Lumican inhibits cell migration through α2β1 integrin

Lumican, an extracellular matrix protein of the small leucine-rich proteoglycan family, has been shown to impede melanoma progression by inhibiting cell migration. In the present study, we show that lumican targets α2β1 integrin thereby inhibiting cell migration. A375 melanoma cells were transfected with siRNA directed against the α2 integrin subunit. Compared to A375 control cells, the anti-migratory effect of lumican was abrogated on transfected A375 cells. Moreover, lumican inhibited the chemotactic migration of Chinese hamster ovary (CHO) cells stably transfected with α2 integrin subunit (CHO-A2) but not that of wild-type CHO cells (CHO-WT) lacking this subunit. In contrast to CHO-WT cells, we observed in time-lapse microscopy a decrease of CHO-A2 cell migration speed in presence of lumican. Focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) and total FAK were analysed in CHO-WT and CHO-A2 cells. A significant decrease of the ratio pFAK/FAK was shown in presence of recombinant human lumican. Using solid phase assays, a direct binding between lumican and the α2β1 integrin was demonstrated. This interaction did not involve the glycan moiety of lumican and was cation independent. Lumican was also able to bind the activated I domain of the α2 integrin subunit with a K_d ≥ 200 nM. In conclusion, we demonstrated for the first time that the inhibition of cell migration by lumican depends on a direct binding between the core protein of lumican and the α2β1 integrin.     

Beta-tricalcium phosphate exerts osteoconductivity through α2β1 integrin and down-stream MAPK/ERK signaling pathway

Beta-tricalcium phosphate (β-TCP) has been clinically used as a bone graft substitute for decades because of its excellent osteoconductivity. However, the exact mechanism(s) by which β-TCP exerts osteoconductivity are not fully documented. This study was aimed to investigate the molecular mechanism(s) by which β-TCP modulates the biological response of primary human osteoblasts (HOBs). It was showed that HOBs seeded into the β-TCP scaffolds expressed significantly higher levels of osteogenic genes, compared to those cultured on tissue culture plastic; meanwhile these cells showed 7-fold increase in α2 integrin subunit gene expression and the activation of the mitogen-activated protein kinase (MAPK)/extracellular related kinase (ERK) signaling pathway. In addition, the osteogenic conduction by β-TCP scaffolds was attenuated directly by inhibiting MAPK/ERK or indirectly by blocking the α2β1 integrin signaling pathway. We concluded that β-TCP scaffold exerts osteoconductivity through α2β1 integrin and down-stream MAPK/ERK signaling pathway, suggesting a feasible approach to consider when designing or fabricating the scaffolds for bone tissue engineering.     

The α7β1 integrin in muscle development and disease

The alpha7beta1 integrin is a laminin receptor on the surface of skeletal myoblasts and myofibers. Alternative forms of both the alpha7 and beta1 chains are expressed in a developmentally regulated fashion during myogenesis. These different alpha7beta1 isoforms localize at specific sites on myofibers and appear to have distinct functions in skeletal muscle. These functions include the migration and proliferation of developing myoblasts, the formation and integrity of neuromuscular and myotendinous junctions, and the "gluing" together of muscle fibers that is essential to the generation of contractile force. The alpha7beta1 integrin appears to be both directly and indirectly causally related to several muscle diseases. Enhanced expression of alpha7beta1-mediated linkage of the extracellular matrix is seen in Duchenne muscular dystrophy and may compensate for the absence of the dystrophin-mediated linkage. Downregulation of expression of the integrin may contribute to the development of pathology in congenital laminin deficiencies. Mutations in the alpha7 integrin gene underlie additional congenital muscle diseases. The functional roles of this integrin in the formation and stability of the neuromuscular and myotendinous junctions and its localization between fibers suggest that altered expression or function of this integrin may have widespread involvement in other myopathies. The localization of the alpha7 gene at human chromosome 12q13 is a useful clue for focusing such studies.     

