Regulation of fibronectin receptor distribution [published erratum appears in J Cell Biol 1992 Jul;118(2):491]

Studies on human osteoclast formation have been hampered by lack of a defined isolated progenitor cell population. We describe here the establishment of a human leukemic cell line (designated FLG 29.1) from bone marrow of a patient with acute monoblastic leukemia. The cultured cells are predominantly undifferentiated leukemic blasts, but addition of 12-o-tetradecanoylphorbol 13-acetate (TPA; 0.1 microM) induces irreversible differentiation into adherent, non-dividing, multinucleated cells. TPA-treated cells bear surface antigens typical of fetal osteoclasts, degrade 45Ca-labeled devitalized bone particles, display tartrate-resistant acid phosphatase in both mononuclear and multinuclear cells and receptors for calcitonin. Calcitonin increases intracellular cAMP accumulation in TPA-treated cells. TPA-treated cells show some ultrastructural features of osteoclasts as evidenced by transmission EM. These results indicate that FLG 29.1 cells may represent an osteoclast committed cell population, which upon induction with TPA acquire some morphological, phenotypical, and functional features of differentiated osteoclasts.     

Rat testicular germ cells and sertoli cells release different types of bioactive transforming growth factor beta in vitro

Several in vivo studies have reported the presence of immunoreactive transforming growth factor-β's (TGF-β's) in testicular cells at defined stages of their differentiation. The most pronounced changes in TGF-β₁ and TGF-β₂ immunoreactivity occurred during spermatogenesis. In the present study we have investigated whether germ cells and Sertoli cells are able to secrete bioactive TGF-β's in vitro, using the CCl64 mink lung epithelial cell line as bioassay for the measurement of TGF-β. In cellular lysates, TGF-β bioactivity was only observed following heat-treatment, indicating that within these cells TGF-β is present in a latent form. To our surprise, active TGF-β could be detected in the culture supernatant of germ cells and Sertoli cells without prior heat-treatment. This suggests that these cells not only produce and release TGF-β in a latent form, but that they also release a factor which can convert latent TGF-β into its active form. Following heat-activation of these culture supernatant's, total TGF-β bioactivity increased 6- to 9-fold. Spermatocytes are the cell type that releases most bioactive TGF-β during a 24 h culture period, although round and elongated spermatids and Sertoli cells also secrete significant amounts of TGF-β. The biological activity of TGF-β could be inhibited by neutralizing antibodies against TGF-β₁ (spermatocytes and round spermatids) and TGF-β₂ (round and elongating spermatids). TGF-β activity in the Sertoli cell culture supernatant was inhibited slightly by either the TGF-β₁ and TGF-β₂ neutralizing antibody.These in vitro data suggest that germ cells and Sertoli cells release latent TGF-β's. Following secretion, the TGF-β's are converted to a biological active form that can interact with specific TGF-β receptors. These results strengthen the hypothesis that TGF-β's may play a physiological role in germ cell proliferation/differentiation and Sertoli cell function.     

Characterization and regulation of the latent transforming growth factor-β complex secreted by vascular pericytes

Transforming growth factor-β (TGF-β) stimulates the accumulation of extracellular matrix in renal and hepatic disease. Kidney glomerular mesangial cells (GMC) and liver fat-storing cells (FSC) produce latent or inactive TGF-β. In this study, we characterized the latent TGF-β complexes secreted by these cells. Human FSC produce a single latent TGF-β complex, predominantly of the TGF-β1 isoform, whereas GMC secrete multiple complexes of latent TGF-β, containing β1 and β2 isoforms. At least four forms were identified in GMC using ion exchange chromatography, including a peak not previously described in other cell types which eluted at 0.12 M NaCl, and predominantly of the β2 isoform. Both cell types secrete the latent TGF-β1 binding protein of 190 kDa, as part of a high molecular weight TGF-β complex. Epidermal growth factor stimulates the secretion of latent TGF-β and latent TGF-β binding protein in both cell types. Secretion of the latent TGF-β in both cell types was found to be associated with secretion of decorin. This study shows that vascular pericytes from the kidney and the liver have distinctly different profiles of latent TGF-β complexes, with GMC secreting a unique form of latent TGF-β2. The regulatory effect of epidermal growth factor and platelet-derived growth factor has potential implication for the pathophysiology of liver regeneration and chronic liver and kidney diseases. © 1996 Wiley-Liss, Inc.     

