Search

Metastasis accounts for 90% of cancer-related deaths, yet the mechanisms by which cancer cells colonize secondary organs remain poorly understood. For breast cancer patients, metastasis to the liver is associated with poor prognosis and a median survival of 6 months. Standard of care is chemotherapy, but recurrence occurs in 30% of patients. Systemic chemotherapy has been shown to induce hepatotoxicity and fibrosis, but how chemotherapy impacts the composition of the liver extracellular matrix (ECM) remains unknown. Individual ECM proteins drive tumor cell proliferation and invasion, features that are essential for metastatic outgrowth in the liver. First, we find that the ECM of livers isolated from chemotherapy-treated MMTV-PyMT mice increases the invasion, but not proliferation, of metastatic breast cancer cells. Proteomic analysis of the liver ECM identified Collagen V to be more abundant in paclitaxel-treated livers. We show that Collagen V increases cancer cell invasion via α1β1 integrins and MAPK signaling, while also increasing the alignment of Collagen I, which has been associated with increased invasion. Treatment with obtustatin, an inhibitor specific to α1β1 integrins, inhibits tumor cell invasion in decellularized ECM from paclitaxel-treated livers. Overall, we show chemotherapy treatment alters the liver microenvironment, priming it as a pro-metastatic niche for cancer metastasis., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Integrins mediate cell adhesion to extracellular matrix components. Integrin alpha 1 beta 1 is a collagen receptor expressed on many mesenchymal cells, but mice deficient in alpha 1 integrin (alpha1-KO) have no gross structural defects. Here, the regeneration of a fractured long bone was studied in alpha1-KO mice. These mice developed significantly less callus tissue than the wild-type (WT) mice, and safranin staining revealed a defect in cartilage formation. The mRNA levels of nine extracellular matrix genes in calluses were evaluated by Northern blotting. During the first 9 days the mRNA levels of cartilage-related genes, including type II collagen, type IX collagen, and type X collagen, were lower in alpha1-KO mice than in WT mice, consistent with the reduced synthesis of cartilaginous matrix appreciated in tissue sections. Histological observations also suggested a diminished number of chondrocytes in the alpha 1-KO callus. Proliferating cell nuclear antigen staining revealed a reduction of mesenchymal progenitors at the callus site. Although, the number of mesenchymal stem cells (MSCs) obtained from WT and alpha 1-KO whole marrow was equal, in cell culture the proliferation rate of the MSCs of alpha 1-KO mice was slower, recapitulating the in vivo observation of reduced callus cell proliferation. The results demonstrate the importance of proper collagen-integrin interaction in fracture healing and suggest that alpha1 integrin plays an essential role in the regulation of MSC proliferation and cartilage production.

Keloids and hypertrophic scars are significant symptomatic clinical problems characterized by excess collagen. Although extensive research has focused on fibroblasts and collagen turnover in these aberrant scars, little work has been done on the expression of integrins (cell membrane structures that link cells to extracellular matrix) within these lesions. Integrin-mediated regulation of collagen synthesis has previously been observed in explanted fibroblasts from normal and fibrotic dermis, and integrin alpha1 knockout mice maintain increased collagen synthesis consistent with a role for alpha1beta1 in providing negative feedback on collagen synthesis. These findings suggested the need to evaluate integrin roles in keloids and hypertrophic scars. In this study we examined integrin expression in keloids (n = 11), hypertrophic scars (n = 5), radiation ulcers (n = 2), and normal skin specimens (n = 8). We used a novel approach to analysis by isolating dermal fibroblasts directly from tissue (without explant culture) and determining surface integrin expression by flow cytometry. We found that keloids and hypertrophic scars have marked alterations in fibroblast integrin expression and contain several distinct populations of fibroblasts. One of these populations expresses high levels of alpha1 integrin, and the proportion of these cells is higher in keloids (63% +/- 3.6% SEM) and hypertrophic scars (45% +/- 2.7% SEM) than in normal skin tissues (28% +/- 4.7% SEM). The different populations of fibroblasts defined by integrin expression merge, however, when the cells are serially cultured, suggesting that there may be aspects of the dermal microenvironment that maintain the integrin phenotypic heterogeneity in dermal fibroblasts.

