Kalinin is more efficient than laminin in promoting adhesion of primary keratinocytes and some other epithelial cells and has a different requirement for integrin receptors.

Kalinin was purified from squamous cell carcinoma (SCC25) spent culture media using an immunoaffinity column prepared from the mAb BM165. The affinity-purified material was separated by SDS-PAGE into three bands of 165-155, 140, and 105 kD identical to those obtained from normal human keratinocyte cultures and previously identified as kalinin. Kalinin promoted adhesion of a large number of normal cells and established cell lines with an activity similar to other adhesion molecules such as the laminin-nidogen complex, fibronectin, or collagen IV. However, kalinin was a much better substrate than laminin-nidogen complex for adhesion of cells of epithelial origin including primary human keratinocytes. Adhesion to kalinin was followed by cell shape changes ranging from rounded to fully spread cells depending on the cell types. The adhesion-promoting activity of kalinin was conformation dependent and was abolished by heat denaturation. mAb BM165 prevented cell adhesion to kalinin but not to other extracellular matrix substrates. However, either complete or partial inhibition was observed with different cells suggesting the existence of at least two cell-binding sites on the kalinin molecule. Experiments inhibiting cell adhesion with function-blocking anti-integrin subunit antibodies indicated that both alpha 3 beta 1 and alpha 6 beta 1 integrins are involved in the cellular interactions with kalinin, while for cell adhesion to classical mouse Engelbreth-Holm-Swarm laminin only alpha 6 beta 1 integrins, and not alpha 3 beta 1, appeared to be functional. Altogether, these results suggest that kalinin may fulfill additional functions than laminin, particularly for epithelial cells.

Attachment of cells to basement membrane collagen type IV.

Of ten different cell lines examined, three showed distinct attachment and spreading on collagen IV substrates, and neither attachment nor spreading was enhanced by adding soluble laminin or fibronectin. This reaction was not inhibited by cycloheximide or antibodies to laminin, indicating a direct attachment to collagen IV without the need of mediator proteins. Cell-binding sites were localized to the major triple-helical domain of collagen IV and required an intact triple helical conformation for activity. Fibronectin showed preferential binding to denatured collagen IV necessary to mediate cell binding to the substrate. Fibronectin binding sites of collagen IV were mapped to unfolded structures of the major triple-helical domain and show a similar specificity to fibronectin-binding sites of collagen I. The data extend previous observations on biologically potential binding sites located in the triple helix of basement membrane collagen IV.

Multiple cell surface receptors for the short arms of laminin: alpha 1 beta 1 integrin and RGD-dependent proteins mediate cell attachment only to domains III in murine tumor laminin.

Cell surface molecules that interact with the cross formed by the three short arms of murine tumor laminin were studied using thermal perturbation, antibody and peptide blocking, and affinity chromatography. Several potential receptors for the laminin short arms were revealed that differed from those mediating cell attachment to the E8 (long arm) fragment. Two cell lines, Rugli and L8 attached well to E1-X (short arm) fragments of laminin. This attachment was blocked by antibodies against alpha 1 integrin chains. Other cells were unable to attach strongly to E1-X, but attached to P1. This attachment was unaffected by anti-beta 1 integrin antibodies, but specifically blocked by the peptide GRGDS. By contrast, binding of Rugli cells was RGD independent and blocked by anti-beta 1 integrin antibodies. G7 and C2C12 myoblasts were very sensitive to GRGDS (ID50 approximately 2 micrograms.ml-1) for attachment to P1 which implied that a non-beta 1 series integrin, possibly alpha v beta 3, was involved. On heat denaturation of P1(3) attachment remained sensitive to RGDS and ID50 was unchanged. On heat denaturation of E1-X, attachment remained sensitive to RGDS but the ID50 increased to approximately 200 micrograms.ml-1. Cellular beta 1 integrins were retained on laminin affinity columns. A beta 1 integrin with an approximately 190 kD alpha-chain could be isolated from Rugli cells whose attachment could be blocked by anti-alpha 1 antibodies and not from cells blocked by RGDS peptides. Anti-alpha 1 antibodies blocked Rugli attachment to native laminin, but only when the E8 cell binding sites on laminin were also blocked. Thus, a receptor related to alpha 1 beta 1 integrin can function simultaneously with a receptor for E8. Anti-alpha 1 also blocked attachment to heated laminin, suggesting that the heat-stable attachment activity in laminin involved the E1-X binding site. Thus, at least two putative receptors mediate attachment to the short arms of laminin. One, related to alpha 1 beta 1 integrin, recognizes RGDS-independent sites in E1-X defined by P1 (within domains III, IIIa, IIIb), and one is an RGD-dependent molecule recognizing sites in P1, and is not a beta 1 integrin.

