The human alpha 2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of alpha 2-macroglobulin.

It is assumed that vitronectin and other adhesion molecules induce cell spreading. We found that vascular smooth muscle cells require unidentified plasma components besides adhesion molecules to spread in fibrin gel, a likely provisional matrix at wound sites. By purification, the plasma components were found to be ␣ 1 -proteinase inhibitor, ␣ 1 -antichymotrypsin, and ␣ 2 -macroglobulin. The chemically inactivated ␣ 1 -proteinase inhibitor and ␣ 2 -macroglobulin lose the spreading activity, indicating that these proteins function as proteinase inhibitors but not as adhesion molecules. Not only antiintegrin (␣ v ␤ 3 and ␣ 5 ␤ 1 ) antibodies but also anti-fibronectin antibodies inhibit the cell spreading. The spreading occurs without the addition of fibronectin and integrins, suggesting that cells produce these molecules. In the absence of the proteinase inhibitors, Western blot analysis shows that the fibronectin is degraded in fibrin gel, while it is intact in the presence of the inhibitors. Thus, the proteinase inhibitors prevent adhesion molecules such as fibronectin from being degraded by a cell-derived proteinase(s) and thus play a role in cell spreading.Under physiological states, especially in wound healing and vascular injury, cell spreading has been observed in threedimensional matrices. In most of the studies, however, spreading has been conducted on artificial two-dimensional surfaces rather than in three-dimensional matrices. Cell spreading relates to the cells' ability to migrate, proliferate (1), and resist apoptosis (2). While fibrin is known as a blood coagulant protein, it has also been implicated in several forms of tissue response to injuries including angiogenesis (3), vascular disease (4, 5), wound healing (6), and metastasis of cancer (7,8). During in vitro experiments, we unexpectedly found that human smooth muscle cells do not spread in fibrin gel unless serum or plasma was added. Vitronectin, which is identified as the serum-spreading factor (9, 10), surprisingly did not promote the spreading. We then tested other molecules such as fibronectin, osteopontin, thrombospondin, platelet-derived growth factor, and plasminogen activator inhibitor type-1 (PAI-1) 1 as possible spreading factors. However, none of these factors were able to promote the cell spreading. We then attempted to purify the cell spreading factor(s) from bovine plasma and found that these are serine proteinase inhibitors. MATERIALS AND METHODSCell Spreading in Fibrin Gel-Cultured human vascular smooth muscle cells (HNB18E6E7) were originally derived from the aorta of a newborn infant (2 days old) autopsy (11,12). The cells were detached by brief exposure to 0.05% trypsin (Life Technologies, Inc.), 0.02% EDTA. The remaining trypsin was inactivated by an excess of soybean trypsin inhibitor (Sigma). After washing with 20 mM phosphate, pH 7.4, containing 0.15 M NaCl, the cells were resuspended at 62,500 cells/49 l in serum-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 25 mM HEPES, pH 7.4. The cell ...

Section: Discussionmentioning

confidence: 99%

α1-Proteinase Inhibitor, α1-Antichymotrypsin, or α2-Macroglobulin Is Required for Vascular Smooth Muscle Cell Spreading in Three-dimensional Fibrin Gel

Ikari¹,

Fujikawa²,

Yee³

et al. 2000

“…Concentrations of u-PA and PAI-1 in conditioned medium were determined using enzyme-linked immunosorbent assays, purchased from American Diagnostica (Greenwich, CT). Polyclonal antibodies against LRP/␣ 2 MR, RAP (8,43,44), and the human very low density lipoprotein (VLDL) receptor have been described (45).…”

Section: Methodsmentioning

confidence: 99%

The Low Density Lipoprotein Receptor-related Protein/α2-Macroglobulin Receptor Regulates Cell Surface Plasminogen Activator Activity on Human Trophoblast Cells

