Regulation of cell adhesion and type VII collagen binding by the beta3 chain short arm of laminin-5: effect of its proteolytic cleavage

The basement membrane protein laminin-5 (Lm5), a heterotrimer of alpha3 (or alpha3A), beta3, and gamma2 chains, regulates cellular adhesion and motility. Here we examined the proteolysis and biological function of the laminin beta3 chain. First, we found that the beta3 chain of Lm5 is cleaved at its N-terminal, short arm by an endogenous proteinase(s) in normal human keratinocytes and some other cell lines. To examine the effect of beta3 chain cleavage, we expressed a wild-type Lm5 and two Lm5 mutants with partially deleted beta3 chains in HEK293 cells. Experiments with the purified Lm5 forms demonstrated that the deletion of the beta3 short arm or its N-terminal domain LN decreases the cell adhesion activity of Lm5, but does not significantly affect the motility activity. A recombinant beta3 short arm protein enhanced integrin-mediated cell adhesion to Lm5 by binding to an unidentified cell receptor. It was also found that the laminin EGF-like domain of the beta3 short arm is a binding site for type VII collagen. These results suggest that the beta3 short arm is involved not only in the matrix assembly of Lm5, but also in its cell adhesion activity. The proteolytic cleavage of the beta3 chain may modulate these functions of Lm5 in vivo.     

Recombinant human laminin-10 (alpha5beta1gamma1). Production, purification, and migration-promoting activity on vascular endothelial cells

The laminin (LN) family of large heterotrimeric extracellular matrix glycoproteins has multiple functions: LNs take part in the regulation of processes such as cell migration, differentiation, and proliferation, in addition to contributing to the structure of basement membranes. LN-10, composed of alpha5, beta1, and gamma1 chains, is widely distributed in most basement membranes of both epithelia and endothelia. We determined the complete human cDNA sequence for the LN alpha5 chain and produced recombinant human LN-10 (rLN-10) in HEK293 cells by triple transfection of full-length cDNAs encoding the human LN alpha5, beta1, and gamma1 chains. The rLN-10 was purified using affinity chromatography and had an apparent molecular mass of approximately 800 kDa in SDS-PAGE and a native domain structure in rotary shadowing electron microscopy. By using function-blocking monoclonal antibodies, integrin alpha(3)beta(1) was found to be a major mediator of adhesion of HT-1080 and human saphenous vein endothelial cells. Human saphenous vein endothelial cells adhered more strongly to rLN-10 than to LN-1 and LN-8 and showed better migration on rLN-10, compared with several other matrices. Considering the cell adhesive and migration-promoting properties of rLN-10 on endothelial cells, this molecule could be useful in improving the biocompatibility and endothelialization of vascular grafts.     

Colocalization of multiple laminin isoforms predominantly beneath hemidesmosomes in the upper lamina densa of the epidermal basement membrane.

Multiple laminin isoforms including laminins 5 (alpha3 beta3 gamma2), 6 (alpha3 beta1 gamma1), 10 (alpha5 beta1 gamma1), and possibly laminins 7 (alpha3 beta2 gamma1) and 11 (alpha5 beta2 gamma1) are present in the epidermal basement membrane. However, only the precise epidermal ultrastructural localization of laminin 5 (alpha3 beta3 gamma2) has been elucidated. We therefore determined the precise expression and ultrastructural localization of the alpha5, beta1, beta2, and gamma1 chains in the epidermis. The expression of laminin chains in skin samples was analyzed from patients with epidermolysis bullosa (EB, n=15) that harbor defects in specific hemidesmosome (HD)-associated components. The expression of the alpha5, beta1, and gamma1 chains (present in laminins 10/11) and beta2 chain (laminins 7/11) was unaffected in all intact (unseparated) skin of EB patients including Herlitz junctional EB with laminin-5 defects (n=6). In the basement membrane of human epidermis, the alpha5, beta1, beta2, and gamma1 chains were expressed but also localized to the dermal vessels. Immunogold electron microscopy of normal human epidermis localized the alpha5, beta1, beta2, and gamma1 chains to the upper lamina densa, with between 84% and 92% of labeling restricted to beneath the HDs, similar to laminin 5 (n> or =200 gold particles per sample, sample number n=4) but distinct from collagen IV labeling (with only 63% labeling beneath HDs, p<0.001). Taken together, the majority of the alpha5beta1/beta2gamma1 laminin chains are located beneath HDs. This suggests that laminin-10-associated chains have specific functions or molecular interactions beneath HDs in the epidermal basement membrane.     

