Protection of transforming growth factor β activity by heparin and fucoidan

The transforming growth factor-β (TGF-β) family of proteins exert diverse and potent effects on proliferation, differentiation, and extracellular matrix synthesis. However, relatively little is known about the stability or processing of endogenous TGF-β activity in vitro or in vivo. Our previous work indicated that (1) TGF-β1 has strong heparin-binding properties that were not previously recognized because of neutralization by iodination, and (2) heparin, and certain other polyanions, could block the binding of TGF-β1 to α₂-macroglobulin (α₂-M). The present studies investigated the influence of heparin-like molecules on the stability of the TGF-β1 signal in the pericellular environment. The results indicate that heparin and fucoidan, a naturally occurring sulfated L-fucose polymer, suppress the formation of an initial non-covalent interaction between ₁₂₅I-TGF-β1 and activated α₂-M. Electrophoresis of ₁₂₅I-TGF-β1 showed that fucoidan protects TGF-β1 from proteolytic degradation by plasmin and trypsin. While plasmin caused little, if any, activation of latent TGF-β derived from vascular smooth muscle cells (SMC), plasmin degraded acid-activated TGF-β, and purified TGF-β1, and this degradation was inhibited by fucoidan. In vitro, heparin and fucoidan tripled the half-life of ₁₂₅I-TGF-β1 and doubled the amount of cell-associated ₁₂₅I-TGF-β1. Consistent with this protective effect, heparin- and fucoidan-treated SMC demonstrated elevated levels of active, but not latent, TGF-β activity. © 1994 wiley-Liss, Inc.     

Expression,localization, and function of transforming growth factor-βs in embryonic chick spinal cord,hindbrain, and dorsal root ganglia

We have studied the localizations of transforming growth factor-beta (TGF-β) 2 and 3 immunohistochemically using isoform-specific antibodies and TGF-β3 mRNA by in situ hybridization in the nervous system of the 3- to 15-day-old chick embryo with special reference to spinal cord, hindbrain, and dorsal root ganglia (DRG). At embryonic day (E) 3, TGF-β3 mRNA as well as TGF-β2 and 3 immunoreactivities (IRs) were most prominent in the notochord, wall of the aorta, and dermomyotome. At E5 and E7, strong TGF-β2 and 3 IR were seen in or on radial glia of spinal cord and hindbrain. Radial glia in the floor plate region and ventral commissure gave the most intense signal. In the DRG, fiber strands of intense IRs representing extracellular matrix or satellite cells were seen. Neuronal perikarya did not become IR for TGF-β2 and 3 until E11, but even then the moderate signals for TGF-β3 mRNA could not be specifically localized to the neuronal cell bodies. In E11 and older embryos, spinal cord glial or glial progenitor cells, but not neuronal cell bodies were labeled for TGF-β3 mRNA. Immunocytochemistry and western blot analysis indicated that E8 DRG neurons have the TGF-β receptor type II, and treatment of these cells with NGF induces expression of TGF-β3 mRNA. The TGF-β isoforms 1, 2, and 3 did not promote survival of E8 DRG neurons in dissociated cell cultures. All three TGF-β isoforms, however, promoted neurite growth from E8 DRG explants, but were less potent than nerve growth factor. Our data suggest identical localizations of TGF-β2 and -β3 IR in the developing chick and mammalian nervous systems, underscoring the general importance of TGF-βs in fundamental events of neural development. © 1996 John Wiley & Sons, Inc.     

Novel TGF-beta antagonist inhibits tumor growth and angiogenesis by inducing IL-2 receptor-driven STAT1 activation

Carcinoma derived TGF-β acts as a potent pro-oncogenic factor and suppresses antitumor immunity. To antagonize TGF-β-mediated effects in tandem with a proinflammatory immune stimulus, we generated a chimeric protein borne of the fusion of IL-2 and the soluble extracellular domain of TGF-βR II (FIST). FIST acts as a decoy receptor trapping active TGF-β in solution and interacts with IL-2-responsive lymphoid cells, inducing a distinctive hyperactivation of STAT1 downstream of IL-2R, which in turn promotes SMAD7 overexpression. Consequently, FIST-stimulated lymphoid cells are resistant to TGF-β-mediated suppression and produce significant amounts of proinflammatory cytokines. STAT1 hyperactivation further induces significant secretion of angiostatic CXCL10. Moreover, FIST upregulates T-bet expression in NK cells promoting a potent Th1-mediated antitumor response. As a result, FIST stimulation completely inhibits pancreatic cancer (PANC02) and melanoma (B16) tumor growth in immunocompetent C57BL/6 mice. In addition, melanoma cells expressing FIST fail to form tumors in CD8(-/-), CD4(-/-), B cell-deficient (μMT), and beige mice, but not in NOD-SCID and Rag2/γc knockout mice, consistent with the pivotal role of FIST-responsive, cancer-killing NK cells in vivo. In summary, FIST constitutes a novel strategy of treating cancer that targets both the host's angiogenic and innate immune response to malignant cells.