Past studies have described the serendipitous appearance of peripheral CD4(+)CD8(+) double-positive (DP) T cells in both humans and nonhuman primates usually following a viral infection or resulting from a malignancy. However, understanding the role of DP T cells has been hampered by the lack of their reproducible generation. Herein, we describe DP T cells produced after a single intratracheal or intranasal dose of recombinant adenovirus 2 or 5 vector into mice. In a time-dependent fashion, DP T cells localized only in the deep cervical lymph nodes but not in the lungs or in any of the respiratory lymph nodes. These DP T cells were TCR(alpha)beta(+) and CD8(alpha)beta(+), but not TCR(gamma)delta(+) nor CD8(alpha)alpha(+), suggesting that these cells are unrelated to intestinally derived DP T cells. Upon co-stimulation with anti-CD3 and anti-CD28, DP T cells showed increased expression of VLA-1, VLA-2, and CD69, and were more effective than CD4(+) T cells in T helper cell activity, as evidenced by increased IgA, IgG, and IgM production. Such co-stimulation also favored the production of IFN-gamma and IL-10 where CD4(+) T cells were more inclined to produce IFN-gamma and IL-2.

Angiogenesis is a complex process, involving functional cooperativity between cytokines and endothelial cell (EC) surface integrins. In this study, we investigated the mechanisms through which the alpha(1)beta(1) and alpha(2)beta(1) integrins support angiogenesis driven by vascular endothelial growth factor (VEGF). Dermal microvascular EC attachment through either alpha(1)beta(1) or alpha(2)beta(1) supported robust VEGF activation of the Erk1/Erk2 (p44/42) mitogen-activated protein kinase signal transduction pathway that drives EC proliferation. Haptotactic EC migration toward collagen I was dependent on alpha(1)beta(1) and alpha(2)beta(1) as was VEGF-stimulated chemotaxis of ECs in a uniform collagen matrix. Consistent with the functions of alpha(1)beta(1) and alpha(2)beta(1) in supporting signal transduction and EC migration, antibody antagonism of either integrin resulted in potent inhibition of VEGF-driven angiogenesis in mouse skin. Moreover, combined antagonism of alpha(1)beta(1) and alpha(2)beta(1) substantially reduced tumor growth and angiogenesis of human squamous cell carcinoma xenografts. Collectively, these studies identify critical collaborative functions for the alpha(1)beta(1) and alpha(2)beta(1) integrins in supporting VEGF signal transduction, EC migration, and tumor angiogenesis.

The mechanisms underlying the contraction-dependent apoptosis of primary fibroblasts are of prime importance in understanding anchorage-dependent survival/apoptosis of dermal fibroblasts. As integrins are essential extracellular matrix receptors in fibroblasts, their role in anchorage-dependent apoptosis/survival of fibroblasts was analyzed. Primary human fibroblasts displayed a marked reduction of apoptosis in mechanically relaxed collagen matrices in the presence of adhesion-blocking antibodies against alpha1beta1 or alpha2beta1. Anti-alphavbeta3 antibodies had a considerably weaker effect. In additional experiments RD cells, which lack alpha2 integrin, displayed no apoptosis in mechanically relaxed collagen matrices. Their susceptibility to apoptosis was restored after transfection with functional alpha2 integrin, and it could be blocked again by adhesion-blocking antibodies against alpha2beta1 integrin. Therefore we conclude that apoptosis of human primary fibroblasts in contractile collagen matrices is - at least in part - inhibited by adhesion-blocking anti-integrin antibodies, suggesting that the mode of apoptosis in this case is different from anoikis. Further, apoptosis in a mechanically relaxed collagen matrix could be abrogated by depolymerization of F-actin using cytochalasin D and also by disturbing actin-myosin interaction using 2,3-butanedione monoxime, indicating a possible dependence of apoptosis on mechanical forces and/or cell shape.

