The transcriptional control of TGF-beta in human osteoblast-like cells is distinct from that of IL-1 beta.

Transforming growth factor beta (TGF-beta) and interleukin 1 (IL-1) are among the most potent osteotropic cytokines. The expression of mRNA for both TGF-beta and IL-1 beta was studied in human osteoblast-like cells in vitro. These cells constitutively expressed TGF-beta but not IL-1 beta mRNA. Treatment of the cells with the systemic hormones 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (10(-8) M) and parathyroid hormone (10(-7) M) induced an increase in TGF-beta mRNA but failed to stimulate the production of IL-1-beta mRNA. Retinoic acid (10(-8) M) had no effect on either mRNA species. The cytokines IL-1 alpha (200 pg/ml), tumour necrosis factor alpha (TNF-alpha) (17 ng/ml) and bacterial lipopolysaccharide (LPS) (500 ng/ml) stimulated the production of IL-1 beta mRNA after 6-8 hours. This was followed by an increase in protein production after 24 hours. In contrast, the production of TGF-beta mRNA remained constant after treatment with these agents. Treatment of the cells with hydrocortisone (10(-8) M) resulted in the suppression of both TGF-beta and IL-1 beta mRNA. However, when the stimulating agent 1,25-(OH)2D3 was added in conjunction with hydrocortisone the mRNA expression of TGF-beta mRNA returned to 70% of the stimulated level. In contrast, the addition of the stimulatory agent IL-1 alpha to hydrocortisone-treated cells resulted in no increase in IL-1 beta mRNA. In-situ hybridization demonstrated both TGF-beta and IL-1 beta mRNA at the cellular level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression patterns of PDGF-A, -B, -C and -D and the PDGF-receptors alpha and beta in activated rat hepatic stellate cells (HSC)

The platelet-derived growth factor (PDGF) family, which regulates many physiological and pathophysiological processes has recently been enlarged by two new members, the isoforms PDGF-C and -D. Little is known about the expression levels of these new members in hepatic fibrosis. We therefore investigated by quantitative real time PCR (Taqman) the mRNA expression profiles of all four PDGF isoforms in transdifferentiating primary cultured hepatic stellate cells (HSC), an in vitro model system of hepatic fibrogenesis, either with or without stimulation of the cells with PDGF-BB or TGF-beta1. All four isoforms were expressed in HSC transdifferentiating to myofibroblast-like cells (MFB) albeit with different profiles: while PDGF-A mRNA exhibited minor fluctuations only, PDGF-B was rapidly down-regulated. In contrast, both PDGF-C and -D mRNA were strongly induced: PDGF-C up to 5 fold from day 2 to day 8 and PDGF-D up to 8 fold from day 2 to day 5 of culture. Presence of PDGF-DD in activated HSC was confirmed at the protein level by immunocytochemistry. Stimulation of HSC and MFB with PDGF-BB led to down-regulation of the new isoforms, whereas TGF-beta1 upregulated PDGF-A only. We further show that PDGF receptor-beta (PDGFR-beta) mRNA was rapidly upregulated within the first day of culture and was constantly expressed from day 2 on while the expression profile of PDGFR-alpha mRNA was very similar to that of PDGF-A during transdifferentiation. Given the dramatic changes in PDGF-C and -D expression, which may compensate for down-regulation of PDGF-B, we hypothesize that the new PDGF isoforms may fulfil specific functions in hepatic fibrogenesis.     

