Dorota Szpak

Expression, activation, and function of integrin αMβ2 (Mac-1) on neutrophil-derived microparticles

Leukocyte-derived microparticles (MPs) are markers of cardiovascular diseases and contribute to pathogenesis by their interaction with various cell types. The presence and activation state of a multifunctional leukocyte receptor, integrin alpha(M)beta(2) (CD11b/18), on MPs derived from human neutrophils (PMNs) were examined. alpha(M)beta(2) expression was significantly enhanced on MPs derived from stimulated compared with resting PMNs. Furthermore, alpha(M)beta(2) on MPs from stimulated but not resting PMNs was in an activated conformation because it was capable of binding activation-specific monoclonal antibodies (CBRM1/5 and mAb24) and soluble fibrinogen. MPs expressing active alpha(M)beta(2) interacted with and were potent activators of resting platelets as assessed by induction of P-selectin expression and activation of alpha(IIb)beta(3). With the use of function-blocking antibodies and MPs obtained from alpha(M)(-/-)-deficient mice, we found that engagement of GPIbalpha on platelets by alpha(M)beta(2) on MPs plays a pivotal role in MP binding. Platelet activation by MPs occurs by a pathway dependent on Akt phosphorylation. PSGL-1/P-selectin interaction also is involved in the conjugation of MPs to platelets, and the combination of blocking reagents to both alpha(M)beta(2)/GPIbalpha and to PSGL-1/P-selectin completely abrogates MP-induced platelet activation. Thus, cooperation of these 2 receptor/counterreceptor systems regulates the prothrombotic properties of PMN-derived MPs.

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 αVβ3-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 αVβ3.

Neutrophil Apoptosis: Selective Regulation by Different Ligands of Integrin αMβ2

Neutrophils undergo spontaneous apoptosis, but their survival can be extended during inflammatory responses. αMβ2 is reported either to delay or accelerate neutrophil apoptosis, but the mechanisms by which this integrin can support such diametrically opposed responses are poorly understood. The abilities of closely related αMβ2 ligands, plasminogen and angiostatin, derived from plasminogen, as well as fibrinogen and its two derivative αMβ2 recognition peptides, P1 and P2-C, differed markedly in their effects on neutrophil apoptosis. Plasminogen, fibrinogen, and P2-C suppressed apoptosis via activation of Akt and ERK1/2 kinases, while angiostatin and P1 failed to activate these prosurvival pathways and did not prevent neutrophil apoptosis. Using cells transfected with αMβ2 or its individual αM or β2 subunits, and purified receptors and its constituent chains, we show that engagement of both subunits with prosurvival ligands is essential for induction of the prosurvival response. Hence, engagement of a single integrin by closely related ligands can induce distinct signaling pathways, which can elicit distinct cellular responses.

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 β1 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 β1 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 β1 integrins and Twist to increase VEGF production. This signaling cascade enhances breast cancer cell invasion and tumor angiogenesis and metastasis.—Sossey‐Alaoui, K., Pluskota, E., Davuluri, G., Bialkowska, K., Das, M., Szpak, D., Lindner, D. J., Downs‐Kelly, E., Thompson, C. L., Plow, E. F. Kindlin‐3 enhances breast cancer progression and metastasis by activating Twist‐mediated angiogenesis. FASEB J. 28, 2260–2271 (2014). www.fasebj.org