Characterization of a 60-kDa cell surface-associated transforming growth factor-β binding protein that can interfere with transforming growth factor-β receptor binding

We have characterized a 60-kDa transforming growth factor-β (TGF-β) binding protein that was originally identified on LNCaP adenocarcinoma prostate cells by affinity cross-linking of cell surface proteins by using ¹²⁵I-TGF-β1. Binding of ¹²⁵I-TGF-β1 to the 60-kDa protein was competed by an excess of unlabeled TGF-β1 but not by TGF-β2, TGF-β3, activin, or osteogenic protein-1 (OP-1), also termed bone morphogenetic protein-7 (BMP-7). In addition, no binding of ¹²⁵I-TGF-β2 and ¹²⁵I-TGF-β3 to the 60-kDa binding protein on LNCaP cells could be demonstrated by using affinity labeling techniques. The 60-kDa TGF-β binding protein showed no immunoreactivity with antibodies against the known type I and type II receptors for members of the TGF-β superfamily. Treatment of LNCaP cells with 0.25 M NaCl, 1 μg/ml heparin, or 10% glycerol caused a release of the 60-kDa protein from the cell surface. In addition, we found that the previously described TGF-β type IV receptor on GH3 cells, which does not form a heteromeric complex with TGF-β receptors, could be released from the cell surface by these same treatments. This suggests that the 60-kDa protein and the similarly sized TGF-β type IV receptor are related proteins. The eluted 60-kDa LNCaP protein was shown to interfere with the binding of TGF-β to the TGF-β receptors. Thus, the cell surface-associated 60-kDa TGF-β binding protein may play a role in regulating TGF-β binding to TGF-β receptors. J. Cell. Physiol. 173:447–459, 1997. © 1997 Wiley-Liss, Inc.     

Transforming growth factor-β1 is an autocrine mediator of U937 cell growth arrest and differentiation induced by vitamin D3 and retinoids

Vitamin D and retinoids cooperate to inhibit the proliferation and induce the differentiation of human myelomonocytic U937 leukemia cells. In the present work, we investigated the role of TGF-β as an endogenous mediator of this process. We found that the TGF-β1 precursor began to accumulate in cell culture supernatants soon after the addition of 1α,25 dihydroxyvitamin D₃ (VD) and retinoids. We used neutralizing antibodies (AbTGF-β) and antisense oligonucleotide (AS Oligo) to inhibit its possible effects. Our data demonstrated that AbTGF-β partially inhibit the expression of the differentiated phenotype, as assessed by measurement of phagocytic activity, response to the chemotactic peptide fMLP, and lysozyme secretion. AS Oligo was also inhibitory, and the effects of AS Oligo and AbTGF-β were cumulative. Cell growth inhibition induced by VD and retinoids was completely reversed, and differentiation was reduced by about 75% when both inhibitors were associated. Time course experiments based on the delayed addition of AbTGF-β and AS Oligo showed that TGF-β1 was required for cell differentiation 24 h after the addition of inducers. Studies on TGF-β receptors revealed that, while the expression of type II receptor was stable, the level of type I TGF-β receptor mRNA and the expression of the protein began to decline early during the differentiation process. As a whole, these results support the notion that an autocrine TGF-β pathway, activated by VD and retinoids in U937 cells, is involved in the early steps of the process leading to cell growth arrest and differentiation. J Cell Physiol 178:109–119, 1999. © 1999 Wiley-Liss, Inc.     

