LDL binds to surface-expressed human T-cadherin in transfected HEK293 cells and influences homophilic adhesive interactions.

T-cadherin (T-cad) is an unusual glycosylphosphatidylinositol-anchored member of the cadherin family of cell adhesion molecules. Binding of low density lipoproteins (LDLs) to T-cad can be demonstrated on Western blots of smooth muscle cell lysates, membranes and purified proteins. Using HEK293 cells transfected with human T-cad cDNA (T-cad+), we have investigated the adhesion properties of expressed mature and precursor proteins and examined the postulate that LDL represents a physiologically relevant ligand for T-cad. T-cad+ exhibits an increased Ca(2+)-dependent aggregation (vs. control) that was reduced by selective proteolytic cleavage of precursor T-cad and abolished after either proteolytic or phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of both mature and precursor proteins, indicating that both proteins function in intercellular adhesion. T-cad+ exhibited a significantly increased specific cell surface-binding of [(125)I]-LDL that was sensitive to PI-PLC pre-treatment of cells. Ca(2+)-dependent intercellular adhesion of T-cad+ was significantly inhibited by LDL. Our results support the suggestion that LDL is a physiologically relevant ligand for T-cad.

Identification of 130 kDa cell surface LDL-binding protein from smooth muscle cells as a partially processed T-cadherin precursor.

Atypical cell surface lipoprotein-binding proteins of 105 kDa and 130 kDa are present in membranes of vascular smooth muscle cells. We recently identified the 105 kDa protein from human aortic media as T-cadherin, an unusual glycosylphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. The goal of the present study was to determine the identity of 130 kDa lipoprotein-binding protein of smooth muscle cells. We applied different approaches that included protein sequencing of purified protein from human aortic media, the use of human T-cadherin peptide-specific antisera, and enzymatic treatment of cultured cells with trypsin and GPI-specific phospholipase C. Our results indicate that the 130 kDa protein is a partially processed form of T-cadherin which is attached to the membrane surface of smooth muscle cells via a GPI anchor and contains uncleaved N-terminal propeptide sequence. Our data disclose that, in contrast to classical cadherins, T-cadherin is expressed on the cell surface in both its precursor (130 kDa) and mature (105 kDa) forms.

Density- and proliferation status-dependent expression of T-cadherin, a novel lipoprotein-binding glycoprotein: a function in negative regulation of smooth muscle cell growth?

The atypical low density lipoprotein (LDL) binding proteins (Mr 105 and 130 kDa; p105 and p130) in human aortic medial membranes and cultured human and rat aortic smooth muscle cells (SMC) have recently been identified as the cell adhesion glycoprotein T-cadherin. Although cadherins are generally recognized to be important regulators of morphogenesis, the function of T-cadherin in the vasculature is poorly understood. This study has examined the relationship between expression of T-cadherin and the density and proliferation status of SMC. T-cadherin (p105 and p130) levels in SMC lysates were measured on Western blots using ligand-binding techniques. T-cadherin expression was dependent upon cell density, and maximal levels were achieved at confluency. T-cadherin levels were reversibly modulated by switching cultures between serum-free (upmodulation) and serum-containing (downmodulation) conditions. Platelet-derived growth factor (PDGF)-BB, epidermal growth factor (EGF) or insulin-like growth factor (IGF) elicited a dose- and time-dependent downmodulation that was reversible after transfer of SMC to growth factor-free medium. Our results support the hypothesis that T-cadherin may function as a negative determinant of cell growth.

Characteristics of smooth muscle cell lipoprotein binding proteins (p105/p130) as T-cadherin and regulation by positive and negative growth regulators.

Smooth muscle cells (SMC) express atypical surface low density lipoprotein (LDL) binding proteins of M(r)105 and M(r)130 (p105 and p130) which have been putatively identified as the cell adhesion glycoprotein T-cadherin. Using cultured human and rat aortic SMC and analysis by ligand (LDL)- and immuno-blotting techniques we now confirm identity of p105 and p130 as T-cadherin, as adjudged by sensitivity to PI-PLC cleavage, insensitivity to trypsin degradation in the presence of calcium, and immunoreactivity to anti-T-cadherin peptide antisera. The function of T-cadherin (p105/p130) in the vasculature is unknown. The proteins were downmodulated by the peptide growth factors PDGF-BB, IGF, EGF, and bFGF, but not by vasoactive peptide hormones (angiotensin II, vasopressin, bradykinin, and endothelin). TGF beta, a recognized inhibitor of SMC proliferation, per se had no effect but inhibited growth factor-induced p105/p130 downmodulation. Expression of p105/p130 in quiescent SMC and growth-stimulated SMC (respectively, in serum-free and serum or PDGF-BB containing culture conditions) was increased by forskolin and 8-Br-cyclic GMP, both anti-mitogenic substances, but was unaffected by phorbol ester, calcium ionophores, or calcium antagonists. The findings are compatible with a function for the lipoprotein binding proteins (T-cadherin) in negative regulation of SMC growth.

Atypical low density lipoprotein binding site that may mediate lipoprotein-induced signal transduction.