Regulation of TGF-beta 1-induced connective tissue growth factor expression in airway smooth muscle cells

Transforming growth factor (TGF)-beta may play an important role in airway remodeling, and the fibrogenic effect of TGF-beta may be mediated through connective tissue growth factor (CTGF) release. We investigated the role of MAPKs and phosphatidylinositol 3-kinase (PI3K) and the effects of inflammatory cytokines on TGF-beta-induced CTGF expression in human airway smooth muscle cells (ASMC). We examined whether Smad signal was involved in the regulatory mechanisms. TGF-beta 1 induced a time- and concentration-dependent expression of CTGF gene and protein as analyzed by real-time RT-PCR and Western blot. Inhibition of ERK and c-jun NH(2)-terminal kinase (JNK), but not of p38 MAPK and PI3K, blocked the effect of TGF-beta 1 on CTGF mRNA and protein expression and on Smad2/3 phosphorylation. T helper lymphocyte 2-derived cytokines, IL-4 and IL-13, attenuated TGF-beta 1-stimulated mRNA and protein expression of CTGF and inhibited TGF-beta 1-stimulated ERK1/2 and Smad2/3 activation in ASMC. The proinflammatory cytokines tumor necrosis factor-alpha and IL-1 beta reduced TGF-beta 1-stimulated mRNA expression of CTGF but did not inhibit TGF-beta-induced Smad2/3 phosphorylation. TGF-beta 1-stimulated CTGF expression is mediated by mechanisms involving ERK and JNK pathways and is downregulated by IL-4 and IL-13 through modulation of Smad and ERK signals.     

Regulation of vascular smooth muscle cell integrin expression by transforming growth factor beta1 and by platelet-derived growth factor-BB.

We have examined the ability of transforming growth factor-beta 1 (TGF-beta 1) and platelet-derived growth factor-BB (PDGF-BB) to regulate the expression of various integrins in cultured rabbit vascular smooth muscle cells (SMC). We found that expression of the alpha v beta 3 integrin complex was induced by both growth factors, although TGF-beta 1 appeared to be the more potent inducer. mRNA level of the beta 3 integrin subunit was undetectable in quiescent cells and enhanced by both growth factors, while the alpha v integrin subunit mRNA level did not change with growth factor addition. Therefore, appearance of the alpha v beta 3 integrin protein complex after growth factor stimulation was due to increased expression of the beta 3 integrin subunit mRNA. The TGF-beta 1 induced increase in beta 3 integrin mRNA was delayed, but did not require prior protein synthesis, since cycloheximide was unable to block the increase in beta 3 mRNA level. By contrast, PDGF-BB induced a more rapid increase in beta 3 integrin mRNA level that peaked by 6 h after growth factor addition and no detectable beta 3 integrin mRNA remained after 24 h. Interestingly, the PDGF-BB induced elevation of beta 3 integrin, although more rapid, was completely inhibited by cycloheximide. Expression of the alpha 5 integrin subunit in response to growth factors was very similar to beta 3. However, in contrast to beta 3 and alpha 5, neither TGF-beta 1 nor PDGF-BB were able to alter the expression of the beta 1 integrin subunit in vascular SMC. However, in TGF-beta 1 treated cells, there was a large increase in expression of a 190 kDa polypeptide that was associated with the beta 1 integrin subunit. This 190 kDa polypeptide was not detected in PDGF treated SMC or in TGF-beta 1 treated fibroblasts. The alpha 1 integrin subunit has a MW of approximately 190 kDa and is capable of complexing with beta 1. Analysis of the alpha 1 integrin subunit mRNA level indicated that it was indeed induced by TGF-beta 1, but not by PDGF-BB, suggesting that the 190 kDa polypeptide may be the alpha 1 integrin subunit. These results indicate that TGF-beta 1 and PDGF-BB are potent but distinct activators of integrin expression in vascular SMC.     

Differential expression of transforming growth factor-beta 1, -beta 2, and -beta 3 by glioblastoma cells, astrocytes, and microglia.

