Expression and synthesis of TGFbeta1 is induced in macrophages by 9-oxononanoyl cholesterol, a major cholesteryl ester oxidation product

Within the broad variety of compounds generated via oxidative reactions in low density lipoproteins (LDL) and subsequently found in the atherosclerotic plaque, are aldehydes still esterified to the parent lipid and termed core-aldehydes. The most represented cholesterol core-aldehyde in LDL is 9-oxononanoyl cholesterol (9-ONC), an oxidation product of cholesteryl linoleate. Here we report that 9-ONC, at concentration actually detectable in biological material, significantly up-regulates the expression and the synthesis of the pro-fibrogenic cytokine transforming growth factor beta1 (TGFbeta1) by cultured macrophages. As previously demonstrated for other lipid oxidation products present in LDL, namely a biologically representative mixture of oxysterols and the unesterified aldehyde 4-hydroxynonenal, these effects on TGFbeta1 by 9-ONC further points to LDL lipid oxidation as a powerful source of pro-fibrogenic stimuli.     

TGFβ inhibits GM-CSF-induced phosphorylation of ERK and MEK in human myeloid leukaemia cell lines via inhibition of phosphatidylinositol 3-kinase (PI3-k)

Objectives:  Activation of SMAD-independent p44/42 MAPK (ERK1/2) signalling by TGFβ has been recently reported in various cell types. However, the mechanisms for the linkage between the SMAD-dependent and -independent pathways are poorly understood. In this study, we investigated whether TGF-β activates the ERK pathway and how TGFβ communicates with the MAP kinase signals induced by a mitogen, in human myeloid leukaemia cells.
Materials and methods and results:  TGFβ dramatically suppressed proliferation of MV4–11 and TF-1 cells without detectable phosphorylation of ERK1/2 and MEK1/2 for the duration of 48 h, as detected by MTT assay and Western blot analysis, respectively. In contrast, GM-CSF induced rapid and transient phosphorylation of MEK1/2 and ERK1/2 and up-regulated cell proliferation. Both GM-CSF-induced ERK1/2 activation and cell proliferation were significantly inhibited by TGFβ. GM-CSF also induced transient phosphorylation of the p85 subunit of PI3-kinase. Corresponding to this change, phosphorylated p85 was found to bind to the GM-CSF receptor-α subunit, as detected by immunoprecipitation and Western blot analysis. PD98059, a selective inhibitor of MEK, blocked GM-CSF-induced phosphorylation of MEK and ERK but not p85. However, TGFβ and LY294002, a potent inhibitor of PI3-kinase, significantly inhibited phosphorylation of both p85 and ERK1/2.
Conclusions:  These studies thus indicate that TGFβ does not activate the ERK pathway but turns off the GM-CSF-induced ERK signal via inhibition of the PI3-kinase-Akt pathway, in these human laeukemia cells.     

Follistatin antagonizes transforming growth factor-beta3-induced epithelial-mesenchymal transition in vitro: implications for murine palatal development supported by microarray analysis

Abstract Epithelial–mesenchymal transition (EMT) is involved in normal embryonic development as well as in tumor progression and invasiveness. This process is also known to be a crucial step in palatogenesis during fusion of the bi-lateral palatal processes. Disruption of this step results in a cleft palate, which is among the most frequent birth defects in humans. A number of genes and encoded proteins have been shown to play a role in this developmental stage. The central role is attributed to the cytokine transforming growth factor-β3 (TGF-β3), which is expressed in the medial edge epithelium (MEE) already before the fusion process. The MEE covers the tips of the growing palatal shelves and eventually undergoes EMT or programmed cell death (apoptosis). TGF-β3 is described to induce EMT in embryonic palates. With regard to the early expression of this molecule before the fusion process, it is not well understood which mechanisms prevent the TGF-β3 producing epithelial cells from undergoing differentiation precociously. We used the murine palatal fusion to study the regulation of EMT. Specifically, we analyzed the MEE for the expression of known antagonists of TGF-β molecules using in situ hybridization and detected the gene coding for Follistatin to be co-expressed with TGF-β3. Further, we could show that Follistatin directly binds to TGF-β3 and that it completely blocks TGF-β3-induced EMT of the normal murine mammary gland (NMuMG) epithelial cell line in vitro . In addition, we analyzed the gene expression profile of NMuMG cells during TGF-β3-induced EMT by microarray hybridization, detecting strong changes in the expression of apoptosis-regulating genes.     

