Accelerated re-epithelialization in beta3-integrin-deficient- mice is associated with enhanced TGF-beta1 signaling.

The upregulation of TGF-beta1 and integrin expression during wound healing has implicated these molecules in this process, but their precise regulation and roles remain unclear. Here we report that, notably, mice lacking beta(3)-integrins show enhanced wound healing with re-epithelialization complete several days earlier than in wild-type mice. We show that this effect is the result of an increase in TGF-beta1 and enhanced dermal fibroblast infiltration into wounds of beta(3)-null mice. Specifically, beta(3)-integrin deficiency is associated with elevated TGF-beta receptor I and receptor II expression, reduced Smad3 levels, sustained Smad2 and Smad4 nuclear localization and enhanced TGF-beta1-mediated dermal fibroblast migration. These data indicate that alpha(v)beta(3)-integrin can suppress TGF-beta1-mediated signaling, thereby controlling the rate of wound healing, and highlight a new mechanism for TGF-beta1 regulation by beta(3)-integrins.

Drosophila integrins and their ligands.

The major advance during the past year was the identification of ligands for two of the previously known position-specific integrins in Drosophila. At the same time, two new Drosophila integrin subunits (one alpha and one beta) were discovered, and significant progress was made on developmental genetic analyses of integrin functions, shedding light on the roles of integrins in Drosophila development.

Reduced recruitment of inflammatory cells in a contact hypersensitivity response in P-selectin-deficient mice.

The inflammatory response at sites of contact hypersensitivity induced by oxazolone was examined in the ears of P-selectin-deficient and wild-type mice. Accumulation of CD4+ T lymphocytes, monocytes, and neutrophils was reduced significantly in the mutant mice, as well as mast cell degranulation. In contrast, there was no significant difference in vascular permeability or edema between the two genotypes. The results demonstrate a role for P-selectin in recruitment of CD4+ T lymphocytes and show that P-selectin plays a role in long-term inflammation as well as in acute responses.

Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and vascular cell adhesion molecule 1.

We have used intravital microscopy to study physiologically perfused microvessels in murine bone marrow (BM). BM sinusoids and venules, but not adjacent bone vessels, supported rolling interactions of hematopoietic progenitor cells. Rolling did not involve L-selectin, but was partially reduced in wild-type mice treated with antibodies to P- or E-selectin and in mice that were deficient in these two selectins. Selectin-independent rolling was mediated by alpha4 integrins, which interacted with endothelial vascular cell adhesion molecule (VCAM)-1. Parallel contribution of the endothelial selectins and VCAM-1 is not known to direct blood cell trafficking to other noninflamed tissues. This combination of constitutively expressed adhesion molecules may thus constitute a BM-specific recruitment pathway for progenitor cells analogous to the vascular addressins that direct selective lymphocyte homing to lymphoid organs.

Interaction of dendritic cells with skin endothelium: A new perspective on immunosurveillance.

The goal of this study was to determine the mechanisms by which dendritic cells (DCs) in blood could interact with endothelium, a prerequisite to extravasation into tissues. Our results indicate that DCs express both HECA-452-reactive and nonreactive isoforms of P-selectin glycoprotein ligand 1 (PSGL-1) and can tether and roll efficiently on E- and P-selectin under flow conditions in vitro. Freshly isolated blood DCs were further observed to roll continuously along noninflamed murine dermal endothelium in vivo. This interaction is strictly dependent on endothelial selectins, as shown by experiments with blocking antibodies and with E- and P-selectin-deficient mice. We hypothesize that DCs in blood are constitutively poised at the interface of blood and skin, ready to extravasate upon induction of inflammation, and we showed that cutaneous inflammation results in a rapid recruitment of DCs from the blood to tissues. We propose that this is an important and previously unappreciated element of immunosurveillance.

Cell adhesion: old and new questions.

Metazoans clearly need cell adhesion to hold themselves together, but adhesion does much more than that. Adhesion receptors make transmembrane connections, linking extracellular matrix and adjacent cells to the intracellular cytoskeleton, and they also serve as signal transducers. In this article, I briefly summarize our present understanding of the molecular basis and biological consequences of cell adhesion and discuss how our current knowledge sheds light on questions of specificity of cell adhesion. I offer some thoughts and speculations about the evolution of cell-adhesion molecules and processes, consider their inter-relationships with other forms of cell-cell communication and discuss unresolved questions ripe for investigation as we enter the postgenomic era.

