Motility of fibronectin receptor-deficient cells on fibronectin and vitronectin: collaborative interactions among integrins.

Cells are capable of adhering to and migrating on protein components of the extracellular matrix. These cell-matrix interactions are thought to be mediated largely through a family of cell surface receptors termed integrins. However, the manner in which individual integrins are involved in cell adhesion and motility has not been fully determined. To explore this issue, we previously selected a series of CHO variants that are deficient in expression of the integrin alpha 5 beta 1, the "classical" fibronectin receptor. Two sets of subclones of these variants were defined which respectively express approximately 20% or 2% of fibronectin receptor on the cell surface when compared to wild-type cells (Schreiner, C. L., J. S. Bauer, Y. N. Danilov, S. Hussein, M. M. Sczekan, and R. L. Juliano. 1989. J. Cell Biol. 109:3157-3167). In the current study, the variant clones were tested for haptotactic motility on substrata coated with fibronectin or vitronectin. Data from assays using fibronectin show that cellular motility of the 20% variants was substantially decreased (30-75% of wild type), while the motility of the 2% variants was nearly abolished (2-20% of wild type). Surprisingly, a similar pattern was seen for haptotactic motility of both 2% and 20% variants when vitronectin was used (approximately 20-30% of wild type). The reduced haptotactic motility of the fibronectin receptor-deficient variant clones on vitronectin was shown not to be due to reduced vitronectin receptor (alpha v beta 3) expression nor to a failure of these variants to adhere to vitronectin substrata. Transfection of the deficient variants with a cDNA for the human alpha 5 subunit resulted in normal levels of fibronectin receptor expression (as a human alpha 5/hamster beta 1 chimera) and restored the motility of the CHO variants on fibronectin and vitronectin. This indicates that expression of the alpha 5 subunit is required for normal haptotactic motility on vitronectin substrata and suggests that the fibronectin receptor (alpha 5 beta 1) plays a cooperative role with vitronectin receptors in cell motility.     

Hydrocortisone stimulates fibronectin synthesis in cultured fibroblasts

The effect of hydrocortisone on fibronectin synthesis was investigated in cultured skin fibroblasis. Confluent cells were treated with hydrocortisone (10^?7 M to 10^?5 M) for 2 days and labeled with [³H]proline for 24 h. Fibronectin levels in both the culture medium and the cell layer were studied by gelatin-Sepharose affinity chromatography and SDS-polyacrylamide gel electrophoresis. In control cultures of human fetal skin fibroblasts, fibronectin constituted 8% of the total labeled proteins in the medium. The proportion of fibronectin increased to 13.1% at 10^?7 M hydrocortisone, 15.5% at 10^?6 M and to 19.4% at 10^?5 M. The proportion of fibronectin associated with the cell layer remained at 2-3% of total [³H]prolne-labeled proteins and did not increase with hydrocortisone exposure. The stimulating effect of hydrocortisone on medium fibronectin was also demonstrated in cultured human newborn foreskin fibroblasts and in rabbit skin fibroblasts.     

Molecular interactions between fibronectin and integrins during mouse blastocyst outgrowth

