Integrin alpha IIb beta 3 mediates binding of the Lyme disease agent Borrelia burgdorferi to human platelets

Lyme disease is a chronic, multisystemic infection caused by the tick-borne spirochete Borrelia burgdorferi. Attachment of the spirochete to host cells via specific receptors is likely to be important in the establishment of infection. B. burgdorferi have previously been shown to bind to a variety of mammalian cells in vitro. Here we demonstrate that binding of B. burgdorferi to human platelets is mediated by the integrin alpha IIb beta 3 (glycoprotein IIb-IIIa), a critical receptor in thrombosis and hemostasis. Functional expression of this receptor requires platelet activation, and binding of the spirochete was observed only to activated platelets. Binding was inhibited by a synthetic Arg-Gly-Asp peptide that blocks ligand interaction with many integrins and by a synthetic peptide based on the gamma chain of fibrinogen that blocks binding to alpha IIb beta 3. In addition, attachment of the spirochete to platelets was inhibited by monoclonal antibodies directed against alpha IIb beta 3 that are known to block ligand-receptor interaction. No inhibition was seen with control peptides or with antibodies directed against other platelet receptors. B. burgdorferi bound efficiently to purified alpha IIb beta 3 but did not bind to platelets deficient in this integrin. Efficient platelet binding was displayed by a cloned, infectious B. burgdorferi strain, whereas a cloned noninfectious strain did not bind to platelets. Binding to integrins may be important for the ability of B. burgdorferi to establish infection in the diverse tissues affected by Lyme disease.     

Integrin alpha 2 beta 1 is a positive regulator of collagenase (MMP-1) and collagen alpha 1(I) gene expression

A classical model for studying the effects of extracellular matrix is to culture cells inside a three-dimensional collagen gel. When surrounded by fibrillar collagen, many cell types decrease the production of type I collagen, and the expression of interstitial collagenase (matrix metalloproteinase-1; MMP-1) is simultaneously induced. To study the role of the collagen-binding integrins alpha 1 beta 1 and alpha 2 beta 1 in this process, we used three different osteogenic cell lines with distinct patterns of putative collagen receptors: HOS cells, which express only alpha 1 beta 1 integrin, MG-63 cells, which express only alpha 2 beta 1 integrin, and KHOS-240 cells, which express both. Inside collagen gels, alpha 1 (I) collagen mRNA levels were decreased in HOS and KHOS-240 cells but not in MG-63 cells. In contrast, MMP-1 expression was induced in KHOS-240 and MG-63 cells but not in HOS cells. Transfection of MG-63 cells with alpha 2 integrin cDNA in an antisense orientation reduced the expression level of alpha 2 integrin. These cell clones showed induction and reduction of mRNA levels for MMP-1, respectively. HOS cells normally lacking alpha 2 beta 1 integrin were forced to express it, and this prevented the down-regulation in the levels of alpha 1 (I) collagen mRNA when cells were grown inside collagen gels. The data indicate that the level of MMP-1 expression is regulated by the collagen receptor alpha 2 beta 1 integrin. The down-regulation of collagen alpha 1 (I) is mediated by another receptor. Integrin alpha 2 beta 1 may compete with it and thus be a positive regulator of collagen synthesis.     

alpha3beta1 Integrin is required for normal development of the epidermal basement membrane

