Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (Mr 600 000) containing the three short chains (Mr 200 000) but lacking the long chain (Mr 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (M_r 600 000) containing the three short chains (M_r 200 000) but lacking the long chain M_r 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

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.     

Formation of a supramolecular complex is involved in the reconstitution of basement membrane components

Basement membrane macromolecules, including type IV collagen, laminin, and heparan sulfate proteoglycan, do not aggregate when incubated alone. Rather, precipitation occurs in the presence of equimolar amounts of laminin and type IV collagen but variable amounts of heparan sulfate proteoglycan. This interaction requires native laminin and type IV collagen. Heparan sulfate proteoglycan increases the precipitation of laminin particularly in the presence of type IV collagen. Fibronectin does not cause type IV collagen to precipitate. These studies show that the components of basement membrane interact in a highly specific manner and suggest that such interactions may be involved in the deposition of basement membrane in situ.     

Targeting perlecan in human keratinocytes reveals novel roles for perlecan in epidermal formation

Heparin-binding growth factors are crucial for the formation of human epidermis, but little is known about the role of heparan sulfate proteoglycans in this process. Here we investigated the role of the heparan sulfate proteoglycan, perlecan, in the formation of human epidermis, by utilizing in vitro engineered human skin. By disrupting perlecan expression either in the dermis or the epidermis, we found that epidermally derived perlecan is essential for epidermal formation. Perlecan-deficient keratinocytes formed a strikingly thin and poorly organized epidermis because of premature apoptosis and failure to complete their stratification program. Exogenous perlecan fully restored epidermal formation. Perlecan deposition in the basement membrane zone correlated with formation of multilayered epidermis. Perlecan deficiency, however, had no effect on the lining and deposition of major basement membrane components as was evident by a continuous linear staining of laminin and collagen IV. Similarly, perlecan deficiency did not affect the distribution of beta1 integrin. Addition of the perlecan ligand, fibroblast growth factor 7, protected perlecan-deficient keratinocytes from cell death and improved the thickness of the epidermis. Taken together, our results revealed novel roles for perlecan in epidermal formation. Perlecan regulates both the survival and terminal differentiation steps of keratinocytes. Our results suggested a model whereby perlecan regulates these processes via controlling the bioavailability of perlecan-binding soluble factors involved in epidermal morphogenesis.     

Laminins in basement membrane assembly

The heterotrimeric laminins are a defining component of all basement membranes and self-assemble into a cell-associated network. The three short arms of the cross-shaped laminin molecule form the network nodes, with a strict requirement for one α, one β and one γ arm. The globular domain at the end of the long arm binds to cellular receptors, including integrins, α-dystroglycan, heparan sulfates and sulfated glycolipids. Collateral anchorage of the laminin network is provided by the proteoglycans perlecan and agrin. A second network is then formed by type IV collagen, which interacts with the laminin network through the heparan sulfate chains of perlecan and agrin and additional linkage by nidogen. This maturation of basement membranes becomes essential at later stages of embryo development.     

Keratinocyte-Targeted Expression of Human Laminin γ2 Rescues Skin Blistering and Early Lethality of Laminin γ2 Deficient Mice

Laminin-332 is a heterotrimeric basement membrane component comprised of the α3, ß3, and γ2 laminin chains. Laminin-332 modulates epithelial cell processes, such as adhesion, migration, and differentiation and is prominent in many embryonic and adult tissues. In skin, laminin-332 is secreted by keratinocytes and is a key component of hemidesmosomes connecting the keratinocytes to the underlying dermis. In mice, lack of expression of any of the three Laminin-332 chains result in impaired anchorage and detachment of the epidermis, similar to that seen in human junctional epidermolysis bullosa, and death occurs within a few days after birth. To bypass the early lethality of laminin-332 deficiency caused by the knockout of the mouse laminin γ2 chain, we expressed a dox-controllable human laminin γ2 transgene under a keratinocyte-specific promoter on the laminin γ2 (Lamc2) knockout background. These mice appear similar to their wild-type littermates, do not develop skin blisters, are fertile, and survive >1.5 years. Immunofluorescence analyses of the skin showed that human laminin γ2 colocalized with mouse laminin α3 and ß3 in the basement membrane zone underlying the epidermis. Furthermore, the presence of “humanized” laminin-332 in the epidermal basement membrane zone rescued the alterations in the deposition of hemidesmosomal components, such as plectin, collagen type XVII/BP180, and integrin α6 and ß4 chains, seen in conventional Lamc2 knockout mice, leading to restored formation of hemidesmosomes. These mice will be a valuable tool for studies of organs deficient in laminin-332 and the role of laminin-332 in skin, including wound healing.     

