Localization of Lutheran, a novel laminin receptor, in normal, knockout, and transgenic mice suggests an interaction with laminin alpha5 in vivo.

Laminins are major components of all basement membranes. One laminin that has garnered particular interest, due to its widespread expression pattern and importance during development, is the laminin alpha5 chain. In vitro studies have suggested that the Lutheran blood group glycoprotein/basal cell adhesion molecule (Lu), an Ig superfamily transmembrane protein, is a receptor for laminins containing the alpha5 chain. However, there are no in vivo studies showing that these proteins are capable of interacting in tissues. We have isolated the mouse ortholog of Lu and characterized its expression and localization in mouse tissues. Lu was primarily found on the basal surface of epithelial cells and on muscle cells adjacent to basement membranes containing laminin alpha5. In addition, there was both a dramatic reduction in the basal concentration of Lu in mice lacking laminin alpha5, and a significant increase in Lu protein in transgenic mice overexpressing laminin alpha5. Together, these data provide the first in vivo evidence for an interaction between Lu and laminin alpha5 and support the hypothesis that Lu is a laminin alpha5 receptor. We propose that laminin alpha5 is involved in concentrating Lu on the basal surface of epithelial cells. This may be one mechanism by which basement membrane signals are transmitted to the cell.

Clusterin regulates vascular smooth muscle cell nodule formation and migration.

Vascular smooth muscle cells (VSMC) are the principal cellular component of the blood vessel wall where they exist in a differentiated state to maintain vascular tone. However, VSMC are not terminally differentiated and can be induced to dediffentiate, proliferate, and migrate. In fact, smooth muscle cell migration from the vascular wall into the lumen of the vessel is a central feature of occlusive vascular pathologies including atherosclerosis and intimal hyperplasia. In vitro, in the presence of an extracellular matrix, cultured vascular smooth muscle cells can migrate and invade the underlying gelatinous matrix, form multicellular nodular aggregations, and secrete the glycoprotein clusterin. Nodular cultures appear to mimic some of the properties of differentiated VSMC, in vivo. Here, to test the hypothesis that clusterin functions to modulate the formation of VSMC nodules and to facilitate cell migration a clusterin negative VSMC clone, SM-CLU13AS (Moulson and Millis, 1999, J Cell Physiol 180:355), was transiently transfected with plasmid pRcCMVCLU that contains the full-length porcine clusterin cDNA sequence under control of the CMV promoter. The transiently transfected VSMC culture expressed and secreted clusterin and formed nodules. To determine if clusterin regulates VSMC migration we used modified Boyden chamber assays. Clusterin, at 10 microg/ml, clearly promotes VSMC migration. In addition, a 15 amino acid synthetic peptide, representing amino acids 118-132 [KQTCMKFYARVCRSG] of the mature clusterin polypeptide, inhibits VSMC attachment to gelatinous substrate. Finally, clusterin appears to have a role in regulating endogenous clusterin expression in the clusterin negative clone. These results clearly establish that clusterin has functional role in VSMC nodule formation and support the conclusion that clusterin is a critical component of smooth muscle cell phenotypic modulation.

Clusterin (Apo J) regulates vascular smooth muscle cell differentiation in vitro.

Previously we reported a significant and substantial increase in the synthesis and secretion of clusterin in cultured porcine vascular smooth muscle cells (VSMC) during the time when the VSMC culture modulates from a proliferating monolayer morphology to a nodular cell culture morphology. That in vitro process appears to recapitulate some aspects of in vivo vascular remodeling in response to injury and is facilitated by the presence of a well-developed extracellular matrix. To directly test the hypothesis that clusterin regulates VSMC phenotypic modulation, cultured VSMC were stably transfected with an expression plasmid containing the full-length murine clusterin sequence in antisense orientation. Twenty-four clones were selected on the basis of neomycin resistance and characterized for clusterin expression and culture morphology. In contrast to clone SM-CLU18AS, which expresses a high level of clusterin and forms multicellular nodules, clone SM-CLU13AS expresses a low level of clusterin and does not form nodules even in the presence of a preformed collagen gel. Importantly, clusterin-negative SM-CLU13AS retains the ability to form nodules in an environment containing exogenous clusterin. SM-CLU13AS forms nodules when cultured in Matrigel (which contains clusterin) and in the presence of clusterin-containing conditioned media prepared from nodular SMC cultures or SM-CLU18AS cultures. These results demonstrate that clusterin is required for VSMC nodule formation and suggest that it may play a role in smooth muscle cell reorganization in the vascular wall.