characteristics, formation and function. Nat. Rev. Mol. Cell Biol. 12, 413–426 (2011). · DOI
  12. Reyes-Reyes, M., Mora, N., Zentella, A. & Rosales, C. Phosphatidylinositol 3-kinase mediates integrin-dependent NF-(κ) B and MAPK activation through separate signaling pathways. J. Cell Sci. 114, 1579–1589 (2001). · DOI
  13. Kawai, T. & Akira, S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11, 373–384 (2010). · DOI · Paper page
  14. Sabbah, A. et al. Activation of innate immune antiviral responses by Nod2. Nat. Immunol. 10, 1073–1080 (2009). · DOI
  15. Coutermarsh-Ott, S., Eden, K. & Allen, I. C. Beyond the inflammasome: regulatory NOD-like receptor modulation of the host immune response following virus exposure. J. Gen. Virol. 97, 825–838 (2016). · DOI
  16. Lupfer, C., Thomas, P. G. & Kanneganti, T. -D. Nucleotide oligomerization and binding domain 2-dependent dendritic cell activation is necessary for innate immunity and optimal CD8+ T cell responses to influenza A virus infection. J. Virol. 88, 8946–8955 (2014). · DOI
  17. Lupfer, C. et al. Receptor interacting protein kinase 2-mediated mitophagy regulates inflammasome activation during virus infection. Nat. Immunol. 14, 480–488 (2013). · DOI · Paper page
  18. Kim, Y. G. et al. Viral infection augments Nod1/2 signaling to potentiate lethality associated with secondary bacterial infections. Cell Host Microbe 9, 496–507 (2011). · DOI
  19. Kapoor, A., Forman, M. & Arav-Boger, R. Activation of nucleotide oligomerization domain 2 (NOD2) by human cytomegalovirus initiates innate immune responses and restricts virus replication. PLoS ONE 9, e92704 (2014). · DOI
  20. Davoli-Ferreira, M. et al. Nucleotide-binding oligomerization domain-containing protein 2 prompts potent inflammatory stimuli during Neospora caninum infection. Sci. Rep. 6, 29289 (2016). · DOI
  21. Ley, K., Rivera-Nieves, J., Sandborn, W. J. & Shattil, S. Integrin-based therapeutics: biological basis, clinical use and new drugs. Nat. Rev. Drug. Discov. 15, 173–183 (2016). · DOI
  22. Nieberler, M. et al. Exploring the role of RGD-recognizing integrins in cancer. Cancers (Basel) 9, 116 (2017). · DOI
  23. Cyster, J. G., Dang, E. V., Reboldi, A. & Yi, T. 25-Hydroxycholesterols in innate and adaptive immunity. Nat. Rev. Immunol. 14, 731–743 (2014). · DOI · Paper page
  24. Lund, E. G., Kerr, T. A., Sakai, J., Li, W. -P. & Russell, D. W. cDNA cloning of mouse and human cholesterol 25-hydroxylases, polytopic membrane proteins that synthesize a potent oxysterol regulator of lipid metabolism. J. Biol. Chem. 273, 34316–34327 (1998). · DOI · Paper page
  25. Gold, E. S. et al. 25-Hydroxycholesterol acts as an amplifier of inflammatory signaling. Proc. Natl Acad. Sci. 111, 10666–10671 (2014). · DOI · Paper page
  26. Chen, J. et al. αvβ3 integrins mediate flow-induced NF-κB activation, proinflammatory gene expression, and early atherogenic inflammation. Am. J. Pathol. 185, 2575–2589 (2015). · DOI
  27. Takagi, J., Strokovich, K., Springer, T. A. & Walz, T. Structure of integrin alpha5beta1 in complex with fibronectin. EMBO J. 22, 4607–4615 (2003). · DOI
  28. Calderwood, D. A., Tuckwell, D. S., Eble, J., Kuhn, K. & Humphries, M. J. The integrin alpha1 A-domain is a ligand binding site for collagens and laminin. J. Biol. Chem. 272, 12311–12317 (1997). · DOI
  29. Lechner, A. M. et al. RGD-dependent binding of procathepsin X to integrin alphavbeta3 mediates cell-adhesive properties. J. Biol. Chem. 281, 39588–39597 (2006). · DOI
  30. Saegusa, J. et al. The direct binding of insulin-like growth factor-1 (IGF-1) to integrin αvβ3 is involved in IGF-1 signaling. J. Biol. Chem. 284, 24106–24114 (2009). · DOI