To investigate the mechanism of trophoblast adhesion to fibronectin, we cultured blastocysts in serum-free medium on proteolytic fibronectin fragments containing its major functional domains, and localized fibronectin-binding integrins in outgrowing trophoblast cells by immunofluorescent staining. Outgrowth comparable to that obtained with intact fibronectin was observed using a 120 kD chymotryptic fragment containing the central cell-binding domain (FN-120) and the Arg-Gly-Asp (RGD) recognition sequence. A 40 kD COOH-terminal chymotryptic fragment of fibronectin containing both a heparin-binding region and an alternate (non-RGD) cell-binding site was inactive in supporting trophoblast adhesion. Three synthetic peptides derived from the heparin-binding domain, including the CS1 alternate cell-binding site, were also unable to promote trophoblast cell adhesion. A 75 kD recombinant protein, ProNectin F, containing 13 copies of the cell recognition epitope of fibronectin, Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser, vigorously supported blastocyst outgrowth. Blastocyst outgrowth was not significantly different when surfaces were precoated with cellular fibronectin, which contains an alternatively spliced type III repeat and is the form actually encountered in vivo. Several putative fibronectin receptors were localized in trophoblast outgrowths by immunofluorescent labeling. Antibodies reactive with integrin subunits α3, α5, αllb, αv, β1 and β3, but not α4, all bound to trophoblast cells. Antibodies raised against either the β1 or β3 integrin subunits significantly inhibited fibronectin-mediated outgrowth. These findings demonstrate the key role of the central cell-binding domain of fibronectin in trophoblast adhesion, and suggest four RGD-binding integrins, α3β1, α5β1, αllbβ3, and αvβ3, that could mediate trophoblast adhesion in vitro and may play an important role during implantation. © 1995 Wiley-Liss, Inc.     

Butyrate stimulates fibronectin synthesis in cultured rabbit cornea

To understand corneal wound healing, in which fibronectin plays an important role, we investigated the regulatory mechanism of fibronectin synthesis in cultured rabbit corneal blocks in situ. The amount of fibronectin was determined by enzyme-linked immunosorbent assay. We found that butyrate stimulated fibronectin synthesis in a dose-response fashion, and that butyrate had a greater stimulatory effect than did any of its derivatives examined. This suggests that butyrate stimulates fibronectin synthesis specifically; 8Br-cAMP also stimulated fibronectin synthesis. Additivity of stimulation by butyrate and by 8Br-cAMP was observed at the saturated concentration of each; the present result indicated an independent mechanism of action for these two compounds.     

Involvement of gangliosides and glycoprotein fibronectin receptors in cellular adhesion to fibronectin

We have used a rat neural cell line, B65, to investigate the relative contributions of gangliosides and glycoprotein receptors in adhesion to fibronectin. Monoclonal antibodies against two neuroectoderm-associated gangliosides, D1.1 and GD3, inhibit the rate of B65 attachment to fibronectin, suggesting that these gangliosides are involved in the adhesion process. Adhesion to fibronectin is not affected by a third monoclonal antibody against a separate, unidentified cell-surface component of B65 cells. Furthermore, B65 cells lacking D1.1 adhere to fibronectin at a slower rate than B65 cells that express D1.1. The involvement of glycoprotein receptors in adhesion is demonstrated by the ability of antibodies against human fibronectin receptor to inhibit B65 attachment to fibronectin. In addition, adhesion is blocked by a hexapeptide containing the Arg-Gly-Asp fibronectin sequence which is necessary for binding to the receptor. Trypsin treatment of B65 cells in the absence of divalent cations results in proteolysis of the fibronectin receptor with an accompanying loss of ability of the cells to attach to fibronectin. D1.1 and GD3 expression is not affected by this trypsinization, indicating that the gangliosides alone are incapable of mediating attachment. The glycoprotein receptors must be primarily responsible for adhesion to fibronectin with the gangliosides playing a secondary role as enhancers or modulators.     

