Localized alpha4 integrin phosphorylation directs shear stress-induced endothelial cell alignment

Abstract

Vascular endothelial cells respond to laminar shear stress by aligning in the direction of flow, a process which may contribute to atheroprotection. Here we report that localized alpha4 integrin phosphorylation is a mechanism for establishing the directionality of shear stress-induced alignment in microvascular endothelial cells. Within 5 minutes of exposure to a physiological level of shear stress, endothelial alpha4 integrins became phosphorylated on Ser(988). In wounded monolayers, phosphorylation was enhanced at the downstream edges of cells relative to the source of flow. The shear-induced alpha4 integrin phosphorylation was blocked by inhibitors of cAMP-dependent protein kinase A (PKA), an enzyme involved in the alignment of endothelial cells under prolonged shear. Moreover, shear-induced localized activation of the small GTPase Rac1, which specifies the directionality of endothelial alignment, was similarly blocked by PKA inhibitors. Furthermore, endothelial cells bearing a nonphosphorylatable alpha4(S(988)A) mutation failed to align in response to shear stress, thus establishing alpha4 as a relevant PKA substrate. We thereby show that shear-induced PKA-dependent alpha4 integrin phosphorylation at the downstream edge of endothelial cells promotes localized Rac1 activation, which in turn directs cytoskeletal alignment in response to shear stress.

Figure 1. α4 integrin is phosphorylated at downstream cell edges in response to shear stress

A and C. HMECs were seeded onto CS-1-coated glass slides, left confluent (A) or scratch wounded (C) and fixed either without any exposure to flow (T=0) or immediately after 5 minute exposure to flow of culture media at 12 dynes/cm² (T=5). Fixed samples were labeled with PSα4 antibodies (A, upper panels in C) or total α4 integrin (lower panel in C). The black arrow indicates the direction of laminar flow. White arrowheads point to regions of concentrated α4 integrin phosphorylation or total α4 integrin. Bar, 10 μm. B. Western blots of α4 immunoprecipitates from HMECs subjected to shear for 0 or 5 minutes, blotted with antibodies to total α4 or PSα4. Densitometric ratios of band intensities (PSα4/total, shear:static) are indicated. D. HMECs plated on CS-1-coated slides were scratch wounded (top-to-bottom) and subjected to flow (left-to-right) for 5, 15 or 30 minutes, then stained with PSα4 antibodies. Cells at the wound margin were scored by blinded observers for phospho-α4 staining at the cell periphery. The black arrow indicates the direction of laminar flow. White arrows indicate the wound margins generated by a scratch, and small white arrowheads point of regions of concentrated α4 integrin phosphorylation.

Figure 1. α4 integrin is phosphorylated at downstream cell edges in response to shear stress

A and C. HMECs were seeded onto CS-1-coated glass slides, left confluent (A) or scratch wounded (C) and fixed either without any exposure to flow (T=0) or immediately after 5 minute exposure to flow of culture media at 12 dynes/cm² (T=5). Fixed samples were labeled with PSα4 antibodies (A, upper panels in C) or total α4 integrin (lower panel in C). The black arrow indicates the direction of laminar flow. White arrowheads point to regions of concentrated α4 integrin phosphorylation or total α4 integrin. Bar, 10 μm. B. Western blots of α4 immunoprecipitates from HMECs subjected to shear for 0 or 5 minutes, blotted with antibodies to total α4 or PSα4. Densitometric ratios of band intensities (PSα4/total, shear:static) are indicated. D. HMECs plated on CS-1-coated slides were scratch wounded (top-to-bottom) and subjected to flow (left-to-right) for 5, 15 or 30 minutes, then stained with PSα4 antibodies. Cells at the wound margin were scored by blinded observers for phospho-α4 staining at the cell periphery. The black arrow indicates the direction of laminar flow. White arrows indicate the wound margins generated by a scratch, and small white arrowheads point of regions of concentrated α4 integrin phosphorylation.

Figure 2. Protein kinase A activity is required for shear-induced α4 phosphorylation

Monolayers of HMECs seeded on CS-1 were scratch wounded and subjected to flow in medium with or without 100 μM H-89 for 5 minutes. Cells were fixed and stained with PSα4 antibodies (upper panels) or antibodies to α4 (lower panel). The black arrow indicates the direction of laminar flow. White arrowheads point to areas at the cell periphery where α4 is phosphorylated. Bar, 10 μm.

Figure 3. Rac1 activation following shear stress requires PKA activity

HMECs were transfected with Rac1(wt)-GFP and seeded on CS-1-coated slides, shear-loaded with Alexa-PBD and scratch wounded. The cells were subjected to flow for 5 minutes in the presence or absence of H-89. Rac1 activation was assessed by FRET. White arrowheads point to the cell periphery. The black arrow indicates the direction of laminar flow for all samples. Bar, 10 μm.

Figure 4. Shear-induced stress fiber alignment requires PKA activity

A. HMECs were plated on CS-1 and subjected to flow for 20 hours in the continuous presence or absence of 100 μM H-89, then fixed and labeled with rhodamine-phalloidin. Bar, 10 μm. B. Average angles of stress fibers (mapped onto a 0-90° range) from a line parallel to the direction of flow and the S.D. of the angles are shown as the angular and radial positions of the corresponding bullets.

Figure 5. α4 integrin is required for shear-induced alignment

A. HMECs were seeded on fibronectin and subjected for 20 hours to shear flow with growth medium containing control IgG or blocking antibodies to human α2, α4 or α5 integrins. Cells were fixed and labeled with rhodamine-phalloidin. Bars, 10 μm. Average angles of stress fibers (mapped onto a 0-90° range) from a line parallel to the direction of flow and the S.D. of the angles are shown as the angular and radial positions of the corresponding bullets. B. Pulmonary lung endothelial cells isolated from wild type mice treated with vector (wt) or wild type and α4 fl/fl mice treated with Cre recombinase (wt CRE and α4 fl/fl CRE, respectively) were plated on fibronectin and subjected to flow for 20 hours, then labeled with rhodamine-phalloidin. Bar, 10 μm.

Figure 6. Phosphorylation of α4 integrin is necessary for alignment

A. Pulmonary lung endothelial cells isolated from wild type and α4(S⁹⁸⁸A) mice were plated on fibronectin and subjected to flow for 20 hours, then labeled with rhodamine-phalloidin. Bar, 10 μm. B. Average angles of stress fibers (mapped onto a 0-90° range) from a line parallel to the direction of flow and the standard deviations of the angles are shown as the angular and radial positions of the corresponding bullets.

Figure 7. Non-phosphorylatable α4 integrin inhibits shear-induced Rac1 activation

HMECs co-transfected with Rac1(wt)-GFP and α4(wt) or α4(S⁹⁸⁸A) were shear-loaded with recombinant PAK-1-binding domain of p21 coupled to Alexa dye (Alexa-PBD), plated on CS-1-coated slides, scratch wounded, and subjected to 12 dynes/cm² shear stress for five minutes. Fixed cells were assayed for Rac FRET. A. Representative images of cells at the wound edge after exposure to shear stress. Bar, 25 μm. B. Quantification of polarized Rac1 activation by shear. Images of cells at the wound edges were divided into quadrants and the FRET signal at the cell periphery was assessed by visual inspection for at least 15 cells per sample. Shown are the percentages of cells in which FRET was observed in the quadrant facing the wound (“Toward” in A.) in cells over-expressing α4(wt) or α4(S⁹⁸⁸A) as indicated, +/- SEM. **, p < 0.0002; *, p < 0.00001.