A cardiac amino-terminal GRK2 peptide inhibits insulin resistance yet enhances maladaptive cardiovascular and brown adipose tissue remodeling in females during diet-induced obesity.

Obesity and metabolic disorders are increasing in epidemic proportions, leading to poor outcomes including heart failure. With a growing recognition of the effect of adipose tissue dysfunction on heart disease, it is less well understood how the heart can influence systemic metabolic homeostasis. Even less well understood is sex differences in cardiometabolic responses. Previously, our lab investigated the role of the amino-terminus of GRK2 in cardiometabolic remodeling using transgenic mice with cardiac restricted expression of a short peptide, ßARKnt. Male mice preserved insulin sensitivity, enhanced metabolic flexibility and adipose tissue health, elicited cardioprotection, and improved cardiac metabolic signaling. To examine the effect of cardiac ßARKnt expression on cardiac and metabolic function in females in response to diet-induced obesity, we subjected female mice to high fat diet (HFD) to trigger cardiac and metabolic adaptive changes. Despite equivalent weight gain, ßARKnt mice exhibited improved glucose tolerance and insulin sensitivity. However, ßARKnt mice displayed a progressive reduction in energy expenditure during cold challenge after acute and chronic HFD stress. They also demonstrated reduced cardiac function and increased markers of maladaptive remodeling and tissue injury, and decreased or aberrant metabolic signaling. ßARKnt mice exhibited reduced lipid deposition in the brown adipose tissue (BAT), but delayed or decreased markers of BAT activation and function suggested multiple mechanisms contributed to the decreased thermogenic capacity. These data suggest a non-canonical cardiac regulation of BAT lipolysis and function that highlights the need for studies elucidating the mechanisms of sex-specific responses to metabolic dysfunction.

A peptide of the amino-terminus of GRK2 induces hypertrophy and yet elicits cardioprotection after pressure overload.

G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression and activity are elevated early on in response to several forms of cardiovascular stress and are a hallmark of heart failure. Interestingly, though, in addition to its well-characterized role in regulating GPCRs, mounting evidence suggests a GRK2 "interactome" that underlies a great diversity in its functional roles. Several such GRK2 interacting partners are important for adaptive and maladaptive myocyte growth; therefore, an understanding of domain-specific interactions with signaling and regulatory molecules could lead to novel targets for heart failure therapy. Herein, we subjected transgenic mice with cardiac restricted expression of a short, amino terminal fragment of GRK2 (ßARKnt) to pressure overload and found that unlike their littermate controls or previous GRK2 fragments, they exhibited an increased left ventricular wall thickness and mass prior to cardiac stress that underwent proportional hypertrophic growth to controls after acute pressure overload. Importantly, despite this enlarged heart, ßARKnt mice did not undergo the expected transition to heart failure observed in controls. Further, ßARKnt expression limited adverse left ventricular remodeling and increased cell survival signaling. Proteomic analysis to identify ßARKnt binding partners that may underlie the improved cardiovascular phenotype uncovered a selective functional interaction of both endogenous GRK2 and ßARKnt with AKT substrate of 160 kDa (AS160). AS160 has emerged as a key downstream regulator of insulin signaling, integrating physiological and metabolic cues to couple energy demand to membrane recruitment of Glut4. Our preliminary data indicate that in ßARKnt mice, cardiomyocyte insulin signaling is improved during stress, with a coordinate increase in spare respiratory activity and ATP production without metabolite switching. Surprisingly, these studies also revealed a significant decrease in gonadal fat weight, equivalent to human abdominal fat, in male ßARKnt mice at baseline and following cardiac stress. These data suggest that the enhanced AS160-mediated signaling in the ßARKnt mice may ameliorate pathological cardiac remodeling through direct modulation of insulin signaling within cardiomyocytes, and translate these to beneficial effects on systemic metabolism.

The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation.