Cloning and expression of a rat Smad1: Regulation by TGFß and modulation by the ras/MEK pathway

A new family of signaling intermediates for TGFß superfamily members and other growth factors has recently been identified and termed Smads. It has been suggested that the Smad1 subfamily is regulated primarily by the TGFß superfamily member bone morphogenetic protein (BMP). Here we demonstrate that TGFß induced phosphorylation of endogenous Smad1 in untransformed IECs and that the RI and RII TGFß receptors were detectable in Smad1 immunocomplexes. Expression of a dominant-negative mutant of Ras inhibited the ability of TGFß to phosphorylate endogenous Smad1. In a separate series of experiments, we have cloned a rat homologue of the drosophila mad gene (termed RSmad1) by screening an intestinal epithelial cell (IEC) cDNA library. By using an in vitro kinase assay with RSmad1 as the substrate, we demonstrate that the TGFß receptor complex can directly phosphorylate RSmad1. We show, further, that a dominant-negative mutant of MEK1 inhibited the ability of RSmad1 to induce the TGFß-responsive reporter p3TP-Lux in a human breast cancer cell line. Collectively, our data demonstrate that TGFß can regulate Smad1 and that the Ras and MEK signaling components are partially required for the ability of TGFß to regulate Smad1. J. Cell. Physiol. 178:387–396, 1999. © 1999 Wiley-Liss, Inc.     

Bovine Chromaffin Cells Release a Transforming Growth Factor-β-Like Molecule Contained Within Chromaffin Granules

Abstract: Bovine chromaffin cells contain within their storage vesicles and release upon cholinergic stimulation a complex mixture of proteins and peptides. We present data suggesting that one of these proteins resembles transforming growth factor (TGF)-β in terms of its biological activity. The assay used to assess the activity of TGF-β is based on cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. The assay is highly specific in detecting TGF-β1, -β2, and -β3 but does not detect several cytokines and growth factors, such as fibroblast growth factor-2, transforming growth factor-α, platelet-derived growth factor-AB, insulin-like growth factor-I, or neurotrophin-3 or -4. Moreover, we show that this assay does not detect a wide range of TGF-β superfamily members (activin A, bone morphogenetic protein-2, -4, -6, and -7, growth/differentiation factor-5, and glial cell line-derived neurotrophic factor). Chromaffin granules contain ∼1 ng of TGF-β/10 mg of protein. The biological activity elicited by the chromaffin granule component can be neutralized by using an antibody against TGF-β1/β2/β3. TGF-β is releasable from cultured chromaffin cells stimulated with the cholinergic agonist carbachol (10⁻⁵ M ). These data suggest that TGF-β is stored in chromaffin granules and can be released by exocytosis.     

Activation of extracellular signal-regulated kinase by TGF-β1 via TβRII and Smad7 dependent mechanisms in human bronchial epithelial BEP2D cells

