TGF-beta is a potent inhibitor of hepatocyte growth factor secretion by human fibroblasts.

Transforming growth factor-beta 1 (TGF-beta 1) inhibited secretion of human hepatocyte growth factor (hHGF), which is also known as scatter factor or fibroblast-derived tumor cytotoxic factor, by MRC-5 cells. The effect was detectable at as little as 10 pg/ml and was more potent than that of dexamethasone. Complete inhibition was observed after 12 h in the presence of 5 ng/ml of TGF-beta 1. Phorbol 12-myristate 13-acetate-induced secretion of hHGF from human skin fibroblasts was also suppressed by TGF-beta 1. TGF-beta 2 inhibited hHGF secretion by MRC-5 cells to the same extent as TGF-beta 1, but other growth factors such as epidermal growth factor and acidic and basic fibroblast growth factors had only a slight or null inhibitory effect.     

Hepatocyte growth factor is a potent mitogen for cultured rabbit renal tubular epithelial cells.

Hepatocyte growth factor (HGF), which is a potent growth factor of adult rat hepatocytes in primary culture, also strongly stimulated DNA synthesis of rabbit renal tubular epithelial cells in secondary culture. Its mitogenic activity was dose-dependent, being detectable at 3 ng/ml and maximal at 30 ng/ml. Over 20% of the cells were shifted to the S-phase by HGF alone, judging by the labeling index. HGF had additive effects with EGF, acidic fibroblast growth factor (a-FGF), and insulin. Transforming growth factor-beta 1 (TGF-beta 1) strongly inhibited DNA synthesis of renal tubular cells stimulated by HGF. The growth of renal tubular epithelial cells was also regulated by cell density: DNA synthesis stimulated by HGF was high at lower cell density and was strongly suppressed at high cell density. These results suggest that HGF may act as a renotropic factor in compensatory renal growth or renal regeneration in vivo.     

Up-regulation of hepatocyte growth factor gene expression by interleukin-1 in human skin fibroblasts.

Hepatocyte growth factor (HGF) functions as a hepatotrophic and renotrophic factor for regeneration of the liver and kidney. When 1 ng/ml of interleukin-1 alpha (IL-1 alpha) or interleukin-1 beta (IL-1 beta) was added to cultures of human skin fibroblasts, the production of HGF was 5-6 fold higher than levels in the controls. HGF mRNA level in the cells was increased to 4-fold higher levels at 6 h after exposure to IL-1 alpha. Tumor necrosis factor-alpha and interferon-gamma but no other cytokine tested had slightly stimulatory effects on HGF production. The tumor promoter, tetradecanoylphorbol 13-acetate (TPA) markedly enhanced the stimulatory effect of IL-1 alpha and IL-1 beta on the production of HGF. The stimulatory effect of both IL-1 alpha and IL-1 beta and the synergistical stimulation with TPA were completely abrogated by 10 ng/ml TGF-beta 1 or 1 microM dexamethasone. These results suggest that IL-1 alpha and IL-1 beta are positive regulators for expression of the HGF gene and are likely have a role in regeneration of tissues following the occurrence of inflammatory diseases.     

Basic fibroblast growth factor stimulates hepatocyte growth factor/scatter factor secretion by human mesenchymal cells

Basic fibroblast growth factor (bFGF) together with other pleiotropic factors plays an important role in many complex physiological processes such as embryonic development, angiogenesis, and wound repair. Among these factors, hepatocyte growth factor/scatter factor (HGF/SF) which is secreted by cells of mesodermal origin exerts its mito- and motogenic activities on cells of epithelial and endothelial origin. Knowledge of the regulatory mechanisms of HGF/SF may contribute to the understanding of its role in physio-pathological processes. We observed that the secretion of HGF/SF by MRC-5 cells and by other fibroblast-derived cell cultures in conditioned media was enhanced by exposure to bFGF. HGF/SF was measured by the scatter assay, a bioassay for cell motility, and was further characterized by Western blot analysis with anti-HGF/SF antibodies. Exposure of MRC-5 cultures to 10 ng/ml of bFGF resulted already 6 h posttreatment in a threefold higher amount of scatter factor secreted into the medium as compared to untreated cultures. HGF/SF secretion was sustained after bFGF treatment for the following 72 h when increased amounts of HGF/SF were detected both in conditioned media as well as associated to the extracellular matrix. The secretion of HGF/SF in cell supernatants increased dose dependently upon treatment with bFGF starting from basal levels of 6 U/ml and reaching 27 U/ml at 30 ng/ml bFGF, plateauing thereafter. Upregulation of HGF/SF by IL-1, already described by others, was confirmed in this study. Based on our findings an articulated interaction can be speculated for bFGF, HGF/SF, and IL-1, e.g., in tissue regeneration during inflammatory processes or in wound healing. © 1996 Wiley-Liss, Inc.     

Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

Transforming growth factor-beta down-regulates apolipoprotein M in HepG2 cells

Apolipoprotein M (apoM) is a novel apolipoprotein presented mostly in high-density lipoprotein (HDL) in human plasma, and is exclusively expressed in liver and in kidney. The pathophysiological function of apoM has not yet been elucidated. Apolipoprotein B (apoB), the characteristic apolipoprotein of low-density lipoprotein (LDL), is like apoM, a very hydrophobic protein, and thereafter they both must co-circulate with lipoprotein particles in plasma. The cytokine, transforming growth factor-beta (TGF-beta), has been shown to decreased apoB secretion in HepG2 cells, and we hypothesized that TGF-beta may have the same effects on apoM expression in HepG2 cells. In the present study, we used real-time RT-PCR to analyze apoM and apoB mRNA levels during administration of TGF-beta, as well as TGF-alpha, epidermal growth factor (EGF) and hepatic growth factor (HGF). TGF-beta significantly inhibited both apoM and apoB mRNA expression in HepG2 cells. The inhibitory effects of TGF-beta were dose-dependent, i.e. 1 ng/ml of TGF-beta decreased apoM mRNA levels by 30%, and 10 or 100 ng/ml of TGF-beta decreased apoM mRNA levels more than 65%. The effect of TGF-beta on apoB mRNA expression was slightly weaker than that of apoM, with a maximum effect at 10 or 100 ng/ml TGF-beta where apoB mRNA levels decreased about 55%. The inhibitory effects of TGF-beta on apoM and apoB mRNA levels also increased with increasing incubation time, where the maximum effect was obtained at 24 h. Moreover TGF-alpha, EGF and HGF all decreased both apoM and apoB mRNA levels, but to a less extent than TGF-beta. Further, all four cytokines had more pronounced effects on apoM mRNA expression than apoB mRNA expression. The present study suggested that apoM, like apoB, may be involved in the hepatic lipoprotein assembly in vivo.     

Effect of transforming growth factor beta on cell proliferation and glycosaminoglycan synthesis by rabbit growth-plate chondrocytes in culture

The effects of the transforming growth factor beta (TGF-beta) on the growth and glycosaminoglycan synthesis of rabbit growth plate-chondrocytes in culture were studied. In serum-free medium, TGF-beta caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [3H]thymidine incorporation (ED50 = 0.1-0.3 ng/ml). The inhibitory effect was maximal at a dose of 1 ng/ml, and extended for a duration of 16-42 h. In contrast, TGF-beta potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-beta (1 ng/ml) to cultures containing 10% FCS increased [3H]thymidine incorporation to 1.6-times that in cultures with 10% FCS alone. Consistent with this finding, TGF-beta potentiated DNA synthesis stimulated by the purified growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF). The maximal stimulation of DNA synthesis by FGF (0.4 ng/ml) was further potentiated dose dependently by TGF-beta (ED50 = 0.1 ng/ml, maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-beta (1 ng/ml) and FGF (0.4 ng/ml), [3H]thymidine incorporation was 3-times higher than that of cultures treated with FGF alone. This TGF-beta-induced potentiation of DNA synthesis was associated with replication of chondrocytes, as shown by a marked increase in the amount of DNA during treatment of sparse cultures of the cells with the growth factors for 5 days. In contrast, TGF-beta caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of growth-plate chondrocytes (ED50 = 0.3 ng/ml, maximum at 1 ng/ml). This stimulatory effect of TGF-beta was greater than that of insulin-like growth factor I (IGF-I) or PDGF. Furthermore, TGF-beta stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-beta, whereas epiphyseal growth cartilage is not. Thus, the present data indicate that TGF-beta may be important in bone formation by modulating growth and phenotypic expression of chondrocytes in the growth plate, possibly via a paracrine mechanism.     

