William B. Kiosses

Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton.

Soluble factors from serum such as lysophosphatidic acid (LPA) are thought to activate the small GTP‐binding protein Rho based on their ability to induce actin stress fibers and focal adhesions in a Rho‐dependent manner. Cell adhesion to extracellular matrices (ECM) has also been proposed to activate Rho, but this point has been controversial due to the difficulty of distinguishing changes in Rho activity from the structural contributions of ECM to the formation of focal adhesions. To address these questions, we established an assay for GTP‐bound cellular Rho. Plating Swiss 3T3 cells on fibronectin‐coated dishes elicited a transient inhibition of Rho, followed by a phase of Rho activation. The activation phase was greatly enhanced by serum. In serum‐starved adherent cells, LPA induced transient Rho activation, whereas in suspended cells Rho activation was sustained. Furthermore, suspended cells showed higher Rho activity than adherent cells in the presence of serum. These data indicate the existence of an adhesion‐dependent negative‐feedback loop. We also observed that both cytochalasin D and colchicine trigger Rho activation despite their opposite effects on stress fibers and focal adhesions. Our results show that ECM, cytoskeletal structures and soluble factors all contribute to regulation of Rho activity.

A mechanosensory complex that mediates the endothelial cell response to fluid shear stress.

Shear stress is a fundamental determinant of vascular homeostasis, regulating vascular remodelling, cardiac development and atherogenesis, but the mechanisms of transduction are poorly understood. Previous work showed that the conversion of integrins to a high-affinity state mediates a subset of shear responses, including cell alignment and gene expression. Here we investigate the pathway upstream of integrin activation. PECAM-1 (which directly transmits mechanical force), vascular endothelial cell cadherin (which functions as an adaptor) and VEGFR2 (which activates phosphatidylinositol-3-OH kinase) comprise a mechanosensory complex. Together, these receptors are sufficient to confer responsiveness to flow in heterologous cells. In support of the relevance of this pathway in vivo, PECAM-1-knockout mice do not activate NF-κB and downstream inflammatory genes in regions of disturbed flow. Therefore, this mechanosensing pathway is required for the earliest-known events in atherogenesis.

Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI

The proper function of Rho GTPases requires precise spatial and temporal regulation of effector interactions. Integrin-mediated cell adhesion modulates the interaction of GTP-Rac with its effectors by controlling GTP-Rac membrane targeting. Here, we show that the translocation of GTP-Rac to membranes is independent of effector interactions, but instead requires the polybasic sequence near the carboxyl terminus. Cdc42 also requires integrin-mediated adhesion for translocation to membranes. A recently developed fluorescence resonance energy transfer (FRET)-based assay yields the surprising result that, despite its uniform distribution, the interaction of activated V12-Rac with a soluble, cytoplasmic effector domain is enhanced at specific regions near cell edges and is induced locally by integrin stimulation. This enhancement requires Rac membrane targeting. We show that Rho-GDI, which associates with cytoplasmic GTP-Rac, blocks effector binding. Release of Rho-GDI after membrane translocation allows Rac to bind to effectors. Thus, Rho-GDI confers spatially restricted regulation of Rac–effector interactions.

Binding of paxillin to α4 integrins modifies integrin-dependent biological responses

The α4 integrins are indispensable for embryogenesis, haematopoiesis and immune responses, possibly because α4 regulates cellular functions differently from other integrins through its cytoplasmic tail. We used novel mimics of the α4 tail to identify molecules that could account for α4-specific signalling. Here we report that the α4 tail, but not several other α-subunit tails, binds tightly to the signalling adaptor paxillin. Paxillin physically associated with α4 integrins in Jurkat T cells at high stoichiometry, and joining the α4 tail to αIIb resulted in a complex of integrin αIIbβ3 with paxillin. This association markedly enhanced the rates of αIIbβ3-dependent phosphorylation of focal adhesion kinase and cell migration. It also reduced cell spreading, focal adhesion and stress fibre formation. A point mutation within the α4 tail that disrupts paxillin binding reversed all of these effects. Furthermore, α4β1-dependent adhesion to VCAM-1 led to spreading of mouse embryonic fibroblasts derived from paxillin-null but not from wild-type mice. Thus, the tight association of paxillin with the α4 tail leads to distinct biochemical and biological responses to integrin-mediated cell adhesion.

Activation of Rac1 by shear stress in endothelial cells mediates both cytoskeletal reorganization and effects on gene expression

Hemodynamic shear stress is a fundamental determinant of vascular remodeling and atherogenesis. Changes in focal adhesions, cytoskeletal organization and gene expression are major responses of endothelial cells to shear stress. Here, we show that activation of the small GTPase Rac is essential for gene expression and for providing spatial information for shear stress‐induced cell alignment. Fluorescence resonance energy transfer (FRET) localizes activated Rac1 in the direction of flow. This directional Rac1 activation is downstream of shear‐induced new integrin binding to extracellular matrix. Additionally, Rac1 mediates flow‐induced stimulation of nuclear factor κB (NF‐κB) and the subsequent expression of intercellular cell adhesion molecule 1 (ICAM‐1), an adhesion receptor involved in the recruitment of leukocytes to atherosclerotic plaque. These studies provide a unifying model linking three of the main responses to shear stress that mediate both normal adaptation to hemodynamic forces and inflammatory dysfunction of endothelial cells in atherosclerosis.

