Fibronectin dependent upregulation of matrix metalloproteinases in hepatic stellate cells

Activation and transition of hepatic stellate cells (HSCs) to myofibroblast (MFB)-like cells is influenced by growth factors, cytokines and matrix proteins like fibronectin (FN). To examine whether the FN-dependent transition of HSCs is mediated through FN receptor, a marker function, such as matrix metallo-proteinase (MMP) production by HSCs in primary culture was studied. An upregulation of MMP production by HSCs maintained on FN was observed. FN-dependent upregulation of MMPs was significantly reduced when cells were pre-treated with antibodies to alpha5beta1 integrin. Treatment of cells with genistein, a protein kinase C inhibitor completely blocked the gelatinase production by HSCs, indicating that the FN-dependent upregulation of MMPs is mediated through integrins and it involves tyrosine phosphorylation dependent signaling pathways.     

Accessibility to the fibronectin synergy site in a 3D matrix regulates engagement of alpha5beta1 versus alphavbeta3 integrin receptors

Cell adhesion and migration on fibronectin (FN) extracellular matrix are mediated by integrin receptors. Integrins alpha5beta1 and alphavbeta3 require the RGD cell-binding sequence in FN, but alpha5beta1 also requires the nearby synergy site for maximal binding. In this study, we investigated how differences in the numbers of RGD or synergy sites within a three-dimensional (3D) FN-rich matrix influence cell adhesion and migration. CHO cell adhesion, spreading, and migration were reduced on 3D chimeric matrix containing FN lacking RGD (FN(RGD-)). Incorporation of FN with mutation of the synergy site (FN(syn-)), however, resulted in selective usage of integrins. CHO cells expressing alpha5beta1 showed decreased interactions with FN(syn-) chimeric matrix. In contrast, the presence of FN(syn-) had no effect on CHOalphavbeta3 cell migration. Interestingly, CHOalpha5/alphavbeta3 cells expressing both integrins selectively used alpha5beta1 for migration on wild type FN matrix but preferred alphavbeta3 for migration on FN(syn-) chimeric matrix. Thus sequestration or exposure of the FN synergy site within a 3D matrix may represent a novel mechanism for regulating cell functions through differential usage of integrin receptors. [Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resource: a video recording shows migration of HT1080 cells on 3D matrix. HT1080 cells were allowed to attach to the matrix in serum-free DMEM for 2 h. FBS was then added to the medium to a final concentration of 10% and video recording was started. Images were taken every 5 min for 2 h. The video plays at 6 frames/s.].     

Ligand-dependent and -independent integrin focal contact localization: the role of the alpha chain cytoplasmic domain.

Many integrin receptors localize to focal contact sites upon binding their ligand. However, unoccupied integrin receptors do not localize to focal contact sites. Because the integrin beta 1 cytoplasmic domain appears to have a focal contact localization signal, there must be a mechanism by which this domain is kept inactive in the unoccupied state and becomes exposed or activated in the occupied receptor. We considered that this mechanism involves the alpha subunit cytoplasmic domain. To test this hypothesis, we have established two NIH 3T3 cell lines that express either the human alpha 1 wild-type subunit (HA1 cells) or the cytoplasmic domain deleted alpha 1 subunit (CYT cells). Both cell lines express similar levels of the human alpha 1 subunit, and there is no significant effect of the deletion on the dimerization and surface expression of the receptor. Furthermore, the deletion had no effect on the binding or adhesion via alpha 1 beta 1 to its ligand collagen IV. However, when these two cell lines are plated on fibronectin (FN), which is a ligand for alpha 5 beta 1 but not for alpha 1 beta 1, there is a striking difference in the cellular localization of alpha 1 beta 1. The HA1 cells show only alpha 5 in focal contacts, without alpha 1, demonstrating that all of the integrin localization is ligand dependent. In contrast, when the CYT cells are plated on FN, the mutant alpha 1 appears in focal contacts along with the alpha 5/beta 1. Thus, there is both ligand-dependent (alpha 5/beta 1) and ligand-independent (alpha 1/beta 1) focal contact localization in these cells. The truncated alpha 1 also localized to focal contacts in a ligand-independent manner on vitronectin. We conclude that the mutant alpha 1 no longer requires ligand occupancy for focal contact localization. These data strongly suggest that the alpha cytoplasmic domain plays a role in the normal ligand-dependent integrin focal contact localization.     

