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Kindlin-2 is a cytoskeletal adapter protein that is present in many different cell types. By virtue of its interaction with multiple binding partners, Kindlin-2 intercalates into numerous signaling pathways and cytoskeletal nodes. A specific interaction of Kindlin-2 that is of paramount importance in many cellular responses is its direct binding to the cytoplasmic tails of integrins, an interaction that controls many of the adhesive, migratory and signaling responses mediated by members of the integrin family of cell-surface heterodimers. Kindlin-2 is highly expressed in many cancers and is particularly prominent in prostate cancer cells. CRISPR/cas9 was used as a primary approach to knockout expression of Kindlin-2 in both androgen-independent and dependent prostate cancer cell lines, and the effects of Kindlin-2 suppression on oncogenic properties of these prostate cancer cell lines was examined. Adhesion to extracellular matrix proteins was markedly blunted, consistent with the control of integrin function by Kindlin-2. Migration across matrices was also affected. Anchorage independent growth was markedly suppressed. These observations indicate that Kindlin-2 regulates hallmark features of prostate cancer cells. In androgen expressing cells, testosterone-stimulated adhesion was Kindlin-2-dependent. Furthermore, tumor growth of a prostate cancer cell line lacking Kindlin-2 and implanted into the prostate gland of immunocompromised mice was markedly blunted and was associated with suppression of angiogenesis in the developing tumor. These results establish a key role of Kindlin-2 in prostate cancer progression and suggest that Kindlin-2 represents an interesting therapeutic target for treatment of prostate cancer., (© 2024. The Author(s).)

Highly aggressive, metastatic, neuroendocrine prostate cancer, which typically develops from prostate cancer cells acquiring resistance to androgen deprivation therapy, is associated with limited treatment options and hence poor prognosis. We have previously demonstrated that the αVβ3 integrin is over-expressed in neuroendocrine prostate cancer. We now show that LM609, a monoclonal antibody that specifically targets the human αVβ3 integrin, hinders the growth of neuroendocrine prostate cancer patient-derived xenografts in vivo. Our group has recently identified a novel αVβ3 integrin binding partner, NgR2, responsible for regulating the expression of neuroendocrine markers and for inducing neuroendocrine differentiation in prostate cancer cells. Through in vitro functional assays, we here demonstrate that NgR2 is crucial in promoting cell adhesion to αVβ3 ligands. Moreover, we describe for the first time co-fractionation of αVβ3 integrin and NgR2 in small extracellular vesicles derived from metastatic prostate cancer patients' plasma. These prostate cancer patient-derived small extracellular vesicles have a functional impact on human monocytes, increasing their adhesion to fibronectin. The monocytes incubated with small extracellular vesicles do not show an associated change in conventional polarization marker expression and appear to be in an early stage that may be defined as "adhesion competent". Overall, these findings allow us to better understand integrin-directed signaling and cell-cell communication during cancer progression. Furthermore, our results pave the way for new diagnostic and therapeutic perspectives for patients affected by neuroendocrine prostate cancer., Competing Interests: Declaration of Competing Interest FQ is an employee/contractor at Janssen Pharmaceutical. EC is a consultant of Dotquant and received funding under institutional SRA from Janssen Research, Bayer Pharmaceuticals, Forma Therapeutics, Foghorn, Kronos Bios, Genentech, Astra Zeneca, and MacroGenics. No potential conflict of interest was reported by the other authors., (Copyright © 2023. Published by Elsevier B.V.)

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 β1 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The communication of talin-activated integrin αIIbβ3 with the cytoskeleton (integrin outside-in signaling) is essential for platelet aggregation, wound healing, and hemostasis. Filamin, a large actin crosslinker and integrin binding partner critical for cell spreading and migration, is implicated as a key regulator of integrin outside-in signaling. However, the current dogma is that filamin, which stabilizes inactive αIIbβ3, is displaced from αIIbβ3 by talin to promote the integrin activation (inside-out signaling), and how filamin further functions remains unresolved. Here, we show that while associating with the inactive αIIbβ3, filamin also associates with the talin-bound active αIIbβ3 to mediate platelet spreading. Fluorescence resonance energy transfer-based analysis reveals that while associating with both αIIb and β3 cytoplasmic tails (CTs) to maintain the inactive αIIbβ3, filamin is spatiotemporally rearranged to associate with αIIb CT alone on activated αIIbβ3. Consistently, confocal cell imaging indicates that integrin α CT-linked filamin gradually delocalizes from the β CT-linked focal adhesion marker-vinculin likely because of the separation of integrin α/β CTs occurring during integrin activation. High-resolution crystal and nuclear magnetic resonance structure determinations unravel that the activated integrin αIIb CT binds to filamin via a striking α-helix→β-strand transition with a strengthened affinity that is dependent on the integrin-activating membrane environment containing enriched phosphatidylinositol 4,5-bisphosphate. These data suggest a novel integrin αIIb CT-filamin-actin linkage that promotes integrin outside-in signaling. Consistently, disruption of such linkage impairs the activation state of αIIbβ3, phosphorylation of focal adhesion kinase/proto-oncogene tyrosine kinase Src, and cell migration. Together, our findings advance the fundamental understanding of integrin outside-in signaling with broad implications in blood physiology and pathology., (© 2023 by The American Society of Hematology.)

