Integrin Cytoplasmic Domain Research Articles

Kindlin-2 interacts with and stabilizes EGFR and is required for EGF-induced breast cancer cell migration

Epidermal growth factor receptor (EGFR) mediates multiple signaling pathways that regulate cell proliferation, migration and tumor invasion. Kindlin-2 has been known as a focal adhesion molecule that binds to integrin to control cell migration and invasion. However, molecular mechanisms underlying the role of Kindlin-2 in breast cancer progression remain elusive. Here we report that Kindlin-2 interacts with EGFR and mediates EGF-induced breast cancer cell migration. We found that EGF treatment dramatically increases Kindlin-2 expression at both mRNA and protein levels in a variety of cancer cells. Inhibitors specific for EGFR or PI3K blocked Kindlin-2 induction by EGF. Importantly, Kindlin-2 interacted with EGFR kinase domain, which was independent of Kindlin-2 binding to integrin cytoplasmic domain. Intriguingly, Kindlin-2 stabilized EGFR protein by blocking its ubiquitination and degradation. Depletion of Kindlin-2 impaired EGF-induced cell migration. Our results demonstrated that Kindlin-2 participates in EGFR signaling and regulates breast cancer progression.

The Dual Structural Roles of the Membrane Distal Region of α Integrin Cytoplasmic Tail in Integrin inside-out Activation

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. DisclosuresNo relevant conflicts of interest to declare.

VPS33B, a Member of the Sec1/Munc18 Protein Family, Binds to Integrin Beta Subunits and Is Required for αIIbβ3 Outside-in Signaling

Integrins play fundamental roles in many biological processes such as development, immunity, cancer, wound healing, hemostasis, and thrombosis. Integrin activation is essential for cell adhesion, spreading, survival, proliferation, and migration. Integrins are heterodimeric transmembrane glycoproteins composed of a and b subunits. The function of integrins is modulated by bi-directional transmembrane signaling: inside-out and outside-in signaling, which are mediated through the interactions between integrin a or b subunit cytoplasmic tails and intracellular proteins and can be regulated by many different biochemical signaling pathways. αIIbβ3 is a major integrin expressed in megakaryocytes and platelets. Antagonists of αIIbβ3 are potent anti-thrombotic drugs and new inhibitors targeting αIIbβ3 are under preclinical testing or large patient trials to treat acute coronary syndromes. Integrin αIIbβ3 activation involves binding of proteins, including talin, kindlins, Src kinase, and Gα13 to the cytoplasmic domain of its β subunit. To gain insight into αIIbβ3 signaling and to identify new proteins that regulate αIIbβ3 activation, agarose beads conjugated with glutathione S-transferase (GST)-β3 integrin cytoplasmic domain fusion protein (GST-β3CD) or GST-αIIb integrin cytoplasmic domain fusion protein (GST-αIIbCD) were incubated with human platelet lysates. Proteins pulled down by the GST-β3CD or GST-αIIbCD beads were subjected to mass spectrometric analysis. We found that the GST-β3CD but not the GST-αIIbCD beads specifically pulled down a previously unreported protein, the vacuolar protein sorting-associated protein 33B (VPS33B), encoded by the VPS33B gene. To verify that VPS33B is in a complex with αIIbβ3, we infected Chinese hamster ovary (CHO) cells stably expressing αIIbβ3 (A5 cells) with an adenovirus containing FLAG epitope-tagged human VPS33B cDNA. Cells were allowed to adhere and spread on fibrinogen. FLAG-VPS33B exhibited a significant intracellular colocalization with aIIbb3. Immunoblotting analysis revealed a specific association of b3 with precipitated FLAG-VPS33B. The specific interaction was also confirmed by reciprocal immunoprecipitation using a specific β3 antibody. Furthermore, in an in vitro binding assay, we were able to pull down VPS33B from the lysates of CHO cells overexpressing VPS33B by the GST-β3CD beads. The interaction appears to be direct, since we were also able to pull down purified FLAG-VPS33B with GST-β3CD and GST-β1CD proteins. The pulldown assay showed that the β3 fragment spanning residues 716~730 bound efficiently to FLAG-VPS33B. In contrast, the fragments containing either residues 729~762 or residues 744~762 fragments failed to do the same. More importantly, using the rabbit polyclonal antibody against VPS33B, we were able to coimmunoprecipitate endogenous b3 subunit with VPS33B from human platelet lysate. Thus, we identified a novel binding protein of integrin β3.Next, we investigated the role of VPS33B in integrin activation using a recombinant integrin activation model of CHO cells. Overexpression of VPS33B in CHO cells expressing αIIbβ3 (A5 cells) markedly potentiated cell spreading on fibrinogen and F-actin formation. To establish a role of VPS33B in integrin activation in platelets, we created a mouse model with megakaryocyte- and platelet-specific deletion of VPS33B. Platelets lacking VPS33B were defective in spreading on fibrinogen. VPS33B-/- platelets failed to support clot retraction. On the other hand, thrombin-induced fibrinogen binding to platelets and platelet aggregation were not affected by the loss of VPS33B. Collectively, these results demonstrate an essential role of VPS33B in αIIbβ3 outside-in signaling but is not requried for integrin inside-out signaling. We further demonstrate that VPS33B promotes αIIbβ3 outside-in signaling through RhoA and Rac1 activation, leading to clot retraction and cell spreading, respectively. Therefore, our results for the first time establish vesicle trafficking proteins as an important novel class of modifiers of integrin function in platelets and cells. DisclosuresNo relevant conflicts of interest to declare.

