skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling

Abstract

The transforming growth factor β isoforms, TGF-β1, -β2, and -β3, 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-β pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-βs in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-β monomer, lacking the heel helix, a structural motif essential for binding the TGF-β type I receptor (TβRI) but dispensable for binding the other receptor required for TGF-β signaling, the TGF-β type II receptor (TβRII), as an alternative therapeutic modality for blocking TGF-β signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-β monomers and bound TβRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-β signaling with a Ki of 20–70 nM. Investigation of the mechanism showed that the high affinity of the engineered monomer for TβRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TβRI,more » enabled it to bind endogenous TβRII but prevented it from binding and recruiting TβRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-β signaling and may inform similar modifications of other TGF-β family members.« less

Authors:
; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »; « less
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
OTHER U.S. STATESUNIVERSITYNCINIHAHA
OSTI Identifier:
1355043
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; Journal Volume: 292; Journal Issue: 17
Country of Publication:
United States
Language:
ENGLISH
Subject:
60 APPLIED LIFE SCIENCES

Citation Formats

Kim, Sun Kyung, Barron, Lindsey, Hinck, Cynthia S., Petrunak, Elyse M., Cano, Kristin E., Thangirala, Avinash, Iskra, Brian, Brothers, Molly, Vonberg, Machell, Leal, Belinda, Richter, Blair, Kodali, Ravindra, Taylor, Alexander B., Du, Shoucheng, Barnes, Christopher O., Sulea, Traian, Calero, Guillermo, Hart, P. John, Hart, Matthew J., Demeler, Borries, and Hinck, Andrew P.. An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling. United States: N. p., 2017. Web. doi:10.1074/jbc.M116.768754.
Kim, Sun Kyung, Barron, Lindsey, Hinck, Cynthia S., Petrunak, Elyse M., Cano, Kristin E., Thangirala, Avinash, Iskra, Brian, Brothers, Molly, Vonberg, Machell, Leal, Belinda, Richter, Blair, Kodali, Ravindra, Taylor, Alexander B., Du, Shoucheng, Barnes, Christopher O., Sulea, Traian, Calero, Guillermo, Hart, P. John, Hart, Matthew J., Demeler, Borries, & Hinck, Andrew P.. An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling. United States. doi:10.1074/jbc.M116.768754.
Kim, Sun Kyung, Barron, Lindsey, Hinck, Cynthia S., Petrunak, Elyse M., Cano, Kristin E., Thangirala, Avinash, Iskra, Brian, Brothers, Molly, Vonberg, Machell, Leal, Belinda, Richter, Blair, Kodali, Ravindra, Taylor, Alexander B., Du, Shoucheng, Barnes, Christopher O., Sulea, Traian, Calero, Guillermo, Hart, P. John, Hart, Matthew J., Demeler, Borries, and Hinck, Andrew P.. Wed . "An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling". United States. doi:10.1074/jbc.M116.768754.
@article{osti_1355043,
title = {An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling},
author = {Kim, Sun Kyung and Barron, Lindsey and Hinck, Cynthia S. and Petrunak, Elyse M. and Cano, Kristin E. and Thangirala, Avinash and Iskra, Brian and Brothers, Molly and Vonberg, Machell and Leal, Belinda and Richter, Blair and Kodali, Ravindra and Taylor, Alexander B. and Du, Shoucheng and Barnes, Christopher O. and Sulea, Traian and Calero, Guillermo and Hart, P. John and Hart, Matthew J. and Demeler, Borries and Hinck, Andrew P.},
abstractNote = {The transforming growth factor β isoforms, TGF-β1, -β2, and -β3, 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-β pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-βs in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-β monomer, lacking the heel helix, a structural motif essential for binding the TGF-β type I receptor (TβRI) but dispensable for binding the other receptor required for TGF-β signaling, the TGF-β type II receptor (TβRII), as an alternative therapeutic modality for blocking TGF-β signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-β monomers and bound TβRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-β signaling with a Ki of 20–70 nM. Investigation of the mechanism showed that the high affinity of the engineered monomer for TβRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TβRI, enabled it to bind endogenous TβRII but prevented it from binding and recruiting TβRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-β signaling and may inform similar modifications of other TGF-β family members.},
doi = {10.1074/jbc.M116.768754},
journal = {Journal of Biological Chemistry},
number = 17,