Structural and functional characterization of soluble endoglin receptor

Endoglin, an accessory membrane receptor of transforming growth factor-β (TGF-β)1, modulates the cellular response to TGF-β via its interaction with type I and II TGF-β receptors. It has been considered a promising target for the development of therapeutics and cancer markers. We have established stable CHO cell lines that efficiently secrete soluble endoglin (s-endoglin) fused with human growth hormone. Two oligomeric forms were observed in a homogeneous preparation of s-endoglin, as a dimer and a tetramer. The dimeric s-endoglin enhanced TGF-β responsiveness in U937 cells, thus proving its potential for therapeutic applications. Small angle X-ray scattering (SAXS) experiments revealed elongated conformations of both dimeric and tetrameric s-endoglins in solution, suggesting that s-endoglin might undergo conformational adaptations upon TGF-β binding. The current results provide important references and material for high-resolution structural studies and for medical applications of s-endoglin.     

Modulation of the effects of osteoprotegerin (OPG) ligand in a human leukemic cell line by OPG and calcitonin

The discovery of osteoprotegerin (OPG), osteoprotegerin ligand (OPGL), and RANK has elucidated the mechanism by which osteoblasts and stromal cells regulate osteoclastic differentiation and function and mediate the effects exerted by other hormones and cytokines. We have investigated the effects of these novel cytokines on the preosteoclastic cell line FLG 29.1. We show that OPGL alone and in combination with macrophage colony-stimulating factor (CSF-1) dramatically reduced replication and increased tartrate-resistant acid phosphatase activity. However, although FLG29.1 cells appear to adhere to the bone surface, they are not able to form resorption lacunae. OPG and calcitonin completely abolished the differentiation induced by OPGL. RANK was detectable in FLG 29.1 and the number of positive cells was increased by OPGL/CSF-1 treatment while reduced by calcitonin. We propose that calcitonin could interact with the OPG/OPGL, and its effects on RANK may explain in part the action of this hormone in suppressing bone resorption.     

Extracellular heat shock protein HSP90β secreted by MG63 osteosarcoma cells inhibits activation of latent TGF-β1

Transforming growth factor-beta 1 (TGF-β1) is secreted as a latent complex, which consists of latency-associated peptide (LAP) and the mature ligand. The release of the mature ligand from LAP usually occurs through conformational change of the latent complex and is therefore considered to be the first step in the activation of the TGF-β signaling pathway. So far, factors such as heat, pH changes, and proteolytic cleavage are reportedly involved in this activation process, but the precise molecular mechanism is still far from clear. Identification and characterization of the cell surface proteins that bind to LAP are important to our understanding of the latent TGF-β activation process. In this study, we have identified heat shock protein 90 β (HSP90β) from the cell surface of the MG63 osteosarcoma cell line as a LAP binding protein. We have also found that MG63 cells secrete HSP90β into extracellular space which inhibits the activation of latent TGF-β1, and that there is a subsequent decrease in cell proliferation. TGF-β1-mediated stimulation of MG63 cells resulted in the increased cell surface expression of HSP90β. Thus, extracellular HSP90β is a negative regulator for the activation of latent TGF-β1 modulating TGF-β signaling in the extracellular domain.     

Relationship between actions of transforming growth factor (TGF)-β and cell surface expression of its receptors in clonal osteoblastic cells