Alport syndrome is a genetic disorder resulting from mutations in type IV collagen genes. The defect results in pathological changes in kidney glomerular and inner-ear basement membranes. In the kidney, progressive glomerulonephritis culminates in tubulointerstitial fibrosis and death. Using gene knockout-mouse models, we demonstrate that two different pathways, one mediated by transforming growth factor (TGF)-beta1 and the other by integrin alpha1beta1, affect Alport glomerular pathogenesis in distinct ways. In Alport mice that are also null for integrin alpha1 expression, expansion of the mesangial matrix and podocyte foot process effacement are attenuated. The novel observation of nonnative laminin isoforms (laminin-2 and/or laminin-4) accumulating in the glomerular basement membrane of Alport mice is markedly reduced in the double knockouts. The second pathway, mediated by TGF-beta1, was blocked using a soluble fusion protein comprising the extracellular domain of the TGF-beta1 type II receptor. This inhibitor prevents focal thickening of the glomerular basement membrane, but does not prevent effacement of the podocyte foot processes. If both integrin alpha1beta1 and TGF-beta1 pathways are functionally inhibited, glomerular foot process and glomerular basement membrane morphology are primarily restored and renal function is markedly improved. These data suggest that integrin alpha1beta1 and TGF-beta1 may provide useful targets for a dual therapy aimed at slowing disease progression in Alport glomerulonephritis.

Background: Glomerular mesangial cell (MC) proliferation, hypertrophy, and abnormal matrix remodeling characterized by increased expression of fibronectin, laminin and collagen type IV, and neoexpression of collagen I and III are the main biological features of progressive glomerulonephritis (GN). Especially, persistent pathological matrix remodeling may lead to glomerular scar formation (glomerular scarring). We reported recently that alpha1beta1 integrin, a major collagen receptor for MCs, may be a potential adhesion molecule for MC-mediated pathological collagen matrix remodeling in GN., Methods: To address further the direct role of alpha1beta1 integrin in MC behavior, such as cell growth and matrix remodeling, alpha1beta1 integrin was overexpressed in MCs by transfecting an expression vector containing a full-length rat alpha1 integrin cDNA. Flow cytometry and immunoprecipitation analysis were applied for selection of transfectants with a stable expression of the alpha1 integrin subunit. The effect of alpha1beta1 integrin overexpression on MC biology was examined with a 3H-thymidine incorporation assay, flow cytometric analysis of cell size and DNA content, Western blot analysis of a cyclin-dependent-kinase inhibitor, p27Kip1, alpha-smooth muscle actin expression, and a collagen gel contraction assay., Results: The alpha1 transfectants displayed a dramatic inhibition of 3H-thymidine incorporation as compared with the mock transfectants. Increased expression of the alpha1 subunit inversely correlated with cell cycle progression and paralleled the expression of p27Kip1 and alpha-smooth muscle actin, as well as the cell size in MCs. In addition, the alpha1-transfectants were able to enhance collagen matrix reorganization effectively., Conclusion: These results indicate that MC-alpha1beta1 integrin expression is a critical determinant of MC phenotypes, including cell growth, cell size, and collagen matrix remodeling ability, and thereby contributes to scar matrix remodeling (sclerosis) in GN.

In mice, monoclonal antibody (mAb) to the alpha1 integrin abrogate gastro-intestinal damage during graft-versus-host-disease (GVHD), suggesting anti alpha1 mAb as candidates for treatment in humans as well. Our current data show that one such reagent, mAb 1B3.1, when immobilized to plastic wells via rabbit- anti murine (ram) immunoglobulin (Ig) induces a protein kinase-dependent spreading of activated human T cells. Furthermore, it significantly increases the proliferative response, and expression of interleukin-2 (IL-2) receptors (R) and CD69, of resting T cells, expressing minimal integrin on the cell surface, to sub-optimal stimulation by anti-CD3 mAb. We found, in addition, that mAb 1B3.1 a) immuno-precipitates alpha1beta1 integrins from cell-surface iodinated canine epithelial cells b) is highly reactive with canine T cells after their activation and c) inhibits adhesion of canine T cells to collagen IV. Despite the potential ability of the mAb to co-activate T cells in vitro, two dogs that received 4 injections of 0.5-0.3 mg/Kg of mAb 1B3.1 remained healthy, developing only marginal transient lymphopenia. Injection of 0.75mg/Kg in a third dog induced a more marked lymphopenia, and an additional dose of 1.0 mg/Kg 2 weeks later was followed by gastrointestinal hemorrhage. importantly, the lymphopenia was associated with a greater and more persistent decrease of CD8+ than of CD4+ T cells, leading to an increase in the CD4/CD8 ratio 24 hours after the injection. Thus, despite it's co-activating effects in vitro, administration of this mAb in vivo is feasible when appropriately dosed, and may have immuno-modulatory effects.

