Biological roles of laminins 8, 9 and 10

Abstract: In order to form multicellular organisms, cells need attachment, either directly to each other through cell-cell interactions, or indirectly via a complex mixture of macro-molecules called the extracellular matrix. Basement membranes are specialized forms of this matrix. They can be seen in the electron microscope as a thin flat structure underlying epithelial or endothelial cells, or surrounding certain individual cells such as muscle, nerve and fat cells. The basement membranes provide a structural framework for individual cells, or groups of cells, delineating them from the surrounding tissues, and anchor the cells through specific interactions with cell surface receptors. These interactions are of vital importance for the development of a cell as well as for its maintenance, and in many cases, even for its survival. Basement membranes are evolutionary old inventions and can be found in very primitive organisms. Most mammalian basement membranes have a type IV collagen- network and a lamininnetwork that are thought to be interconnected via a linker molecule called nidogen. Dystroglycan and the integrin family of receptors are the most well characterized cell surface receptors for laminins. Laminins are large glycoproteins comprised of three individual polypeptide chains (alpha-beta-gamma) and to date, 5 alpha, 4 P and 3 V genetically distinct variants are known. Different chain variants combine into the laminin isoforms, and 14 have been identified in vivo so far. The different isoforms have partially overlapping expression pattems, which are complex both temporally and spatially. This study mainly focuses on the biological roles of laminin 8, (alpha4-beta1- gamma1) laminin 9, (alpha4-beta2-gamma1) and laminin 10 (alpha5-beta1-gamma1). In order to study the biological role of the laminin alpha 4 chain, its gene was targeted in mouse embryonic stein cells. The Lama 4 null mice were viable and fertile but displayed transient hemorrhages at birth, a subtle motor impairment as adult mice, as well as resistance to obesity. The hemorrhages in the newborn null animals were located in soft tissues. Mild fetal hemorrhages were also seen, but they were never as extensive as in the newborn. We therefore hypothesized that the hemorrhages were the result of microvessel damage during delivery. Electron microscopy analysis revealed defects in the basement membranes of microvessels in muscle from newborn animals. Furthermore, immunostaining of endothelial basement membranes showed that the amount of type IV collagen and nidogen were significantly reduced. It was concluded that laminin alpha 4 is important for endothelial basement membrane formation. The Lama 4 null mice also exhibited a mild motor impairment. Using neurophysiologic testing, we found muscle function to be intact and muscle histology revealed no obvious signs of dystrophy or myopathy. A detailed analysis of the neuromuscular junctions (NMJs) found these to be generally properly formed in Lama 4 null mice, but to have interesting specific defects in the apposition of the active zones to the junctional folds. In the NMJ, the laminin beta 2 and V1 chains are expressed; leading to the conclusion that laminin 9 (alpha 4-beta 2-gamma 1) is of importance for the localization of synaptic specializations in the NMJ. Resistance to diet-induced as well as age-related obesity was another finding in Lama 4 null mice. The difference in the weight of males became significant after 10 weeks on a high fat containing diet and was not due to a decrease in food intake. The adipocyte size was reduced to half of that of controls. The null animals had normal levels of serum lipids, except for cholesterol, which was not elevated to the same extent as in control animals on high-fat diet. Furthermore, mills showed increased spontaneous activity but no increase in resting metabolic rate. The hyperactivity could be part of a neurological syndrome, and is likely to contribute to the lean phenotype. The cDNA encoding the human laminin alpha 5 chain was obtained through screening of 2, phage cDNA libraries and PCR amplification of cDNA mixes. The cDNA sequence was used to make a full-length laminin alpha 5 expression vector, which was used to transfect a human embryonic kidney cell line. This cell line was also transfected with vectors expressing the laminin beta 1 and gamma 1 chains, enabling the cells to produce all the three chain components of laminin 10. The resulting cell line was shown to produce high amounts of recombinant laminin 10, which could be purified using affinity chromatography. The protein was shown to promote cell adhesion as well as cell migration. Using function-blocking antibodies, integrin alpha 3-beta 1 was identified as a main mediator of adhesion to laminin 10 in HT- 1080 cells, as well as in human saphenous vein endothelial cells.