The type beta transforming growth factors (TGF) are potent regulators of the growth and functions of lymphocytes and macrophages. Recently the human glioblastoma cell line 308 was shown to produce TGF-beta 2. The relevance of this finding was evaluated further by comparing human glioblastoma cells with their nontransformed animal counterpart, astrocytes, with regard to the production of the three TGF-beta isoforms observed so far in mammals. In this report astrocytes are demonstrated to secrete also TGF-beta 2 and to express TGF-beta 1, -beta 2, and -beta 3 mRNA in vitro. In contrast, cultured murine brain macrophages release TGF-beta 1 and are positive for TGF-beta 1 mRNA only. Glia cell-derived TGF-beta 1 and -beta 2 are detected in latent form whereas both latent and active TGF-beta are identified in the supernatant of three human glioblastoma cell lines tested. These cell lines, however, show heterogeneity in regard to the isoform of TGF-beta expressed but share with astrocytes the inability to release TGF-beta 3. Provided production and activation of latent TGF-beta occur in vivo, astrocytes and microglia may then be expected to exert regulatory influences on immune mediated diseases of the central nervous system.     

During Drosophila spermatogenesis beta 1, beta 2 and beta 3 tubulin isotypes are cell-type specifically expressed but have the potential to coassemble into the axoneme of transgenic flies

alpha and beta Tubulins exist in a number of different isotypes with distinct expression patterns during development. We have shown by immunofluorescent staining that beta 1, beta 2 and beta 3 tubulins are distributed very specifically in the testes of Drosophila. beta 3 Tubulin is present exclusively in cytoplasmic microtubules of cells somatic in origin, while the beta 1 isotype is localized in the somatic cells and in early germ cells of both the microtubules of the cytoskeleton as well as in the mitotic spindle. In contrast, beta 2 tubulin is present in all microtubular arrays (cytoskeleton, meiotic spindles, axoneme) of germ cells from meiotic prophase onward, though not detectable in somatic cells. Thus, a switch of beta tubulin isotypes from beta 1 to beta 2 occurs during male germ cell differentiation. This switch is also observed in the distantly related species Drosophila hydei. By fusing beta 1 or beta 3 amino acid coding regions to the control region of the beta 2 tubulin gene and performing germ line transformation experiments, we have examined the copolymerization properties of the different tubulin isotypes. Neither beta 1 nor beta 3 are detectable in the axoneme in the wild-type situation. Analysis of transgenic flies carrying beta 2-beta 1 fusion genes or beta 2-beta 3 fusion genes revealed that both beta 1 and beta 3 tubulin isotypes have the potential to co-incorporate with beta 2 tubulin into microtubules of the sperm axoneme. Male flies homozygous for the fusion genes (beta 2-beta 1 or beta 2-beta 3) remain fertile, despite the mixture of beta tubulin isotypes in the axoneme.     

Upregulation of fibronectin but not of entactin, collagen IV and smooth muscle actin by anaphylatoxin C5a in rat hepatic stellate cells

Rat Kupffer cells (KC), hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC) all express the C5a receptor (C5aR) constitutively in contrast to hepatocytes (HC). HSC showed an unexpectedly high level of expression of the C5aR. As these cells are known to play a key role in the induction of liver fibrosis we hypothesized that C5a may possibly induce fibrogenetic proteins in these cells. HSC are known to express the extracellular matrix (ECM) proteins collagen IV, fibronectin, entactin and the structure protein smooth muscle actin (SMA) which is regarded as a marker for the fibrotic conversion of HSC to myofibroblast-like cells. We investigated the effect of recombinant rat C5a (rrC5a) on the upregulation of these ECM-proteins and of SMA, all of which are known to be expressed by HSC. The profibrotic cytokine TGF-beta1 (2 ng/ml), which was used as a control, clearly upregulated the three matrix proteins but not SMA. In the absence of any stimulus HSC upregulated the three ECM-proteins as well as SMA during their conversion into myofibroblast-like cells. This resulted in a high stimulus-independent plateau of the mRNA expressions for all four proteins after four to five days of culture. Readouts were therefore taken at 72 h after the isolation of the HSC when the investigated mRNA levels had not yet reached their maxima due to the conversion of the cells. The first 24 h of culture were performed without stimulus and the following 48 h in the presence of 100 nM rrC5a (1 micro g/ml) or TGF-beta1 (2 ng/ml). Only fibronectin-specific mRNA was clearly upregulated by C5a whereas entactin, collagen IV and SMA were not affected by C5a. By competitive-quantitative PCR the upregulation of fibronectin-specific mRNA was determined to be about five-fold. As TGF-beta1 upregulated all of the three investigated ECM-proteins but not SMA it was checked as to whether C5a might act indirectly by upregulating the expression of TGF-beta1 in KC and HSC, as both cell types are known to be sources of this profibrotic cytokine. However, using RT-PCR, such an effect was not detectable in either cell type after 3, 10 or 24 h.     