Various osteoblastic cell lines were examined for the relationship between the presence of cell-surface transforming growth factor (TGF)-β receptors and the synthesis of matrix proteins with their responsiveness to TGF-β. Treatment with TGF-β1 inhibited proliferation and stimulated proteoglycan and fibronectin synthesis in MC3T3-E1 and MG 63 cells. The major proteoglycans synthesized by these cells were decorin and biglycan, and TGF-β1 markedly stimulated the synthesis of decorin in MC3T3-E1 and of biglycan in MG 63 cells. SaOS 2 and UMR 106 cells synthesized barely detectable amounts of decorin or biglycan, and TGF-β1 did not stimulate the synthesis of these proteoglycans. In SaOS 2 cells, however, TGF-β1 enhanced fibronectin synthesis. TGF-β1 did not show any of these effects in UMR 106 cells. Receptor cross-linking studies revealed that only MC3T3-E1 and MG 63 cells had both types I and II signal-transducing receptors for TGF-β in addition to betaglycan. SaOS 2 cells possessed type I but no type II receptor on the cell surface. In contrast, SaOS 2 as well as MC3T3-E1 and MG 63 cells expressed type II receptor mRNA by Northern blot analysis, and cell lysates contained type II receptor by Western blot analysis. Thus, it appears that type II receptor present in SaOS 2 cells is not able to bind TGF-β1 under these conditions. UMR 106 cells with no response to TGF-β1 had neither of the signal-transducing receptors by any of the analyses. These observations using clonal osteoblastic cell lines demonstrate that the ability of osteoblastic cells to synthesize bone matrix proteoglycans is associated with the responsiveness of these cells to TGF-β1, that the responsiveness of osteoblastic cells to TGF-β1 in cell proliferation and proteoglycan synthesis correlates with the presence of both types I and II receptors, and that the effect of TGF-β1 on fibronectin synthesis can develop with little binding of TGF-β1 to type II receptor if type I receptor is present. It is suggested that the combination of cell-surface receptors for TGF-β determines the responsiveness of osteoblastic cells to TGF-β and that changes in cell-surface TGF-β receptors may play a role in the regulation of matrix protein synthesis and bone formation in osteoblasts. © 1995 Wiley-Liss, Inc.     

Heparanase is a key player in renal fibrosis by regulating TGF-β expression and activity

Epithelial–mesenchymal transition (EMT) of tubular cells is one of the mechanisms which contribute to renal fibrosis and transforming growth factor-β (TGF-β) is one of the main triggers. Heparanase (HPSE) is an endo-β-D-glucuronidase that cleaves heparan-sulfate thus regulating the bioavailability of growth factors (FGF-2, TGF-β). HPSE controls FGF-2-induced EMT in tubular cells and is necessary for the development of diabetic nephropathy in mice.The aim of this study was to investigate whether HPSE can modulate the expression and the effects of TGF-β in tubular cells.First we proved that the lack of HPSE or its inhibition prevents the increased synthesis of TGF-β by tubular cells in response to pro-fibrotic stimuli such as FGF-2, advanced glycosylation end products (AGE) and albumin overload.Second, since TGF-β may derive from sources different from tubular cells, we investigated whether HPSE modulates tubular cell response to exogenous TGF-β. HPSE does not prevent EMT induced by TGF-β although it slows its onset; indeed in HPSE-silenced cells the acquisition of a mesenchymal phenotype does not develop as quickly as in wt cells. Additionally, TGF-β induces an autocrine loop to sustain its signal, whereas the lack of HPSE partially interferes with this autocrine loop.Overall these data confirm that HPSE is a key player in renal fibrosis since it interacts with the regulation and the effects of TGF-β. HPSE is needed for pathological TGF-β overexpression in response to pro-fibrotic factors. Furthermore, HPSE modulates TGF-β-induced EMT: the lack of HPSE delays tubular cell transdifferentiation, and impairs the TGF-β autocrine loop.     

Actions of TGF-β as tumor suppressor and pro-metastatic factor in human cancer

Transforming growth factor-β (TGF-β) is a secreted polypeptide that signals via receptor serine/threonine kinases and intracellular Smad effectors. TGF-β inhibits proliferation and induces apoptosis in various cell types, and accumulation of loss-of-function mutations in the TGF-β receptor or Smad genes classify the pathway as a tumor suppressor in humans. In addition, various oncogenic pathways directly inactivate the TGF-β receptor-Smad pathway, thus favoring tumor growth. On the other hand, all human tumors overproduce TGF-β whose autocrine and paracrine actions promote tumor cell invasiveness and metastasis. Accordingly, TGF-β induces epithelial–mesenchymal transition, a differentiation switch that is required for transitory invasiveness of carcinoma cells. Tumor-derived TGF-β acting on stromal fibroblasts remodels the tumor matrix and induces expression of mitogenic signals towards the carcinoma cells, and upon acting on endothelial cells and pericytes, TGF-β regulates angiogenesis. Finally, TGF-β suppresses proliferation and differentiation of lymphocytes including cytolytic T cells, natural killer cells and macrophages, thus preventing immune surveillance of the developing tumor. Current clinical approaches aim at establishing novel cancer drugs whose mechanisms target the TGF-β pathway. In conclusion, TGF-β signaling is intimately implicated in tumor development and contributes to all cardinal features of tumor cell biology.     

