Inhibitory Action Of TGF-beta Research Articles

SMAD3 inhibits SF-1-dependent activation of the CYP17 promoter in H295R cells

Cytochrome P450c17, encoded by the CYP17 gene, is a component of 17alpha-hydroxylase/17,20 lyase which catalyses 17alpha-hydroxylation of pregnenolone or progesterone, required for glucocorticosteroid and androgen synthesis. It has been reported that transforming growth factor beta (TGF-beta) decreases both basal and cAMP-stimulated levels of CYP17 mRNA, but the mechanism of TGF-beta action on CYP17 expression remains unknown. We investigated an inhibitory effect of TGF-beta on CYP17 expression in H295R cells using constructs containing the CYP17 promoter region fused with the luciferase gene. In the H295R cells, TGF-beta decreased endogenous SF-1 level and inhibited activity of the 300 bp fragment of CYP17 promoter, which was stimulated by coexpression of SF-1. Overexpression of SMAD3 caused an inhibition of SF-1-stimulated CYP17 promoter activity, whereas overexpression of SMAD7 was ineffective. In conclusion, our results suggest that the inhibitory action of TGF-beta on CYP17 transcription involve at least two mechanisms: SMAD3 dependent inactivation of CYP17 promoter activity and repression of SF-1 expression.

Transforming Growth Factor β Affects Osteoclast Differentiation via Direct and Indirect Actions

Transforming growth factor beta (TGF-beta) is abundant in bone and has complex effects on osteolysis, with both positive and negative effects on osteoclast differentiation, suggesting that it acts via more than one mechanism. Osteoclastogenesis is determined primarily by osteoblast (OB) expression of the tumor necrosis factor (TNF)-related molecule receptor activator of NF-kappaB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), which are increased and decreased, respectively, by osteolytic factors. A RANKL-independent osteoclastogenic mechanism mediated by TNF-alpha has also been shown. Therefore, we investigated TGF-beta effects on osteoclast formation in culture systems in which osteoclastogenic stimulus is dependent on OBs and culture systems where it was provided by exogenously added RANKL or TNF-alpha. Both OPG and TGF-beta inhibited osteoclast formation in hemopoietic cell/OB cocultures, but the kinetics of their action differed. TGF-beta also inhibited osteoclastogenesis in cocultures of cells derived from OPG null (opg-/-) mice. TGF-beta strongly decreased RANKL messenger RNA (mRNA) expression in cultured osteoblasts, and addition of exogenous RANKL to TGFbeta-inhibited cocultures of opg-/- cells partially restored osteoclastogenesis. Combined, these data indicate that the inhibitory actions of TGF-beta were mediated mainly by decreased OB production of RANKL. In contrast, in the absence of OBs, TGF-beta greatly increased osteoclast formation in recombinant RANKL- or TNF-alpha-stimulated cultures of hemopoietic cells or RAW 264.7 macrophage-like cells to levels several-fold greater than attainable by maximal stimulation by RANKL or TNF-alpha. These data suggest that TGF-beta may increase osteoclast formation via action on osteoclast precursors. Therefore, although RANKL (or TNF-alpha) is essential for osteoclast formation, factors such as TGF-beta may powerfully modify these osteoclastogenic stimuli. Such actions may be critical to the control of physiological and pathophysiological osteolysis.

Serum transforming growth factor-beta 1 is related to the degree of immunoparesis in patients with multiple myeloma.

The expansion of myeloma cells is regulated by cytokines, among which IL-6 is a major growth factor. It has been recently suggested that serum transforming growth factor beta 1 (TGF beta 1), a cytokine found in large amounts in alpha-granules of platelets, might play a role in multiple myeloma (MM). It was the purpose of this study to determine serum TGF beta 1 levels in MM patients and to seek a correlation with disease parameters. Measurements were done by ELISA. We studied 35 MM patients (19 stage II, 16 stage III, 20 IgG, 8 IgA and 6 BJ, 1 IgD) in different phases of the disease, 27 healthy individuals and 17 thrombocytopenic patients with other haematological diseases (three MDS, three congenital thrombocytopenia, 11 ITP). Overall samples from MM patients were included: 10 at diagnosis, 18 in remission and 32 in relapse. In normal controls TGF beta 1 serum levels ranged from 1 to 33 ng/ml (median 16.5 ng/ml). In both thrombocytopenic controls with other diseases and thrombocytopenic MM patients (seven samples), TGF beta 1 serum levels were very low (median 3.2 and 4.5 ng/ml respectively). In MM patients with PLT > 100 x 10(9)/L (53 samples), TGF beta 1 serum levels were in the normal range in patients without immunoparesis (1 to 27 ng/ml, median 16.6 ng/ml), whereas they were higher in patients with immunoparesis (polyclonal immunoglobulins (Igs) below lower normal reference values) ranging from 10.2 to 45 ng/ml (median 26.8 ng/ml) (P < 0.01). Serum TGF beta 1 levels fluctuated in the same patient at different times but not according to relapse or remission. Correlation was found only between serum TGF beta 1 levels and immunoparesis and not between serum TGF beta 1 levels and disease stage or Ig subtype nor with prognostic factors for MM (serum CRP, beta 2M or IL-6). This finding suggests that the remaining normal plasma cells are sensitive to the inhibitory action of TGF beta 1 on Ig production. In conclusion TGF beta 1 serum levels are very low in thrombocytopenic patients confirming that platelets are the major source of this cytokine. Furthermore, a strong correlation was found between TGF beta 1 serum levels and immunoparesis in MM patients.

