ALBERT

Description: Abstract 4 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. Our previous study demonstrated that reduction of kindlin-2 levels in human endothelial cells results in defective adhesive and migratory responses, suggesting that kindlin-2 may be implicated in angiogenesis. We tested this hypothesis in the kindlin-2+/− mice utilizing murine RM1 prostate tumor and Matrigel implant models. Staining of tumor sections for EC with CD31 showed shorter and thinner blood vessels and reduced vascular area by 3.5-fold in tumors grown in kindlin-2+/− mice compared to WT mice (P=0.0186, n=6). The vessels that did form in the kindlin-2+/− mice were immature as they lacked smooth muscle cells and pericytes, had thinner basement membrane, and were leaky as evidenced by increased by 2-fold area for plasma-derived fibrin in tumor sections (P=0.0006, n=5). Consistent with the blunted angiogenic response and vascular leakiness in the kindlin-2+/− mice, the tumors grown in kindlin-2+/− animals had 2-fold larger necrotic core and were 2.5-fold smaller than those derived in WT mice (P=0.042, n=7). Also, the permeability of preexisting blood vessels in ear and dorsal skin of WT and kindlin-2+/− mice was compared after injection of Evans blue dye. Baseline permeability of vasculature in ear and dorsal skin was enhanced by ∼70–100 % in kindlin-2+/− mice as compared WT mice (P

Description: Natural Killer (NK) cells are innate lymphocytes that play a central role in anti-viral and anti-tumor responses through direct cytotoxicity and production of inflammatory cytokines. Tumors can evade T-cell mediated immune-surveillance by down-regulation of the Class I Major Histocompatibility Complex (MHC-I) (Haworth et al, Ped Blood and Cancer, 2015). However, this lack of 'self' MHC-I serves as an activation stimulus for NK cells to recognize tumor cells. The molecular mechanism for 'self' recognition and destruction of 'missing self' are poorly understood. Integrins facilitate cell-to-cell interactions and are hypothesized to play a role in the 'self' versus 'missing self' recognition (Crozat et al, Blood, 2011). One of the critical regulators of integrin activation is Kindlin-3, which helps in their 'inside-out' signaling. Kindlin-3 binds to the cytoplasmic tail of β2-integrin and induces a conformational change increasing ligand affinity (Ye et al, Curr Biol, 2013). Consequently, Kindlin-3 is localized at the immunologic synapse and has been shown to interact with Adhesion and Degranulation Adaptor Protein (ADAP) (Kasirer-Friede et al, Blood 2014). As our group has shown, ADAP plays a central role in the signaling transduction for inflammatory cytokine production in NK cells (Rajasekaran et al, Nat Immunol, 2013). Clinically, defects in Kindlin-3 functions in humans are manifested by severe immune deficiency and bleeding disorder defined as Leukocyte Adhesion Deficiency-III (LAD-III). Based on these observations, we hypothesized Kindlin-3-dependent integrin function is critical for NK cell-mediated anti-tumor cytotoxicity and production of inflammatory cytokines. To define the role of Kindlin-3 in NK cell effector functions, we utilized a murine model. Kindlin-3 knock-in (K3KI) mice carry a double substitution mutation disrupting the binding of Kindlin-3 to β2-integrin (Xu et al, Arterioscler Thromb Vac Biol, 2014). NK cells from K3KI mice were evaluated for development and effector functions. Flow cytometry was utilized to identify maturation and developmental populations. Inflammatory cytokine production was assessed by Interferon-γ release following NK cell and tumor co-culture as well as plate-bound antibody activation. Cytotoxicity was assessed by 51Cr-release assay and the following tumor cells were used: cells representing, 1) 'self' (RMA and EL4 thymomas with autologous MHC-I); 2) 'missing-self' (RMA/S with decreased MHC expression relative to RMA); 3) 'induced self' (EL4 thymomas stably expressing H60, an activating ligand for NKG2D; 4) 'non-self' (YAC1 lymphoma with allogeneic MHC). Our results show an overall increase in the peripheral NK populations collected from spleens of K3KI mice, as seen in patients with LAD-III. However, no significant maturational defects were noted in the bone marrow of the K3KI mice. In vitro analyses reveal that K3KI NK cells have significantly impaired anti-tumor cytotoxicity relative to wild type controls (Figure 1). There was a significant reduction in the cytotoxic ability of K3KI NK cells towards 'induced self' or 'missing self' recognition (p

Description: Key Points Kindlin-2 regulates hemostasis in vivo by limiting CD39 and CD73 expression on the surface of endothelial cells. Kindlin-2 interacts directly with CHC and controls clathrin-dependent CD39 and CD73 endocytosis/recycling in endothelial cells.

Description: Key Points Kindlin-3–β2-integrin signaling in neutrophils is involved in regulation of both neutrophil recruitment and NET release. Disrupting the crosstalk between kindlin-3 and β2-integrins in neutrophils with a blocking peptide preferentially attenuates NET release.