Transforming growth factor-β1 (TGF-β1) can activate mitogen-activated protein kinases (MAPKs) in many types of cells. The mechanism of this activation is not well elucidated. Here, we explore the role of TGF-β/Smads signaling compounds in TGF-β1-mediated activation of extracellular signal-regulated kinase (ERK) MAPK in human papillomavirus (HPV)-18 immortalized human bronchial epithelial cell line BEP2D and the role of TGF-β1-induced phosphorylation of ERK in proliferation and apoptosis of BEP2D. The cell models of siRNA-mediated silencing of TGF-β receptor type II (TβRII), Smad2, Smad3, Smad4, and Smad7 were employed in this study. Our results demonstrate that TGF-β1 activates ERK in a time-dependent manner with a maximum effect at 60 min; overexpression of Smad7 increased this TGF-β1-mediated phosphorylation of the ERK; and siRNA-mediated silencing of TβRII, Smad3, Smad4, and Smad7 abrogated this effect. Moreover, we observed that overexpression of Smad7 restored TGF-β1-mediated ERK phosphorylation in Smad4 knockdown cells but not in TβRII knockdown cells. In BEP2D cells, TGF-β1 treatment effectively inhibited cells’ proliferation and induced their apoptosis. Pretreatment with U0126, an inhibitor of ERK1/2, significantly enhanced the TGF-β1-mediated antiproliferative and apoptosis induction effects in BEP2D cells. These data revealed that TβRII and Smad7 play the critical roles in TGF-β1-mediated activation of ERK; Smad3 and Smad4 can play an indirect role through up-regulating Smad7 expression; and TGF-β1-induced phosphorylation of ERK may participate in BEP2D cell proliferation and apoptosis regulation.     

Macrophage-enhanced formation of cholesteryl ester-core aldehydes during oxidation of low density lipoprotein.

Oxidation of low density lipoproteins (LDL) results in changes to the lipoprotein particle that are potentially pro-atherogenic. To investigate mechanisms contributing to the formation of cholesteryl ester (CE)-core aldehydes (9-oxononanoyl- and 5-oxovaleroyl-cholesterol; 9-ONC and 5-OVC, respectively) LDL was incubated in the presence of mouse macrophages (J774 cells) under different culture conditions. Here we demonstrate that the formation of core aldehydes occurs only in transition metal-containing HAM's F10 medium but not in Dulbecco's modified Eagle's medium (DMEM), independent of supplementation with iron and copper at concentrations up to ten times higher than present in HAM's F10. The antioxidative properties of DMEM could be ascribed to the higher amino acid and vitamin content as compared to HAM's F10 medium. Supplementation with these components efficiently inhibited LDL oxidation in HAM's F10. Stimulation of J774 cells with phorbol ester (PMA) resulted in significantly enhanced 9-ONC and 5-OVC formation rates that were accompanied by increased consumption of LDL cholesteryl linoleate (Ch18:2) and cholesteryl arachidonate (Ch20:4) in the cellular supernatant. In PMA (10 ng/ml) activated cells, approximately 5% of Ch18:2 contained in LDL was converted to 9-ONC and 4% of Ch20:4 was converted to 5-OVC. With respect to core aldehyde formation, lipopolysaccharide (LPS, 10 microg/ml) was a less effective stimulant as compared to PMA. Part of the core aldehydes accumulated within the cells.Our study demonstrates that i) J774 macrophages are able to promote/accelerate core aldehyde formation in HAM's F10 medium, and ii) that core aldehyde formation rates can be increased by stimulation of the cells with PMA, and, although to a lesser extent, with LPS. Finally we could show that iii) a small amount of the core aldehydes is internalized by J774 macrophages.     

Covalent binding of oxidized cholesteryl esters to protein: implications for oxidative modification of low density lipoprotein and atherosclerosis

It has been proposed that plasma low density lipoproteins (LDL) undergo oxidative modification before they can produce foam cells in atherosclerosis. The oxidation of LDL generates a variety of reactive aldehydic products, which covalently bind to the LDL apolipoprotein B-100 (apoB). In the present study, to investigate the mechanisms contributing to the modification of LDL, we analyzed oxidized cholesteryl esters generated during the autoxidation of LDL and characterized their covalent binding to the lysine residues of LDL apoB. In addition, we raised a monoclonal antibody specific to a lysine-bound oxidized cholesteryl ester and determined its production in human atherosclerotic lesions. The peroxidation of LDL with Cu2+ produced 9-oxononanoylcholesterol (9-ONC) and 5-oxovaleroylcholesterol as the major oxidized cholesteryl esters. We observed that the levels of 9-ONC and 5-oxovaleroylcholesterol peaked at 12 h and significantly decreased thereafter. The reduction of the core aldehyde levels was accompanied by (i) the formation of free 7-ketocholesterol and 7-ketocholesteryl ester core aldehydes and (ii) an increase in the amounts of apoB-bound cholesterol and 7-ketocholesterol, suggesting that the cholesteryl ester core aldehydes were further converted to their 7-ketocholesterol- and apoB-bound derivatives. To detect the protein-bound 9-ONC, we raised the monoclonal antibody 2A81, directed against 9-ONC-modified protein, and found that it extensively recognized protein-bound cholesteryl ester core aldehydes. Agarose gel electrophoresis followed by immunoblot analysis of the oxidized LDL clearly demonstrated the formation of antigenic structures. Furthermore, immunohistochemical analysis of the atherosclerotic lesions from the human aorta showed that immunoreactive materials with mAb 2A81 were indeed present in the lesions, in which the intense immunoreactivity was mainly located in the macrophage-derived foam cells and the thickening neointima of the arterial walls. The results of this study suggest that the binding of cholesteryl ester core aldehydes to LDL might represent the process common to the oxidative modification of lipoproteins.     