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 α4β1 and macrophage αVβ3. 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., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

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 αVβ3 integrin expression is increased during PrCa progression toward NEPrCa. Here, we show a novel pathway activated by αVβ3 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 αVβ3, 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 αVβ3 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., (© 2022. The Author(s).)

Talin-induced integrin binding to extracellular matrix ligands (integrin activation) is the key step to trigger many fundamental cellular processes including cell adhesion, cell migration, and spreading. Talin is widely known to use its N-terminal head domain (talin-H) to bind and activate integrin, but how talin-H operates in the context of full-length talin and its surrounding remains unknown. Here we show that while being capable of inducing integrin activation, talin-H alone exhibits unexpectedly low potency versus a constitutively activated full-length talin. We find that the large C-terminal rod domain of talin (talin-R), which otherwise masks the integrin binding site on talin-H in inactive talin, dramatically enhances the talin-H potency by dimerizing activated talin and bridging it to the integrin co-activator kindlin-2 via the adaptor protein paxillin. These data provide crucial insight into the mechanism of talin and its cooperation with kindlin to promote potent integrin activation, cell adhesion, and signaling., (© 2022. The Author(s).)

Cancer metabolism, including in mitochondria, is a disease hallmark and therapeutic target, but its regulation is poorly understood. Here, we show that many human tumors have heterogeneous and often reduced levels of Mic60, or Mitofilin, an essential scaffold of mitochondrial structure. Despite a catastrophic collapse of mitochondrial integrity, loss of bioenergetics, and oxidative damage, tumors with Mic60 depletion slow down cell proliferation, evade cell death, and activate a nuclear gene expression program of innate immunity and cytokine/chemokine signaling. In turn, this induces epithelial-mesenchymal transition (EMT), activates tumor cell movements through exaggerated mitochondrial dynamics, and promotes metastatic dissemination in vivo. In a small-molecule drug screen, compensatory activation of stress response (GCN2) and survival (Akt) signaling maintains the viability of Mic60-low tumors and provides a selective therapeutic vulnerability. These data demonstrate that acutely damaged, "ghost" mitochondria drive tumor progression and expose an actionable therapeutic target in metastasis-prone cancers., Competing Interests: The authors declare no competing interest.

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 (K2 lox/lox ) locus, followed by tissue-specific deletion of Kindlin-2 in the myoepithelial compartment of the mammary glands by crossing the K2 lox/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-β/ERK MAP kinase signaling axis. Thus, Kindlin-2 within the mammary gland microenvironment facilitates the progression and metastasis of BC.

Plasminogen and its multiple receptors have been implicated in the responses of many different cell types. Among these receptors, histone 2B (H2B) has been shown to play a prominent role in macrophage responses. The contribution of H2B to plasminogen-induced endothelial migration, an event relevant to wound healing and angiogenesis, is unknown. Plasminogen enhanced the migration of endothelial cells, which was inhibited by both Protease-Activated Receptor-1 (PAR1) and 2 (PAR2) antagonists. H2B was detected on viable endothelial cells of venous and arterial origin, and an antibody to H2B that blocks plasminogen binding also inhibited the plasminogen-dependent migration by these cells. The antibody blockade was as effective as PAR1 or PAR2 antagonists in inhibiting endothelial cell migration. In pull-down experiments, H2B formed a complex with both PAR1 and PAR2 but not β3 integrin, another receptor implicated in endothelial migration in the presence of plasminogen. H2B was found to be associated with clathrin adapator protein, AP2µ (clathrin AP2µ) and β-arrestin2, which are central to the internationalization/signaling machinery of the PARs. These associations with PAR1-clathrin adaptor AP2µ- and PAR2-β-arrestin2-dependent internalization/signaling pathways provide a mechanism to link plasminogen to responses such as wound healing and angiogenesis.

