Microglia control vascular architecture via a TGFß1 dependent paracrine mechanism linked to tissue mechanics.

Tissue microarchitecture and mechanics are important in development and pathologies of the Central Nervous System (CNS); however, their coordinating mechanisms are unclear. Here, we report that during colonization of the retina, microglia contacts the deep layer of high stiffness, which coincides with microglial bipolarization, reduction in TGFß1 signaling and termination of vascular growth. Likewise, stiff substrates induce microglial bipolarization and diminish TGFß1 expression in hydrogels. Both microglial bipolarization in vivo and the responses to stiff substrates in vitro require intracellular adaptor Kindlin3 but not microglial integrins. Lack of Kindlin3 causes high microglial contractility, dysregulation of ERK signaling, excessive TGFß1 expression and abnormally-patterned vasculature with severe malformations in the area of photoreceptors. Both excessive TGFß1 signaling and vascular defects caused by Kindlin3-deficient microglia are rescued by either microglial depletion or microglial knockout of TGFß1 in vivo. This mechanism underlies an interplay between microglia, vascular patterning and tissue mechanics within the CNS.

Integrin-Kindlin3 requirements for microglial motility in vivo are distinct from those for macrophages.

Microglia play a critical role in the development and homeostasis of the CNS. While mobilization of microglia is critical for a number of pathologies, understanding of the mechanisms of their migration in vivo is limited and often based on similarities to macrophages. Kindlin3 deficiency as well as Kindlin3 mutations of integrin-binding sites abolish both integrin inside-out and outside-in signaling in microglia, thereby resulting in severe deficiencies in cell adhesion, polarization, and migration in vitro, which are similar to the defects observed in macrophages. In contrast, while Kindlin3 mutations impaired macrophage mobilization in vivo, they had no effect either on the population of microglia in the CNS during development or on mobilization of microglia and subsequent microgliosis in a model of multiple sclerosis. At the same time, acute microglial response to laser-induced injury was impaired by the lack of Kindlin3-integrin interactions. Based on 2-photon imaging of microglia in the brain, Kindlin3 is required for elongation of microglial processes toward the injury site and formation of phagosomes in response to brain injury. Thus, while Kindlin3 deficiency in human subjects is not expected to diminish the presence of microglia within CNS, it might delay the recovery process after injury, thereby exacerbating its complications.

Emergence and subsequent functional specialization of kindlins during evolution of cell adhesiveness.

Kindlins are integrin-interacting proteins essential for integrin-mediated cell adhesiveness. In this study, we focused on the evolutionary origin and functional specialization of kindlins as a part of the evolutionary adaptation of cell adhesive machinery. Database searches revealed that many members of the integrin machinery (including talin and integrins) existed before kindlin emergence in evolution. Among the analyzed species, all metazoan lineages­but none of the premetazoans­had at least one kindlin-encoding gene, whereas talin was present in several premetazoan lineages. Kindlin appears to originate from a duplication of the sequence encoding the N-terminal fragment of talin (the talin head domain) with a subsequent insertion of the PH domain of separate origin. Sequence analysis identified a member of the actin filament-associated protein 1 (AFAP1) superfamily as the most likely origin of the kindlin PH domain. The functional divergence between kindlin paralogues was assessed using the sequence swap (chimera) approach. Comparison of kindlin 2 (K2)/kindlin 3 (K3) chimeras revealed that the F2 subdomain, in particular its C-terminal part, is crucial for the differential functional properties of K2 and K3. The presence of this segment enables K2 but not K3 to localize to focal adhesions. Sequence analysis of the C-terminal part of the F2 subdomain of K3 suggests that insertion of a variable glycine-rich sequence in vertebrates contributed to the loss of constitutive K3 targeting to focal adhesions. Thus emergence and subsequent functional specialization of kindlins allowed multicellular organisms to develop additional tissue-specific adaptations of cell adhesiveness.

Integrin function in vascular biology: a view from 2013.

