The type I collagen induction of MT1-MMP-mediated MMP-2 activation is repressed by alphaVbeta3 integrin in human breast cancer cells.

The influence of alphaVbeta3 integrin on MT1-MMP functionality was studied in human breast cancer cells of differing beta3 integrin status. Overexpression of beta3 integrin caused increased cell surface expression of alphaV integrin and increased cellular adhesion to extracellular matrix (ECM) substrates in BT-549, MDA-MB-231 and MCF-7 cells. beta3 integrin expression also enhanced the migration of breast cancer cells on ECM substrates and enhanced collagen gel contraction. In vivo, alphaVbeta3 cooperated with MT1-MMP to increase the growth of MCF-7 cells after orthotopic inoculation in immunocompromised mice, but had no influence on in vitro proliferation. Despite these stimulatory effects, overexpression of beta3 integrin suppressed the type I collagen (Col I) induced MMP-2 activation in all breast cancer cell lines analyzed. This was also evident in extracts from the MCF-7 tumors in vivo, where MMP-2 activation was stimulated by MT1-MMP transfection, but attenuated with beta3 integrin expression. Although our studies confirm important biological effects of alphaVbeta3 integrin on enhancing cell adhesion and migration, ECM remodeling and tumor growth, beta3 integrin caused reduced MMP-2 activation in response to Col I in vitro, which appears to be physiologically relevant, as it was also seen in tumor xenografts in vivo. The reduction of MMP-2 activation (and thus MT1-MMP activity) by alphaVbeta3 in response to Col I may be important in scenarios where cells which are activated for matrix degradation need to preserve some pericellular collagen, perhaps as a substrate for cell adhesion and migration, thus maintaining a balanced level of proteolysis required for efficient tumor growth.     

MT1-MMP promotes cell growth and ERK activation through c-Src and paxillin in three-dimensional collagen matrix

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is essential for tumor invasion and growth. We show here that MT1-MMP induces extracellular signal-regulated kinase (ERK) activation in cancer cells cultured in collagen gel, which is indispensable for their proliferation. Inhibition of MT1-MMP by MMP inhibitor or small interfering RNA suppressed activation of focal adhesion kinase (FAK) and ERK in MT1-MMP-expressing cancer cells, which resulted in up-regulation of p21^WAF1 and suppression of cell growth in collagen gel. Cell proliferation was also abrogated by the inhibitor against ERK pathway without affecting FAK phosphorylation. MT1-MMP and integrin α_vβ₃ were shown to be involved in c-Src activation, which induced FAK and ERK activation in collagen gel. These MT1-MMP-mediated signal transductions were paxillin dependent, as knockdown of paxillin reduced cell growth and ERK activation, and co-expression of MT1-MMP with paxillin induced ERK activation. The results suggest that MT1-MMP contributes to proliferation of cancer cells in the extracellular matrix by activating ERK through c-Src and paxillin.     

Syndecan-2 overexpression regulates adhesion and migration through cooperation with integrin α2

Syndecan-2, a transmembrane heparan sulfate proteoglycan, is known to serve as an adhesion receptor, but details of the regulatory mechanism governing syndecan-2 cell adhesion and migration remain unclear. Here, we examined this regulatory mechanism, showing that overexpression of syndecan-2 enhanced collagen adhesion, cell migration and invasion of normal rat intestinal epithelial cells (RIE1), and increased integrin α2 expression levels. Interestingly, RIE1 cells transfected with either syndecan-2 or integrin α2 showed similar adhesion and migration patterns, and a function-blocking anti-integrin α2 antibody abolished syndecan-2-mediated adhesion and migration. Consistent with these findings, transfection of integrin α2 siRNA diminished syndecan-2-induced cell migration in HCT116 human colon cancer cells. Taken together, these results demonstrate a novel cooperation between syndecan-2 and integrin α2β1 in adhesion-mediated cell migration and invasion. This interactive dynamic might be a possible mechanism underlying the tumorigenic activities of colon cancer cells.     