Susceptibility to doxorubicin-induced nephropathy (DIN), a toxic model for the induction of proteinuria in mice, is related to the single-nucleotide polymorphism (SNP) C6418T of the Prkdc gene encoding for the DNA-repair enzyme DNA-PKcs. In addition, plasminogen (Plg) has been reported to play a role in glomerular damage. Here, we investigated the interdependence of both factors for the development of DIN. Genotyping confirmed the SNP of the Prkdc gene in C57BL/6 (PrkdcC6418/C6418) and 129S1/SvImJ (PrkdcT6418/T6418) mice. Intercross of heterozygous 129SB6F1 mice led to 129SB6F2 hybrids with Mendelian inheritance of the SNP. After doxorubicin injection, only homozygous F2 mice with PrkdcT6418/T6418 developed proteinuria. Genetic deficiency of Plg (Plg-/-) in otherwise susceptible 129S1/SvImJ mice led to resistance to DIN. Immunohistochemistry revealed glomerular binding of Plg in Plg+/+ mice after doxorubicin injection involving histone H2B as Plg receptor. In doxorubicin-resistant C57BL/6 mice, Plg binding was absent. In conclusion, susceptibility to DIN in 129S1/SvImJ mice is determined by a hierarchical two-hit process requiring the C6418T SNP in the Prkdc gene and subsequent glomerular binding of Plg. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Integrins serve as conduits for the transmission of information between cells and their extracellular environment. Signaling across integrins is bidirectional, transducing both inside-out and outside-signaling. Integrin activation, a transition from a low affinity/avidity state to a high affinity/avidity state for cognate ligands, is an outcome of inside-signaling. Such activation is particularly important for the recognition of soluble ligands by blood cells but also influences cell-cell and cell-matrix interactions. Integrin activation depends on a complex series of interactions, which both accelerate and inhibit their interconversion from the low to the high affinity/avidity state. There are three components regarded as being most proximately involved in integrin activation: the integrin cytoplasmic tails, talins and kindlins. The participation of each of these molecules in integrin activation is highly regulated by post-translation modifications. The importance of targeted phosphorylation of integrin cytoplasmic tails and talins in integrin activation is well-established, but much less is known about the role of post-translational modification of kindlins. The kindlins, a three-member family of 4.1-ezrin-radixin-moesin (FERM)-domain proteins in mammals, bind directly to the cytoplasmic tails of integrin beta subunits. This commentary provides a synopsis of the emerging evidence for the role of kindlin phosphorylation in integrin regulation.

Objective: Plasminogen/plasmin is a serine protease system primarily responsible for degrading fibrin within blood clots. Plasminogen mediates its functions by interacting with plasminogen receptors on the cell surface. H2B, one such plasminogen receptor, is found on the surface of several cell types including macrophages. Both basic and clinical studies support the role of plasminogen in the process of foam cell formation (FCF), a hallmark of atherosclerosis. Growing evidence also implicates serine protease-activated receptors (PARs) in atherosclerosis. These receptors are also found on macrophages, and plasmin is capable of activating PAR1 and PAR4. The goal of this study was to determine the extent of H2B's contribution to plasminogen-mediated FCF by macrophages and if PARs are involved in this process., Approach and Results: Treating macrophages with plasminogen increases their oxidized low-density lipoprotein uptake and plasminogen-mediated foam cell formation (Plg-FCF) significantly. The magnitude of Plg-FCF correlates with cell-surface expression of the H2B level. H2B blockade or downregulation reduces Plg-FCF, whereas its overexpression or high endogenous levels increases Plg-FCF. Modulating PAR1 level in mouse macrophages affects Plg-FCF. Activation/overexpression of PAR1 increases and its blockade/knockdown reduces this response. Confocal imaging indicates that both H2B and PAR1 colocalize with clathrin coated pits on the surface of macrophages, and reducing expression of clathrin or interfering with the clathrin-coated pits integrity reduces Plg-FCF., Conclusion: Our data indicate that the magnitude of Plg-FCF by macrophages is proportional to the H2B levels and demonstrate for the first time that PAR1 is involved in this process and that the integrity of clathrin-coated pits is required for the full effect of Plg-induced FCF., (© 2021 International Society on Thrombosis and Haemostasis.)

Focal adhesions (FAs) are integrin-containing protein complexes regulated by a network of hundreds of protein-protein interactions. They are formed in a spatiotemporal manner upon the activation of integrin transmembrane receptors, which is crucial to trigger cell adhesion and many other cellular processes including cell migration, spreading and proliferation. Despite decades of studies, a detailed molecular level understanding on how FAs are organized and function is lacking due to their highly complex and dynamic nature. However, advances have been made on studying key integrin activators, talin and kindlin, and their associated proteins, which are major components of nascent FAs critical for initiating the assembly of mature FAs. This review will discuss the structural and functional findings of talin and kindlin and their immediate interaction network, which will shed light upon the architecture of nascent FAs and how they act as seeds for FA assembly to dynamically regulate diverse adhesion-dependent physiological and pathological responses., (© 2020 The Protein Society.)

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.

