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Focal Adhesion Kinase Activity and Localization is Critical for TNF-α-Induced Nuclear Factor-κB Activation

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Abstract

While sustained nuclear factor-κB (NF-κB) activation is critical for proinflammatory molecule expression, regulators of NF-κB activity during chronic inflammation are not known. We investigated the role of focal adhesion kinase (FAK) on sustained NF-κB activation in tumor necrosis factor-α (TNF-α)–stimulated endothelial cells (ECs) both in vitro and in vivo. We found that FAK inhibition abolished TNF-α-mediated sustained NF-κB activity in ECs by disrupting formation of TNF-α receptor complex-I (TNFRC-I). Additionally, FAK inhibition diminished recruitment of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and the inhibitor of NF-κB (IκB) kinase (IKK) complex to TNFRC-I, resulting in elevated stability of IκBα protein. In mice given TNF-α, pharmacological and genetic FAK inhibition blocked TNF-α-induced IKK-NF-κB activation in aortic ECs. Mechanistically, TNF-α activated and redistributed FAK from the nucleus to the cytoplasm, causing elevated IKK-NF-κB activation. On the other hand, FAK inhibition trapped FAK in the nucleus of ECs even upon TNF-α stimulation, leading to reduced IKK-NF-κB activity. Together, these findings support a potential use for FAK inhibitors in treating chronic inflammatory diseases.

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Abbreviations

ECs:

Endothelial cells

FAK:

Focal adhesion kinase

HAoEC:

Human aortic endothelial cell

HUVEC:

Human umbilical vein endothelial cell

IκBα:

Inhibitor of NF-κBα

IKK:

IκB kinase

KD:

Kinase-dead

NEMO:

Nuclear factor-κB essential modulator (IKKγ)

NF-κB:

Nuclear factor-κB

pS536:

Phospho-serine 536 NF-κB

pY397:

Autophosphorylation at tyrosine 397 of FAK

RIPK1:

Receptor-interacting serine/threonine-protein kinase 1

TNF-α:

Tumor necrosis factor-α

TNFR1:

Tumor necrosis factor-α receptor 1

TNFRC-I:

TNF-α receptor complex-I

VCAM-1:

Vascular cell adhesion molecule-1

WT:

Wild-type

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Acknowledgments

We thank Dr. Elly Trepman for critical reading of the manuscript, and Drs. Mary I. Townsley and John V. Marymont for editorial support.

Funding

This work was supported by American Heart Association grants 12SDG10970000 and 16GRNT30960007 to SL, National Institutes of Health grants R01CA190688 to EA and R01HL136432 to SL, and 2019 College of Medicine Intramural grant from University of South Alabama to SL. The confocal microscope was supported by National Institutes of Health grant S10RR027535.

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Author notes

  1. James M. Murphy and Kyuho Jeong contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of South Alabama College of Medicine, 5851 N. USA Drive, Room 2366, Mobile, AL, 36688, USA

    James M. Murphy, Kyuho Jeong, Donna L. Cioffi & Ssang-Taek Steve Lim

  2. Department of Pathology, University of South Alabama College of Medicine, Mobile, AL, 36617, USA

    Pamela Moore Campbell

  3. Wallace H. Coulter Department of Bioengineering, Emory University and Georgia Institute of Technology, Atlanta, GA, 30322, USA

    Hanjoong Jo

  4. Department of Pathology, O’Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, 35294, USA

    Eun-Young Erin Ahn

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Contributions

J.M. Murphy, E-Y.E. Ahn, H. Jo, and S-T.S. Lim designed the research; J.M. Murphy and K. Jeong performed the research; J.M. Murphy, K. Jeong, P.M. Campbell, E-Y.E. Ahn, and S-T.S. Lim analyzed data; D.L. Cioffi contributed new reagents; J.M. Murphy, K. Jeong, and S-T.S. Lim wrote the paper.

