Transforming growth factor (TGF) β1, β2, and β3 (TGF-β1–TGF-β3, respectively) are small secreted signaling proteins that each signal through the TGF-β type I and type II receptors (TβRI and TβRII, respectively). However, TGF-β2, which is well-known to bind TβRII several hundred-fold more weakly than TGF-β1 and TGF-β3, has an additional requirement for betaglycan, a membrane-anchored nonsignaling receptor. Betaglycan has two domains that bind TGF-β2 at independent sites, but how it binds TGF-β2 to potentiate TβRII binding and how the complex with TGF-β, TβRII, and betaglycan undergoes the transition to the signaling complex with TGF-β, TβRII, and TβRI are not understood. To investigate the mechanism, the binding of the TGF-βs to the betaglycan extracellular domain, as well as its two independent binding domains, either directly or in combination with the TβRI and TβRII ectodomains, was studied using surface plasmon resonance, isothermal titration calorimetry, and size-exclusion chromatography. These studies show that betaglycan binds TGF-β homodimers with a 1:1 stoichiometry in a manner that allows one molecule of TβRII to bind. These studies further show that betaglycan modestly potentiates the binding of TβRII and must be displaced to allow TβRI to bind. These findings suggest that betaglycan functions to bind and concentrate TGF-β2 on the cell surface and thus promote the binding of TβRII by both membrane-localization effects and allostery. These studies further suggest that the transition to the signaling complex is mediated by the recruitment of TβRI, which simultaneously displaces betaglycan and stabilizes the bound TβRII by direct receptor–receptor contact.
Edited by Norma AllewellThe transforming growth factor  isoforms, TGF-1, -2, and -3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF- pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-s in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF- monomer, lacking the heel helix, a structural motif essential for binding the TGF- type I receptor (TRI) but dispensable for binding the other receptor required for TGF- signaling, the TGF- type II receptor (TRII), as an alternative therapeutic modality for blocking TGF- signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF- monomers and bound TRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF- signaling with a K i of 20 -70 nM. Investigation of the mechanism showed that the high affinity of the engineered monomer for TRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TRI, enabled it to bind endogenous TRII but prevented it from binding and recruiting TRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF- signaling and may inform similar modifications of other TGF- family members.The transforming growth factor  isoforms, TGF-1, -2, and -3, are small secreted signaling proteins. Their overall structures are similar and consist of two cystine-knotted monomers tethered together by a single inter-chain disulfide bond (1). They coordinate wound healing, modulate immune cell function, maintain the extracellular matrix, and regulate epithelial and endothelial cell growth and differentiation (2). The TGF-s are synthesized as pre-pro-proteins, and after maturation, secretion, and release from their pro-domains (3), the mature homodimeric growth factors (GFs) 3 bind and bring together two single-pass transmembrane receptors, known as TRI and TRII, to form the signaling-competent TRI 2 -TRII 2 heterotetramer (4, 5). TGF- GFs assemble TRI 2 -TRII 2 heterotetramer in a sequential manner, first by binding TRII followed by recruitment of TRI (6, 7). The stepwise assembly of TRII and TRI into a heterotetramer is driven by binding of TRI to a composite TGF-/TRII interface (Fig. 1A) (8, 9).The disruption or dysregulation of the TGF- pathway is responsible for several human diseases. These include connec-
It has been recently demonstrated that progranulin is overexpressed in ovarian cancer and that this protein is involved in the stimulation of cell proliferation, malignancy, and chemoresistance in ovarian cancer. The goal of the present study was to establish the differences in progranulin expression among normal, benign, and malignant ovarian tissues and to identify the signal transduction pathways activated by progranulin in an ovarian cancer cell line. Compared with benign tumors and normal ovarian tissue, progranulin mRNA and protein were overexpressed in malignant tumors. Survival analysis by the Kaplan-Meier method showed a correlation between high mRNA expression levels with poor survival outcome. Progranulin activated the MAPK-signaling pathway in NIH-OVCAR-3 cells. Progranulin expression may be potentially involved in the pathogenesis and malignant progression of ovarian cancer, and thus may represent a therapeutic target for this particular malignancy.
The effects of transforming growth factor beta (TGF-β) signaling on prostate tumorigenesis has been shown to be strongly dependent on the stage of development, with TGF-β functioning as a tumor suppressor in early stages of disease and as a promoter in later stages. To study in further detail the paradoxical tumor-suppressive and tumor-promoting roles of the TGF-β pathway, we investigated the effect of systemic treatment with a TGF-β inhibitor on early stages of prostate tumorigenesis. To ensure effective inhibition, we developed and employed a novel trivalent TGF-β receptor trap, RER, comprised of domains derived from the TGF-β type II and type III receptors. This trap was shown to completely block TβRII binding, to antagonize TGF-β1 and TGF-β3 signaling in cultured epithelial cells at low picomolar concentrations, and it showed equal or better anti-TGF-β activities than a pan TGF-β neutralizing antibody and a TGF-β receptor I kinase inhibitor in various prostate cancer cell lines. Systemic administration of RER inhibited prostate tumor cell proliferation as indicated by reduced Ki67 positive cells and invasion potential of tumor cells in high grade prostatic intraepithelial neoplasia (PIN) lesions in the prostate glands of Pten conditional null mice. These results provide evidence that TGF-β acts as a promoter rather than a suppressor in the relatively early stages of this spontaneous prostate tumorigenesis model. Thus, inhibition of TGF-β signaling in early stages of prostate cancer may be a novel therapeutic strategy to inhibit the progression as well as the metastatic potential in patients with prostate cancer.
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