Role of the I-domain in collagen binding specificity and activation of the integrins α1β1 and α2β1

Adhesion to collagens by most cell types is mediated by the integrins α1β1 and α2β1. Both integrin α subunits belong to a group which is characterized by the presence of an I domain in the N-terminal half of the molecule, and this domain has been implicated in the ligand recognition. Since purified α1β1 and α2β1 differ in their binding to collagens I and IV and recognize different sites within the major cell binding domain of collagen IV, we investigated the potential role of the α1 and α2 I domains in specific collagen adhesion. We find that introducing the α2 I domain into α1 results in surface expression of a functional collagen receptor. The adhesion mediated by this chimeric receptor (α1-2-1β1) is similar to the adhesion profile conferred by α2β1, not α1β1. The presence of α2 or α1-2-1 results in preferential binding to collagen I, whereas α1 expressing cells bind better to collagen IV. In addition, α1 containing cells bind to low amounts of a tryptic fragment of collagen IV, whereas α2 or α1-2-1 bearing cells adhere only to high concentrations of this substrate. We also find that collagen adhesion of NIH-3T3 mediated by α2β1 or α1-2-1β1, but not by α1, requires the presence of Mn²⁺ ions. This ion requirement was not found in CHO cells, implicating the I domain in cell type-specific activation of integrins. J. Cell. Physiol. 176:634–641, 1998. © 1998 Wiley-Liss, Inc.     

MT1-MMP Initiates Activation of pro-MMP-2 and Integrin αvβ3 Promotes Maturation of MMP-2 in Breast Carcinoma Cells

We evaluated cellular mechanisms involved in the activation pathway of matrix prometalloproteinase-2 (pro-MMP-2), an enzyme implicated in the malignant progression of many tumor types. Membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves the N-terminal prodomain of pro-MMP-2 thus generating the activation intermediate that then matures into the fully active enzyme of MMP-2. Our results provide evidence on how a collaboration between MT1-MMP and integrin αvβ3 promotes more efficient activation and specific, transient docking of the activation intermediate and, further, the mature, active enzyme of MMP-2 at discrete regions of cells. We show that coexpression of MT1-MMP and integrin αvβ3 in MCF7 breast carcinoma cells specifically enhances in trans autocatalytic maturation of MMP-2. The association of MMP-2′s C-terminal hemopexin-like domain with those molecules of integrin αvβ3 which are proximal to MT1-MMP facilitates MMP-2 maturation. Vitronectin, a specific ligand of integrin αvβ3, competitively blocked the integrin-dependent maturation of MMP-2. Immunofluorescence and immunoprecipitation studies supported clustering of MT1-MMP and integrin αvβ3 at discrete regions of the cell surface. Evidently, the identified mechanisms appear to be instrumental to clustering active MMP-2 directly at the invadopodia and invasive front of αvβ3-expressing cells or in their close vicinity, thereby accelerating tumor cell locomotion.     

Membrane type-1 matrix metalloproteinase (MT1-MMP) processing of pro-alphav integrin regulates cross-talk between alphavbeta3 and alpha2beta1 integrins in breast carcinoma cells

We have recently demonstrated that in breast carcinoma MCF7 cells MT1-MMP processes the alphav, alpha3, and alpha5 integrin precursors generating the respective mature S-S-linked heavy and light alpha-chains. The precursor of alpha2 integrin subunit was found resistant to MT1-MMP proteolysis. The processing of the alphav subunit by MT1-MMP facilitated alphavbeta3-dependent adhesion, activation of FAK signaling pathway, and migration of MCF7 cells on vitronectin. To elucidate further the effects of MT1-MMP on cellular integrins, we examined the functional activity of alpha5beta1 and alpha2beta1 integrins in MCF7 cells expressing MT1-MMP. Either expression of MT1-MMP alone or its coexpression with alphavbeta3 failed to affect the functionality of alpha5beta1 integrin, and adhesion of cells to fibronectin. MT1-MMP, however, profoundly affected the cross-talk involving alphavbeta3 and alpha2beta1 integrins. In MT1-MMP-deficient cells, integrin alphavbeta3 suppressed the functional activity of the collagen-binding alpha2beta1 integrin receptor and diminished cell adhesion to type I collagen. Coexpression of MT1-MMP with integrin alphavbeta3 restored the functionality of alpha2beta1 integrin and, consequently, the ability of MCF7 cells to adhere efficiently to collagen. We conclude that the MT1-MMP-controlled cross-talk between alphavbeta3 and alpha2beta1 integrins supports binding of aggressive, MT1-MMP-, and alphavbeta3 integrin-expressing malignant cells on type I collagen, the most common substratum of the extracellular matrix.     