Recognition of the laminin E8 cell-binding site by an integrin possessing the alpha 6 subunit is essential for epithelial polarization in developing kidney tubules.

It has been previously shown that A-chain and domain(E8)-specific antibodies to laminin that inhibit cell adhesion also interfere with the establishment of epithelial cell polarity during kidney tubule development (Klein, G., M. Langegger, R. Timpl, and P. Ekblom. 1988. Cell. 55:331-341). A monoclonal antibody specific for the integrin alpha 6 subunit, which selectively blocks cell binding to E8, was used to study the receptors involved. Immunofluorescence staining of embryonic kidneys and of organ cultures of metanephric mesenchyme demonstrated coappearance of the integrin alpha 6 subunit and the laminin A-chain in regions where nonpolarized mesenchymal cells convert into polarized epithelial cells. Both epitopes showed marked colocalization in basal areas of tubules, while an exclusive immunostaining for alpha 6 was observed in lateral and apical cell surfaces of the tubular epithelial cells. Organ culture studies demonstrated a consistent inhibition of kidney epithelium development by antibodies against the alpha 6 subunit. The data suggest that the recognition of E8 cell-binding site of laminin by a specific integrin is crucial for the formation of kidney tubule epithelium from undifferentiated mesenchymal stem cells. In some other cell types (endothelium, some ureter cells) an exclusive expression of alpha 6 with no apparent colocalization of laminin A-chain in the corresponding basement membrane was seen. Thus, in these cells, integrins possessing the alpha 6 subunit may bind to laminin isoforms that differ from those synthesized by developing tubules.

Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8.

The involvement of integrins in mediating interaction of cells to well-characterized proteolytic fragments (P1, E3, and E8) of laminin was assessed by antibody blocking studies. Cell adhesion to fragment P1 was affected by mAbs against the integrin beta 1 and beta 3 subunits and furthermore could be prevented completely by a synthetic peptide containing the Arg-Gly-Asp sequence. Because the beta 3 antibody-sensitive cell lines expressed the vitronectin receptor (alpha v beta 3) at high levels, the involvement of this receptor in cell adhesion to P1 is strongly suggested. Integrin-mediated cell adhesion to E3 is of low affinity and was inhibited by antibodies against the integrin beta 1 subunit. In contrast, adhesion of some cell types to E3 was not or only partially sensitive to inhibition by anti-integrin subunit antibodies. Cell adhesion to E8 was blocked completed by integrin alpha 6 or beta 1 antibodies. The alpha 6-specific antibody did not inhibit cell adhesion to E3 or P1. Furthermore, the antibody only blocked adhesion to laminin of those cells that adhered exclusively to the E8 fragment. In addition, expression of alpha 6 beta 1 was closely correlated with the ability of cells to bind to the E8 fragment of laminin. These results indicate that the alpha 6 beta 1 integrin is a specific receptor for the E8 fragment of laminin. Many cell types expressed, instead of or in addition to alpha 6 beta 1 the recently described integrin alpha 6 beta 4. Although the ligand of alpha 6 beta 4 was not identified, it must be different from that of alpha 6 beta 1, because cells that express alpha 6 beta 4, but not alpha 6 beta 1, do not adhere to E8, and cell adhesion to E8 was specifically blocked by beta 1 specific antibodies. In conclusion, the data indicate that distinct integrin receptors belonging to the beta 1 or beta 3 subfamily are involved in adhesion of cells to the various laminin fragments. Adhesion to E3 may also be brought about by other receptor molecules, possibly proteoglycans, not belonging to the integrin family.

Hyaluronan reduces migration and proliferation in CHO cells.

Expression of the hyaluronan synthase gene in hyaluronan-deficient CHO cells changed the cell morphology from a spindle shape to a flattened epithelial-type form. Hyaluronan producing CHO cells showed reduced initial cell adhesion, migration, proliferation and density at contact inhibition, but no difference in random migration determined by the Boyden chamber assay. Addition of hyaluronan to the medium of CHO cells reduced migration, proliferation and initial cell adhesion. In contrast, coating the plastic dish with hyaluronan enhanced initial cell adhesion. These results are discussed in the context of the perplexing properties of hyaluronan on cellular functions.