Aim: Sodium retention is the hallmark of nephrotic syndrome (NS) and mediated by the proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases. Plasmin is highly abundant in nephrotic urine and has been proposed to be the principal serine protease responsible for ENaC activation in NS. However, a proof of the essential role of plasmin in experimental NS is lacking., Methods: We used a genetic mouse model of NS based on an inducible podocin knockout (Bl6-Nphs2 tm3.1Antc *Tg(Nphs1-rtTA*3G) 8Jhm *Tg(tetO-cre) 1Jaw or nphs2 Δipod ). These mice were crossed with plasminogen deficient mice (Bl6-Plg tm1Jld or plg -/- ) to generate double knockout mice (nphs2 Δipod *plg -/- ). NS was induced after oral doxycycline treatment for 14 days and mice were followed for subsequent 14 days., Results: Uninduced nphs2 Δipod *plg -/- mice had normal kidney function and sodium handling. After induction, proteinuria increased similarly in both nphs2 Δipod *plg +/+ and nphs2 Δipod *plg -/- mice. Western blot revealed the urinary excretion of plasminogen and plasmin in nphs2 Δipod *plg +/+ mice which were absent in nphs2 Δipod *plg -/- mice. After the onset of proteinuria, amiloride-sensitive natriuresis was increased compared to the uninduced state in both genotypes. Subsequently, urinary sodium excretion dropped in both genotypes leading to an increase in body weight and development of ascites. Treatment with the serine protease inhibitor aprotinin prevented sodium retention in both genotypes., Conclusions: This study shows that mice lacking urinary plasminogen are not protected from ENaC-mediated sodium retention in experimental NS. This points to an essential role of other urinary serine proteases in the absence of plasminogen., (© 2020 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

The crosstalk between tumor cells and the adjacent normal epithelium contributes to cancer progression, but its regulators have remained elusive. Here, we show that breast cancer cells maintained in hypoxia release small extracellular vesicles (sEVs) that activate mitochondrial dynamics, stimulate mitochondrial movements, and promote organelle accumulation at the cortical cytoskeleton in normal mammary epithelial cells. This results in AKT serine/threonine kinase (Akt) activation, membrane focal adhesion turnover, and increased epithelial cell migration. RNA sequencing profiling identified integrin-linked kinase (ILK) as the most upregulated pathway in sEV-treated epithelial cells, and genetic or pharmacologic targeting of ILK reversed mitochondrial reprogramming and suppressed sEV-induced cell movements. In a three-dimensional (3D) model of mammary gland morphogenesis, sEV treatment induced hallmarks of malignant transformation, with deregulated cell death and/or cell proliferation, loss of apical-basal polarity, and appearance of epithelial-to-mesenchymal transition (EMT) markers. Therefore, sEVs released by hypoxic breast cancer cells reprogram mitochondrial dynamics and induce oncogenic changes in a normal mammary epithelium., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (© 2020 Federation of American Societies for Experimental Biology.)

Studies of isolated cells, mice, and humans have demonstrated the vital role of the FERM domain protein kindlin-3 in integrin activation in certain hematopoietic and non-hematopoietic cells, consequent to binding to integrin β-subunits. To explore regulatory mechanisms, we developed a monoclonal antibody that selectively recognizes the phosphorylated form of Ser 484 (pS 484 ) in kindlin-3. Activation of platelets, HEL megakaryocytic-like cells and BT549 breast cancer cells led to enhanced expression of pS 484 as assessed by immunofluorescence or Western blotting. In platelets, pS 484 rose rapidly and transiently upon stimulation. When a mutant form of kindlin-3, T 482 S 484 /AA kindlin-3, was transduced into mouse megakaryocytes, it failed to support activation of integrin α IIb β 3 , whereas wild-type kindlin-3 did. In MDA-MB231 breast cancer cells, expression of T 482 S 484 /AA kindlin-3 suppressed cell spreading, migration, invasion, and VEGF production. Wild-type kindlin-3 expressing cells markedly increased tumor growth in vivo, whereas T 482 S 484 /AA kindlin-3 significantly blunted tumor progression. Thus, our data establish that a unique phosphorylation event in kindlin-3 regulates its cellular functions., (© 2020 Bialkowska et al.)

Thrombospondin-4 (TSP-4) attracted renewed attention recently as a result of assignment of new functions to this matricellular protein in cardiovascular, muscular, and nervous systems. We have previously reported that TSP-4 promotes local vascular inflammation in a mouse atherosclerosis model. A common variant of TSP-4, P387-TSP-4, was associated with increased cardiovascular disease risk in human population studies. In a mouse atherosclerosis model, TSP-4 had profound effect on accumulation of macrophages in lesions, which prompted us to examine its effects on macrophages in more detail. We examined the effects of A387-TSP-4 and P387-TSP-4 on mouse macrophages in cell culture and in vivo in the model of LPS-induced peritonitis. In tissues and in cell culture, TSP-4 expression was associated with inflammation: TSP-4 expression was upregulated in peritoneal tissues in LPS-induced peritonitis, and pro-inflammatory signals, INFγ, GM-CSF, and LPS, induced TSP-4 expression in macrophages in vivo and in cell culture. Deficiency in TSP-4 in macrophages from Thbs4 -/- mice reduced the expression of pro-inflammatory macrophage markers, suggesting that TSP-4 facilitates macrophage differentiation into a pro-inflammatory phenotype. Expression of TSP-4, especially more active P387-TSP-4, was associated with higher cellular apoptosis. Cultured macrophages displayed increased adhesion to TSP-4 and reduced migration in presence of TSP-4, and these responses were further increased with P387 variant. We concluded that TSP-4 expression in macrophages increases their accumulation in tissues during the acute inflammatory process and supports macrophage differentiation into a pro-inflammatory phenotype. In a model of acute inflammation, TSP-4 supports pro-inflammatory macrophage apoptosis, a response that is closely related to their pro-inflammatory activity and release of pro-inflammatory signals. P387-TSP-4 was found to be the more active form of TSP-4 in all examined functions.

