Requirement for receptor-bound urokinase in plasmin-dependent cellular conversion of latent TGF-β to TGF-β

The role of receptor-bound urokinase-type plasminogen activator (uPA) in cellular activation of latent transforming growth factor-beta (LTGF-β) was investigated in a model system of mouse LB6 cells transfected with either a human uPA receptor cDNA (LhuPAR⁺). a human prouPA cDNA (LhuPA), or a control neomycinresistance cDNA (Lneo). When LhuPAR⁺ cells were co-cultured with LhuPA cells, the plasmin-dependent fibrinolytic activity generated was more than that observed in either homotypic cultures with fivefold greater number of LhuPA cells or co-cultures containing LhuPA and Lneo cells instead of the LhuPAR⁺ cells. The preferential activation of TGF-β by co-cultures with the greatest plasmin-generation potential, LhuPAR⁺ and LhuPA cells, was confirmed by three independent bioassays. In the first assay, a 48% decrease in PA activity, a measure of active TGF-β production, was observed with BAE cells treated with conditioned medium (CM) from co-cultures of LhuPA and LhuPAR⁺ cells. Inclusion of neutralizing antibodies to TGF-β abrogated the inhibitory effect of CM on PA activity demonstrating that the inhibitory molecule was TGF-β. Addition of the amino terminal fragment of uPA (ATF) or omission of plasminogen from co-cultures blocked both the fibrinolytic activity and the generation of TGF-β activity in the CM. In the second assay, CM from co-cultures of LhuPA and LhuPAR⁺ cells inhibited the migration of BAE cells in a wound assay. Controls with anti-TGF-β IgG indicated that the inhibition was due to TGF-β. In the third assay, proliferation of mink lung epithelial cells was inhibited by CM generated by co-cultures of LhuPA and LhuPAR⁺ cells as compared to CM from the same cells cultured in the absence of plasminogen or to CM from a co-culture of LhuPA with LhuPAR^? cells. Excess mannose-6-phosphate (M6P) blocked the generation of TGF-β as assayed by both the BAE migration and PA assays, presumably because it interfered with cellsurface localization of LTGF-β. Additionally, small numbers of LhuPA and LhuPAR⁺ cells co-cultured with BAE cells inhibited the BAE cell PA activity via the paracrine action of TGF-β. These results support the conclusion that plasmindependent activation LTGF-β by LB6 cells is promoted by the surface localization of uPA by its receptor. © 1994 Wiley-Liss, Inc.     

TGF-β1 regulation of dendritic cells

Dendritic cells (DCs) represent antigen-presenting cell (APC) populations in lymphoid and nonlymphoid organs which are considered to play key roles in the initiation of antigen-specific T-cell proliferation. According to current knowledge, the net outcome of T-cell immune responses seems to be significantly influenced by the activation stage of antigen-presenting DCs. Several studies have shown that transforming growth factor-beta 1 (TGF-β1) inhibits in vitro activation and maturation of DCs. TGF-β1 inhibits upregulation of critical T-cell costimulatory molecules on the surface of DCs and reduces the antigen-presenting capacity of DCs. Thus, in addition to direct inhibitory effects of TGF-β1 on effector T lymphocytes, inhibitory effects of TGF-β1 at the level of APCs may critically contribute to previously characterized immunosuppressive effects of TGF-β1. In contrast to these negative regulatory effects of TGF-β1 on function and maturation of lymphoid tissue type DCs, certain subpopulations of immature DCs in nonlymphoid tissues are positively regulated by TGF-β1 signaling. In particular, epithelial-associated DC populations seem to critically require TGF-β1 stimulation for development and function. Recent studies established that TGF-β1 stimulation is absolutely required for the development of epithelial Langerhans cells (LCs) in vitro and in vivo. Furthermore, TGF-β1 seems to enhance antigen processing and costimulatory functions of epithelial LCs.     

