Albumin endocytosis in endothelial cells induces TGF-β receptor II signaling

Abstract

Vascular endothelial cells undergo albumin endocytosis using a set of albumin binding proteins. This process is important for maintaining cellular homeostasis. We showed by several criteria that the previously described 73-kDa endothelial cell surface albumin binding protein is the 75-kDa transforming growth factor (TGF)-beta receptor type II (TbetaRII). Albumin coimmunoprecipitated with TbetaRII from a membrane fraction from rat lung microvascular endothelial cells. Albumin endocytosis-negative COS-7 cells became albumin endocytosis competent when transfected with wild-type TbetaRII but not when transfected with a domain-negative kinase mutant of TbetaRII. An antibody specific for TbetaRII inhibited albumin endocytosis. A mink lung epithelial cell line, which expresses both the TGF-beta receptor type I (TbetaRI) and the TbetaRII receptor, exhibited albumin binding to the cell surface and endocytosis. In contrast, mutant L-17 and DR-26 cells lacking TbetaRI or TbetaRII, respectively, each showed a dramatic reduction in binding and endocytosis. Albumin endocytosis induced Smad2 phosphorylation and Smad4 translocation as well as increased protein expression of the inhibitory Smad, Smad7. We identified regions of significant homology between amino acid sequences of albumin and TGF-beta, suggesting a structural basis for the interaction of albumin with the TGF-beta receptors and subsequent activation of TbetaRII signaling. The observed albumin-induced internalization of TbetaRII signaling may be an important mechanism in the vessel wall for controlling TGF-beta responses in endothelial cells.

p38 MAPK activation coupled to endocytosis is a determinant of endothelial monolayer integrity

“…TGF-␤ signaling is triggered by its binding to a heteromeric complex consisting of two transmembrane serine/threonine kinase receptors, type I and type II (T␤RI and T␤RII) (8,16,26). In serum-deprived RLMVEC, we observed that supplementation with albumin induced activation of caveolaemediated endocytosis (11) and subsequent T␤RII signaling that resulted in Smad2 phosphorylation and Smad4 translocation (21,24). These studies did not examine the downstream consequences of activation of endocytosis in promoting endothelial cell proliferation and survival.…”

“…RLMVEC were fixed with 4% paraformaldehyde and stained with mouse anti-caveolin-1 monoclonal antibody and polyclonal rabbit anti-p38-P MAPK antibody as described previously (24). Cells were washed with PBSTriton (0.1%) and labeled with Alexa 488 or Alexa 594 secondary antibodies (1:200 in PBS) for 1 h at room temperature, washed, and mounted with Prolong antifade mounting medium (Molecular Probes).…”

“…We have shown (24) that albumin activation of transforming growth factor (TGF)-␤ receptor (T␤R)II stimulates endocytosis in rat lung microvessel endothelial cell (RLMVEC) monolayers. TGF-␤ signaling is triggered by its binding to a heteromeric complex consisting of two transmembrane serine/threonine kinase receptors, type I and type II (T␤RI and T␤RII) (8,16,26).…”

“…Fluorescence measurements in live cells (RLMVEC) were performed as described previously (24) with a Nikon Eclipse 800 fluorescence microscope equipped with differential interference contrast (DIC). Fluorescence and DIC images were acquired with a charge-coupled device camera (Hamamatsu Photonics, Hamamatsu, Japan) and processed with Metamorph imaging analysis software (Universal Imaging, West Chester PA).…”

“…Endocytosis was assayed by the uptake of albumin with FITC-BSA or TRITC-BSA as the tracer (24). In RLM-VEC treated with the MAPK p38 inhibitor SB-203580 (6,7,25) (Calbiochem, San Diego, CA), the drug (1-20 M) was incubated in increasing concentrations in confluent cell monolayers for 4 h at 37°C.…”