Parkin ubiquitination of Kindlin-2 enables mitochondria-associated metastasis suppression.

Mitochondria are signaling organelles implicated in cancer, but the mechanisms are elusive. Here, we show that Parkin, an E3 ubiquitination (Ub) ligase altered in Parkinson's disease, forms a complex with the regulator of cell motility, Kindlin-2 (K2), at mitochondria of tumor cells. In turn, Parkin ubiquitinates Lys581 and Lys582 using Lys48 linkages, resulting in proteasomal degradation of K2 and shortened half-life from ∼5 h to ∼1.5 h. Loss of K2 inhibits focal adhesion turnover and ß1 integrin activation, impairs membrane lamellipodia size and frequency, and inhibits mitochondrial dynamics, altogether suppressing tumor cell-extracellular matrix interactions, migration, and invasion. Conversely, Parkin does not affect tumor cell proliferation, cell cycle transitions, or apoptosis. Expression of a Parkin Ub-resistant K2 Lys581Ala/Lys582Ala double mutant is sufficient to restore membrane lamellipodia dynamics, correct mitochondrial fusion/fission, and preserve single-cell migration and invasion. In a 3D model of mammary gland developmental morphogenesis, impaired K2 Ub drives multiple oncogenic traits of EMT, increased cell proliferation, reduced apoptosis, and disrupted basal-apical polarity. Therefore, deregulated K2 is a potent oncogene, and its Ub by Parkin enables mitochondria-associated metastasis suppression.

VEGFR2 survival and mitotic signaling depends on joint activation of associated C3ar1/C5ar1 and IL-6R-gp130.

Purified vascular endothelial cell (EC) growth factor receptor-2 (VEGFR2) auto-phosphorylates upon VEGF-A occupation in vitro, arguing that VEGR2 confers its mitotic and viability signaling in and of itself. Herein, we show that, in ECs, VEGFR2 function requires concurrent C3a/C5a receptor (C3ar1/C5ar1) and IL-6 receptor (IL-6R)-gp130 co-signaling. C3ar1/C5ar1 or IL-6R blockade totally abolished VEGFR2 auto-phosphorylation, downstream Src, ERK, AKT, mTOR and STAT3 activation, and EC cell cycle entry. VEGF-A augmented production of C3a/C5a/IL-6 and their receptors via a two-step p-Tyk2/p-STAT3 process. Co-immunoprecipitation analyses, confocal microscopy, ligand pulldown and bioluminescence resonance energy transfer assays all indicated that the four receptors are physically interactive. Angiogenesis in murine day 5 retinas and in adult tissues was accelerated when C3ar1/C5ar1 signaling was potentiated, but repressed when it was disabled. Thus, C3ar1/C5ar1 and IL-6R-gp130 joint activation is needed to enable physiological VEGFR2 function.

The P387 thrombospondin-4 variant promotes accumulation of macrophages in atherosclerotic lesions.

Thrombospondin-4 (TSP4) is a pro-angiogenic protein that has been implicated in tissue remodeling and local vascular inflammation. TSP4 and, in particular, its SNP variant, P387 TSP4, have been associated with cardiovascular disease. Macrophages are central to initiation and resolution of inflammation and development of atherosclerotic lesions, but the effects of the P387 TSP4 on macrophages remain essentially unknown. We examined the effects of the P387 TSP4 variant on macrophages in cell culture and in vivo in a murine model of atherosclerosis. Furthermore, the levels and distributions of the two TSP4 variants were assessed in human atherosclerotic arteries. In ApoE-/- /P387-TSP4 knock-in mice, lesions size measured by Oil Red O did not change, but the lesions accumulated more macrophages than lesions bearing A387 TSP4. The levels of inflammatory markers were increased in lesions of ApoE-/- /P387-TSP4 knock-in mice compared to ApoE-/- mice. Lesions in human arteries from individuals carrying the P387 variant had higher levels of TSP4 and higher macrophage accumulation. P387 TSP4 was more active in supporting adhesion of cultured human and mouse macrophages in experiments using recombinant TSP4 variants and in cells derived from P387-TSP4 knock-in mice. TSP4 supports the adhesion of macrophages and their accumulation in atherosclerotic lesions without changing the size of lesions. P387 TSP4 is more active in supporting these pro-inflammatory events in the vascular wall, which may contribute to the increased association of P387 TSP4 with cardiovascular disease.