Molecular analysis of laminin N-terminal domains mediating self-interactions

The ability of laminins to self-polymerize is crucial for the formation of basement membranes. Development of this polymerized network has profound effects upon tissue architecture as well as on the intracellular organization and differentiation of neighboring cells. The laminin N-terminal (LN) domains have been shown to mediate this interaction and studies using proteolytic fragments derived from laminin-1 led to the theory that network assembly depends on the formation of a heterotrimeric complex between LN domains derived from alpha, beta, and gamma chains in different laminin molecules with homologous interactions being insignificant. The laminin family consists of 15 known isoforms formed from five alpha, three beta, and three gamma chains, of which some are truncated and lack the N-terminal LN domain. To address whether the model of heterotrimeric complex formation is applicable to laminin isoforms other than laminin-1, eight LN domains found in the laminin protein family were recombinantly expressed and tested in three different assays for homologous and heterologous interactions. The results showed that the lack of homologous interactions is an exception, with such interactions being seen for LN domains derived from all alpha chains and from the beta2 and beta3 subunits. The gamma chain-derived LN domains showed a far more limited binding repertoire, particularly in the case of the gamma3 chain, which is found present in a range of non-basement membrane locations. Further, whereas the interactions depended upon Ca2+ ions, with EDTA reversibly abrogating binding, EDTA-induced conformational changes were not reversible. Together these results demonstrate that the assembly model proposed on the basis of laminin-1 may be a simplification, with the assembly of naturally occurring laminin networks being far more complex and highly dependent upon which laminin isoforms are present.     

The beta3 chain short arm of laminin-332 (laminin-5) induces matrix assembly and cell adhesion activity of laminin-511 (laminin-10)

The basement membrane (BM) protein laminin-332 (Lm332) (laminin-5) has unique activity and structure as compared with other laminins: it strongly promotes cellular adhesion and migration, and its alpha3, beta3, and gamma2 chains are all truncated in their N-terminal regions (short arms). In the present study, we investigated the biological function of the laminin beta3 chain. When the beta3 chain short arm (beta3SA) was overexpressed in HEK293 cells (beta3SA-HEK), they deposited a large amount of beta3SA and a small amount of laminin-511 (Lm511) (laminin-10) on culture plates. Control HEK293 cells secreted Lm511 but failed to deposit it. The extracellular matrix (ECM) deposited by beta3SA-HEK cells strongly promoted cell attachment and spreading. The beta3SA-HEK ECM did not directly bind Lm511, but it stimulated control HEK293 cells to deposit Lm511 on the culture plates. Although purified beta3SA did not support cell adhesion by itself, it enhanced the cell adhesion activity of Lm511. Experiments with anti-integrin antibodies also suggested that the strong cell adhesion activity of the beta3SA-HEK ECM was derived from the synergistic action of beta3SA and Lm511. It has previously been found that beta3SA binds an unknown cell surface receptor. Taken together, the present study suggests that the short arm of the laminin beta3 chain enhances the matrix assembly of Lm511 and its cell adhesion activity by interacting with its receptor.     

Characterization of laminin 5B and NH2-terminal proteolytic fragment of its alpha3B chain: promotion of cellular adhesion, migration, and proliferation

Various laminin isoforms have specific biological functions depending on their structures. Laminin 5A, which consists of the three truncated chains alpha3A, beta3, and gamma2, is known to have strong activity to promote cell adhesion and migration, whereas a laminin 5 variant consisting of a full-sized alpha3 chain (alpha3Beta) and the beta3 and gamma2 chains, laminin 5B, has not been characterized yet. In the present study, we for the first time cloned a full-length human laminin alpha3B cDNA and isolated the human laminin 5B protein. The molecular size of the mature alpha3B chain (335 kDa) was approximately twice as large as the mature alpha3A chain in laminin 5A. Laminin 5B had significantly higher cell adhesion and cell migration activities than laminin 5A. In addition, laminin 5B potently stimulated cell proliferation when added into the culture medium directly. Furthermore, we found that the alpha3B chain undergoes proteolytic cleavage releasing a 190-kDa NH(2)-terminal fragment. The 190-kDa fragment had activities to promote cellular adhesion, migration, and proliferation through its interaction with integrin alpha(3)beta(1). These activities of the NH(2)-terminal structure of the alpha3B chain seem to contribute to the prominent biological activities and the physiological functions of laminin 5B.     