Kindlin-2 (K2), a 4.1R-ezrin-radixin-moesin (FERM) domain adaptor protein, mediates numerous cellular responses, including integrin activation. The C-terminal 15-amino acid sequence of K2 is remarkably conserved across species but is absent in canonical FERM proteins, including talin. In CHO cells expressing integrin αIIbß3, co-expression of K2 with talin head domain resulted in robust integrin activation, but this co-activation was lost after deletion of as few as seven amino acids from the K2 C terminus. This dependence on the C terminus was also observed in activation of endogenous αIIbß3 in human erythroleukemia (HEL) cells and ß1 integrin activation in macrophage-like RAW264.1 cells. Kindlin-1 (K1) exhibited a similar dependence on its C terminus for integrin activation. Expression of the K2 C terminus as an extension of membrane-anchored P-selectin glycoprotein ligand-1 (PSGL-1) inhibited integrin-dependent cell spreading. Deletion of the K2 C terminus did not affect its binding to the integrin ß3 cytoplasmic tail, but combined biochemical and NMR analyses indicated that it can insert into the F2 subdomain. We suggest that this insertion determines the topology of the K2 FERM domain, and its deletion may affect the positioning of the membrane-binding functions of the F2 subdomain and the integrin-binding properties of its F3 subdomain. Free C-terminal peptide can still bind to K2 and displace the endogenous K2 C terminus but may not restore the conformation needed for integrin co-activation. Our findings indicate that the extreme C terminus of K2 is essential for integrin co-activation and highlight the importance of an atypical architecture of the K2 FERM domain in regulating integrin activation.

Kindlin-2 interacts with endothelial adherens junctions to support vascular barrier integrity.

KEY POINTS: A reduction in Kindlin-2 levels in endothelial cells compromises vascular barrier function. Kindlin-2 is a previously unrecognized component of endothelial adherens junctions. By interacting directly and simultaneously with ß- or γ-catenin and cortical actin filaments, Kindlin-2 stabilizes adherens junctions. The Kindlin-2 binding sites for ß- and γ-catenin reside within its F1 and F3 subdomains. Although Kindlin-2 does not associate directly with tight junctions, its downregulation also destabilizes these junctions. Thus, impairment of both adherens and tight junctions may contribute to enhanced leakiness of vasculature in Kindlin-2+/- mice. ABSTRACT: Endothelial cells (EC) establish a physical barrier between the blood and surrounding tissue. Impairment of this barrier can occur during inflammation, ischaemia or sepsis and cause severe organ dysfunction. Kindlin-2, which is primarily recognized as a focal adhesion protein in EC, was not anticipated to have a role in vascular barrier. We tested the role of Kindlin-2 in regulating vascular integrity using several different approaches to decrease Kindlin-2 levels in EC. Reduced levels of Kindlin-2 in Kindlin-2+/- mice aortic endothelial cells (MAECs) from these mice, and human umbilical ECs (HUVEC) treated with Kindlin-2 siRNA showed enhanced basal and platelet-activating factor (PAF) or lipopolysaccharide-stimulated vascular leakage compared to wild-type (WT) counterparts. PAF preferentially disrupted the Kindlin-2+/- MAECs barrier to BSA and dextran and reduced transendothelial resistance compared to WT cells. Kindlin-2 co-localized and co-immunoprecipitated with vascular endothelial cadherin-based complexes, including ß- and γ-catenin and actin, components of adherens junctions (AJ). Direct interaction of Kindlin-2 with ß- and γ-catenin and actin was demonstrated in co-immunoprecipitation and surface plasmon resonance experiments. In thrombin-stimulated HUVECs, Kindlin-2 and cortical actin dissociated from stable AJs and redistributed to radial actin stress fibres of remodelling focal AJs. The ß- and γ-catenin binding site resides within the F1 and F3 subdomains of Kindlin-2 but not the integrin binding site in F3. These results establish a previously unrecognized and vital role of Kindlin-2 with respect to maintaining the vascular barrier by linking Vascuar endothelial cadherin-based complexes to cortical actin and thereby stabilizing AJ.