Effect of Calcitriol in Combination with Corticosterone, Interleukin-1β, and Transforming Growth Factor-β1 on Nerve Growth Factor Secretion in an Astroglial Cell Line

Abstract: In astrocytes, nerve growth factor (NGF) synthesis has been described to be stimulated by the cytokines interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) and inhibited by corticosterone. As all three factors are present in the brain under certain conditions, we investigated the effect of their combined application on NGF secretion in the astroglial cell line RC7 and, in addition, studied the effect of calcitriol (1α,25-dihydroxyvitamin D₃). Calcitriol stimulated NGF secretion, whereas corticosterone reduced basal levels of NGF secretion as well as inhibited the NGF secretion induced by IL-1β, calcitriol, and TGF-β1. Calcitriol had an additive effect when applied together with IL-1β and a synergistic effect when applied with TGF-β1. Moreover, calcitriol not only counteracted the inhibitory effect of corticosterone on NGF secretion stimulated by TGF-β1 but even augmented it to a level more than threefold higher than that reached with TGF-β1 alone. Due to the trophic effect of NGF on basal forebrain cholinergic neurons, these findings might be of therapeutic relevance under conditions where cholinergic function is impaired and the endogenous levels of corticosterone, IL-1β, or TGF-β1 are elevated.     

Cellular Localization and Temporal Elevation of Tumor Necrosis Factor-α, Interleukin-1α, and Transforming Growth Factor-β1 mRNA in Hippocampal Injury Response Induced by Trimethyltin

Abstract: In certain pathologic states, cytokine production may become spatially and temporally dysregulated, leading to their inappropriate production and potentially detrimental consequences. Tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, and transforming growth factor-β (TGF-β) mediate a range of host responses affecting multiple cell types. To study the role of cytokines in the early stages of brain injury, we examined alterations in the 17-day-old mouse hippocampus during trimethyltin-induced neurodegeneration characterized by neuronal necrosis, microglia activation in the dentate, and astrocyte reactivity throughout the hippocampus. By 24 h after dosing, elevations in mRNA levels for TNF-α, IL-1α, IL-1β, and IL-6 mRNA were seen. TGF-β1 mRNA was elevated at 72 h. In situ hybridization showed that TNF-α and IL-1α were localized to the microglia, whereas TGF-β1 was expressed predominantly in hippocampal pyramidal cells. Intercellular adhesion molecule-1, EB-22, Mac-1, and glial fibrillary acidic protein mRNA levels were elevated within the first 3 days of exposure in the absence of increased inducible nitric oxide synthetase and interferon-γ mRNA. These data suggest that pro-inflammatory cytokines contribute to the progression and pattern of neuronal degeneration in the hippocampus.     

