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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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p38 inhibits shear stress–induced NF-κB activation on Coll. (A) BAE cells plated on Coll, LN, FN, or FG were sheared for the indicated times. Phosphorylation of p38 was assessed by Western blotting with phosphorylation site-specific antibodies, quantified by densitometry, and normalized to total p38. Values are means ± SD (n = 3). A representative Western blot is shown for each condition. (B) Cells were treated with the pharmacological p38 inhibitor SB202190 (1 μM for 1 h), and NF-κB nuclear translocation was assessed with or without shear stress for 30 min. Greater than 100 cells were counted per condition per experiment; n = 3; *, P < 0.05; **, P < 0.01. (C) Cells were preincubated with SB202190 and shear stress–induced p65 phosphorylation (Ser536) was assessed as previously described. Densitometric quantification was normalized to total p65. n = 3. (D) Cells were transiently transfected with dominant-negative p38 (p38-AGF) and sheared for 60 min, and p65 nuclear translocation was assessed. Transfected cells were identified by costaining for total p38. Greater than 100 cells were counted per condition per experiment; n = 3; *, P < 0.05. A representative stain is shown.
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fig3: p38 inhibits shear stress–induced NF-κB activation on Coll. (A) BAE cells plated on Coll, LN, FN, or FG were sheared for the indicated times. Phosphorylation of p38 was assessed by Western blotting with phosphorylation site-specific antibodies, quantified by densitometry, and normalized to total p38. Values are means ± SD (n = 3). A representative Western blot is shown for each condition. (B) Cells were treated with the pharmacological p38 inhibitor SB202190 (1 μM for 1 h), and NF-κB nuclear translocation was assessed with or without shear stress for 30 min. Greater than 100 cells were counted per condition per experiment; n = 3; *, P < 0.05; **, P < 0.01. (C) Cells were preincubated with SB202190 and shear stress–induced p65 phosphorylation (Ser536) was assessed as previously described. Densitometric quantification was normalized to total p65. n = 3. (D) Cells were transiently transfected with dominant-negative p38 (p38-AGF) and sheared for 60 min, and p65 nuclear translocation was assessed. Transfected cells were identified by costaining for total p38. Greater than 100 cells were counted per condition per experiment; n = 3; *, P < 0.05. A representative stain is shown.

Mentions: In addition to NF-κB, p38 MAP kinase is also involved in stimulating inflammatory protein expression in a variety of systems. However, p38 shows differential effects on NF-κB, either enhancing or suppressing NF-κB depending on the stimulus (Schwenger et al., 1998; Alpert et al., 1999; Bowie and O'Neill, 2000; Bradbury et al., 2001). To examine p38 in this system, BAE cells were plated on different ECM proteins for 4 h and effects of shear were tested. Western blotting with an antibody against the phosphorylated and activated form of p38 showed that shear stress stimulated a transient increase in p38 phosphorylation in cells on Coll but not on other ECM proteins (Fig. 3 A). This result is consistent with previous reports showing that the Coll-binding integrin α2β1 stimulates p38 activation (Ivaska et al., 1999). To test if this Coll-specific p38 activation mediates inhibition of shear stress–induced NF-κB activation, cells were preincubated with the p38 inhibitor SB202190 (1 μM for 1 h) before fluid flow. Inhibition of p38 with SB202190 restored shear stress–induced NF-κB nuclear translocation and NF-κB phosphorylation in cells on Coll but had no effect on cells on FN (Fig. 3, B and C). To confirm this result, endothelial cells were transiently transfected with a dominant-negative mutant of p38 (p38-AGF). This construct did not alter baseline levels of NF-κB activity but enhanced flow-induced NF-κB nuclear translocation in cells on Coll (Fig. 3 D). Thus, selective activation of p38 mediates suppression of NF-κB in cells on Coll.


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)

p38 inhibits shear stress–induced NF-κB activation on Coll. (A) BAE cells plated on Coll, LN, FN, or FG were sheared for the indicated times. Phosphorylation of p38 was assessed by Western blotting with phosphorylation site-specific antibodies, quantified by densitometry, and normalized to total p38. Values are means ± SD (n = 3). A representative Western blot is shown for each condition. (B) Cells were treated with the pharmacological p38 inhibitor SB202190 (1 μM for 1 h), and NF-κB nuclear translocation was assessed with or without shear stress for 30 min. Greater than 100 cells were counted per condition per experiment; n = 3; *, P < 0.05; **, P < 0.01. (C) Cells were preincubated with SB202190 and shear stress–induced p65 phosphorylation (Ser536) was assessed as previously described. Densitometric quantification was normalized to total p65. n = 3. (D) Cells were transiently transfected with dominant-negative p38 (p38-AGF) and sheared for 60 min, and p65 nuclear translocation was assessed. Transfected cells were identified by costaining for total p38. Greater than 100 cells were counted per condition per experiment; n = 3; *, P < 0.05. A representative stain is shown.
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Related In: Results  -  Collection

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fig3: p38 inhibits shear stress–induced NF-κB activation on Coll. (A) BAE cells plated on Coll, LN, FN, or FG were sheared for the indicated times. Phosphorylation of p38 was assessed by Western blotting with phosphorylation site-specific antibodies, quantified by densitometry, and normalized to total p38. Values are means ± SD (n = 3). A representative Western blot is shown for each condition. (B) Cells were treated with the pharmacological p38 inhibitor SB202190 (1 μM for 1 h), and NF-κB nuclear translocation was assessed with or without shear stress for 30 min. Greater than 100 cells were counted per condition per experiment; n = 3; *, P < 0.05; **, P < 0.01. (C) Cells were preincubated with SB202190 and shear stress–induced p65 phosphorylation (Ser536) was assessed as previously described. Densitometric quantification was normalized to total p65. n = 3. (D) Cells were transiently transfected with dominant-negative p38 (p38-AGF) and sheared for 60 min, and p65 nuclear translocation was assessed. Transfected cells were identified by costaining for total p38. Greater than 100 cells were counted per condition per experiment; n = 3; *, P < 0.05. A representative stain is shown.
Mentions: In addition to NF-κB, p38 MAP kinase is also involved in stimulating inflammatory protein expression in a variety of systems. However, p38 shows differential effects on NF-κB, either enhancing or suppressing NF-κB depending on the stimulus (Schwenger et al., 1998; Alpert et al., 1999; Bowie and O'Neill, 2000; Bradbury et al., 2001). To examine p38 in this system, BAE cells were plated on different ECM proteins for 4 h and effects of shear were tested. Western blotting with an antibody against the phosphorylated and activated form of p38 showed that shear stress stimulated a transient increase in p38 phosphorylation in cells on Coll but not on other ECM proteins (Fig. 3 A). This result is consistent with previous reports showing that the Coll-binding integrin α2β1 stimulates p38 activation (Ivaska et al., 1999). To test if this Coll-specific p38 activation mediates inhibition of shear stress–induced NF-κB activation, cells were preincubated with the p38 inhibitor SB202190 (1 μM for 1 h) before fluid flow. Inhibition of p38 with SB202190 restored shear stress–induced NF-κB nuclear translocation and NF-κB phosphorylation in cells on Coll but had no effect on cells on FN (Fig. 3, B and C). To confirm this result, endothelial cells were transiently transfected with a dominant-negative mutant of p38 (p38-AGF). This construct did not alter baseline levels of NF-κB activity but enhanced flow-induced NF-κB nuclear translocation in cells on Coll (Fig. 3 D). Thus, selective activation of p38 mediates suppression of NF-κB in cells on Coll.

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