Dual role of the leukocyte integrin αMß2 in angiogenesis.

Polymorphonuclear neutrophils (PMNs) and macrophages are crucial contributors to neovascularization, serving as a source of chemokines, growth factors, and proteases. α(M)ß(2)(CD11b/CD18) and α(L)ß(2)(CD11a/CD18) are expressed prominently and have been implicated in various responses of these cell types. Thus, we investigated the role of these ß2 integrins in angiogenesis. Angiogenesis was analyzed in wild-type (WT), α(M)-knockout (α(M)(-/-)), and α(L)-deficient (α(L)(-/-)) mice using B16F10 melanoma, RM1 prostate cancer, and Matrigel implants. In all models, vascular area was decreased by 50-70% in α(M)(-/-) mice, resulting in stunted tumor growth as compared with WT mice. In contrast, α(L) deficiency did not impair angiogenesis and tumor growth. The neovessels in α(M)(-/-) mice were leaky and immature because they lacked smooth muscle cell and pericytes. Defective angiogenesis in the α(M)(-/-) mice was associated with attenuated PMN and macrophage recruitment into tumors. In contrast to WT or the α(L)(-/-) leukocytes, the α(M)(-/-) myeloid cells showed impaired plasmin (Plm)-dependent extracellular matrix invasion, resulting from 50-75% decrease in plasminogen (Plg) binding and pericellular Plm activity. Surface plasmon resonance verified direct interaction of the α(M)I-domain, the major ligand binding site in the ß(2) integrins, with Plg. However, the α(L)I-domain failed to bind Plg. In addition, endothelial cells failed to form tubes in the presence of conditioned medium collected from TNF-α-stimulated PMNs derived from the α(M)(-/-) mice because of severely impaired degranulation and secretion of VEGF. Thus, α(M)ß(2) plays a dual role in angiogenesis, supporting not only Plm-dependent recruitment of myeloid cells to angiogenic niches, but also secretion of VEGF by these cells.

Molecular basis of kindlin-2 binding to integrin-linked kinase pseudokinase for regulating cell adhesion.

Integrin-linked kinase (ILK) is a distinct intracellular adaptor essential for integrin-mediated cell-extracellular matrix adhesion, cell spreading, and migration. Acting as a major docking platform in focal adhesions, ILK engages many proteins to dynamically link integrins with the cytoskeleton, but the underlying mechanism remains elusive. Here, we have characterized the interaction of ILK with kindlin-2, a key regulator for integrin bidirectional signaling. We show that human kindlin-2 binds to human ILK with high affinity. Using systematic mapping approaches, we have identified a major ILK binding site involving a 20-residue fragment (residues 339-358) in kindlin-2. NMR-based analysis reveals a helical conformation of this fragment that utilizes its leucine-rich surface to recognize the ILK pseudokinase domain in a mode that is distinct from another ILK pseudokinase domain binding protein, α-parvin. Structure-based mutational experiments further demonstrate that the kindlin-2 binding to ILK is crucial for the kindlin-2 localization to focal adhesions and cell spreading (integrin outside-in signaling) but dispensable for the kindlin-2-mediated integrin activation (integrin inside-out signaling). These data define a specific mode of the kindlin-2/ILK interaction with mechanistic implications as to how it spatiotemporally mediates integrin signaling and cell adhesion.

Kindlin-2 regulates hemostasis by controlling endothelial cell-surface expression of ADP/AMP catabolic enzymes via a clathrin-dependent mechanism.