Regulation of biological activity and matrix assembly of laminin-5 by COOH-terminal, LG4-5 domain of alpha3 chain

The basement membrane protein laminin-5 (LN5; alpha3beta3gamma2) undergoes specific proteolytic processing of the 190-kDa alpha3 chain to the 160-kDa form after the secretion, releasing its COOH-terminal, LG4-5 domain. To clarify the biological significance of this processing, we tried to express a recombinant precursor LN5 with a 190-kDa alpha3 chain (pre-LN5), in which the cleavage sequence Gln-Asp was changed to Ala-Ala by point mutation. When the wild-type and mutated LN5 heterotrimers were expressed in HEK293 cells, the wild-type alpha3 chain was completely cleaved, whereas the mutated alpha3 chain was partially cleaved at the same cleavage site (Ala-Ala). pre-LN5 was preferentially deposited on the extracellular matrix, but this deposition was effectively blocked by exogenous heparin. This suggests that interaction between the LG4-5 domain and heparan sulfate proteoglycans on the cell surface and/or extracellular matrix is important in the matrix assembly of LN5. Next, we purified both pre-LN5 and the mature LN5 with the processed, 160-kDa alpha3 chain (mat-LN5) from the conditioned medium of the HEK293 cells and compared their biological activities. mat-LN5 showed higher activities to promote cell adhesion, cell scattering, cell migration, and neurite outgrowth than pre-LN5. These results indicate that the proteolytic removal of LG4-5 from the 190-kDa alpha3 chain converts the precursor LN5 from a less active form to a fully active form. Furthermore, the released LG4-5 fragment stimulated the neurite outgrowth in the presence of mat-LN5, suggesting that LG4-5 synergistically enhances integrin signaling as it is released from the precursor LN5.     

Expression of laminin chains by central neurons: analysis with gene and protein trapping techniques

Laminins exert numerous effects on neurons in vitro, but expression of laminin subunit genes by neurons in vivo remains controversial. To reexamine this issue, we generated mice from ES cells in which the laminin alpha1, alpha5, beta1, and gamma1 genes had been "trapped" by insertion of a histochemically detectable selectable marker, betageo (beta-galactosidase fused to neomycin phosphotransferase). The presence of laminin-betageo fusion proteins was assayed histochemically and immunochemically, revealing expression of laminin beta1 and gamma1 genes, but not alpha chain genes, by defined subsets of neurons in brain and retina. We also used the gene traps in a novel way to assay expression of endogenous laminin subunits, which were barely detectable by ordinary immunohistochemical methods. The trapping vector included a transmembrane domain that anchors proteins otherwise destined for secretion. Laminin alpha/beta/gamma heterotrimers are assembled intracellularly, and we show that the trapped laminin gamma1 fusion protein "co-trapped" endogenous beta1 intracellularly. The laminin gamma1 fusion was also able to co-trap transgene-derived alpha chains, but we detected no co-trapped endogenous alpha chains. The co-trapping method may be generally useful for identifying proteins or isolating protein complexes associated with trapped gene products.     

Laminins 2 (alpha2beta1gamma1, Lm-211) and 8 (alpha4beta1gamma1, Lm-411) are synthesized and secreted by tooth pulp fibroblasts and differentially promote neurite outgrowth from trigeminal ganglion sensory neurons

The tooth pulp innervation originates from the trigeminal ganglion (TG) and represents an illustrative example of tissue targeting by sensory nerves. Pulpal fibroblasts strongly promote neurite outgrowth from TG neurons in vitro. In the present study, we have investigated the possible participation of laminins (LNs), potent neuritogenic extracellular matrix components. Immunohistochemistry of human tooth pulp demonstrated expression of LN alpha1, alpha2, alpha4, alpha5, beta1 and gamma1, and laminin-binding integrin alpha3, alpha6, beta1 and beta4 chains in nerves. Though faintly stained for laminins in situ, pulpal fibroblasts reacted, once cultured and permeabilized, with antibodies to LN alpha2, alpha4, beta1 and gamma1 chains by flow cytometry. The cells also expressed the corresponding mRNAs and were able to assemble and secrete LN-2 (alpha2beta1gamma1, Lm-211) and LN-8 (alpha4beta1gamma1, Lm-411). LN-8 displayed a chondroitin sulphate (CS) modification in its alpha4 chain. In functional assays, mouse LN-1 (alpha1beta1gamma1, Lm-111) and recombinant human (rh) LN-8, but not native or rhLN-2, strongly promoted neurite outgrowth from TG neurons, mimicking the effect of cultured pulp fibroblast. Altogether, the results indicate that LN-2 and LN-8 are synthesized by tooth pulp fibroblasts and differentially promote neurite outgrowth from TG neurons. LN-8 may contribute to sensory innervation of teeth and other tissues during development and/or regeneration.     