Antagonistic effects of transforming growth factor-beta on vitamin D3 enhancement of osteocalcin and osteopontin transcription: reduced interactions of vitamin D receptor/retinoid X receptor complexes with vitamin E response elements.

Osteocalcin and osteopontin are noncollagenous proteins secreted by osteoblasts and regulated by a complex interplay of systemic and locally produced factors, including growth factors and steroid hormones. We investigated the mechanism by which transforming growth factor-beta (TGF beta) inhibits 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-enhanced expression of the osteocalcin (OC) and osteopontin (OP) genes. ROS 17/2.8 cells, in which both genes are expressed, were transfected with reporter constructs driven by native (i.e. wild-type) rat OC and mouse OP promoters. TGF beta abrogated the 1,25-(OH)2D3 enhanced transcription of both the OC and OP genes. The inhibitory TGF beta response for each requires vitamin D response element (VDRE) sequences, although there are additional contributions from proximal basal regulatory elements. These transcriptional effects were further investigated for contribution of the trans-activating factors, which interact with OC and OP VDREs, involving the vitamin D receptor (VDR) and retinoid X receptor (RXR). Gel mobility shift assays show that TGF beta significantly reduces induction of the heterodimers VDR/RXR complexes in 1,25-(OH)2D3-treated ROS 17/2.8 cells. However, Western blot and ligand binding analysis reveal that TGF beta does not affect nuclear availability of the VDR. We also show that activator protein-1 activity is up-regulated by TGF beta; thus, activator protein-1 binding sites in the OC promoter may potentially contribute to inhibitory effects of TGF beta on basal transcription. Our studies demonstrate that the inhibitory action of TGF beta on the 1,25-(OH)2D3 enhancement of OC and OP transcription in osteoblastic cells results from modulations of protein-DNA interactions at the OC and OP VDRE, which cannot be accounted for by changes in VDR protein levels. As OC and OP participate in bone turnover, our results provide insight into the contributions of TGF beta and 1,25-(OH)2D3 to VDR-mediated gene regulatory mechanism operative in bone formation and/or resorption events.

Expression of transforming growth factor-beta (TGF beta) isoforms and TGF beta type II receptor messenger ribonucleic acid and protein, and the effect of TGF beta s on endometrial stromal cell growth and protein degradation in vitro.

Reverse transcription-polymerase chain reaction analysis of total RNA and immunocytochemical observations revealed that human endometrial glandular epithelial and stromal cells in primary culture express messenger RNAs and proteins for transforming growth factor-beta 1 (TGF beta 1), TGF beta 2, and TGF beta 3 as well as TGF beta type II receptor. The epithelial and stromal cells synthesize and secrete into their culture-conditioned medium 2.6 +/- 0.3 and 1.4 +/- 0.2 ng TGF beta 1/10(6) cells, respectively; after transient acidification of the medium, the TGF beta 1 levels were 18.1 +/- 0.4 and 7.8 +/- 0.7 ng/10(6) cells. These cells also contain specific binding sites for [125I]TGF beta 1, indicated by light microscope autoradiography. TGF beta s at 0.01-10 ng/ml neither stimulated or inhibited subconfluent quiescent stromal cells under serum-free condition nor altered the mitogenic action of 10% fetal bovine serum. However, in the presence of 2% fetal bovine serum, which induced half-maximal stimulation of [3H]thymidine incorporation, TGF beta 1 and TGF beta 2 at 0.1-0.5 ng/ml and TGF beta 3 at 0.1-2.5 ng/ml significantly stimulated the rate of [3H]thymidine incorporation into quiescent stromal cells (P < 0.005); they were ineffective at higher concentrations. TGF beta s did not have any effect on cell proliferation, as determined by cell counting; however, at 0.1 ng/ml and higher concentrations, TGF beta s significantly reduced the metabolic activity of stromal cells, as determined by colorimetric 3-(4,5-dimethylthiazol-2-yl)2, 5-diphenyltetrazolium bromide assay (P < 0.05). The stimulatory and inhibitory actions of TGF beta s in both assays were reversible using 5-10 micrograms/ml TGF beta 1- and TGF beta 2- and 3-6 micrograms/ml TGF beta 3-specific neutralizing antibodies. TGF beta 1 at 1 ng/ml had no significant effect on long-lived protein degradation, assayed by incorporation of [14C]valine into newly synthesized protein by stromal cells, and was similar to the effect of epidermal growth factor or platelet-derived growth factor-BB (10 ng/ml). The data suggest that the TGF beta expression by various endometrial cell types in an autocrine/paracrine manner acts as a negative regulator essential for restraining endometrial growth and transition from proliferation to differentiation stages during the secretory phase after mitogenic stimulation during the proliferative phase of the menstrual cycle.