Surface distribution of LETS protein in relation to the cytoskeleton of normal and transformed cells.

The organization of LETS protein on the surface of NIL8 hamster cells has been examined by immunofluorescence staining. The distribution of LETS protein was found to depend on the culture conditions; in subconfluent, low-serum arrested cultures the LETS protein is predominantly located at the cell-substrate interface and also in regions of cell-cell contact, whereas in dense cultures the cells are surrounded by a network of LETS protein fibrils. Transformed derivatives of these cells exhibit only sporadic staining for LETS protein, in the form of short intercellular bridges. Agents that cause alterations in cell shape and cytoplasmic filaments have been used to explore the relationship of LETS protein to the internal cytoskeletal elements. Reciprocally, perturbations of the cell surface were examined for their effects on internal filaments. The arrangement of microtubules seems to be unrelated to the presence of LETS protein in the cells studied. Actin microfilament bundles and LETS protein respond in a coordinate fashion to some perturbants but independently with respect to others. The patterns of staining for LETS protein are consistent with an involvement in cell-to-cell and cell-to-substrate adhesion.

The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins.

Thrombospondin is one of a class of adhesive glycoproteins that mediate cell-to-cell and cell-to-matrix interactions. We have used two monoclonal antibodies to isolate cDNA clones of thrombospondin from a human endothelial cell cDNA library and have determined the complete nucleotide sequence of the coding region. Three regions of known amino acid sequence of human platelet thrombospondin confirm that the clones are authentic. Three types of repeating amino acid sequence are present in thrombospondin. The first is 57 amino acids long and shows homology with circumsporozoite protein from Plasmodium falciparum. The second is 50-60 amino acids long and shows homology with epidermal growth factor precursor. The third occurs as a continuous eightfold repeat of a 38-residue sequence; structural homology with parvalbumin and calmodulin indicates that these repeats constitute the multiple calcium-binding sites of thrombospondin. The amino acid sequence arg-gly-asp-ala is included in the last type 3 repeat. This sequence is probably the site for the association of thrombospondin with cells. In addition, localized homologies with procollagen, fibronectin, and von Willebrand factor are present in one region of the thrombospondin molecule.

Cell attachment to thrombospondin: the role of ARG-GLY-ASP, calcium, and integrin receptors.

Thrombospondin is a 420,000-D glycoprotein that has recently been shown to have several properties in common with the members of a class of adhesive proteins. To characterize further the adhesive properties of thrombospondin, we have studied its ability to support cell attachment. Thrombospondin adsorbed to plastic dishes supports the attachment of human endothelial and smooth muscle cells and the monocyte-like cell line (U937) as well as normal rat kidney cells. The majority of attached cells do not spread on the solid-phase thrombospondin. The attachment of all four cell types to thrombospondin is abolished if the assay is performed in the presence of EGTA, although the cells still attach to fibronectin. If thrombospondin is adsorbed to the dishes in the presence of EGTA and then washed with buffer containing calcium before addition of the cells, attachment is still markedly inhibited, indicating that calcium affects the conformation and function of thrombospondin. Attachment of all four cell types is also markedly inhibited by the synthetic peptides gly-arg-gly-asp-ser-pro (GRG-DSP) and gly-arg-gly-asp-ala-cys (GRGDAC) but not by the control peptide gly-arg-gly-glu-ser-pro (GRG-ESP). Affinity chromatography of n-octylglucoside extracts of surface-labeled endothelial cells or smooth muscle cells on thrombospondin-Sepharose and GRG-DSP-Affigel columns was used to identify an integrin complex related to glycoprotein IIb-IIIa as an RGD-dependent receptor for thrombospondin. In addition, a monoclonal antibody (LM609) that blocks attachment of endothelial cells to vitronectin, fibrinogen, and von Willebrand factor also inhibits attachment of endothelial cells to thrombospondin. These data indicate that the attachment of cells to thrombospondin is mediated by RGD and calcium-dependent mechanisms and is consistent with the hypothesis that the GRGDAC sequence in thrombospondin is a site for interaction with an integrin receptor of the beta 3 subclass.

