Most cultured cell types secrete small latent transforming growth factor-β (TGF-β) as a disulfide-bonded complex with a member of the latent TGF-β binding protein (LTBP) family. Using the baculovirus expression system, we have mapped the domain of LTBP-1 mediating covalent association with small latent TGF-β1. Coexpression in Sf9 cells of small latent TGF-β1 with deletion mutants of LTBP-1 showed that the third eight-cysteine repeat of LTBP-1 is necessary and sufficient for covalent interaction with small latent TGF-β1. Analysis by mass spectrometry of this eight-cysteine repeat, produced as a recombinant peptide in Sf9 cells, confirmed that it was N-glycosylated, as expected from the primary sequence. No other post-translational modifications of this domain were detected. Alkylation of the recombinant peptide with vinyl pyridine failed to reveal any free cysteines, indicating that, in the absence of small latent TGF-β, the eight cysteines of this domain are engaged in intramolecular bonds. These data demonstrate that the third LTBP-1 eight-cysteine repeat recognizes and associates covalently with small latent TGF-β1 through a mechanism that does not require any specific post-translational modification of this domain. They also suggest that this domain adopts different conformations depending on whether it is free or bound to small latent TGF-β. Most cultured cell types secrete small latent transforming growth factor-β (TGF-β) as a disulfide-bonded complex with a member of the latent TGF-β binding protein (LTBP) family. Using the baculovirus expression system, we have mapped the domain of LTBP-1 mediating covalent association with small latent TGF-β1. Coexpression in Sf9 cells of small latent TGF-β1 with deletion mutants of LTBP-1 showed that the third eight-cysteine repeat of LTBP-1 is necessary and sufficient for covalent interaction with small latent TGF-β1. Analysis by mass spectrometry of this eight-cysteine repeat, produced as a recombinant peptide in Sf9 cells, confirmed that it was N-glycosylated, as expected from the primary sequence. No other post-translational modifications of this domain were detected. Alkylation of the recombinant peptide with vinyl pyridine failed to reveal any free cysteines, indicating that, in the absence of small latent TGF-β, the eight cysteines of this domain are engaged in intramolecular bonds. These data demonstrate that the third LTBP-1 eight-cysteine repeat recognizes and associates covalently with small latent TGF-β1 through a mechanism that does not require any specific post-translational modification of this domain. They also suggest that this domain adopts different conformations depending on whether it is free or bound to small latent TGF-β.
Byproducts of cytokine activation are sometimes useful as surrogate biomarkers for monitoring cytokine generation in patients. Transforming growth factor (TGF)-β plays a pivotal role in pathogenesis of hepatic fibrosis. TGF-β is produced as part of an inactive latent complex, in which the cytokine is trapped by its propeptide, the latency-associated protein (LAP). Therefore, to exert its biological activity, TGF-β must be released from the latent complex. Several proteases activate latent TGF-β by cutting LAP. We previously reported that Camostat Mesilate, a broad spectrum protease inhibitor, which is especially potent at inhibiting plasma kallikrein (PLK), prevented liver fibrosis in the porcine serum-induced liver fibrosis model in rats. We suggested that PLK may work as an activator of latent TGF-β during the pathogenesis of liver diseases in the animal models. However, it remained to be elucidated whether this activation mechanism also functions in fibrotic liver in patients. Here, we report that PLK cleaves LAP between R(58) and L(59) residues. We have produced monoclonal antibodies against two degradation products of LAP (LAP-DP) by PLK, and we have used these specific antibodies to immunostain LAP-DP in liver tissues from both fibrotic animals and patients. The N-terminal side LAP-DP ending at R(58) (R(58) LAP-DP) was detected in liver tissues, while the C-terminal side LAP-DP beginning at L(59) (L(59) LAP-DP) was not detectable. The R(58) LAP-DP was seen mostly in α-smooth muscle actin-positive activated stellate cells. These data suggest for the first time that the occurrence of a PLK-dependent TGF-β activation reaction in patients and indicates that the LAP-DP may be useful as a surrogate marker reflecting PLK-dependent TGF-β activation in fibrotic liver both in animal models and in patients.