C型产气荚膜梭菌β1、β2毒素基因的融合

利用PCR技术 ,从C型产气荚膜梭菌染色体DNA中扩增出 β1 和 β2 毒素基因 ,构建了含 β1 - β2 融合基因表达质粒的重组菌株BL2 1(DE3) (pETXB1_2 )。经酶切鉴定和序列测定证实 ,构建的重组质粒pETXB1_2含有 β1 - β2 融合基因 ,且基因序列和阅读框架正确。经ELISA检测 ,重组菌株表达的 β1 - β2 融合蛋白能够被 β1 、β2 毒素抗体识别。免疫实验结果表明 ,用β1 - β2 融合蛋白免疫的小鼠可以抵抗 1MLD的C型产气荚膜梭菌C5 9_4 4毒素攻击 ,表明构建的重组菌株可以作为预防仔猪红痢基因工程亚单位苗的候选菌株。     

Differential regulation of the expression of transforming growth factor-beta mRNAs by growth factors and retinoic acid in chicken embryo chondrocytes, myocytes, and fibroblasts.

Transforming growth factor-beta (TGF-beta) autoregulates its expression in several mammalian cell types. We now report that addition of TGF-beta s 1, 2, and 3 to primary chicken embryo cells differentially affects expression of the messenger RNAs for the different TGF-beta isoforms depending on the cell type. In cultured sternal chondrocytes, addition of TGF-beta s 1, 2, or 3 results in an increase in the steady-state levels of the messenger RNAs for TGF-beta s 2 and 3, but does not change expression of TGF-beta 4 mRNA. In contrast, in cultured cardiac myocytes, addition of TGF-beta s 1, 2, or 3 results in an increase in expression of TGF-beta s 3 and 4 mRNAs, but does not change expression of TGF-beta 2 mRNA. Moreover, expression of TGF-beta s 2, 3, and 4 mRNAs is not affected by addition of any of the TGF-beta s to fibroblasts. Addition of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or interleukin-1 (IL-1) to these chicken cells also has differential effects on expression of the different TGF-beta mRNAs depending on the cell type. Retinoic acid also has contrasting effects on chondrocytes and myocytes either increasing or decreasing, respectively, expression of TGF-beta s 2 and 3 mRNAs and TGF-beta 2 protein. Our results indicate a complex pattern of regulation of the different TGF-beta genes by themselves as well as by PDGF, EGF, IL-1, dexamethasone, TPA, and retinoic acid in chicken embryo cells.     

Signaling to the epithelium is not sufficient to mediate all of the effects of transforming growth factor beta and bone morphogenetic protein 4 on murine embryonic lung development.