Identification of fibronectin receptors on T lymphocytes

We report the identification of fibronectin receptors on thymocytes and T lymphoma cells. Affinity chromatography of extracts of the T cell lymphoma, WR16.1, on a fibronectin-Sepharose column combined with specific elution using a synthetic peptide containing the cell attachment-promoting sequence, arginine-glycine-aspartic acid, yielded two polypeptide components having apparent molecular masses of approximately 160 kD reduced and 175 and 150 kD nonreduced. Immunoprecipitations from surface-iodinated WR16.1 cells or fibronectin-adherent thymocytes using a rabbit antiserum raised against the fibronectin receptor that is present on human fibroblasts revealed, in each case, the same two radiolabeled components. In contrast, immunoprecipitation from fibronectin-nonadherent T lymphoma cells, designated WR2.3, revealed the presence of only the smaller subunit. Although the lymphocyte receptor and the fibronectin receptor identified on fibroblasts share immunologic determinants, they differ in that the molecular mass of the lymphocyte protein is larger. Moreover, trypsinization of either thymocytes or the WR16.1 T lymphoma cells resulted in a subsequent loss of their ability to adhere to fibronectin-coated substrates and a reduction in the electrophoretic mobility of each of the polypeptide chains of the fibronectin receptor present on their surfaces. These changes, however, were not observed with normal rat kidney fibroblasts or mouse 3T3 fibroblasts in response to trypsinization. The data establish the existence on normal lymphocytes of fibronectin receptors that are quite similar to those found on fibroblasts. The possible function of this molecule on thymocytes is discussed.     

Matrix metalloproteinase 1 interacts with neuronal integrins and stimulates dephosphorylation of Akt

Several studies have demonstrated that matrix metalloproteinases (MMPs) are cytotoxic. The responsible mechanisms, however, are not well understood. MMPs may promote cytotoxicity through their ability to disrupt or degrade matrix proteins that support cell survival, and MMPs may also cleave substrates to generate molecules that stimulate cell death. In addition, MMPs may themselves act on cell surface receptors that affect cell survival. Among such receptors is the alpha(2)beta(1) integrin, a complex that has previously been linked to leukocyte death. In the present study we show that human neurons express alpha(2)beta(1) and that pro-MMP-1 interacts with this integrin complex. We also show that stimulation of neuronal cultures with MMP-1 is associated with a rapid reduction in the phosphorylation of Akt, a kinase that can influence caspase activity and cell survival. Moreover, MMP-1-associated dephosphorylation of Akt is inhibited by a blocking antibody to the alpha(2) integrin, but not by batimastat, an inhibitor of MMP-1 enzymatic activity. Such dephosphorylation is also stimulated by a catalytic mutant of pro-MMP-1. Additional studies show that MMP-1 causes neuronal death, which is significantly diminished by both a general caspase inhibitor and anti-alpha(2) but not by batimastat. Together, these results suggest that MMP-1 can stimulate dephosphorylation of Akt and neuronal death through a non-proteolytic mechanism that involves changes in integrin signaling.     

Hyposialylation of neprilysin possibly affects its expression and enzymatic activity in hereditary inclusion-body myopathy muscle

Autosomal recessive hereditary inclusion-body myopathy (h-IBM) is caused by mutations of the UDP- N -acetylglucosamine 2-epimerase/ N -acetylmannosamine kinase gene, a rate-limiting enzyme in the sialic acid metabolic pathway. Previous studies have demonstrated an abnormal sialylation of glycoproteins in h-IBM. h-IBM muscle shows the abnormal accumulation of proteins including amyloid-β (Aβ). Neprilysin (NEP), a metallopeptidase that cleaves Aβ, is characterized by the presence of several N-glycosylation sites, and changes in these sugar moieties affect its stability and enzymatic activity. In the present study, we found that NEP is hyposialylated and its expression and enzymatic activity reduced in all h-IBM muscles analyzed. In vitro , the experimental removal of sialic acid by Vibrio Cholerae neuraminidase in cultured myotubes resulted in reduced expression of NEP. This was most likely because of a post-translational modification consisting in an abnormal sialylation of the protein that leads to its reduced stability. Moreover, treatment with Vibrio Cholerae neuraminidase was associated with an increased immunoreactivity for Aβ mainly in the form of distinct cytoplasmic foci within myotubes. We hypothesize that, in h-IBM muscle, hyposialylated NEP has a role in hampering the cellular Aβ clearing system, thus contributing to its abnormal accumulation within vulnerable fibers and possibly promoting muscle degeneration.     