RGD cadherins and α2β1 integrin in cancer metastasis: A dangerous liaison

We propose a new cadherin family classification comprising epithelial cadherins (cadherin 17 [CDH17], cadherin 16, VE-cadherin, cadherin 6 and cadherin 20) containing RGD motifs within their sequences. Expression of some RGD cadherins is associated with aggressive forms of cancer during the late stages of metastasis, and CDH17 and VE-cadherin have emerged as critical actors in cancer metastasis. After binding to α2β1 integrin, these cadherins promote integrin β1 activation, and thereby cell adhesion, invasion and proliferation, in liver and lung metastasis. Activation of α2β1 integrin provokes an affinity increase for type IV collagen, a major component of the basement membrane and a critical partner for cell anchoring in liver and other metastatic organs. Activation of α2β1 integrin by RGD motifs breaks an old paradigm of integrin classification and supports an important role of this integrin in cancer metastasis. Recently, synthetic peptides containing the RGD motif of CDH17 elicited highly specific and selective antibodies that block the ability of CDH17 RGD to activate α2β1 integrin. These monoclonal antibodies inhibit metastatic colonization in orthotopic mouse models of liver and lung metastasis for colorectal cancer and melanoma, respectively. Hopefully, blocking the cadherin RGD ligand capacity will give us control over the integrin activity in solid tumors metastasis, paving the way for development of new agents of cancer treatment.     

The αMβ2 integrin and its role in neutrophil function

Neutrophils are the first cell type to arrive at the injury sites and play a critical role in host defense,by virtue of its ability to adhere and transmigrate through endothelium,to phagocytose foreign pathogens,and to produce free oxygen radicals and proteolytic enzymes.Yet,inappropriate neutrophil activation causes tissue damage and various inflammatory diseases.These physiological and pathological functions of neutrophils depend on the engagement of certain surface receptors,especially αMβ2,the major β2 integrin receptor present on neutrophil surface.Understanding of the molecular mechanisms underlying ligand binding by αMβ2,as well as the rolea of αMβ2ligand interactions in neutrophil functions will enable us to regulate more precisely neutrophil activities:that is,to promote their host defense functions,and at the same time to minimize their deleterious effects of normal cells.     

Hepatocyte growth factor upregulates α2β1 integrin in Madin-Darby canine kidney cells: Implications in tubulogenesis

It has been well established that hepatocyte growth factor (HGF) induces branching tubule formation of Madin-Darby canine kidney (MDCK) cells cultured in collagen gel. Tubulogenesis per se requires the involvement of cell proliferation, migration, focalization proteolysis, cell-cell interaction and differentiation. However, signaling pathways and proteins involved in HGF-induced tubulogenesis by MDCK cells have not been thoroughly studied. Because cell-matrix interactions play important roles in tubulogenesis, we analyzed whether HGF altered the expression of extracellular matrix receptor (alpha2, alpha3, beta1 and alphavbeta3 integrin). We found that among those proteins examined, alpha2beta1 integrin levels were enhanced by HGF. HGF-induced upregulation of alpha2beta1 integrin was mediated via upregulation of alpha2 integrin mRNA abundance. Cycloheximide blocked the HGF-induced increase in alpha2 integrin mRNA expression. To understand the signaling pathways leading to an HGF-induced increase in alpha2beta1 integrin levels, PD98059 (MEK1 inhibitor), LY294002 (PI3-kinase inhibitor), and GF109203X (PKC inhibitor) were used. We found that PD98059 blocked the HGF-induced increase in alpha2beta1 integrin expression. Furthermore, 5E8 (specific anti-alpha2beta1 integrin antibody) was employed to elucidate the potential role of HGF-induced upregulation of alpha2beta1 integrin in branching morphogenesis. 5E8 did not alter HGF-induced scattering effects but disrupted HGF-induced branching tubulogenesis in collagen gel via inhibition of cell-cell interactions and growth. Taken together, HGF upregulates alpha2beta1 integrin expression via an indirect pathway, the results of which contribute to the regulation of cell-cell interactions and cell growth during branching morphogenesis in collagen gel.     

Mechanical force regulates integrin turnover in Drosophila in vivo

Regulated assembly and disassembly,?or?turnover,?of integrin-mediated cell-extracellular matrix?(ECM)?adhesions is essential for dynamic cell movements?and?long-term tissue maintenance. For example, in Drosophila,?misregulation of integrin turnover disrupts muscle-tendon?attachment at myotendinous junctions (MTJs). We demonstrate that?mechanical force, which modulates integrin activity, also regulates integrin and intracellular adhesion complex (IAC) turnover in vivo. Using conditional mutants to alter the tensile force on MTJs, we found that the proportion of IAC components undergoing turnover inversely correlated with the force applied on MTJs. This effect was disrupted by point mutations in β-integrin that interfere with ECM-induced conformational changes and activation of β-integrin or integrin-mediated cytoplasmic signalling. These mutants also disrupted integrin dynamics at MTJs during larval development. Together, these data suggest that specific β-integrin-mediated signals regulate adhesion turnover in response to tension during tissue?formation. We propose that integrin-ECM adhesive stability is continuously controlled by force in vivo through integrin-dependent auto-regulatory feedback mechanisms so that tissues can quickly adapt to and withstand mechanical stresses.     