Integrin dynamics and matrix assembly: tensin-dependent translocation of alpha(5)beta(1) integrins promotes early fibronectin fibrillogenesis

Fibronectin matrix assembly is a multistep, integrin-dependent process. To investigate the role of integrin dynamics in fibronectin fibrillogenesis, we developed an antibody-chasing technique for simultaneous tracking of two integrin populations by different antibodies. We established that whereas the vitronectin receptor alpha(v)beta(3) remains within focal contacts, the fibronectin receptor alpha(5)beta(1) translocates from focal contacts into and along extracellular matrix (ECM) contacts. This escalator-like translocation occurs relative to the focal contacts at 6.5 +/- 0.7 microm/h and is independent of cell migration. It is induced by ligation of alpha(5)beta(1) integrins and depends on interactions with a functional actin cytoskeleton and vitronectin receptor ligation. During cell spreading, translocation of ligand-occupied alpha(5)beta(1) integrins away from focal contacts and along bundles of actin filaments generates ECM contacts. Tensin is a primary cytoskeletal component of these ECM contacts, and a novel dominant-negative inhibitor of tensin blocked ECM contact formation, integrin translocation, and fibronectin fibrillogenesis without affecting focal contacts. We propose that translocating alpha(5)beta(1) integrins induce initial fibronectin fibrillogenesis by transmitting cytoskeleton-generated tension to extracellular fibronectin molecules. Blocking this integrin translocation by a variety of treatments prevents the formation of ECM contacts and fibronectin fibrillogenesis. These studies identify a localized, directional, integrin translocation mechanism for matrix assembly.     

Beta 1 and beta 3 integrins have different roles in the adhesion and migration of vascular smooth muscle cells on extracellular matrix.

Extracellular matrix receptors on ductus arteriosus smooth muscle cells (SMC) must enable the cells to migrate through both interstitial and basement membrane matrices to form intimal mounds during postnatal ductus closure. We examined the role of beta 1 and beta 3 integrin receptors on SMC adhesion and migration. Using a new assay to measure cell migration, we found that lamb ductus arteriosus SMC attach to and migrate over surfaces coated with fibronectin (FN), laminin (LN), vitronectin (VN), and collagens I (I) and IV (IV). Blocking antibodies, specific to different integrin complexes, showed that SMC adhesion to FN, LN, I, and IV depended exclusively on functioning beta 1 integrins with little, if any, contribution by the alpha V beta 3 integrin; on the other hand, cell migration over these substrates depended to a large extent on the alpha V beta 3 receptor. Immunofluorescent staining demonstrated that during the early phase of SMC migration, the beta 1 integrins organized rapidly into focal plaques that, with time, gradually covered the cell's basal surface; on the other hand, the beta 3 receptor remained concentrated at all times at the cell's margins. Ligand affinity chromatography and immunoprecipitation techniques identified a unique series of beta 1 integrins binding to each matrix component: FN (alpha 5 beta 1, alpha 3 beta 1, alpha V beta 1), LN (alpha 1 beta 1, alpha 7 beta 1), VN (alpha V beta 1), I (alpha 1 beta 1, alpha 2 beta 1), and IV (alpha 1 beta 1). In contrast, the beta 3 integrin, alpha V beta 3, bound to all the substrates tested: FN, LN, VN, I, and IV. The results indicate that beta 1 and beta 3 integrins may play different roles in attachment and migration as SMC move through the vascular extracellular matrix to produce obliteration of the ductus arteriosus lumen.     

