Integrin-mediated Cellular Adhesion Research Articles

Coupling of a specific photoreactive triple-helical peptide to crosslinked collagen films restores binding and activation of DDR2 and VWF

Collagen-based scaffolds may require chemical crosslinking to achieve mechanical properties suitable for tissue engineering. Carbodiimide treatment, often used for this purpose, consumes amino acid side chains required for receptor recognition, thus reducing cell–collagen interaction. Here, we restore recognition and function of both von Willebrand Factor (VWF) and Discoidin Domain Receptor 2 (DDR2) to crosslinked collagen films by derivatisation with a specific triple-helical peptide (THP), an approach previously applied to integrin-mediated cellular adhesion. The THP contained the collagen III-derived active sequence, GPRGQOGVNleGFO, conjugated to a photoreactive moiety, diazirine, allowing UV-dependent covalent coupling to collagen films. Crosslinking of collagen films attenuated the binding of recombinant VWF A3 domain and of DDR2 (as the GST and Fc fusions, respectively), and coupling of the specific THP restored their attachment. These derivatised films supported activation of DDR2 expressed in either COS-7 or HEK293 cells, reflected by phosphorylation of tyrosine 740, and VWF-mediated platelet deposition from flowing blood was restored. Further, such films were able to increase low-density lipoprotein uptake in vascular endothelial cells, a marker for endothelial phenotype. Thus, covalent linkage of specific THPs to crosslinked collagen films i) restores their cognate protein binding, ii) triggers the corresponding cellular responses, and iii) demonstrates the broad applicability of the approach to a range of receptors for applications in regenerative medicine.

Abstract 5091: Synergistic apoptosis induced by combined PI3 kinase and bcl-xl inhibition in genotypic subsets of prostate cancer is attenuated by a feedback signaling loop via the alpha-5 integrin

Abstract Background: Integrin-mediated cellular adhesion to extracellular matrix components is a crucial regulator of tumor cell survival and chemoresistance. The integrin alpha 5 (ITGA5) plays a vital role in the migration and adhesion of prostate cancer (PCa) cells to fibronectin secreted by bone-derived mesenchymal stromal cells (Joshi, 2015), architects of a metastatic niche (Shiozawa, 2011). Hypothesis: The mechanism(s) whereby ITGA5 regulates the survival and chemoresistance in PCa may define novel therapeutic approaches. Methods and Results: To assess the pro-survival function of ITGA5 in PCa, we found that ITGA5 shRNA reduced bcl-2 and bcl-xl expression, and induced apoptosis in PC-3 cells. In these PTEN-mutant (mt) AR-negative(-) cells, pharmacological inhibition of the PI3K signaling pathway (BKM120) in combination with ITGA5 knockdown synergistically induced apoptosis as assessed by induction of cleaved caspase-3 and cleaved PARP. Similarly, BKM120 in combination with pharmacological inhibition of bcl-2/bcl-xL (ABT263) also induced apoptosis in synergistic fashion in PC-3 cells. Synergistic apoptosis with PI3-kinase inhibitors (BKM120, pictilisib) and ABT-263 was restricted to PTEN-mt PC-3 and LNCaP cells and was not observed in PTEN-wt, AR- DU145 cells. The pan-Akt-inhibitor GDC-0068 in combination with ABT263 induced apoptosis specifically in PTEN-mt AR+ LNCaP and C4-2B cells. By contrast, there was diminished evidence of synergy of PI3-Kinase inhibitors and bcl-2 specific inhibition with ABT199 pointing toward the requirement of bcl-xl inhibition. However, resistance to longer-term therapy with PI3K and bcl-xl/bcl-2 inhibition was observed in PC-3 but not LNCaP cells. Although single-agent PI3K inhibitor therapy downregulates ITGA5 expression in PC-3 cells, by contrast bcl-2/bcl-xl inhibitor therapy, alone or in combination with PI3K inhibitor therapy, strongly upregulates ITGA5 expression together with Akt activation suggesting that ITGA5 represents a pathway of resistance to combinatorial therapy through feedback loop signals generated with bcl-xl inhibition. In support of this hypothesis, ITGA5 knockdown together with BKM120 and ABT263 combinatorial therapy blocked the upregulation of ITGA5 and associated Akt activation, further reduced bcl-xl expression, and enhanced apoptosis in PC-3 cells. In PTEN-wild type AR-positive VCAP cells, evidence for synergistic apoptosis with PI3K and bcl-2/bcl-xl inhibition was also obtained suggesting that the TMPRSS2-ERG fusion may phenocopy the pro-survival pathways of PTEN-deficient cells. Conclusions: These data point toward a personalized pharmacological strategy combining PI3-kinase or Akt inhibition with bcl-2/bcl-xL inhibition in genotypic subsets of prostate cancer and identify a potential resistance pathway mediated by ITGA5-mediated signaling. Citation Format: Wenying Ren, Raghav Joshi, Paul Mathew. Synergistic apoptosis induced by combined PI3 kinase and bcl-xl inhibition in genotypic subsets of prostate cancer is attenuated by a feedback signaling loop via the alpha-5 integrin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5091.

