Primary structure, expression and function of laminin [alpha]4

Abstract

Basement membranes (BMs) are thin extracellular sheet-like structures that compartmentalize tissues. They are found beneath epithelia and endothelia, and surrounding individual cells, such as muscle fibers, neurons and adipocytes. Laminins, type IV collagens, perlecan and nidogens are some of the main components of mature basement membranes. Laminins are composed of [alpha], [beta] and [gamma] chains. They are not only major structural elements of basement membranes, but they also serve as cell-matrix linkers and signaling molecules through their interactions with cell surface receptors such as integrins. Laminins are multidomain glycoproteins with five a, four b and three g chains identified in mammals so far. The chains combine to at least twelve heterotrimeric isoforms that have different tissue distributions and functions. Laminin a4 is recently described laminin chain, the role of which has been unknown. This study was part of a project aimed at characterizing laminin a4 and the laminin-8 isoform containing this polypeptide chain.

The complete primary structure of the mouse laminin a4 chain was derived from cDNA clones. Northern analyses of whole mouse embryo mRNAs revealed weak expression at day 7, but it later increased and peaked at day 15. In adult tissues the strongest expression was observed in lung and, in cardiac and skeletal muscles. Weak expression was also seen in other adult tissues, such as brain, spleen, liver, kidney, and testis. By in situ hybridization of fetal and newborn tissues, expression of the laminin [alpha]4 chain was mainly localized to mesenchymal cells. Strong expression was seen in the villi and submucosa of the developing intestine, the mesenchymal stroma surrounding the branching lung epithelia, and the external root sheath of vibrissae follicles, as well as in cardiac and skeletal muscle fibers. In the developing kidney, intense but transient expression was associated with the differentiation of epithelial kidney tubules from the nephrogenic mesenchyme. Immunohistological staining localized the laminin a4 chain primarily to lung septa, heart, capillaries, perineurium, and developing skeletal muscle.

To study the properties of laminin-8, recombinant laminin-8 heterotrimer ([alpha]4[beta]1[gamma]1) was produced in a mammalian expression system using triply transfected HEK-293 cells. The protein was purified using affinity chromatography aided by a fusion epitope tag followed by ion-exchange chromatography. The purified recombinant laminin-8 was shown to form the expected Y-shaped molecules in rotary shadowing electron microscopy. Recombinant laminin-8 supported cell adhesion, and integrins receptors mediating cell adhesion to laminin-8 were identified using function-blocking monoclonal antibodies. Integrin [alpha]6[beta]1 was found to be a major mediator of adhesion of HT-1080 fibrosarcoma cells and cultured capillary endothelial cells to laminin-8. Integrin [alpha]6[beta]4 was also able to mediate cell adhesion to laminin. Antibodies to integrins [alpha]1, [alpha]2, [alpha]3 and [alpha]5 did not affect binding. Considering the expression patterns of laminin-8 and integrin [alpha]6[beta]1 it is likely that the former is a ligand for the latter in vivo as well. Platelets were found to contain laminin-8, and secrete some of it after stimulation with either thrombin or phorbol ester. Laminin-8 was purified from platelets using monoclonal anti-laminin b1 antibody column, and the identity of the three chains was determined with antibodies and amino-acid sequencing. Platelets adhered to purified platelet laminin-8 and recombinant laminin-8 using integrin a[alpha]6[beta]1.

To further explore the biological role of the laminin [alpha]4 chain, mice carrying inactivated Lama4 alleles were generated. The laminin [alpha]4 null mice presented neonatal hemorrhages and a motorical dysfunction as adults. The hemorrhages were observed especially in the soft tissues of hind limbs, the head, lower back and neck regions, but also in the heart and meningies. As a consequence of the bleedings, the newborn mice suffered from anemia. There were also extensive bleedings and deterioration of microvessel growth in experimental angiogenesis in the cornea. Histological examination of newborn skeletal muscle revealed lack of other laminin chains and delayed deposition of type IV collagen and nidogen into capillary BMs. In contrast, perlecan deposition appeared normal. Electron microscopy showed discontinuities and absence of distinct capillary basement membranes, as compared to wild-type littermates, while other basement membranes appeared normal. In adult null mice, immunostaining for the BM components in muscle capillaries and muscle fibers were identical to those of controls, except for laminin [alpha]4. The fact that the vascular phenotype in null mice disappeared during the first weeks of life could be explained by initiation of expression and deposition of laminin-10 ([alpha]5[beta]1[gamma]1) into the capillary BMs, which probably then facilitates type IV collagen and nidogen deposition, restoring BM stability and structure. The results demonstrate a central role for the laminin a4 chain during microvessel growth and, in the absence of other laminin [alpha] chains, in the assembly of the type IV collagen network.