Many studies have suggested that transforming growth factor beta (TGF-beta) and bone morphogenetic protein 4 (Bmp4) regulate early development of the lung. In this study, administration of growth factors directly into the lumen of lungs grown in organ culture was used to limit their activity to the epithelium and test the hypothesis that signaling to the epithelium is sufficient to mediate the known effects of TGF-beta and BMP-4 on early lung development. Addition of TGF-beta1, beta2, or beta3 to the medium surrounding lungs grown in organ culture resulted in decreased branching, reduced cell proliferation, accumulation of alpha-smooth muscle actin protein (alpha-SMA) in the mesenchyme, and decreased expression of a marker for respiratory epithelium, surfactant protein-C (Sp-C). When TGF-beta1 was restricted to the epithelium, accumulation of alpha-SMA and inhibition of Sp-C expression were not observed but branching and proliferation were inhibited. In contrast, branching was not inhibited in lungs where TGF-beta2 or TGF-beta3 were restricted to the epithelium suggesting differences in the mechanism of signaling by TGF-beta1, TGF-beta2 or TGF -beta3 in lung. Addition of Bmp4 to the medium surrounding lungs grown in organ culture stimulated cell proliferation and branching morphogenesis; however, direct injection of Bmp4 into the lung lumen had no effect on proliferation or branching. Based on these data and data from mesenchyme-free cultures, we propose that the mesenchyme influences growth factor signaling in the lung.     

Inhibition of glycogen synthase kinase-3beta is sufficient for airway smooth muscle hypertrophy

We examined the role of glycogen synthase kinase-3beta (GSK-3beta) inhibition in airway smooth muscle hypertrophy, a structural change found in patients with severe asthma. LiCl, SB216763, and specific small interfering RNA (siRNA) against GSK-3beta, each of which inhibit GSK-3beta activity or expression, increased human bronchial smooth muscle cell size, protein synthesis, and expression of the contractile proteins alpha-smooth muscle actin, myosin light chain kinase, smooth muscle myosin heavy chain, and SM22. Similar results were obtained following treatment of cells with cardiotrophin (CT)-1, a member of the interleukin-6 superfamily, and transforming growth factor (TGF)-beta, a proasthmatic cytokine. GSK-3beta inhibition increased mRNA expression of alpha-actin and transactivation of nuclear factors of activated T cells and serum response factor. siRNA against eukaryotic translation initiation factor 2Bepsilon (eIF2Bepsilon) attenuated LiCl- and SB216763-induced protein synthesis and expression of alpha-actin and SM22, indicating that eIF2B is required for GSK-3beta-mediated airway smooth muscle hypertrophy. eIF2Bepsilon siRNA also blocked CT-1- but not TGF-beta-induced protein synthesis. Infection of human bronchial smooth muscle cells with pMSCV GSK-3beta-A9, a retroviral vector encoding a constitutively active, nonphosphorylatable GSK-3beta, blocked protein synthesis and alpha-actin expression induced by LiCl, SB216763, and CT-1 but not TGF-beta. Finally, lungs from ovalbumin-sensitized and -challenged mice demonstrated increased alpha-actin and CT-1 mRNA expression, and airway myocytes isolated from ovalbumin-treated mice showed increased cell size and GSK-3beta phosphorylation. These data suggest that inhibition of the GSK-3beta/eIF2Bepsilon translational control pathway contributes to airway smooth muscle hypertrophy in vitro and in vivo. On the other hand, TGF-beta-induced hypertrophy does not depend on GSK-3beta/eIF2B signaling.     

Beta ig-h3 induces keratinocyte differentiation via modulation of involucrin and transglutaminase expression through the integrin alpha3beta1 and the phosphatidylinositol 3-kinase/Akt signaling pathway

