Betaglycan Inhibits TGF-β Signaling by Preventing Type I-Type II Receptor Complex Formation

Abstract

Transforming growth factor (TGF)-␤ is a multifunctional growth factor with important roles in development, cell proliferation, and matrix deposition. It signals through the sequential activation of two serine/ threonine kinase receptors, the type I and type II receptors. A third cell surface receptor, betaglycan, serves as a co-receptor for TGF-␤ in some cell types, enhancing TGF-␤-mediated signaling. We have examined the function of betaglycan in renal epithelial LLC-PK 1 cells that lack endogenous betaglycan. We demonstrate that the expression of betaglycan in LLC-PK 1 cells results in inhibition of TGF-␤ signaling as measured by reporter gene expression, thymidine incorporation, collagen production, and phosphorylation of the downstream signaling effectors Smad2 and Smad3. In comparison, the expression of betaglycan in L6 myoblasts enhances TGF-␤ signaling, which is consistent with the published literature. The effects of betaglycan in LLC-PK 1 cells are not mediated by ligand sequestration or increased production of a soluble form of the receptor, which has been reported to serve as a ligand antagonist. We demonstrate instead that in LLC-PK 1 cells, unlike L6 cells, expression of betaglycan prevents association between the type I and type II TGF-␤ receptors, which is required for signaling. This is a function of the glycosaminoglycan modifications of betaglycan. Betaglycan in LLC-PK 1 cells exhibits higher molecular weight glycosaminoglycan (GAG) chains than in L6 cells, and a GAG ؊ betaglycan mutant does not inhibit TGF-␤ signaling or type I/type II receptor association in LLC-PK 1 cells. Our data indicate that betaglycan can function as a potent inhibitor of TGF-␤ signaling by a novel mechanism and provide support for an essential but complex role for proteoglycan co-receptors in growth factor signaling. TGF-␤1 is an essential regulator of development, cell proliferation, and matrix deposition (1-3). Biological effects of TGF-␤ are achieved through the sequential activation of two high affinity serine/threonine kinase receptors, the type II (T␤RII) and the type I (T␤RI) TGF-␤ receptors. TGF-␤ signaling is initiated by the binding of TGF-␤ to T␤RII followed by the formation or stabilization of T␤RI-T␤RII complexes, transphosphorylation of T␤RI by T␤RII, and phosphorylation of the receptor-associated cytoplasmic effector molecules Smad2 and Smad3 by T␤RI (1, 3). A third TGF-␤ transmembrane receptor, the type III receptor or betaglycan, is a widely expressed heparan and chondroitin sulfate proteoglycan that binds TGF-␤ with high affinity through its protein core but has no apparent intrinsic signaling activity (4 -7).Betaglycan is believed to be a co-receptor for TGF-␤. In several cell lines, including rat L6 myoblasts lacking endogenous betaglycan, it significantly increases the affinity of T␤RI and T␤RII for TGF-␤, thereby enhancing the response to TGF-␤ (4, 5). In addition, betaglycan eliminates differences in efficacy among the three TGF-␤ isoforms. TGF-␤2, which binds to T␤RI and T␤RII with low affinity ...

Regulation of TGF-β ligand and receptor expression in neonatal rat lungs exposed to chronic hypoxia

“…Equal protein loading was assessed with an antibody against constitutive HSC70. The 110-kDa band of T␤RIII represents the core protein, whereas the Ͼ200-kDa band represents the mature protein, which has been subjected to posttranslational modifications, including glycosylation (9,30). Of note, densitometry measurements for T␤RIII reflect integration of the Ͼ200-and 110-kDa bands, because both are specific for T␤RIII.…”

“…This, in combination with the increased levels of bioactive TGF-␤, would lead to an overall upregulation of the signal transducing components of this system. In addition, although there is significant controversy surrounding the exact role of T␤RIII (or betaglycan), there is evidence that, in certain cell types, especially epithelial cells, this receptor may function as an inhibitor of TGF-␤ signaling (9). In this respect, it has become increasingly clear that the cell surface expression of T␤Rs, especially T␤RI and T␤RII, is able to regulate not only the cellular response to TGF-␤, but also the activation of specific downstream signaling molecules (6)(7)(8).…”

Signaling Receptors for TGF-β Family Members

“…The extracellular domain of betaglycan has two lobular subdomains separated by a linker domain; each lobular subdomain separately contributes to ligand binding and together forms a high-affinity ligand-binding site (Mendoza et al 2009). The polysaccharide chains are not necessary for TGF-b binding; in contrast, large polysaccharide chains may perturb TbRI -TbRII interaction and thus inhibit TGF-b signaling (Eickelberg et al 2002). Betaglycan has been shown to promote both Smad and non-Smad signaling (You et al 2007).…”

The Shedding of Betaglycan Is Regulated by Pervanadate and Mediated by Membrane Type Matrix Metalloprotease-1

“…Although the glycosaminoglycan chains are not required for all of these functions (10,17), they may modulate them. It has been reported that in some cell lines the nature of the glycosaminoglycan attached to membrane betaglycan core protein may sterically prevent its association with the TGF-␤ type II receptor, turning it into an inhibitor of TGF-␤ (18). Betaglycan is also required for the epithelial-mesenchymal transition of cardiac endothelial cells which leads to heart valve formation.…”