LDL induces intracellular signalling and cell migration via atypical LDL-binding protein T-cadherin

Abstract

Cadherins are a superfamily of adhesion molecules that mediate Ca(2+)-dependent cell-cell adhesion. T-cadherin (T-cad), a unique glycosylphosphatidylinositol-anchored member of the cadherin superfamily, was initially identified by immunoblotting of vascular cell membranes as an atypical low affinity low density lipoprotein (LDL)-binding protein. It is not known whether this heterophilic interaction is physiologically relevant. Expression of T-cadherin is upregulated in vascular cells during atherosclerosis, restenosis and tumour angiogenesis, conditions characterized by enhanced cell migration and growth. Elevated levels of serum low density lipoproteins (LDL), which result in cholesterol accumulation in vascular wall, is a widely accepted risk factor in atherosclerosis development. Additionally to its metabolic effects, LDL can produce hormone-like effects in a number of cell types. This study has utilized HEK293 cells and L929 cells stably transfected with T-cadherin cDNA to investigate T-cad-dependent responses to LDL. Stable expression of T-cad in both HEK293 and L929 cells results in significantly (p < 0.05) elevated specific surface binding of [I125]-LDL. Compared with mock-transfectants, cells expressing T-cad exhibit significantly (p < 0.01) enhanced LDL-induced mobilization of intracellular Ca(2+)-stores and a significantly (p < 0.01) increased migration toward an LDL gradient (0.1% BSA + 60 microg/ml LDL) in Boyden chamber migration assay. Thus LDL-binding to T-cad is capable of activating physiologically relevant intracellular signaling and functional responses.

Fig. 1

Overexpression of human T-cadherin in HEK 293 and L929 cell lines and constitutive expression of T-cadherin in HUVEC. Whole cell lysates (50 μg/lane) from T-cad-transfected L929 cells (clone TC3-lane 3) and HEK293 cells (clone T5-lane 5, clone T8-lane 6) and mock transfected cells HEK293 (clone HEK/GFP-lane 4) and L929 (clone K9-lane 2) were analyzed using immunoblot method. Lysates of HUVEC, which contain both 105 kDa and 130 kDa forms of T-cadherin (lane 1) served as a positive control.

Fig. 2

LDL binding capacity is increased in T-cad expressing HEK293 and L929 cell lines. Panel A: HEK293 cells. Panel B: L929 cells. Specific surface-binding of [¹²⁵I]-LDL to T-cad expressing cells (closed symbols) and mock-transfected cells (open symbols) was determined as described in ‘Materials and Methods’. Non-linear regression analysis was applied to calculate the binding parameters B_max and K_d. Data (means ± S.E.M.) were obtained from six independent experiments. * indicates significant difference (p < 0.05) between T-cad+ cells and mock cells.

Fig. 3

T-cad-expressing HEK293 exhibit increased calcium responsiveness to LDL. Alterations in [Ca²⁺]_in concentration in response to the addition of 60 μg/ml LDL was monitored in Fura-2 loaded HEK293 cells as described in ‘Materials and Methods’. Individual kinetic profiles and mean amplitude (mean ± S.E.M.) of the Ca²⁺ response to LDL in mock cells (n = 21) and T-cad+ cells (n = 28) are shown. * indicates significant difference (p < 0.01) between T-cad+ and mock cells.

Fig. 4

T-cad-expressing L929 exhibit increased calcium responsiveness to LDL. Alterations in [Ca²⁺]_in concentration in response to the addition of 60 μg/ml LDL was monitored in Fura-2 loaded L929 cells as described in ‘Materials and Methods’. Individual kinetic profiles and mean amplitude (mean ± S.E.M.) of the Ca²⁺ response to LDL in mock cells (n = 22) and T-cad+ cells (n = 22) are shown. * indicates significant difference (p < 0.01) between T-cad+ and mock cells.

Fig. 5

Intracellular calcium release response to LDL is absent after depletion of intracellular calcium stores. Preincubation of T-cad+ and mock-transfected L929 cells with 20 μM CPA in the Ca²⁺-containing medium for 5 min resulted in the slow and transient Ca²⁺ rise. Within 5 min the [Ca²⁺]_in concentration gradually decreased. LDL (60 μg/ml) added in that conditions had no effect on [Ca²⁺]_in and there was no significant difference in Ca²⁺-response of T-cad+ cell compared to mock cells. Data (mean ± S.E.M.) were obtained from three independent experiments.

Fig. 6

T-cadherin overexpression stimulates cell migration in response to LDL. The Boyden chamber assay was used to determine migration of cells in control conditions (DMEM/0.1% BSA), and in DMEM/0.1% BSA containing LDL (LDL), DMEM/0. 1% BSA containing LDL pre-incubated with Fab fragments of goat antibody against LDL (LDL+Fab), or DMEM/0.1% BSA with Fab fragments (Fab) as described in Methods. Data (mean ± S.E.M.) were obtained from three independent experiments. * indicates significant migration difference (p < 0.001) between control and LDL-containing conditions. ζ indicates significant difference in LDL-stimulated migration between T-cad+ cells and mock cells (p < 0.01).