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The subendothelial extracellular matrix modulates NF-kappaB activation by flow: a potential role in atherosclerosis.

Orr AW, Sanders JM, Bevard M, Coleman E, Sarembock IJ, Schwartz MA - J. Cell Biol. (2005)

Bottom Line: Flow-induced NF-kappaB activation is downstream of conformational activation of integrins, resulting in new integrin binding to the subendothelial extracellular matrix and signaling.Whereas endothelial cells plated on fibronectin or fibrinogen activate NF-kappaB in response to flow, cells on collagen or laminin do not.Furthermore, altering the extracellular matrix to promote p38 activation in cells on fibronectin suppresses NF-kappaB activation, suggesting a novel therapeutic strategy for treating atherosclerosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA.

ABSTRACT
Atherosclerotic plaque forms in regions of the vasculature exposed to disturbed flow. NF-kappaB activation by fluid flow, leading to expression of target genes such as E-selectin, ICAM-1, and VCAM-1, may regulate early monocyte recruitment and fatty streak formation. Flow-induced NF-kappaB activation is downstream of conformational activation of integrins, resulting in new integrin binding to the subendothelial extracellular matrix and signaling. Therefore, we examined the involvement of the extracellular matrix in this process. Whereas endothelial cells plated on fibronectin or fibrinogen activate NF-kappaB in response to flow, cells on collagen or laminin do not. In vivo, fibronectin and fibrinogen are deposited at atherosclerosis-prone sites before other signs of atherosclerosis. Ligation of integrin alpha2beta1 on collagen prevents flow-induced NF-kappaB activation through a p38-dependent pathway that is activated locally at adhesion sites. Furthermore, altering the extracellular matrix to promote p38 activation in cells on fibronectin suppresses NF-kappaB activation, suggesting a novel therapeutic strategy for treating atherosclerosis.

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Integrin α2β1 mediates p38 activation and NF-κB inhibition. (A) BAE cells were incubated for 30 min with either the α2β1 blocking (R2-8C8) or the α2β1 nonblocking antibody (12F1) and sheared for 5 or 30 min. Lysates were analyzed for p38 phosphorylation by immunoblotting. Bands were quantified by densitometry and normalized for total p38 protein. Values are means ± SD (n = 4). *, P < 0.05; **, P < 0.01. (B) Cells on Coll were incubated with 12F1 or R2-8C8 and sheared for 5 or 30 min, and the phosphorylation of p65 was analyzed by immunoblotting. Bands were quantified by densitometry and normalized for total p65. **, P < 0.01 (n = 5). (C) Cells plated on Coll I or FN were pretreated with the p38 inhibitor SB202190 (1 μM for 1 h) and integrin ligation was induced with the β1 integrin–activating antibody TS2/16 (15 μg/ml for 1 h). p65 localization was assessed by immunostaining, and the percentage of cells showing nuclear localization was scored (n = 3; *, P < 0.05). (D) TS2/16-induced p65 phosphorylation of p65 on Ser536 was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p65 (n = 3–6).
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fig4: Integrin α2β1 mediates p38 activation and NF-κB inhibition. (A) BAE cells were incubated for 30 min with either the α2β1 blocking (R2-8C8) or the α2β1 nonblocking antibody (12F1) and sheared for 5 or 30 min. Lysates were analyzed for p38 phosphorylation by immunoblotting. Bands were quantified by densitometry and normalized for total p38 protein. Values are means ± SD (n = 4). *, P < 0.05; **, P < 0.01. (B) Cells on Coll were incubated with 12F1 or R2-8C8 and sheared for 5 or 30 min, and the phosphorylation of p65 was analyzed by immunoblotting. Bands were quantified by densitometry and normalized for total p65. **, P < 0.01 (n = 5). (C) Cells plated on Coll I or FN were pretreated with the p38 inhibitor SB202190 (1 μM for 1 h) and integrin ligation was induced with the β1 integrin–activating antibody TS2/16 (15 μg/ml for 1 h). p65 localization was assessed by immunostaining, and the percentage of cells showing nuclear localization was scored (n = 3; *, P < 0.05). (D) TS2/16-induced p65 phosphorylation of p65 on Ser536 was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p65 (n = 3–6).

Mentions: Although Coll binds both integrins α2β1 and α1β1, expression of α1β1 is restricted to microvascular endothelial cells (Defilippi et al., 1991; Heino, 2000). To test whether integrin α2β1 mediates Coll-specific signaling after shear, endothelial cells were pretreated with the α2β1 integrin–blocking antibody R2-8C8 (20 μg/ml for 30 min) or, as a control, with the nonblocking α2β1 antibody 12F1 (20 μg/ml for 30 min). With this short exposure to blocking antibodies, we can inhibit new integrin binding without significantly affecting existing adhesions or cytoskeletal structure (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200410073/DC1). Blocking α2β1 integrin ligation prevented the shear stress–induced activation of p38 in cells on Coll (Fig. 4 A) and rescued NF-κB phosphorylation (Fig. 4 B). The control antibody had no effect. Preventing new ligation of integrin α6β1 with the blocking antibody GoH3 (20 μg/ml for 30 min) did not enhance flow-induced p65 nuclear translocation or phosphorylation in cells on LN, instead, the already low level of NF-κB was reduced further. These results suggest α6β1 is not suppressive (Fig. S2, A–C, available at http://www.jcb.org/cgi/content/full/jcb.200410073/DC1).


The subendothelial extracellular matrix modulates NF-kappaB activation by flow: a potential role in atherosclerosis.