In cancer, cellular senescence is a complex process that leads to inhibition of proliferation of cells that may develop a neoplastic phenotype. A plethora of signaling pathways, when dysregulated, have been shown to elicit a senescence response. Two well-known tumor suppressor pathways, controlled by the p53 and retinoblastoma proteins, have been implicated in maintaining the cellular senescence phenotype. Kindlin-2, a member of an actin cytoskeleton organizing and integrin activator proteins, has been shown to play a key role in the regulation of several hallmarks of several cancers, including breast cancer (BC). The molecular mechanisms whereby Kindlin-2 regulates cellular senescence in BC tumors remains largely unknown. Here we show that Kindlin-2 regulates cellular senescence in part through its interaction with p53, whereby it regulates the expression of the p53-responsive genes; i.e., SerpinB2 and p21, during the induction of senescence. Our data show that knockout of Kindlin-2 via CRISPR/Cas9 in several BC cell lines significantly increases expression levels of both SerpinB2 and p21 resulting in the activation of hallmarks of cellular senescence. Mechanistically, interaction between Kindlin-2 and p53 at the promotor level is critical for the regulated expression of SerpinB2 and p21. These findings identify a previously unknown Kindlin-2/p53/SerpinB2 signaling axis that regulates cellular senescence and intervention in this axis may serve as a new therapeutic window for BCs treatment.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Thrombospondin-4 (TSP-4) belongs to the thrombospondin protein family that consists of five highly homologous members. A number of novel functions have been recently assigned to TSP-4 in cardiovascular and nervous systems, inflammation, cancer, and the motor unit, which have attracted attention to this extracellular matrix (ECM) protein. These newly discovered functions set TSP-4 apart from other thrombospondins. For example, TSP-4 promotes angiogenesis while other TSPs either prevent it or have no effect on new blood vessel growth; TSP-4 reduces fibrosis and collagen production while TSP-1 and TSP-2 promote fibrosis in several organs; unlike other TSPs, TSP-4 appears to have some structural functions in ECM. The current information about TSP-4 functions in different organs and physiological systems suggests that this evolutionary conserved protein is a major regulator of the extracellular matrix (ECM) organization and production and tissue remodeling during the embryonic development and response to injury. In this review article, we summarize the properties and functions of TSP-4 and discuss its role in tissue remodeling., (Copyright © 2017. Published by Elsevier B.V.)

Dynamic communication between integrin-containing complexes (focal adhesions, FAs) and actin filaments is critical for regulating cell adhesion. Pseudokinase ILK plays a key role in this process but the underlying mechanism remains highly elusive. Here we show that by recruiting FA adaptors PINCH and Parvin into a heterotrimeric complex (IPP), ILK triggers F-actin filament bundling - a process known to generate force/mechanical signal to promote cytoskeleton reassembly and dynamic cell adhesion. Structural, biochemical, and functional analyses revealed that the F-actin bundling is orchestrated by two previously unrecognized WASP-Homology-2 actin binding motifs within IPP, one from PINCH and the other from Parvin. Strikingly, this process is also sensitized to Mg-ATP bound to the pseudoactive site of ILK and its dysregulation severely impairs stress fibers formation, cell spreading, and migration. These data identify a crucial mechanism for ILK, highlighting its uniqueness as a pseudokinase to transduce non-catalytic signal and regulate cell adhesion.

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.

Current antithrombotic drugs, including widely used antiplatelet agents and anticoagulants, are associated with significant bleeding risk. Emerging experimental evidence suggests that the molecular and cellular mechanisms of hemostasis and thrombosis can be separated, thereby increasing the possibility of new antithrombotic therapeutic targets with reduced bleeding risk. We review new coagulation and platelet targets and highlight the interaction between integrin α M β 2 (Mac-1, CD11b/CD18) on leukocytes and GPIbα on platelets that seems to distinguish thrombosis from hemostasis., (© 2018 by The American Society of Hematology.)