Hepatocyte growth Factor/Scatter factor (HGF/SF) is a regulator of fibronectin splicing in MDCK cells: comparison between the effects of HGF/SF and TGF-beta1 on fibronectin splicing at the EDA region.

EDA-containing fibronectin (EDA + FN) is selectively produced under several physiological and pathological conditions requiring tissue remodeling, where cells actively proliferate and migrate. Only a few growth factors, such as transforming growth factor (TGF)-beta1, have been reported to regulate FN splicing at the EDA region. In the present study, we showed for the first time that hepatocyte growth factor/scatter factor (HGF/SF), which is mainly produced by mesenchymal cells and functions as a motogenic and mitogenic factor for epithelial cells, modulates FN splicing at the EDA region in MDCK epithelial cells. HGF/SF treatment increased the ratio of EDA + FN mRNA to mRNA of FN that lacks EDA (EDA - FN) (EDA+/EDA- ratio) more than TGF-beta1 treatment did: at a range from 0.02 to 20 ng/ml, HGF/SF increased the ratio in a dose-dependent manner by up to 2. 1-fold compared with nontreated control, while TGF-beta1 stimulated the EDA+/EDA- ratio by 1.5-fold at the optimum dose of 10 ng/ml. However, TGF-beta1 increased total FN mRNA levels by 3-fold at 10 ng/ml, but HGF/SF did not. We previously demonstrated that fibroblasts cultured at low cell density expressed more EDA + FN than those at high cell density. The same effect of cell density was also observed in MDCK cells. Furthermore, at low cell density, HGF/SF stimulated EDA inclusion into FN mRNA more effectively than did TGF-beta1, whereas at high cell density, TGF-beta1 was more potent than HGF/SF. Simultaneous treatment of cells with HGF/SF and TGF-beta1 synergistically stimulated EDA inclusion into FN mRNA. This stimulation of EDA inclusion into FN mRNA by HGF/SF led to increased EDA + FN protein production and secretion by cells, which was demonstrated by immunoblotting. Thus, our studies have shown that HGF/SF is an enhancer of EDA inclusion into FN mRNA as is TGF-beta1. However, these two factors were different in their effects at low and high cell densities and also in their effects on total FN mRNA levels.     

Effect of transforming growth factor-beta on human chorionic gonadotropin induced testosterone production by cultured rat testicular cells

The potential role of transforming growth factor-beta (TGF-beta) as an intragonadal regulator in the testis was investigated by studying the effect of TGF-beta on testosterone (T) production by neonatal rat testis cells in primary cultures. After 3 days of preincubation in serum-free medium, testis cells were treated with hormones for 3 additional days. Human chorionic gonadotropin (hCG) treatment (0.3-30 ng/ml) of testis cells elicited a dose-dependent increase of T levels with maximum values greater than 9-fold over baseline. Although TGF-beta alone did not affect T levels, a dose-dependent inhibition of hCG-stimulated T production was observed when cells were cotreated with TGF-beta. Maximal inhibition was greater than 85%, and the IC50 value was 5 ng/ml (2 x 10(-10) M; n = 5 experiments). This inhibitory effect was evident 48 h after the initiation of treatment and could be reversed 1 day after the cessation of TGF-beta exposure of cells. TGF-beta also reduced forskolin and (Bu)2cAMP-induced T production (greater than 85% decrease), indicating that TGF-beta can inhibit steroidogenesis distal to the formation of cAMP. The conversion of exogenously added androgen precursors (progesterone (P) and 17 alpha-hydroxyprogesterone) to T by hCG-stimulated cells was suppressed by the addition of TGF-beta. In contrast, endogenous P accumulation did not change in cultures treated with TGF-beta. Because TGF-beta-like activity has been found in the testis, the observed inhibitory effect of TGF-beta suggests a potential intratesticular regulatory role of this growth factor.     