Reconstitution of the epidermal basement membrane after enzymatic dermal-epidermal separation of embryonic mouse skin

Pieces of trypsin-isolated 14-day embryonic mouse epidermis were recombined with various living or non-living dermal or non-dermal substrates, in order to analyse the reconstruction of the dermal-epidermal junction. The constitution and ultrastructure of the epidermal basement membrane were characterized by immunolabelling of laminin, type IV collagen and bullous pemphigoid antigen, and by transmission electron microscopy. Trypsin treatment of dorsal skin followed by dermal-epidermal separation does not visibly damage the epidermal basement membrane, which remains attached to the lower face of epidermis. When freshly isolated epidermis is reassociated with dermis, the basement membrane is first degraded during the first 4 h of culture, then reconstituted within 24 h. When epidermis is cultured in isolation the basement membrane disappears within 4 h and is not reconstructed. Epidermis, precultured for 4 h and thus deprived of its basement membrane prior to reassociation, is able to reconstruct an antigenically and ultrastructurally normal basement membrane, when recombined with living or frozen-killed (-20 degrees C) dermis, with muscle tissue, or with a film of fibrous type I collagen. No basement membrane is reconstituted when the epidermis is recombined with heat (100 degrees C) killed dermis. It is concluded that, in the reconstituted epidermal basement membrane, laminin, type IV collagen, bullous pemphigoid antigen, and lamina densa are of exclusive epidermal origin.     

Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma

We have studied the extractability of type IV collagen, laminin, and heparan sulfate proteoglycan from EHS tumor tissue growth in normal and lathyritic animals. Laminin and heparan sulfate proteoglycan were readily extracted with chaotropic solvents from both normal and lathyritic tissue. The collagenous component was only solubilized from lathyritic tissue in the presence of a reducing agent. These results indicate that lysine-derived cross-links and disulfide bonds stabilize the collagenous component in the matrix but not the laminin or the heparan sulfate proteoglycan. The majority of the collagen present in the extracts had a native triple helix based upon the pattern of peptides resistant to pepsin digestion and visualization in the electron microscope by the rotary shadow technique. This protein was composed of chains (Mr 185000 and 170000) identical in migration to the chains of newly synthesized type IV procollagen. This finding confirms earlier work that indicates that the biosynthetic form, type IV procollagen, is incorporated as such in the basement membrane matrix. Material with smaller chains (Mr 160000 and 140000) appeared on storage in acetic acid solutions. These results indicate that the lower molecular weight collagen in acid extracts of basement membrane arises artifactually due to an endogenous acid-active protease.     

The incubation of laminin, collagen IV, and heparan sulfate proteoglycan at 35 degrees C yields basement membrane-like structures

Three basement membrane components, laminin, collagen IV, and heparan sulfate proteoglycan, were mixed and incubated at 35 degrees C for 1 h, during which a precipitate formed. Centrifugation yielded a pellet which was fixed in either potassium permanganate for ultrastructural studies, or in formaldehyde for Lowicryl embedding and immunolabeling with protein A-gold or anti-rabbit immunoglobulin-gold. Three types of structures were observed and called types A, B, and C. Type B consisted of 30-50-nm-wide strips that were dispersed or associated into a honeycomb-like pattern, but showed no similarity with basement membranes. Immunolabeling revealed that type B strips only contained heparan sulfate proteoglycan. The structure was attributed to self-assembly of this proteoglycan. Type A consisted of irregular strands of material that usually accumulated into semisolid groups. Like basement membrane, the strands contained laminin, collagen IV, and heparan sulfate proteoglycan, and, at high magnification, they appeared as a three-dimensional network of cord-like elements whose thickness averaged approximately 3 nm. But, unlike the neatly layered basement membranes, the type A strands were arranged in a random, disorderly manner. Type C structures were convoluted sheets composed of a uniform, dense, central layer which exhibited a few extensions on both surfaces and was similar in appearance and thickness to the lamina densa of basement membranes. Immunolabeling showed that laminin, collagen IV, and proteoglycan were colocalized in the type C sheets. At high magnification, the sheets appeared as a three-dimensional network of cords averaging approximately 3 nm. Hence, the organization, composition, and ultrastructure of type C sheets made them similar to the lamina densa of authentic basement membranes.     