Adhesion to fibronectin induces p27Kip1 nuclear accumulation through down-regulation of Jab1 and contributes to cell adhesion-mediated drug resistance (CAM-DR) in RPMI 8,226 cells

Mounting evidence has been shown that integrin-mediated cellular adhesion confers resistance to chemotherapy of multiple myeloma. The molecular mechanism underlying cell adhesion-mediated drug resistance (CAM-DR) is, however, poorly understood. In this report, we demonstrated that RPMI 8,226 cells accumulated p27(Kip1) in the nucleus when they were adhered to fibronectin (FN). The adhesion-mediated p27(Kip1) nuclear recruitment was regulated via the down-regulation of Jab1, a negative regulator of cell cycle. Overexpression of Jab1 reversed the elevated p27(Kip1) in the nucleus, which needed phosphorylation of p27(Kip1) on Serine 10, whereas inhibition of Jab1 by siRNA further increased the elevated p27(Kip1). Furthermore, we found overexpression of Jab1 did not affect 8,226 cells adhesion to FN, but reversed doxorubicin or mitoxantrone-induced CAM-DR phenotype. In conclusion, our data suggest that Jab1 plays an important role in CAM-DR, which depends on pSer10-p27(Kip1)-mediated subcellular localization of p27(Kip1). The understanding of this novel molecular mechanism may prove valuable in designing new therapeutic approaches for CAM-DR in Multiple myeloma.

Focal adhesions in osteoneogenesis

As materials technology and the field of tissue engineering advance, the role of cellular adhesive mechanisms, in particular, interactions with implantable devices, becomes more relevant in both research and clinical practice. A key tenet of medical device technology is to use the exquisite ability of biological systems to respond to the material surface or chemical stimuli in order to help to develop next-generation biomaterials. The focus of this review is on recent studies and developments concerning focal adhesion formation in osteoneogenesis, with an emphasis on the influence of synthetic constructs on integrin-mediated cellular adhesion and function.

Flightless I Regulates Hemidesmosome Formation and Integrin-Mediated Cellular Adhesion and Migration during Wound Repair

Flightless I (Flii), a highly conserved member of the gelsolin family of actin-remodelling proteins associates with actin structures and is involved in cellular motility and adhesion. Our previous studies have shown that Flii is an important negative regulator of wound repair. Here, we show that Flii affects hemidesmosome formation and integrin-mediated keratinocyte adhesion and migration. Impaired hemidesmosome formation and sparse arrangements of keratin cytoskeleton tonofilaments and actin cytoskeleton anchoring fibrils were observed in Flii(Tg/+) and Flii(Tg/Tg) mice with their skin being significantly more fragile than Flii(+/-) and WT mice. Flii(+/-) primary keratinocytes showed increased adhesion on laminin and collagen I than WT and Flii(Tg/Tg) primary keratinocytes. Decreased expression of CD151 and laminin-binding integrins alpha3, beta1, alpha6 and beta4 were observed in Flii overexpressing wounds, which could contribute to the impaired wound re-epithelialization observed in these mice. Flii interacts with proteins directly linked to the cytoplasmic domain of integrin receptors suggesting that it may be a mechanical link between ligand-bound integrin receptors and the actin cytoskeleton driving adhesion-signaling pathways. Therefore Flii may regulate wound repair through its effect on hemidesmosome formation and integrin-mediated cellular adhesion and migration.