Beta 1 integrin-dependent and -independent polymerization of fibronectin

The mouse cell line GD25, which lacks expression of the beta 1 family of integrin heterodimers due to disruption of the beta 1 integrin subunit gene, was used for expression of full-length cDNA coding for splice variant A of the mouse beta 1 integrin subunit. In a stably transformed clone (GD25-beta 1A), the expressed protein was found to form functional heterodimeric receptors together with the subunits alpha 3, alpha 5, and alpha 6. Both GD25 and GD25-beta 1A attached to fibronectin and formed focal contacts which contained alpha v beta 3, but no detectable alpha 5 beta 1A. The presence of GRGDS peptide allowed alpha 5 beta 1A to locate to focal contacts of GD25-beta 1A cultured on fibronectin, while the beta 1-null GD25 cells were unable to attach under these conditions. Affinity chromatography revealed that alpha 5 beta 1A and alpha v beta 3 could bind to a large cell-binding fragment of fibronectin. alpha 5 beta 1A strongly promoted polymerization of fibronectin into a fibrillar network on top of the cells. Whereas little alpha v beta 3 was colocalized with the fibronectin fibrils in GD25-beta 1A cells, this integrin was able to support fibronectin fibril polymerization in GD25 cells. However, the alpha v beta 3-induced polymerization was less efficient and occurred mainly in dense cultures of the GD25 cells. Thus, while both alpha 5 beta 1A and alpha v beta 3 are able to support adhesion to fibronectin, alpha v beta 3 dominates in the formation of focal contacts, and alpha 5 beta 1A has a prime function in fibronectin matrix assembly. This is the first report on fibronectin matrix assembly in the absence of beta 1 integrins.     

Evidence for a differential functional regulation of the two beta(3)-integrins alpha(V)beta(3) and alpha(IIb)beta(3)

The functional regulation of integrins is a major determinant of cell adhesion, migration and tissue maintenance. The binding of cytoskeletal proteins to various sites of integrin cytoplasmic domains is a key mechanism of this functional regulation. Expression of recombinant integrin alpha(IIb)beta(3) and alpha(M)beta(2) lacking the GFFKR-region in CHO cells results in constitutively activated integrins. In contrast, CHO cells stably expressing either a GFFKR-deleted alpha(V(del))beta(3) or a FF to AA-substituted alpha(V(AA))beta(3) do not reveal a constitutively activated integrin. Adhesion to immobilized fibrinogen is strongly impaired in alpha(V(del))beta(3) or alpha(V(AA))beta(3)-expressing cells, whereas it is not impaired in alpha(IIb)beta(3) and alpha(M)beta(2), both lacking the GFFKR-region. In a parallel plate flow chamber assay, alpha(V)beta(3)-expressing cells adhere firmly to fibrinogen and spread even at shear rates of 15 to 20 dyn/cm(2), whereas alpha(V(del))beta(3) or alpha(V(AA))beta(3) cells are detached at 15 dyn/cm(2). Actin stress fiber formation and focal adhesion plaques containing alpha(V)beta(3) are observed in alpha(V)beta(3) cells but not in alpha(V(del))beta(3) or alpha(V(AA))beta(3)-expressing cells. As an additional manifestation of impaired outside-in signaling, phosphorylation of pp125(FAK) was reduced in these cells. In summary, we report that the GFFKR-region of the alpha(V)-cytoplasmic domain and in particular two phenylalanines are essential for integrin alpha(V)beta(3) function, especially for outside-in signaling. Our results suggest that the two beta(3)-integrins alpha(IIb)beta(3) and alpha(V)beta(3) are differentially regulated via their GFFKR-region.     