PURPOSE OF REVIEW: This review considers recent developments concerning the role of integrins in vascular biology with a specific emphasis on integrin activation, and the crosstalk between integrins and growth factor receptors. RECENT FINDINGS: Recent studies have shown leukocytes can mediate direct transfer of molecules into endothelial cells, how specific integrins can be used to transduce signaling events, in particular in vascular beds, and how endothelial cell integrins can be targeted with specific ligands for the delivery of therapeutics. Kindlin and talin are both essential for integrin activation based on in-vivo studies of mice and humans in which the genes encoding for these proteins have been inactivated. Recent studies have attempted to translate these in-vivo realities into in-vitro models with mixed results. SUMMARY: Mechanisms and consequences of integrin-ligand interactions on blood and vascular cells remain a major topic of hematological research. Crucial to the ligand binding function of integrins are two intracellular binding partners, talin and kindlin. In seeking to define the molecular basis for 'integrin activation', a mechanism must be envisioned in which both proteins talin and kindlin are required to produce a productive functional response, be it platelet aggregation or leukocyte extravasation. On endothelial cells, integrins and vascular endothelial growth factor receptor 2 influence the activation of one another by virtue of their direct physical interaction. It has been shown that this bidirectional communication is subject to regulation during angiogenesis.

Regulation of cell adhesion and migration by Kindlin-3 cleavage by calpain.

Integrin activation on hematopoietic cells is essential for platelet aggregation, leukocyte adhesion, and transmigration through endothelium and extracellular matrix into inflamed tissues. To migrate through matrix, leukocyte integrin adhesion complexes undergo dynamic changes. Here we show that Kindlin-3, a main activator and binding partner of integrins in hematopoietic cells, can be cleaved by calpain in an activation-dependent manner. This calpain-mediated cleavage occurs in platelets and leukocytes as well as in endothelial cells. We determined the calpain I cleavage site in Kindlin-3 at tyrosine 373 in the N-terminal part of Kindlin-3 pleckstrin homology domain. Expression of the calpain-resistant Y373N mutant of Kindlin-3 promotes stronger cell adhesion to extracellular matrix under flow as well as to activated endothelium. In contrast, Y373N mutation in Kindlin-3 hinders cell migration. Mechanistically, calpain-resistant Y373N mutant of Kindlin-3 exhibited an activation-independent association with ß integrin cytoplasm domain. Thus, cleavage of Kindlin-3 by calpain controls the dynamics of integrin-Kindlin-3 interaction and as a result, integrin-dependent adhesion and migration of hematopoietic cells. This represents a novel mechanism regulating reversibility of integrin adhesion complexes in leukocytes, which, in turn, is critical for their successful transmigration through the extracellular matrix.

Integrin ß3 crosstalk with VEGFR accommodating tyrosine phosphorylation as a regulatory switch.

Integrins mediate cell adhesion, migration, and survival by connecting intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the importance of the interaction between ß(3) integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. Here we present in vitro evidence of the direct association between the cytoplasmic tails (CTs) of ß(3) and VEGFR2. Specifically, the membrane-proximal motif around (801)YLSI in VEGFR2 mediates its binding to non-phosphorylated ß(3)CT, accommodating an α-helical turn in integrin bound conformation. We also show that Y(747) phosphorylation of ß(3) enhances the above interaction. To demonstrate the importance of ß(3) phosphorylation in endothelial cell functions, we synthesized ß(3)CT-mimicking Y(747) phosphorylated and unphosphorylated membrane permeable peptides. We show that a peptide containing phospho-Y(747) but not F(747) significantly inhibits VEGF-induced signaling and angiogenesis. Moreover, phospho-Y(747) peptide exhibits inhibitory effect only in WT but not in ß(3) integrin knock-out or ß(3) integrin knock-in cells expressing ß(3) with two tyrosines substituted for phenylalanines, demonstrating its specificity. Importantly, these peptides have no effect on fibroblast growth factor receptor signaling. Collectively these data provide novel mechanistic insights into phosphorylation dependent cross-talk between integrin and VEGFR2.

Hemodynamic activation of beta-catenin and T-cell-specific transcription factor signaling in vascular endothelium regulates fibronectin expression.