Human periodontal fibroblast response to a nanostructured hydroxyapatite bone replacement graft in vitro

Objective The efficacy of nanostructured hydroxyapatite (NHA) for the treatment of osseous defects has been demonstrated in recent studies, even though the underlining biological mechanism is still poorly known. This study examined the alterations in cellular adhesion and mitogenic responses in human periodontal ligament (PDL) cells treated with a novel nanostructured hydroxyapatite bone graft substitute and characterized associated changes in cellular signalling pathways. Methods Cultured PDL cells were stimulated with NHA in a surface coated form. Proliferation was determined by bromodeoxyuridine (BrdU) incorporation and cell adhesion was analysed by a colorimetric assay. In order to understand altered adhesion properties of PDL fibroblasts their integrin profile was analysed and the phosphorylation status of focal adhesion kinase (FAK) and β1 integrin was determined by immunoblotting. In order to understand the signalling mechanisms of increased cell proliferation of PDL cells caused by NHA, the phosphorylation status of the serine/threonine protein kinase Akt, of the signal regulated kinases ERK1/2 and of the epidermal growth factor receptor (EGFR) was analysed by western blot using phospho-specific antibodies. Results The results indicated that NHA is a strong stimulator of PDL cell attachment and proliferation. Mechanistically, α5β1 integrin-mediated cellular adhesion of PDL fibroblasts, which resulted in altered phosphorylation and activation levels of FAK. Proliferation mediated by NHA was mechanistically caused by activation of the epidermal growth factor receptor (EGFR) pathway and its downstream targets ERK1/2 and Akt. Conclusions In sum, our findings present evidence that α5β1 integrin-mediated cellular adhesion of NHA to PDL fibroblasts, whereas proliferation was caused by activation of the epidermal growth factor receptor (EGFR) and the MAP kinase (ERK1/2) and Akt pathways.

Extracellular Signal-Regulated Kinase Activation during Renal Ischemia/Reperfusion Mediates Focal Adhesion Dissolution and Renal Injury

Acute renal failure due to ischemia/reperfusion involves disruption of integrin-mediated cellular adhesion and activation of the extracellular signal-regulated kinase (ERK) pathway. The dynamics of focal adhesion organization and phosphorylation during ischemia/reperfusion in relation to ERK activation are unknown. In control kidneys, protein tyrosine-rich focal adhesions, containing focal adhesion kinase, paxillin, and talin, were present at the basolateral membrane of tubular cells and colocalized with short F-actin stress fibers. Unilateral renal ischemia/reperfusion caused a reversible protein dephosphorylation and loss of focal adhesions. The focal adhesion protein phosphorylation rebounded in a biphasic manner, in association with increased focal adhesion kinase, Src, and paxillin tyrosine phosphorylation. Preceding phosphorylation of these focal adhesion proteins, reperfusion caused increased phosphorylation of ERK. The specific mitogen-activated protein kinase kinase 1/2 inhibitor U0126 prevented ERK activation and attenuated focal adhesion kinase, paxillin, and Src phosphorylation, focal adhesion restructuring, and ischemia/reperfusion-induced renal injury. We propose a model whereby ERK activation enhanced protein tyrosine phosphorylation during ischemia/reperfusion, thereby driving the dynamic dissolution and restructuring of focal adhesions and F-actin cytoskeleton during reperfusion and renal injury.