Integrin-based therapeutics have garnered considerable interest in the medical treatment of inflammation. Integrins mediate the fast recruitment of monocytes and neutrophils to the site of inflammation, but are also required for host defense, limiting their therapeutic use. Here, we report a novel monoclonal antibody, anti-M7, that specifically blocks the interaction of the integrin Mac-1 with its pro-inflammatory ligand CD40L, while not interfering with alternative ligands. Anti-M7 selectively reduces leukocyte recruitment in vitro and in vivo. In contrast, conventional anti-Mac-1 therapy is not specific and blocks a broad repertoire of integrin functionality, inhibits phagocytosis, promotes apoptosis, and fuels a cytokine storm in vivo. Whereas conventional anti-integrin therapy potentiates bacterial sepsis, bacteremia, and mortality, a ligand-specific intervention with anti-M7 is protective. These findings deepen our understanding of ligand-specific integrin functions and open a path for a new field of ligand-targeted anti-integrin therapy to prevent inflammatory conditions.

Activation of transmembrane receptor integrin by talin is essential for inducing cell adhesion. However, the pathway that recruits talin to the membrane, which critically controls talin's action, remains elusive. Membrane-anchored mammalian small GTPase Rap1 is known to bind talin-F0 domain but the binding was shown to be weak and thus hardly studied. Here we show structurally that talin-F0 binds to human Rap1b like canonical Rap1 effectors despite little sequence homology, and disruption of the binding strongly impairs integrin activation, cell adhesion, and cell spreading. Furthermore, while being weak in conventional binary binding conditions, the Rap1b/talin interaction becomes strong upon attachment of activated Rap1b to vesicular membranes that mimic the agonist-induced microenvironment. These data identify a crucial Rap1-mediated membrane-targeting mechanism for talin to activate integrin. They further broadly caution the analyses of weak protein-protein interactions that may be pivotal for function but neglected in the absence of specific cellular microenvironments.

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., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest to disclose.

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 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., 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 β- and γ-catenin and actin, components of adherens junctions (AJ). Direct interaction of Kindlin-2 with β- 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 β- 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., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

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 ., (©2017 American Association for Cancer Research.)

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 αIIbβ3, 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 αIIbβ3 in human erythroleukemia (HEL) cells and β1 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 β3 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., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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.

It is well established that the gene expression patterns are substantially altered in cardiac hypertrophy and heart failure, however, less is known about the reasons behind such global differences. MicroRNAs (miRNAs) are short non-coding RNAs that can target multiple molecules to regulate wide array of proteins in diverse pathways. The goal of the study was to profile alterations in miRNA expression using end-stage human heart failure samples with an aim to build signaling network pathways using predicted targets for the altered miRNA and to determine nodal molecules regulating individual networks. Profiling of miRNAs using custom designed microarray and validation with an independent set of samples identified eight miRNAs that are altered in human heart failure including one novel miRNA yet to be implicated in cardiac pathology. To gain an unbiased perspective on global regulation by top eight altered miRNAs, functional relationship of predicted targets for these eight miRNAs were examined by network analysis. Ingenuity Pathways Analysis network algorithm was used to build global signaling networks based on the targets of altered miRNAs which allowed us to identify participating networks and nodal molecules that could contribute to cardiac pathophysiology. Majority of the nodal molecules identified in our analysis are targets of altered miRNAs and known regulators of cardiovascular signaling. Cardio-genomics heart failure gene expression public data base was used to analyze trends in expression pattern for target nodal molecules and indeed changes in expression of nodal molecules inversely correlated to miRNA alterations. We have used NF kappa B network as an example to show that targeting other molecules in the network could alter the nodal NF kappa B despite not being a miRNA target suggesting an integrated network response. Thus, using network analysis we show that altering key functional target proteins may regulate expression of the myriad signaling pathways underlying the cardiac pathology.

Integrins α M β 2 and α X β 2 are homologous adhesive receptors that are expressed on many of the same leukocyte populations and bind many of the same ligands. Although α M β 2 was extensively characterized and implicated in leukocyte inflammatory and immune functions, the roles of α X β 2 remain largely obscure. Here, we tested the ability of mice deficient in integrin α M β 2 or α X β 2 to deal with opportunistic infections and the capacity of cells derived from these animals to execute inflammatory functions. The absence of α M β 2 affected the recruitment of polymorphonuclear neutrophils (PMN) to bacterial and fungal pathogens as well as to model inflammatory stimuli, and α M β 2 -deficient PMN displayed defective inflammatory functions. In contrast, deficiency of α X β 2 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 α X β 2 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 α M β 2 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 α X β 2 , but not of α M β 2 , increased survival against a septic challenge with lipopolysaccharide (LPS) by 2-fold. Together, these results suggest that α M β 2 plays a primary role in PMN inflammatory functions and regulates the anti-inflammatory functions of Mϕ, whereas α X β 2 is central in the regulation of inflammatory functions of recruited and tissue-resident Mϕ., (Copyright © 2016 American Society for Microbiology.)