Role of receptor complexes in resistance or sensitivity to growth inhibition by TGFβ in intestinal epithelial cell clones

Untransformed rat intestinal epithelial cells (IEC-18) were chemically mutagenized, selected in the presence of TGFβ₁, and cloned by limiting dilution. Two clones (4–5, 4–6) were resistant to growth inhibition by both TGFβ₁ and TGFβ₂. Another clone (4–1) was more sensitive to both TGFβ isoforms (relative to parental IEC-18 cells). IC₅₀ values for TGFβ_{1 and 2} in the 4–1 cells were at least 1/9 those of the parental cells; growth rates were reduced by 49% for TGFβ₁ and by 26% for TGFβ₂ in this clone. This increased sensitivity to TGFβ was explained by the 5- to 10-fold increase, relative to parental cells, in binding of TGFβ₁ and TGFβ₂ to both the type I and II receptors. In contrast, the resistance to growth inhibition by TGFβ in the 4–5 and 4–6 cells could not be explained by a decrease in either TGFβ binding affinities or in total number of receptors expressed, by the presence of serum binding components, or by occupation of receptor binding sites with autocrine TGF-β₁. However, in comparison to TGFβ-sensitive cells (IEC-18, 4–1), the resistant cells displayed a higher ratio of type II relative to type I receptor binding by TGF-β₁. Thus, a critical ratio of binding to receptor subtypes correlated with growth inhibition by TGF-β₁. Resistance to TGF-β₂ in the same clones did not appear to be receptor related. Thus different mechanisms for resistance to TGF-β₁ and TGF-β₂ were observed within a given clone. © 1993 Wiley-Liss, Inc.     

The role of transforming growth factor-β1, -β2, and -β3 in androgen-responsive growth of NRP-152 rat prostatic epithelial cells

We have investigated the role of autocrine/paracrine TGF-β secretion in the regulation of cell growth by androgens as demonstrated by its inhibition by two androgen response modifiers; the nonsteroidal antiandrogen hydroxyflutamide (OHF), believed to act by inhibiting androgen binding to androgen receptors, or finasteride, an inhibitor of 5α-reductase, the enzyme necessary for the conversion of testosterone to 5α-dihydrotestosterone (DHT), using the nontumorigenic rat prostatic epithelial cell line NRP-152. Growth of these cells was stimulated three- to sixfold over control by either testosterone or DHT under serum-free culture conditions. This was accompanied by a two- to threefold decrease in the secretion rate of TGF-β1, -β2, and -β3. Finasteride reversed the ability of testosterone but not DHT to stimulate growth and downregulate expression of TGF-β1, -β2, and -β3 in a dose-dependent fashion, suggesting that this activity of testosterone required its conversion to DHT. OHF antagonized the stimulatory effects of DHT on NRP-152 cell growth but could reverse the inhibitory effects of DHT only on TGF-β2 and TGF-β3 and not TGF-β1 secretion. This suggests that either TGF-β1 regulation by DHT or the androgen antagonism of OHF occurs independent of androgen receptor binding. Neutralizing antibodies to TGF-β (pantropic and isoform-specific) were able to block the ability of finasteride to antagonize the effects of testosterone nearly completely while only partially inhibiting the antiandrogenic effects of OHF. Thus, the ability of androgens to stimulate growth of NRP-152 cells involves the downregulation of the production of TGF-β1, -β2, and -β3 in addition to other growth-stimulatory mechanisms. J. Cell. Physiol. 175:184–192, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