Rac recruits high-affinity integrin αvβ3 to lamellipodia in endothelial cell migration

Integrin αvβ3 has an important role in the proliferation, survival, invasion and migration of vascular endothelial cells. Like other integrins, αvβ3 can exist in different functional states with respect to ligand binding. These changes involve both affinity modulation, by which conformational changes in the integrin heterodimer govern affinity for individual extracellular matrix proteins, and avidity modulation, by which changes in lateral mobility and integrin clustering affect the binding of cells to multivalent matrices. Here we have used an engineered monoclonal antibody Fab (antigen-binding fragment) named WOW-1, which binds to activated integrins αvβ3 and αvβ5 from several species, to investigate the role of αvβ3 activation in endothelial cell behaviour. Because WOW-1 is monovalent, it is insensitive to changes in integrin clustering and therefore reports only changes in affinity. WOW-1 contains an RGD tract in its variable region and binds only to unoccupied, high-affinity integrins. By using WOW-1, we have identified the selective recruitment of high-affinity integrins as a mechanism by which lamellipodia promote formation of new adhesions at the leading edge in cell migration.

Differential αv integrin–mediated Ras-ERK signaling during two pathways of angiogenesis

Antagonists of αvβ3 and αvβ5 disrupt angiogenesis in response to bFGF and VEGF, respectively. Here, we show that these αv integrins differentially contribute to sustained Ras-extracellular signal–related kinase (Ras-ERK) signaling in blood vessels, a requirement for endothelial cell survival and angiogenesis. Inhibition of FAK or αvβ5 disrupted VEGF-mediated Ras and c-Raf activity on the chick chorioallantoic membrane, whereas blockade of FAK or integrin αvβ3 had no effect on bFGF-mediated Ras activity, but did suppress c-Raf activation. Furthermore, retroviral delivery of active Ras or c-Raf promoted ERK activity and angiogenesis, which anti-αvβ5 blocked upstream of Ras, whereas anti-αvβ3 blocked downstream of Ras, but upstream of c-Raf. The activation of c-Raf by bFGF/αvβ3 not only depended on FAK, but also required p21-activated kinase-dependent phosphorylation of serine 338 on c-Raf, whereas VEGF-mediated c-Raf phosphorylation/activation depended on Src, but not Pak. Thus, integrins αvβ3 and αvβ5 differentially regulate the Ras-ERK pathway, accounting for distinct vascular responses during two pathways of angiogenesis.

TLR2 is constitutively expressed within the kidney and participates in ischemic renal injury through both MyD88-dependent and -independent pathways.

TLRs are an evolutionarily conserved family of cell membrane proteins believed to play a significant role in innate immunity and the response to tissue injury, including that induced by ischemia. TLR signaling pathways activate transcription factors that regulate expression of prosurvival proteins, as well as proinflammatory cytokines and chemokines through one of two proximal adapter proteins, MyD88 or Toll/IL-1R domain-containing adaptor-inducing IFN-β (Trif). Our study defines the constitutive protein expression of TLR2 in kidneys of humans and mice, and provides insight into the signaling mechanisms by which a deficiency of TLR2 protects from ischemic organ injury. Our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in TLR2, MyD88, Trif, and MyD88 × Trif. TLR2 protein was evident in many cell types in the kidney, including renal tubules of the outer stripe of the medulla, glomeruli, and in the renal vasculature. The pattern of protein expression was similar in humans and mice. The absence of TLR2, MyD88, and MyD88 × Trif conferred both physiologic and histologic protection against sublethal ischemia at 24 h. Interestingly, TLR2-deficient mice were better protected from ischemic renal injury than those deficient for the adapter protein MyD88, raising the intriguing possibility that TLR-2-dependent/MyD88-independent pathways also contribute to kidney injury. We conclude that TLR2 protein is constitutively expressed in the kidney and plays an important role in the pathogenesis of acute ischemic injury by signaling both MyD88-dependent and MyD88-independent pathways.

Increased filamin binding to β-integrin cytoplasmic domains inhibits cell migration

Multicellular animal development depends on integrins. These adhesion receptors link to the actin cytoskeleton, transmitting biochemical signals and force during cell migration and interactions with the extracellular matrix. Many integrin–cytoskeleton connections are formed by filamins and talin. The β7 integrin tail binds strongly to filamin and supports less migration, fibronectin matrix assembly and focal adhesion formation than either the β1D tail, which binds strongly to talin, or the β1A tail, which binds modestly to both filamin and talin. To probe the role of filamin binding, we mapped the filamin-binding site of integrin tails and identified amino acid substitutions that led to selective loss of filamin binding to the β7 tail and gain of filamin binding to the β1A tail. These changes affected cell migration and membrane protrusions but not fibronectin matrix assembly or focal adhesion formation. Thus, tight filamin binding restricts integrin-dependent cell migration by inhibiting transient membrane protrusion and cell polarization.

Effects of cell tension on the small GTPase Rac

Cells in the body are subjected to mechanical stresses such as tension, compression, and shear stress. These mechanical stresses play important roles in both physiological and pathological processes; however, mechanisms transducing mechanical stresses into biochemical signals remain elusive. Here, we demonstrated that equibiaxial stretch inhibited lamellipodia formation through deactivation of Rac. Nearly maximal effects on Rac activity were obtained with 10% strain. GAP-resistant, constitutively active V12Rac reversed this inhibition, supporting a critical role for Rac inhibition in the response to stretch. In contrast, activation of endogenous Rac with a constitutively active nucleotide exchange factor did not, suggesting that regulation of GAP activity most likely mediates the inhibition. Uniaxial stretch suppressed lamellipodia along the sides lengthened by stretch and increased it at the adjacent ends. A fluorescence assay for localized Rac showed comparable changes in activity along the sides versus the ends after uniaxial stretch. Blocking polarization of Rac activity by expressing V12Rac prevented subsequent alignment of actin stress fibers. Treatment with Y-27632 or ML-7 that inhibits myosin phosphorylation and contractility increased lamellipodia through Rac activation and decreased cell polarization. We hypothesize that regulation of Rac activity by tension may be important for motility, polarization, and directionality of cell movement.