A Novel β3 Intracytoplasmic Domain Mutation Associated with Macrothrombocytopenia: Structural Analysis in Comparison with Previously Reported Cases

Background: Glanzmann thrombasthenia (GT) is a moderate to severe inherited bleeding syndrome associating a lack of platelet aggregation with quantitative or qualitative deficiencies of the αIIbβ3 integrin. Rare patients with mutations in ITGA2B or ITGB3 genes show a variable GT-like phenotype associated with macrothrombocytopenia (MTP). Gain-of-function mutations that localize to the integrin cytoplasmic domains cause autosomal dominant MTP. We now report a novel heterozygous ΔLeu in ITGB3 causing a structural modification of the cytoplasmic domain of β3 in a family with moderate MTP. In silico modeling was performed to obtain structural insights into this and other known mutations affecting cytoplasmic domains of αIIb and β3 and impacting megakaryopoiesis.Aim: To help define structural changes in αIIbβ3 linked to MTP.Results: A novel in-frame heterozygous 3bp deletion (c.2230_2232delCTC) was identified by exome sequencing performed as part of the BRIDGE-BPD consortium project in a French pedigree with lifelong mucocutaneous bleeding. The consequence is the loss of β3 Leu718 (ΔL718) the second L of a LLITIHD motif of the mature protein (nomenclature used throughout). Platelets of the proband have (i) moderate expression of αIIbβ3, (ii) a major reduction in fibrinogen (Fg) and PAC-1 binding after ADP stimulation yet (iii) spontaneous Fg or PAC-1 binding to platelets was not seen. Western blotting showed a slightly slower migration of β3 and normal αIIb. The proband had a moderate MTP with platelet size heterogeneity and frequent enlarged α-granules regrouping several of normal size with Fg bound to the membrane of the α-granules as revealed by immunogold staining and electron microscopy. Platelet aggregation was much reduced with all physiologic agonists including TRAP with a reduced surface expression of P-selectin and diminished secretion. The same 3bp del in ITGB3 co-segregates with the platelet phenotype and bleeding across 3 generations in the family. We then performed molecular modeling (PyMol Molecular Graphics System version 1.3) to compare the structural effects of this mutation on αIIbβ3 with other cytoplasmic tail mutations described in the literature giving GT-like phenotypes and/or MTP. Firstly αIIb-R995Q/W or β3-D723H were confirmed to disrupt a salt bridge between the integrin subunits and induce the separation of their tails. PyMol modeling showed that the DL718 results in D723 being no longer in phase with R995 thereby disrupting the salt bridge. Significantly, introduction of a β-turn by an activating β3 L718P substitution again results in the spontaneous separation of the tails, once more through a conformational change of the mutated β3. In contrast, a previously reported ITGB3 C2268T variant resulting in Arg724*, a truncation coming after the deletion in our patient, or a β3 S752P substitution that completely blocks platelet aggregation and PAC-1 binding (and specific loss of kindlin-3 binding) despite a capacity to secrete a storage pool of Fg bound α-granule αIIbβ3 failed to give MTP despite resulting in a GT-like syndrome.Conclusions: The novel ITGB3 ΔL718 of β3 showed an intermediate phenotype with MTP, a much decreased platelet aggregation, a reduced activity of αIIbβ3 at the platelet surface but a progressive fusion of α-granules. This is despite the theoretical retention of the talin and kindlin-3 binding sites suggesting that transmembrane signaling is the principle defect. Our studies highlight how considerable phenotypic heterogeneity exists in patients with genetic variants affecting αIIb and β3 cytoplasmic domains and suggest different signaling mechanisms for surface and intracellular αIIbβ3 pools.*PN and ET; WHO and ATN made equal contributions,On behalf of the BRIDGE-Bleeding and Platelet Disorder Consortium DisclosuresNo relevant conflicts of interest to declare.