αMß2 Is Antiatherogenic in Female but Not Male Mice.

Atherosclerosis is a complex inflammatory process characterized by monocyte recruitment into the arterial wall, their differentiation into macrophages, and lipid accumulation. Because integrin αMß2 (CD11b/CD18) mediates multiple diverse functions of leukocytes, we examined its role in atherogenesis. αM-/-/ApoE-/- and ApoE-/- mice were fed a control or high fat diet for 3 or 16 wk to induce atherogenesis. Unexpectedly, αM deficiency accelerated development of atherosclerosis in female but not in male mice. The size of aortic root lesions was 3-4.5-fold larger in female αM-/-/ApoE-/- than in ApoE-/- mice. Monocyte and macrophage content within the lesions was increased 2.5-fold in female αM-/-/ApoE-/- mice due to enhanced proliferation. αMß2 elimination promoted gender-dependent foam cell formation due to enhanced uptake of cholesterol by αM-/-/ApoE-/- macrophages. This difference was attributed to enhanced expression of lipid uptake receptors, CD36 and scavenger receptor A1 (SR-A1), in female mice. Macrophages from female αM-/-/ApoE-/- mice showed dramatically reduced expression of FoxM1 transcription factor and estrogen receptors (ER) α and ß. As their antagonists inhibited the effect of 17ß-estradiol (E2), E2 decreased CD36, SR-A1, and foam cell formation in ApoE-/- macrophages in an ERα- and ERß-dependent manner. However, female αM-/-/ApoE-/- macrophages failed to respond to E2 and maintained elevated CD36, SR-A1, and lipid accumulation. FoxM1 inhibition in ApoE-/- macrophages reduced ERs and enhanced CD36 and SR-A1 expression, whereas FoxM1 overexpression in αM-/-/ApoE-/- macrophages reversed their proatherogenic phenotype. We demonstrate a new, surprising atheroprotective role of αMß2 in female ApoE-/- mice. αMß2 maintains ER expression in macrophages and E2-dependent inhibition of foam cell formation.

The Upregulation of Integrin αDß2 (CD11d/CD18) on Inflammatory Macrophages Promotes Macrophage Retention in Vascular Lesions and Development of Atherosclerosis.

Macrophage accumulation is a critical step during development of chronic inflammation, initiating progression of many devastating diseases. Leukocyte-specific integrin αDß2 (CD11d/CD18) is dramatically upregulated on macrophages at inflammatory sites. Previously we found that CD11d overexpression on cell surfaces inhibits in vitro cell migration due to excessive adhesion. In this study, we have investigated how inflammation-mediated CD11d upregulation contributes to macrophage retention at inflammatory sites during atherogenesis. Atherosclerosis was evaluated in CD11d-/-/ApoE-/- mice after 16 wk on a Western diet. CD11d deficiency led to a marked reduction in lipid deposition in aortas and isolated macrophages. Macrophage numbers in aortic sinuses of CD11d-/- mice were reduced without affecting their apoptosis and proliferation. Adoptive transfer of fluorescently labeled wild-type and CD11d-/- monocytes into ApoE-/- mice demonstrated similar recruitment from circulation, but reduced accumulation of CD11d-/- macrophages within the aortas. Furthermore, CD11d expression was significantly upregulated on macrophages in atherosclerotic lesions and M1 macrophages in vitro. Interestingly, expression of the related ligand-sharing integrin CD11b was not altered. This difference defines their distinct roles in the regulation of macrophage migration. CD11d-deficient M1 macrophages demonstrated improved migration in a three-dimensional fibrin matrix and during resolution of peritoneal inflammation, whereas migration of CD11b-/- M1 macrophages was not affected. These results prove the contribution of high densities of CD11d to macrophage arrest during atherogenesis. Because high expression of CD11d was detected in several inflammation-dependent diseases, we suggest that CD11d/CD18 upregulation on proinflammatory macrophages may represent a common mechanism for macrophage retention at inflammatory sites, thereby promoting chronic inflammation and disease development.

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.

Distinct Effects of Integrins αXß2 and αMß2 on Leukocyte Subpopulations during Inflammation and Antimicrobial Responses.