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice. In the present study, we tested whether hemostasis might be perturbed in kindlin-2(+/-) mice. Bleeding time and carotid artery occlusion time were significantly prolonged in kindlin-2(+/-) mice. Whereas plasma concentrations/activities of key coagulation/fibrinolytic proteins and platelet counts and aggregation were similar in wild-type and kindlin-2(+/-) mice, kindlin-2(+/-) endothelial cells (ECs) showed enhanced inhibition of platelet aggregation induced by adenosine 5'-diphosphate (ADP) or low concentrations of other agonists. Cell-surface expression of 2 enzymes involved in ADP/adenosine 5'-monophosphate (AMP) degradation, adenosine triphosphate (ATP) diphosphohydrolase (CD39) and ecto-5'-nucleotidase (CD73) were increased twofold to threefold on kindlin-2(+/-) ECs, leading to enhanced ATP/ADP catabolism and production of adenosine, an inhibitor of platelet aggregation. Trafficking of CD39 and CD73 at the EC surface was altered in kindlin-2(+/-) mice. Mechanistically, this was attributed to direct interaction of kindlin-2 with clathrin heavy chain, thereby controlling endocytosis and recycling of CD39 and CD73. The interaction of kindlin-2 with clathrin was independent of its integrin binding site but still dependent on a site within its F3 subdomain. Thus, kindlin-2 regulates trafficking of EC surface enzymes that control platelet responses and hemostasis.

Integrin αIIbß3: from discovery to efficacious therapeutic target.

From the initial description of platelets in 1882, their propensity to aggregate and to contribute to thrombosis was apparent. Indeed, excessive platelet aggregation is associated with myocardial infarction and other thrombotic diseases whereas Glanzmann thrombasthenia, in which platelet aggregation is reduced, is a bleeding syndrome. Over the last half of the 20th century, many investigators have provided insights into the cellular and molecular basis for platelet aggregation. The major membrane protein on platelets, integrin αIIbß3, mediates this response by rapidly transiting from its resting to an activated state in which it serves as a receptor for ligands that can bridge platelets together. Monoclonal antibodies, natural products, and small peptides were all shown to inhibit αIIbß3 dependent platelet aggregation, and these inhibitors became the forerunners of antagonists that proceeded through preclinical testing and into large patient trials to treat acute coronary syndromes, particularly in the context of percutaneous coronary interventions. Three such αIIbß3 antagonists, abciximab, eptifibatide, and tirofiban, received Food and Drug Administration approval. Over the past 15 years, millions of patients have been treated with these αIIbß3 antagonists and many lives have been saved by their administration. With the side effect of increased bleeding and the development of new antithrombotic drugs, the use of αIIbß3 antagonists is waning. Nevertheless, they are still widely used for the prevention of periprocedural thrombosis during percutaneous coronary interventions. This review focuses on the biology of αIIbß3, the development of its antagonists, and some of the triumphs and shortcomings of αIIbß3 antagonism.

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.

Spatial coordination of kindlin-2 with talin head domain in interaction with integrin ß cytoplasmic tails.

Both talin head domain and kindlin-2 interact with integrin ß cytoplasmic tails, and they function in concert to induce integrin activation. Binding of talin head domain to ß cytoplasmic tails has been characterized extensively, but information on the interaction of kindin-2 with this integrin segment is limited. In this study, we systematically examine the interactions of kindlin-2 with integrin ß tails. Kindlin-2 interacted well with ß(1) and ß(3) tails but poorly with the ß(2) cytoplasmic tail. This binding selectivity was determined by the non-conserved residues, primarily the three amino acids at the extreme C terminus of the ß(3) tail, and the sequence in ß(2) was non-permissive. The region at the C termini of integrin ß(1) and ß(3) tails recognized by kindlin-2 was a binding core of 12 amino acids. Kindlin-2 and talin head do not interact with one another but can bind simultaneously to the integrin ß(3) tail without enhancing or inhibiting the interaction of the other binding partner. Kindlin-2 itself failed to directly unclasp integrin α/ß tail complex, indicating that kindlin-2 must cooperate with talin to support the integrin activation mechanism.