Integrins alpha3beta1 and alpha6beta4 are abundant receptors on keratinocytes for laminin-5, a major component of the basement membrane between the epidermis and the dermis in skin. These integrins are recruited to distinct adhesion structures within keratinocytes; alpha6beta4 is present in hemidesmosomes, while alpha3beta1 is recruited into focal contacts in cultured cells. To determine whether differences in localization reflect distinct functions of these integrins in the epidermis, we studied skin development in alpha3beta1-deficient mice. Examination of extracellular matrix by immunofluorescence microscopy and electron microscopy revealed regions of disorganized basement membrane in alpha3beta1-deficient skin. Disorganized matrix was first detected by day 15.5 of embryonic development and became progressively more extensive as development proceeded. In neonatal skin, matrix disorganization was frequently accompanied by blistering at the dermal-epidermal junction. Laminin-5 and other matrix proteins remained associated with both the dermal and epidermal sides of blisters, suggesting rupture of the basement membrane itself, rather than detachment of the epidermis from the basement membrane as occurs in some blistering disorders such as epidermolysis bullosa. Consistent with this notion, primary keratinocytes from alpha3beta1-deficient skin adhered to laminin-5 through alpha6 integrins. However, alpha3beta1-deficient keratinocytes spread poorly compared with wild-type cells on laminin-5, demonstrating a postattachment requirement for alpha3beta1 and indicating distinct roles for alpha3beta1 and alpha6beta4. Our findings support a novel role for alpha3beta1 in establishment and/or maintenance of basement membrane integrity, while alpha6beta4 is required for stable adhesion of the epidermis to the basement membrane through hemidesmosomes.     

Integrin alpha 8 beta 1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin

The integrin alpha 8 subunit, isolated by low stringency hybridization, is a novel integrin subunit that associates with beta 1. To identify ligands, we have prepared a function-blocking antiserum to the extracellular domain of alpha 8, and we have established by transfection K562 cell lines that stably express alpha 8 beta 1 heterodimers on the cell surface. We demonstrate here by cell adhesion and neurite outgrowth assays that alpha 8 beta 1 is a fibronectin receptor. Studies on fibronectin fragments using RGD peptides as inhibitors show that alpha 8 beta 1 binds to the RGD site of fibronectin. In contrast to the endogenous alpha 5 beta 1 fibronectin receptor in K562 cells, alpha 8 beta 1 not only promotes cell attachment but also extensive cell spreading, suggesting functional differences between the two receptors. In chick embryo fibroblasts, alpha 8 beta 1 is localized to focal adhesions. We conclude that alpha 8 beta 1 is a receptor for fibronectin and can promote attachment, cell spreading, and neurite outgrowth on fibronectin.     

The alpha7beta1 integrin mediates adhesion and migration of skeletal myoblasts on laminin

Many aspects of myogenesis are believed to be regulated by myoblast interactions with specific components of the extracellular matrix. For example, laminin has been found to promote adhesion, migration, and proliferation of mammalian myoblasts. Based on affinity chromatography, the alpha7beta1 integrin has been presumed to be the major receptor mediating myoblast interactions with laminin. We have prepared a monoclonal antibody, O26, that specifically reacts with both the X1 and the X2 extracellular splice variants of the alpha7 integrin chain. This antibody completely and selectively blocks adhesion and migration of rat L8E63 myoblasts on laminin-1, but not on fibronectin. In contrast, a polyclonal antibody to the fibronectin receptor, alpha5beta1 integrin, blocks myoblast adhesion on fibronectin, but not on laminin-1. The alpha7beta1 integrin also binds to a mixture of laminin-2 and laminin-4, the major laminin isoforms in developing and adult skeletal muscle, but O26 is a much less potent inhibitor of myoblast adhesion on the laminin-2/4 mixture than on laminin-1. Based on affinity chromatography, we suggest that this may be due to higher affinity binding of alpha7X1 to laminin-2/4 than to laminin-1.     

Integrin alpha 6 beta 4 forms a complex with the cytoskeletal protein HD1 and induces its redistribution in transfected COS-7 cells