Integrins αMß2 and αXß2 are homologous adhesive receptors that are expressed on many of the same leukocyte populations and bind many of the same ligands. Although αMß2 was extensively characterized and implicated in leukocyte inflammatory and immune functions, the roles of αXß2 remain largely obscure. Here, we tested the ability of mice deficient in integrin αMß2 or αXß2 to deal with opportunistic infections and the capacity of cells derived from these animals to execute inflammatory functions. The absence of αMß2 affected the recruitment of polymorphonuclear neutrophils (PMN) to bacterial and fungal pathogens as well as to model inflammatory stimuli, and αMß2-deficient PMN displayed defective inflammatory functions. In contrast, deficiency of αXß2 abrogated intraperitoneal recruitment and adhesive functions of monocytes and macrophages (Mϕ) and the ability of these cells to kill/phagocytose Candida albicans or Escherichia coli cells both ex vivo and in vivo During systemic candidiasis, the absence of αXß2 resulted in the loss of antifungal activity by tissue Mϕ and inhibited the production of tumor necrosis factor alpha (TNF-α)/interleukin-6 (IL-6) in infected kidneys. Deficiency of αMß2 suppressed Mϕ egress from the peritoneal cavity, decreased the production of anti-inflammatory IL-10, and stimulated the secretion of IL-6. The absence of αXß2, but not of αMß2, increased survival against a septic challenge with lipopolysaccharide (LPS) by 2-fold. Together, these results suggest that αMß2 plays a primary role in PMN inflammatory functions and regulates the anti-inflammatory functions of Mϕ, whereas αXß2 is central in the regulation of inflammatory functions of recruited and tissue-resident Mϕ.

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.

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.

Kindlin-3 enhances breast cancer progression and metastasis by activating Twist-mediated angiogenesis.

The FERM domain containing protein Kindlin-3 has been recognized as a major regulator of integrin function in hematopoietic cells, but its role in neoplasia is totally unknown. We have examined the relationship between Kindlin-3 and breast cancer in mouse models and human tissues. Human breast tumors showed a ∼7-fold elevation in Kindlin-3 mRNA compared with nonneoplastic tissue by quantitative polymerase chain reaction. Kindlin-3 overexpression in a breast cancer cell line increased primary tumor growth and lung metastasis by 2.5- and 3-fold, respectively, when implanted into mice compared with cells expressing vector alone. Mechanistically, the Kindlin-3-overexpressing cells displayed a 2.2-fold increase in vascular endothelial growth factor (VEGF) secretion and enhanced ß1 integrin activation. Increased VEGF secretion resulted from enhanced production of Twist, a transcription factor that promotes tumor angiogenesis. Knockdown of Twist diminished VEGF production, and knockdown of ß1 integrins diminished Twist and VEGF production by Kindlin-3-overexpressing cells, while nontargeting small interfering RNA had no effect on expression of these gene products. Thus, Kindlin-3 influences breast cancer progression by influencing the crosstalk between ß1 integrins and Twist to increase VEGF production. This signaling cascade enhances breast cancer cell invasion and tumor angiogenesis and metastasis.

Direct interaction of kindlin-3 with integrin αIIbß3 in platelets is required for supporting arterial thrombosis in mice.

OBJECTIVE: Kindlin-3 is a critical supporter of integrin function in platelets. Lack of expression of kindlin-3 protein in patients impairs integrin αIIbß3-mediated platelet aggregation. Although kindlin-3 has been categorized as an integrin-binding partner, the functional significance of the direct interaction of kindlin-3 with integrin αIIbß3 in platelets has not been established. Here, we evaluated the significance of the binding of kindlin-3 to integrin αIIbß3 in platelets in supporting integrin αIIbß3-mediated platelet functions. APPROACH AND RESULTS: We generated a strain of kindlin-3 knockin (K3KI) mice that express a kindlin-3 mutant that carries an integrin-interaction defective substitution. K3KI mice could survive normally and express integrin αIIbß3 on platelets similar to their wild-type counterparts. Functional analysis revealed that K3KI mice exhibited defective platelet function, including impaired integrin αIIbß3 activation, suppressed platelet spreading and platelet aggregation, prolonged tail bleeding time, and absence of platelet-mediated clot retraction. In addition, whole blood drawn from K3KI mice showed resistance to in vitro thrombus formation and, as a consequence, K3KI mice were protected from in vivo arterial thrombosis. CONCLUSIONS: These observations demonstrate that the direct binding of kindlin-3 to integrin αIIbß3 is involved in supporting integrin αIIbß3 activation and integrin αIIbß3-dependent responses of platelets and consequently contributes significantly to arterial thrombus formation.

