ALK1 regulates the internalization of endoglin and the type III TGF-ß receptor.

Complex formation and endocytosis of transforming growth factor-ß (TGF-ß) receptors play important roles in signaling. However, their interdependence remained unexplored. Here, we demonstrate that ALK1, a TGF-ß type I receptor prevalent in endothelial cells, forms stable complexes at the cell surface with endoglin and with type III TGF-ß receptors (TßRIII). We show that ALK1 undergoes clathrin-mediated endocytosis (CME) faster than ALK5, type II TGF-ß receptor (TßRII), endoglin, or TßRIII. These complexes regulate the endocytosis of the TGF-ß receptors, with a major effect mediated by ALK1. Thus, ALK1 enhances the endocytosis of TßRIII and endoglin, while ALK5 and TßRII mildly enhance endoglin, but not TßRIII, internalization. Conversely, the slowly endocytosed endoglin has no effect on the endocytosis of either ALK1, ALK5, or TßRII, while TßRIII has a differential effect, slowing the internalization of ALK5 and TßRII, but not ALK1. Such effects may be relevant to signaling, as BMP9-mediated Smad1/5/8 phosphorylation is inhibited by CME blockade in endothelial cells. We propose a model that links TGF-ß receptor oligomerization and endocytosis, based on which endocytosis signals are exposed/functional in specific receptor complexes. This has broad implications for signaling, implying that complex formation among various receptors regulates their surface levels and signaling intensities.

Endoglin interacts with VEGFR2 to promote angiogenesis.

Endoglin, a TGF-ß coreceptor predominantly expressed in endothelial cells, plays an important role in vascular development and tumor-associated angiogenesis. However, the mechanism by which endoglin regulates angiogenesis, especially during tip cell formation, remains largely unknown. In this study, we report that endoglin promoted VEGF-induced tip cell formation. Mechanistically, endoglin interacted with VEGF receptor (VEGFR)-2 in a VEGF-dependent manner, which sustained VEGFR2 on the cell surface and prevented its degradation. Endoglin mutants deficient in the ability to interact with VEGFR2 failed to sustain VEGFR2 on the cell surface and to promote VEGF-induced tip cell formation. Further, an endoglin-targeting monoclonal antibody (mAb), TRC105, cooperated with a VEGF-A targeting mAb, bevacizumab, to inhibit VEGF signaling and tip cell formation in vitro and to inhibit tumor growth, metastasis, and tumor-associated angiogenesis in a murine tumor model. This study demonstrate a novel mechanism by which endoglin initiates and regulates VEGF-driven angiogenesis while providing a rationale for combining anti-VEGF and anti-endoglin therapy in patients with cancer.-Tian, H., Huang, J. J., Golzio, C., Gao, X., Hector-Greene, M., Katsanis, N., Blobe, G. C. Endoglin interacts with VEGFR2 to promote angiogenesis.

TßRIII independently binds type I and type II TGF-ß receptors to inhibit TGF-ß signaling.

Transforming growth factor-ß (TGF-ß) receptor oligomerization has important roles in signaling. Complex formation among type I and type II (TßRI and TßRII) TGF-ß receptors is well characterized and is essential for signal transduction. However, studies on their interactions with the type III TGF-ß coreceptor (TßRIII) in live cells and their effects on TGF-ß signaling are lacking. Here we investigated the homomeric and heteromeric interactions of TßRIII with TßRI and TßRII in live cells by combining IgG-mediated patching/immobilization of a given TGF-ß receptor with fluorescence recovery after photobleaching studies on the lateral diffusion of a coexpressed receptor. Our studies demonstrate that TßRIII homo-oligomerization is indirect and depends on its cytoplasmic domain interactions with scaffold proteins (mainly GIPC). We show that TßRII and TßRI bind independently to TßRIII, whereas TßRIII augments TßRI/TßRII association, suggesting that TßRI and TßRII bind to TßRIII simultaneously but not as a complex. TßRIII expression inhibited TGF-ß-mediated Smad2/3 signaling in MDA-MB-231 cell lines, an effect that depended on the TßRIII cytoplasmic domain and did not require TßRIII ectodomain shedding. We propose that independent binding of TßRI and TßRII to TßRIII competes with TßRI/TßRII signaling complex formation, thus inhibiting TGF-ß-mediated Smad signaling.

Regulation of TGF-ß receptor hetero-oligomerization and signaling by endoglin.

Complex formation among transforming growth factor-ß (TGF-ß) receptors and its modulation by coreceptors represent an important level of regulation for TGF-ß signaling. Oligomerization of ALK5 and the type II TGF-ß receptor (TßRII) has been thoroughly investigated, both in vitro and in intact cells. However, such studies, especially in live cells, are missing for the endothelial cell coreceptor endoglin and for the ALK1 type I receptor, which enables endothelial cells to respond to TGF-ß by activation of both Smad2/3 and Smad1/5/8. Here we combined immunoglobulin G-mediated immobilization of one cell-surface receptor with lateral mobility studies of a coexpressed receptor by fluorescence recovery after photobleaching (FRAP) to demonstrate that endoglin forms stable homodimers that function as a scaffold for binding TßRII, ALK5, and ALK1. ALK1 and ALK5 bind to endoglin with differential dependence on TßRII, which plays a major role in recruiting ALK5 to the complex. Signaling data indicate a role for the quaternary receptor complex in regulating the balance between TGF-ß signaling to Smad1/5/8 and to Smad2/3.

Type III TGF-ß receptor downregulation generates an immunotolerant tumor microenvironment.

Cancers subvert the host immune system to facilitate disease progression. These evolved immunosuppressive mechanisms are also implicated in circumventing immunotherapeutic strategies. Emerging data indicate that local tumor-associated DC populations exhibit tolerogenic features by promoting Treg development; however, the mechanisms by which tumors manipulate DC and Treg function in the tumor microenvironment remain unclear. Type III TGF-ß receptor (TGFBR3) and its shed extracellular domain (sTGFBR3) regulate TGF-ß signaling and maintain epithelial homeostasis, with loss of TGFBR3 expression promoting progression early in breast cancer development. Using murine models of breast cancer and melanoma, we elucidated a tumor immunoevasion mechanism whereby loss of tumor-expressed TGFBR3/sTGFBR3 enhanced TGF-ß signaling within locoregional DC populations and upregulated both the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in plasmacytoid DCs and the CCL22 chemokine in myeloid DCs. Alterations in these DC populations mediated Treg infiltration and the suppression of antitumor immunity. Our findings provide mechanistic support for using TGF-ß inhibitors to enhance the efficacy of tumor immunotherapy, indicate that sTGFBR3 levels could serve as a predictive immunotherapy biomarker, and expand the mechanisms by which TGFBR3 suppresses cancer progression to include effects on the tumor immune microenvironment.