The characteristics of low density lipoprotein (LDL) binding in quiescent cultures of human vascular smooth muscle cells (VSMC) have been further investigated and compared with the characteristics of high affinity LDL binding in human fibroblasts [via the apolipoprotein (apo) B/E receptor] and with the properties of LDL-induced phosphoinositide catabolism in VSMC. In VSMC the bulk of specific 125I-LDL binding occurs at a low affinity site, several characteristics of which are distinct from those of 125I-LDL binding to the apo B/E receptor in fibroblasts. (a) The affinity of LDL binding in VSMC is 25-50 times lower than that in fibroblasts (Kd approximately 50 micrograms/ml versus Kd approximately 2 micrograms/ml). (b) The kinetics of LDL association and dissociation in VSMC are more rapid than those in fibroblasts. (c) In contrast to apo B/E receptor-mediated binding of LDL in fibroblasts, binding of LDL to VSMC is insensitive to heparin, chemical modification of lysine residues, and chelation (with EDTA) of divalent cations. (d) Apo E-free high density lipoprotein 3 displaces labeled LDL more effectively in VSMC than in fibroblasts. (e) The ratio of bound/internalized LDL to degraded LDL differs markedly between fibroblasts and VSMC. LDL-stimulated phosphoinositide catabolism in VSMC, which occurs with an activation constant similar to the Kd for low affinity LDL binding, is insensitive to heparin, modification of lysine and arginine residues in LDL, and chelation of divalent cations. Thus, the atypical low affinity receptor in these cells may mediate the effects of LDL on signal transduction.

Characterization of an atypical lipoprotein-binding protein in human aortic media membranes by ligand blotting.

By use of ligand-blotting techniques, this study investigated lipoprotein-binding proteins in human aortic smooth muscle. PAGE was performed under non-reducing conditions, and, using low-density lipoprotein (LDL) as ligand, with rabbit anti-apolipoprotein (apo) B and 125I-labelled goat anti-rabbit IgG as primary and secondary antibodies respectively, we demonstrate that membranes from human aortic media (and cultured human smooth-muscle cells) contain a major lipoprotein-binding protein with an apparent molecular mass of 105 kDa. Anionized preparations (carbamoyl- and acetyl-) of LDL, which did not displace 125I-LDL bound to the apo B,E receptor of cultured fibroblasts, were also recognized as ligands for the 105 kDa protein in aortic media membranes. LDL binding to 105 kDa protein was decreased in the presence of high density lipoprotein (HDL), although more than 100-fold molar excess of HDL was required to achieve 50% displacement of bound LDL. The LDL-binding activity of 105 kDa protein was inhibited by EDTA, and was also significantly decreased when samples were reduced by beta-mercaptoethanol before electrophoresis. Monoclonal antibodies against apo B,E receptor reacted with partially purified bovine adrenal apo B,E receptor, but not with 105 kDa protein of human aortic media membranes. The spectrum of properties of this vascular smooth-muscle lipoprotein-binding protein binding are clearly distinct from those of other previously characterized lipoprotein-binding molecules.

Characteristics of low and high density lipoprotein binding and lipoprotein-induced signaling in quiescent human vascular smooth muscle cells.

Low density lipoprotein (LDL) and high density lipoprotein (HDL) have been shown to stimulate signal transduction events in a number of cell types, including cultured vascular smooth muscle cells (VSMC), but it is not known whether these events are mediated through distinct lipoprotein receptors for transmembrane signaling. This study has used confluent quiescent cultures of human microarteriolar VSMC to investigate the relationship between the characteristics of 125I-LDL and 125I-HDL3 binding and those of LDL- and HDL3-stimulated cell signaling. Two distinct binding sites for LDL (Kd1 approximately 2 micrograms/ml and Kd2 approximately 40 micrograms/ml) and a single class of sites for HDL3 (Kd approximately 30 micrograms/ml) were identified. The Kd1 for high affinity 125I-LDL binding in quiescent VSMC was comparable to the value for heparin-sensitive binding of 125I-LDL to apolipoprotein B/E receptors in fibroblasts (Kd approximately 1 microgram/ml). Concentrations of lipoproteins required for half-maximal stimulation (EC50) of phosphoinositide catabolism and intracellular calcium mobilization in VSMC were approximately 35 micrograms/ml for HDL3 and approximately 40 micrograms/ml for LDL. Both LDL- and HDL3-stimulated signaling responses in VSMC, as well as 125I-HDL3 binding and low affinity 125I-LDL binding to VSMC, were insensitive to heparin. Competition binding studies (with unlabeled lipoproteins at 2.5-200 micrograms/ml) showed partial displacement of 125I-LDL by HDL3 and of 125I-HDL3 by LDL, whereas complete displacement of 125I-LDL or 125I-HDL3 by their homologous lipoproteins was achieved. Thus, the binding sites for HDL3 are distinct from those for LDL. Because the response of VSMC to combinations of LDL and HDL3 was additive, LDL and HDL3 also exert their signaling effects through distinct sites. Further investigation is required to unequivocally demonstrate that the heparin-insensitive HDL3 and low affinity LDL binding sites in VSMC are those through which LDL and HDL3 stimulate transmembrane signaling.