Focal adhesion kinase (pp125 FAK) phosphorylation is linked to force generation in F9 cells

Acute renal failure due to ischemia/reperfusion involves disruption of integrin-mediated cellular adhesion and activation of the extracellular signal-regulated kinase (ERK) pathway. The dynamics of focal adhesion organization and phosphorylation during ischemia/reperfusion in relation to ERK activation are unknown. In control kidneys, protein tyrosine-rich focal adhesions, containing focal adhesion kinase, paxillin, and talin, were present at the basolateral membrane of tubular cells and colocalized with short F-actin stress fibers. Unilateral renal ischemia/reperfusion caused a reversible protein dephosphorylation and loss of focal adhesions. The focal adhesion protein phosphorylation rebounded in a biphasic manner, in association with increased focal adhesion kinase, Src, and paxillin tyrosine phosphorylation. Preceding phosphorylation of these focal adhesion proteins, reperfusion caused increased phosphorylation of ERK. The specific mitogen-activated protein kinase kinase 1/2 inhibitor U0126 prevented ERK activation and attenuated focal adhesion kinase, paxillin, and Src phosphorylation, focal adhesion restructuring, and ischemia/reperfusion-induced renal injury. We propose a model whereby ERK activation enhanced protein tyrosine phosphorylation during ischemia/reperfusion, thereby driving the dynamic dissolution and restructuring of focal adhesions and F-actin cytoskeleton during reperfusion and renal injury.

Inhibitory effects of OK-432 (Picibanil) on cellular proliferation and adhesive capacity of breast carcinoma cells.

We investigated the potent inhibitory effects of OK-432 (Picibanil) on both cellular adhesion and cell proliferation of estrogen-dependent (MCF-7) or estrogen-independent (MDA-MB-231) breast carcinoma cells. Cellular proliferation of both MCF-7 and MDA-MB-231 cells was markedly inhibited in a dose-dependent manner, when the carcinoma cells were exposed to OK-432. Cell attachment assay demonstrated that incubation with OK-432 for 24 h reduced integrin-mediated cellular adhesion of both cell types. However, fluorescence activated cell sorter (FACS) analysis revealed that incubation with OK-432 for 24 h did not decrease the cell surface expressions of any integrins. These results suggest that the binding avidity of integrins is reduced by OK-432 without alteration of the integrin expression. We conclude that OK-432 inhibits integrin-mediated cellular adhesion as well as cell proliferation of breast carcinoma cells regardless of estrogen-dependence, and that these actions of OK-432 contribute to prevention or inhibition of breast carcinoma invasion and metastasis.

Heparin Modulates Integrin-Mediated Cellular Adhesion: Specificity of Interactions with α and β Integrin Subunits

Heparin Modulates Integrin-Mediated Cellular Adhesion: Specificity of Interactions with α and β Integrin Subunits

Heparin Modulates Integrin-Mediated Cellular Adhesion: Specificity of Interactions with α and β Integrin Subunits

Heparin is known to influence the growth, proliferation, and migration of vascular cells, but the precise mechanisms are unknown. We previously demonstrated that unfractionated heparin (UH) binds to the platelet integrin alpha(IIb)beta(3), and enhances ligand binding. To help define the specificity and site(s) of heparin-integrin interactions, we employed the erythroleukemic K562 cell line, transfected to express specific integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)). By comparing K562 cells expressing a common alpha subunit (Kalpha(v)beta(3), Kalpha(v)beta(5)) with cells expressing a common beta subunit (Kalpha(v)beta(3), Kalpha(IIb)beta(3)), we observed that heparin differentially modulated integrin-mediated adhesion to vitronectin. UH at 0.5-7.5 microg/ml consistently enhanced the adhesion of beta(3) expressing cells (Kalpha(v)beta(3),Kalpha(IIb)beta(3)). In contrast, UH at 0.5-7.5 microg/ml inhibited Kalpha(v)beta(5) adhesion. Experiments using integrin-blocking antibodies, appropriate control ligands, and nontransfected native K562 cells revealed that heparin's actions were mediated by the specific integrins under study. Preincubation of heparin with Kalpha(v)beta(3) cells enhanced adhesion, while preincubation of heparin with the adhesive substrate (vitronectin) had minimal effect. There was a structural specificity to heparin's effect, in that a low molecular weight heparin and chondroitin sulfate showed significantly less enhancement of adhesion. These findings suggest that heparin's modulation of integrin-ligand interactions occurs through its action on the integrin. The inhibitory or stimulatory effects of heparin depend on the beta subunit type, and the potency is dictated by structural characteristics of the glycosaminoglycan.