Bioactive TGF-β can associate with lipoproteins and is enriched in those containing apolipoprotein E3

Transforming growth factor-β1 (TGF-β1) has central functions in development, tissue maintenance, and repair and has been implicated in major diseases. We discovered that TGF-β1 contains several amphipathic helices and hydrophobic domains similar to apolipoprotein E (apoE), a protein involved in lipoprotein metabolism. Indeed, TGF-β1 associates with lipoproteins isolated from human plasma, cultured liver cells, or astrocytes, and its bioactivity was highest in high-density lipoprotein preparations. Importantly, lipoproteins containing the apoE3 isoform had higher TGF-β levels and bioactivity than those containing apoE4, a major genetic risk factor for atherosclerosis and Alzheimer's disease. Because TGF-β1 can be protective in these diseases an association with apoE3 may be beneficial. Association of TGF-β with different types of lipoproteins may facilitate its diffusion, regulate signaling, and offer additional specificity for this important growth factor.     

Expression of interleukin-1 receptors and their role in interleukin-1 actions in murine microglial cells

Interleukin (IL)-1 is an important mediator of acute brain injury and inflammation, and has been implicated in chronic neurodegeneration. The main source of IL-1 in the CNS is microglial cells, which have also been suggested as targets for its action. However, no data exist demonstrating expression of IL-1 receptors [IL-1 type-I receptor (IL-1RI), IL-1 type-II receptor (IL-1RII) and IL-1 receptor accessory protein (IL-1RAcP)] on microglia. In the present study we investigated whether microglia express IL-1 receptors and whether they present target or modulatory properties for IL-1 actions. RT-PCR analysis demonstrated lower expression of IL-1RI and higher expression of IL-1RII mRNAs in mouse microglial cultures compared with mixed glial or pure astrocyte cultures. Bacterial lipopolysaccharide (LPS) caused increased expression of IL-1RI, IL-1RII and IL-1RAcP mRNAs, induced the release of IL-1beta, IL-6 and prostaglandin-E2 (PGE2), and activated nuclear factor kappaB (NF-kappaB) and the mitogen-activated protein kinases (MAPKs) p38, and extracellular signal-regulated protein kinase (ERK1/2), but not c-Jun N-terminal kinase (JNK) in microglial cultures. In comparison, IL-1beta induced the release of PGE2, IL-6 and activated NF-kappaB, p38, JNK and ERK1/2 in mixed glial cultures, but failed to induce any of these responses in microglial cell cultures. IL-1beta also failed to affect LPS-primed microglial cells. Interestingly, a neutralizing antibody to IL-1RII significantly increased the concentration of IL-1beta in the medium of LPS-treated microglia and exacerbated the IL-1beta-induced IL-6 release in mixed glia, providing the first evidence that microglial IL-1RII regulates IL-1beta actions by binding excess levels of this cytokine during brain inflammation.     

Specificity in the crosstalk of TGFbeta/GDNF family members is determined by distinct GFR alpha receptors

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) are neurotrophic factors for parasympathetic neurons including ciliary ganglion (CG) neurons. Recently, we have shown that survival and signaling mediated by GDNF in CG neurons essentially requires transforming growth factor β (TGFβ). We have provided evidence that TGFβ regulates the availability of the glycosyl phosphatidylinositol (GPI)-anchored GDNF receptor alpha 1 (GFRα1) by promoting the recruitment of the receptor to the plasma membrane. We report now that in addition to GDNF, NRTN, but not persephin (PSPN) or artemin (ARTN), is able to promote survival of CG neurons. Interestingly, in contrast to GDNF, NRTN is not dependent on cooperation with TGFβ, but efficiently promotes neuronal survival and intracellular signaling in the absence of TGFβ. Additional treatment with TGFβ does not further increase the NRTN response. Both NRTN and GDNF exclusively bind to and activate their cognate receptors, GFRα2 and GFRα1, respectively, as shown by the use of receptor-specific neutralizing antibodies. Immunocytochemical staining for the two receptors on the surface of CG neurons reveals that, in contrast to the effect on GFRα1, TGFβ is not required for recruitment of GFRα2 to the plasma membrane. Moreover, binding of radioactively labeled GDNF but not NRTN is increased upon treatment of CG neurons with TGFβ. Disruption of TGFβ signaling does interfere with GDNF-, but not NRTN-mediated signaling and survival. We propose a model taking into account data from GFRα1 crystallization and ontogenetic development of the CG that may explain the differences in TGFβ-dependence of GDNF and NRTN.     