Beta ig-h3 is an extracellular matrix protein whose expression is highly induced by transforming growth factor (TGF)-beta1. Whereas beta ig-h3 is known to mediate keratinocyte adhesion and migration, its effects on keratinocyte differentiation remain unclear. In the present study, it was demonstrated that expression of both beta ig-h3 and TGF-beta1 was enhanced during keratinocyte differentiation and that expression of the former was strongly induced by that of the latter. This study also asked whether changes in beta-h3 expression would affect keratinocyte differentiation. Indeed, down-regulation of beta ig-h3 by transfection with antisense beta ig-h3 cDNA constructs effectively inhibited keratinocyte differentiation by decreasing the promoter activities and thus expression of involucrin and transglutaminase. The result was an approximately 2-fold increase in mitotic capacity of the cells. Conversely, overexpression of beta ig-h3, either by transfection with beta ig-h3 expression plasmids or by exposure to recombinant beta ig-h3, enhanced keratinocyte differentiation by inhibiting cell proliferation and concomitantly increasing involucrin and transglutaminase expression. Recombinant beta ig-h3 also promoted keratinocyte adhesion through interaction with integrin alpha3beta1. Changes in beta ig-h3 expression did not affect intracellular calcium levels. Subsequent analysis revealed not only induction of Akt phosphorylation by recombinant beta ig-h3 but also blockage of Akt phosphorylation by LY294002, an inhibitor of phosphatidylinositol 3-kinase. Taken together, these findings indicate that enhanced beta ig-h3, induced by enhanced TGF-beta during keratinocyte differentiation, provoked cell differentiation by enhancing involucrin and transglutaminase expression through the integrin alpha3beta1 and phosphatidylinositol 3-kinase/Akt signaling pathway. Lastly, it was observed that beta ig-h3-mediated keratinocyte differentiation was caused by promotion of cell adhesion and not by calcium regulation.     

IL-1beta, BK, and TGF-beta1 attenuate PGI2-mediated cAMP formation in human pulmonary artery smooth muscle cells by multiple mechanisms involving p38 MAP kinase and PKA

We have previously shown that interleukin (IL)-1beta, transforming growth factor (TGF)-beta1, or bradykinin (BK) impair cAMP generation in response to prostacyclin analogs in human pulmonary artery smooth muscle (PASM), suggesting that inflammation can impair the effects of prostacyclin analogs on PASM in pulmonary hypertension. Here we explored the biochemical mechanisms involved. We found that IL-1beta, BK, and TGF-beta1 reduced adenylyl cyclase isoform 1, 2, and 4 mRNA, increased Galphai protein levels, and reduced prostacyclin receptor (IP receptor) mRNA expression. In contrast, Galphas protein levels were unchanged. Protein kinase A (PKA) (H-89, KT-2750, PKIm) and p38 mitogen-activated protein (MAP) kinase (SB-202190) inhibitors attenuated these effects, but protein kinase C (bisindolylmaleide) or phosphoinositol 3-kinase (LY-294002) inhibitors did not. Fluorescent kemptide assay and Western blotting confirmed that PKA and p38 MAP kinase were activated by IL-1beta, BK, and TGF-beta1. These studies suggest that IL-1beta, BK, and TGF-beta1 impair IP receptor-mediated cAMP accumulation by multiple effects on different components of the signaling pathway and that these effects are PKA and p38 MAP kinase dependent.     

Discordant transforming growth factor beta 1 RNA and protein localization during chemical carcinogenesis of the skin.

Transforming growth factor beta (TGF-beta) inhibits proliferation of normal keratinocytes, and this response is retained, to variable extents, in benign tumors of the skin (S. Haddow, D. J. Fowlis, K. Parkinson, R. J. Akhurst, and A. Balmain, Oncogene, 6: 1465-1470, 1991). To investigate the profile of TGF-beta biosynthesis during various stages of chemical carcinogenesis of the skin, we used a combination of ribonuclease protection assay, in situ hybridization with gene-specific probes for TGF-beta 1, -beta 2, and -beta 3, and immunohistochemistry with isoform-specific antibodies against TGF-beta 1. Following 12-O-tetradecanoylphorbol-13-acetate treatment of adult mouse skin, there was a rapid induction of TGF-beta 1 protein. Intracellular TGF-beta 1 protein was localized to suprabasal keratinocytes, and the extracellular form was localized predominantly to the dermis. Despite ubiquitous induction of TGF-beta 1 protein by 12-O-tetradecanoylphorbol-13-acetate in various mouse strains, we noted strain-specific differences in the quantitative induction of TGF-beta 1 RNA. Papillomas and carcinomas induced in vivo had elevated levels of TGF-beta 1 RNA within the basal keratinocyte compartment but did not contain significant levels of TGF-beta 1 protein within the tumor. We postulate that the tumor evades TGF-beta 1-controlled negative growth regulation by altered translational and/or posttranslational processing mechanisms of this growth factor. Levels of TGF-beta 2 and -beta 3 RNA were not elevated at any stage of chemical carcinogenesis of the skin.     

