Ying-Li Hu

Fluid Shear Stress Activation of Focal Adhesion Kinase LINKING TO MITOGEN-ACTIVATED PROTEIN KINASES

Shear stress, the tangential component of hemodynamic forces, activates the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) signal transduction pathways in cultured vascular endothelial cells to induce the transcriptional activation of many immediate early genes. It appears that integrins, protein-tyrosine kinases, and the structural integrity of actin are important factors involved in these shear stress-induced responses. The underlying molecular events were investigated by the application of a shear stress of 12 dyn/cm2 on bovine aortic endothelial cells (BAEC). We found that such a shear stress increased the tyrosine phosphorylation and the kinase activity of focal adhesion kinase (FAK) and its association with growth factor receptor binding protein 2 (Grb2) in a rapid and transient manner, suggesting that FAK may be linked to these mitogen-activated protein kinase signaling pathways through a Grb2·Son of sevenless (Sos) complex. FAK(F397Y), which encodes a dominant negative mutant of FAK, attenuated the shear stress-induced kinase activity of Myc epitope-tagged ERK2 and hemagglutinin epitope-tagged JNK1. ΔmSos1, encoding a dominant negative mutant of Sos in which the guanine nucleotide exchange domain has been deleted, also attenuated shear stress activation of Myc-ERK2 and hemagglutinin-JNK1. Pretreating the confluent BAEC monolayers with a blocking type anti-vitronectin receptor monoclonal antibody had similar inhibitory effects in these shear stress-activated ERKs and JNKs. Confocal microscopic observation further demonstrated that FAK tended to cluster with vitronectin receptor near the abluminal side of the sheared BAEC. These results demonstrate that FAK signaling is critical in the shear stress-induced dual activation of ERK and JNK.

The role of the dynamics of focal adhesion kinase in the mechanotaxis of endothelial cells.

The migration of vascular endothelial cells (ECs) is critical in vascular remodeling. We showed that fluid shear stress enhanced EC migration in flow direction and called this “mechanotaxis.” To visualize the molecular dynamics of focal adhesion kinase (FAK) at focal adhesions (FAs), FAK tagged with green fluorescence protein (GFP) was expressed in ECs. Within 10 min of shear stress application, lamellipodial protrusion was induced at cell periphery in the flow direction, with the recruitment of FAK at FAs. ECs under flow migrated with polarized formation of new FAs in flow direction, and these newly formed FAs subsequently disassembled after the rear of the cell moved over them. The cells migrating under flow had a decreased number of FAs. In contrast to shear stress, serum did not significantly affect the speed of cell migration. Serum induced lamellipodia and FAK recruitment at FAs without directional preference. FAK(Y397) phosphorylation colocalized with GFP-FAK at FAs in both shear stress and serum experiments. The total level of FAK(Y397) phosphorylation after shear stress was lower than that after serum treatment, suggesting that the polarized change at cell periphery rather than the total level of FAK(Y397) phosphorylation is important for directional migration. Our results demonstrate the dynamics of FAK at FAs during the directional migration of EC in response to mechanical force, and suggest that mechanotaxis is an important mechanism controlling EC migration.

Roles of Microtubule Dynamics and Small GTPase Rac in Endothelial Cell Migration and Lamellipodium Formation under Flow

Endothelial cell (EC) migration is required for vascular development and wound healing. We investigated the roles of microtubule (MT) dynamics and the small GTPase Rac in the fluid shear stress-induced protrusion of lamellipodia and enhancement of migration of bovine aortic ECs (BAECs). Shear stress increased lamellipodial protrusion and cell migration. Treating BAECs with paclitaxel (Taxol), an MT-stabilizing agent, inhibited lamellipodial protrusion and reduced migration speed in both the static and sheared groups. After Taxol washout, both lamellipodial protrusion and cell migration increased in the flow direction. Taxol treatment also decreased the shear-induced Rac activation. Transfection of BAECs with a dominant negative mutant of Rac1 inhibited lamellipodial protrusion and cell migration under static and shear conditions. Transfection with an activated mutant of Rac1 induced lamellipodia in all directions and attenuated the shear-induced migration, suggesting that an appropriate level of Rac activity and a polarized lamellipodial protrusion are important for cell migration under static and shear conditions. Our findings suggest that MT dynamics and optimum Rac activation are required for the polarized protrusion of lamellipodia that drives the directional EC migration under flow.

Sustained JNK activation induces endothelial apoptosis: studies with colchicine and shear stress

The disruption of microtubules by treating bovine aortic endothelial cells with 10-7-10-5M colchicine caused apoptosis, as evidenced by DNA laddering and TdT-mediated dUTP nick end labeling fluorescence staining. Colchicine treatment also induced a sustained activation of c-Jun NH2-terminal kinase (JNK) that lasted for ≥12 h. The blockade of JNK activity by using the negative interfering mutant JNK(K-R) markedly decreased the apoptosis induced by colchicine. Exposure of bovine aortic endothelial cells to laminar shear stress (12 dyn/cm2) caused a transient (<2 h) activation of JNK, and there was no induction of apoptosis. The sustained activation of JNK may play a significant role in the apoptosis induced by colchicine.