Localization of binding sites for laminin, heparan sulfate proteoglycan and fibronectin on basement membrane (type IV) collagen.

Rotary shadowing electron microscopy was used to examine complexes formed by incubating combinations of the basement membrane components: type IV collagen, laminin, large heparan sulfate proteoglycan and fibronectin. Complexes were analyzed by length measurement from the globular (COOH) domain of type IV collagen, and by examination of the four arms of laminin and the two arms of fibronectin. Type IV collagen was found to contain binding sites for laminin, heparan sulfate proteoglycan and fibronectin. With laminin the most frequent site was centered approximately 81 nm from the carboxy end of type IV collagen. Less frequent sites appeared to be present at approximately 216 nm and approximately 291 nm, although this was not apparent when the sites were expressed as a fraction of the length of type IV collagen to which they were bound. For heparan sulfate proteoglycan the most frequent site occurred at approximately 206 nm with a less frequent site at approximately 82 nm. For fibronectin, a single site was present at approximately 205 nm. Laminin bound to type IV collagen through its short arms, particularly through the end of the lateral short arms and to heparan sulfate proteoglycan mainly through the end of its long arm. Fibronectin bound to type IV collagen through the free end region of its arms. Using a computer graphics program, the primary laminin binding sites of two adjacent type IV collagen molecules were found to align in the "polygonal" model of type IV collagen, whereas with the "open network" model, a wide meshed matrix is predicted. It is proposed that basement membrane may consist of a lattice of type IV collagen coated with laminin, heparan sulfate proteoglycan and fibronectin.

Structure and biological activity of the extracellular matrix.

The extracellular matrix is formed by complex and intricate networks within which molecules are precisely organized. These molecular networks determine the specific histoarchitecture of tissues and provide cells with information and a scaffold. Most of the structural extracellular matrix molecules - collagens, noncollagenous glycoproteins, and proteoglycans - are chimeric and share common domains. Studies of the interactions between extracellular matrix molecules and mapping of the interaction sites to defined structural modules have led to the concept that the function of the extracellular matrix relies largely in the polymers that they form. Furthermore, determination of the tertiary structure of protein motifs involved either in the assembly of the various molecules into polymers or in cell-extracellular matrix interactions has recently opened the field of structural biology of the extracellular matrix.

Structural basis of beta 1 integrin-mediated cell adhesion to a large heparan sulfate proteoglycan from basement membranes.

In a cell attachment assay, several cell lines were found to adhere and spread on heparan sulfate proteoglycan purified from a basement membrane-producing tumor. This adhesion was clearly distinct from that on laminin. Cell adhesion to the proteoglycan was completely inhibited by three different antibodies against integrin beta 1 subunit, while inhibitory antibodies against beta 3 and alpha 2 to alpha 6 subunits were without strong effects. Removal of heparan sulfate from the proteoglycan diminished cell attachment, but addition of heparin to the cells did not affect adhesion to the proteoglycan. This suggests that both the heparan sulfate side chains and core protein structures are required for efficient cell adhesion. Studies with proteolytic fragments and synthetic RGD peptides showed that the single RGD sequence of mouse proteoglycan is not involved in cellular recognition. Characterization of fragments also allowed to localize cell adhesion and heparan sulfate attachment sites to the same 160 kDa core protein structure but not to fragments derived from the N-terminal portion of the proteoglycan.

Laminins: a family of diverse multifunctional molecules of basement membranes.

Laminins represent a growing family of disulfide-linked heterotrimers constituted by the association of three genetically different polypeptides, the alpha, beta, and gamma chains. Laminins are endowed with structural and biological functions. They play a direct critical role in the control of cellular behavior by providing cells with specific information through interactions with cell surface receptors. Because of their structural properties, they represent crucial building blocks for tissue assembly, architecture, and stability. The expression of laminin chain variants is spatio-temporally regulated, which suggests that laminin isoforms might have different functions responsible for the biological and morphological polymorphism of basement membranes. The different cells present in the skin express several laminin chains, which lead to the deposition of various laminin isoforms, whose mechanical and biological functions are likely to be adapted to the properties of the dermo-epidermal junction. Recently, defective laminin isoforms have been shown to be associated with several inborn and acquired diseases, illustrating a major structural function for laminins in skin integrity.

