Jarkko Kortesmaa

Deletion of the Laminin α4 Chain Leads to Impaired Microvessel Maturation

ABSTRACT The laminin α4 chain, a component of laminin-8 and -9, is expressed in basement membranes, such as those beneath endothelia, the perineurium of peripheral nerves, and around developing muscle fibers. Laminin α4-null mice presented with hemorrhages during the embryonic and neonatal period and had extensive bleeding and deterioration of microvessel growth in experimental angiogenesis, as well as mild locomotion defects. Histological examination of newborn mice revealed delayed deposition of type IV collagen and nidogen into capillary basement membranes, and electron microscopy showed discontinuities in the lamina densa. The results demonstrate a central role for the laminin α4 chain in microvessel growth and, in the absence of other laminin α chains, in the composition of endothelial basement membranes.

Properly formed but improperly localized synaptic specializations in the absence of laminin alpha4.

Precise apposition of pre- to postsynaptic specializations is required for optimal function of chemical synapses, but little is known about how it is achieved. At the skeletal neuromuscular junction, active zones (transmitter release sites) in the nerve terminal lie directly opposite junctional folds in the postsynaptic membrane. Few active zones or junctional folds form in mice lacking the laminin β2 chain, which is normally concentrated in the synaptic cleft. β2 and the broadly expressed γ1 chain form heterotrimers with α chains, three of which, α2, α4 and α5, are present in the synaptic cleft. Thus, α2β2γ1, α4β2γ1 and α5β2γ1 heterotrimers are all lost in β2 mutants. In mice lacking laminin α4, active zones and junctional folds form in normal numbers, but are not precisely apposed to each other. Thus, formation and localization of synaptic specializations are regulated separately, and α4β2γ1 (called laminin-9) is critical in the latter process.

Localization of Laminin α4-Chain in Developing and Adult Human Tissues

Recent studies suggest important functions for laminin-8 (Ln-8; α4β1γ1) in vascular and blood cell biology, but its distribution in human tissues has remained elusive. We have raised a monoclonal antibody (MAb) FC10, and by enzyme-linked immunoassay (EIA) and Western blotting techniques we show that it recognizes the human Ln α4-chain. Immunoreactivity for the Ln α4-chain was localized in tissues of mesodermal origin, such as basement membranes (BMs) of endothelia, adipocytes, and skeletal, smooth, and cardiac muscle cells. In addition, the Ln α4-chain was found in regions of some epithelial BMs, including epidermis, salivary glands, pancreas, esophageal and gastric glands, intestinal crypts, and some renal medullary tubules. Developmental differences in the distribution of Ln α4-chain were detected in skeletal muscle, walls of vessels, and intestinal crypts. Ln α4- and Ln α2-chains co-localized in BMs of fetal skeletal muscle cells and in some epithelial BMs, e.g., in gastric glands and acini of pancreas. Cultured human pulmonary artery endothelial (HPAE) cells produced Ln α4-chain as Mr 180,000 and 200,000 doublet and rapidly deposited it to the growth substratum. In cell-free extracellular matrices of human kidney and lung, Ln α4-chain was found as Mr 180,000 protein.

Impeded Interaction between Schwann Cells and Axons in the Absence of Laminin α4

The Schwann cell basal lamina (BL) is required for normal myelination. Loss or mutations of BL constituents, such as laminin-2 (α2β1γ1), lead to severe neuropathic diseases affecting peripheral nerves. The function of the second known laminin present in Schwann cell BL, laminin-8 (α4β1γ1), is so far unknown. Here we show that absence of the laminin α4 chain, which distinguishes laminin-8 from laminin-2, leads to a disturbance in radial sorting, impaired myelination, and signs of ataxia and proprioceptive disturbances, whereas the axonal regenerative capacity is not influenced. In vitro studies show poor axon growth of spinal motoneurons on laminin-8, whereas it is extensive on laminin-2. Schwann cells, however, extend longer processes on laminin-8 than on laminin-2, and, in contrast to the interaction with laminin-2, solely use the integrin receptor α6β1 in their interaction with laminin-8. Thus, laminin-2 and laminin-8 have different critical functions in peripheral nerves, mediated by different integrin receptors.