We have investigated the potential regulatory role of TGF-beta in the interactions of neurons and Schwann cells using an in vitro myelinating system. Purified populations of neurons and Schwann cells, grown alone or in coculture, secrete readily detectable levels of the three mammalian isoforms of TGF-beta; in each case, virtually all of the TGF-beta activity detected is latent. Expression of TGF-beta 1, a major isoform produced by Schwann cells, is specifically and significantly downregulated as a result of axon/Schwann cell interactions. Treatment of Schwann cells or Schwann cell/neuron cocultures with TGF-beta 1, in turn, has dramatic effects on proliferation and differentiation. In the case of purified Schwann cells, treatment with TGF-beta 1 increases their proliferation, and it promotes a pre- or nonmyelinating Schwann cell phenotype characterized by increased NCAM expression, decreased NGF receptor expression, inhibition of the forskolin-mediated induction of the myelin protein P0, and induction of the Schwann cell transcription factor suppressed cAMP-inducible POU protein. Addition of TGF-beta 1 to the cocultures inhibits many of the effects of the axon on Schwann cells, antagonizing the proliferation induced by contact with neurons, and, strikingly, blocking myelination. Ultrastructural analysis of the treated cultures confirmed the complete inhibition of myelination and revealed only rudimentary ensheathment of axons. Associated defects of the Schwann cell basal lamina and reduced expression of laminin were also detected. These effects of TGF-beta 1 on Schwann cell differentiation are likely to be direct effects on the Schwann cells themselves which express high levels of TGF-beta 1 receptors when cocultured with neurons. The regulated expression of TGF-beta 1 and its effects on Schwann cells suggest that it may be an important autocrine and paracrine mediator of neuron/Schwann cell interactions. During development, TGF-beta 1 could serve as an inhibitor of Schwann cell proliferation and myelination, whereas after peripheral nerve injury, it may promote the transition of Schwann cells to a proliferating, nonmyelinating phenotype, and thereby enhance the regenerative response.
Background Breast to bone metastases frequently induce a "vicious cycle" in which osteoclast mediated bone resorption and proteolysis results in the release of bone matrix sequestered factors that drive tumor growth. While osteoclasts express numerous proteinases, analysis of human breast to bone metastases unexpectedly revealed that bone forming osteoblasts were consistently positive for the proteinase, MMP-2. Given the role of MMP-2 in extracellular matrix degradation and growth factor/cytokine processing, we tested whether osteoblast derived MMP-2 contributed to the vicious cycle of tumor progression in the bone microenvironment. Methodology/Principal Findings To test our hypothesis, we utilized murine models of the osteolytic tumor-bone microenvironment in immunocompetent wild type and MMP-2 null mice. In longitudinal studies, we found that host MMP-2 significantly contributed to tumor progression in bone by protecting against apoptosis and promoting cancer cell survival (caspase-3; immunohistochemistry). Our data also indicate that host MMP-2 contributes to tumor induced osteolysis (μCT, histomorphometry). Further ex vivo/in vitro experiments with wild type and MMP-2 null osteoclast and osteoblast cultures identified that 1) the absence of MMP-2 did not have a deleterious effect on osteoclast function (cd11B isolation, osteoclast differentiation, transwell migration and dentin resorption assay); and 2) that osteoblast derived MMP-2 promoted tumor survival by regulating the bioavailability of TGFβ, a factor critical for cell-cell communication in the bone (ELISA, immunoblot assay, clonal and soft agar assays). Conclusion/Significance Collectively, these studies identify a novel "mini-vicious cycle" between the osteoblast and metastatic cancer cells that is key for initial tumor survival in the bone microenvironment. In conclusion, the findings of our study suggest that the targeted inhibition of MMP-2 and/or TGFβ would be beneficial for the treatment of bone metastases.
The majority of human skeletal dysplasias are caused by dysregulation of growth plate homeostasis. As TGF-beta signaling is a critical determinant of growth plate homeostasis, skeletal dysplasias are often associated with dysregulation of this pathway. The context-dependent action of TFG-beta signaling is tightly controlled by numerous mechanisms at the extracellular level and downstream of ligand-receptor interactions. However, TGF-beta is synthesized as an inactive precursor that is cleaved to become mature in the Golgi apparatus, and the regulation of this posttranslational intracellular processing and trafficking is much less defined. Here, we report that a cysteine-rich protein, E-selectin ligand-1 (ESL-1), acts as a negative regulator of TGF-beta production by binding TGF-beta precursors in the Golgi apparatus in a cell-autonomous fashion, inhibiting their maturation. Furthermore, ESL-1 inhibited the processing of proTGF-beta by a furin-like protease, leading to reduced secretion of mature TGF-beta by primary mouse chondrocytes and HEK293 cells. In vivo loss of Esl1 in mice led to increased TGF-beta/SMAD signaling in the growth plate that was associated with reduced chondrocyte proliferation and delayed terminal differentiation. Gain-of-function and rescue studies of the Xenopus ESL-1 ortholog in the context of early embryogenesis showed that this regulation of TGF-beta/Nodal signaling was evolutionarily conserved. This study identifies what we believe to be a novel intracellular mechanism for regulating TGF-beta during skeletal development and homeostasis.