Cytoplasmic Domain Interactions of Syndecan-1 and Syndecan-4 with α6β4 Integrin Mediate Human Epidermal Growth Factor Receptor (HER1 and HER2)-dependent Motility and Survival

Epithelial cells are highly dependent during wound healing and tumorigenesis on the α6β4 integrin and its association with receptor tyrosine kinases. Previous work showed that phosphorylation of the β4 subunit upon matrix engagement depends on the matrix receptor syndecan (Sdc)-1 engaging the cytoplasmic domain of the β4 integrin and coupling of the integrin to human epidermal growth factor receptor-2 (HER2). In this study, HER2-dependent migration activated by matrix engagement is compared with migration stimulated by EGF. We find that whereas HER2-dependent migration depends on Sdc1, EGF-dependent migration depends on a complex consisting of human epidermal growth factor receptor-1 (HER1, commonly known as EGFR), α6β4, and Sdc4. The two syndecans recognize distinct sites at the extreme C terminus of the β4 integrin cytoplasmic domain. The binding motif in Sdc1 is QEEXYX, composed in part by its syndecan-specific variable (V) region and in part by the second conserved (C2) region that it shares with other syndecans. A cell-penetrating peptide containing this sequence competes for HER2-dependent epithelial migration and carcinoma survival, although it is without effect on the EGFR-stimulated mechanism. β4 mutants bearing mutations specific for Sdc1 and Sdc4 recognition act as dominant negative mutants to block cell spreading or cell migration that depends on HER2 or EGFR, respectively. The interaction of the α6β4 integrin with the syndecans appears critical for it to be utilized as a signaling platform; migration depends on α3β1 integrin binding to laminin 332 (LN332; also known as laminin 5), whereas antibodies that block α6β4 binding are without effect. These findings indicate that specific syndecan family members are likely to have key roles in α6β4 integrin activation by receptor tyrosine kinases.

Membrane tension drives ligand-independent integrin signaling.

Integrins anchor cells to the extracellular matrix (ECM) and control a multitude of essential cellular functions by activating a variety of signaling pathways. In this issue of The EMBO Journal, a study conducted by Ferraris and colleagues report that binding of urokinase-type plasminogen activator receptor (uPAR) to vitronectin is sufficient to trigger ligand-independent β1 and β3 integrin signaling. The coupling of uPAR and integrins occurs independent of lateral interaction between the two receptors, but relies on membrane tension (Ferraris et al, 2014).

Sorting Nexin 31 Binds Multiple β Integrin Cytoplasmic Domains and Regulates β1 Integrin Surface Levels and Stability

Trafficking of α5β1 integrin to lysosomes and its subsequent degradation is influenced by ligand occupancy and the binding of SNX17 via its protein 4.1, ezrin, radixin, moesin (FERM) domain to the membrane-distal NPxY motif in the cytoplasmic domain of β1 integrin in early endosomes. Two other sorting nexin (SNX) family members, namely SNX27 and SNX31, share with SNX17 next to their obligate phox domain a FERM domain, which may enable them to bind β integrin tails. Here we report that, in addition to SNX17, SNX31 but not SNX27 binds several β integrin tails in early endosomes in a PI3 (phosphatidylinositide 3)-kinase-dependent manner. Similarly like SNX17, binding of SNX31 with β1 integrin tails in early endosomes occurs between the FERM domain and the membrane-distal NPxY motif in the β1 integrin cytoplasmic domain. Furthermore, expression of SNX31 rescues β1 integrin surface levels and stability in SNX17-depleted cells. In contrast to SNX17, expression of SNX31 is restricted and found highly expressed in bladder and melanoma tissue. Altogether, these results demonstrate that SNX31 is an endosomal regulator of β integrins with a restricted expression pattern.