The integrin alpha 6 beta 4 is a major component of hemidesmosomes, in which it is linked to intermediate filaments. Its presence in these structures is dependent on the beta 4 cytoplasmic domain but it is not known whether beta 4 interacts directly with keratin filaments or by interaction with other proteins. In this study, we have investigated the interaction of GST-cyto beta 4A fusion proteins with cellular proteins and demonstrate that a fragment of beta 4A, consisting of the two pairs of fibronectin type III repeats, separated by the connecting segment, forms a specific complex containing a 500-kDa protein that comigrates with HD1, a hemidesmosomal plaque protein. A similar protein was also bound by a glutathione S-transferase fusion protein containing the cytoplasmic domain of a variant beta 4 subunit (beta 4B), in which a stretch of 53 amino acids is inserted in the connecting segment. Subsequent immunoblot analysis revealed that the 500-kDa protein is in fact HD1. In COS-7 cells, which do not express alpha 6 beta 4 or the hemidesmosomal components BP230 and BP180, HD1 is associated with the cytoskeleton, but after transfecting the cells with cDNAs for human alpha 6 and beta 4, it was, instead, colocalized with alpha 6 beta 4 at the basal side of the cells. The organization of the vimentin, keratin, actin, and tubulin cytoskeletal networks was not affected by the expression of alpha 6 beta 4 in COS-7 cells. The localization of HD1 at the basal side of the cells depends on the same region of beta 4 that forms a complex containing HD1 in vitro, since the expression of alpha 6 with a mutant beta 4 subunit that lacks the four fibronectin type III repeats and the connecting segment did not alter the distribution of HD1. The results indicate that for association of alpha 6 beta 4 with HD1, the cytoplasmic domain of beta 4 is essential. We suggest that this association may be crucial for hemidesmosome assembly.     

Modulation of in vivo migratory function of alpha 2 beta 1 integrin in mouse liver

We report herein that expression of alpha 2 beta 1 integrin increased human erythroleukemia K562 transfectant (KX2C2) cell movement after extravasation into liver parenchyma. In contrast, a previous study demonstrated that alpha 2 beta 1 expression conferred a stationary phenotype to human rhabdomyosarcoma RD transfectant (RDX2C2) cells after extravasation into the liver. We therefore assessed the adhesive and migratory function of alpha 2 beta 1 on KX2C2 and RDX2C2 cells using a alpha 2 beta 1-specific stimulatory monoclonal antibody (mAb), JBS2, and a blocking mAb, BHA2.1. In comparison with RDX2C2 cells, KX2C2 were only weakly adherent to collagen and laminin. JBS2 stimulated alpha 2 beta 1-mediated interaction of KX2C2 cells with both collagen and laminin resulting in increases in cell movement on both matrix proteins. In the presence of Mn2+, JBS2-stimulated adhesion on collagen beyond an optimal level for cell movement. In comparison, an increase in RDX2C2 cell movement on collagen required a reduction in its adhesive strength provided by the blocking mAb BHA2.1. Consistent with these in vitro findings, in vivo videomicroscopy revealed that alpha 2 beta 1-mediated postextravasation cell movement of KX2C2 cells in the liver tissue could also be stimulated by JBS2. Thus, results demonstrate that alpha 2 beta 1 expression can modulate postextravasation cell movement by conferring either a stationary or motile phenotype to different cell types. These findings may be related to the differing metastatic activities of different tumor cell types.     

Integrin alpha 6A beta 1 induces CD81-dependent cell motility without engaging the extracellular matrix migration substrate

It is well established that integrins and extracellular matrix (ECM) play key roles in cell migration, but the underlying mechanisms are poorly defined. We describe a novel mechanism whereby the integrin alpha 6 beta 1, a laminin receptor, can affect cell motility and induce migration onto ECM substrates with which it is not engaged. By using DNA-mediated gene transfer, we expressed the human integrin subunit alpha 6A in murine embryonic stem (ES) cells. ES cells expressing alpha 6A (ES6A) at the surface dimerized with endogenous beta 1, extended numerous filopodia and lamellipodia, and were intensely migratory in haptotactic assays on laminin (LN)-1. Transfected alpha 6A was responsible for these effects, because cells transfected with control vector or alpha 6B, a cytoplasmic domain alpha 6 isoform, displayed compact morphology and no migration, like wild-type ES cells. The ES6A migratory phenotype persisted on fibronectin (Fn) and Ln-5. Adhesion inhibition assays indicated that alpha 6 beta 1 did not contribute detectably to adhesion to these substrates in ES cells. However, anti-alpha 6 antibodies completely blocked migration of ES6A cells on Fn or Ln-5. Control experiments with monensin and anti-ECM antibodies indicated that this inhibition could not be explained by deposition of an alpha 6 beta 1 ligand (e.g., Ln-1) by ES cells. Cross-linking with secondary antibody overcame the inhibitory effect of anti-alpha 6 antibodies, restoring migration or filopodia extension on Fn and Ln-5. Thus, to induce migration in ES cells, alpha 6A beta 1 did not have to engage with an ECM ligand but likely participated in molecular interactions sensitive to anti-alpha 6 beta 1 antibody and mimicked by cross-linking. Antibodies to the tetraspanin CD81 inhibited alpha 6A beta 1-induced migration but had no effect on ES cell adhesion. It is known that CD81 is physically associated with alpha 6 beta 1, therefore our results suggest a mechanism by which interactions between alpha 6A beta 1 and CD81 may up-regulate cell motility, affecting migration mediated by other integrins.     