Role of the α1β1 integrin complex in collagen gel contraction in vitro by fibroblasts

Matrix remodeling, critical to embryonic morphogenesis and wound healing, is dependent on the expression of matrix components, their receptors, and matrix proteases. The collagen gel assay has provided an effective model for the examination of the functional role(s) of each of these groups of molecules in matrix remodeling. Previous investigations have indicated that collagen gel contraction involves the β1 integrin family of matrix receptors and is stimulated by several growth factors, including TGF-β, PDGF, and angiotensin II. In particular, collagen gel remodeling by human cells involves the α2β1 and, to a lesser extent the α1β1 integrin complexes. The present studies were undertaken to determine the role of the α1 integrin chain, a collagen/laminin receptor, in collagen gel contration by rodent and avian fibroblasts. A high degree of correlation was found between the expression of the α1β1 integrin complex and the relative ability of cells to contract collagen gels. Further studies using antibodies and antisense oligonucleotides against the α1 integrin indicated a significant role for this integrin chain in contraction of collagen gels by rat cardiac fibroblasts. In addition, antibodies to the α1 integrin chain inhibited migration of these fibroblasts on a collagen substratum, suggesting that at least one role of this integrin is in migration of cells in collagen gels. These results indicate that the α1β integrin complex plays a significant role in cellular interactions with interstital collagen that are involved in matrix remodeling such as is seen during morphogenesis and wound healing. © 1995 Wiley-Liss, Inc.     

α11β1 integrin‐mediated MMP‐13‐dependent collagen lattice contraction by fibroblasts: Evidence for integrin‐coordinated collagen proteolysis

We have previously determined that integrin α11β1 is required on mouse periodontal ligament (PDL) fibroblasts to generate the force needed for incisor eruption. As part of the phenotype of α11^?/? mice, the incisor PDL (iPDL) is thickened, due to disturbed matrix remodeling. To determine the molecular mechanism behind the disturbed matrix dynamics in the PDL we crossed α11^?/? mice with the Immortomouse and isolated immortalized iPDL cells. Microarray analysis of iPDL cells cultured inside a 3D collagen gel demonstrated downregulated expression of a number of genes in α11‐deficient iPDL cells, including matrix metalloproteinase‐13 (MMP‐13) and cathepsin K. α11^?/? iPDL cells in vitro displayed disturbed interactions with collagen I during contraction of attached and floating collagen lattices and furthermore displayed reduced MMP‐13 protein expression levels. The MMP‐13 specific inhibitor WAY 170523 and the Cathepsin K Inhibitor II both blocked part of the α11 integrin‐mediated collagen remodeling. In summary, our data demonstrate that in iPDL fibroblasts the mechanical strain generated by α11β1 integrin regulates molecules involved in collagen matrix dynamics. The positive regulation of α11β1‐dependent matrix remodeling, involving MMP‐13 and cathepsin K, might also occur in other types of fibroblasts and be an important regulatory mechanism for coordinated extracellular and intracellular collagen turnover in tissue homeostasis. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Expression and regulation of α1β1 integrin in Schwann cells