Overexpression of the muscle-specific protein, melusin, protects from cardiac ischemia/reperfusion injury.

Melusin is a muscle-specific protein which interacts with β1 integrin cytoplasmic domain and acts as chaperone protein. Its overexpression induces improved resistance to cardiac overload delaying left ventricle dilation and reducing the occurrence of heart failure. Here, we investigated possible protective effect of melusin overexpression against acute ischemia/reperfusion (I/R) injury with or without Postconditioning cardioprotective maneuvers. Melusin transgenic (Mel-TG) mice hearts were subjected to 30-min global ischemia followed by 60-min reperfusion. Interestingly, infarct size was reduced in Mel-TG mice hearts compared to wild-type (WT) hearts (40.3 ± 3.5 % Mel-TG vs. 59.5 ± 3.8 % WT hearts; n = 11 animals/group; P < 0.05). The melusin protective effect was also demonstrated by measuring LDH release, which was 50 % lower in Mel-TG compared to WT. Mel-TG hearts had a higher baseline level of AKT, ERK1/2 and GSK3β phosphorylation, and displayed increased phospho-kinases level after I/R compared to WT mice. Post-ischemic Mel-TG hearts displayed also increased levels of the anti-apoptotic factor phospho-BAD. Importantly, pharmacological inhibition of PI3K/AKT (Wortmannin) and ERK1/2 (U0126) pathways abrogated the melusin protective effect. Notably, HSP90, a chaperone known to protect heart from I/R injury, showed high levels of expression in the heart of Mel-TG mice suggesting a possible collaboration of this molecule with AKT/ERK/GSK3β pathways in the melusin-induced protection. Postconditioning, known to activate AKT/ERK/GSK3β pathways, significantly reduced IS and LDH release in WT hearts, but had no additive protective effects in Mel-TG hearts. These findings implicate melusin as an enhancer of AKT and ERK pathways and as a novel player in cardioprotection from I/R injury.

Regulated Splicing of the α6 Integrin Cytoplasmic Domain Determines the Fate of Breast Cancer Stem Cells

Although the α6β1 integrin has been implicated in thefunction of breast and other cancer stem cells (CSCs), little is known about its regulation and relationship to mechanisms involved in the genesis of CSCs. We report that a CD44(high)/CD24(low) population, enriched for CSCs, is comprised of distinct epithelial and mesenchymal populations that differ in expression of the two α6 cytoplasmic domain splice variants: α6A and α6B. α6Bβ1 expression defines the mesenchymal population and is necessary for CSC function, a function that cannot be executed by α6Aintegrins. The generation of α6Bβ1 is tightly controlled and occurs as a consequence of an autocrine vascular endothelial growth factor (VEGF) signaling that culminates in the transcriptional repression of a key RNA-splicing factor. These data alter our understanding of how α6β1 contributes tobreast cancer, and they resolve ambiguities regarding the use of total α6 (CD49f) expression as a biomarker for CSCs.

IGF-IR Promotes Prostate Cancer Growth by Stabilizing α5β1 Integrin Protein Levels

Dynamic crosstalk between growth factor receptors, cell adhesion molecules and extracellular matrix is essential for cancer cell migration and invasion. Integrins are transmembrane receptors that bind extracellular matrix proteins and enable cell adhesion and cytoskeletal organization. They also mediate signal transduction to regulate cell proliferation and survival. The type 1 insulin-like growth factor receptor (IGF-IR) mediates tumor cell growth, adhesion and inhibition of apoptosis in several types of cancer. We have previously demonstrated that β1 integrins regulate anchorage-independent growth of prostate cancer (PrCa) cells by regulating IGF-IR expression and androgen receptor-mediated transcriptional functions. Furthermore, we have recently reported that IGF-IR regulates the expression of β1 integrins in PrCa cells. We have dissected the mechanism through which IGF-IR regulates β1 integrin expression in PrCa. Here we report that IGF-IR is crucial for PrCa cell growth and that β1 integrins contribute to the regulation of proliferation by IGF-IR. We demonstrate that β1 integrin regulation by IGF-IR does not occur at the mRNA level. Exogenous expression of a CD4 - β1 integrin cytoplasmic domain chimera does not interfere with such regulation and fails to stabilize β1 integrin expression in the absence of IGF-IR. This appears to be due to the lack of interaction between the β1 cytoplasmic domain and IGF-IR. We demonstrate that IGF-IR stabilizes the β1 subunit by protecting it from proteasomal degradation. The α5 subunit, one of the binding partners of β1, is also downregulated along with β1 upon IGF-IR knockdown while no change is observed in the expression of the α2, α3, α4, α6 and α7 subunits. Our results reveal a crucial mechanistic role for the α5β1 integrin, downstream of IGF-IR, in regulating cancer growth.