CCN2 promotes keratinocyte adhesion and migration via integrin α5β1

Background

CCN2, (a.k.a. connective tissue growth factor and CTGF) has emerged as a regulator of cell migration. While the importance of CCN2 for the fibrotic process in wound healing has been well studied, the effect of CCN2 on keratinocyte function is not well understood. In this study, we investigated the mechanism behind CCN2-driven keratinocyte adhesion and migration.Materials and methods: Adhesion assays were performed by coating wells with 10 μg/ml fibronectin (FN) or phosphate-buffered saline (PBS). Keratinocytes were seeded in the presence or absence of 200 ng/ml CCN2, 5 mmol/l ethylenediaminetetraacetic acid, 10 mmol/l cations, 500 μl arginine–glycine–aspartic acid (RGD), 500 μM arginine–glycine–glutamate–serine (RGES), and 10 μg/ml anti-integrin blocking antibodies. Migration studies were performed using a modified Boyden chamber assay. Quantitative PCR was used to study the effect of CCN2 on integrin subunit mRNA expression. To block intracellular pathways, keratinocytes were pretreated with 20 μM PD98059 (MEK-1 inhibitor) or 20 μM PF573228 (FAK inhibitor) for 60 min prior the addition of CCN2. Western blot was used to measure CCN2, p-ERK1/2, and ERK1/2.Results: CCN2 enhanced keratinocyte adhesion to fibronectin via integrin α5β1. The addition of anti-integrin α5β1 antibodies reduced CCN2-mediated keratinocyte migration. In addition, CCN2 regulated mRNA and protein expression of integrin subunits α5 and β1. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1-specific inhibitor PD98059 markedly reduced CCN2-induced keratinocyte migration.Conclusions: Our results demonstrate that CCN2 enhances keratinocyte adhesion and migration through integrin α5β1 and activation of the FAK-MAPK signaling cascade.     

Discovery of orally available integrin α5β1 antagonists

Previous research within our laboratories identified the 3-hydroxypyrrolidine scaffold 1 as a new and selective integrin α5β1 inhibitor class which was designed for local administration. Herein the discovery of new orally available integrin α5β1 inhibitor scaffolds for potential systemic treatment is described.     

Mena binds α5 integrin directly and modulates α5β1 function

Mena is an Ena/VASP family actin regulator with roles in cell migration, chemotaxis, cell-cell adhesion, tumor cell invasion, and metastasis. Although enriched in focal adhesions, Mena has no established function within these structures. We find that Mena forms an adhesion-regulated complex with α5β1 integrin, a fibronectin receptor involved in cell adhesion, motility, fibronectin fibrillogenesis, signaling, and growth factor receptor trafficking. Mena bound directly to the carboxy-terminal portion of the α5 cytoplasmic tail via a 91-residue region containing 13 five-residue "LERER" repeats. In fibroblasts, the Mena-α5 complex was required for "outside-in" α5β1 functions, including normal phosphorylation of FAK and paxillin and formation of fibrillar adhesions. It also supported fibrillogenesis and cell spreading and controlled cell migration speed. Thus, fibroblasts require Mena for multiple α5β1-dependent processes involving bidirectional interactions between the extracellular matrix and cytoplasmic focal adhesion proteins.     

αv integrin processing interferes with the cross-talk between αvβ5/β6 and α2β1 integrins

Background information. Previous studies have reported that cross‐talk between integrins may be an important regulator of integrin—ligand binding and subsequent signalling events that control a variety of cell functions in many tissues. We previously demonstrated that αvβ5/β6 integrin represses α2β1‐dependent cell migration. The αv subunits undergo an endoproteolytic cleavage by protein convertases, whose role in tumoral invasion has remained controversial. Results. Inhibition of convertases by the convertase inhibitor α1‐PDX (α1‐antitrypsin Portland variant), leading to the cell‐surface expression of an uncleaved form of the αv integrin, stimulated cell migration toward type I collagen. Under convertase inhibition, α2β1 engagement led to enhanced phosphorylation of both FAK (focal adhesion kinase) and MAPK (mitogen‐activated protein kinase). This outside‐in signalling stimulation was associated with increased levels of activated β1 integrin located in larger than usual focal‐adhesion structures and a cell migration that was independent of the PI3K (phosphoinositide 3‐kinase)/Akt (also called protein kinase B) pathway. Conclusions. The increase in cell migration observed upon convertases inhibition appears to be due to the up‐regulation of β1 integrins and to their location in larger focal‐adhesion structures. The endoproteolytic cleavage of αv subunits is necessary for αvβ5/β6 integrin to control α2β1 function and could thus play an essential role in colon cancer cell migration.     