volume = 292,
place = {United States},
year = {Wed Feb 22 00:00:00 EST 2017},
month = {Wed Feb 22 00:00:00 EST 2017}
}
  • Purpose: To examine a relationship between serum transforming growth factor β -1 (TGF-β1) values and radiation-induced fibrosis (RIF). Methods and Materials: We conducted a prospective analysis of the development of RIF in 39 women with American Joint Committee on Cancer stage 0-I breast cancer treated with lumpectomy and accelerated partial breast irradiation via intracavitary brachytherapy (IBAPBI). An enzyme-linked immunoassay (Quantikine, R and D, Minneapolis, MN) was used to measure serum TGF-β1 before surgery, before IBAPBI, and during IBAPBI. Blood samples for TGF-β1 were also collected from 15 healthy, nontreated women (controls). The previously validated tissue compliance meter (TCM) was usedmore » to objectively assess RIF. Results: The median time to follow-up for 39 patients was 44 months (range, 5-59 months). RIF was graded by the TCM scale as 0, 1, 2, and 3 in 5 of 20 patients (25%), 6 of 20 patients (30%), 5 of 20 patients (25%), and 4 of 20 patients (20%), respectively. The mean serum TGF-β1 values were significantly higher in patients before surgery than in disease-free controls, as follows: all cancer patients (30,201 ± 5889 pg/mL, P=.02); patients with any type of RIF (32,273 ± 5016 pg/mL, P<.0001); and women with moderate to severe RIF (34,462 ± 4713 pg/mL, P<0.0001). Patients with moderate to severe RIF had significantly elevated TGF-β1 levels when compared with those with none to mild RIF before surgery (P=.0014) during IBAPBI (P≤0001), and the elevation persisted at 6 months (P≤.001), 12 months (P≤.001), 18 months (P≤.001), and 24 months (P=.12). A receiver operating characteristic (ROC) curve of TGF-β1 values predicting moderate to severe RIF was generated with an area under the curve (AUC){sub ROC} of 0.867 (95% confidence interval 0.700-1.000). The TGF-β1 threshold cutoff was determined to be 31,000 pg/mL, with associated sensitivity and specificity of 77.8% and 90.0%, respectively. Conclusions: TGF-β1 levels correlate with the development of moderate to severe RIF. The pre-IBAPBI mean TGF-β1 levels can serve as an early biomarker for the development of moderate to severe RIF after IBAPBI.« less
  • Fibroblast growth factor 21 (FGF-21) is a secreted protein, which has anti-diabetic and lipocaic effects, but its ability to protect against hepatic fibrosis has not been studied. In this study, we investigated the ability of FGF-21 to attenuate dimethylnitrosamine (DMN)-induced hepatic fibrogenesis in mice and the mechanism of its action. Hepatic fibrosis was induced by injection of DMN, FGF-21 was administered to the mice once daily in association with DMN injection till the end of the experiment. Histopathological examination, tissue 4-hydroxyproline content and expressions of smooth muscle α-actin (α-SMA) and collagen I were measured to assess hepatic fibrosis. Ethanol/PDGF-BB-activated hepaticmore » stellate cells (HSCs) were used to understand the mechanisms of FGF-21 inhibited hepatic fibrogenesis. Results showed that FGF-21 treatment attenuated hepatic fibrogenesis and was associated with a significant decrease in intrahepatic fibrogenesis, 4-hydroxyproline accumulation, α-SMA expression and collagen I deposition. FGF-21 treatment inhibited the activation of HSCs via down-regulating the expression of TGF-β, NF-κB nuclear translocation, phosphorylation levels of smad2/3 and IκBα. Besides, FGF-21 treatment caused activated HSC apoptosis with increasing expression of Caspase-3, and decreased the ratio of Bcl-2 to Bax. In conclusion, FGF-21 attenuates hepatic fibrogenesis and inhibits the activation of HSC warranting the use of FGF-21 as a potential therapeutic agent in the treatment of hepatic fibrosis. - Highlights: • Fibroblast growth factor 21 attenuates hepatic fibrogenesis. • Fibroblast growth factor 21 attenuates hepatic fibrogenesis via TGF-β/smad2/3 signaling pathways. • Fibroblast growth factor 21 attenuates hepatic fibrogenesis via NF-κB signaling pathways.« less