Integrin αXß2 is a leukocyte receptor for Candida albicans and is essential for protection against fungal infections.

The opportunistic fungus Candida albicans is one of the leading causes of infections in immunocompromised patients, and innate immunity provides a principal mechanism for protection from the pathogen. In the present work, the role of integrin α(X)ß2 in the pathogenesis of fungal infection was assessed. Both purified α(X)ß2 and α(X)ß2-expressing human epithelial kidney 293 cells recognized and bound to the fungal hyphae of SC5314 strain of C. albicans but not to the yeast form or to hyphae of a strain deficient in the fungal mannoprotein, Pra1. The binding of the integrin to the fungus was inhibited by ß-glucans but not by mannans, implicating a lectin-like activity in recognition but distinct in specificity from that of α(M)ß2. Mice deficient in α(X)ß2 were more prone to systemic infection with the LD50 fungal inoculum decreasing 3-fold in α(X)ß2-deficient mice compared with wild-type mice. After challenging i.v. with 1.5 × 104 cell/g, 60% of control C57BL/6 mice died within 14 d compared with 100% mortality of α(X)ß2-deficient mice within 9 d. Organs taken from α(X)ß2-deficient mice 16 h postinfection revealed a 10-fold increase in fungal invasion into the brain and a 2-fold increase into the liver. These data indicate that α(X)ß2 is important for protection against systemic C. albicans infections and macrophage subsets in the liver, Kupffer cells, and in the brain, microglial cells use α(X)ß2 to control fungal invasion.

The integrin coactivator kindlin-2 plays a critical role in angiogenesis in mice and zebrafish.

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice and causes severe developmental defects in zebrafish. Knockdown of kindlin-2 levels in endothelial cells resulted in defective adhesive and migratory responses, suggesting that angiogenesis might be aberrant even with partial reduction of kindlin-2. This hypothesis has now been tested in the kindlin-2(+/-) mice. RM1 prostate tumors grown in kindlin-2(+/-) mice had fewer blood vessels, which were thinner and shorter and supported less tumor growth compared with wild-type littermates. The vessels that did form in the kindlin-2(+/-) mice lacked smooth muscle cells and pericytes and had thinner basement membranes, indicative of immature vessels. VEGF-induced angiogenesis in matrigel implants was also abnormal in the kindlin-2(+/-) mice. Vessels in the kindlin-2(+/-) mice were leaky, and BM transplantation from kindlin-2(+/-) to WT mice did not correct this defect. Endothelial cells derived from kindlin-2(+/-) mice had integrin expression levels similar to WT mice but reduced αVß3-dependent signaling, migration, adhesion, spreading, and tube formation. Developmental angiogenesis was markedly impaired by kindlin-2 morpholinos in zebrafish. Taken together, kindlin-2 plays an important role in pathologic and developmental angiogenesis, which arises from defective activation of integrin αVß3.

αMß2 integrin activation prevents alternative activation of human and murine macrophages and impedes foam cell formation.

RATIONALE: The alternative activation of monocytes by interleukin (IL)-13 and IL-4 is a significant component of the inflammatory response. The consequences of alternative activation in inflammatory diseases remain to be determined. OBJECTIVE: In this report, we explored how integrins, receptors important for monocyte migration to inflammatory sites, regulate IL-13-mediated monocyte activation. We focused on the analysis of 2 proteins, which are upregulated during the alternative activation and are important for the development of atherosclerosis, an oxidative enzyme 15-lipoxygenase (15-LO) and a scavenger receptor CD36. METHODS AND RESULTS: We found that adhesion of resting monocytes through ß(2) integrins and inside-out activation of ß(2) integrins by monocyte chemoattractant protein-1 did not change IL-13-stimulated 15-LO upregulation; however, preincubation of monocytes with the antibody MEM48, which generates full activation of ß(2) integrins, significantly inhibited 15-LO mRNA and protein expression. In contrast, activation of ß(1) integrins had no effect on 15-LO expression. Analysis of integrin clustering through α(M), α(L), α(X), and α(D) subunits demonstrated the pivotal role for integrin α(M)ß(2) in inhibiting 15-LO expression. IL-13 treatment upregulates 15-LO-dependent CD36 expression on human monocytes; our studies showed that ß(2) integrin activation and α(M) integrin clustering significantly inhibited IL-13-dependent CD36 mRNA and protein expression, as well as CD36-related foam cell formation. Moreover, IL-13 stimulation of α(M)-deficient peritoneal macrophages demonstrated an upregulated level of 15-LO induction, CD36 expression, and lipid accumulation as compared with wild-type controls. CONCLUSIONS: The adhesion of monocytes/macrophages through activated integrin α(M)ß(2) has a regulatory and potential atheroprotective function during the alternative activation of macrophages.