Loss of the α2β1 integrin alters human papilloma virus-induced squamous carcinoma progression in vivo and in vitro

Expression of the α2β1 integrin, a receptor for collagens and laminin, is altered during tumor progression. Recent studies have linked polymorphisms in the α2 integrin gene with oral, squamous cell carcinoma (SCC). To determine the α2β1 integrin's role in SCC progression, we crossed α2-null mice with K14-HPV16 transgenic animals. Pathological progression to invasive carcinoma was evaluated in HPV-positive, α2-null (HPV/KO) and HPV-positive, wild-type (HPV/WT) animals. α2β1 integrin expression stimulated progression from hyperplasia and papillomatosis to dysplasia with concomitant dermal mast cell infiltration. Moreover, lymph node metastasis was decreased by 31.3% in HPV/KO, compared to HPV/WT, animals. To evaluate the integrin-specific impact on the malignant epithelium versus the microenvironment, we developed primary tumor cell lines. Although transition from dysplasia to carcinoma was unaltered during spontaneous tumor development, isolated primary HPV/KO SCC cell lines demonstrated decreased migration and invasion, compared to HPV/WT cells. When HPV/WT and HPV/KO SCC cells were orthotopically injected into WT or KO hosts, tumor α2β1 integrin expression resulted in decreased tumor latency, regardless of host integrin status. HPV/WT SCC lines failed to demonstrate a proliferative advantage in vitro, however, the HPV/WT tumors demonstrated increased growth compared to HPV/KO SCC lines in vivo. Although contributions of the integrin to the microenvironment cannot be excluded, our studies indicate that α2β1 integrin expression by HPV-transformed keratinocytes modulates SCC growth and progression.     

Phosphoenolpyruvate-dependent inhibition of collagen biosynthesis, α2β1 integrin and IGF-I receptor signaling in cultured fibroblasts

The mechanism of collagen biosynthesis regulation is not fully understood. The finding that prolidase plays an important role in collagen biosynthesis and phosphoenolpyruvate inhibits prolidase activity "in vitro" led to evaluate its effect on collagen biosynthesis in cultured human skin fibroblasts. Confluent fibroblasts were treated with millimolar concentrations (1-4 mM) of phosphoenolpyruvate monopotassium salt (PEP) for 24 h. It was found that PEP-dependent decrease in prolidase activity and expression was accompanied by parallel decrease in collagen biosynthesis. However, the experiments with inhibitor of PEP production, 3-mercaptopicolinate revealed no direct correlation between collagen biosynthesis and prolidase activity and expression. Since insulin-like growth factor (IGF-I) is the most potent stimulator of both collagen biosynthesis and prolidase activity, and prolidase is regulated by beta(1) integrin signaling, the effect of PEP on IGF-I receptor (IGF-IR) and beta(1) integrin receptor expressions were evaluated. It was found that the exposure of the cells to 4 mM PEP contributed to a decrease in IGF-IR and beta(1) integrin receptor expressions. The data suggest that PEP-dependent decrease of collagen biosynthesis in cultured human skin fibroblasts may undergo through depression of alpha(2)beta(1) integrin and IGF-IR signaling. The hypothetical mechanism of the role of prolidase in IGF-IR, beta(1) integrin receptor expressions, and clinical significance of the process are discussed.     

17β-Estradiol protects against apoptosis induced by levofloxacin in rat nucleus pulposus cells by upregulating integrin α2β1

Levofloxacin has been reported to have cytotoxicity to chondrocytes in vitro. And 17β-estradiol has been widely studied for its protective effects against cell apoptosis. Based on apoptotic cell model induced by levofloxacin, the purpose of this study was to explore the mechanism by which 17β-estradiol protects rat nucleus pulposus cells from apoptosis. Inverted phase-contrast microscopy, flow cytometry, and caspase-3 activity assay were used to find that levofloxacin induced marked apoptosis, which was abolished by 17β-estradiol. Interestingly, estrogen receptor antagonist, ICI182780, and functional blocking antibody to α₂β₁ integrin, both prohibited the effect of 17β-estradiol. Simultaneously, levofloxacin decreased cellular binding ability to type II collagen, which was also reversed by 17β-estradiol. Furthermore, western blot and real-time quantitative PCR were used to find that integrin α₂β₁ was responsible for estrogen-dependent anti-apoptosis, which was time–response and dose–response effect. 17β-estradiol was proved for the first time to protect rat nucleus pulposus cells against levofloxacin-induced apoptosis by upregulating integrin α₂β₁ signal pathway.     