Characterization of the external proteins of hamster fibroblasts.

The results of metabolic labeling studies and enzymatic treatments followed by analysis on polyacrylamide gels show that the external proteins of hamster fibroblast cell lines, which have been identified by lactoperoxidase-catalyzed iodination, do not contain sulphated mucopolysaccharides or hyaluronic acid and are probably unrelated to collagen. Several of the iodinated species comigrate with carbohydrate-containing molecules. In particular, the major iodine-labeled polypeptide of normal fibroblasts appears to be a glycoprotein. This glycoprotein is absent or much reduced in virus-transformed cells, as detected both by iodination and by metabolic labeling. We conclude that the major iodinated polypeptide is not detected on transformed cells because it is absent rather than because it is masked. Approximate molecular weights of the external proteins are also reported.

Antibodies to the conserved cytoplasmic domain of the integrin beta 1 subunit react with proteins in vertebrates, invertebrates, and fungi.

The integrin family of cell surface receptors can be divided into three groups on the basis of their homologous beta subunits: beta 1, beta 2, and beta 3. We have raised an antibody against a synthetic peptide corresponding to the COOH-terminal domain of the chicken integrin beta 1 subunit that reacts with beta subunits from a variety of vertebrates, invertebrates, and fungi, demonstrating strong evolutionary conservation of sequences in this domain. In Drosophila cells, the antibody recognizes integrin alpha beta complexes that appear to be identical with position-specific antigens. Cross-reactive proteins are also detected in Caenorhabditis elegans and Candida albicans. The antiserum is specific for beta 1 subunits and does not recognize other integrin beta subunits in humans. In immunofluorescence analyses of cultured cells, the antibody reacts only with permeabilized cells confirming that this highly conserved COOH-terminal segment is a cytoplasmic domain.

Layilin, a novel talin-binding transmembrane protein homologous with C-type lectins, is localized in membrane ruffles.

Changes in cell morphology and motility are mediated by the actin cytoskeleton. Recent advances in our understanding of the regulators of microfilament structure and dynamics have shed light on how these changes are controlled, and efforts continue to define all the structural and signaling components involved in these processes. The actin cytoskeleton-associated protein talin binds to integrins, vinculin, and actin. We report a new binding partner for talin that we have named layilin, which contains homology with C-type lectins, is present in numerous cell lines and tissue extracts, and is expressed on the cell surface. Layilin colocalizes with talin in membrane ruffles, and is recruited to membrane ruffles in cells induced to migrate in in vitro wounding experiments and in peripheral ruffles in spreading cells. A ten-amino acid motif in the layilin cytoplasmic domain is sufficient for talin binding. We have identified a short region within talin's amino-terminal 435 amino acids capable of binding to layilin in vitro. This region overlaps a binding site for focal adhesion kinase.

Retroviral expression of alternatively spliced forms of rat fibronectin.

We describe the construction in retroviral vectors and the expression of recombinant rat fibronectin (FN) cDNAs corresponding with the various alternatively spliced forms of FN. In NIH 3T3 cells, the exogenous rat FN subunits are efficiently secreted as heterodimers with endogenous mouse subunits. In contrast, in lymphoid WEHI231 cells, there is no endogenous FN synthesis and the recombinant FNs are secreted and can be purified as homogeneous proteins. We show that the purified recombinant FNs are biochemically and biologically functional. In basic assays for adhesion, spreading, cytoskeletal organization, and migration using various established adherent cell lines, different forms of FNs containing the different alternatively spliced segments show no marked differences in activity. We have used these recombinant FNs to investigate three systems in which earlier results had suggested potential differences between different forms of FN. First, all forms tested appear equally active in restoring normal morphology to a transformed cell line. Second, we detect minor differences in their ability to assemble into preexisting extracellular matrices. Finally, we report that only those forms of FN that contain the V segment will promote the spreading of a lymphoid cell line indicating that this segment confers additional biological functions for some cell types, a result that confirms and extends earlier data. These homogeneous, biologically active recombinant FNs will allow further studies of the role of the alternatively spliced segments of FN.

Reappearance of an embryonic pattern of fibronectin splicing during wound healing in the adult rat.

