BackgroundThe Lutheran blood group glycoprotein (Lu), an Ig superfamily (IgSF) transmembrane receptor, is also known as basal cell adhesion molecule (B-CAM). Lu/B-CAM is a specific receptor for laminin α5, a major component of basement membranes in various tissues. Previous reports have shown that Lu/B-CAM binding to laminin α5 contributes to sickle cell vaso-occlusion. However, as there are no useful tools such as function-blocking antibodies or drugs, it is unclear how epithelial and sickled red blood cells adhere to laminin α5 via Lu/B-CAM.Methodology/Principal FindingsIn this study, we discovered a function-blocking antibody that inhibits Lu binding to laminin α5 using a unique binding assay on tissue sections. To characterize the function-blocking antibody, we identified the site on Lu/B-CAM recognized by this antibody. The extracellular domain of Lu/B-CAM contains five IgSF domains, D1-D2-D3-D4-D5. The antibody epitope was localized to D2, but not to the D3 domain containing the major part of the laminin α5 binding site. Furthermore, mutagenesis studies showed that Arg175, the LU4 blood group antigenic site, was crucial for forming the epitope and the antibody bound sufficiently close to sterically hinder the interaction with α5. Cell adhesion assay using the antibody also showed that Lu/B-CAM serves as a secondary receptor for the adhesion of carcinoma cells to laminin α5.Conclusion/SignificanceThis function-blocking antibody against Lu/B-CAM should be useful for not only investigating cell adhesion to laminin α5 but also for developing drugs to inhibit sickle cell vaso-occlusion.
Human dental pulp cells (DPCs), adherent cells derived from dental pulp tissues, are potential tools for cell transplantation therapy. However, little work has been done to optimize such transplantation. In this study, DPCs were treated with fibroblast growth factor-2 (FGF2) for 5–6 consecutive serial passages and were transplanted into the injury site immediately after complete transection of the rat spinal cord. FGF2 priming facilitated the DPCs to promote axonal regeneration and to improve locomotor function in the rat with spinal cord injury (SCI). Additional analyses revealed that FGF2 priming protected cultured DPCs from hydrogen-peroxide–induced cell death and increased the number of DPCs in the SCI rat spinal cord even 7 weeks after transplantation. The production of major neurotrophic factors was equivalent in FGF2-treated and untreated DPCs. These observations suggest that FGF2 priming might protect DPCs from the post-trauma microenvironment in which DPCs infiltrate and resident immune cells generate cytotoxic reactive oxygen species. Surviving DPCs could increase the availability of neurotrophic factors in the lesion site, thereby promoting axonal regeneration and locomotor function recovery.
Hepatocytes rapidly lose hepatic functions upon isolation from liver, perhaps due to disrupted cell/matrix interactions. The matrix macromolecule laminin-111 consists of three chains, α1, β1, and γ1; it is a major component of Matrigel, which can maintain hepatic differentiation. We previously showed that the A13 peptide (RQVFQVAYIIIKA, α1 chain 121-133) derived from mouse laminin α1 exhibits hepatocyte attachment activity and maintains hepatic differentiation. Here, we sought to identify hepatocyte adhesive sequences from the mouse laminin β1 and γ1 chains using 22 synthetic peptides that show biological activity for fibrosarcoma cells. Nine peptides showed hepatocyte attachment activity. Of these, B160 (VILQQSAADIAR, β1 chain 1607-1618), and C16 (KAFDITYVRLKF γ1 chain 139-150) exhibited the most potent activity. Hepatocytes cultured on both peptides also maintained expression of albumin, tyrosine aminotransferase, tryptophan-2,3-dioxygenase, and cytochrome P450. The morphology of hepatocytes on both peptides was a rounded shape typical for hepatic differentiation. We also characterized the nature of adhesion to the peptides. Heparin and EDTA inhibited cell attachment to both peptides, suggesting that hepatocyte attachment to the peptides was mediated by multiple receptors. The identification of active sequences regulating hepatic functions may facilitate the design of hepatocyte culture substrata that can regulate specific cellular behaviors in the context of a bioartificial liver.
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