Lack of heparan sulfate proteoglycan in a discontinuous and irregular placental basement membrane

A discontinuous basement membrane of variable width that surrounds spongiotrophoblast cells of rat placenta was examined for the presence of type IV collagen, laminin, a heparan sulfate proteoglycan, entactin, and fibronectin using monospecific antibodies or antisera and the indirect peroxidase technique. At the level of the light microscope, the basement membrane was immunostained for type IV collagen, laminin, entactin, and fibronectin. Heparan sulfate proteoglycan immunostaining, however, was virtually absent even after pretreatment of sections with 0.1 N acetic acid, pepsin (0.1 microgram/ml) or 0.13 M sodium borohydride. Examination in the electron microscope confirmed the lack of immunostaining for heparan sulfate proteoglycan, whereas the other substances were mainly localized to the lamina densa part of the basement membrane. The absence of heparan sulfate proteoglycan in this discontinuous and irregular basement membrane even though type IV collagen, laminin, entactin, and fibronectin are present, suggests that heparan sulfate proteoglycan may have a structural role in the formation of basement membrane.     

Light microscopic immunolocalization of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin in the basement membranes of a variety of rat organs

Immunohistochemical methods were used to determine whether type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan were present in diverse basement membranes. Antisera or antibodies against each substance were prepared, tested by enzyme-linked immunosorbent assay, and exposed to frozen sections of duodenum, trachea, kidney, spinal cord, cerebrum, and incisor tooth from rats aged 20 days to 34 months. Bound antibodies were then localized by indirect or direct peroxidase methods for examination in the light microscope. Immunostaining for type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan was observed in all of the basement membranes encountered. Fibronectin was also found in connective tissue. In general, the intensity of immunostaining was strong for type IV collagen and laminin, moderate for heparan sulfate proteoglycan, and weak for fibronectin. The pattern was similar in the age groups under study. Very recently the sulfated glycoprotein, entactin, was also detected in the basement membranes of the listed tissues in 20-day-old rats. It is accordingly proposed that, at least in the organs examined, type IV collagen, laminin, fibronectin, heparan sulfate proteoglycan, and entactin are present together in basement membranes.     

Molecular architecture of basement membranes

Basement membranes are specialized extracellular matrices with support, sieving, and cell regulatory functions. The molecular architectures of these matrices are created through specific binding interactions between unique glycoprotein and proteoglycan protomers. Type IV collagen chains, using NH2-terminal, COOH-terminal, and lateral association, form a covalently stabilized polygonal framework. Laminin, a four-armed glycoprotein, self-assembles through terminal-domain interactions to form a second polymer network, Entactin/nidogen, a dumbbell-shaped sulfated glycoprotein, binds laminin near its center and interacts with type IV collagen, bridging the two. A large heparan sulfate proteoglycan, important for charge-dependent molecular sieving, is firmly anchored in the basement membrane and can bind itself through a core-protein interaction to form dimers and oligomers and bind laminin and type IV collagen through its glycosaminoglycan chains. Heterogeneity of structure and function occur in different tissues, in development, and in response to different physiological needs. The molecular architecture of these matrices may be regulated during or after primary assembly through variations in compositions, isoform substitutions, and the modifying influence of exogenous macromolecules such as heparin and heparan sulfate.     

Immunohistochemical study of basement membrane reconstruction by an epidermis-dermis recombination experiment using cultured chick embryonic skin: induction of tenascin.

The production of extracellular matrix components such as laminin, Type IV collagen, fibronectin, and tenascin during the formation of basement membrane in cultured epidermis-dermis recombinant skin of 13-day-old chick embryo was analyzed immunohistochemically. The epidermis and dermis were separated from each other by treatment with EDTA and/or dispase. The basal lamina of the basement membrane was thus removed from both epidermis and dermis. The isolated epidermis was overlaid onto the isolated dermis, i.e., recombined, and then cultured for 1-7 days in a chemically defined medium (BGJb) on a Millipore filter. Immunofluorescence labeling was used for light microscopy and HRP or colloidal gold labeling for electron microscopy. In specimens from 2-day cultures, positive sites of anti-laminin and anti-fibronectin reaction were observed light microscopically as patches which, at the electron microscopic level, corresponded to fragments of the basal lamina located immediately beneath and in the vicinity of the attachment plaques of the hemidesmosomes. The staining pattern became continuous 7 days after recombination. Fluorescence labeling of laminin and fibronectin appeared somewhat earlier than that of Type IV collagen and tenascin. All of the four components were found localized primarily in the basal lamina. Furthermore, fibronectin and tenascin were also distributed in the extracellular matrix of the dermis. The expression of tenascin, which does not exist in the basement membrane of 13-day-old intact embryonic skin, was induced in vitro. These results suggest that hemidesmosomes may play an important role in the reconstruction of the basement membrane and that various components of the basement membrane appeared at different times during the reconstruction.     