Integrin alpha(v)beta1 is an adenovirus coreceptor

The human embryonic kidney (HEK293) cell line, commonly used for recombinant adenovirus (Ad) propagation, does not express the Ad coreceptor alpha(v)beta3 or alpha(v)beta5 integrins, yet these cells are efficiently infected by Ad vectors. Here we demonstrate that Ad binds to HEK293 cells via the fiber receptor CAR and is subsequently internalized via interaction with integrin alpha(v)beta1. Function-blocking antibodies directed against alpha(v) or beta1, but not beta3, beta5, or alpha5, integrin subunits block Ad infection and viral endocytosis. Therefore, alpha(v)beta1 serves as a coreceptor for Ad infection, and the lack of beta3 and/or beta5 but the relatively high expression of alpha(v)beta1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.     

The LG1-3 tandem of laminin alpha5 harbors the binding sites of Lutheran/basal cell adhesion molecule and alpha3beta1/alpha6beta1 integrins

The laminin-type globular (LG) domains of laminin alpha chains have been implicated in various cellular interactions that are mediated through receptors such as integrins, alpha-dystroglycan, syndecans, and the Lutheran blood group glycoprotein (Lu). Lu, an Ig superfamily transmembrane receptor specific for laminin alpha5, is also known as basal cell adhesion molecule (B-CAM). Although Lu/B-CAM binds to the LG domain of laminin alpha5, the binding site has not been precisely defined. To better delineate this binding site, we produced a series of recombinant laminin trimers containing modified alpha chains, such that all or part of alpha5LG was replaced with analogous segments of human laminin alpha1LG. In solid phase binding assays using a soluble Lu (Lu-Fc) composed of the Lu extracellular domain and human IgG1 Fc, we found that Lu bound to Mr5G3, a recombinant laminin containing alpha5 domains LN through LG3 fused to human laminin alpha1LG4-5. However, Lu/B-CAM did not bind other recombinant laminins containing alpha5LG3 unless alpha5LG1-2 was also present. A recombinant alpha5LG1-3 tandem lacking the laminin coiled coil (LCC) domain did not reproduce the activity of Lu/B-CAM binding. Therefore, proper structure of the alpha5LG1-3 tandem with the LCC domain was essential for the binding of Lu/B-CAM to laminin alpha5. Our results also suggest that the binding site for Lu/B-CAM on laminin alpha5 may overlap with that of integrins alpha3beta1 and alpha6beta1.     

Detection and binding properties of GABA(A) receptor assembly intermediates

Density gradient centrifugation of native and recombinant gamma-aminobutyric acid, type A (GABA(A)) receptors was used to detect assembly intermediates. No such intermediates could be identified in extracts from adult rat brain or from human embryonic kidney (HEK) 293 cells transfected with alpha(1), beta(3), and gamma(2) subunits and cultured at 37 degrees C. However, subunit dimers, trimers, tetramers, and pentamers were found in extracts from the brain of 8-10-day-old rats and from alpha(1)beta(3)gamma(2) transfected HEK cells cultured at 25 degrees C. In both systems, alpha(1), beta(3), and gamma(2) subunits could be identified in subunit dimers, indicating that different subunit dimers are formed during GABA(A) receptor assembly. Co-transfection of HEK cells with various combinations of full-length and C-terminally truncated alpha(1) and beta(3) or alpha(1) and gamma(2) subunits and co-immunoprecipitation with subunit-specific antibodies indicated that even subunits containing no transmembrane domain can assemble with each other. Whereas alpha(1)gamma(2), alpha(1)Ngamma(2), alpha(1)gamma(2)N, and alpha(1)Ngamma(2)N, combinations exhibited specific [(3)H]Ro 15-1788 binding, specific [(3)H]muscimol binding could only be found in alpha(1)beta(3) and alpha(1)beta(3)N, but not in alpha(1)Nbeta(3) or alpha(1)Nbeta(3)N combinations. This seems to indicate that a full-length alpha(1) subunit is necessary for the formation of the muscimol-binding site and for the transduction of agonist binding into channel gating.     