Fibronectin fragmentation promotes alpha4beta1 integrin-mediated contraction of a fibrin-fibronectin provisional matrix

In injured tissues, the fibrin-fibronectin (FN) provisional matrix provides a framework for cell adhesion, migration, and repair. Effective repair and remodeling require a proper balance between extracellular matrix (ECM) deposition, contraction, and turnover. We utilized a three-dimensional (3D) fibrin-FN provisional matrix model to determine the contributions of the FN-binding integrin receptors alpha5beta1 and alpha4beta1 to matrix contraction. CHOalpha5 cells expressing alpha5beta1, a receptor for FN's RGD cell-binding domain, were highly contractile, and cells were well spread on a 3D fibrin-FN matrix. In contrast, CHOalpha4 cells expressing the alpha4beta1 receptor for FN's alternatively spliced V region attached less efficiently to FN and were deficient in fibrin-FN matrix contraction. Surprisingly, cell adhesion and matrix contraction by CHOalpha4 cells were dramatically enhanced, to levels equivalent to CHOalpha5 cells, when proteolyzed FN was used in place of intact FN in the fibrin-FN matrix. Similar enhancement was observed when ligand binding by alpha4beta1 integrins was activated by treatment with Mn(++), but not by stimulation of actin organization with LPA. Therefore, alpha4beta1-dependent cell responses to the provisional matrix are modulated by cleavage of matrix components.     

Human fibroblasts with mutations in COL5A1 and COL3A1 genes do not organize collagens and fibronectin in the extracellular matrix, down-regulate alpha2beta1 integrin, and recruit alphavbeta3 Instead of alpha5beta1 integrin

Dermal fibroblasts derived from types I and IV Ehlers-Danlos syndrome (EDS) patients, carrying mutations in COL5A1 and COL3A1 genes, respectively, synthesize aberrant types V and III collagen (COLL) and show defective organization of these proteins into the extracellular matrix (ECM) and high reduction of their functional receptor, the alpha(2)beta(1) integrin, compared with control fibroblasts. EDS cells also show reduced levels of fibronectin (FN) in the culture medium and lack an FN fibrillar network. Finally, EDS cells prevalently organize alpha(v)beta(3) integrin instead of alpha(5)beta(1) integrin. The alpha(v)beta(3) integrin, distributed on the whole EDS cell surface, shows FN binding and assembly properties when the cells are treated with purified FN. Treatment of EDS cells with purified COLLV or COLLIII, but not with FN, restores the control phenotype (COLL(+), FN(+), alpha(v)beta(3)(-), alpha(5)beta(1)(+), alpha(2)beta(1)(+)). Function-blocking antibodies to COLLV, COLLIII, or alpha(2)beta(1) integrin induce in control fibroblasts an EDS-like phenotype (COLL(-), FN(-), alpha(v)beta(3)(+), alpha(5)beta(1)(-), alpha(2)beta(1)(-)). These results show that in human fibroblasts alpha(2)beta(1) integrin organization and function are controlled by its ligand, and that the alpha(2)beta(1)-COLL interaction, in turn, regulates FN integrin receptor recruitment: high alpha(2)beta(1) integrin levels induce alpha(5)beta(1) integrin organization, while low alpha(2)beta(1) integrin levels lead to alpha(v)beta(3) integrin organization.     

The role of alpha3beta1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes

Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the incorporation of laminin-5 into its proper higher-order structure within the extracellular matrix of keratinocytes and (2) that the organizational state of laminin-5 has an influence on laminin-5 matrix function.     

The FN13 peptide inhibits human tumor cells invasion through the modulation of alpha v beta 3 integrins organization and the inactivation of ILK pathway

We report the effect of the stable expression of a 13 amino acid human fibronectin (FN) peptide (FN13) on the organization of the FN extracellular matrix (ECM) and of FN integrin receptors (FNRs), in relationship with the inhibition of cellular invasion, in three FN-ECM defective human tumor-derived cell lines: SK-Hep1C3, hepatoma, ACN, neuroblastoma, and SK-OV-3, ovary carcinoma. All these cell lines stably expressing the FN13 peptide, organized an FN-ECM, disorganized alpha v beta 1 integrins and inactivated the ILK pathway, with the loss of secretion of MMP-9. This was associated with the inhibition of cell invasion in Matrigel matrix only in SK-Hep1C3 and ACN, but not in SK-OV-3 cells. Analysis of the integrin receptors organization showed that the FN13 expressing cells SK-Hep1C3 and ACN organized alpha v beta 3 integrins, whereas SK-OV-3 organized alpha v beta 5 dimers. The functional block of alpha v beta 5 integrins, with an inactivating anti-alpha v beta 5 antibody, led to the induction of alpha v beta 3 integrins also in SK-OV-3 cells, and to the inhibition of cell invasion. These data show that in the human tumor cells studied FN13 inhibits the in vitro invasion through the dissociation of alpha v beta 1 dimers, leading to ILK pathway inactivation, only when the organization of alpha v beta 3 integrins is induced in the plasma membrane.     