Dual Role of the Leukocyte Integrin αMβ2 in Angiogenesis

Polymorphonuclear neutrophils (PMNs) and macrophages are crucial contributors to neovascularization, serving as a source of chemokines, growth factors, and proteases. αMβ2(CD11b/CD18) and αLβ2(CD11a/CD18) are expressed prominently and have been implicated in various responses of these cell types. Thus, we investigated the role of these β2 integrins in angiogenesis. Angiogenesis was analyzed in wild-type (WT), αM-knockout (αM−/−), and αL-deficient (αL−/−) mice using B16F10 melanoma, RM1 prostate cancer, and Matrigel implants. In all models, vascular area was decreased by 50–70% in αM−/− mice, resulting in stunted tumor growth as compared with WT mice. In contrast, αL deficiency did not impair angiogenesis and tumor growth. The neovessels in αM−/− mice were leaky and immature because they lacked smooth muscle cell and pericytes. Defective angiogenesis in the αM−/− mice was associated with attenuated PMN and macrophage recruitment into tumors. In contrast to WT or the αL−/− leukocytes, the αM−/− 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 αMI-domain, the major ligand binding site in the β2 integrins, with Plg. However, the αLI-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 αM−/− mice because of severely impaired degranulation and secretion of VEGF. Thus, αMβ2 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.

The Kindlin-2 regulation of epithelial-to-mesenchymal transition in breast cancer metastasis is mediated through miR-200b

Metastasis is the main cause of death in cancer patients, including breast cancer (BC). Despite recent progress in understanding the biological and molecular determinants of BC metastasis, effective therapeutic treatments are yet to be developed. Among the multitude of molecular mechanisms that regulate cancer metastasis, the epithelial-to-mesenchymal transition (EMT) program plays a key role in the activation of the biological steps leading to the metastatic phenotype. Kindlin-2 has been associated with the pathogenesis of several types of cancers, including BC. The role of Kindlin-2 in the regulation of BC metastasis, and to a lesser extent in EMT is not well understood. In this study, we show that Kindlin-2 is closely associated with the development of the metastatic phenotype in BC. We report that knockout of Kindlin-2 in either human or mouse BC cells, significantly inhibits metastasis in both human and mouse models of BC metastasis. We also report that the Kindlin-2-mediated inhibition of metastasis is the result of inhibition of expression of key molecular markers of the EMT program. Mechanistically, we show that miR-200b, a master regulator of EMT, directly targets and inhibits the expression of Kindlin-2, leading to the subsequent inhibition of EMT and metastasis. Together, our data support the targeting of Kindlin-2 as a therapeutic strategy against BC metastasis.

Kindlin-2 regulates the growth of breast cancer tumors by activating CSF-1–mediated macrophage infiltration

Interplay between tumor cells and host cells in the tumor microenvironment dictates the development of all cancers. In breast cancer, malignant cells educate host macrophages to adopt a protumorigenic phenotype. In this study, we show how the integrin-regulatory protein kindlin-2 (FERMT2) promotes metastatic progression of breast cancer through the recruitment and subversion of host macrophages. Kindlin-2 expression was elevated in breast cancer biopsy tissues where its levels correlated with reduced patient survival. On the basis of these observations, we used CRISPR/Cas9 technology to ablate Kindlin-2 expression in human MDA-MB-231 and murine 4T1 breast cancer cells. Kindlin-2 deficiency inhibited invasive and migratory properties in vitro without affecting proliferation rates. However, in vivo tumor outgrowth was inhibited by >80% in a manner associated with reduced macrophage infiltration and secretion of the macrophage attractant and growth factor colony-stimulating factor-1 (CSF-1). The observed loss of CSF-1 appeared to be caused by a more proximal deficiency in TGFβ-dependent signaling in Kindlin-2-deficient cells. Collectively, our results illuminate a Kindlin-2/TGFβ/CSF-1 signaling axis employed by breast cancer cells to capture host macrophage functions that drive tumor progression. Cancer Res; 77(18); 5129-41. ©2017 AACR.

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

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 β‐ or γ‐catenin and cortical actin filaments, Kindlin‐2 stabilizes adherens junctions. The Kindlin‐2 binding sites for β‐ 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.

αMβ2 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 αMβ2 (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. αMβ2 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 β. As their antagonists inhibited the effect of 17β-estradiol (E2), E2 decreased CD36, SR-A1, and foam cell formation in ApoE−/− macrophages in an ERα- and ERβ-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 αMβ2 in female ApoE−/− mice. αMβ2 maintains ER expression in macrophages and E2-dependent inhibition of foam cell formation.