TCR V alpha 24 and V beta 11 coexpression defines a human NK1 T cell analog containing a unique Th0 subpopulation

Murine NK1 natural T (NT) cells are a population of alphabeta T cells that express NK cell receptors and an invariant TCR rearrangement. These cells rapidly produce large amounts of IL-4 upon activation and have been suggested to promote Th2 differentiation. We sought to determine whether a human NK1 T cell analogue could be detected in PBMC, and if so, characterize the TCR usage, cytokine expression, and surface phenotype of this subset. Using flow cytometry, we have demonstrated a distinct population of V alpha24+, V beta11+, CD56+ T cells consistent with NT cells. Upon sequencing, these cells expressed an invariant V alpha24-J alphaQ TCR rearrangement, verifying their identity as a human NK1 T cell analogue. NT cells demonstrated increased frequencies of both IFN-gamma and IL-4 production. Strikingly, 30 to 45% of CD4+ NT cells expressed IL-4, a sixfold greater frequency than that seen in mainstream CD4+ alphabeta T cells. Contrary to the pattern seen with mainstream T cells, virtually all IL-4-producing NT cells coexpressed IFN-gamma, indicating that this subset of NT cells has a unique Th0 phenotype. These data establish that V alpha24+ NT cells are a potent source of IL-4 and as such, may play a role in Th2 priming in human immune responses. This work demonstrates that human NT cells can be phenotypically identified and functionally studied in the blood of healthy or diseased subjects.     

Biochemical characterization of human osteoclast integrins. Osteoclasts express alpha v beta 3, alpha 2 beta 1, and alpha v beta 1 integrins

The study of osteoclast integrins has been previously hampered by the lack of a source of large numbers of purified osteoclasts. Osteoclastoma, a human giant cell tumor of bone, supplied a rich source of osteoclasts within a tissue containing many diverse cell types. Osteoclastoma integrin immunostaining confirmed the presence of the integrin alpha v beta 3 complex and the alpha 2 and beta 1 integrin subunits on osteoclasts. However, weak integrin expression, for example with alpha v beta 5, was difficult to interpret. Purification with magnetic beads coated with vitronectin receptor monoclonal antibody (13C2) enabled osteoclast membranes to be isolated with high purity and yield (57%) from osteoclastoma tissue. Positively (osteoclast-enriched) selected membranes were biochemically assessed for integrin expression by immunoprecipitation and visualization by non-radioactive enhanced chemiluminescence. alpha 1, alpha 4, alpha 6, alpha 8, alpha M, alpha X, gpIIb, beta 4, beta 6, and beta 8 integrin chains were undetectable at a sensitivity of 1 ng. alpha 3, alpha 5, alpha L, beta 2, and alpha v beta 5 were found in the negatively selected osteoclastoma tissue but not in the positively purified osteoclast membranes. The presence of alpha v beta 1 and alpha 2 beta 1 dimers was demonstrated biochemically on the immunoisolated osteoclast membranes. Osteoclast alpha v beta 3 isolation by Arg-Gly-Asp (RGD) affinity chromatography for NH2-terminal amino acid sequencing confirmed that the osteoclast vitronectin receptor was identical to that previously characterized on other cell types. In situ hybridization using human alpha v riboprobes in osteoclasts from human and rodent bone further demonstrated the high level and specificity of expression of alpha v vitronectin receptor in osteoclasts.     