Background: Extracellular matrix molecules profoundly regulate cell behavior, including proliferation. In glomerulonephritis, type I collagen accumulates in the mesangium and is constantly structurally modified and degraded during the course of the disease., Methods: We studied how two structurally distinct forms of type I collagen, monomer versus polymerized fibrils, affect cell proliferation, mitogen-activated protein kinase (MAPK) activation, and expression of G1-phase regulatory proteins in cultured rat mesangial cells (MCs). To analyze the possible involvement of collagen-binding integrins in type I collagen-derived growth signals further, distribution patterns of integrin chains were examined by immunocytochemistry., Results: Polymerized type I collagen completely prevented the increase of DNA synthesis and cell replication induced by 5% fetal calf serum (FCS) or 25 ng/mL platelet-derived growth factor (PDGF) in MCs on monomer type I collagen. Protein expression of cyclins D1 and E was markedly down-regulated in MCs plated on polymerized type I collagen for eight hours in 5% FCS, as compared with MCs on monomer type I collagen. Incubation with 5% FCS reduced expression of the cdk-inhibitor protein p27Kip1 on monomer but not on polymerized type I collagen. Moreover, polymerized type I collagen markedly reduced cyclin E-associated kinase activity in the presence of 5% FCS. Polymerized type I collagen diminished the PDGF-induced phosphorylation and nuclear translocation of p42/p44 MAPK, but did not affect phosphorylation of PDGF beta-receptors. In MCs plated on monomer type I collagen, alpha1, alpha2, and beta1 integrin chains were recruited into focal contacts. However, on polymerized type I collagen, alpha2 and beta1, but not alpha1, integrin chains were condensed into focal contacts., Conclusions: The growth-inhibitory effect of polymerized type I collagen is characterized by rapid changes of expression and/or activation of MAPK and G1-phase regulators and could result from the lack of alpha1beta1 integrin signaling in MCs on polymerized type I collagen. Conceivably, deposition of polymerized type I collagen might reflect a reparative response to control MC replication in glomerular inflammation.

Distinct collagen subtypes are recognized by specific cell surface receptors. Two of the best known collagen receptors are members of the integrin family and are named alpha1beta1 and alpha2beta1. Integrin alpha1beta1 is abundant on smooth muscle cells, whereas the alpha2beta1 integrin is the major collagen receptor on epithelial cells and platelets. Many cell types, such as fibroblasts, osteoblasts, chondrocytes, endothelial cells, and lymphocytes may concomitantly express both of the receptors. We have studied the cell biology of these integrins at two levels. First, we have analyzed their ligand binding mechanism and specificity. Second, we have studied their signaling function inside three-dimensional collagen gels. This mini-review summarizes our most recent results. In conclusion, our data indicate that alpha1beta1 and alpha2beta1 integrins have differences in their ligand binding specificity. Furthermore, the two receptors are connected to distinct signaling pathways and their ligation may lead to opposite cellular responses.

Very late antigen 1 (VLA1) is an integrin collagen receptor that is expressed by lymphocytes in several disease states. VLA1 blockade has been shown to ameliorate gut disease in experimental graft-versus-host disease. Here we show that in the VLA1 null mouse, which is generally healthy, there is a 50% reduction in gut intraepithelial lymphocytes (IELs) despite an otherwise normal lymphocyte distribution in peripheral blood and lymphoid organs. The gammadelta to alphabeta ratios of IELs are unchanged. We also find that IL2-stimulated splenocytes from VLA1 null animals show a deficiency in adhesion to fibrillar and basement membrane collagen as well as reduced proliferation in response to collagen substratum. These results suggest that some, but not all, intraepithelial lymphocytes require VLA1 to survive or proliferate within the gut epithelium or possibly to traverse the basement membrane., (Copyright 2000 Academic Press.)