Glycated human serum albumin enhances macrophage inflammatory protein-1beta mRNA expression through protein kinase C-delta and NADPH oxidase in macrophage-like differentiated U937 cells

BACKGROUND: In a previous report (Higai K et al., Biol Pharm Bull, 2007), glycated human serum albumin (Glc-HSA) was found to induce interleukin-8 (IL-8) mRNA expression in human monocyte-derived U937 cells through a reactive oxygen species (ROS)-dependent pathway; however, Glc-HSA signaling has not been elucidated in macrophages. METHODS: U937 cells were differentiated by treatment with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) for 2 days and the macrophage-like differentiated U937 (differentiated U937) cells were stimulated with Glc-HSA and glycolaldehyde dimer-modified HSA (GA-HSA) in the presence of various signaling inhibitors. Macrophage inflammatory protein-1beta (MIP-1beta) mRNA expression was determined by real-time PCR. Intracellular ROS generation was estimated by confocal laser microscopy. RESULTS: Glc-HSA and GA-HSA markedly enhanced MIP-1beta mRNA expression in differentiated U937 cells. Enhanced MIP-1beta mRNA expression was completely suppressed by the ROS scavenger N-acetyl-l-cysteine, the NADPH oxidase inhibitors diphenylene iodonium and apocynin, and the protein kinase C (PKC)-delta inhibitor rottlerin. Furthermore, ROS generation was suppressed completely by rottlerin but not by the PKC-gamma inhibitor Ro318425 or the PKC-alpha, -beta1 and -micro inhibitor Go6976. CONCLUSION: Glc-HSA and GA-HSA enhance MIP-1beta mRNA expression in differentiated U937 cells through PKC-delta-dependent activation of NADPH oxidase.     

Expression of TGF-beta signaling genes in the normal, premalignant, and malignant human trophoblast: loss of smad3 in choriocarcinoma cells

We had earlier shown that TGF-beta controls proliferation, migration, and invasiveness of normal human trophoblast cells, whereas premalignant and malignant trophoblast cells are resistant to TGF-beta. To identify signaling defects responsible for TGF-beta resistance in premalignant and malignant trophoblasts, we have compared the expression of TGF-beta signaling molecules in a normal trophoblast cell line (HTR-8), its premalignant derivative (RSVT2/C), and two choriocarcinoma cell lines (JAR and JEG-3). RT-PCR analysis revealed that all these cell lines expressed the mRNA of TGF-beta1, -beta2, and -beta3, TGF-beta receptors type I, II, and III, and post-receptor signaling genes smad2, smad3, smad4, smad6, and smad7 with the exception that TGF-beta2 and smad3 were undetectable in JAR and JEG-3 cells. Immunoblot analysis confirmed the absence of smad3 protein in choriocarcinoma cells. Treatment with TGF-beta1 induced smad3 phosphorylation and smad3 translocation to the nucleus in the normal and premalignant trophoblast cells. These results suggest that loss of smad3 may account for a functional disruption in the TGF-beta signaling pathway in choriocarcinomas, but not in the premalignant trophoblast.     