  31. Saegusa, J. et al. Pro-inflammatory secretory phospholipase A2 type IIA binds to integrins alphavbeta3 and alpha4beta1 and induces proliferation of monocytic cells in an integrin-dependent manner. J. Biol. Chem. 283, 26107–26115 (2008). · DOI
  32. Fujita, M., Takada, Y. K. & Takada, Y. Integrins αvβ3 and α4β1 act as coreceptors for fractalkine, and the integrin-binding defective mutant of fractalkine is an antagonist of CX3CR1. J. Immunol. 189, 5809–5819 (2012). · DOI
  33. Fujita, M., Takada, Y. K. & Takada, Y. The chemokine fractalkine can activate integrins without CX3CR1 through direct binding to a ligand-binding site distinct from the classical RGD-binding site. PLoS ONE 9, e96372 (2014). · DOI
  34. Fujita, M. et al. Proinflammatory secreted phospholipase A2 type IIA (sPLA-IIA) induces integrin activation through direct binding to a newly identified binding site (site 2) in integrins αvβ3, α4β1, and α5β1. J. Biol. Chem. 290, 259–271 (2015). · DOI
  35. Bissantz, C., Kuhn, B. & Stahl, M. A medicinal chemist’s guide to molecular interactions. J. Med. Chem. 53, 5061–5084 (2010). · DOI
  36. Takada, Y., Kamata, T., Irie, A., Puzon-McLaughlin, W. & Zhang, X. -P. Structural basis of integrin-mediated signal transduction. Matrix Biol. 16, 143–151 (1997). · DOI
  37. Yokoyama, K., Zhang, X. -P., Medved, L. & Takada, Y. Specific binding of integrin αvβ3 to the fibrinogen γ and αE chain C-terminal domains. Biochemistry 38, 5872–5877 (1999). · DOI
  38. Takagi, J., DeBottis, D. P., Erickson, H. P. & Springer, T. A. The role of the specificity-determining loop of the integrin β subunit I-like domain in autonomous expression, association with the α subunit, and ligand binding. Biochemistry 41, 4339–4347 (2002). · DOI
  39. Berendsen, H. J. & Hayward, S. Collective protein dynamics in relation to function. Curr. Opin. Struct. Biol. 10, 165–169 (2000). · DOI
  40. Xiao, T., Takagi, J., Coller, B. S., Wang, J. -H. & Springer, T. A. Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics. Nature 432, 59–67 (2004). · DOI
  41. Lee, C. C., Carette, J. E., Brummelkamp, T. R. & Ploegh, H. L. A reporter screen in a human haploid cell line identifies CYLD as a constitutive inhibitor of NF-κB. PLoS ONE 8, e70339 (2013). · DOI
  42. Liu, S. Y. et al. Interferon-inducible cholesterol-25-hydroxylase broadly inhibits viral entry by production of 25-hydroxycholesterol. Immunity 38, 92–105 (2013). · DOI · Paper page
  43. Grassmé, H. et al. β1-Integrin accumulates in cystic fibrosis luminal airway epithelial membranes and decreases sphingosine, promoting bacterial infections. Cell Host Microbe 21, 707–718.e708 (2017). · DOI
  44. Walsh, C. et al. Oral delivery of PND-1186 FAK inhibitor decreases tumor growth and spontaneous breast to lung metastasis in pre-clinical models. Cancer Biol. Ther. 9, 778–790 (2010). · DOI
  45. Humphries, J. D., Byron, A. & Humphries, M. J. Integrin ligands at a glance. J. Cell. Sci. 119, 3901–3903 (2006). · DOI
  46. Hubbard, A. K. & Rothlein, R. Intercellular adhesion molecule-1 (ICAM-1) expression and cell signaling cascades. Free Radic. Biol. Med. 28, 1379–1386 (2000). · DOI
  47. Maile, L. A. et al. Insulin-like growth factor-I signaling in smooth muscle cells is regulated by ligand binding to the 177CYDMKTTC184 sequence of the β3-subunit of αVβ3. Mol. Endocrinol. 20, 405–413 (2006). · DOI
  48. Artoni, A. et al. Integrin β3 regions controlling binding of murine mAb 7E3: Implications for the mechanism of integrin αIIbβ3 activation. Proc. Natl Acad. Sci. USA 101, 13114–13120 (2004). · DOI