The adhesive extracellular matrix glycoprotein fibronectin (FN) is thought to play an important role in the cell migration associated with wound healing. Immunolocalization studies show abundant FN in healing wounds; however, these studies cannot define the cellular site(s) of FN synthesis, nor do they distinguish the different and potentially functionally distinct forms of FN that can arise from alternative splicing of the primary gene transcript. To examine these questions of FN synthesis and splicing during wound healing, we have performed in situ hybridization with segment-specific probes on healing wounds in adult rat skin. We find that the FN gene is expressed at increased levels after wounding both in the cells at the base of the wound and in subjacent muscle and dermis lateral to the wound. Interestingly, however, the pattern of splicing of FN mRNA was different in these areas. In adjacent dermis and muscle, the splicing pattern remains identical with that seen in normal adult rat skin, with two of the three spliced segments (EIIIA and EIIIB) excluded from FN mRNA. In contrast, these two segments are included in the FN mRNA present in the cells at the base of the wound. As a result, the mRNA in this region is spliced in a pattern identical with that found during early embryogenesis. The finding that the pattern of FN splicing during wound healing resembles an embryonic pattern suggests that alternative splicing may be used during wound healing as a mechanism to generate forms of FN that may be functionally more appropriate for the cell migration and proliferation associated with tissue repair.

Phagocytosis of gelatin-latex particles by a murine macrophage line is dependent on fibronectin and heparin.

It has been suggested that fibronectin plays a role in clearing particles from the circulation by promoting binding to phagocytes of the reticuloendothelial system. By use of a well-defined system to investigate the possible opsonic role of fibronectin, we have studied the uptake of gelatin-coated latex particles by a murine macrophage cell line (P388D1). Fibronectin promotes binding of gelatin-coated beads to these cells in both suspension and monolayer cultures. In both cases there is a requirement for heparin as a cofactor. Other glycosaminoglycans (chondroitin sulfates A and C, dermatan sulfate, and keratan sulfate) were inactive, whereas heparan sulfate was somewhat active. Proof that beads were actually endocytosed was obtained by electron microscopy, which showed beads internalized in membrane-bounded vesicles, and by immunofluorescence analyses, using antibodies to fibronectin to stain external beads. Two rapid assays for the opsonic activity of fibronectin were developed based on differential centrifugation of cell-associated beads and on the immunofluorescence procedure. Binding and endocytosis were time- and temperature-dependent and varied with the amount of gelatin on the beads and with the concentrations of fibronectin and heparin added, and could be inhibited by F(ab')2 antifibronectin. These studies provide a sound basis for a detailed analysis of the interaction of fibronectin with the cell surface and of its involvement in endocytosis.

Fibronectin localization in the rat glomerulus.

Fibronectin (FN) has been localized in the rat glomerulus using indirect immunolabeling. It was demonstrated in frozen sections by immunofluorescence, in sections of fixed kidneys by both peroxidase and ferritin-labeled antibodies, and in isolated glomerular basement membranes (GBM) with ferritin-labeled antibodies. Complementary and convergent results were obtained with these approaches. FN was most abundant in the mesangial matrix where it was especially concentrated at the interface between the endothelial and mesangial cells. In the peripheral capillary loop, FN was also detected in the laminae rarae (interna and externa) of the GBM--i.e., between the endothelial and epithelial cells, respectively, and the GBM. These findings indicate that FN is an important constituent of the glomerulus, and they are compatible with the assumption that, in the glomerulus, as in cultured cells, FN is involved in cell-to-cell (mesangial-mesangial, mesangial-endothelial) and cell-to-substrate (mesangial cell-mesangial matrix, epithelium-GBM, endothelium-GBM) attachment.

Integrin-mediated signals regulated by members of the rho family of GTPases.