Calcium and Calmodulin-dependent Serine/Threonine Protein Kinase Type II (CaMKII)-mediated Intramolecular Opening of Integrin Cytoplasmic Domain-associated Protein-1 (ICAP-1α) Negatively Regulates β1 Integrins

Focal adhesion turnover during cell migration is an integrated cyclic process requiring tight regulation of integrin function. Interaction of integrin with its ligand depends on its activation state, which is regulated by the direct recruitment of proteins onto the β integrin chain cytoplasmic domain. We previously reported that ICAP-1α, a specific cytoplasmic partner of β1A integrins, limits both talin and kindlin interaction with β1 integrin, thereby restraining focal adhesion assembly. Here we provide evidence that the calcium and calmodulin-dependent serine/threonine protein kinase type II (CaMKII) is an important regulator of ICAP-1α for controlling focal adhesion dynamics. CaMKII directly phosphorylates ICAP-1α and disrupts an intramolecular interaction between the N- and the C-terminal domains of ICAP-1α, unmasking the PTB domain, thereby permitting ICAP-1α binding onto the β1 integrin tail. ICAP-1α direct interaction with the β1 integrin tail and the modulation of β1 integrin affinity state are required for down-regulating focal adhesion assembly. Our results point to a molecular mechanism for the phosphorylation-dependent control of ICAP-1α function by CaMKII, allowing the dynamic control of β1 integrin activation and cell adhesion.

Integrin inactivators: balancing cellular functions in vitro and in vivo

Integrins mediate cell-matrix and cell-cell interactions and integrate extracellular cues to the cytoskeleton and cellular signalling pathways. Integrin function on the cell surface is regulated by their activity switching such that intracellular proteins interacting with the integrin cytoplasmic domains increase or decrease integrin-ligand binding affinity. It is widely accepted that integrin activation by specific proteins is essential for cell adhesion and integrin linkage to the actin cytoskeleton. However, there is also increasing evidence that integrin-inactivating proteins are crucial for appropriate integrin function in vitro and in vivo and that the regulation of integrin-ligand interactions is a fine-tuned balancing act between inactivation and activation.

Osteoblast mineralization requires β1 integrin/ICAP-1–dependent fibronectin deposition

The morphogenetic and differentiation events required for bone formation are orchestrated by diffusible and insoluble factors that are localized within the extracellular matrix. In mice, the deletion of ICAP-1, a modulator of β1 integrin activation, leads to severe defects in osteoblast proliferation, differentiation, and mineralization and to a delay in bone formation. Deposition of fibronectin and maturation of fibrillar adhesions, adhesive structures that accompany fibronectin deposition, are impaired upon ICAP-1 loss, as are type I collagen deposition and mineralization. Expression of β1 integrin with a mutated binding site for ICAP-1 recapitulates the ICAP-1-null phenotype. Follow-up experiments demonstrated that ICAP-1 negatively regulates kindlin-2 recruitment onto the β1 integrin cytoplasmic domain, whereas an excess of kindlin-2 binding has a deleterious effect on fibrillar adhesion formation. These results suggest that ICAP-1 works in concert with kindlin-2 to control the dynamics of β1 integrin-containing fibrillar adhesions and, thereby, regulates fibronectin deposition and osteoblast mineralization.