  49. Hermanowski-Vosatka, A., Van Strijp, J. A., Swiggard, W. J. & Wright, S. D. Integrin modulating factor-1: a lipid that alters the function of leukocyte integrins. Cell 68, 341–352 (1992). · DOI
  50. Blanc, M. et al. The transcription factor STAT-1 couples macrophage synthesis of 25-hydroxycholesterol to the interferon antiviral response. Immunity 38, 106–118 (2013). · DOI
  51. McDonald, J. G. & Russell, D. W. Editorial: 25-Hydroxycholesterol: a new life in immunology. J. Leukoc. Biol. 88, 1071–1072 (2010). · DOI
  52. Tsai, S. Y. et al. DAMP molecule S100A9 acts as a molecular pattern to enhance inflammation during influenza A virus infection: role of DDX21-TRIF-TLR4-MyD88 pathway. PLoS Pathog. 10, e1003848 (2014). · DOI
  53. Kligys, K. R. et al. α6β4 integrin, a master regulator of expression of integrins in human keratinocytes. J. Biol. Chem. 287, 17975–17984 (2012). · DOI
  54. Colburn, Z. T. & Jones, J. C. R. Complexes of alpha6beta4 integrin and vimentin act as signaling hubs to regulate epithelial cell migration. J. Cell Sci. 131, pii: jcs214593 (2018). · DOI
  55. Horzum, U., Ozdil, B. & Pesen-Okvur, D. Step-by-step quantitative analysis of focal adhesions. MethodsX 1, 56–59 (2014). · DOI
  56. Marjomaki, V. et al. α2β1-integrin clustering and internalization protocol. Bio-Protoc. 4, e1088 (2014). · DOI
  57. Trad, A. et al. The disintegrin domain of ADAM17 antagonises fibroblastcarcinoma cell interactions. Int. J. Oncol. 42, 1793–1800 (2013). · DOI
  58. Xiong, J. -P. et al. Crystal structure of the extracellular segment of integrin αVβ3 in complex with an Arg-Gly-Asp ligand. Science 296, 151–155 (2002). · DOI
  59. Xiong, J. -P. et al. Crystal structure of the complete integrin αVβ3 ectodomain plus an α/β transmembrane fragment. J. Cell Biol. 186, 589–600 (2009). · DOI
  60. Labute, P. The generalized Born/volume integral implicit solvent model: estimation of the free energy of hydration using London dispersion instead of atomic surface area. J. Comput. Chem. 29, 1693–1698 (2008). · DOI
  61. Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A. & Case, D. A. Development and testing of a general amber force field. J. Comput. Chem. 25, 1157–1174 (2004). · DOI
  62. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983). · DOI
  63. Bakan, A., Meireles, L. M. & Bahar, I. ProDy: protein dynamics inferred from theory and experiments. Bioinformatics 27, 1575–1577 (2011). · DOI
  64. Koukos, P. I. & Glykos, N. M. Grcarma: A fully automated task-oriented interface for the analysis of molecular dynamics trajectories. J. Comput. Chem. 34, 2310–2312 (2013). · DOI

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Authors
Pokharel, S. M., Shil, N. K., GC, J. B., Colburn, Z. T., Tsai, S.-Y., Segovia, J. A., Chang, T.-H., Bandyopadhyay, S., Natesan, S., Jones, J. C. R., & Bose, S.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2019-04-01
Volume
10
Issue
1
ISSN
2041-1723
DOI
10.1038/s41467-019-09453-x
PMC
PMC6443809
Cited by
73
References
5
Language
en
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CoChat. (2026). Citation check: Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response. https://app.cochat.ai/papers/10.1038/s41467-019-09453-x
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Pokharel, S. M., Shil, N. K., GC, J. B., Colburn, Z. T., Tsai, S.-Y., Segovia, J. A., Chang, T.-H., Bandyopadhyay, S., Natesan, S., Jones, J. C. R., & Bose, S. (2019). Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response. Nature Communications. https://doi.org/10.1038/s41467-019-09453-x