Description: P-selectin glycoprotein ligand 1 (PSGL-1, CD162) and integrin αMβ2 (Mac-1, CD11bCD18) are leukocyte adhesion molecules essential for innate immunity and inflammation. The interaction of PSGL-1 with P-selectin (CD62P) mediates tethering, rolling, and weak adhesion of leukocytes, during which they become sufficiently activated in situ by locally released or displayed cytokines and chemoattractants for integrin-mediated firm adhesion. However, communication between P-selectin and the integrin, whether P-selectin can trigger β2-integrin activation, remains controversial. We found that P-selectin immunoglobulin chimera and PSGL-1 monoclonal antibodies (mAbs) increased adhesion of human neutrophils to immobilized, but not soluble, fibrinogen. This intermediate state of neutrophil adhesion was defined by moderate clustering of integrin αMβ2, no increase in CBRM1/5 (a mAb specific for the activation epitope on the αM subunit) recognition, and no increase in surface expression of αMβ2, whereas phorbol myristate acetate (PMA) induced extensive changes in these 3 parameters. Furthermore, platelet-activating factor or interleukin 8 acted in concert with P-selectin for further enhancing the activation of αMβ2. We thus propose a model in which P-selectin induces an intermediate state of integrin activation and then cooperates with other extracellular stimuli to support maximal adhesion of human neutrophils.

Description: 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.

Description: Integrin inside-out activation is essential for platelet aggregation mediated by αIIbβ3 and leukocytes migration and arresting mediated by αLβ2. How integrin is activated by the inside-out stimulation is not completely understood. Integrin activation from inside the cell is regulated through the transmembrane and cytoplasmic domains. Mutagenesis and structural studies revealed that the inactive integrin conformation is maintained by the specific interactions at the transmembrane and cytoplasmic domains. Inside-out signals impinging on integrin cytoplasmic domain disturb the transmembrane and cytoplasmic associations, resulting in conformational change of extracellular domain that is required for binding ligands. Studies on the mechanism of integrin inside-out activation have been focused on β cytoplasmic tail that is relatively conserved and bears binding sites for the common intracellular activators including talin and kindlin. The integrin α cytoplasmic tails only share a conserved GFFKR motif at the membrane-proximal region that forms specific interface with the membrane-proximal region of β cytoplasmic tail. The membrane-distal regions after the GFFKR motif are diverse significantly both in length and sequence. Their roles in integrin activation have not been well characterized. In this study, by comprehensive mutagenesis, we defined the role of the membrane-distal region of α integrin cytoplasmic tail in maintaining integrin in the resting state and in integrin inside-out activation. We found that complete deletion of the αIIb cytoplasmic membrane-distal region greatly enhances αIIbβ3 activation induced by the active mutations such as β3-K716A and β3-G708L, indicating that the missing of membrane-distal region facilitates integrin activation, i.e. the αIIb membrane-distal region contributes to the inactive integrin conformation. On the other hand, complete deletion of the αIIb membrane-distal region abolished integrin activation induced by the active mutations of αIIb-R995 and β3-D723, indicating that the αIIb membrane-distal region also contributes to integrin inside-out activation. We demonstrated that deletion of the membrane-distal region of αIIb, αV, or αL integrin greatly diminished ligand binding induced by overexpression of talin-1 head and/or kindlin-2 or -3 in 293FT cells. We further confirmed the effect of α cytoplasmic membrane-distal region on integrin inside-out activation in K562 cells. In the absence of αIIb cytoplasmic membrane-distal region, PMA failed to induce ligand binding to αIIbβ3 integrin expressed in K562 cells. This effect was due to the lack of talin-1-head and kindlin-induced integrin conformational change (ectodomain extension and headpiece opening) in the absence of α cytoplasmic membrane-distal region as reported by the conformation-dependent monoclonal antibodies. Structural superposition of αIIbβ3 transmembrane-cytoplasmic heterodimer and talin-1-head/β-tail complex reveals steric clashes between talin-1 head and the αIIb membrane-distal residues (NR997) immediately follow the GFFKR motif, which has been suggested to play a role in talin-mediated integrin activation. To test this possibility, we retained two native residues, NR997 for the αIIb membrane-distal region and found that it partially restores talin-1-head-induced integrin activation. Replacing the NR997 with small amino acids, GG997 or AA997 has little effect, while with the bulky residues YY997 significantly reduced talin-1-head-induced αIIbβ3 activation. Interestingly, retaining two native residues for the membrane-distal region of αV or αL integrin failed to restore talin-1-head-induced αVβ3 or αLβ2 activation. Retaining as long as 8 native residues for the αL membrane-distal region is not sufficient to restore talin-1-head-induced αLβ2 activation to the level of intact αL. These data demonstrate that a steric clash might play a role but is not the sole mechanism by which the α cytoplasmic membrane-distal region participates in integrin inside-out activation. A proper length and amino acids of the membrane-distal region is required for talin-induced integrin activation. Our data established an essential role of the α integrin cytoplasmic membrane-distal region in integrin activation and provide new insight of how talin and kindlin induce the high affinity integrin conformation that is required for fully functional integrins. Disclosures No relevant conflicts of interest to declare.