Zhang¹,

Sakthivel²,

Kniss³

et al. 1998

The low density lipoprotein receptor-related protein/ ␣ 2 -macroglobulin receptor (LRP/␣ 2 MR) mediates the internalization of numerous ligands, including prourokinase (pro-UK) and complexes between two-chain urokinase (tc-u-PA) and plasminogen activator inhibitor type-1 (PAI-1). It has been suggested that through its ability to internalize these ligands, LRP/␣ 2 MR may regulate the expression of plasminogen activator activity on cell surfaces; this hypothesis, however, has not been experimentally confirmed. To address this issue, we assessed the ability of LRP/␣ 2 MR to regulate plasminogen activator activity on human trophoblast cells, which express both LRP/␣ 2 MR and the urokinase receptor (uPAR). Trophoblasts internalized and degraded exogenous 125 I-pro-UK (primarily following its conversion to tc-u-PA and incorporation into tc-u-PA⅐PAI complexes) in an LRP/␣ 2 MR-dependent manner, which was inhibited by the LRP/␣ 2 MR receptor-associated protein. Receptor-associated protein also caused a ϳ50% reduction in cell surface plasminogen activator activity and delayed the regeneration of unoccupied uPAR by cells on which uPAR were initially saturated with pro-UK. Identical effects were caused by anti-LRP/␣ 2 MR antibodies. These results demonstrate that LRP/␣ 2 MR promotes the expression of cell surface plasminogen activator activity on trophoblasts by facilitating the clearance of tc-u-PA⅐PAI complexes and regeneration of unoccupied cell surface uPAR.

“…Polyclonal rabbit antihuman LRP antibody Rb777 was produced as described previously (7). Human ␣ 2 M was isolated and activated by methylamine treatment as described previously (32). Monoclonal antibodies and ␣ 2 M were 125 I-labeled using IODO-GEN (Pierce) as described previously (18).…”

Section: Methodsmentioning

confidence: 99%

Lipoprotein Lipase Binds to Low Density Lipoprotein Receptors and Induces Receptor-mediated Catabolism of Very Low Density Lipoproteins

Medh

Bowen

Fry

et al. 1996

Lipoprotein lipase (LPL)1 catalyzes the hydrolysis of triglycerides in plasma chylomicrons and very low density lipoproteins (VLDL) (1-4). In humans, deficiency of either LPL or apolipoprotein (apo) CII, an essential cofactor for LPL's lipolytic activity, results in severe elevation in plasma chylomicrons and large VLDL (5). In addition to its lipolytic actions, LPL is also a ligand for receptors belonging to the family of low density lipoprotein (LDL) receptors, including LDL receptorrelated protein (LRP)/␣ 2 -macroglobulin (␣ 2 M) receptor, GP330/ LRP-2, and VLDL receptors (6 -13). LPL can influence lipoprotein catabolism by simultaneously binding to both lipoproteins and these cell surface receptors (6 -13). LPL promotes VLDL catabolism in LDL receptor-lacking mutant fibroblasts, and ligands specific for LRP inhibit LPL-stimulated VLDL degradation (9). LPL promotes lipoprotein binding to purified LRP (9) and GP330/LRP-2 (11) in solid-phase assays. Argraves et al. (12) demonstrated that transfection of PEA13 cells with VLDL receptors enhances their ability to internalize and degrade 125 I-LPL. Takahashi et al. (13) showed that in chinese hamster ovary cells co-transfected with the VLDL receptor and LPL, binding and degradation of 125 I-VLDL is much greater than in cells transfected with VLDL receptor alone. These studies demonstrate that LPL-dependent VLDL catabolism is mediated by LRP (6 -10), GP330/LRP-2 (10, 11), and VLDL receptors (12, 13). LPL also binds to cell surface heparan sulfate proteoglycans (3, 14 -16). LPL binding to either heparan sulfate proteoglycans or receptors can be separated from its lipolytic activity as indicated by studies with LPLC, the carboxyl-terminal, noncatalytic domain of LPL (8,17,18).