Expression of type I and II receptors for transforming growth factor β in the adult rat testis

 After having established the specificity of the antibodies for the rat testis by western blot analysis, the potential target cells for transforming growth factors (TGFβs) were identified by immunohistochemical detection of both type I (TβRI) and type II (TβRII) transducing receptors for TGFβs in the adult rat testis in situ. Leydig cells showed a strong TβRII immunoreactivity whereas the TβRI staining was weak. Only TβRII was detectable in Sertoli cells. In germ cells, staining for TβRI was stronger than for TβRII and the expression of both receptors depended on the seminiferous cycle stage. TβRI first appeared in pachytene spermatocytes and was absent in elongated spermatids from stage XIV onwards. Labelling for TβRII was observed as early as the spermatogonia stage; it increased in pachytene spermatocytes at the onset of TβRI and disappeared in elongating spermatids from stage XI onwards. These results show that TGFβs can affect somatic cells functions and suggest that these factors are involved in the control of meiosis and early spermiogenesis, exerting a direct effect on germ cells. Accepted: 18 June 1998     

Cytokine-Regulated Expression of Platelet-Derived Growth Factor Gene and Protein in Cultured Human Astrocytes

Abstract: To elucidate mechanisms regulating the production of platelet-derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte-enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF AB protein was detected in serum-free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor-β₁ (TGF-β₁) or tumor necrosis factor-α (TNF-α); the largest increase was detected after combined treatment with the two cytokines. Interleukin-1β (IL-1β) by itself had little or no effect but synergized with TGF-β₁ in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A- and PDGF B-chain mRNAs were detected in untreated astrocytes. PDGF B-chain mRNA levels were increased by TGF-β₁, TNF-α, TNF-α/TGF-β₁, or IL-1β/TGF-β₁, whereas PDGF A-chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF-β₁, TNF-α, and IL-1β are able to stimulate astrocyte PDGF production. This cytokine network could play a role in CNS development and repair after injury or inflammation.     

Up-regulation of IL-1 receptor type I and tyrosine hydroxylase in the rat carotid body following intraperitoneal injection of IL-1β

It is well established that reciprocal modulation exists between the central nervous system and immune system. Interleukin (IL)-1β, a proinflammatory cytokine secreted at early stage of immune challenge, has been recognized as one of the informational molecules in immune-to-brain communication. However, how this large molecule is transmitted to the brain is still unknown. In recent years it has been reported that the cranial nerves, especially the vagus, may play a pivotal role in this regard. It is proposed that IL-1β may bind to its corresponding receptors located in the glomus cells of the vagal paraganglia and then elicit action potentials in the nerve. The existence of IL-1 receptor type I (IL-1RI) in the vagal paraganglia has been shown. The carotid body, which is the largest peripheral chemoreceptive organ, is also a paraganglion. We hypothesize that the carotid body might play a role similar to the vagal paraganglia because they are architectonically similar. Recently we verified the presence of IL-1RI in the rat carotid body and observed increase firing in the carotid sinus nerve following IL-1β stimulation. The aim of this study was to observe the changes in expression of IL-1RI and tyrosine hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis, in the glomus cells of the rat carotid body following intraperitoneal injection of IL-1β. The radioimmunoassay result showed that the blood IL-1β level was increased after the intraperitoneal injection of rmIL-1β (750 ng/kg) from 0.48 ± 0.08 to 0.78 ± 0.07 ng/ml (P < 0.05). Immunofluorescence and Western blot analysis showed that the expression of IL-1RI and TH in the rat carotid body was increased significantly following peritoneal IL-1β stimulation. In addition, double immunofluorescence labeling for TH and PGP9.5, a marker for glomus cells, or TH immunofluoresence with hematoxylin-eosin (HE) counterstaining revealed that a considerable number of glomus cells did not display TH immunoreactivity. These data provide morphological evidence for the response of the carotid body to proinflammatory cytokine stimulation. The results also indicate that not all of the glomus cells express detectable TH levels either in normal or in some abnormal conditions. Xi-Jing Zhang and Xi Wang are co-first authors.