Mesoderm induction and the control of gastrulation in Xenopus laevis: the roles of fibronectin and integrins

Exposure of isolated Xenopus animal pole ectoderm to the XTC mesoderm-inducing factor (XTC-MIF) causes the tissue to undergo gastrulation-like movements. In this paper, we take advantage of this observation to investigate the control of various aspects of gastrulation in Xenopus. Blastomeres derived from induced animal pole regions are able, like marginal zone cells, but unlike control animal pole blastomeres, to spread and migrate on a fibronectin-coated surface. Dispersed animal pole cells are also able to respond to XTC-MIF in this way; this is one of the few mesoderm-specific responses to induction that has been observed in single cells. The ability of induced animal pole cells to spread on fibronectin is abolished by the peptide GRGDSP. However, the elongation of intact explants is unaffected by this peptide. This may indicate that fibronectin-mediated cell migration is not required for convergent extension. We have investigated the molecular basis of XTC-MIF-induced gastrulation-like movements by measuring rates of synthesis of fibronectin and of the integrin beta 1 chain in induced and control explants. No significant differences were observed, and this suggests that gastrulation is not initiated simply by control of synthesis of these molecules. In future work, we intend to investigate synthesis of other integrin subunits and to examine possible post-translational modifications to fibronectin and the integrins.     

Potential proteolytic activity of human plasma fibronectin: fibronectin gelatinase

Human plasma fibronectin contains a latent proteinase that after activation cleaves gelatin and fibronectin. The autoactivation propensity of the two purified cathepsin D-produced fragments of fibronectin (190 and 120 kDa) was compared. Both polypeptides were spontaneously activated in the presence of Ca2+. This activation was inhibited by EDTA. The active gelatinase was isolated from the autodigest of the 190-kDa fragment. Among various protein substrates, including laminin and native type I and IV collagens, the purified enzyme degraded only gelatin and fibronectin. We have named this proteinase FN-gelatinase. FN-gelatinase is inhibited by phenylmethanesulfonyl fluoride and also by pepstatin A like retroviral aspartic proteinases. The amino-acid composition of the purified enzyme (35 kDa) was compared with the entire fibronectin sequence using the computer programme FIT. The optimal fit indicated that the 35-kDa fragment corresponds to the stretch # 1043-1404. This sequence contains a 93-residue segment (# 1140-1233) analogous to retroviral aspartic proteinases, comprising the sequence DTG of their putative active site.     

The combined action of hyaluronic acid and fibronectin stimulates neutrophil migration

Previous investigations have demonstrated that the chemotactic response of polymorphonuclear leukocytes (PMN) was stimulated by hyaluronic acid (HA) when serum was present. The aim of the present investigation was to identify the serum factor necessary for the stimulation of PMN chemotaxis by HA. By means of gel filtration, the m.w. of the serum component was shown to be greater than 350,000. Immunoprecipitation of serum with anti-fibronectin, but not with anti-IgM and anti-alpha 2-macroglobulin, inhibited the stimulation of PMN chemotaxis by HA. Preincubation of PMN with HA (10 to 500 micrograms/L) and isolated fibronectin (0.1 to 100 mg/L) significantly stimulated the chemotactic response of PMN. Also, random migration of PMN was significantly increased by preincubation of the cells with HA (10 to 500 micrograms/L) and isolated fibronectin (50 to 200 mg/L). Additionally, PMN preincubated with HA (10 to 50 micrograms/L) and with fibronectin (10 to 50 mg/L) added afterwards, and PMN preincubated with fibronectin (10 mg/L) and with HA (5 to 10 micrograms/L) added after the preincubation, showed a significant stimulation of the chemotactic response. PMN preincubated with serum and chondroitin sulfate, or with fibrinogen and HA, demonstrated no stimulation of the chemotactic response. The present investigation suggests that the combined action of HA and fibronectin, which probably takes place at the cellular membrane, is a major mechanism in the HA-mediated stimulation of PMN migration.     