Signal Transduction in Matrix Contraction and the Migration of Vascular Smooth Muscle Cells in Three-Dimensional Matrix

The interaction of vascular smooth muscle cells (SMCs) and extracellular matrix plays important roles in vascular remodeling. We investigated the signaling pathways involved in SMC-induced matrix contraction and SMC migration in three-dimensional (3D) collagen matrix. Matrix contraction is inhibited by the disruption of actin filaments but not microtubules. Therefore, we investigated the roles of signaling pathways related to actin filaments in matrix contraction. SMC-induced matrix contraction was markedly blocked (–80%) by inhibiting the Rho-p160ROCK pathway and myosin light chain kinase, and was decreased to a lesser extent (30–40%) by a negative mutant of Rac and inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) or p38 mitogen-activated protein kinase (MAPK), but it was not affected by the inhibition of Ras and Cdc42-Wiskott-Aldrich syndrome protein (WASP) pathways. Inhibition of extracellular-signal-regulated kinase (ERK) decreased SMC-induced matrix contraction by only 15%. The migration speed and persistence of SMCs in the 3D matrix were decreased by the inhibition of p160ROCK, PI 3-kinase, p38 MAPK or WASP to different extents, and p160ROCK inhibitor had the strongest inhibitory effect. Our results suggest that the SMC-induced matrix contraction and the migration of SMCs in 3D matrix share some signaling pathways leading to force generation at cell-matrix adhesions and that various signaling pathways have different relative importance in the regulations of these processes in SMCs.

FAK and paxillin dynamics at focal adhesions in the protrusions of migrating cells

Cell migration requires the fine spatiotemporal integration of many proteins that regulate the fundamental processes that drive cell movement. Focal adhesion (FA) dynamics is a continuous process involving coordination between FA and actin cytoskeleton, which is essential for cell migration. We studied the spatiotemporal relationship between the dynamics of focal adhesion kinase (FAK) and paxillin at FAs in the protrusion of living endothelial cells. Concurrent dual-color imaging showed that FAK was assembled at FA first, which was followed by paxillin recruitment to the FA. By tracking and quantifying FAK and paxillin in migrating cells, the normalized FAK/Paxillin fluorescence intensity (FI) ratio is > 1 (≈4 fold) at cell front, ≈1 at cell center, and < 1 at cell rear. The significantly higher FAK FI than paxillin FI at cell front indicates that the assembly of FAK-FAs occurs ahead of paxillin at cell front. To determine the time difference between the assemblies of FAK and paxillin at nascent FAs, FAs containing both FAK and paxillin were quantified by image analysis and time correlation. The results show that FAK assembles at the nascent FAs earlier than paxillin in the protrusions at cell front.

RhoA and membrane fluidity mediates the spatially polarized Src/FAK activation in response to shear stress.

While Src plays crucial roles in shear stress-induced cellular processes, little is known on the spatiotemporal pattern of high shear stress (HSS)-induced Src activation. HSS (65 dyn/cm2) was applied on bovine aortic endothelial cells to visualize the dynamic Src activation at subcellular levels utilizing a membrane-targeted Src biosensor (Kras-Src) based on fluorescence resonance energy transfer (FRET). A polarized Src activation was observed with higher activity at the side facing the flow, which was enhanced by a cytochalasin D-mediated disruption of actin filaments but inhibited by a benzyl alcohol-mediated enhancement of membrane fluidity. Further experiments revealed that HSS decreased RhoA activity, with a constitutively active RhoA mutant inhibiting while a negative RhoA mutant enhancing the HSS-induced Src polarity. Cytochalasin D can restore the polarity in cells expressing the active RhoA mutant. Further results indicate that HSS stimulates FAK activation with a spatial polarity similar to Src. The inhibition of Src by PP1, as well as the perturbation of RhoA activity and membrane fluidity, can block this HSS-induced FAK polarity. These results indicate that the HSS-induced Src and subsequently FAK polarity depends on the coordination between intracellular tension distribution regulated by RhoA, its related actin structures and the plasma membrane fluidity.

3-dimensional forces and molecular dynamics of live cells

The forces exerted by an adherent cell on a substrate were studied previously only in the two-dimensions (2D) tangential to the substrate surface. We used a novel technique to measure the three-dimensional (3D) stresses exerted by live bovine aortic endothelial cells (BAECs) on polyacrylamide deformable substrate, with particular emphasis on the 3D forces of focal adhesions. On 3D images acquired by confocal microscopy, displacements were determined with imageprocessing programs, and stresses in tangential (XY) and normal (Z) directions were computed by finite element method (FEM). BAECs generated stress in normal direction (Tz) with an order of magnitude comparable to that in tangential direction (Txy). Tz is upward at the cell edge and downward under the nucleus, changing continuously with a sign reversal between cell edge and nucleus edge. With the use of green fluorescent protein (GFP) labeled paxillin, the dynamics of this intracellular molecule were studied concurrently with the measurement of 3D forces. In the dynamic region, including the new lamellapodium forming region in the front and the retracting region in the rear, the tangential forces (Fxy) are correlated with the size of the focal adhesions (FAs) much more strongly than those in the stable region under the nucleus. In the dynamic region, normal force (Fz) was upward and positively correlated with FA size, while Fz in the stable region was downward and negatively correlated with FA size. These findings show the influence of the size of FAs on the 3D forces they exert on the substrate. This technique can be applied to study any adherent type of live cells to assess their biomechanical dynamics in conjunction with biochemical and functional activities, thus elucidating cellular functions in health and disease.