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Interaction between integrin α5 and PDE4D regulates endothelial inflammatory signalling

Nature Cell Biology volume 18, pages 1043–1053 (2016)Cite this article

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Abstract

Atherosclerosis is primarily a disease of lipid metabolism and inflammation; however, it is also closely associated with endothelial extracellular matrix (ECM) remodelling, with fibronectin accumulating in the laminin–collagen basement membrane. To investigate how fibronectin modulates inflammation in arteries, we replaced the cytoplasmic tail of the fibronectin receptor integrin α5 with that of the collagen/laminin receptor integrin α2. This chimaera suppressed inflammatory signalling in endothelial cells on fibronectin and in knock-in mice. Fibronectin promoted inflammation by suppressing anti-inflammatory cAMP. cAMP was activated through endothelial prostacyclin secretion; however, this was ECM-independent. Instead, cells on fibronectin suppressed cAMP via enhanced phosphodiesterase (PDE) activity, through direct binding of integrin α5 to phosphodiesterase-4D5 (PDE4D5), which induced PP2A-dependent dephosphorylation of PDE4D5 on the inhibitory site Ser651. In vivo knockdown of PDE4D5 inhibited inflammation at athero-prone sites. These data elucidate a molecular mechanism linking ECM remodelling and inflammation, thereby identifying a new class of therapeutic targets.

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Figure 1: Integrin α subunit cytoplasmic tails determine ECM-specific inflammatory signalling.
Figure 2: Integrin chimaera knock-in mouse showed reduced inflammation in artery.
Figure 3: Prostacyclin mediates shear-dependent PKA activation but is ECM-independent.
Figure 4: Involvement of PDE4D in ECM-dependent inflammatory signalling.
Figure 5: Mapping the integrin binding site on PDE4D5.
Figure 6: ECM-dependent regulation of PDE4D phosphorylation.
Figure 7: In vivo PDE4D knockdown reduces flow-dependent inflammation.

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Acknowledgements

We thank K. Yamada (NIH, USA), D. Calderwood (Yale University, USA), H. Kim (POSTECH, Korea) and A. Jayaraman (Texas A&M University, USA) for providing reagents, and J. Hwa (Yale University, USA) for advice on prostacyclin experiments. Lipid analysis was done by the Yale Mouse Phenotypic Center, supported by a U24 DK059635 grant. This work was funded by a National Institutes of Health grant 5R01HL75092 to M.A.S. G.B. is funded by an MRC project grant (MR/J007412/1). We are grateful to R. Webber and N. Copeland for maintaining the mouse colonies used in this study.

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Authors and Affiliations

  1. Department of Internal Medicine, Yale Cardiovascular Research Center, Yale University, New Haven, Connecticut 06520, USA

    Sanguk Yun, Madhusudhan Budatha, Brian G. Coon & Martin A. Schwartz

  2. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    James E. Dahlman, Robert Langer & Daniel G. Anderson

  3. Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    James E. Dahlman, Robert Langer & Daniel G. Anderson

  4. Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    James E. Dahlman, Robert Langer & Daniel G. Anderson

  5. Institute of Cardiovascular and Medical Science, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK

    Ryan T. Cameron & George Baillie

  6. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    Robert Langer & Daniel G. Anderson

  7. Department of Biomedical Engineering, Yale University, New Haven, Connecticut 06520, USA

    Martin A. Schwartz

  8. Department of Cell Biology, Yale University, New Haven, Connecticut 06520, USA

    Martin A. Schwartz

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Contributions

S.Y. and M.A.S. designed the project. S.Y. performed in vitro experiments and M.B. designed and performed in vivo experiments with the aid of S.Y. J.E.D. prepared and provided nanoparticles. B.G.C. contributed PDE4D5 imaging. R.T.C. performed in vitro PDE assay. R.L. and D.G.A. provided advice on nanoparticle formulation. S.Y. and M.A.S. wrote the manuscript with the contribution of all the authors.

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Correspondence to Martin A. Schwartz.

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Integrated supplementary information

Supplementary Figure 1 Characterization of α5/2 chimera endothelial cells.

(a) BAECs expressing human integrin wild type α5 or the α5/2 chimera were stained with mAb16 that recognizes human integrin α5 extracellular domain. (b) Wild type α5 or the α5/2 chimera cells were lysed and immunoprecipitated with mAb16 to isolate exogenous human integrin α proteins. Western blots with antibodies against the α5 and α2 cytoplasmic tails confirm the sequences (upper and middle panels), and show similar pairing with the β1 subunit (lower panel). (c,d) Wild type α5 and α5/2 chimera cells spreading on fibronectin. BAECs expressing wild type integrin α5 or the chimera were detached and replated on dishes coated with fibronectin (10 μg/ml) for the indicated times. The cells were either fixed and stained with wheat germ agglutinin (c) or lysed and subjected to immunoblotting for FAK phosphorylation (Y397) (d). (e) Wild type α5 or α5/2 chimera cells were plated on fibronectin and grown to monolayer and then kept in low-serum media (1% FBS) for two days to induce fibronectin fibrillogenesis. The cells were fixed and stained for fibronectin. For each condition, n = 30 images pooled across three independent experiments were averaged. The box plot shows the median, with upper and lower percentiles, and the bars show maxima and minima values. (f) Wild type α5 or α5/2 chimera cells were plated on fibronectin and sheared for 36 hrs (20dynes/cm2). Cells were stained with phalloidin and Hoechst and alignment in the direction of flow was quantified (±30°). Scale bars: 50 μm. n = 10 images (60-100 cells/field) were used for quantification for each condition. Error bars are SEM.