The alpha 5 beta 1 integrin associates with a dystrophin-containing lattice during muscle development.

The organization of the alpha 5 beta 1 integrin on skeletal muscle was studied in culture and in sections from adult and embryonic tissue using monoclonal antibodies specific for the alpha 5 subunit. The alpha 5 beta 1 integrin showed changes in organization and in the molecules with which it colocalizes. On early myoblasts, possessing a fibroblast-like morphology, the alpha 5 integrin organization was indistinguishable from that on fibroblasts; it was expressed prominently and localized in numerous focal contacts around the cell periphery. In bipolar myoblasts and early myotubes, the alpha 5 integrin was expressed only weakly and localized in a small number of focal contact-like structures. As myogenesis proceeded there was an apparent increase in integrin expression and a change in organization. In addition to the focal contact-like structures that persist throughout myogenesis in vitro, a dense lattice-like structure of integrin appeared. Fibrillar fibronectin, talin, and non-muscle alpha-actinin did not colocalize with the alpha 5 beta 1 integrin in the lattice structure as they did in the focal contact-like structures. However, dystrophin, which displayed a diffuse distribution earlier, now colocalized with the alpha 5 beta 1 integrin in the punctate lattice. Coincident with the registration of myofibrils into visible sarcomeres, the prominent dense, lattice structure disappeared leaving the focal contact-like structures as the only regions of organized alpha 5 beta 1 integrin. Despite the presence of the beta 1 integrin in neuromuscular or myotendinous junctions in vivo and on myotubes in vitro, the alpha 5 beta 1 integrin was not present in either junction. These observations suggest that the alpha 5 beta 1 integrin is involved in the adhesion of muscle to the extracellular matrix, the organization of the dystrophin-containing lattice, and the organization of nascent myofibrils which emanate from the focal contact- and stress fiber-like structures in muscle. Other integrins appear to anchor myofibrils at the myotendinous and neuromuscular junctions.     

Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin

Rabbit synovial fibroblasts (RSF) express basal levels of the metalloproteinases (MMP) collagenase, stromelysin-1 and 92-kD gelatinase when plated on intact fibronectin (FN), but elevated levels when plated on either the central RGD-containing cell-binding region of FN (120FN) or antibody against the alpha 5 beta 1 integrin, suggesting that domains outside 120FN may suppress the induction of MMP (Werb, Z., P. M. Tremble, O. Behrendtsen, E. Crowley, and C.H. Damsky. 1989. J. Cell Biol. 109:877-889). We therefore attempted to reconstitute the basal signaling of intact FN by plating RSF on 120FN together with domains of FN outside this region. Large COOH-terminal fragments containing both the heparin-binding and HICS domains suppressed MMP when combined with 120FN. To map the active sequences, peptides from this region and larger fragments that did, or did not, include the CS-1 portion of IIICS were tested. Only CS-1 peptide, or larger fragments containing CS-1, suppressed MMP expression induced by 120FN. In contrast, peptide V from the heparin-binding region, shown previously to stimulate focal contact formation, further enhanced MMP expression by RSF when present on the substrate with 120FN. RSF expressed alpha 4 beta 1 integrin, the receptor for CS-1, and the anti-alpha 4 mAb blocked the ability of CS-1 to suppress MMP induction by 120FN. These results show that signals modulating MMP expression and focal contact assembly are regulated independently, and that cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins plays a dominant role in regulating expression of these extracellular matrix-remodeling genes in response to FN. This work demonstrates directly the modular way in which information in the extracellular matrix is detected and processed by cell surface receptors.     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.     