Integrin-linked kinase at a glance

Integrins are a large family of cell–extracellular matrix (ECM) and cell–cell adhesion molecules that regulate development and tissue homeostasis by controlling cell migration, survival, proliferation and differentiation ([Hynes, 2002][1]). They are non-covalently associated heterodimers

Abstract 2459: Membrane type 1 matrix metalloproteinase and integrin-linked kinase: A characterization of potential partners in ovarian cancer metastasis

Abstract Epithelial ovarian cancer (EOC) is the fifth leading cause of overall cancer death among American women. In 2010, 15,520 deaths were directly attributed to EOC; and 21,650 additional cases were diagnosed. When EOC is diagnosed prior to metastatic dissemination, the overall 5 year survival rate is 93%; however, over 75% of women with EOC are diagnosed with metastasis already present, dropping the survival rate to less than 20%. The process of EOC metastasis is unique in that epithelial cells detach from the primary tumor and shed into the peritoneal cavity as single cells and multicellular aggregates (MCA), which adhere intraperitoneally and invade the interstitial collagen-rich submesothelial matrix, where they proliferate to anchor secondary lesions. Proteolytic activity promotes the migration and collagen invasion crucial to metastatic success. Also key to cell migratory activity are focal adhesions (FA), integrin- and protease-rich cell adhesion sites that physically link the actin cytoskeleton to the extracellular matrix (ECM) and transduce signals into the intracellular compartment. Sites of adhesive contacts with collagen fibers have been associated with beta-1 integrin clustering and various matrix metalloproteinases. Membrane type 1 matrix metalloproteinase (MT1-MMP), an ECM degrading protease localized to the cell surface, has been shown to promote a collagen-invasive phenotype in ovarian carcinomas. Additionally, collagen-activated integrin-linked kinase (ILK), a beta-1 integrin cytoplasmic domain interactor, has been shown to regulate several biological processes that promote invasion and suppress anoikis. As initial steps to assess the contributions and potential inter-relationships of tumor cell invasion at FAs, presented here is a characterization of MT1-MMP and ILK expression in select ovarian cancer cell lines and in human EOC tumor tissue microarrays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2459. doi:1538-7445.AM2012-2459

β1 Integrin Gene Excision in the Adult Murine Cardiac Myocyte Causes Defective Mechanical and Signaling Responses

How mechanical signals are transmitted in the cardiac myocyte is poorly understood. In this study, we produced a tamoxifen-inducible mouse model in which β1 integrin could be reduced specifically in the adult cardiomyocyte, so that the function of this integrin could be assessed in the postnatal and mechanically stressed heart. The expression of β1 integrin was reduced to 35% of control levels, but function remained normal at baseline. With aortic constriction, the knockout mice survived but had a blunted hypertrophic response. Integrin knockout myocytes, in contrast to controls, showed reduced integrin-linked kinase expression both at baseline and after hemodynamic stress; focal adhesion kinase expression was reduced after stress. Alterations in multiple signaling pathways were detected in the integrin knockout group after acute and chronic hemodynamic stress. Most remarkably, when we challenged the knockout mice with short-term loading, the robust responses of several kinases (extracellular signal-regulated kinase 1/2, p38, and Akt) evident in control mice were essentially abolished in the knockout mice. We also found that reduction of myocyte β1 integrin expression modified adrenergic-mediated signaling through extracellular signal-regulated kinase, p38, and Akt. Reduction of β1 integrin expression in the mature cardiac myocyte leads to a varied response compared with when this protein is reduced during either the embryonic or perinatal period. These results show that β1 integrin expression is required for proper mechanotransductive and adrenergic responses of the adult heart.

Focal adhesion proteins Zyxin and Vinculin are co-distributed at tubulobulbar complexes

Tubulobulbar complexes (TBCs) are actin-related double-membrane invaginations formed at intercellular junctions in the seminiferous epithelium of mammalian testis. They occur at basal junction complexes between neighboring Sertoli cells and at apical junctions between Sertoli cells and spermatids. They are proposed to internalize intercellular junctions during the translocation of spermatocytes from basal to adluminal compartments of the seminiferous epithelium, and during sperm release from Sertoli cells. Although TBCs are specific to the seminiferous epithelium, they morphologically resemble podosomes in osteoclasts. Previously, we have reported that a key group of proteins consisting of N-WASp, Arp2/3, cortactin and dynamin that occur at podosomes also is present at TBCs. Here we explore the prediction that zyxin, a focal adhesion protein known to be present at podosomes, also is present at apical TBCs. A rabbit polyclonal anti-zyxin antibody (B71) was used to label fixed fragments and frozen sections of testis. In both fragments and sections, B71 labeled tubular regions of TBCs at apical sites of attachment between Sertoli cells and spermatids, in addition to being localized at actin related intercellular adhesion junctions termed ectoplasmic specializations. Although the function of zyxin at TBCs has yet to be determined, the protein is known to interact with the cytoplasmic domain of integrins at focal adhesions, and integrins are known to be present in TBCs.