Increased adhesion of fibroblasts from patients with scleroderma to extracellular matrix components: in vitro modulation by IFN-gamma but not by TGF-beta.

A characteristic feature of systemic scleroderma is fibrosis of the skin and eventually of internal organs resulting from an overproduction of collagen and other connective tissue components by the resident fibroblasts. The balance between the cells and the amount of the surrounding extracellular matrix is then altered. Because cellular metabolism depends to a large extent on cellular contacts and communications with connective tissue molecules, we have therefore investigated the interactions with extracellular matrix components of fibroblasts obtained from skin of patients affected with scleroderma. In comparison to fibroblasts from healthy skin, all fibroblasts from scleroderma patients had an increased adhesion capacity to collagens I, IV, VI, fibronectin, and laminin. In addition, whereas adhesion of control fibroblasts was stimulated by a pre-treatment with transforming growth factor-beta, adhesion patterns of scleroderma fibroblasts remained unchanged. However, pre-incubation of the cells with interferon-gamma decreased the adhesion of both scleroderma and control fibroblasts.

Activation of collagen gene expression in keloids: co-localization of type I and VI collagen and transforming growth factor-beta 1 mRNA.

Untreated, clinically active keloids were examined as model system to study the spatial expression of extracellular matrix and transforming growth factor-beta 1 (TGF-beta 1) genes in fibrotic skin diseases. In situ hybridizations localized active expression of type I and VI collagen genes to the areas containing an abundance of fibroblasts and apparently representing the expanding border of the lesions. Within this zone, microvascular endothelial cells also expressed the type I collagen genes, as evaluated by simultaneous use of in situ hybridization for collagen gene expression and immunolocalization for factor VIII-related antigen, a marker for endothelial cell differentiation. Slot-blot hybridizations of RNA isolated from this zone suggested that the expression of type I and IV collagen genes was selectively enhanced, as compared to type III collagen gene expression. TGF-beta 1 protein and mRNA were also detected in areas active in type I and type VI collagen gene expression, indicating that TGF-beta 1 gene is transcribed and the corresponding protein is deposited in areas of elevated collagen gene expression, including microvascular endothelial cells. We conclude that the initial step in the development of fibrotic reaction in keloids involves the expression of the TGF-beta 1 gene by the neovascular endothelial cells, thus activating the adjacent fibroblasts to express markedly elevated levels of TGF-beta 1, as well as type I and VI collagen genes.

Laminins of the dermo-epidermal junction.

Laminins are the most abundant structural non-collagenous glycoproteins ubiquitously present in basement membranes. They are multidomain molecules constituting a family of possibly more than 50 members. Some members such as laminins 5, 6 and 10 are specific of the basal lamina present under stratified epithelia. Although only few intact laminin isoforms have been purified from cultivated cells or tissues, genetic engineering has opened the way for a rapid development of laminin structural biology. Moreover, the phenotypes resulting from gene targeting in mouse or from laminin defects in acquired or inherited human diseases highlight the pivotal role of laminins in morphogenesis, development, and physiology. Indeed, the laminins display a remarkable repertoire of functions, most importantly as structural elements forming a network throughout the basement membrane to which other collagenous or non-collagenous glycoproteins and proteoglycans attach. Furthermore, they are signaling molecules providing adjacent cells with diverse information by interacting with cell surface components.

Cell adhesion to a population of laminin isoforms isolated from normal renal tissue.

To assess whether cells react differently towards a population of several laminin isoforms, as found in vivo, vs. a single isoform, we have compared the biological activity of kidney laminins to that of pure laminin 1. The kidney laminin preparation contained laminin 1 and further isoforms. Both substrates induced adhesion of a large spectrum of cell types, with kidney laminins being the most active. Unfolding of the coil-coiled conformation of the kidney isoforms negatively affected cell adhesion-promoting activity, which indicated that conformation-dependent cell binding is a characteristic feature of many or all laminins. Cellular interactions with kidney laminins were mediated by alpha3beta1 and alpha6beta1 integrins, with the contribution of alpha3beta1 being apparently lower than that of alpha6beta1 integrins. Immunofluorescence staining of vinculin and integrin subunits decorated focal adhesions on kidney laminins which differed in morphology from those formed on laminin 1 alone, in spite of the presence of the latter in the kidney preparation. These observations collectively indicate that tissue specific but often overlapping expression of laminin isoforms might modulate cell behavior by the activation of distinct sets of integrins and by the induction of distinct molecular assemblies within the cell adhesion signaling complexes.