Integrin signaling in vascular function.

PURPOSE OF REVIEW: In the current review, we summarize recent progress on vasculature-specific function and regulation of integrins and integrin-associated proteins, including advances in our understanding of inside-out integrin activation. The studies on regulation of integrin activation received new impulse in 2009 with the identification of kindlin protein family members as crucial mediators of integrin inside-out signaling. In the current review, we outline the recent findings on the role of kindlins in the vascular system, as well as new studies that have begun shaping the mechanistic model of kindlins' function. RECENT FINDINGS: Several tissue-specific knockout models for integrins and genes associated with the integrin functions have been recently presented, including smooth muscle-specific integrin-linked kinase and endothelial-specific focal adhesion kinase and talin-1 ablation. In the heterozygous animal knockout model, kindlin-2 has been demonstrated as a crucial modulator of angiogenesis and vascular permeability. As a number of articles have advanced our understanding of kindlin function, they are reviewed and discussed in further detail. New findings include an additional lipid-binding site within the kindlin molecule and preferential binding of the nonphosphorylated form of ß-integrins. SUMMARY: The role of integrins in angiogenesis has been demonstrated to include, in addition to cell adhesion and mechanotransduction, specific signaling functions. The importance of integrin inside-out pathway in vascular physiology has been unequivocally proven, and endothelial permeability is directly regulated by this process. Inhibition of kindlin-dependent steps in the inside-out pathway as an approach to block platelet aggregation should be paralog-specific, as it may have adverse effects on vascular permeability.

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.

Thrombospondin-4 regulates vascular inflammation and atherogenesis.

RATIONALE: Thrombospondin (TSP)-4 is an extracellular protein that has been linked to several cardiovascular pathologies. However, a role for TSP-4 in vascular wall biology remains unknown. OBJECTIVE: We have examined the effects of TSP-4 gene (Thbs4) knockout on the development of atherosclerotic lesions in ApoE(-/-) mice. METHODS AND RESULTS: Deficiency in TSP-4 reduced atherosclerotic lesions: at 20 weeks of age, the size of the aortic root lesions in Thbs4(-/-)/ApoE(-/-) mice was decreased by 48% in females and by 39% in males on chow diets; in mice on Western diets, lesions in the descending aorta were reduced by 30% in females and 33% in males. In ApoE(-/-) mice, TSP-4 was abundant in vessel areas prone to lesion development and in the matrix of the lesions themselves. TSP-4 deficiency reduced the number of macrophages in lesions in all groups by ≥ 2-fold. In addition, TSP-4 deficiency reduced endothelial cell activation (expression of surface adhesion molecules) and other markers of inflammation in the vascular wall (decreased production of monocyte chemoattractant protein-1 and activation of p38). In vitro, both the adhesion and migration of wild-type macrophages increased in the presence of purified recombinant TSP-4 in a dose-dependent manner (up to 7- and 4.7-fold, respectively). These responses led to p38-MAPkinase activation and were dependent on ß(2) and ß(3) integrins, which recognize TSP-4 as a ligand. CONCLUSIONS: TSP-4 is abundant in atherosclerotic lesions and in areas prone to development of lesions and may influence the recruitment of macrophages by activating endothelial cells and directly interacting with macrophages to increase their adhesion and migration. Our observations suggest an important role for this matricellular protein in the local regulation of inflammation associated with atherogenesis.

Targeted Deletion of Kindlin-2 in Mouse Mammary Glands Inhibits Tumor Growth, Invasion, and Metastasis Downstream of a TGF-ß/EGF Oncogenic Signaling Pathway.