Cell adhesion-mediated drug resistance (CAM-DR) protects the K562 chronic myelogenous leukemia cell line from apoptosis induced by BCR/ABL inhibition, cytotoxic drugs, and gamma-irradiation.

Integrin-mediated cellular adhesion to extracellular matrix (ECM) components is an important determinant of chemotherapeutic response of human myeloma cells. Here, we demonstrate that when K562 chronic myelogenous leukemia (CML) cells are adhered to fibronectin (FN), they become resistant to apoptosis induced by the BCR/ABL inhibitors AG957 and STI-571, as well as DNA damaging agents and gamma-irradiation. This phenomenon, termed cell adhesion-mediated drug resistance (CAM-DR), was induced by adhesion through the alpha5beta1 (VLA-5) integrin. Phosphotyrosine analysis demonstrates that anti-apoptotic signaling through integrins in K562 cells is independent of the tyrosine kinases activated by BCR/ABL, with the possible exception of an unknown 80 kDa protein. Cytoprotection of FN-adhered CML cells indicates that tumor-ECM interactions may be critical for the emergence of drug-resistant tumor populations and treatment failure in this disease. Antagonists of beta1 integrin-mediated adhesion or corresponding signal transduction elements may sensitize CML cells to chemotherapy and prevent resistance to the novel BCR/ABL kinase inhibitors being used for the treatment of this disease.

Reelin binds alpha3beta1 integrin and inhibits neuronal migration.

Mice that are mutant for Reelin or Dab1, or doubly mutant for the VLDL receptor (VLDLR) and ApoE receptor 2 (ApoER2), show disorders of cerebral cortical lamination. How Reelin and its receptors regulate laminar organization of cerebral cortex is unknown. We show that Reelin inhibits migration of cortical neurons and enables detachment of neurons from radial glia. Recombinant and native Reelin associate with alpha3beta1 integrin, which regulates neuron-glia interactions and is required to achieve proper laminar organization. The effect of Reelin on cortical neuronal migration in vitro and in vivo depends on interactions between Reelin and alpha3beta1 integrin. Absence of alpha3beta1 leads to a reduction of Dab1, a signaling protein acting downstream of Reelin. Thus, Reelin may arrest neuronal migration and promote normal cortical lamination by binding alpha3beta1 integrin and modulating integrin-mediated cellular adhesion.

Moving Fer for Less Adhesion

Cadherin and integrin are proteins that mediate cellular adhesion at sites of cell-cell contact (cadherin) or sites of contact with the extracellular matrix (integrins). Two papers describe how cadherin- and integrin-mediated cellular adhesion are coordinately regulated. Li et al . cloned the ligand, neurocan, for the cell-surface galactosyltransferase that is associated with N-cadherin. Treatment of retinal cells with either full-length neurocan or an NH 2 fragment of neurocan inhibited cadherin- and integrin-mediated cell adhesion and neurite outgrowth and increased phosphorylation of β-catenin. The ability of neurocan to inhibit adhesion was dependent on the presence of the cell-surface galactosyltransferase. Arregui et al. disrupted cadherin- and integrin-mediated adhesion and neurite outgrowth with membrane-permeable peptides and found that the peptide mimicking the juxtamembrane domain of cadherin (JMP) caused the dissociation of the nonreceptor protein kinase Fer from the cadherin complex and its association with the integrin complex. The ability of JMP to inhibit integrin-mediated adhesion was dependent on the presence of N-cadherin and could be blocked with a Fer peptide that blocked the association of Fer with the integrin complex. Li et al . found that treatment of cells with neurocan also caused the release of Fer from the cadherin complex and association with the integrin complex. Thus, neurocan may be an endogenous ligand that promotes the cross-talk between cadherin and integrin complexes through relocalization of Fer. Li, H., Leung, T.-C., Hoffman, S., Balsamo, J., and Lilien, J. (2000) Coordinate regulation of cadherin and integrin function by the chondroitin sulfate proteoglycan neurocan. J. Cell Biol. 149 : 1275-1288. [Abstract] [Full Text] Arregui, C., Pathre, P., Lilien, J., and Balsamo, J. (2000) The nonreceptor tyrosine kinase Fer mediates cross-talk between the N-cadherin and β1-integrins. J. Cell Biol. 149 : 1263-1273. [Abstract] [Full Text]