An alpha 5 beta 1-like integrin receptor mediates the binding of less pathogenic Candida species to fibronectin

The present study was undertaken to investigate whether less pathogenic Candida species (C. tropicalis, C. stellatoidea, C. krusei and C. glabrata) express a fibronectin receptor (FNr) antigenically related to alpha 5 beta 1 integrin, which mediates their binding to fibronectin (FN). By flow cytometric analysis, a monoclonal antibody (MAb) directed against human alpha 5 integrin subunit (clone SAM-1) and two different antisera to FNr positively stained C. tropicalis, C. stellatoidea and C. glabrata, with the greatest expression observed for C. tropicalis. No or only marginal immunoreactivity was found on C. krusei. C. tropicalis, C. stellatoidea, C. glabrata, but not C. krusei yeasts specifically adhered to FN; higher levels of adhesion were found for C. tropicalis and C. stellatoidea with respect to C. glabrata. Less pathogenic Candida spp. bound to the Arg-Gly-Asp (RGD) containing 120-kDa fragment of FN and adhesion to intact FN was markedly inhibited by Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), but not by Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) peptides. In addition, anti-alpha 5 SAM-1 MAb and both anti-FNr antisera strongly blocked binding of less pathogenic Candida spp. to FN. Overall, these results indicate that less pathogenic Candida spp., including C. tropicalis, C. stellatoidea and C. glabrata, express a receptor antigenically related to alpha 5 beta 1 integrin which mediates their adhesion to FN.     

Post-translational modifications of alpha5beta1 integrin by glycosaminoglycan chains. The alpha5beta1 integrin is a facultative proteoglycan

Cell-fibronectin interactions, mediated through several different receptors, have been implicated in a wide variety of cellular properties. Among the cell surface receptors for fibronectin, integrins are the best characterized, particularly the prototype alpha5beta1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified alpha5beta1 integrin as the only sulfated integrin among beta1 integrin heterodimers expressed by the human melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and chemical reduction. The covalent nature of alpha5beta1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 M NaCl, 4 M MgCl2, 8 M urea, and 6 M guanidine HCl. Based on deglycosylation procedures as chemical beta-elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the alpha5beta1 heterodimer and alpha5 and beta1 integrin subunits were proteoglycans. The importance of alpha5beta1 sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells.     

The muscle-specific laminin receptor alpha7 beta1 integrin negatively regulates alpha5 beta1 fibronectin receptor function

alpha7 beta1 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the alpha7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with alpha7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the alpha7 beta1. alpha7 transfectants, however, showed a 70% reduction in the ability to adhere to fibronectin and were unable to assemble a fibronectin matrix. The degree of reduction was inversely related to the level of alpha7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the alpha5 beta1 fibronectin receptor. Although cell surface expression of alpha5 beta1 was reduced by a factor of 20-25% in alpha7 transfectants compared to control untransfected cells, this slight reduction was not sufficient to explain the dramatic reduction in cell adhesion (70%) and matrix assembly (close to 100%). Binding studies showed that the affinity of 125I-fibronectin for its surface receptor was decreased by 50% in alpha7 transfectants, indicating that the alpha5 beta1 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn2+, a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn2+ restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in alpha7 transfectants. These data indicate that alpha7 expression leads to the functional down regulation of alpha5beta1 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of a negative cooperativity between alpha7 and alpha5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.     

Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors-the alpha1beta1 and alpha2beta1 integrins-through induction of mRNAs encoding the alpha1 and alpha2 subunits. In contrast, VEGF did not induce increased expression of the alpha3beta1 integrin, which also has been implicated in collagen binding. Integrin alpha1-blocking and alpha2-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and alpha1-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of alpha1beta1 and alpha2beta1 expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of alpha1-blocking and alpha2-blocking Abs. In vivo, a combination of alpha1-blocking and alpha2-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of alpha1beta1 and alpha2beta1 expression by EC is an important mechanism by which VEGF promotes angiogenesis and that alpha1beta1 and alpha2beta1 antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.     