Epithelial development and differentiation in the mammary gland is not dependent on alpha 3 or alpha 6 integrin subunits

In the mammary gland, both laminin and integrins have been shown to be required for normal ductal morphogenesis during development in vivo, and for functional differentiation in culture models. Major integrin receptors for laminins in the mammary gland are alpha 3 beta 1, alpha 6 beta 1, and alpha 6 beta 4. However, the specific subunits that contribute to laminin-mediated mammary cell function and development have not been identified. In this study, we use a genetic approach to test the hypothesis that laminin-binding integrins are required for the function of the mammary gland in vivo. Rudiments of embryonic mammary gland were shown to develop in the absence of these integrin subunits. Postnatal development of the mammary gland was studied in integrin null tissue that had been transplanted into the mammary fat pads of syngeneic hosts. In mammary epithelium lacking alpha 6 integrin, the beta 4 subunit was not apparent and hemidesmosome formation was only rudimentary. However, despite this deficiency, normal ductal morphogenesis and branching of the mammary gland occurred and myoepithelial cells were distributed normally with respect to luminal cells. Mammary alveoli devoid of alpha 3 or alpha 6 integrin formed in pregnancy and were histologically and functionally identical to those in wild-type mammary gland. The tissue underwent full morphological differentiation, and the epithelial cells retained the ability to synthesize beta-casein. This work demonstrates that mammary tissue genetically lacking major laminin-binding integrin receptors is still able to develop and function.     

Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen

Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of beta1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of beta1-null platelets to native fibrillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, beta1-null platelets adhere to fibrillar, but not soluble collagen under static as well as low (150 s(-1)) and high (1000 s(-1)) shear flow conditions, probably through binding of alphaIIbbeta3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on fibrillar as well as soluble collagen. These data show that GPVI plays the central role in platelet-collagen interactions by activating different adhesive receptors, including alpha2beta1 integrin, which strengthens adhesion without being essential.     

Integrins in point contacts mediate cell spreading: factors that regulate integrin accumulation in point contacts vs. focal contacts

We have studied the function and distribution of the alpha 1 beta 1, alpha 5 beta 1 and alpha 6 beta 1 heterodimers on type-1 astrocytes with antibodies specific for integrin subunits (alpha 1, alpha 5, alpha 6, and beta 1). The alpha 1 beta 1 heterodimer mediates adhesion to laminin and collagen, the alpha 5 beta 1 to fibronectin in an RGD- dependent manner. The alpha 5 beta 1 integrin is found in focal contacts in long-term cultures of well-spread astrocytes colocalizing with vinculin and the termini of actin stress fibers. alpha 1 beta 1 heterodimers can occasionally be found as small aggregates within focal contacts but they do not accumulate there. Instead, alpha 1 beta 1 integrins are found in punctate deposits called point contacts which are distributed over the upper and the lower cell surfaces whether laminin, collagen, fibronectin or polylysine is used as a substratum. Unlike focal contacts, point contacts contain clathrin but rarely codistribute with actin or vinculin. Two observations indicate that these point contacts are functional. First, mAb 3A3, directed against the rat alpha 1 subunit, inhibits the attachment of astrocytes to laminin and collagen. Second, during the spreading of astrocytes, a band of point contacts forms around the cell perimeter at a time when no focal contacts are visible. While alpha 1 beta 1 integrins are found only in point contacts in astrocytes, the alpha 6 beta 1 integrin, another laminin receptor, is localized within focal contacts. Moreover, alpha 1 beta 1 heterodimers accumulate in focal contacts in fibroblasts. Thus, the alpha subunit contributes, independent of its ligand, to functional integrin heterodimer accumulation in focal contacts or in point contacts. This accumulation varies among different cell types with apparently identical heterodimers as well as with the motile state (spreading vs. flattened) of the same cells.     