Modulation of gene expression in the preimplantation mouse embryo by TGF-α and TGF-β

The effect of growth factors on regulating gene expression in the preimplantation mouse embryo was examined, since results of previous experiments revealed a stimulatory effect of exogenously-added growth factors on preimplantation development in vitro. Treatment of early cavitating blastocysts with either 250 pM TGF-α or TGF-β results in changes in the pattern of total protein synthesis as assessed by high-resolution two-dimensional gel electrophoresis. In some cases, the synthesis of a particular polypeptide is either up- or downregulated by each growth factor, whereas in other instances the synthesis of a polypeptide is modulated by one but not the other growth factor. Use of the mRNA differential display method permitted the identification of genes whose expression is either up- or downregulated by these growth factors. Treatment of mouse blastocysts with either TGF-α or TGF-β results in the increased expression of the b subunit of the F₀ ATPase. TGF-β also stimulates the expression of the DNA polymerase α. TGF-α treatment results in the increase in expression of a gene homologous to the human HEPG2 cDNA, as well as in a decrease in expression of fibronectin. © 1995 Wiley-Liss, Inc.     

Genomics of TGF-β1 signaling in stem cell commitment and dendritic cell development

Dendritic cells are professional antigen presenting cells and central for establishing and maintaining immunity and immunological tolerance. They develop from hematopoietic stem cells through successive steps of lineage commitment and differentiation. Dendritic cell development and function are regulated by specific cytokines, including transforming growth factor type β1 (TGF-β1). Our previous work demonstrated the importance of TGF-β1 signaling for dendritic cell development and subset specification. Here, we used genome-wide gene expression profiling with DNA microarrays to investigate the activity of TGF-β1 on gene expression in dendritic cell development. This study identified specific gene categories induced by TGF-β1 with an impact on dendritic cell biology.     

Retinoic acid down-regulates VPAC1 receptors and TGF-β3 but up-regulates TGF-β2 in lung cancer cells

The effects of retinoic acid (RA) on lung cancer cells were investigated. Both all-trans (t-RA) and 13-cis RA (c-RA) decreased specific ¹²⁵I-VIP binding to NCI-H1299 cells in a time- and concentration-dependent manner. After 20 hr, 30 μM t-RA decreased specific ¹²⁵I-VIP binding by 60%. By Scatchard analysis, the density of VIP binding sites but not the affinity was reduced by 42%. NCI-H1299 VPAC₁ receptor mRNA was reduced by 48%. VIP caused a 3-fold elevation in the NCI-H1299 cAMP, and the increase in cAMP caused by VIP was reduced by 38% if the NCI-H1299 cells were treated with t-RA. Using the MTT assay, 3 μM t-RA and 3 μM c-RA inhibited NCI-H1299 proliferation by 60 and 23% respectively. Also, transforming growth factor (TGF)-β2 increased after treatment of NCI-H1299 cells with t-RA whereas TGF-β1 mRNA was unaffected and TGF-β3 mRNA was decreased. These results suggest that RA may inhibit lung cancer growth by down-regulating VPAC₁ receptor and TGF-β3 mRNA but up-regulating TGF-β2 mRNA.     

TGF-β1: immunosuppressant and viability factor for T lymphocytes

Transforming growth factor-beta (TGF-β) is a multifunctional cytokine with multiple roles in the immune system. To date, it has been difficult to develop a comprehensive picture of the effect of TGF-β on T lymphocytes, because TGF-β not only acts directly on T lymphocytes, but also acts indirectly by regulating the function of antigen-presenting cells. In early studies, it was mostly the inhibitory function of TGF-β that was demonstrated; recently, however TGF-β was recognized as an antiapoptotic survival factor for T lymphocytes. The outcome of the TGF-β effect on T lymphocytes was shown to strongly depend on their stage of differentiation and on the cytokine milieu. TGF-β cannot be classified as a classical Th1 or Th2 cytokine. However, recently the existence of the TGF-β-producing Th3 subset was described which might play an important regulatory role during an immune response. A better understanding of the molecular mechanism of how TGF-β inhibits or stimulates T lymphocytes will help to predict the complex functions of this cytokine.     

