Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2-associated disorders.

Congenital nephrotic syndrome is clinically and genetically heterogeneous. The majority of cases can be attributed to mutations in the genes NPHS1, NPHS2, and WT1. By homozygosity mapping in a consanguineous family with isolated congenital nephrotic syndrome, we identified a potential candidate region on chromosome 3p. The LAMB2 gene, which was recently reported as mutated in Pierson syndrome (microcoria-congenital nephrosis syndrome; OMIM #609049), was located in the linkage interval. Sequencing of all coding exons of LAMB2 revealed a novel homozygous missense mutation (R246Q) in both affected children. A different mutation at this codon (R246W), which is highly conserved through evolution, has recently been reported as causing Pierson syndrome. Subsequent LAMB2 mutational screening in six additional families with congenital nephrotic syndrome revealed compound heterozygosity for two novel missense mutations in one family with additional nonspecific ocular anomalies. These findings demonstrate that the spectrum of LAMB2-associated disorders is broader than previously anticipated and includes congenital nephrotic syndrome without eye anomalies or with minor ocular changes different from those observed in Pierson syndrome. This phenotypic variability likely reflects specific genotypes. We conclude that mutational analysis in LAMB2 should be considered in congenital nephrotic syndrome, if no mutations are found in NPHS1, NPHS2, or WT1.

Modulation of in vivo migratory function of alpha 2 beta 1 integrin in mouse liver.

We report herein that expression of alpha 2 beta 1 integrin increased human erythroleukemia K562 transfectant (KX2C2) cell movement after extravasation into liver parenchyma. In contrast, a previous study demonstrated that alpha 2 beta 1 expression conferred a stationary phenotype to human rhabdomyosarcoma RD transfectant (RDX2C2) cells after extravasation into the liver. We therefore assessed the adhesive and migratory function of alpha 2 beta 1 on KX2C2 and RDX2C2 cells using a alpha 2 beta 1-specific stimulatory monoclonal antibody (mAb), JBS2, and a blocking mAb, BHA2.1. In comparison with RDX2C2 cells, KX2C2 were only weakly adherent to collagen and laminin. JBS2 stimulated alpha 2 beta 1-mediated interaction of KX2C2 cells with both collagen and laminin resulting in increases in cell movement on both matrix proteins. In the presence of Mn2+, JBS2-stimulated adhesion on collagen beyond an optimal level for cell movement. In comparison, an increase in RDX2C2 cell movement on collagen required a reduction in its adhesive strength provided by the blocking mAb BHA2.1. Consistent with these in vitro findings, in vivo videomicroscopy revealed that alpha 2 beta 1-mediated postextravasation cell movement of KX2C2 cells in the liver tissue could also be stimulated by JBS2. Thus, results demonstrate that alpha 2 beta 1 expression can modulate postextravasation cell movement by conferring either a stationary or motile phenotype to different cell types. These findings may be related to the differing metastatic activities of different tumor cell types.

An epitope on VLA-6 (alpha6beta1) integrin involved in migration but not adhesion is required for extravasation of murine melanoma B16F1 cells in liver.

VLA-6 (alpha6beta1) integrin represents the major receptor for interaction with laminin substrate. It has been proposed that VLA-6 mediates tumor cell adhesion to the endothelium during extravasation. We have further explored this possibility using mouse melanoma B16F1 cells, which express VLA-6 as the principal laminin receptor, and two VLA-6 monoclonal antibodies (mAbs), MA6 and GoH3. Adhesion is a prerequisite of cell movement on matrix proteins. Thus, GoH3, which inhibited VLA-6-mediated adhesion, blocked cell movement on laminin. The recently prepared alpha6 integrin-specific mAb MA6 bound to an epitope in close proximity to GoH3, but it had no effect on VLA-6-mediated cell adhesion. We report here that although MA6 did not affect adhesion, it blocked mouse melanoma B16F1 cell movement on laminin to the same extent as GoH3. Results therefore demonstrate an active role of VLA-6 in providing cell movement as well as the initial adhesive event on laminin. In addition, mAb MA6 had no effect on the induction of tyrosine phosphorylation of focal adhesion kinase upon adhesion of B16F1 cells to laminin. Therefore, inhibition of cell movement by MA6 involved mechanism(s) other than an interference of VLA-6 signaling events leading to phosphorylation of focal adhesion kinase. The epitopes of GoH3 and MA6 may represent spatially and temporally related sites on VLA-6 that are involved during cell movement, or, alternatively, MA6 may inhibit the interaction of VLA-6 with associated cell surface molecules required for cell movement. In vivo videomicroscopy experiments also revealed that an inhibition of VLA-6 migratory function by MA6 resulted in a reduction in the ability of B16F1 to extravasate during hematogenous metastasis in the liver.

Integrin VLA-2 (alpha2beta1) function in postextravasation movement of human rhabdomyosarcoma RD cells in the liver.

It is now known that members of the selectin and integrin families are critical in the initial interaction of cells in circulation with endothelial surfaces. Also, platelet/endothelial cell adhesion molecule-1 has been shown to be involved in transendothelial migration of extravasating cells. Little is known about adhesion molecules involved in subsequent postextravasation events. In this study, the significance of VLA-2 (alpha2beta1) integrin in the movement of human rhabdomyosarcoma RD cells in the liver was characterized by in vivo videomicroscopy. Results show that after extravasation, the mock-transfected RDpF cells were able to migrate to the subcapsular region of the liver. Although the RDX2C2 transfectant expressing VLA-2 integrin extravasated equally well, a majority of RDX2C2 cells remained in close proximity to blood vessels and failed to reach the subcapsular region. The functional involvement of VLA-2 in affecting the ability of RD cells to reach the subcapsular region was verified by the preparation of an RD transfectant [RDX2C2(I-)] expressing a nonfunctional variant of VLA-2 lacking the inserted (I)-domain of alpha2 subunit. In vivo microscopy showed that RDX2C2(I-) cells migrated in a manner similar to control RDpF cells. To demonstrate that RDX2C2 cells that remained in dose proximity to blood vessels were due to VLA-2 function, a blocking monoclonal antibody against VLA-2 (BHA2.1) was prepared. Mice were injected with BHA2.1 or control monoclonal antibody P3 at the time when RDX2C2 cells completed their extravasation. Treatment with BHA2.1 increased the number of RDX2C2 cells that reached the subcapsular region and subsequently formed tumor foci. Therefore, VLA-2 integrin expression has major roles in postextravasation movement and affects tumor foci formation at the liver surface.