Expression and chain assembly of human laminin-332 in an insect cell-free translation system

Laminins are a family of large heterotrimeric glycoproteins comprising alpha, beta, and gamma chains. To determine the molecular mechanisms underlying chain assembly in vitro, we expressed human laminin-332 subunits in an insect cell-free translation system. We successfully produced the beta3-gamma2 heterodimer and the alpha3-beta3-gamma2 heterotrimer of the laminin coiled-coil (LCC) domain following co-translation of each chain. The alpha3-beta3 and the alpha3-gamma2 heterodimer were not detected, suggesting that the alpha3 chain can assemble with only beta3-gamma2 heterodimer to form a heterotrimer via disulfide bonds. These results are consistent with those of a previous report indicating that laminin chain assembly proceeds through the beta-gamma heterodimer to the alpha-beta-gamma heterotrimer in vivo. We suggest that the cell-free translation system is a valid system with which to study the mechanisms underlying laminin chain assembly.     

Laminins in normal,keratoconus, bullous keratopathy and scarred human corneas

The laminin composition (LMalpha1-alpha5, beta1-beta3, gamma1 and gamma2 chains) of normal corneas and corneal buttons from keratoconus, bullous keratopathy (BKP), Fuchs' dystrophy + BKP, Fuchs' dystrophy without BKP and scar after deep lamellar keratoplasty (DLKP) was investigated with immunohistochemistry. The epithelial basement membranes (BMs) of both normal and diseased corneas contained LMalpha3, alpha5, beta1, beta3, gamma1 and gamma2 chains. The epithelial BM morphology was altered in the different diseases. Scarring was associated with irregular BM and ectopic stromal localization of different laminin chains. The Descemet's membrane (DM) contained LMalpha5, beta1 and gamma1 chains in all cases and additionally LMbeta3 and gamma2 chains in the majority of keratoconus corneas. The interface in the DLKP cornea had patches of LMalpha3, alpha4, alpha5, beta1 and beta2 chains, and an extra BM-like structure under the Bowman's membrane. These results suggest that laminin chains participate in the process of corneal scarring and in the pathogenesis of some corneal diseases. The novel finding of LMalpha3, beta3 and gamma2 in the DM of keratoconus buttons indicates that this membrane is also involved in the disease and that some cases of keratoconus may have a congenital origin, without normal downregulation of the LMbeta3 chain.     

The beta subunit increases Ca2+ currents and gating charge movements of human cardiac L-type Ca2+ channels.

The properties of the gating currents (nonlinear charge movements) of human cardiac L-type Ca2- channels and their relationship to the activation of the Ca2+ channel (ionic) currents were studied using a mammalian expression system. Cloned human cardiac alpha1 + rabbit alpha 2 subunits or human cardiac alpha 1 + rabbit alpha 2 + human beta 3 subunits were transiently expressed in HEK293 cells. The maximum Ca2+ current density increased from -3.9 +/- 0.9 pA/pF for the alpha 1 + alpha 2 subunits to -11.6 +/- 2.2 pA/pF for alpha 1 + alpha 2 + beta 3 subunits. Calcium channel gating currents were recorded after the addition of 5 mM Co2+, using a -P/5 protocol. The maximum nonlinear charge movement (Qmax) increased from 2.5 +/- 0.3 nC/muF for alpha 1 + alpha 2 subunit to 12.1 +/- 0.3 nC/muF for alpha 1 + alpha 2 + beta 3 subunit expression. The QON was equal to the QOFF for both subunit combinations. The QON-Vm data were fit by a sum of two Boltzmann expressions and ranged over more negative potentials, as compared with the voltage dependence for activation of the Ca2+ conductance. We conclude that 1) the beta subunit increases the number of functional alpha 1 subunits expressed in the plasma membrane of these cells and 2) the voltage-dependent activation of the human cardiac L-type calcium channel involves the movements of at least two nonidentical and functionally distinct gating structures.     