Roles of integrins and fibronectin in the entry of Streptococcus pyogenes into cells via protein F1

Entry of group A streptococcus (GAS) into cells has been suggested as an important trait in GAS pathogenicity. Protein F1, a fibronectin (Fn) binding protein, mediates GAS adherence to cells and the extracellular matrix, and efficient cell internalization. We demonstrate that the cellular receptors responsible for protein F1-mediated internalization of GAS are integrins capable of Fn binding. In HeLa cells, bacterial entry is blocked by anti-β1 integrin monoclonal antibody. In the mouse cell line GD25, a β1 null mutant, the αvβ3 integrin promotes GAS entry. Internalization of these cells by GAS is blocked by a peptide that specifically binds to αvβ3 integrin. In both cell lines, entry of GAS requires the occupancy of protein F1 by Fn. Neither the 29 kDa nor the 70 kDa N-terminal fragments or the 120 kDa cell-binding fragment of Fn promote bacterial entry. Fn-coated beads are taken up efficiently by HeLa cells. Both the entry of GAS via protein F1 and the uptake of Fn-coated beads are blocked by anti-β1 antibody but are unaffected by a large excess of soluble Fn. Internalization of HeLa cells by bacteria bearing increasing amounts of prebound Fn to protein F1 reveals a sigmoidal ultrasensitive curve. These suggest that the ability of particles to interact via Fn with multiple integrin sites plays a central role in their ability to enter cells.     

Preferential expression of fibronectin receptors on immature thymocytes

Fibronectin-adherent (FNR+) thymocytes are enriched for immature (CD4-8-) and large (CD4+8+) cells, and depleted of mature (CD4-8+ and CD4+8-) and nonmature small (CD4+8+) cells. Among purified CD4-8- thymocytes, cells with the surface marker J11d and the IL-2 receptor, which can give rise to all other thymocyte subsets, showed selective attachment to fibronectin. Analysis of FNR+ thymocytes showed that such cells are greatly enriched for cells in cycle. Additionally, FNR+ cells expressed low levels of T cell receptor. These results suggest a role for the fibronectin receptor during the early, proliferative phase of thymocyte differentiation. The data suggest that loss of the fibronectin receptor is a hallmark of cells that have become committed either to functional maturation or to programmed cell death.     

A protein kinase C/Ras/ERK signaling pathway activates myeloid fibronectin receptors by altering beta1 integrin sialylation

Here we report that myeloid cells differentiating along the monocyte/macrophage lineage down-regulate the ST6Gal-I sialyltransferase via a protein kinase C/Ras/ERK signaling cascade. In consequence, the beta1 integrin subunit becomes hyposialylated, which stimulates the ligand binding activity of alpha5beta1 fibronectin receptors. Pharmacologic inhibitors of protein kinase C, Ras, and MEK, but not phosphoinositide 3-kinase, block ST6Gal-I down-regulation, integrin hyposialylation, and fibronectin binding. In contrast, constitutively active MEK stimulates these same events, indicating that ERK is both a necessary and sufficient activator of hyposialylation-dependent integrin activation. Consistent with the enhanced activity of hyposialylated cell surface integrins, purified alpha5beta1 receptors bind fibronectin more strongly upon enzymatic desialylation, an effect completely reversed by resialylation of these integrins with recombinant ST6Gal-I. Finally, we have mapped the N-glycosylation sites on the beta1 integrin to better understand the potential effects of differential sialylation on integrin structure/function. Notably, there are three N-glycosylated sites within the beta1 I-like domain, a region that plays a crucial role in ligand binding. Our collective results suggest that variant sialylation, induced by a specific signaling cascade, mediates the sustained increase in cell adhesiveness associated with monocytic differentiation.     