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Correspondence to Ssang-Taek Steve Lim.

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Supplementary Information

Supplementary Fig. 1

FAK activity is important for TNF-α and IL-1β activation of NF-κB a, HAoECs were treated for 1 h with DMSO or PF-271 (2.5 μmol/L) prior to TNF-α (10 ng/mL) stimulation. Western blot analysis of pY397 FAK, FAK, pS536 NF-κB, NF-κB, IκBα, and β-actin (n=3). b, HAoECs were treated for 1 h with DMSO or PF-271 (2.5 μmol/L) prior to TNF-α (10 ng/ml) stimulation. NF-κB chromatin immunoprecipitation (ChIP) was performed (Life Technologies #51-0500) and NF-κB binding to IκBα promoter was determined via RT-qPCR and calculated as percent of input (n=3). **P<0.01, n.s.: not significant. c, After HAoECs were treated for 1 h with DMSO or a Src inhibitor (Dasatinib, 1 μmol/L), they were stimulated with TNF-α (10 ng/mL) for the times shown. Western blot analysis of pY397 FAK, FAK, pY418 Src, Src, pS536 NF-κB, IκBα, and β-actin (n=3). d, HAoECs were treated for 1 h with DMSO or PF-271 (2.5 μmol/L) prior to TNF-α (10 ng/mL) stimulation. Western blot analysis of pS176/177 IKKα/β, IKKα/β, pS32/36-IκBα, and IκBα. Same lysate samples as in Fig. 2c (n=3). (PNG 5255 kb)

High resolution image (TIF 1123 kb)

Supplementary Fig. 2

FAK inhibition reduces FAK association with TNFR1 and RIPK1 in HAoECs a and b, HAoECs were for 1 h with DMSO or PF-271 (2.5 μmol/L) prior to TNF-α (10 ng/ml) stimulation for 0.5 h. a, Immunostaining of HAoECs for FAK (green; mouse) and TNFR1 (red; rabbit). b, Immunostaining of HAoECs for FAK (green; mouse) and RIPK1 (red; rabbit). Scale bar, 20 μm (n=4). (PNG 5140 kb)

High resolution image (TIF 2473 kb)

Supplementary Fig. 3

Nuclear-localized FAK decreases TNF-α-induced NF-κB activation in mice a and b, C57BL/6 mice were treated with vehicle or PF-271 (35 mg/kg) for 2 days, and the last dose was 3 h before injection with PBS or mouse TNF-α (0.02 mg/kg) for 0.5 h. a, Immunostaining of mouse aorta for pS536 NF-κB (red; rabbit), vWF (green, EC marker; mouse), and DAPI (blue). b, Immunostaining of mouse aorta for pS176/177 IKKα/β (red; rabbit), vWF (green, EC marker; mouse), and DAPI (blue). Scale bar, 10 μm (n=4). (PNG 5029 kb)

High resolution image (TIF 743 kb)

Supplementary Fig. 4

EC-specific FAK inhibition decreases TNF-α-induced NF-κB activation in mice FAK flox/WT SCL-Cre and FAK flox/KD SCL-Cre mice were treated with tamoxifen (75 mg/kg) every other day for 2 weeks to generate FAK-WT and FAK-KD EC mice. After rest for 1 week, mice were injected with PBS or mouse TNF-α (0.02 mg/kg) for 0.5 h. Immunostaining of mouse aorta for pS536 NF-κB (red; rabbit), vWF (green, EC marker; mouse), and DAPI (blue). Scale bar, 10 μm (n=4). (PNG 5046 kb)

High resolution image (TIF 609 kb)

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Murphy, J.M., Jeong, K., Cioffi, D.L. et al. Focal Adhesion Kinase Activity and Localization is Critical for TNF-α-Induced Nuclear Factor-κB Activation. Inflammation 44, 1130–1144 (2021). https://doi.org/10.1007/s10753-020-01408-5

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