Effects of interferon-alpha on cellular proliferation and adhesion of breast carcinoma cells.

We examined the potent inhibitory effects of interferon-alpha (IFN-alpha) on both cellular adhesion and cell proliferation of MCF-7 breast carcinoma cells. When MCF-7 cells were exposed to IFN-alpha at a concentration of 5x10(3) IU/ml for 5 days, cell proliferation was markedly inhibited. Cell attachment assay demonstrated that incubation with IFN-alpha for up to 48 h reduced alpha2beta1 integrin-mediated cellular adhesion. However, fluorescence activated cell sorter (FACS) analysis revealed that incubation with IFN-alpha for 24 h had no effect upon the cell surface expressions of either alpha2 and beta1 integrin on MCF-7 cells. These antiproliferative and antiadhesive actions of IFN-alpha may be applied to treatment for patients with metastatic breast carcinoma.

The anti-platelet and anti-thrombotic effects of FK633, a peptide-mimetic [formula omitted] antagonist

The anti-platelet and anti-thrombotic properties of FK633, a peptide mimetic GPIIb IIIa antagonist were studied. In human platelet rich plasma, FK633 inhibited ADP-, collagen-, thrombin-, and PAF-induced platelet aggregation with IC50 values of 103, 87, 98, and 239 nM, respectively. RGDS acted similarly, but it's potency was about 1,000 times weaker than FK633. FK633 inhibited 125I-fibrinogen binding to human washed platelet with an IC50 of 88 nM. FK633 did not inhibit α vβ 3, α 5β 1, and α vβ 1 integrin-mediated cellular adhesion up to 1.0mM, while RGDS inhibited all these interactions. In dogs, bolus injection of FK633 at 0.1 mg/kg significantly suppressed ex vivo ADP-induced platelet aggregation (>40% inhibition) and thrombus formation at stenosed and injured coronary artery, but did not prolong template bleeding time. However, FK633 inhibited >90% ADP-induced aggregation at 0.32 mg/kg, causing significant prolongation of the bleeding time. Thus, FK633 is a specific GPIIb IIIa antagonist with potent anti-thrombotic effect in vivo, but careful dosing study might be necessary to avoid the bleeding complications in the clinic.

Molecular Analysis and Developmental Expression of the Focal Adhesion Kinase pp125 FAK in Xenopus laevis

Integrin-mediated cellular adhesion to components of the extracellular matrix (ECM) is important in a number of morphogenetic events that occur during vertebrate embryogenesis. Recent studies suggest that the focal adhesion kinase pp125 FAK is involved in the regulation of integrin-dependent signaling processes triggered by cell adhesion to the ECM. We report the cDNA cloning and sequence analysis of the Xenopus homolog of pp125 FAK. We also describe temporal and spatial patterns of FAK expression during early development. Xenopus FAK shares greater than 90% identity with its avian and mammalian homologs. FAK mRNA and protein are present in the fertilized egg and in cleavage stage embryos. During gastrulation, FAK protein expression increases significantly and is detected in mesoderm, marginal zone ectoderm, and cells of the blastocoel roof. Later in development, FAK is prominently expressed at intersomitic junctions, in the brain, and in several cranial nerves. Phosphotyrosyl-FAK is first detected during gastrulation, suggesting that the phosphorylation of FAK on tyrosine is developmentally regulated. These data indicate that FAK is likely to participate in a variety of integrin-ECM-dependent signaling events during morphogenesis.