Syndecan-1 regulates cell migration and fibronectin fibril assembly

Corneal scarring is a major cause of blindness worldwide and can result from the deposition of abnormal amounts of collagen fibers lacking the correct size and spacing required to produce a clear cornea. Collagen fiber formation requires a preformed fibronectin (FN) matrix. We demonstrate that the loss of syndecan1 (sdc1) in corneal stromal cells (CSC) impacts cell migration rates, the sizes and composition of focal and fibrillar adhesions, the activation of integrins, and the assembly of fibronectin into fibrils. Integrin and fibronectin expression are not altered on sdc1-null CSCs. Cell adhesion, spreading, and migration studies using low compared to high concentrations of FN and collagen I (CNI) or vitronectin (VN) with and without activation of integrins by manganese chloride show that the impact of sdc1 depletion on integrin activation varies depending on the integrin-mediated activity evaluated. Differences in FN fibrillogenesis and migration in sdc1-null CSCs are reversed by addition of manganese chloride but cell spreading differences remain. To determine if our findings on sdc1 were specific to the cornea, we compared the phenotypes of sdc1-null dermal fibroblasts with those of CSCs. We found that without sdc1, both cell types migrate faster; however, cell-type-specific differences in FN expression and its assembly into fibrils exist between these two cell types. Together, our data demonstrate that sdc1 functions to regulate integrin activity in multiple cell types. Loss of sdc1-mediated integrin function results in cell-type specific differences in matrix assembly. A better understanding of how different cell types regulate FN fibril formation via syndecans and integrins will lead to better treatments for scarring and fibrosis.     

Purification and characterization of mammalian integrins expressed by a rat neuronal cell line (PC12): evidence that they function as alpha/beta heterodimeric receptors for laminin and type IV collagen

Cells of the rat neuronal line, PC12, adhere well to substrates coated with laminin and type IV collagen, but attach poorly to fibronectin. Adhesion and neurite extension in response to these extracellular matrix proteins are inhibited by Fab fragments of an antiserum (anti-ECMR) that recognizes PC12 cell surface integrin subunits of Mr 120,000, 140,000, and 180,000 (Tomaselli, K. J., C. H. Damsky, and L. F. Reichardt. 1987. J. Cell Biol. 105:2347-2358). Here we extend our study of integrin structure and function in PC12 cells using integrin subunit-specific antibodies prepared against synthetic peptides corresponding to the cytoplasmic domains of the human integrin beta 1 and the fibronectin receptor alpha (alpha FN) subunits. Anti-integrin beta 1 immunoprecipitated a 120-kD beta 1 subunit and two noncovalently associated integrin alpha subunits of 140 and 180 kD from detergent extracts of surface-labeled PC12 cells. Immunodepletion studies using anti-integrin beta 1 demonstrated that these two putative alpha/beta heterodimers are identical to those recognized by the adhesion-perturbing ECMR antiserum. Anti-alpha FN immunoprecipitated fibronectin receptor heterodimers in human and rat fibroblastic cells, but not in PC12 cells. Thus, low levels of expression of the integrin alpha FN subunit can explain the poor attachment of PC12 cells to FN. The PC12 cell integrins were purified using a combination of lectin and ECMR antibody affinity chromatography. The purified integrins: (a) completely neutralize the ability of the anti-ECMR serum to inhibit PC12 cell adhesion to laminin and collagen IV; (b) have hydrodynamic properties that are very similar to those of previously characterized integrin alpha/beta heterodimeric receptors for ECM proteins; and (c) can be incorporated into phosphatidylcholine vesicles that then bind specifically to substrates coated with laminin or collagen IV but not fibronectin. Thus, the ligand-binding specificity of the liposomes containing the purified PC12 integrins closely parallels the substrate-binding preference of intact PC12 cells. These results demonstrate that mammalian integrins purified from a neuronal cell line can, when incorporated into lipid vesicles, function as receptors for laminin and type IV collagen.     

Expression of normal and mutant avian integrin subunits in rodent cells [published erratum appears in J Cell Biol 1989 Oct;109(4 Pt 1):1187]

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.