The interaction of collagen IV with cells is mediated mainly by the integrin alpha1beta1. The recognition site has been located to a segment of the triple-helical domain 100 nm away from the N terminus of the collagen molecule. The three essential amino acid residues of the alpha1beta1 binding site, arginine alpha2(IV)461 and the two aspartate residues alpha1(IV)461, are all located on different chains. Since the spatial array of the three residues depends on the stagger of the chains within the triple helix, the stagger has been elucidated using fluorescence resonance energy transfer with phenylalanine alpha1(IV)473 and tryptophan alpha2(IV)479 as the fluorescent donor/acceptor pair. The distance R between phenylalanine and tryptophan was determined by analysis of the energy transfer efficiency, E, and the orientation factor, kappa(2). In parallel, distance R and orientation factor, kappa(2 )were also calculated from the coordinates of the triple helix. Comparison of the calculated and empirically determined values unequivocally showed the stagger to be alpha1'alpha1alpha2. This arrangement of the three alpha chains describes the conformation of the alpha1beta1 integrin recognition site, that is the distinct orientation of the side-chains of the essential residues aspartate and arginine in respect to the helix axis., (Copyright 2000 Academic Press.)

Type VIII collagen is a matrix protein expressed in a number of tissues undergoing active remodeling, including injured arteries during neointimal formation and in human atherosclerotic plaques; however, very little is known about its function. We have investigated whether the type VIII collagen stimulates smooth muscle cell (SMC) migration and invasion by binding to integrin receptors and up-regulating matrix metalloproteinase (MMP) production. SMCs attached to plates coated with type VIII collagen in a dose-dependent manner, with maximal attachment occurring with coating solutions containing 25 microgram/ml collagen. Type VIII collagen at 100 microgram/ml stimulated an 83-fold increase in the migration of SMCs in a chemotaxis chamber. Antibodies against beta1 integrin receptors prevented attachment and migration of SMCs. Antibodies against alpha1 or alpha2 integrins reduced attachment of SMCs to type VIII collagen by 29% and 77%, respectively. We found that SMCs grown from the rat neointima, but not medial SMCs, increased their production of MMP-2 and -9 on adherence to type VIII collagen. This suggests that there is an important difference in phenotype between intimal and medial SMCs and that intimal SMCs have distinct matrix-dependent signaling mechanisms. Our findings suggest that type VIII collagen deposited in vascular lesions functions to promote SMC attachment and chemotaxis, and signals through integrin receptors to stimulate MMP synthesis, all of which are important mechanisms used in cell migration and invasion.

An enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of soluble alpha1beta1 integrins (salpha1) in human serum samples was developed. Solid phase-bound anti-alpha1 integrin monoclonal antibody (mAb) TS2/7 was used to capture salpha1, and mAb 1B3.1 was used to detect the immobilized integrin. An extract of human placenta (PE) containing 340 ng/mL of VLA-1 molecules served as a positive control, and serum samples from normal donors and patients were assayed. Optimal binding of anti-alpha1 integrin mAb 1B3.1, expressed as specific optical density (OD), was obtained when a 5 microng/mL solution of anti-alpha1 integrin "capture" mAb TS2/7 was immobilized to the wells and the PE was added. Solutions of albumin or collagen, in contrast, did not result in binding, confirming the specificity of the assay for sal. Furthermore, the specific OD of the wells correlated directly with the concentration of PE. A concentration of salpha1 above that of a 1:100 dilution of PE--that is, >3.4 ng/mL of integrin, in which the intra-assay correlation of variance was <5.7%, was found in 5 of 8, 3 of 8, and 6 of 9 serum samples from normal individuals, patients with connective tissue diseases (CTD), and patients with liver diseases (LD), respectively. These results suggest, for the first time, that salpha1 are present in healthy and diseased human serum.