Early response to tumor necrosis factor of human promyelocytic leukemia cell lines sensitive and resistant to the factor

Early cellular events with respect to protein synthesis and the steady-state level of cellular myc (c-myc) mRNA were analyzed in the tumor necrosis factor (TNF)-sensitive human promyelocytic leukemia cell line HL-60 and in its TNF-resistant variant HL-60R after their exposure to TNF. Addition of TNF at 100 units (U)/ml induced de novo synthesis of two proteins with apparent molecular masses of 100 kDa and 40 kDa in HL-60 cells. The induced synthesis of the 100 kDa protein continued for 6 h, while that of the 40 kDa protein was transient. The 100 kDa protein was detectable in HL-60R cells which were maintained in medium containing 1,000 U/ml TNF, whereas the synthesis of the 40 kDa protein could be transiently induced by TNF at 10(5) U/ml. Dot blot hybridization revealed that the steady-state level of c-myc mRNA in HL-60 cells was transiently reduced by TNF at 100 U/ml but remained at a reduced level for 6 h when 10(5) U/ml TNF was present. In HL-60R cells, TNF at 10(5) U/ml could transiently reduce the c-myc mRNA level. These results showed that induction of the synthesis of a 40 kDa protein and a reduction in the steady-state level of c-myc mRNA were concomitant with cellular sensitivity to the cytostatic action of TNF in HL-60 cells.     

Overexpression of regucalcin suppresses cell response for tumor necrosis factor-alpha or transforming growth factor-beta1 in cloned normal rat kidney proximal tubular epithelial NRK52E cells

The regulatory role of regucalcin on cell responses for tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1) was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2-transfected cells (transfectant) were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture, cells were further cultured for 24-72 h in medium without BS containing either vehicle, TNF-alpha (0.1 or 1.0 ng/ml of medium), or TGF-beta1 (1.0 or 5.0 ng/ml). Culture with TNF-alpha or TGF-beta1 caused a significant decrease in the number of wild-type cells. This decrease was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with TNF-alpha (1.0 ng/ml) or TGF-beta1 (5.0 ng/ml). This DNA fragmentation was significantly suppressed in transfectants. TNF-alpha- or TGF-beta1-induced cell death was significantly prevented in culture with caspase-3 inhibitor (10(-8) M). Nitric oxide (NO) synthase activity in wild-type cells was significantly increased by addition of calcium chloride (10 microM) and calmodulin (5 microg/ml) into the enzyme reaction mixture. This increase was significantly suppressed in transfectants. Culture with TNF-alpha caused a significant increase in NO synthase activity in wild-type cells. The effect of TNF-alpha was not seen in transfectants. Culture with TGF-beta1 did not cause a significant increase in NO synthase activity in wild-type cells and transfectants. Culture with TNF-alpha or TGF-beta1 caused a remarkable increase in alpha-smooth muscle actin in wild-type cells. This increase was significantly prevented in transfectants. The expression of Smad 2 or NF-kappaB mRNAs was significantly increased in transfectants as compared with that of wild-type cells. Smad 3 or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expression was not significantly changed in transfectants. NF-kappaB mRNA expression in wild-type cells was significantly increased with culture of TNF-alpha. Smad 2 mRNA expression was significantly enhanced in wild-type cells cultured with TGF-beta1. These effects of TNF-alpha or TGF-beta1 were not significantly enhanced in transfectants. This study demonstrates that overexpression of regucalcin has suppressive effects on cell responses which are mediated through intracellular signaling pathways of TNF-alpha or TGF-beta1 in kidney NRK52E cells.     

Differential inhibitory effects of TGF-beta on EGF-, PDGF-, and HBGF-1-stimulated MG63 human osteosarcoma cell growth: possible involvement of growth factor interactions at the receptor and postreceptor levels

The growth of MG63 human osteosarcoma cell line in 5% serum is stimulated by epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or heparin-binding growth factor-1 (HBGF-1). The mitogenic effect of EGF and PDGF is completely blocked by TFG-beta at 1 ng per ml and the effect of HBGF-1 is attenuated by 75-80%. Treatment of MG63 cells with TGF-beta reduces HBGF-1 receptor binding affinity from 1.24 x 10(-11) M to 3.51 x 10(-11) M with no change on the receptor number (1.1 x 10(3) per cell). The receptor-binding affinity of EGF and PDGF is not altered by TGF-beta treatment; however, the number of EGF receptor is increased by 25%. Both EGF and PDGF stimulate MG63 cellular tyrosine kinase activity, and such stimulation is inhibited by TGF-beta pretreatment. No change in the cellular protein tyrosine phosphorylation pattern can be detected in HBGF-1-stimulated cells with and without TGF-beta pretreatment. These data suggest that TGF-beta inhibits EGF and PDGF mitogenicity by blocking EGF- and PDGF-stimulated tyrosine kinase activity and attenuates HBGF-1 mitogenicity by decreasing its receptor affinity.     