Regulation of cell adhesion receptors by transforming growth factor-beta. Concomitant regulation of integrins that share a common beta 1 subunit

Cell adhesion to extracellular matrices is mediated by a set of heterodimeric cell surface receptors called integrins that might be the subject of regulation by growth and differentiation factors. We have examined the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of the very late antigens or alpha beta 1 group of integrins in human cell lines. The six known members of this family share a common beta 1 subunit but have distinct alpha subunits that confer selective affinity toward type I collagen, fibronectin, laminin, and other as yet unknown cell adhesion proteins. Using a panel of specific antibodies and cDNA probes, we show that in WI-38 lung fibroblasts TGF-beta 1 elevates concomitantly the expression of alpha 1, alpha 2, alpha 3, alpha 5, and beta 1 integrin subunits at the protein and/or mRNA level, their assembly into the corresponding alpha beta 1 complexes, and their exposure on the cell surface. The rate of synthesis of total alpha subunits relative to beta 1 subunit is higher in TGF-beta 1-treated cells than in control cells. The characteristically slow (t1/2 approximately 10 h) rate of beta 1 conversion from precursor form to mature glycoprotein in untreated cells increases markedly (to t1/2 approximately 3 h) in response to TGF-beta 1. The results suggest that in WI-38 fibroblasts the beta 1 subunit is synthesized in excess over alpha subunits, and assembly of beta 1 subunits with rate-limiting alpha subunits is required for transit through the Golgi and exposure of alpha beta 1 complex on the cell surface. TGF-beta 1 does not induce the synthesis of integrin subunits that are not expressed in unstimulated cells, such as alpha 4 and alpha 6 subunits in WI-38 fibroblasts. However, alpha 4 and alpha 6 subunits can be regulated by TGF-beta in those cells that express them. The results suggest that TGF-beta regulates the expression of individual integrin subunits by parallel but independent mechanisms. By modifying the balance of individual alpha beta 1 integrins, TGF-beta 1 might modulate those aspects of cell migration, positioning, and development that are guided by adhesion to extracellular matrices.     

Transforming growth factor-beta 1, -beta 2 and -beta 3 in cartilage and bone cells during endochondral ossification in the chick.

The localization of TGF-beta 1, -beta 2 and -beta 3 was studied in the growth plate, epiphysis and metaphysis of the tibiotarsus of three-week-old chicks. The different TGF-beta isoforms were localized to hypertrophic chondrocytes, chondroclasts, osteoblasts and osteoclasts using immunohistochemical staining analysis with specific TGF-beta antibodies. TGF-betas in osteoclasts and chondroclasts were restricted to those cells located on the respective matrices. TGF-beta 3 localization was mainly cytoplasmic in the transitional (early hypertrophic) chondrocytes, but nuclear staining was also detected in some proliferating chondrocytes. The cell-specific localization of these TGF-beta isoforms supports the hypothesis that TGF-beta has a role in the coupling of new bone formation to bone and cartilage matrix resorption during osteochondral development and suggests that TGF-beta may be a marker of chondrocyte differentiation. TGF-beta localization preceded a marked increase in type II collagen mRNA expression in transitional chondrocytes, suggesting a role for TGF-beta in the induction of synthesis of extracellular matrix.     

Development of a retroviral vector for inducible expression of transforming growth factor beta 1.

A retroviral vector system for the expression of exogenous genes under the control of an inducible promoter was developed. By utilizing this system, the cDNA for human transforming growth factor beta 1 (TGF-beta 1) was inserted into a retroviral vector under the control of an internal mouse metallothionein promoter and introduced via infection into normal rat kidney fibroblasts (NRK-49F) and epithelial cells (NRK-52E), Chinese hamster ovary cells (CHO), and the human monocytic cell line U937. Control of TGF-beta 1 expression, achieved by Cd2+ induction of vector-encoded TGF-beta 1 mRNA, was cell line specific and resulted in a concomitant increase in neutralizable TGF-beta 1 production by the cells. Autocrine stimulation of vector-containing cells by vector-encoded TGF-beta 1 was detected by an increase in soft-agar colony formation of NRK-49F infectants compared with that of the control cells. In addition, the use of a second internal promoter in a retroviral vector of similar design allowed isolation of stable infectants from a cell line (CHO) in which the viral long terminal repeat does not function efficiently.