OBJECTIVE: The goal of this study was to assess the activity of ß-catenin/T-cell-specific transcription factor (TCF) signaling in atherosclerosis development and its regulation of fibronectin in vascular endothelium. METHODS AND RESULTS: Histological staining identified preferential nuclear localization of ß-catenin in the endothelium of atheroprone aorta before and during lesion development. Transgenic reporter studies revealed that increased levels of TCF transcriptional activity in endothelium correlated anatomically with ß-catenin nuclear localization and fibronectin deposition. Exposure of endothelial cells to human-derived atheroprone shear stress induced nuclear localization of ß-catenin, transcriptional activation of TCF, and expression of fibronectin. Activation of fibronectin expression required ß-catenin, TCF, and the transcriptional coactivator CRBP-binding protein. Finally, we identified platelet endothelial cell adhesion molecule-1 as a critical regulator of constitutive ß-catenin and glycogen synthase kinase-3ß activities. CONCLUSIONS: These data reveal novel constitutive activation of the endothelial ß-catenin/TCF signaling pathway in atherosclerosis and regulation of fibronectin through hemodynamic shear stress.

Suppression of RhoG activity is mediated by a syndecan 4-synectin-RhoGDI1 complex and is reversed by PKCalpha in a Rac1 activation pathway.

Fibroblast growth factor 2 (FGF2) is a major regulator of developmental, pathological, and therapeutic angiogenesis. Its activity is partially mediated by binding to syndecan 4 (S4), a proteoglycan receptor. Angiogenesis requires polarized activation of the small guanosine triphosphatase Rac1, which involves localized dissociation from RhoGDI1 and association with the plasma membrane. Previous work has shown that genetic deletion of S4 or its adapter, synectin, leads to depolarized Rac activation, decreased endothelial migration, and other physiological defects. In this study, we show that Rac1 activation downstream of S4 is mediated by the RhoG activation pathway. RhoG is maintained in an inactive state by RhoGDI1, which is found in a ternary complex with synectin and S4. Binding of S4 to synectin increases the latter's binding to RhoGDI1, which in turn enhances RhoGDI1's affinity for RhoG. S4 clustering activates PKCalpha, which phosphorylates RhoGDI1 at Ser(96). This phosphorylation triggers release of RhoG, leading to polarized activation of Rac1. Thus, FGF2-induced Rac1 activation depends on the suppression of RhoG by a previously uncharacterized ternary S4-synectin-RhoGDI1 protein complex and activation via PKCalpha.

Endogenous RhoG is dispensable for integrin-mediated cell spreading but contributes to Rac-independent migration.

Rac activation by integrins is essential for cell spreading, migration, growth and survival. Based mainly on overexpression of dominant-negative mutants, RhoG has been proposed to mediate integrin-dependent Rac activation upstream of ELMO and Dock180. RhoG-knockout mice, however, display no significant developmental or functional abnormalities. To clarify the role of RhoG in integrin-mediated signaling, we developed a RhoG-specific antibody, which, together with shRNA-mediated knockdown, allowed analysis of the endogenous protein. Despite dramatic effects of dominant-negative constructs, nearly complete RhoG depletion did not substantially inhibit cell adhesion, spreading, migration or Rac activation. Additionally, RhoG was not detectably activated by adhesion to fibronectin. Using Rac1(-/-) cells, we found that constitutively active RhoG induced membrane ruffling via both Rac-dependent and -independent pathways. Additionally, endogenous RhoG was important for Rac-independent cell migration. However, RhoG did not significantly contribute to cell spreading even in these cells. These data therefore clarify the role of RhoG in integrin signaling and cell motility.

RhoG regulates endothelial apical cup assembly downstream from ICAM1 engagement and is involved in leukocyte trans-endothelial migration.

During trans-endothelial migration (TEM), leukocytes use adhesion receptors such as intercellular adhesion molecule-1 (ICAM1) to adhere to the endothelium. In response to this interaction, the endothelium throws up dynamic membrane protrusions, forming a cup that partially surrounds the adherent leukocyte. Little is known about the signaling pathways that regulate cup formation. In this study, we show that RhoG is activated downstream from ICAM1 engagement. This activation requires the intracellular domain of ICAM1. ICAM1 colocalizes with RhoG and binds to the RhoG-specific SH3-containing guanine-nucleotide exchange factor (SGEF). The SH3 domain of SGEF mediates this interaction. Depletion of endothelial RhoG by small interfering RNA does not affect leukocyte adhesion but decreases cup formation and inhibits leukocyte TEM. Silencing SGEF also results in a substantial reduction in RhoG activity, cup formation, and TEM. Together, these results identify a new signaling pathway involving RhoG and its exchange factor SGEF downstream from ICAM1 that is critical for leukocyte TEM.