Transforming growth factor beta stimulates the expression of fibronectin and of both subunits of the human fibronectin receptor by cultured human lung fibroblasts

Transforming growth factors of the beta-class (TGFs-beta) stimulate extracellular matrix synthesis and have been implicated in embryogenesis, wound healing, and fibroproliferative responses to tissue injury. Because cells communicate with several extracellular matrix components via specific cell membrane receptors, we hypothesized that TGFs-beta may also regulate the expression of such receptors. We confirmed that TGF-beta 1 increases the expression of fibronectin, an adhesive glycoprotein expressed during embryogenesis and tissue remodeling. Based upon the 48-72-h period required for a maximal fibroproliferative response to dermal injections of TGF-beta 1, we exposed human fetal lung fibroblasts (IMR-90) to TGF-beta 1 for periods up to 48 h in vitro. We observed as much as 6-fold increases in fibronectin synthesis by 24 h as previously reported for fibroblastic cells (Ignotz, R. A., and Massagué, J. (1986) J. Biol. Chem. 261, 4337-4345; Ignotz, R. A., Endo, T., and Massagué, J. (1987) J. Biol. Chem. 262, 6443-6446; Raghow, R., Postlethwaithe, A. E., Keski-Oja, J., Moses, H. L., and Kang, A. H. (1987) J. Clin. Invest. 79, 1285-1288), but up to 30-fold increases by 48 h. These increases are accompanied by similar increases in fibronectin mRNA levels which are prevented by actinomycin D treatment. Using a monospecific antibody raised to the human placental fibronectin receptor complex, we found that TGF-beta 1 stimulated fibronectin receptor synthesis up to 20-40-fold and increases mRNA levels encoding both the alpha- and beta-subunits up to 3-fold, compared to control IMR-90 in serum-free medium. Actinomycin D blocks TGF-beta 1-mediated increases in receptor mRNA levels. The earliest detectable TGF-beta 1-mediated increases in fibronectin receptor complex protein synthesis and mRNA levels occur at 8 h, whereas the earliest increases in fibronectin protein synthesis and mRNA levels occur at 12 h. These results demonstrate that TGF-beta 1 stimulates fibronectin receptor synthesis, extending the diverse stimulatory activities of this polypeptide to matrix receptors. In addition, because fibronectin matrix assembly may involve the fibronectin cell adhesive receptor complex, increased receptor expression may help drive fibronectin deposition into matrix.     

Different integrins mediate cell spreading,haptotaxis and lateral migration of HaCaT keratinocytes on fibronectin

Collaborative role of various fibronectin-binding integrins (α5β1, αvβ1 and αvβ6) as mediators of cell adhesion and migration on fibronectin was studied using cultured HaCaT keratinocytes. This cell line spontaneously expressed all three fibronectin-binding integrins. In addition, the expression of αvβ6 integrin was strongly and specifically upregulated by transforming growth factor-β1 (TGFβ1) whereas the amount of other integrins remained practically unchanged on the cell surface. Adhesion, spreading and motility of HaCaT keratinocytes on fibronectin were promoted by TGFβ1. Based on antibody blocking experiments, both untreated and TGFβ1-treated HaCaT cells used αvβ6 integrin as their main fibronectin receptor for cell spreading. In contrast to TGFβ1-treated cells, the untreated cells also needed α5β1 integrin for maximal cell spreading on fibronectin. Combinations of antibodies blocking both of these receptors totally prevented spreading of both untreated and TGFβ1-treated cells. Haptotactic motility of individual HaCaT cells through fibronectin-coated membranes was again mainly dependent on αvβ6 integrin, while αvβ1 and α5β1 integrins played a lesser role both in untreated and TGFβ1-treated HaCaT cells. However, unlike haptotaxis, lateral migration of HaCaT cell sheet was mainly mediated by β1 integrins, and αvβ6 integrin showed a minor role. The migration process appeared to involve a number of β1 integrins that could adaptively replace each other when blocking antibodies were present. Thus, keratinocytes appear to use different fibronectin receptors for different functions, such as cell spreading, haptotaxis and lateral migration. The cells can also adapt to a situation where one receptor is unfunctional by switching to another receptor of the same ligand.     