Supplementary Figure 2 PDE4D5 is responsible for ECM-dependent inflammation in endothelial cells.

(a) BAECs on FN or collagen were treated with rolipram (1 μM) and assayed for AMPK activation. (b) Immunoblotting HUVEC lysate with pan PDE4D antibody detects the major band co-migrating with reference PDE4D5 (left panel). The band was diminished by transfection with PDE4D siRNA, demonstrating specificity. Similarly, PDE4D5 was the only isoform detectable in BAEC lysate with immunobotting using pan PDE4D antibody or PDE4D5 specific antibody (right two panels). ∗ indicates non-specific bands. (c) BAECs expressing wild type PDE4D5 or FAT-PDE4D5 were plated on MG for 1hr to monitor GFP signal.

Supplementary Figure 3 PP2A regulates PDE4D5 phosphorylation and inflammation.

(a) Identification of PP2A as a PDE4D5 binding protein. FLAG-PDE4D5 expressing BAECs on FN were lysed and immunoprecipitated with FLAG antibody. Bound protein with size of 35 kDa was submitted for mass analysis and identified as PP2A catalytic subunit. (b) BAECs expressing FLAG-tagged PDE4D5 were plated on either FN or matrigel for indicated times. PDE4D5 was immunoprecipitated with FLAG antibody and eluted with FLAG peptides. Similar results were obtained in 3 experiments. (c) BAECs expressing PDE4D5 wild type were kept in suspension for 90 min then replated on FN-coated dishes for the indicated times. For okadaic acid treatment, cells in suspension were added with 5 nM OA for last 20 min before replating on FN. S651 phosphorylation was assayed by Western blotting (n = 4 independent experiments). (d) BAECs expressing PDE4D5 were plated on FN for 5 hr then pretreated with DMSO or okadaic acid (OA, 5 nM) and then stimulated with IL1β for 30 min. S651 phosphorylation was assayed by Western blotting (n = 3 independent experiments). (e) BAECs expressing PDE4D5 were transfected with a siRNAs targeting PP2A catalytic subunit. The cells were replated on FN then subject to laminar shear for 30 min. (f) BAECs were plated on FN for 5 hr then pretreated with DMSO or okadaic acid (OA, 5 nM) and then stimulated with IL1β for 30 min. NFκB activity was assayed by Western blotting (n = 3 independent experiments). (g) BAECs were transfected with two different siRNAs targeting the PP2A catalytic subunit. The cells were replated on FN then subject to oscillatory shear for 2 hrs (n = 3 independent experiments). Data are represented as means ± SEM. ∗p < 0.05 by one way ANOVA (d,g) or two-tailed t-test (c,f). Source data are provided in Supplementary Table 1.

Supplementary Figure 4 Effect of PP2A inhibition on endothelial cell adhesion.

(a) BAECs were transfected with PP2A siRNA or treated with okadaic acid (OA, 5 nM, 1hr) and plated on FN (10 μg/ml) for 1hr. Cell adhesion was quantified as described in Supplementary Fig. 2 (n = 3 independent experiments) and cell morphology was examined after fixation and phalloidin and nuclear staining. Error bars are SEM. Scale bar: 100 μm. (b) BAECs treated as (a) were plated on FN (10 μg/ml) for indicated times and FAK phosphorylation was measured by Western blotting.

Supplementary Figure 5 In vivo knock-down of endothelial PDE4D.

(a) NIH3T3 cells were transfected with either luciferase siRNA or mouse PDE4D siRNA used for in vivo knock-down. After 72h, PDE4D5 was assayed by Western blotting with tubulin as a loading control. (b) Nano-particles containing PDE4D siRNA or luciferase siRNA (1 mg/kg) were injected intravenously. After two weeks, mouse aortas were isolated, and endothelial expression of PDE4D was assayed by qPCR (n = 4). (c) Serum lipid profile of α5/2; ApoE(-/-) mice after high fat diet (n = 6 mice). Data are represented as means ± SEM. ∗p < 0.05 by two-tailed t-test. Source data are provided in Supplementary Table 1.

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Yun, S., Budatha, M., Dahlman, J. et al. Interaction between integrin α5 and PDE4D regulates endothelial inflammatory signalling. Nat Cell Biol 18, 1043–1053 (2016). https://doi.org/10.1038/ncb3405

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