Novel Anti-Cancer Peptides Comprising Three Amino Acids

Background: The NPxY motif common to all β integrin cytoplasmic domains forms part of a canonical recognition sequence for phosphotyrosine-binding domains which are protein modules present in a wide variety of signaling and cytoskeletal proteins. We have recently reported that a non-naturally occurring peptide, RSKAKNPLYR, derived from the β6 integrin cytoplasmic domain inhibits cancer cell growth in vitro and proposed that this may be due, at least in part, to the inhibition of c-Src activity [1]. In the present study we examined the role of the NPLY motif within RSKAKNPLYR in terms of its requirement for inhibition of cancer cell growth. Materials and Methods: The effects of peptide modifications to RSKAKNPLYR on in vitro proliferation of human cancer cell lines (colorectal HT29, prostate DU145, breast MCF-7 and ovarian A2780) were evaluated using the MTT cell growth assay. Passage of peptide across the plasma membrane was assessed by means of confocal microscopy using FITC-labelled peptide. The effect of peptide on kinase activity was assessed in cell-free in vitro kinase assays. Results: The NPLY motif within RSKAKNPLYR was found to be essential for the growth inhibitory effect of this peptide. However, modified forms of this peptide in which all amino acids except the charged residues arginine and lysine were replaced by single non-polar amino acids such as alanine or valine were equally effective at inhibiting cancer cell proliferation. Moreover, these peptides inhibited not only c-Src activity as seen for RSKAKNPLYR but also the activity of members of the PKB/Akt kinase family. Conclusion: Novel decapeptides comprising only three amino acids have anti-cancer effects without the requirement for an integrin-based NPLY motif. These peptides inhibit the activity of not only c-Src but also members of the Akt family of kinases and may be useful as potential anti-cancer agents when used either alone or in combination with compounds previously reported to inhibit c-Src kinase activity.

Kindlin

The modulation of integrin affinity is central to platelet and leukocyte function. Two proteins, talin and kindlin, that interact with distinct regions of integrin cytoplasmic domains, have been shown to play essential roles in inducing the high affinity integrin conformation required for platelet and leukocyte adhesive interactions.Here we highlight some of the key studies that have described roles for talin and kindlin in integrin function and discuss several models that explain how talin and kindlins might work together to regulate integrin activation. Genetic deletion of kindlin-3 in mice results in platelet and leukocyte adhesive dysfunction associated with profoundly impaired activation of multiple classes of integrins, a phenotype similar to that observed in talin-deficient platelets and leukocytes. Since this initial report three years ago, numerous studies have provided important clues to how kindlins activate integrins and, in some cases, the relationship between kindlins and talin in integrin activation. Clearly, talin and kindlins are key regulators of integrin affinity. Future experiments that define precisely how these molecules work in concert should provide important insights into the terminal signaling events that activate integrins.

Osteoblast mineralization requires β1 integrin/ICAP-1–dependent fibronectin deposition

The morphogenetic and differentiation events required for bone formation are orchestrated by diffusible and insoluble factors that are localized within the extracellular matrix. In mice, the deletion of ICAP-1, a modulator of β1 integrin activation, leads to severe defects in osteoblast proliferation, differentiation, and mineralization and to a delay in bone formation. Deposition of fibronectin and maturation of fibrillar adhesions, adhesive structures that accompany fibronectin deposition, are impaired upon ICAP-1 loss, as are type I collagen deposition and mineralization. Expression of β1 integrin with a mutated binding site for ICAP-1 recapitulates the ICAP-1–null phenotype. Follow-up experiments demonstrated that ICAP-1 negatively regulates kindlin-2 recruitment onto the β1 integrin cytoplasmic domain, whereas an excess of kindlin-2 binding has a deleterious effect on fibrillar adhesion formation. These results suggest that ICAP-1 works in concert with kindlin-2 to control the dynamics of β1 integrin–containing fibrillar adhesions and, thereby, regulates fibronectin deposition and osteoblast mineralization.