Collagen fibrillogenesis: fibronectin, integrins, and minor collagens as organizers and nucleators

Collagens are triple helical proteins that occur in the extracellular matrix (ECM) and at the cell-ECM interface. There are more than 30 collagens and collagen-related proteins but the most abundant are collagens I and II that exist as D-periodic (where D=67nm) fibrils. The fibrils are of broad biomedical importance and have central roles in embryogenesis, arthritis, tissue repair, fibrosis, tumor invasion, and cardiovascular disease. Collagens I and II spontaneously form fibrils in vitro, which shows that collagen fibrillogenesis is a selfassembly process. However, the situation in vivo is not that simple; collagen I-containing fibrils do not form in the absence of fibronectin, fibronectin-binding and collagen-binding integrins, and collagen V. Likewise, the thin collagen II-containing fibrils in cartilage do not form in the absence of collagen XI. Thus, in vivo, cellular mechanisms are in place to control what is otherwise a protein self-assembly process. This review puts forward a working hypothesis for how fibronectin and integrins (the organizers) determine the site of fibril assembly, and collagens V and XI (the nucleators) initiate collagen fibrillogenesis.     

Different integrins mediate cell spreading, haptotaxis and lateral migration of HaCaT keratinocytes on fibronectin

Collaborative role of various fibronectin-binding integrins (alpha5beta1, alphavbeta1 and alphavbeta6) as mediators of cell adhesion and migration on fibronectin was studied using cultured HaCaT keratinocytes. This cell line spontaneously expressed all three fibronectin-binding integrins. In addition, the expression of alphavbeta6 integrin was strongly and specifically upregulated by transforming growth factor-beta1 (TGFbeta1) whereas the amount of other integrins remained practically unchanged on the cell surface. Adhesion, spreading and motility of HaCaT keratinocytes on fibronectin were promoted by TGFbeta1. Based on antibody blocking experiments, both untreated and TGFbeta1-treated HaCaT cells used alphavbeta6 integrin as their main fibronectin receptor for cell spreading. In contrast to TGFbeta1-treated cells, the untreated cells also needed alpha5beta1 integrin for maximal cell spreading on fibronectin. Combinations of antibodies blocking both of these receptors totally prevented spreading of both untreated and TGFbeta1-treated cells. Haptotactic motility of individual HaCaT cells through fibronectin-coated membranes was again mainly dependent on alphavbeta6 integrin, while alphavbeta1 and alpha5beta1 integrins played a lesser role both in untreated and TGFbeta1-treated HaCaT cells. However, unlike haptotaxis, lateral migration of HaCaT cell sheet was mainly mediated by beta1 integrins, and alphavbeta6 integrin showed a minor role. The migration process appeared to involve a number of beta1 integrins that could adaptively replace each other when blocking antibodies were present. Thus, keratinocytes appear to use different fibronectin receptors for different functions, such as cell spreading, haptotaxis and lateral migration. The cells can also adapt to a situation where one receptor is unfunctional by switching to another receptor of the same ligand.     

S100A9 mediates neutrophil adhesion to fibronectin through activation of beta2 integrins

Neutrophil migration from the blood to inflammatory sites follows a cascade of events, in which adhesion to endothelial cells and extracellular matrix proteins is essential. S100A8, S100A9, and S100A12 are small abundant proteins found in human neutrophil cytosol and presumed to be involved in leukocyte migration. Here we investigated the S100 proteins' activities in neutrophil tissue migration by evaluating their effects on neutrophil adhesion to certain extracellular matrix proteins. S100A9 induced adhesion only to fibronectin and was the only S100 protein that stimulated neutrophil adhesion to this extracellular matrix protein. Experiments with blocking antibodies revealed that neither beta1 nor beta3 integrins were strongly involved in neutrophil adhesion to fibronectin, contrary to what the literature predicted. In contrast, neutrophil adhesion to fibronectin was completely inhibited by anti-beta2 integrins, suggesting that S100A9-induced specific activation of beta2 integrin is essential to neutrophil adhesion.