  • Affinity-labeling techniques have been used to identify three types of high-affinity receptors for transforming growth factor {beta} (TGF-{beta}) on the surface of many cells in culture. Here the authors demonstrate that membrane preparations from tissue sources may also be used as an alternative system for studying the binding properties of TGF-{beta} receptors. Using a chemical cross-linking technique with {sup 125}I-TGF-{beta}1 and {sup 125}I-TGF-{beta}2 and bis(sulfosuccinimidyl)suberate (BS{sup 3}), they have identified and characterized two high-affinity binding components in membrane preparations derived from human term placenta. The larger species, which migrates as a diffuse band of molecular mass 250-350 kDa on sodiummore » dodecyl sulfate-polyacrylamide electrophoresis gels, is characteristic of the TGF-{beta} receptor type III, a proteoglycan containing glycosaminoglycan (GAG) chains of chondroitin and heparan sulfate. The smaller species of molecular mass 140 kDa was identified as the core glycoprotein of this type III receptor by using the techniques of enzymatic deglycosylation and peptide mapping. Competition experiments, using {sup 125}I-TGF-{beta}1 or {sup 125}I-TGF-{beta}2 and varying amounts of competing unlabeled TGF-{beta}1 or TGF-{beta}2, revealed that both the placental type III proteoglycan and its core glycoprotein belong to a novel class of type III receptors that exhibit a greater affinity for TGF-{beta}2 than for TGF-{beta}1. This preferential binding of TGF-{beta}2 to placental type III receptors suggests differential roles for TGF-{beta}2 and TGF-{beta} 1 in placental function.« less
  • Most cell types have receptors for transforming growth factor-beta (TGF-beta) and respond similarly to TGF-beta 1 and TGF-beta 2. We have demonstrated the presence of a single class of high-affinity receptors (approximately 10,000 sites/cell) for TGF-beta 1 (Kd = 23 pM) and TGF-beta 2 (Kd = 41 pM) on early-passage rat lung fibroblasts (RLF). Incubation with unlabeled TGF-beta 1 and TGF-beta 2 resulted in concentration-dependent inhibition of binding of 15 pM ({sup 125}I)TGF-beta 1 (ED50, 20 and 28 pM, respectively) and ({sup 125}I)TGF-beta 2 (ED50, 36 and 56 pM, respectively). TGF-beta receptors affinity-cross-linked with 100 pM ({sup 125}I)TGF-beta 1 ormore » ({sup 125}I)TGF-beta 2 were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and exhibited labeled protein bands of 68, 88, and 286 kD. Densitometric analysis of the resulting autoradiograms showed that the different molecular weight TGF-beta binding proteins exhibited separate affinities for the two forms of TGF-beta. Both TGF-beta 1 and TGF-beta 2 altered the morphology and cytoskeleton of RLF in a similar manner, but TGF-beta 1 was more potent than TGF-beta 2 in the inhibition of RLF growth and colony formation, with 50% inhibition by 0.12 pM TGF-beta 1 and 4.4 pM TGF-beta 2. Different affinities for the TGF-beta s may indicate selectivity among the receptor subtypes with regard to the biologic responsiveness of RLF to TGF-beta s. We believe this to be the first demonstration of biologically responsive TGF-beta receptors with different affinities for TGF-beta 1 and TGF-beta 2 on cells derived from normal, nonimmortal RLF. In establishing the basic mechanisms of pulmonary fibrosis, it will be essential to understand the biology and biochemistry of the receptors that may control cell division and production of extracellular matrix components by fibroblasts.« less
  • We have studied the consequences of introducing human recombinant transforming growth factor beta 1 (hrTGF-beta 1) into synovial tissue of the rat, to begin to better understand the significance of the fact that biologically active TGF-beta is found in human arthritic synovial effusions. Within 4-6 h after the intra-articular injection of 1 microgram of hrTGF-beta 1 into rat knee joints, extensive recruitment of polymorphonuclear leukocytes (PMNs) was observed. Cytochemistry and high resolution histological techniques were used to quantitate the influx of PMNs, which peaked 6 h post-injection. In a Boyden chamber assay, hrTGF-beta 1 at 1-10 fg/ml elicited a chemotacticmore » response from PMNs greater in magnitude than that evoked by FMLP, establishing that TGF-beta 1 is an effective chemotactic agent for PMNs in vitro as well as in vivo. That PMNs may represent an important source of TGF-beta in inflammatory infiltrates was strongly suggested by a demonstration that stored TGF-beta 1 was secreted during phorbol myristate acetate-stimulated degranulation in vitro. Acid/ethanol extracts of human PMNs assayed by ELISA contained an average of 355 ng of TGF/beta 1 per 10(9) cells potentially available for secretion during degranulation of PMNs. ({sup 3}H)Thymidine incorporation in vivo and autoradiography of tissue sections revealed that widespread cell proliferation was triggered by TGF-beta 1 injection. Synovial lining cells and cells located deep within the subsynovial connective tissue were identified as sources of at least some of the new cells that contribute to TGF-beta 1-induced hyperplasia. Our results demonstrate that TGF-beta is capable of exerting pathogenic effects on synovial tissue and that PMNs may represent a significant source of the TGF-beta present in synovial effusions.« less