Nonparenchymal cells from regenerating rat liver generate interleukin-1alpha and -1beta: a mechanism of negative regulation of hepatocyte proliferation

Fish skeletal muscle prokallikrein was purified from black sea bass, Centropritis striata, and used for the production of polyclonal antiserum. Tissue proteins from primitive fish and teleosts, an alligator, and an insectivore were resolved by sodium dodecylsulfate-polyacrylamide gel electrophoresis, Western blotted, and probed with fish muscle prokallikrein antiserum. A recurring theme was the presence of approximately 36 and 72 kDa kallikrein-like proteins in skeletal muscle, heart, gill, kidney, and spleen of higher teleosts and in selected tissues of sturgeon, shark, alligator, and mole. The presence of immunoreactive kallikreins in osmoregulatory organs such as the gills of teleosts and the rectal gland of sharks signifies a potential role for these proteins in osmoregulation. Black sea bass, rock bass, and sturgeon contained many immunoreactive kallikreins in their swimbladders, which implicates a role for kallikreins in the regulation of blood flow and vascular permeability to facilitate gas exchange within the bladder. Kallikreins were consistently identified in skeletal muscle and heart of all the species evaluated and may regulate local blood flow, muscle contraction or relaxation, or participate in various transport processes. The antiserum to fish prokallikrein recognized immunoreactive kallikreins from pancreatic tissues from fish and lower vertebrates, but not from the pyloric caecum of sea bass. The wide distribution of tissue kallikrein in lower vertebrates suggests that it may participate in a variety of physiological functions.     

Transforming growth factor beta 1 suppresses transformation in hepatocytes by regulating alpha 1 beta 1 integrin expression

We previously reported that (a) treatment of the ras-transformed hepatocyte cell line NR4 with transforming growth factor (TGF) beta 1 suppresses many characteristics associated with the transformed phenotype including altered morphology, actin cytoskeleton reorganization, and anchorage-independent growth such that the cells more closely resemble the immortalized CWSV1 parent cell line; (b) transformed NR4 cells expressed significantly less alpha 1 integrin RNA than the immortalized CWSV1 cells; and (c) TGF-beta 1 treatment of NR4 cells stimulated the expression of alpha 1 and beta 1 integrin RNAs. In this report, the role of the alpha 1 beta 1 integrin in TGF-beta 1-mediated suppression of the ras-transformed phenotype was investigated. We determined that (a) the cell surface integrin that increased in response to TGF-beta 1 treatment of NR4 cells was alpha 1 integrin; (b) TGF-beta 1 altered the ability of NR4 cells to attach to collagen and laminin, the extracellular matrix components that interact with the alpha 1 beta 1 integrin receptor; (c) TGF-beta 1 treatment resulted in relocalization of the alpha 1 integrin on the NR4 cell surface; and (d) TGF-beta 1-mediated inhibition of anchorage-independent growth was blocked by the presence of alpha 1 integrin antibody. A cell line that overexpresses alpha 1 integrin was derived from NR4 cells; characterization of these cells indicated that they continued to express H-ras RNA but were less transformed than the parent NR4 cells. Specifically, they had an altered morphology, an organized actin cytoskeleton, and reduced ability to demonstrate anchorage-independent growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of tumour necrosis factor alpha and interleukin 1 beta on the proliferation of cultured glomerular epithelial cells