Adaptor proteins play a critical role in the assembly of signalling complexes after engagement of platelet receptors by agonists such as collagen, ADP and thrombin. Recently, using proteomics, the Dok (downstream of tyrosine kinase) adapter proteins were identified in human and mouse platelets. In vitro studies suggest that Dok-1 binds to platelet integrin β3, but the underlying effects of Dok-1 on αIIbβ3 signalling, platelet activation and thrombosis remain to be elucidated. In the present study, using Dok-1-deficient (Dok-1-/-) mice, we determined the phenotypic role of Dok-1 in αIIbβ3 signalling. We found that platelets from Dok-1-/- mice displayed normal aggregation, activation of αIIbβ3 (assessed by binding of JON/A), P-selectin surface expression (assessed by anti-CD62P), and soluble fibrinogen binding. These findings indicate that Dok-1 does not affect "inside-out" platelet signalling. Compared with platelets from wild-type (WT) mice, platelets from Dok-1-/- mice exhibited increased clot retraction (p < 0.05 vs WT), increased PLCγ2 phosphorylation, and enhanced spreading on fibrinogen after thrombin stimulation (p < 0.01 vs WT), demonstrating that Dok-1 negatively regulates αIIbβ3 "outside-in" signalling. Finally, we found that Dok-1-/- mice exhibited significantly shortened bleeding times and accelerated carotid artery thrombosis in response to photochemical injury (p < 0.05 vs WT mice). We conclude that Dok-1 modulates thrombosis and haemostasis by negatively regulating αIIbβ3 outside-in signalling.

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 αIIbβ3 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., (© 2016 Bledzka et al.)

Kindlins are 4.1-ezrin-ridixin-moesin (FERM) domain containing proteins. There are three kindlins in mammals, which share high sequence identity. Kindlin-1 is expressed primarily in epithelial cells, kindlin-2 is widely distributed and is particularly abundant in adherent cells, and kindlin-3 is expressed primarily in hematopoietic cells. These distributions are not exclusive; some cells express multiple kindlins, and transformed cells often exhibit aberrant expression, both in the isoforms and the levels of kindlins. Great interest in the kindlins has emerged from the recognition that they play major roles in controlling integrin function. In vitro studies, in vivo studies of mice deficient in kindlins, and studies of patients with genetic deficiencies of kindlins have clearly established that they regulate the capacity of integrins to mediate their functions. Kindlins are adaptor proteins; their function emanate from their interaction with binding partners, including the cytoplasmic tails of integrins and components of the actin cytoskeleton. The purpose of this review is to provide a brief overview of kindlin structure and function, a consideration of their binding partners, and then to focus on the relationship of each kindlin family member with cancer. In view of many correlations of kindlin expression levels and neoplasia and the known association of integrins with tumor progression and metastasis, we consider whether regulation of kindlins or their function would be attractive targets for treatment of cancer.

Unlabelled: Prostate cancer is the second leading cause of cancer-related death in men, second only to lung cancer, mainly due to disease reoccurrence as a result to lack of response to androgen deprivation therapies (ADT) after castration. Patients with metastatic castration-resistant prostate cancer (mCRPC) have very limited treatment options, with docetaxel as the first-line standard of care, for which resistance to this chemotherapeutic ultimately develops. Therefore, finding ways to sensitize tumors to chemotherapies and to limit chemoresistance provides a viable strategy to extend the survival of mCRPC patients. This study investigated the role of Kindlin-2 (FERMT2/K2), a member of the Kindlin family of FERM domain proteins and key regulators of the adhesive functions mediated by integrin, in the sensitization of mCRPC to chemotherapeutics. Loss of K2, which is overexpressed in prostate cancer cells derived from mCRPC tumors, compared with those cells derived from androgen-dependent tumors, significantly enhanced apoptosis and cell death of docetaxel-treated PC3 cells. Furthermore, it was determined that K2-mediated sensitization to docetaxel treatment is the result of inhibition of β1-integrin signaling. Finally, miR-138 specifically targeted K2 and inhibited its expression, thereby regulating a miR-138/K2/β1-integrin signaling axis in mCRPC that is critical for the modulation of sensitivity to chemotherapeutics. Thus, these data identify a novel signaling axis where K2 in combination with chemotherapeutics provides a new target for the treatment of mCRPC., Implications: Targeted inhibition of Kindlin-2 in combination with chemotherapy represents an effective treatment option for mCRPC., (©2015 American Association for Cancer Research.)

Activation of heterodimeric (αβ) integrin is crucial for regulating cell adhesion. Binding of talin to the cytoplasmic face of integrin activates the receptor, but how integrin is maintained in a resting state to counterbalance its activation has remained obscure. Here, we report the structure of the cytoplasmic domain of human integrin αIIbβ3 bound to its inhibitor, the immunoglobin repeat 21 of filamin A (FLNa-Ig21). The structure reveals an unexpected ternary complex in which FLNa-Ig21 not only binds to the C terminus of the integrin β3 cytoplasmic tail (CT), as previously predicted, but also engages N-terminal helices of αIIb and β3 CTs to stabilize an inter-CT clasp that helps restrain the integrin in a resting state. Combined with functional data, the structure reveals a new mechanism of filamin-mediated retention of inactive integrin, suggesting a new framework for understanding regulation of integrin activation and adhesion.