Molecular dynamics simulations to the bidirectional adhesion signaling pathway of integrin αVβ3

The bidirectional force transmission process of integrin through the cell membrane is still not well understood. Several possible mechanisms have been discussed in literature on the basis of experimental data, and in this study, we investigate these mechanisms by free and steered molecular dynamics simulations. For the first time, constant velocity pulling on the complete integrin molecule inside a dipalmitoyl-phosphatidylcholine membrane is conducted. From the results, the most likely mechanism for inside-out and outside-in signaling is the switchblade model with further separation of the transmembrane helices.     

Modeling the molecular basis for α4β1 integrin antagonism

We report a 3D QSAR study of almost 300 structurally diverse small molecule antagonists of the integrin α4β1 whose biological activity spans six orders of magnitude. The alignment of the molecules was based on the conformation of a structurally related ligand bound to the αIIBβ3 and αvβ3 integrins in X-ray crystallographic studies. The molecular field method, CoMSIA, was used to generate the 3D QSAR models. The resulting models showed that the lipophilic properties were the most important, with hydrogen bond donor and steric properties less relevant. The models were highly significant (r(2)=0.89, q2(LOO)=0.67, r(2) (test set)=0.76), and could make robust predictions of the data (SEE=0.46, SEP=0.78, SEP (test set)=0.66). We predicted the antagonist activities of a further ten compounds with useful accuracy. The model appears capable of predicting α4β1 integrin antagonist activity to within a factor of five for compounds within its domain of applicability. The implications for design of improved integrin antagonists will be discussed.     

IGF-I enhances α5β1 integrin expression and cell motility in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Integrins are the major adhesive molecules in mammalian cells and have been associated with metastasis of cancer cells. Insulin-like growth factor-I (IGF)-I plays an important role in regulating cell growth, proliferation, survival, and metabolism. However, the effects of IGF-I in migration and integrin expression in chondrosarcoma cells are largely unknown. In this study, we found that IGF-I increased the migration and the expression of α5β1 integrin in human chondrosarcoma cells. Pretreatment of cells with IGF-I receptor antibody reduced IGF-I-induced cell migration and integrin expression. Activations of phosphatidylinositol 3-kinase (PI3K), Akt, and nuclear factor-κB (NF-κB) pathways after IGF-I treatment were demonstrated, and IGF-I-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of PI3K, Akt, and NF-κB cascades. Taken together, our results indicated that IGF-I enhances the migration of chondrosarcoma cells by increasing α5β1 integrin expression through the IGF-I receptor/PI3K/Akt/NF-κB signal transduction pathway.     

Modulation of α2β1 integrin changes during mammary gland development by β-oestradiol

In order to study the role of cell–matrix interactions in mammary gland function, temporal changes in α₂β₁ integrin, the major receptor for collagen and the influence of β-oestradiol on its level and distribution in rat mammary gland at different stages of development were studied. The level of α₂β₁ integrin determined by ELISA, was found to be high during different days of pregnancy, while in the lactating stage, it was significantly reduced. By immunocytochemical analysis, α₂β₁ integrin was found to be localized towards the luminal side of acinar cells, both in the virgin and midpregnant stage, while it was not detected in the lactating stage. The possible role of hormones in modulating the level of integrin was examined in both in vitro and in vivo experiments using β-oestradiol. Supplementing β-oestradiol to isolated mammary epithelial cells from both virgin and lactating glands caused a concentration dependent increase in the incorporation of [³⁵S]methionine into α₂β₁ integrin associated with the cells. Administration of β-oestradiol to virgin and lactating glands caused about 1.4–4-fold increase in the level of α₂ integrin, indicating that upregulation of integrin during pregnancy may be due to oestrogen and as the oestrogen level falls during lactating phase, downregulation of α₂β₁ integrin occurs. Treatment with β-oestradiol also resulted in the appearance of α₂β₁ integrin in the acinar region of the lactating tissue, while in the untreated controls no staining for integrin was seen. These results indicate that oestrogen, apart from directly affecting the cellular activity, can influence mammary tissue function by affecting cell–ECM interactions through the modulation of integrin receptors for matrix proteins.