The interaction of cells with the extracellular matrix plays a critical role in morphogenesis and cell differentiation. To define how Schwann cells might interact with the extracellular matrix, we chose to study the expression of the laminin/collagen receptor α1β1 integrin during nerve development in the rat from embryonic day 14 to maturity. We found that this integrin is expressed predominantly on mature non-myelin-forming cells and only at very low levels on myelin-forming cells. Significant levels of this integrin were not detected on Schwann cell precursors or embryonic Schwann cells in vivo. Experiments using transected and crushed sciatic nerve showed that α1β1 integrin expression is regulated at least in part by axonal contact. Furthermore, Schwann cell culture experiments showed that α1β1 integrin levels are strongly upregulated by transforming growth factor-βs and phorbol esters. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 914–928, 1997     

Cyclic strain induces reorganization of integrin α5β1 and α2β1 in human umbilical vein endothelial cells

Cyclic strain has been shown to modulate endothelial cell (EC) morphology, proliferation, and function. We have recently reported that the focal adhesion proteins focal adhesion kinase (pp125^FAK) and paxillin, are tyrosine phosphorylated in EC exposed to strain and these events regulate the morphological change and migration induced by cyclic strain. Integrins are also localized on focal adhesion sites and have been reported to induce tyrosine phosphorylation of pp125^FAK under a variety of stimuli. To study the involvement of different integrins in signaling induced by cyclic strain, we first observed the redistribution of α and β integrins in EC subjected to 4 h cyclic strain. Human umbilical vein endothelial cells (HUVEC) seeded on either fibronectin or collagen surfaces were subjected to 10% average strain at a frequency 60 cycles/min. Confocal microscopy revealed that β₁ integrin reorganized in a linear pattern parallel with the long axis of the elongated cells creating a fusion of focal adhesion plaques in EC plated on either fibronectin (a ligand for α₅β₁) or collagen (a ligand for α₂β₁) coated plates after 4 h exposure to cyclic strain. β₃ integrin, which is a vitronectin receptor, did not redistribute in EC exposed to cyclic strain. Cyclic strain also led to a reorganization of α₅ and α₂ integrins in a linear pattern in HUVEC seeded on fibronectin or collagen, respectively. The expression of integrins α₅, α₂, and β₁ did not change even after 24 h exposure to strain when assessed by immunoprecipitation of these integrins. Cyclic strain-induced tyrosine phosphorylation of pp125^FAK occurred concomitant with the reorganization of β₁ integrin. We concluded that α₅β₁ and α₂β₁ integrins play an important role in transducing mechanical stimuli into intracellular signals. J. Cell. Biochem. 64:505–513. © 1997 Wiley-Liss, Inc.     

TGF-β1 facilitates MT1-MMP-mediated proMMP-9 activation and invasion in oral squamous cell carcinoma cells

Matrix metalloproteinase (MMP)-2 and MMP-9, also known as gelatinases or type IV collagenases, are recognized as major contributors to the proteolytic degradation of extracellular matrix during tumor invasion. Latent MMP-2 (proMMP-2) is activated by membrane type 1 MMP (MT1-MMP) on the cell surface of tumor cells. We previously reported that cell-bound proMMP-9 is activated by the MT1-MMP/MMP-2 axis in HT1080 cells treated with concanavalin A in the presence of exogenous proMMP-2. However, the regulatory mechanism of proMMP-9 activation remains largely unknown. Transforming growth factor (TGF)-β1 is frequently overexpressed in tumor tissues and is associated with tumor aggressiveness and poor prognosis. In this study, we examined the role of TGF-β1 on MT1-MMP-mediated proMMP-9 activation using human oral squamous cell carcinoma cells. TGF-β1 significantly increased the expression of MMP-9. By adding exogenous proMMP-2, TGF-β1-induced proMMP-9 was activated during collagen gel culture, which was suppressed by the inhibition of TGF-β1 signaling or MT1-MMP activity. This MT1-MMP-mediated proMMP-9 activation was needed to facilitate TGF-β1-induced cell invasion into collagen gel. Thus, TGF-β1 may facilitate MT1-MMP-mediated MMP-9 activation and thereby stimulate invasion of tumor cells in collaboration with MT1-MMP and MMP-2.     

Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.     