Orr AW, Sanders JM, Bevard M, Coleman E, Sarembock IJ, Schwartz MA - J. Cell Biol. (2005)

Integrin α2β1 mediates p38 activation and NF-κB inhibition. (A) BAE cells were incubated for 30 min with either the α2β1 blocking (R2-8C8) or the α2β1 nonblocking antibody (12F1) and sheared for 5 or 30 min. Lysates were analyzed for p38 phosphorylation by immunoblotting. Bands were quantified by densitometry and normalized for total p38 protein. Values are means ± SD (n = 4). *, P < 0.05; **, P < 0.01. (B) Cells on Coll were incubated with 12F1 or R2-8C8 and sheared for 5 or 30 min, and the phosphorylation of p65 was analyzed by immunoblotting. Bands were quantified by densitometry and normalized for total p65. **, P < 0.01 (n = 5). (C) Cells plated on Coll I or FN were pretreated with the p38 inhibitor SB202190 (1 μM for 1 h) and integrin ligation was induced with the β1 integrin–activating antibody TS2/16 (15 μg/ml for 1 h). p65 localization was assessed by immunostaining, and the percentage of cells showing nuclear localization was scored (n = 3; *, P < 0.05). (D) TS2/16-induced p65 phosphorylation of p65 on Ser536 was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p65 (n = 3–6).
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Related In: Results  -  Collection

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fig4: Integrin α2β1 mediates p38 activation and NF-κB inhibition. (A) BAE cells were incubated for 30 min with either the α2β1 blocking (R2-8C8) or the α2β1 nonblocking antibody (12F1) and sheared for 5 or 30 min. Lysates were analyzed for p38 phosphorylation by immunoblotting. Bands were quantified by densitometry and normalized for total p38 protein. Values are means ± SD (n = 4). *, P < 0.05; **, P < 0.01. (B) Cells on Coll were incubated with 12F1 or R2-8C8 and sheared for 5 or 30 min, and the phosphorylation of p65 was analyzed by immunoblotting. Bands were quantified by densitometry and normalized for total p65. **, P < 0.01 (n = 5). (C) Cells plated on Coll I or FN were pretreated with the p38 inhibitor SB202190 (1 μM for 1 h) and integrin ligation was induced with the β1 integrin–activating antibody TS2/16 (15 μg/ml for 1 h). p65 localization was assessed by immunostaining, and the percentage of cells showing nuclear localization was scored (n = 3; *, P < 0.05). (D) TS2/16-induced p65 phosphorylation of p65 on Ser536 was assessed by Western blotting. Bands were quantified by densitometry and normalized to total p65 (n = 3–6).
Mentions: Although Coll binds both integrins α2β1 and α1β1, expression of α1β1 is restricted to microvascular endothelial cells (Defilippi et al., 1991; Heino, 2000). To test whether integrin α2β1 mediates Coll-specific signaling after shear, endothelial cells were pretreated with the α2β1 integrin–blocking antibody R2-8C8 (20 μg/ml for 30 min) or, as a control, with the nonblocking α2β1 antibody 12F1 (20 μg/ml for 30 min). With this short exposure to blocking antibodies, we can inhibit new integrin binding without significantly affecting existing adhesions or cytoskeletal structure (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200410073/DC1). Blocking α2β1 integrin ligation prevented the shear stress–induced activation of p38 in cells on Coll (Fig. 4 A) and rescued NF-κB phosphorylation (Fig. 4 B). The control antibody had no effect. Preventing new ligation of integrin α6β1 with the blocking antibody GoH3 (20 μg/ml for 30 min) did not enhance flow-induced p65 nuclear translocation or phosphorylation in cells on LN, instead, the already low level of NF-κB was reduced further. These results suggest α6β1 is not suppressive (Fig. S2, A–C, available at http://www.jcb.org/cgi/content/full/jcb.200410073/DC1).

Bottom Line: Flow-induced NF-kappaB activation is downstream of conformational activation of integrins, resulting in new integrin binding to the subendothelial extracellular matrix and signaling.Whereas endothelial cells plated on fibronectin or fibrinogen activate NF-kappaB in response to flow, cells on collagen or laminin do not.Furthermore, altering the extracellular matrix to promote p38 activation in cells on fibronectin suppresses NF-kappaB activation, suggesting a novel therapeutic strategy for treating atherosclerosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA.

ABSTRACT
Atherosclerotic plaque forms in regions of the vasculature exposed to disturbed flow. NF-kappaB activation by fluid flow, leading to expression of target genes such as E-selectin, ICAM-1, and VCAM-1, may regulate early monocyte recruitment and fatty streak formation. Flow-induced NF-kappaB activation is downstream of conformational activation of integrins, resulting in new integrin binding to the subendothelial extracellular matrix and signaling. Therefore, we examined the involvement of the extracellular matrix in this process. Whereas endothelial cells plated on fibronectin or fibrinogen activate NF-kappaB in response to flow, cells on collagen or laminin do not. In vivo, fibronectin and fibrinogen are deposited at atherosclerosis-prone sites before other signs of atherosclerosis. Ligation of integrin alpha2beta1 on collagen prevents flow-induced NF-kappaB activation through a p38-dependent pathway that is activated locally at adhesion sites. Furthermore, altering the extracellular matrix to promote p38 activation in cells on fibronectin suppresses NF-kappaB activation, suggesting a novel therapeutic strategy for treating atherosclerosis.

Show MeSH
Related in: MedlinePlus