TGF-β1 perturbation of the fibroblast cell cycle during exponential growth: switching between negative and positive regulation*

Abstract. We have demonstrated previously that SV40 T antigen and serum regulate the length of G₁ in exponentially growing NIH-3T3 cells in part by inhibiting density dependent negative cell cycle regulation. In these studies it was suggested that T antigen positively regulated G₁ in a density independent manner as well. In this report we show that, 24 h after treatment, TGF-β1 perturbs the cell cycle of exponentially growing fibroblasts in a manner similar to T antigen. However, prior to 24 h, TGF-β1 produced a negative response, elongating the Gi phase of the cell cycle that was followed by a positive response, both of which were density independent. This biphasic response was measured between 0 and 12 h post-treatment and was relative to responses from serum. This switch from an early inhibitory effect to a late stimulatory effect was associated with changes in Rb phosphorylation, the timing and magnitude of which indicated that Rb may be directly regulating T_G1 rather than reporting changes in the population. This is further substantiated by abrogation of the inhibitory effect by expression of wild-type SV40 T antigen and retention of the effect in cells that express an Rb-binding mutant of T antigen (K1). The biphasic regulatory effects of TGF-β1 were also displayed in WI-38 and IMR-90 human fibroblasts. This suggests that this biphasic effect is a property of fibroblasts.     

Role for autocrine TGF-β1 in regulating differentiation of a human leukemic cell line toward osteoclast-like cells

Increasing evidence suggests that transforming growth factor-β (TGF-β) is involved in bone formation during remodeling. Using a recently cloned human leukemic cell line (FLG 29.1 cells) we demonstrate that these cells synthesize and secrete TGF-β1 and that exogenous or autocrine TGF-β1 can induce the same features of osteoclastic-like cells, exerting its effects through the binding to TGF-β specific receptors. Scatchard analysis of ¹²⁵I-labeled TGF-β1 to FLG 29.1 cells revealed the presence of a single high affinity binding site with a Kd value of ～25 pM and a binding capacity of ～900 sites/cell. Affinity labeling experiments showed that FLG 29.1 cells express type I and type II TGF-β receptors. Stimulation of FLG 29.1 cells with low TGF-β1 doses reduced cell proliferation and increased cell adhesion and tartrate resistant acid phosphatase (TRAcP) activity. Pretreatment of FLG 29.1 cells with TGF-β1 caused a significant and dose-dependent response to calcitonin. Northern blot of total mRNA and analysis of the conditioned media (CM) showed that TGF-β1 was synthesized by FLG 29.1 cells. TPA treatment, which induces partial differentiation of these cells, markedly increased TGF-β1 mRNA expression and growth factor release. The majority of TGF-β1 secreted by TPA-treated cells was in its latent form. However, anti-TGF-β antibodies inhibited TGF-β1 and TPA-induced growth inhibition, calcitonin responsiveness, and TRAcP activity, suggesting that the TPA effect is mediated in part by autocrine TGF-β1 and indicating that the cells can activate and respond to the TGF-β that they secrete. These findings support a potential autocrine role for TGF-β1 in osteoclast differentiation. © 1994 Wiley-Liss, Inc.     

Osteogenic protein-1, a bone morphogenic protein member of the TGF-β superfamily,shares chemotactic but not fibrogenic properties with TGF-β

We have previously shown that recombinant human osteogenic protein-1 (rhOP-1), a bone morphogenetic protein member of the TGF-β superfamily, can induce new bone formation when implanted with an appropriate carrier at subcutaneous sites in rats and can restore completely large diaphyseal segmental defects in laboratory animals. The role of OP-1 in the early events of bone induction viz, chemotaxis of phagocytic leukocytes, and fibroblastic mesenchymal cells is currently unknown. In the present study, we examined the effect of rhOP-1 on chemotaxis of phagocytic leukocytes (human neutrophils and monocytes) and fibroblastic mesenchymal cells (infant foreskin fibroblasts). Since OP-1 is structurally related to TGF-β1, we assessed the effects of OP-1 on several other fibroblast functions (in addition to chemotaxis) known to be modulated by TGF-β1. Our results demonstrated that rhOP-1, like TGF-β1, is a potent chemoattractant for human neutrophils, monocytes, and fibroblasts. However, in contrast to TGF-β1, OP-1 does not to stimulate fibroblast mitogenesis, matrix synthesis [collagen and hyaluronic acid (hyaluronan)], or production of tissue inhibitor of metalloproteinase (TIMP), i.e., fibroblast functions associated with fibrogenesis. These results clearly demonstrate a dichotomy between these two members of the TGF-β superfamily with regard to fibrogenic effects on fibroblasts but a similarity in their chemotactic properties. © 1994 Wiley-Liss, Inc.     