The hemopexin domain of membrane-type matrix metalloproteinase-1 (MT1-MMP) Is not required for its activation of proMMP2 on cell surface but is essential for MT1-MMP-mediated invasion in three-dimensional type I collagen

Membrane-type matrix metalloproteinase-1 (MT1-MMP) plays a key role in tumor invasion and metastasis by degrading the extracellular matrix and activating proMMP2. Here we show that the conserved hemopexin domain is required for MT1-MMP-mediated invasion and growth in three-dimensional type I collagen matrix but not proMMP2 activation. Deletion of the hemopexin domains in MT1-, MT2-, MT3-, MT5-, and MT6-MMP does not impair their abilities to activate proMMP2. In fact, hemopexin-less MT5- and MT6-MMP activate proMMP2 better than their wild type counterparts. On the other hand, hemopexin-less MT1-MMP fails to promote cell invasion into type I collagen but retains the capacity to enhance the growth of Madin-Darby canine kidney cells as cysts in three-dimensional collagen matrix. Moreover, the hemopexin domain is also required for MT1-MMP-mediated invasion/scattering of MCF-7 cells in three-dimensional collagen matrix. Because growth and invasion in a three-dimensional model may correlate with tumor invasiveness in vivo, our data suggest that the hemopexin domains of MT-MMPs should be targeted for the development of anti-cancer therapies by employing screening assays developed for three-dimensional models rather than their enzymatic activity toward proMMP2.     

Syndecan-1 Signals Independently of β1 Integrins during Raji Cell Spreading

Syndecan-1-expressing Raji lymphoid cells (Raji-S1 cells) bind and spread rapidly when attaching to matrix ligands that contain heparan sulfate-binding domains. However, these ligands also contain binding sites for integrins, which are widely known to signal, raising the question of whether the proteoglycan core protein participates in generation of the signal for spreading. To address this question, the spreading of the Raji-S1 cells is examined on ligands specific for either β1 integrins, known to be present on the Raji cells, or the syndecan-1 core protein. The cells adhere and spread on invasin, a ligand that activates β1 integrins, the IIICS fragment of fibronectin, which is a specific ligand for the α4β1 integrin, or mAb281.2, an antibody specific for the syndecan-1 core protein. The signaling resulting from adhesion to the syndecan-specific antibody appears integrin independent as (i) the morphology of the cells spreading on the antibody is distinct from spreading initiated by the integrins alone; (ii) spreading on the syndecan or integrin ligands is affected differently by the kinase inhibitors tyrphostin 25, genistein, and staurosporine; and (iii) spreading on the syndecan-specific antibody is not disrupted by blocking β1 integrin activation with mAb13, a β1 inhibitory antibody. These data demonstrate that ligation of syndecan-1 initiates intracellular signaling and suggest that this signaling occurs when cells expressing syndecan-1 adhere to matrix ligands containing heparan sulfate-binding domains.     

Bone sialoprotein supports breast cancer cell adhesion proliferation and migration through differential usage of the αvβ3 and αvβ5 integrins

Bone sialoprotein (BSP), a secreted glycoprotein found in bone matrix, has been implicated in the formation of mammary microcalcifications and osteotropic metastasis of human breast cancer (HBC). BSP possesses an integrin-binding RGD (Arg-Gly-Asp) domain, which may promote interactions between HBC cells and bone extracellular matrix. Purified BSP, recombinant human BSP fragments and BSP-derived RGD peptides are shown to elicit migratory, adhesive, and proliferative responses in the MDA-MB-231 HBC cell line. Recombinant BSP fragment analysis localized a significant component of these activities to the RGD domain of the protein, and synthetic RGD peptides with BSP flanking sequences (BSP-RGD) also conferred these responses. The fibronectin-derived RGD counterpart, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), could not support these cellular responses, emphasizing specificity of the BSP configuration. Although most of the proliferative and adhesive responses could be attributed to RGD interactions, these interactions were only partly responsible for the migrational responses. Experiments with integrin-blocking antibodies demonstrated that BSP-RGD-induced migration utilizes the αvβ3 vitronectin receptor, whereas adhesion and proliferation responses were αvβ5-mediated. Using fluorescence activated cell sorting, we selected two separate subpopulations of MDA-MB-231 cells enriched for αvβ3 or αvβ5 respectively. Although some expression of the alternate αv integrin was still retained, the αvβ5-enriched MDA-MB-231 cells showed enhanced proliferative and adhesive responses, whereas the αvβ3-enriched subpopulation was suppressed for proliferation and adhesion, but showed enhanced migratory responses to BSP-RGD. In addition, similar analysis of two other HBC cell lines showed less marked, but similar RGD-dependent trends in adhesion and proliferation to the BSP fragments. Collectively, these data demonstrate BSP effects on proliferative, migratory, and adhesive functions in HBC cells and that the RGD-mediated component differentially employs αvβ3 and αvβ5 integrin receptors. J. Cell. Physiol. 176:482–494, 1998. © 1998 Wiley-Liss, Inc.     