Rat glomerular epithelial cells were cultured with human monocyte supernatant or with recombinant cytokines. A primary glomerular culture and a glomerular epithelial cell culture were made; supernatant from monocyte cultures derived from healthy humans, and recombinant tumour necrosis factor alpha (TNF alpha) or recombinant interleukin 1 beta (IL-1 beta) were added. Cell proliferation rates were assayed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. In serum-free media, consistent proliferation of glomerular epithelial cells (GEC) was observed throughout the 3 week culture period. Significant growth-stimulatory effects were induced by lipopolysaccharide-treated monocyte conditioned medium and by 1-50 ng/ml of TNF alpha, growth being up to 400% more than in the control culture. The effect of TNF alpha depended mainly on its interaction with epidermal growth factor (EGF). In contrast to TNF alpha, IL-1 beta inhibited GEC proliferation; this was due to the early appearance and proliferation of mesangial cells, despite the culture being serum-free. This study showed that activated monocytes secrete growth factors for GEC in vitro, and that interaction between both TNF alpha and IL-1 beta and between TNF alpha and EGF can modulate GEC proliferation. These findings suggest that, under pathological conditions, monocytes or macrophages affect GEC proliferation, probably being involved in crescent formation.     

The Xenopus Chk1 protein kinase mediates a caffeine-sensitive pathway of checkpoint control in cell-free extracts

We have analyzed the role of the protein kinase Chk1 in checkpoint control by using cell-free extracts from Xenopus eggs. Recombinant Xenopus Chk1 (Xchk1) phosphorylates the mitotic inducer Cdc25 in vitro on multiple sites including Ser-287. The Xchk1-catalyzed phosphorylation of Cdc25 on Ser-287 is sufficient to confer the binding of 14-3-3 proteins. Egg extracts from which Xchk1 has been removed by immunodepletion are strongly but not totally compromised in their ability to undergo a cell cycle delay in response to the presence of unreplicated DNA. Cdc25 in Xchk1-depleted extracts remains bound to 14-3-3 due to the action of a distinct Ser-287-specific kinase in addition to Xchk1. Xchk1 is highly phosphorylated in the presence of unreplicated or damaged DNA, and this phosphorylation is abolished by caffeine, an agent which attenuates checkpoint control. The checkpoint response to unreplicated DNA in this system involves both caffeine-sensitive and caffeine-insensitive steps. Our results indicate that caffeine disrupts the checkpoint pathway containing Xchk1.     

Sequential regulation of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis

Leukocyte emigration possibly requires dynamic regulation of integrin adhesiveness for endothelial and extracellular matrix ligands. Adhesion assays on purified vascular cell adhension molecule (VCAM)-1, fibronectin, and fibronectin fragments revealed distinct kinetic patterns for the regulation of very late antigen (VLA)-4 (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) avidity by the CC chemokines monocyte inflammatory protein (MIP)-1 alpha, RANTES (regulated on activation, normal T expressed and secreted), or monocyte chemoattractant protein (MCP)-1 in monocytes. CC chemokines induced early activation and subsequent deactivation of VLA-4, whereas upregulation of VLA-5 avidity occurred later and persisted. Controlled detachment assays in shear flow suggested that adhesive strength of VLA-4 for VCAM-1 or the 40-kD fragment of fibronectin (FN40) is more rapidly increased and subsequently reduced by MCP-1 than by MIP-1 alpha, and confirmed late and sustained activation of the adhesive strength of VLA-5 for the 120-kD fragment of fibronectin (FN120). Mn2+ or the stimulating beta 1 mAb TS2/16 strongly and stably enhanced monocyte binding to VCAM-1 or fibronectin, and locked beta 1 integrins in a high avidity state, which was not further modulated by CC chemokines. Mn2+ and mAb TS2/16 inhibited CC chemokine-induced transendothelial migration, particularly chemotaxis across stimulated endothelium that involved VLA-4 and VCAM-1. VLA-4 on Jurkat cells is of constitutively high avidity and interfered with migration across barriers expressing VCAM-1. Low but not high site densities of VCAM-1 or FN40 promoted, while FN120 impaired, beta 1 integrin-dependent monocyte chemotaxis to MCP-1 across filters coated with these substrates. Thus, we show that CC chemokines can differentially and selectively regulate avidity of integrins sharing common beta subunits. Transient activation and deactivation of VLA-4 may serve to facilitate transendothelial diapedesis, whereas late and prolonged activation of VLA-5 may mediate subsequent interactions with the basement membrane and extracellular matrix.