Breast cancer (BC) is one of the leading causes of cancer-related deaths due in part to its invasive and metastatic properties. Kindlin-2 (FERMT2) is associated with the pathogenesis of several cancers. Although the role of Kindlin-2 in regulating the invasion-metastasis cascade in BC is widely documented, its function in BC initiation and progression remains to be fully elucidated. Accordingly, we generated a floxed mouse strain by targeting the Fermt2 (K2lox/lox) locus, followed by tissue-specific deletion of Kindlin-2 in the myoepithelial compartment of the mammary glands by crossing the K2lox/lox mice with K14-Cre mice. Loss of Kindlin-2 in mammary epithelial cells (MECs) showed no deleterious effects on mammary gland development, fertility, and lactation in mice bearing Kindlin-2-deletion. However, in a syngeneic mouse model of BC, mammary gland, specific knockout of Kindlin-2 inhibited the growth and metastasis of murine E0771 BC cells inoculated into the mammary fat pads. However, injecting the E0771 cells into the lateral tail vein of Kindlin-2-deleted mice had no effect on tumor colonization in the lungs, thereby establishing a critical role of MEC Kindlin-2 in supporting BC tumor growth and metastasis. Mechanistically, we found the MEC Kindlin-2-mediated inhibition of tumor growth and metastasis is accomplished through its regulation of the TGF-ß/ERK MAP kinase signaling axis. Thus, Kindlin-2 within the mammary gland microenvironment facilitates the progression and metastasis of BC.

The NOGO receptor NgR2, a novel αVß3 integrin effector, induces neuroendocrine differentiation in prostate cancer.

Androgen deprivation therapies aimed to target prostate cancer (PrCa) are only partially successful given the occurrence of neuroendocrine PrCa (NEPrCa), a highly aggressive and highly metastatic form of PrCa, for which there is no effective therapeutic approach. Our group has demonstrated that while absent in prostate adenocarcinoma, the αVß3 integrin expression is increased during PrCa progression toward NEPrCa. Here, we show a novel pathway activated by αVß3 that promotes NE differentiation (NED). This novel pathway requires the expression of a GPI-linked surface molecule, NgR2, also known as Nogo-66 receptor homolog 1. We show here that NgR2 is upregulated by αVß3, to which it associates; we also show that it promotes NED and anchorage-independent growth, as well as a motile phenotype of PrCa cells. Given our observations that high levels of αVß3 and, as shown here, of NgR2 are detected in human and mouse NEPrCa, our findings appear to be highly relevant to this aggressive and metastatic subtype of PrCa. This study is novel because NgR2 role has only minimally been investigated in cancer and has instead predominantly been analyzed in neurons. These data thus pave new avenues toward a comprehensive mechanistic understanding of integrin-directed signaling during PrCa progression toward a NE phenotype.

Kindlins as modulators of breast cancer progression.

Kindlin-1 (K1, FERMT1), Kindlin-2 (K2, FERMT2), and Kindlin-3 (K3, FERMT3) are the three members of the kindlin family of adapter proteins found in mammals. One or more kindlins are found in most cell types, K1 primarily in epithelial cells, K3 in primarily hematopoietic cells and also endothelial cells, and K2 is very broadly distributed. The kindlins consist primarily of a 4.1-erzin-radixin-moiesin (FERM) domain, which is transected by a lipid-binding plextrin-homology (PH) domain. Deficiencies of each kindlin in mice and/ or humans have profound pathogenic consequences. The most well-established function of kindlins depends on their ability to participate in the activat integrin adhesion receptors. This function depends on the binding of each kindlin to the beta subunit of integrins where it cooperates with talin to enhance avidity of interactions with cognate extracellular matrix ligands. Deficiencies of many different integrins are lethal, are critical for normal development of mammary tissue, and excessive expression and/or activation of certain integrins are associated with progression and metastasis of breast cancer. However, via its interaction with many other intracellular proteins, kindlins can influence numerous cellular responses. Changes in expression of each of the three kindlins have been reported in association with breast cancer, with several studies indicating that kindlins are among the most upregulated genes in breast cancer. The association of abnormal functions of K2 with breast cancer is particularly extensive with many reports indicating that it is a major driver of breast cancer via its promotion of cancer cell proliferation, survival, adhesion, migration, invasion, the epithelial-to-mesenchymal transition and its influence on macrophage recruitment and phenotype. These associations suggest that the kindlins and their functions represent an intriguing therapeutic target for exploration of breast cancer therapy.