Basement membrane macromolecules: insights from atomic force microscopy

The major macromolecules of basement membranes-collagen IV, laminin-1, and heparan sulfate proteoglycan (HSPG)-have been analyzed by atomic force microscopy (AFM), both individually and in combination with each other. The positions of laminin binding to collagen IV were mapped and compared with the positions of imperfections in the amino acid sequence of collagen IV; the apparent molecular volumes of the HSPG proteoglycans were measured and used to estimate the corresponding molecular weights. Even the thin, thread-like strands of the polyanion heparan sulfate can be visualized with AFM without staining, coating, or fixation. These strands are single polysaccharide chains and are thus thinner than single-stranded DNA. The heparan sulfate strands in HSPG are necessary for protein filtration in kidney basement membranes. We propose that these thin strands filter proteins by functioning as an entropic brush-i.e., that they filter proteins by their constant thermally driven motion in the basement membrane. These AFM analyses in air are a step toward AFM analyses under fluid of basement membrane macromolecules interacting with each other.     

A Mr 80K hepatocyte surface protein(s) interacts with basement membrane components

We have identified a Mr 80K cell surface protein(s) from adult rat hepatocytes that binds basement membrane components, including collagen IV, heparan sulfate proteoglycan, and laminin. Freshly isolated hepatocytes were cell surface-labeled with 125I using the lactoperoxidase-catalyzed method, and detergent-solubilized membrane proteins were chromatographed on affinity columns prepared with purified basement membrane components. A Mr 80K protein was eluted with 0.15-1 M NaCl from a collagen IV column. Two proteins (Mr 80K and 38K) were eluted from a heparan sulfate proteoglycan column. The larger protein was also eluted from a column made with heparan sulfate side chains. Several proteins (Mr 80K, 67K, 45K, and 32K) bound to an affinity chromatography column made with the laminin A chain-derived synthetic peptide PA22-2, which is active for promoting cell attachment. When fractions eluted from these columns were analyzed by two-dimensional gel electrophoresis, the Mr 80K proteins showed similar patterns with a pI ranging from 8 to 9. The Mr 80K protein(s) may have an important role in the interaction of hepatocytes with basement membrane.     

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and alpha 2 beta 1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize alpha 2 beta 1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.     

Basement membrane-like matrix of teratocarcinoma-derived endodermal cells: presence of laminin and heparan sulfate in the matrix at points of attachment to cells

Teratocarcinoma-derived endodermal PYS-2 cells are known to synthesize an extracellular matrix containing the basement membrane molecules laminin, type IV collagen, and heparan sulfate proteoglycan as major constituents (I. Leivo, K. Alitalo, L. Risteli, A. Vaheri, R. Timpl, J. Wartiovaara, Exp Cell Res 137:15-23, 1982). Immunoferritin techniques with specific antibodies were used in the present study to define the ultrastructural localization of the above constituents in the fibrillar network. Laminin was detected in matrix network adjacent to the basal cell membrane and in protruding matrix fibrils that connect the matrix to the cell membrane. Ruthenium red-stainable heparinase-sensitive 10- to 20-nm particles were often present at the junction of the attachment fibrils and the matrix network, or along the attachment fibrils. A corresponding distribution of ferritin label was observed for basement membrane heparan sulfate proteoglycan. Type IV collagen was found in the matrix network but not in the attachment fibrils. The results suggest that the PYS-2 cells are connected to their pericellular matrix by fibrils containing laminin associated with heparan sulfate-containing particles. These results may also have relevance for the attachment of epithelial cells to basement membranes.     

Basement membrane complexes with biological activity

We have studied the reconstitution of basement membrane molecules from extracts prepared from the basement membrane of the EHS tumor. Under physiological conditions and in the presence of added type IV collagen and heparan sulfate proteoglycan, gellike structures form whose ultrastructure appears as interconnected thin sheets resembling the lamina dense zone of basement membrane. The major components of the reconstituted structures include laminin, type IV collagen, heparan sulfate proteoglycan, entactin, and nidogen. These components polymerize in constant proportions on reconstitution, suggesting that they interact in defined proportions. Molecular sieve studies on the soluble extract demonstrate that laminin, entactin, and nidogen are associated in large but dissociable complexes which may be a necessary intermediate in the deposition of basement membrane. The reconstituted matrix was biologically active and stimulated the growth and differentiation of certain cells.     

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and α2β1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize α2β1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.