The contributions of integrins to cellular responses depend upon their activation, which is regulated by binding of proteins to their cytoplasmic tails. Kindlins are integrin cytoplasmic tail binding partners and are essential for optimal integrin activation, and kindlin-3 fulfills this role in hematopoietic cells. Here, we used human platelets and human erythroleukemia (HEL) cells, which express integrin αIIbβ3, to investigate whether phosphorylation of kindlin-3 regulates integrin activation. When HEL cells were stimulated with thrombopoietin or phorbol 12-myristate 13-acetate (PMA), αIIbβ3 became activated as evidenced by binding of an activation-specific monoclonal antibody and soluble fibrinogen, adherence and spreading on fibrinogen, colocalization of β3 integrin and kindlin-3 in focal adhesions, and enhanced β3 integrin-kindlin-3 association in immunoprecipitates. Kindlin-3 knockdown impaired adhesion and spreading on fibrinogen. Stimulation of HEL cells with agonists significantly increased kindlin-3 phosphorylation as detected by mass spectrometric sequencing. Thr(482) or Ser(484) was identified as a phosphorylation site, which resides in a sequence not conserved in kindlin-1 or kindlin-2. These same residues were phosphorylated in kindlin-3 when platelets were stimulated with thrombin. When expressed in HEL cells, T482A/S484A kindlin-3 decreased soluble ligand binding and cell spreading on fibrinogen compared with wild-type kindlin-3. A membrane-permeable peptide containing residues 476-485 of kindlin-3 was introduced into HEL cells and platelets; adhesion and spreading of both cell types were blunted compared with a scrambled control peptide. These data identify a role of kindlin-3 phosphorylation in integrin β3 activation and provide a basis for functional differences between kindlin-3 and the two other kindlin paralogs., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

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 lineages—but none of the premetazoans—had 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., (© 2015 Meller, Rogozin, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Chemoresistance allows for disease to recur and ultimately causes the death of most breast cancer patients. This scenario is particularly relevant in patients harboring triple-negative breast cancer (TNBC) tumors for which there are no effective FDA-approved drugs. However, a recent study determined that TNBCs can be segregated into 6 genetically distinct subtypes that do in fact exhibit differential rates of pathological complete response (pCR) to standard-of-care chemotherapies. Of these, the mesenchymal and mesenchymal stem-like subtypes of TNBCs exhibit the lowest rates of pCR when treated with standard-of-care chemotherapies. WAVE3 is an actin-cytoskeleton remodeling protein, and recent studies have highlighted a potential role for WAVE3 in promoting tumor progression and metastasis in TNBC. However, whether WAVE3 activity is involved in the development of chemoresistance in TNBCs remains unclear. Here we show that loss of WAVE3 expression resensitizes human TNBC cells to doxorubicin and docetaxel, as measured by increased apoptosis and cell death. We also show that WAVE3 knockdown in the chemotherapy-treated TNBC cells results in inhibition of STAT1 phosphorylation, as well as a significant decrease in expression levels of its downstream effector HIF-1α. Since HIF-1α is a major activator of VEGF-A production, and therefore a stimulator of tumor angiogenesis, loss of HIF-1α in the WAVE3-knockdown cells resulted in the inhibition the chemotherapy-mediated VEGF-A secretion and the downstream activation of angiogenesis, a phenomenon that often accompanies chemoresistance. Our data identify a critical role of WAVE3 in sensitizing TNBC to chemotherapy by inhibiting the STAT1→HIF-1α→VEGF-A signaling axis, and support the possibility that WAVE3 inhibition may be a promising target for TNBC cancer therapy.

In this issue of Blood, Stritt et al show that platelet functions dependent on integrin activation are unimpaired in mice lacking the Rap1-GTP-interacting adaptor molecule (RIAM).

The membrane localization and activation of cytoskeletal protein talin are key steps to initiate the integrin transmembrane receptors' activation, which mediates many cellular adhesive responses such as cell migration, spreading and proliferation. RIAM, a membrane anchor and small GTPase RAP1 effector, is known to bind to the C-terminal rod domain of talin (talin-R) and promote localizations of talin to the membrane. Through systematic mapping analysis, we find that RIAM also binds to the N-terminal head of talin (talin-H), a crucial domain involved in binding and activating integrins. We show that the RIAM binding to talin-H sterically occludes the binding of a talin-R domain that otherwise masks the integrin-binding site on talin-H. We further provide functional evidence that such RIAM-mediated steric unmasking of talin triggers integrin activation. Our findings thus uncover a novel role for RIAM in conformational regulation of talin during integrin activation and cell adhesion.

Polymorphonuclear neutrophils (PMNs) and macrophages are crucial contributors to neovascularization, serving as a source of chemokines, growth factors, and proteases. α(M)β(2)(CD11b/CD18) and α(L)β(2)(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), 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 β(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)β(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., (Copyright © 2014 by The American Association of Immunologists, Inc.)