Monocytic Cells Synthesize, Adhere to, and Migrate on Laminin-8 (α4β1γ1)

Laminins, a growing family of large heterotrimeric proteins with cell adhesive and signaling properties, are major components of vascular and other basement membranes. Expression, recognition, and use of laminin isoforms by leukocytes are poorly understood. In monoblastic THP-1 cells, transcripts for laminin γ1-, β1-, and α4-chains were detected by RT-PCR. Following immunoaffinity purification on a laminin β1 Ab-Sepharose column, laminin β1- (220 kDa), γ1- (200 kDa), and α4- (180/200 kDa) chains were detected by Western blotting in THP-1 cells and in two other monoblastic cell lines, U-937 and Mono Mac 6. After cell permeabilization, a mAb to laminin γ1-chain reacted with practically all blood monocytes by immunofluorescence flow cytometry, and laminin-8 (α4β1γ1) could be isolated also from these cells. Monoblastic JOSK-I cells adhered constitutively to immobilized recombinant laminin-8, less than to laminin-10/11 (α5β1γ1/α5β2γ1) but to a higher level than to laminin-1 (α1β1γ1). Compared with the other laminin isoforms, adhesion to laminin-8 was preferentially mediated by α6β1 and β2 integrins. Laminin-8 and, to a lower extent, laminin-1 promoted spontaneous and chemokine-induced migration of blood monocytes, whereas laminin-10/11 was inhibitory. Altogether, the results indicate that leukocytes, as other cell types, are able to synthesize complete laminin molecules. Expression, recognition, and use of laminin-8 by leukocytes suggest a major role of this laminin isoform in leukocyte physiology.

Deletion of Laminin-8 Results in Increased Tumor Neovascularization and Metastasis in Mice

Laminin-8 (α4β1γ1) is one of the major laminin isoforms expressed in vascular endothelial basement membranes. Here we show that deletion of laminin-8 in mice affects angiogenesis under pathological conditions. Murine tumor models used in laminin α4-deficient mice results in hyperneovascularization and significant promotion of tumor growth and metastasis. The higher tumor growth rates in mutant mice correlate with decreased tumor cell apoptosis. Depletion of laminin α4 chain may alter the structure of vascular basement membranes, leading to increased angiogenesis. Our data suggest that the laminin-8 plays a critical role in the regulation of pathological angiogenesis.

Chain specificity assignment of monoclonal antibodies to human laminins by using recombinant laminin β1 and γ1 chains

Abstract In the present study, the chain specificity of 16 commonly used monoclonal antibodies to human laminin(s) was analysed by using recombinant laminin β1 and γ1 chains. By ELISA, all antibodies reacted with purified placenta laminin, and most antibodies recognised either recombinant β1 or γ1 chains. Reactivity and chain specificity was confirmed against the recombinant chains in Western blotting under non-reducing conditions, and only a few antibodies were reactive under reducing conditions. Most antibodies were able to immunoprecipitate associated laminin β1/γ1 chains from platelet lysates. Based on these results and data from the literature, a tentative epitope map is presented.

Chondroitin sulphate modification in the α4 chain of human recombinant laminin-8 (α4β1γ1)

Abstract We have produced human laminin-8 (α4β1γ1) using recombinant technology. Approximately half of the recombinant laminin-8 (rLN-8) molecules were found to have a chondroitin sulphate modification in the α4 chain. The substituted and non-substituted forms were separated and tested for cell adhesion activity. Lower cell adhesion promoting activity was seen for the substituted form, but the integrin receptor utilization was similar. We also found the human rLN-8 to behave identically in cell adhesion assays compared to a human/mouse hybrid variant of rLN-8.

Laminin α4 deficient mice exhibit decreased capacity for adipose tissue expansion and weight gain.

Obesity is a global epidemic that contributes to the increasing medical burdens related to type 2 diabetes, cardiovascular disease and cancer. A better understanding of the mechanisms regulating adipose tissue expansion could lead to therapeutics that eliminate or reduce obesity-associated morbidity and mortality. The extracellular matrix (ECM) has been shown to regulate the development and function of numerous tissues and organs. However, there is little understanding of its function in adipose tissue. In this manuscript we describe the role of laminin α4, a specialized ECM protein surrounding adipocytes, on weight gain and adipose tissue function. Adipose tissue accumulation, lipogenesis, and structure were examined in mice with a null mutation of the laminin α4 gene (Lama4−/ −) and compared to wild-type (Lama4+/+) control animals. Lama4−/ − mice exhibited reduced weight gain in response to both age and high fat diet. Interestingly, the mice had decreased adipose tissue mass and altered lipogenesis in a depot-specific manner. In particular, epididymal adipose tissue mass was specifically decreased in knock-out mice, and there was also a defect in lipogenesis in this depot as well. In contrast, no such differences were observed in subcutaneous adipose tissue at 14 weeks. The results suggest that laminin α4 influences adipose tissue structure and function in a depot-specific manner. Alterations in laminin composition offers insight into the roll the ECM potentially plays in modulating cellular behavior in adipose tissue expansion.