The organization of the actin cytoskeleton can be regulated by soluble factors that trigger signal transduction events involving the Rho family of GTPases. Since adhesive interactions are also capable of organizing the actin-based cytoskeleton, we examined the role of Cdc42-, Rac-, and Rho-dependent signaling pathways in regulating the cytoskeleton during integrin-mediated adhesion and cell spreading using dominant-inhibitory mutants of these GTPases. When Rat1 cells initially adhere to the extracellular matrix protein fibronectin, punctate focal complexes form at the cell periphery. Concomitant with focal complex formation, we observed some phosphorylation of the focal adhesion kinase (FAK) and Src, which occurred independently of Rho family GTPases. However, subsequent phosphorylation of FAK and paxillin occurs in a Rho-dependent manner. Moreover, we found Rho dependence of the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also stimulates membrane ruffling; we show that this ruffling is independent of Rho but is dependent on both Cdc42 and Rac. Furthermore, we observed that Cdc42 controls the integrin-dependent activation of extracellular signal-regulated kinase 2 and of Akt, a kinase whose activity has been demonstrated to be dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42, Rac, and Rho, we observed that all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture. Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior.

Novel roles for alpha3beta1 integrin as a regulator of cytoskeletal assembly and as a trans-dominant inhibitor of integrin receptor function in mouse keratinocytes.

Previously we found that alpha3beta1 integrin-deficient neonatal mice develop micro-blisters at the epidermal-dermal junction. These micro-blisters were associated with poor basement membrane organization. In the present study we have investigated the effect of alpha3beta1-deficiency on other keratinocyte integrins, actin-associated proteins and F-actin organization. We show that the absence of alpha3beta1 results in an increase in stress fiber formation in keratinocytes grown in culture and at the basal face of the basal keratinocytes of alpha3-null epidermis. Moreover, we see a higher concentration of actin-associated proteins such as vinculin, talin, and alpha-actinin at focal contact sites in the alpha3-deficient keratinocytes. These changes in focal contact composition were not due to a change in steady-state levels of these proteins, but rather to reorganization due to alpha3beta1 deficiency. Apart from the loss of alpha3beta1 there is no change in expression of the other integrins expressed by the alpha3-null keratinocytes. However, in functional assays, alpha3beta1 deficiency allows an increase in fibronectin and collagen type IV receptor activities. Thus, our findings provide evidence for a role of alpha3beta1 in regulating stress fiber formation and as a trans-dominant inhibitor of the functions of the other integrins in mouse keratinocytes. These results have potential implications for the regulation of keratinocyte adhesion and migration during wound healing.

Genetic analysis of alpha 4 integrin functions in the development of mouse skeletal muscle.

It has been suggested, on the basis of immunolocalization studies in vivo and antibody blocking experiments in vitro, that alpha 4 integrins interacting with vascular cell adhesion molecule 1 (VCAM-1) are involved in myogenesis and skeletal muscle development. To test this proposal, we generated embryonic stem (ES) cells homozygous null for the gene encoding the alpha 4 subunit and used them to generate chimeric mice. These chimeric mice showed high contributions of alpha 4-null cells in many tissues, including skeletal muscle, and muscles lacking any detectable (< 2%) alpha 4-positive cells did not reveal any gross morphological abnormalities. Furthermore, assays for in vitro myogenesis using either pure cultures of alpha 4-null myoblasts derived from the chimeras or alpha 4-null ES cells showed conclusively that alpha 4 integrins are not essential for muscle cell fusion and differentiation. Taking these results together, we conclude that alpha 4 integrins appear not to play essential roles in normal skeletal muscle development.

Expression of normal and mutant avian integrin subunits in rodent cells.

We describe the expression of the beta 1 subunit of avian integrin in rodent cells with the purpose of examining the structure-function relationships of various domains within this subunit. The exogenous subunit is efficiently and stably expressed in 3T3 cells, and it forms hybrid heterodimers with endogenous murine alpha subunits, including alpha 3 and alpha 5. These heterodimers are exported to the cell surface and localize in focal contacts where both extracellular matrix and cytoskeleton associate with the plasma membrane. Hybrid heterodimers consisting of exogenous beta 1 and endogenous alpha subunits bind effectively and specifically to columns of cell-binding fragments of fibronectin. The exogenous avian beta 1 subunit appears to function as well as its endogenous murine equivalent, consistent with the high degree of conservation noted previously for integrins. In contrast, expression of a mutant form of avian integrin beta 1 subunit lacking the cytoplasmic domain produces hybrid heterodimers which, while efficiently exported to the cell surface and still capable of binding fibronectin, do not localize efficiently in focal contacts. This further implicates the cytoplasmic domain of the beta 1 subunit in interactions required for cytoskeletal organization.