Laminin, a major glycoprotein component of vessel basement membranes, is recognized by beta1- and beta3-integrins expressed on endothelial cells. To determine how endothelial cell integrins might function in multiple sclerosis (MS) lesions, integrin laminin receptors and laminin were analyzed in central nervous system samples from MS patients and controls by immunohistochemistry. In active MS lesions, endothelial cell VLA-6 and beta1 subunits were decreased compared to controls whereas alpha(v) subunit and VLA-1 were increased. In chronic inactive lesions beta1, VLA-6 and alpha(v) were the same as controls but VLA-1 remained increased. Alpha3 subunit was constant in all samples. By immunoelectron microscopy VLA-1, VLA-6, beta1, and laminin were distributed throughout endothelial cells; alpha(v) was adjacent to and on luminal surfaces; alpha(v) and VLA-1 were on intercellular junctions. These results indicate distinct regulation and functions of these integrins in different lesion stages. In active lesions decreased endothelial cell beta1/VLA-6 could result in their detachment from laminin thereby facilitating leukocyte transvascular migration and blood-brain barrier breakdown. Alpha(v) and VLA-1 on intercellular junctions may participate in re-establishing vessel integrity after leukocyte migration. Luminal surface alpha(v) also likely binds intraluminal ligands and cells. In chronic inactive plaques persistently elevated endothelial cell VLA-1 correlates with long-standing endothelial cell and blood-brain barrier dysfunction.

The control of cellular adhesion to extracellular matrix proteins is poorly understood. In the present analyses, we set out to test the hypothesis that high galectin-3 concentration on the cell surface downregulates cellular adhesion to the extracellular matrix proteins. Various tumor cell lines were briefly incubated without or with galectin-3 and then allowed to adhere to wells coated with laminin-1, collagen IV and fibronectin. Our data demonstrated that the cells which were incubated with galectin-3 prior to plating had significantly reduced adhesion to extracellular matrix proteins. This inhibition involved the carbohydrate recognition domain of the lectin because adhesion was achieved in the presence of galectin-3 and lactose but not galectin-3 and sucrose. Furthermore we demonstrated that galectin-3 associates with alpha 1 beta 1 integrin in a lactose dependent manner.

Many membrane proteins display their biological activity in molecular aggregates of interacting counterparts. The analysis of these aggregates remains difficult; especially intermolecular complexes of membrane proteins tend to dissociate or artificially aggregate during detergent extraction out of membranes. Thus, the existence of protein aggregates was investigated by two approaches. First, after modest detergent extraction, the presence of three well characterized rat liver membrane proteins, alpha 1 beta 1-integrin, dipeptidyl aminopeptidase IV (DPP IV) and cell-CAM 105 (CAM = cell adhesion molecule), in aggregates could be demonstrated when investigated by size-exclusion chromatography (SEC) under non-denaturating conditions. However, the applied detergents partially influenced the resolution of the separation reducing the ability to discriminate between native and artificial protein aggregates. To circumvent these problems, a second approach based on covalent cross-linking of native protein complexes by dithiobis(succinimidylpropionate) was combined with the performance of denaturating SEC. Under such optimized some high-molecular-mass complexes of all model proteins consisting of unknown components could also be detected. Taken together, non-denaturating SEC and chemical cross-linking in combination with denaturating SEC represent methodological approaches for the characterization of protein aggregates.

In HeLa cells beta 1 integrin forms heterodimers with alpha 1, alpha 2, alpha 3, alpha 5 and alpha 6 integrin subunits. Integrin alpha v beta 5 can also be detected. A monoclonal antibody SR-84 identified the alpha 1 integrin subunit in immunoprecipitation assays and inhibited alpha 1-related cell adhesion to different matrix proteins, laminin-1 and type I, IV, and V collagens, whereas its effect on adhesion to type II collagen was marginal. HeLa cells do not attach to type VI collagen. The presence of magnesium was essential for HeLa cell adhesion, whereas calcium alone was not sufficient and high concentrations of calcium even counteracted the effect of magnesium. Cell adhesion to type I collagen was sensitive to changes in pH, unlike cell adhesion to type IV collagen. We conclude that SR-84 is a valuable tool to study alpha 1 integrin-related functions, and that in HeLa cells alpha 1 beta 1 integrin is a magnesium-dependent receptor for type I, IV, and V collagens but not for type II and VI collagens.