Binding of CD40L to Mac-1's I-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis--but does not affect immunity and thrombosis in mice.

RATIONALE: CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and hemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor. OBJECTIVE: Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo. METHODS AND RESULTS: CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high-affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the α1 helix and the ß-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. A cyclisized version optimized for in vivo use, cM7, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr(-/-) mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo. CONCLUSIONS: We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L.

Kindlin-3 deficiency leads to impaired erythropoiesis and erythrocyte cytoskeleton.

Kindlin-3 (K3) is critical for the activation of integrin adhesion receptors in hematopoietic cells. In humans and mice, K3 deficiency is associated with impaired immunity and bone development, bleeding, and aberrant erythrocyte shape. To delineate how K3 deficiency (K3KO) contributes to anemia and misshaped erythrocytes, mice deficient in erythroid (K3KO∖EpoR-cre) or myeloid cell K3 (K3KO∖Lyz2cre), knockin mice expressing mutant K3 (Q597W598 to AA) with reduced integrin-activation function (K3KI), and control wild-type (WT) K3 mice were studied. Both K3-deficient strains and K3KI mice showed anemia at baseline, reduced response to erythropoietin stimulation, and compromised recovery after phenylhydrazine (PHZ)-induced hemolytic anemia as compared with K3WT. Erythroid K3KO and K3 (Q597W598 to AA) showed arrested erythroid differentiation at proerythroblast stage, whereas macrophage K3KO showed decreased erythroblast numbers at all developmental stages of terminal erythroid differentiation because of reduced erythroblastic island (EBI) formation attributable to decreased expression and activation of erythroblast integrin α4ß1 and macrophage αVß3. Peripheral blood smears of K3KO∖EpoR-cre mice, but not of the other mouse strains, showed numerous aberrant tear drop-shaped erythrocytes. K3 deficiency in these erythrocytes led to disorganized actin cytoskeleton, reduced deformability, and increased osmotic fragility. Mechanistically, K3 directly interacted with F-actin through an actin-binding site K3-LK48. Taken together, these findings document that erythroid and macrophage K3 are critical contributors to erythropoiesis in an integrin-dependent manner, whereas F-actin binding to K3 maintains the membrane cytoskeletal integrity and erythrocyte biconcave shape. The dual function of K3 in erythrocytes and in EBIs establish an important functional role for K3 in normal erythroid function.

A Cardiac Amino-Terminal GRK2 Peptide Inhibits Maladaptive Adipocyte Hypertrophy and Insulin Resistance During Diet-Induced Obesity.

Heart disease remains the leading cause of death, and mortality rates positively correlate with the presence of obesity and diabetes. Despite the correlation between cardiac and metabolic dysregulation, the mechanistic pathway(s) of interorgan crosstalk still remain undefined. This study reveals that cardiac-restricted expression of an amino-terminal peptide of GRK2 (ßARKnt) preserves systemic and cardiac insulin responsiveness, and protects against adipocyte maladaptive hypertrophy in a diet-induced obesity model. These data suggest a cardiac-driven mechanism to ameliorate maladaptive cardiac remodeling and improve systemic metabolic homeostasis that may lead to new treatment modalities for cardioprotection in obesity and obesity-related metabolic syndromes.

Tyrosine phosphorylation of integrin beta3 regulates kindlin-2 binding and integrin activation.

