Hypercholesterolemia is a major causative factor for atherosclerotic cardiovascular disease. The molecular mechanisms by which cholesterol initiates and facilitates the process of atherosclerosis are not well understood. Here, we demonstrate that cholesterol treatment suppresses or attenuates TGF-β responsiveness in all cell types studied as determined by measuring TGF-β-induced Smad2 phosphorylation and nuclear translocation, TGF-β-induced PAI-1 expression, TGF-β-induced luciferase reporter gene expression and TGF-β-induced growth inhibition. Cholesterol, alone or complexed in lipoproteins (LDL, VLDL), suppresses TGF-β responsiveness by increasing lipid raft and/or caveolae accumulation of TGF-β receptors and facilitating rapid degradation of TGF-β and thus suppressing TGF-β-induced signaling. Conversely, cholesterol-lowering agents (fluvastatin and lovastatin) and cholesterol-depleting agents (β-cyclodextrin and nystatin) enhance TGF-β responsiveness by increasing non-lipid raft microdomain accumulation of TGF-β receptors and facilitating TGF-β-induced signaling. Furthermore, the effects of cholesterol on the cultured cells are also found in the aortic endothelium of ApoE-null mice fed a high-cholesterol diet. These results suggest that high cholesterol contributes to atherogenesis, at least in part, by suppressing TGF-β responsiveness in vascular cells.

Clathrin-dependent endocytosis is believed to be involved in TGFβ-stimulated cellular responses, but the subcellular locus at which TGFβ induces signaling remains unclear. Here, we demonstrate that inhibitors of clathrin-dependent endocytosis, which are known to arrest the progression of endocytosis at coated-pit stages, inhibit internalization of cell-surface-bound TGFβ and promote colocalization and accumulation of TβR-I and SARA at the plasma membrane. These inhibitors enhance TGFβ-induced signaling and cellular responses (Smad2 phosphorylation/nuclear localization and expression of PAI-1). Dynasore, a newly identified inhibitor of dynamin GTPase activity, is one of the most potent inhibitors among those tested and, furthermore, is a potent enhancer of TGFβ. Dynasore ameliorates atherosclerosis in the aortic endothelium of hypercholesterolemic ApoE-null mice by counteracting the suppressed TGFβ responsiveness caused by the hypercholesterolemia, presumably acting through its effect on TGFβ endocytosis and signaling in vascular cells.

CRSBP-1, a membrane glycoprotein, can mediate cell-surface retention of secreted growth factors containing CRS motifs such as PDGF-BB. CRSBP-1 has recently been found to be identical to LYVE-1, a specific marker for lymphatic capillary endothelial cells. The in vivo role of CRSBP-1/LYVE-1 is unknown. CRSBP-1-null mice are overtly normal and fertile but exhibit identifiable morphological and functional alterations of lymphatic capillary vessels in certain tissues, marked by the constitutively increased interstitial-lymphatic flow and lack of typical irregularly-shaped lumens. The CRSBP-1 ligands PDGF-BB and HA enhance interstitial-lymphatic flow in wildtype mice but not in CRSBP-1-null animals.

The type V TGF-beta receptor (TbetaR-V) mediates IGF-independent growth inhibition by IGFBP-3 and mediates growth inhibition by TGF-beta1 in concert with the other TGF-beta receptor types. TbetaR-V was recently found to be identical to LRP-1. Here we find that insulin and (Q3A4Y15L16) IGF-I (an IGF-I analog that has a low affinity for IGFBP-3) antagonize growth inhibition by IGFBP-3 in mink lung epithelial cells (Mv1Lu cells) stimulated by serum. In these cells, IGFBP-3 induces serine-specific dephosphorylation of IRS-1 and IRS-2. The IGFBP-3-induced dephosphorylation of IRS-2 is prevented by cotreatment of cells with insulin, (Q3A4Y15L16) IGF-I, or TbetaR-V/LRP-1 antagonists. The magnitude of the IRS-2 dephosphorylation induced by IGFBP-3 positively correlates with the degree of growth inhibition by IGFBP-3 in Mv1Lu cells and mutant cells derived from Mv1Lu cells. Stable transfection of murine 32D myeloid cells (which lack endogenous IRS proteins and are insensitive to growth inhibition by IGFBP-3) with IRS-1 or IRS-2 cDNA confers sensitivity to growth inhibition by IGFBP-3; this IRS-mediated growth inhibition can be completely reversed by insulin in 32D cells stably expressing IRS-2 and the insulin receptor. These results suggest that IRS-1 and IRS-2 are key molecules for the TbetaR-V/LRP-1-mediated growth inhibitory signaling cascade.