TGF-β receptor expression on human keratinocytes: A 150 kDa GPI-anchored TGF-β1 binding protein forms a heteromeric complex with type I and type II receptors

Keratinocytes play a critical role in re-epithelialization during wound healing, and alterations in keratinocyte proliferation and function are associated with the development of various skin diseases. Although it is well documented that TGF-β has profound effects on keratinocyte growth and function, there is a paucity of information on the types, isoform specificity and complex formation of TGF-β receptors on keratinocytes. Here, we report that in addition to the types I, II, and III TGF-β receptors, early passage adult and neonatal human keratinocytes display a cell surface glycosylphosphatidylinositol (GPI)-anchored 150 kDa TGF-β1 binding protein. The identities of the four proteins were confirmed on the basis of their affinity for TGF-β isoforms, immunoprecipitation with specific anti-receptor antibodies, sensitivity to phosphatidylinositol specific phospholipase C and dithiothreitol, and 2-dimensional electrophoresis. Interestingly, the antitype I TGF-β receptor antibody immunoprecipitated not only the type I receptor, but also the type II receptor and the 150 kDa component, suggesting that the 150 kDa component form heteromeric complexes with the signalling receptors. In addition, two-dimensional (nonreducing/reducing) electrophoresis confirmed the occurrence of a heterotrimeric complex consisting of the 150 kDa TGF-β1 binding protein, the type II receptor, and the type I receptor. This technique also demonstrated the occurrence of types I and II heterodimers and type I homodimers of TGF-β receptors on keratinocytes, supporting the heterotetrameric model of TGF-β signalling proposed using mutant cells and cells transfected to overexpress these receptors. The keratinocytes responded to TGF-β by markedly downregulating all four TGF-β binding proteins and by potently inhibiting DNA synthesis. The demonstration that the 150 kDa GPI-anchored TGF-β1 binding protein forms a heteromeric complex with the TGF-β signalling receptors suggests that this GPI-anchored protein may modify TGF-β signalling in human keratinocytes. J. Cell. Biochem. 70:573–586, 1998. © 1998 Wiley-Liss, Inc.     

Different roles of TGF-β in the multi-lineage differentiation of stem cells

Stem cells are a population of cells that has infinite or long-term self-renewal ability and can produce various kinds of descendent cells.Transforming growth factor β(TGF-β) family is a superfamily of growth factors,including TGF-β1,TGF-β2 and TGF-β3,bone morphogenetic proteins,activin/inhibin,and some other cytokines such as nodal,which plays very important roles in regulating a wide variety of biological processes,such as cell growth,differentiation,cell death.TGF-β,a pleiotropic cytokine,has been proved to be differentially involved in the regulation of multi-lineage differentiation of stem cells,through the Smad pathway,non-Smad pathways including mitogen-activated protein kinase pathways,phosphatidylinositol-3-kinase/AKT pathways and Rholike GTPase signaling pathways,and their cross-talks.For instance,it is generally known that TGF-β promotes the differentiation of stem cells into smooth muscle cells,immature cardiomyocytes,chondrocytes,neurocytes,hepatic stellate cells,Th17 cells,and dendritic cells.However,TGF-β inhibits the differentiation of stem cells into myotubes,adipocytes,endothelial cells,and natural killer cells.Additionally,TGF-β can provide competence for early stages of osteoblastic differentiation,but at late stages TGF-β acts as an inhibitor.The three mammalian isoforms(TGF-β1,2 and 3) have distinct but overlapping effects on hematopoiesis.Understanding the mechanisms underlying the regulatory effect of TGF-β in the stem cell multi-lineage differentiation is of importance in stem cell biology,and will facilitate both basic research and clinical applications of stem cells.In this article,we discuss the current status and progress in our understanding of different mechanisms by which TGF-β controls multi-lineage differentiation of stem cells.