Matrix-specific suppression of integrin activation in shear stress signaling - PubMed (original) (raw)
Matrix-specific suppression of integrin activation in shear stress signaling
A Wayne Orr et al. Mol Biol Cell. 2006 Nov.
Abstract
Atherosclerotic plaque develops at sites of disturbed flow. We previously showed that flow activates endothelial cell integrins, which then bind to the subendothelial extracellular matrix (ECM), and, in cells on fibronectin or fibrinogen, trigger nuclear factor-kappaB activation. Additionally, fibronectin and fibrinogen are deposited into the subendothelial ECM at atherosclerosis-prone sites at early times. We now show that flow activates ECM-specific signals that establish patterns of integrin dominance. Flow induced alpha2beta1 activation in cells on collagen, but not on fibronectin or fibrinogen. Conversely, alpha5beta1 and alphavbeta3 are activated on fibronectin and fibrinogen, but not collagen. Failure of these integrins to be activated on nonpermissive ECM is because of active suppression by the integrins that are ligated. Protein kinase A is activated specifically on collagen and suppresses flow-induced alphavbeta3 activation. Alternatively, protein kinase Calpha is activated on fibronectin and mediates alpha2beta1 suppression. Thus, integrins actively cross-inhibit through specific kinase pathways. These mechanisms may determine cellular responses to complex extracellular matrices.
Figures
Figure 1.
Flow stimulates α2β1 and α5β1 integrin activation. (A) BAE cells plated on Coll I for 4 h were sheared at 12 dynes/cm2 for the indicated times and then treated with the α2β1 activation marker IAC-1 for 30 min. Cells were washed, lysed, and bound IAC-1 was assessed by Western blotting. Bound IAC-1 was normalized for total protein by probing for actin. Values are means ± SD (n = 4–5). *p < 0.05, **p < 0.01. Representative blots for IAC-1 and actin are shown above (B) BAE cells plated on FN for 4 h were sheared at 12 dynes/cm2 for the indicated times and then treated with the α5β1 activation marker GST-FNIII9-11 for 30 min. Cells were washed, lysed, and bound GST-FNIII9-11 assessed by Western blotting for GST. Binding was normalized to actin. Values are means ± SD (n = 3). *p < 0.05, **p < 0.01. Representative blots for GST-FNIII9-11 and actin are shown above.
Figure 2.
Flow activates integrins through PI 3-kinase independent of the ECM. (A) BAE cells plated on Coll I were treated with the PI 3-kinase inhibitors wortmannin (5 nM) or LY294002 (5 μM) for 30 min. Cells were sheared for 10 min, and IAC-1 binding was assessed as described previously. Values are means ± SD normalized for total protein (n = 4–5) ***p < 0.001 (B) BAE cells plated on FN were treated with wortmannin or LY294002, sheared for 5 min, and GST-FNIII9-11 binding was assessed as described previously. Values are means ± SD normalized for total protein (n = 3). *p < 0.05. (C) BAE cells plated on different matrices were sheared for the indicated times, lysed, and Akt phosphorylation (Thr473) was assessed by Western blotting. Values are means ± SD normalized for total Akt (n = 3–4). Representative blots for phospho- and total-Akt are shown.
Figure 3.
Flow-induced integrin activation is matrix specific. (A) BAE cells plated on Coll I, FN, or FG for 4 h in DMEM containing 0.2% FBS were sheared for 10 min, and IAC-1 binding was assessed as in Figure 1A. Values are means ± SD (n = 3–4). **p < 0.01. (B) BAE cells plated on different matrices were sheared for 5 min, and GST-FNIII9-11 binding was assessed as described in Figure 1B. Values are means ± SD (n = 4–6). (C) BAE cells plated on different matrices were sheared for 5 min and allowed to bind the αvβ3 activation-sensitive WOW-1 for 30 min. Cells were washed, lysed, and bound WOW-1 was assessed by Western blotting. Binding was normalized to total protein by probing for actin. Values are means ± SD (n = 4–7). *p < 0.05, **p < 0.01. (D) BAE cells were plated on a fixed amount of Coll with increasing amounts of FN. Cells were sheared for 10 min, and IAC-1 binding was assessed. Values are means normalized to static cells ± SD (n = 3–4). BAE cells plated on increasing amounts of FN with or without Coll were sheared for 5 min, and GST-FNIII9-11 binding (E) or WOW-1 binding (F) was assessed. Values are means normalized to static cells ± SD (n = 3–4).
Figure 4.
Flow reduces FN matrix assembly on Coll, but not FG. BAE cells grown on Coll or FN were sheared for 2 h in the absence or presence of the integrin activating antibody TS2/16 (10 μg/ml). Cells were extracted sequentially with Triton X-100 and deoxycholate, which removes nonmatrix FN, but leaves FN fibrils, as described in Materials and Methods. Isolated matrices were then fixed and stained for fibronectin. Representative images are shown (n = 3–4).
Figure 5.