Distinct distribution of laminin and its integrin receptors in the pancreas.

Tissue function is regulated by the extracellular microenvironment including cell basement membranes, in which laminins are a major component. Previously, we found that laminin-1 promotes differentiation and survival of pancreatic islet cells. Here we characterize the expression pattern of laminins and their integrin receptors in adult pancreas. Although they are expressed in the basement membrane of acinar cells and duct epithelium, no laminin chains examined were detected extracellularly in the pancreatic islets. In contrast to laminin beta(1)- and gamma(1)-chains, the alpha(1)-chain, unique to laminin-1, was not detected. Laminin-10 (alpha(5)beta(1)gamma(1)) was expressed in acinar tissue, whereas laminins-2 (alpha(2)beta(1)gamma(1)) and -10 were expressed in the blood vessels. The laminin connector molecule, nidogen-1, had a distribution similar to that of laminin beta(1) and gamma(1), whereas fibulin-1 and -2, which compete with nidogen-1, were mostly confined to blood vessels. Integrin subunits alpha(6) and alpha(3) were detected in acinar cells and duct epithelial cells, but alpha(6) was absent in islet cells. Integrin alpha(6)beta(4) was detected only in duct cells, alpha(6)beta(1) in both acinar and ductal cells, and alpha(3)beta(1) in acinar, duct, and islet cells. These findings are a basis for further investigation of the role of extracellular matrix molecules and their receptors in pancreas function.     

Immunohistochemical localization of laminin and fibronectin isoforms in human placental villi.

We studied the localization of laminin alpha1, alpha2, alpha3, alpha5, beta1, beta2, and gamma1 chains and extradomain A- (EDA), EDB-, and oncofetal fibronectin by immunohistochemistry in human placental villi during placental development. The laminin alpha2, alpha5, beta1, beta2, and gamma1 chains were detected in the trophoblastic basement membrane (BM) at all stages of gestation, suggesting the presence of laminin-2, -4, -10, and -11 trimers. The laminin alpha1 chain was selectively found at sites where the villous BM was in contact with proliferating cells in trophoblastic islands or columns. EDA-Fn, but not other Fn isoforms, was found in the trophoblastic BM during the first trimester. The laminin alpha2, beta1, beta2, and gamma1 chains were detected in the villous stroma and capillaries throughout placental development, while the laminin alpha5 chain emerged distinctly during development. Extensive EDA-Fn immunoreactivity was found in first-trimester villous stroma, but distinctly fewer Fn isoforms were seen in the villous stroma during the later stages of gestation. Our results also suggest that, during the formation of new villi, laminins are not found in trophoblastic sprouts before the ingrowth of the villous mesenchyme. Rather, laminins may be deposited at the villous epithelial-mesenchymal interface. Furthermore, the results show that distinct changes occur in the localization of various laminin and Fn isoforms during the maturation of villous trophoblastic and capillary BMs.     

Localization of laminin α3B chain in vascular and epithelial basement membranes of normal human tissues and its down-regulation in skin cancers

The basement membrane (BM) proteins laminins, which consist of alpha, beta and gamma chains, play critical roles in the maintenance of tissue structures. One of laminin alpha chains, alpha3 has two isoforms, the truncated form alpha3A and the full-sized form alpha3B. In contrast to alpha3A laminins, little is known about alpha3B laminins. To show the histological distribution of the laminin alpha3B chain, we prepared alpha3B-specific monoclonal antibodies. Immunohistochemical analysis showed that the alpha3B chain was colocalized with the alpha3A, beta3 and gamma2 chains in the epithelial BMs of the skin, esophagus, breast and lung, suggesting the presence of laminin-3B32 (laminin-5B) and laminin-3A32 (laminin-5A). In the lung alveoli, laminin-3B32 was dominant over laminin-3A32, but vice versa in other epithelial BMs. In contrast, the BMs of blood vessels including capillaries were strongly positive for alpha3B, but almost or completely negative for alpha3A, beta3 and gamma2. alpha3B was colocalized with beta1 and gamma1 in these BMs. The alpha3B chain was scarcely detected in the vessels of malignant skin cancers, though the gamma2 and beta3 chains were highly expressed in the cancer cells. These results strongly suggest that the laminin alpha3B chain is widely expressed in vascular BMs of normal tissues, probably as laminin-3B11/3B21 (laminin-6B/7B).