TGF-β1 is an organizer of responses to neurodgeneration

TGF-β1 mRNA and protein were recently found to increase in animal brains after experimental lesions that cause local deafferentation or neuron death. Elevations of TGF-β1 mRNA after lesions are prominent in microglia but are also observed in neurons and astrocytes. Moreover, TGF-β1 mRNA autoinduces its own mRNA in the brain. These responses provide models for studying the increases of TGF-β1 protein observed in βA/amyloid-containing extracellular plaques of Alzheimer's disease (AD) and Down's syndrome (DS) and in brain cells of AIDS victims. Involvement of TGF-β1 in these human brain disorders is discussed in relation to the potent effects of TGF-β1 on wound healing and inflammatory responses in peripheral tissues. We hypothesize that TGF-β1 and possibly other TGF-β peptides have organizing roles in responses to neurodegeneration and brain injury that are similar to those observed in non-neural tissues. Work from many laboratories has shown that activities of TGF-β peptides on brain cells include chemotaxis, modification of extracellular matrix, and regulation of cytoskeletal gene expression and of neurotrophins. Similar activities of the TGF-β's are well established in other tissues.     

Growth plate chondrocytes store latent transforming growth factor (TGF)-β1 in their matrix through latent TGF-β1 binding protein-1

Osteoblasts produce a 100 kDa soluble form of latent transforming growth factor beta (TGF-β) as well as a 290 kDa form containing latent TGF-β binding protein-1 (LTBP1), which targets the latent complex to the matrix for storage. The nature of the soluble and stored forms of latent TGF-β in chondrocytes, however, is not known. In the present study, resting zone and growth zone chondrocytes from rat costochondral cartilage were cultured to fourth passage and then examined for the presence of mRNA coding for LTBP1 protein. In addition, the matrix and media were examined for LTBP1 protein and latent TGF-β. Northern blots, RT-PCR, and in situ hybridization showed that growth zone cells expressed higher levels of LTBP1 mRNA in vitro than resting zone cells. Immunohistochemical staining for LTBP1 revealed fine fibrillar structures around the cells and in the cell matrix. When the extracellular matrix of these cultures was digested with plasmin, LTBP1 was released, as determined by immunoprecipitation. Both active and latent TGF-β1 were found in these digests by TGF-β1 ELISA and Western blotting. Immunoprecipitation demonstrated that the cells also secrete LTBP1 which is not associated with latent TGF-β, in addition to LTBP1 that is associated with the 100 kDa latent TGF-β complex. These studies show for the first time that latent TGF-β is present in the matrix of costochondral chondrocytes and that LTBP1 is responsible for storage of this complex in the matrix. The data suggest that chondrocytes are able to regulate both the temporal and spatial activation of latent TGF-β, even at sites distant from the cell, in a relatively avascular environment. J. Cell. Physiol. 177:343–354, 1998. © 1998 Wiley-Liss, Inc.     

Changes in TGF-β receptors of rat hepatocytes during primary culture and liver regeneration: Increased expression of TGF-β receptors associated with increased sensitivity to TGF-β-mediated growth inhibition