Dexamethasone and cyclosporin A do not inhibit interleukin-15 expression in the human lung carcinoma cell line A549

A549 cells constitutively expressed IL-15 mRNA which could be upregulated by stimulation with TNF-alpha- or IL-1beta. Constitutive and induced levels of IL-15 mRNA were not decreased in the presence of 10- 6 M dexamethasone. Control experiments revealed that 10- 6 M dexamethasone inhibited the TNF-alpha- or IL-1beta-mediated increase of IL-8 mRNA in A549 cells, which showed that the glucocorticoid was functional. A549 cells did not secrete relevant amounts of IL-15 protein. The constitutive expression and the TNF-alpha- or IL-1beta-mediated upregulation of intracellular IL-15 protein was not inhibited by dexamethasone, in contrast, the release of IL-8 protein was inhibited. Also, cyclosporin A at 250 ng/ml did not inhibit the TNF-alpha-induced upregulation of IL-15 mRNA and intracellular IL-15 protein. The data suggest that the synthesis of IL-15 mRNA and protein is not influenced by immunosuppressive glucocorticoids or by cyclosporin A.     

Effect of dexamethasone on nucleolar casein kinase II activity and phosphorylation of nucleolin in lymphosarcoma P1798 cells.

Ribosomal RNA (rRNA) synthesis in murine P1798 lymphosarcoma cells is reversibly inhibited by glucocorticoids. The effects of dexamethasone upon nucleolin phosphorylation and upon the amount and activity of casein kinase II have been examined. P1798 cells were exposed to 0.1 microM dexamethasone for 36 h. Cells were labeled in vivo with [32P]orthophosphate followed by immunoprecipitation with anti-nucleolin antibody. Nucleolin phosphorylation was reduced by 60% in dexamethasone-treated cells. Nucleoli were isolated and labeled with [gamma-32P]ATP in vitro. Nucleolin protein was reduced to 40% of control in nuclei from dexamethasone-treated cells. Nucleolin phosphorylation was reduced to 20% of control. Nucleolar casein kinase II activity and protein were also reduced (30-55% and 35-50% of control, respectively) by treatment with dexamethasone. Cycloheximide (10 micrograms/ml for 3 h) reduced the amount and activity of casein kinase II, but did not cause a decrease in nucleolin protein. These observations are discussed relative to the hypothesis that glucocorticoids regulate the amount or activity of proteins of short biological half-life that are involved in the regulation of rRNA synthesis.     

Enhanced insulin stimulation of sugar transport and DNA synthesis by glucocorticoids in cultured human skin fibroblasts

Glucocorticoids will enhance the growth of cultured human skin fibroblasts in serum-containing medium. In serum-free cultures hydrocortisone (5 X 10(-6) M) will enhance insulin stimulation of sugar transport and DNA synthesis (as measured by thymidine incorporation into trichloroacetic acid-precipitable material). The optimal concentration for the glucocorticoid effect on DNA synthesis was 5 X 10(-8) M for dexamethasone and 5 X 10(-7) M for hydrocortisone. In dexamethasone-treated cells, concentrations of insulin as low as 250 microU/ml (10 ng/ml) were effective in stimulating DNA synthesis. Further, hydrocortisone and dexamethasone (both at 5 X 10(-6) M) exhibited potentiating effects on insulin-stimulated sugar transport. These effects appeared to be mediated via inhibitory actions on the hexose transport system with the preservation of a functional insulin-receptor interaction resulting in insulin stimulation of deoxy-D-glucose transport at physiological insulin concentrations, 250 microU/ml (10 ng/ml). Hydrocortisone also enhanced specific [125I]insulin binding in these cells. The data indicate that the mechanism(s) of glucocorticoid enhancement of two actions of insulin may be different.