Size and domain structure of collagen VI produced by cultured fibroblasts.

Disulfide-bonded forms of collagen VI were analyzed by immunoblotting of fibroblast culture medium and cell extracts. The protein consists of pepsin and collagenase-resistant domains of about equal size indicating a molecular mass of 340 kDa for collagen VI monomers.

Identification of the Arg-Gly-Asp sequence in laminin A chain as a latent cell-binding site being exposed in fragment P1.

A single RGD-containing sequence present within an epidermal growth factor-like repeat of the short arms of laminin is shown by peptide inhibition to block integrin receptors recognizing a latent cell-binding site of laminin. Based on proteolysis data it is proposed that masking occurs by folding of the globular domain IVa over the cell-binding site in the adjacent rod-like structures of laminin A chain.

Effect of transforming growth factor-beta on collagen type VI expression in human dermal fibroblasts.

Steady-state mRNA levels and protein synthesis of collagen type VI were determined after stimulation of human dermal fibroblasts with transforming growth factor-beta (TGF beta). While there was a 227% increase in the alpha 3(VI) subunit mRNA at maximal TGF-beta concentration, alpha 1(VI) and alpha 2(VI) subunit mRNA levels remained unchanged. Concomitantly collagen type VI immuno-reactive material increased up to 172% of controls in cell culture medium and cell layer extracts. Regulation of alpha 3(VI) gene expression is therefore critical for the control of collagen type VI synthesis and determines the deposition of collagen type VI heterotrimeric molecules.

Arg-Gly-Asp constrained within cyclic pentapeptides. Strong and selective inhibitors of cell adhesion to vitronectin and laminin fragment P1.

Cyclic Arg-Gly-Asp-Phe-Val peptides with either D-Phe or D-Val residues were 20- to more than 100-fold better inhibitors of cell adhesion to vitronectin and/or laminin fragment P1 when compared to a linear variant or Gly-Arg-Gly-Asp-Ser. No or only little increase in inhibitory capacity was observed for fibronectin adhesion and for the binding of platelet receptor alpha IIb beta 3 to fibrinogen. NMR studies of the two most active cyclic peptides showed for both an all-trans conformation with a beta II' and gamma turn. Subtle conformational differences, however, exist between both peptides and may contribute to selectivity of inhibition.

Identification of novel interaction partners for the conserved membrane proximal region of alpha-integrin cytoplasmic domains.

The alpha3Abeta1 integrin is a laminin receptor with a broad specificity for different laminin isoforms. Furthermore, it regulates the function of other integrins, like alpha2beta1, alpha5beta1 and alpha6Abeta1. In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the alpha3A integrin subunit. In addition to the cDNA for nucleotide exchange factor Mss4 and the putative tumour suppressor protein BIN1, two novel cDNAs were identified. Association analysis with different integrin subunits revealed them as cDNAs that encode binding proteins which react with a broad spectrum of alpha subunits. The conserved membrane proximal region of the alpha3A chain was identified as the binding site for all four proteins. They, therefore, may be involved in the regulation of general functions of integrins.

Collagen synthesis in organ culture of normal and atherosclerotic aortas.

Using pulse-label experiments in organ culture, collagen synthesis was studied in aortas from healthy and atherosclerotic specimens. Investigations were carried out on human atherosclerotic plaques as well as in the mini-pig in which atherosclerosis occurred spontaneously, and in the rabbit where atherosclerosis was experimentally induced by cholesterol-enriched feeding. When compared to total protein synthesis, the percentage of newly synthesized collagens measured as radioactive pepsin-resistant material, decreased with age in healthy specimens, whereas it remained at a higher level when the aortas were atherosclerotic. Subsequent molecular sieve chromatography of the radioactivity pepsin-resistant material allowed the separation type I collagen from type III collagen and their relative quantification. The results showed that the newly synthesized type III collagen accounted for 16-31% in aortic explants from young animals, for 30-36% when the explants were derived from older specimens and for 35-48% when the tissues were atherosclerotic.