To clarify the role of transforming growth factor-β (TGF-β) and its receptors in hepatocyte growth, we studied the expression of TGF-β1 and its receptors and the sensitivity to growth inhibition by TGF-β1 protein in rat hepatocytes derived from resting and regenerating livers. In hepatocytes derived from resting livers, mRNAs for TGF-β type II receptor (TβR-II), insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M-6-PR), and TGF-β1 increased with time in primary culture. The cell surface TGF-β receptor proteins (TβR-I, II, and III), examined by the receptor affinity-labeling assay using ¹²⁵I-TGF-β1, also increased, especially after 48 hr of culture. Hepatocytes were more sensitive to inhibition of DNA synthesis, when the TGF-β1 protein was added at later times in culture, corresponding to the presence of increased TGF-β receptors. In hepatocytes from regenerating livers after a partial hepatectomy (PH), an increase of TβR-I, TβR-II, TβR-III, IGF-II/M-6-PR, and TGF-β1 mRNAs was found, compared with hepatocytes from resting livers. Similarly, using TGF-β receptor affinity-labeling assay, hepatocytes from PH livers were found to have an increase in TβR-I, II, and III proteins, with a peak at 4 days post-PH, compared with hepatocytes from resting livers. When TGF-β1 protein was added for a short period (6 or 24 hr) after cell attachment to hepatocyte cultures, it inhibited DNA synthesis more effectively in hepatocytes from regenerating compared with resting livers. Our results show that hepatocyte TGF-β receptors and sensitivity to growth inhibition by TGF-β1 protein change together and are modulated during liver regeneration, as well as during the conditions of primary culture. J. Cell. Physiol. 176:612–623, 1998. © 1998 Wiley-Liss, Inc.     

Active transforming growth factor-β in human melanoma cell lines: No evidence for plasmin-related activation of latent TGF-β

Cultured human melanoma cells were found to secrete TGF-β mostly in latent biologically inactive form but in addition five of six melanoma cell lines studied produced in conditioned culture medium active TGF-β in the range from 370 to 610 pg per 10⁶ cells per 24 h. A distinct characteristic of these melanoma cell lines is that they form active surface-bound plasmin by the activation of plasminogen with surface-bound tissue-type plasminogen activator. The present study was performed to assess the role of plasmin in the process of latent TGF-β activation in the melanoma cell lines. No direct correlation was found between cell-associated plasmin activity and the amount of active TGF-β present in the conditioned medium of individual cell lines. The melanoma cell lines exhibited diverse responses to exogenous active TGF-β1; three cell lines were growth-stimulated, two were growth-inhibited, and one had a very low sensitivity to the growth factor. The active TGF-β produced by the melanoma cells was found to inhibit the natural killer cell function of peripheral blood lymphocytes, suggesting that it may have an immunosuppressive effect and a role in the development of melanomas. © 1996 Wiley-Liss, Inc.     

LYMPHOTOXIN-α (TNF-β) DURING SEPSIS

T cell release of lymphotoxin-α (LT-α, or TNF-β) is stimulated by pyrogenic exotoxins of Gram-positive bacteria and mitogens. In contrast to TNF-α, it is unknown whether LT-α plays any role in the pathogenesis of sepsis and, in particular, the pathogenesis of Gram-positive sepsis. Sera from patients with sepsis were examined for LT-α and compared with normal volunteers and pregnant women. LT-α was detected in 33% of sepsis sera (mean 608.4 pg/ml SE 306), 16% of normal sera (mean 167 pg/ml SE 87) and 23% of sera from pregnant women (mean 714 pg/ml SE 191). These differences were not significant and there were no differences within species sera when grouped by the type of causative organism, or disease severity. LT-α detected by immunoassay in serum was not bioactive, in contrast to that produced in cell culture. Recombinant soluble TNF receptors (rSTNFR) neutralized the bioactivity of recombinant LT-α at rSTNFR concentrations which did not interfere with immunoreactivity and which are known to prevailin vivo. Hence, LT-α is unlikely to have a critical role in the pathogenesis of sepsis. Much of the potential bioactivity of this lymphokine may be abrogated by TNFR in serum.     

Bidirectional regulation of macrophage function by TGF-β

The dual role of transforming growth factor-beta (TGF-β) in modulating macrophage function is an important concept gaining increasing recognition. In addition to its role as a ‘macrophage-deactivating' agent, TGF-β functions as a monocyte activator, inducing cytoke production and mediating host defence. These functions are context-dependent, modulated by the differentiation state of the cell, the local cytokine environment, and the local levels of TGF-β in itself. In general, during the initial stages of inflammation, TGF-β locally acts as a proinflammatory agent by recruiting and activating resting monocytes. As these cells differentiate specific immunosuppressive actions of TGF-β predominate, leading to resolution of the inflammatory response. Increasing our understanding of the bidirectional regulation of macrophage function will facilitate prediction of the ultimate outcome of modulating TGF-β levels in vivo.