Integrin suppression requires new integrin ligation. (A) BAE cells plated on Coll I were treated with the α2β1 nonblocking antibody 12F1 or the α2β1-blocking antibody R2–8C8 (20 μg/ml for 60 min). Cells were stimulated with shear stress for 5 min, and GST-FNIII9-11 binding was assessed. Values are means ± SD normalized for total protein (n = 3). **p < 0.01 (B) BAE cells plated on Coll I were treated with 12F1 or R2–8C8, sheared for 5 min, and WOW-1 binding was assessed. Values are means ± SD normalized for total protein (n = 4). *p < 0.05. (C) BAE cells plated on FN were treated with the FN nonblocking antibody 11E5 or the FN-blocking antibody 16G3 (20 μg/ml for 60 min). Cells were stimulated with shear stress for 10 min and binding of biotinylated IAC-1 was assessed by Western blotting with HRP-conjugated streptavidin. Values are means ± SD normalized for total protein (n = 5). **p < 0.01.
Figure 6.
Effects of PIP3. (A) BAE cells plated on Coll or FN were treated with 5 μM PIP3 micelles with IAC-1 added either simultaneously or 10 min after PIP3 stimulation. IAC-1 binding was determined as described previously. Values are means ± SD normalized for total protein (n = 3–4). *p < 0.05, **p < 0.01 (B) BAE cells plated on Coll or FN were treated with PIP3 micelles with GST-FNIII9-11 added either simultaneously or 10 min after PIP3. GST-FNIII9-11 binding was assayed as described previously. Values are means ± SD normalized for total protein (n = 3–4). *p < 0.05, **p < 0.01. (C) BAE cells plated on Coll or FN were treated with PIP3 micelles with WOW-1 added either simultaneously or 10 min after PIP3 stimulation. WOW-1 binding was assayed as described previously. Values are means ± SD normalized for total protein (n = 3–4). *p < 0.05, **p < 0.01.
Figure 7.
Talin overexpression relieves integrin suppression. (A) BAE cells were transfected with HA- or FLAG-tagged talin by nucleofection. After 48 h, cells were plated on either Coll I or FN, stimulated with shear stress for 10 min, and IAC-1 binding was assessed. Values are means ± SD normalized for total protein (n = 4–7). (B) BAE cells transfected with talin were plated on either Coll I or FN, stimulated with shear stress for 5 min, and WOW-1 binding was assessed. Values are means ± SD normalized for total protein (n = 4).
Figure 8.
α2β1 suppression of αvβ3 requires matrix-specific PKA activation. (A) BAE cells plated on Coll I were treated with the AGC family kinase inhibitor H-7 (100 nM for 1 h), the CaMKII inhibitor KN-62 (2.5 μM for 1 h), the p38 inhibitor SB202190 (1 μM for 1 h), the PKA inhibitor PKI (20 μM for 1 h), and the PKC inhibitor Gö6976 (1 μM for 1 h). Cells were sheared for 5 min, and WOW-1 binding was assessed. Values are means ± SD normalized for total protein (n = 3–8). *p < 0.05, ***p < 0.001. (B) BAE cells transfected with either PKA siRNA (50 nM) or PKCα siRNA (200 nM) for 24 h were plated on Coll, and flow-induced WOW-1 binding was assessed. Values are means ± SD normalized for total protein (n = 3). *p < 0.05. Representative blots are shown. (C) BAE cells plated on different matrices were sheared for the indicated times, lysed, and PKA phosphorylation (Thr197) was assessed by Western blotting. Values are means ± SD normalized for total PKA (n = 3). *p < 0.05.
Figure 9.
α5β1/αvβ3 ligation suppresses α2β1 through PKCα. (A) BAE cells plated on FN were treated with the AGC family kinase inhibitor H-7 (100 nM for 1 h), the PKA inhibitor PKI (20 μM for 1 h), the classic PKC inhibitor Gö6976 (1 μM for 1 h), the PKC inhibitor bisindolylmaleimide (100 nM for 1 h), or with the PKCβ-specific inhibitor hispidin (10 μM for 1 h). Cells were sheared for 10 min, and IAC-1 binding was assessed. Values are means ± SD normalized for total protein (n = 3–8). *p < 0.05, **p < 0.01. (B) BAE cells were plated on FN, treated with PMA (100 nM for 16 h), and expression of individual PKC isoforms was determined by Western blotting. (C) BAE cells were plated on FN, treated with PMA (100 nM for 16 h), sheared for 10 min, and IAC-1 binding was assessed. Values are means ± SD normalized for total protein (n = 3). *p < 0.05. (D) BAE cells transfected with either PKA siRNA or PKCα siRNA for 24 h were plated on FN and flow-induced IAC-1 binding was assessed. Values are means ± SD normalized for total protein (n = 5). *p < 0.05. Representative blots are shown. (E) BAE cells plated on Coll or FN were sheared for 0, 10, or 20 min. Cells were lysed and membrane (M) and cytosolic (C) fractions were isolated as described under Materials and Methods. Membrane-associated PKCα was determined by Western blotting. Values are means ± SD normalized for total protein (n = 3). *p < 0.05. Representative blots are shown above.
Figure 10.
Model for flow-mediated integrin suppression. For cells on collagen, onset of flow stimulates the junctional mechanoreceptor complex to trigger activation of integrin α2β1 through PI 3-kinase. Subsequent binding of integrins to collagen stimulates activation of PKA, which initiates a pathway that ultimately acts upon talin to suppress integrin αvβ3. For cells on FN or FG, onset of flow stimulates PI 3-kinase dependent activation of integrins α5β1 and αvβ3. Subsequent binding of these integrins to their ligands stimulates activation of PKCα, which initiates a pathway that ultimately acts upon talin to suppress integrin α2β1.
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