Reversing microtubule‐directed chemotherapeutic drug resistance by co‐delivering α2β1 inhibitor and paclitaxel with nanoparticles in ovarian cancer

Previous reports indicated that integrins associated signals are tightly related to tumor progression. Here, we observed elevated expression of integrin α2β1 in tumor tissues from microtubule‐directed chemotherapeutic drugs (MDCDs) resistant patients compared with the samples from chemosensitive patients. More importantly, we sorted the integrin α2β1⁺ tumor cells and found those cells revealed high MDCDs resistance, whereas MDCDs shows effective cytotoxicity to those integrin α2β1^? tumor cells in vitro and in vivo. Mechanistically, we demonstrated that integrin α2β1 could induce MDCDs resistance through the activation of the PI3K/AKT pathway. Applying MPEG‐PLA to co‐encapsulate the integrin α2β1 inhibitor E7820 and MDCDs could effectively reverse MDCDs resistance, resulting in enhanced anticancer effects while avoiding potential systemic toxicity in vitro and in vivo. In conclusion, the expression of integrin α2β1 contributes to MDCDs resistance, while applying E7820 combination treatment by MPEG‐PLA nanoparticles could reverse the resistance.     

Adhesion and protease activity in cell lines from human salivary gland tumors are regulated by the laminin-derived peptide AG73, syndecan-1 and β1 integrin

We studied the induction of protease activity by the laminin alpha1-derived peptide AG73 in cells from adenoid cystic carcinoma (CAC2) and myoepithelioma (M1), respectively a malignant and a benign salivary gland tumors. Laminin alpha1 chain and MMP9 were immunolocalized in adenoid cystic carcinoma and myoepithelioma in vivo and in vitro. Cells grown inside AG73-enriched laminin-111 exhibited large spaces in the extracellular matrix, suggestive of remodeling. The broad spectrum MMP inhibitor GM6001 decreased spaces induced by AG73 in CAC2 and M1 cells. This result strongly suggests that AG73-mediated matrix remodeling involves matrix metalloproteinases. CAC2 and M1 cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, as detected by zymography. Furthermore, siRNA silencing of MMP9 decreased remodeling in 3D cultures. We searched for AG73 receptors regulating MMP9 activity in our cell lines. CAC2 and M1 cells grown on AG73 exhibited colocalization of syndecan-1 and beta1 integrin. siRNA knockdown of syndecan-1 expression in these cells resulted in decreased adhesion to AG73 and reduced protease and remodeling activity. We investigated syndecan-1 co-receptors in both cell lines. Silencing beta1 integrin inhibited adhesion to AG73, matrix remodeling and protease activity. Double-knockdown experiments were carried out to further explore syndecan-1 and beta1 integrin cooperation. CAC2 cells transfected with both syndecan-1 and beta1 integrin siRNA oligos showed significant decrease in adhesion to AG73. Simultaneous silencing of receptors also induced a decrease in protease activity. Our results suggest that syndecan-1 and beta1 integrin signaling downstream of AG73 regulate adhesion and MMP production by CAC2 and M1 cells.