Kindlins are essential for integrin activation in cell systems and do so by working in a cooperative fashion with talin via their direct interaction with integrin ß cytoplasmic tails (CTs). Kindlins interact with the membrane-distal NxxY motif, which is distinct from the talin-binding site within the membrane-proximal NxxY motif. The Tyr residues in both motifs can be phosphorylated, and it has been suggested that this modification of the membrane-proximal NxxY motif negatively regulates interaction with the talin head domain. However, the influence of Tyr phosphorylation of the membrane-distal NxxY motif on kindlin binding is unknown. Using mutational analyses and phosphorylated peptides, we show that phosphorylation of the membrane-distal NITY(759) motif in the ß(3) CT disrupts kindlin-2 recognition. Phosphorylation of this membrane-distal Tyr also disables the ability of kindlin-2 to coactivate the integrin. In direct binding studies, peptides corresponding to the non-phosphorylated ß(3) CT interacted well with kindlin-2, whereas the Tyr(759)-phosphorylated peptide failed to bind kindlin-2 with measurable affinity. These observations indicate that transitions between the phosphorylated and non-phosphorylated states of the integrin ß(3) CT determine reactivity with kindlin-2 and govern the role of kindlin-2 in regulating integrin activation.

The integrin co-activator Kindlin-3 is expressed and functional in a non-hematopoietic cell, the endothelial cell.

Integrin activation is crucial for numerous cellular responses, including cell adhesion, migration, and survival. Recent studies in mice have specifically emphasized the vital role of kindlin-3 in integrin activation. Kindlin-3 deficiency in humans also has now been documented and includes symptoms of bleeding, frequent infections, and osteopetrosis, which are consequences of an inability to activate beta1, beta2, and beta3 integrins. To date, kindlin-3 was thought to be restricted to hematopoietic cells. In this article, we demonstrate that kindlin-3 is present in human endothelial cells derived from various anatomical origins. The mRNA and protein for KINDLIN-3 was detected in endothelial cells by reverse transcription-PCR and Western blots. When subjected to sequencing by mass spectrometry, the protein was identified as authentic kindlin-3 and unequivocally distinguished from KINDLIN-1 and KINDLIN-2 or any other known protein. By quantitative real time PCR, the level of kindlin-3 in endothelial cells was 20-50% of that of kindlin-2. Using knockdown approaches, we show that kindlin-3 plays a role in integrin-mediated adhesion of endothelial cells. This function depends upon the integrin and substrate and is distinct from that of kindlin-2. Formation of tube-like structures in Matrigel also was impaired by kindlin-3 knockdown. Mechanistically, the distinct functions of the kindlins can be traced to differences in their subcellular localization in integrin-containing adhesion structures. Thus, the prevailing view that individual kindlins exert their functions in a cell type-specific manner must now be modified to consider distinct functions of the different family members within the same cell type.

The WAVE3-YB1 interaction regulates cancer stem cells activity in breast cancer.

Resistance to therapy is the main cause of tumor recurrence and metastasis and cancer stem cells (CSCs) play a crucial role in this process, especially in triple-negative breast cancers (TNBCs). Unfortunately, no FDA-approved treatment is currently available for this subtype of BC, which explains the high rate of mortality in patients with TNBC tumors. WAVE3, a member of the WASP/WAVE actin-cytoskeleton remodeling family of protein, has been established as a major driver of tumor progression and metastasis of several solid tumors, including those originating in the breast. Our recently published studies found WAVE3 to mediate the process of chemoresistance in TNBCs. The molecular mechanisms whereby WAVE3 regulates chemoresistance in TNBC tumors remains largely unknown, as does the role of WAVE3 in CSC maintenance. Here we show that WAVE3 promotes CSC self-renewal and regulates transcription of CSC-specific genes, which, in part, provides a mechanistic explanation for the function of WAVE3 in chemoresistance in TNBCs. Our data show that WAVE3 is enriched in the CSC-subpopulation of TNBC cell lines. Knockout of WAVE3 via CRISPR/Cas9 significantly attenuates the CSC-subpopulation and inhibits transcription of CSC transcription factors. Mechanistically, we established a link between WAVE3 and the Y-box-binding protein-1 (YB1), a transcription factor and CSC-maintenance gene. Indeed, the interaction of WAVE3 with YB1 is required for YB1 translocation to the nucleus of cancer cells, and activation of transcription of CSC-specific genes. Our findings identify a new WAVE3/YB1 signaling axis that regulates the CSC-mediated resistance to therapy and opens a new therapeutic window for TNBCs treatment.

Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIbα.

Inflammation and thrombosis occur together in many diseases. The leukocyte integrin Mac-1 (also known as integrin αMß2, or CD11b/CD18) is crucial for leukocyte recruitment to the endothelium, and Mac-1 engagement of platelet GPIbα is required for injury responses in diverse disease models. However, the role of Mac-1 in thrombosis is undefined. Here we report that mice with Mac-1 deficiency (Mac-1-/-) or mutation of the Mac-1-binding site for GPIbα have delayed thrombosis after carotid artery and cremaster microvascular injury without affecting parameters of haemostasis. Adoptive wild-type leukocyte transfer rescues the thrombosis defect in Mac-1-/- mice, and Mac-1-dependent regulation of the transcription factor Foxp1 contributes to thrombosis as evidenced by delayed thrombosis in mice with monocyte-/macrophage-specific overexpression of Foxp1. Antibody and small-molecule targeting of Mac-1:GPIbα inhibits thrombosis. Our data identify a new pathway of thrombosis involving leukocyte Mac-1 and platelet GPIbα, and suggest that targeting this interaction has anti-thrombotic therapeutic potential with reduced bleeding risk.

Corrigendum: Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIbα.

This corrects the article DOI: 10.1038/ncomms15559.

Kindlin-2 directly binds actin and regulates integrin outside-in signaling.

Reduced levels of kindlin-2 (K2) in endothelial cells derived from K2(+/-)mice or C2C12 myoblastoid cells treated with K2 siRNA showed disorganization of their actin cytoskeleton and decreased spreading. These marked changes led us to examine direct binding between K2 and actin. Purified K2 interacts with F-actin in cosedimentation and surface plasmon resonance analyses and induces actin aggregation. We further find that the F0 domain of K2 binds actin. A mutation, LK(47)/AA, within a predicted actin binding site (ABS) of F0 diminishes its interaction with actin by approximately fivefold. Wild-type K2 and K2 bearing the LK(47)/AA mutation were equivalent in their ability to coactivate integrin αIIbß3 in a CHO cell system when coexpressed with talin. However, K2-LK(47)/AA exhibited a diminished ability to support cell spreading and actin organization compared with wild-type K2. The presence of an ABS in F0 of K2 that influences outside-in signaling across integrins establishes a new foundation for considering how kindlins might regulate cellular responses.

The specificity and function of the metal-binding sites in the integrin beta3 A-domain.

The A-domains within integrin beta subunits contain three metal sites termed the metal ion-dependent adhesion site (MIDAS), site adjacent to the metal ion-dependent adhesion site (ADMIDAS), and ligand-induced metal-binding site (LIMBS), and these sites are involved in ligand engagement. The selectivity of these metal sites and their role in ligand binding have been investigated by expressing a fragment corresponding to the beta3 A-domain, beta3-(109-352), and single point mutants in which each of the cation-binding sites has been disabled. Equilibrium dialysis experiments identified three Mn2+- and two Ca2+-binding sites with the LIMBS being the site that did not bind Ca2+. Although the ADMIDAS could bind Ca2+, it did not bind Mg2+. These results indicate that the Ca2+-specific site that inhibits ligand binding is the ADMIDAS. Two different assay systems, surface plasmon resonance and a microtiter plate assay, demonstrated that the beta3 A-domain fragment bound fibrinogen in the presence of 0.1 mm Ca2+ but not in 3 mm Ca2+. This behavior recapitulated the effects of Ca2+ on fibrinogen binding to alphavbeta3 but not alphaIIbbeta3. Disabling any of the three cation-binding sites abrogated fibrinogen binding. These results indicate that the specificities of the three metal-binding sites for divalent cations are distinct and that each site can regulate the ligand binding potential of the beta3 A-domain.