Developmentally regulated transforming growth factor-beta 1 action on vascular smooth muscle growth in the SHR.

1. This study examined the effects of transforming growth factor-beta 1 (TGF-beta 1) on platelet-derived growth factor-BB (PDGF-BB)-stimulated proliferation of vascular smooth muscle cells (VSMC) isolated from aortic tissue of 1, 4 and 12 week old spontaneously hypertensive rats (SHR). 2. In 1 week old SHR, TGF-beta 1 inhibited by about 60% VSMC proliferation stimulated by PDGF-BB; this inhibitory action of TGF-beta 1 was absent in VSMC isolated from 4 week old, prehypertensive SHR. In contrast, TGF-beta 1 potentiated by about 125% the mitogenic activity of PDGF-BB in VSMC cultures from adult SHR. 3. Age-dependent alterations in the action of TGF-beta 1 suggests an important role for TGF-beta 1 in the development of vascular hypertrophy from adolescence onwards in the SHR.

Transforming growth factor beta and cyclosporin A inhibit the inducible activity of the interleukin-2 gene in T cells through a noncanonical octamer-binding site.

Transforming growth factor beta (TGF-beta) has a growth-inhibitory effect on numerous different cell types of the immune system, including T lymphocytes. We show in this study that the inhibitory action of TGF-beta on T lymphocytes is accompanied by a block of interleukin 2 (IL-2) gene expression which is mediated, at least in part, by inhibition of IL-2 promoter/enhancer activity. The functional analysis of cis-regulatory (proto-enhancer) elements of the IL-2 enhancer/promoter region showed that the most TGF-beta-responsive element maps to its so-called upstream promoter site. The proto-enhancer activity of the upstream promoter site element is also inhibited by cyclosporin A. The upstream promoter site DNA harbors two noncanonical, closely linked binding sequences for octamer and AP-1-like factors. Both sites are involved in the establishment of IL-2 enhancer activity. Since the activity of genuine octamer sites but not that of AP-1-binding sites is also impaired by TGF-beta and cyclosporin A in El4 T lymphoma cells, we conclude that both immunosuppressives interfere with the activity but not the DNA binding of octamer factors in T lymphocytes.

Transforming growth factor beta and cyclosporin A inhibit the inducible activity of the interleukin-2 gene in T cells through a noncanonical octamer-binding site.

Transforming growth factor beta (TGF-beta) has a growth-inhibitory effect on numerous different cell types of the immune system, including T lymphocytes. We show in this study that the inhibitory action of TGF-beta on T lymphocytes is accompanied by a block of interleukin 2 (IL-2) gene expression which is mediated, at least in part, by inhibition of IL-2 promoter/enhancer activity. The functional analysis of cis-regulatory (proto-enhancer) elements of the IL-2 enhancer/promoter region showed that the most TGF-beta-responsive element maps to its so-called upstream promoter site. The proto-enhancer activity of the upstream promoter site element is also inhibited by cyclosporin A. The upstream promoter site DNA harbors two noncanonical, closely linked binding sequences for octamer and AP-1-like factors. Both sites are involved in the establishment of IL-2 enhancer activity. Since the activity of genuine octamer sites but not that of AP-1-binding sites is also impaired by TGF-beta and cyclosporin A in El4 T lymphoma cells, we conclude that both immunosuppressives interfere with the activity but not the DNA binding of octamer factors in T lymphocytes.

Transforming growth factor-beta 1 messenger RNA and protein expression in the pituitary gland: its action on prolactin secretion and lactotropic growth.

Transforming growth factor-beta 1 (TGF-beta 1) is known to inhibit cell growth and proliferation of many estrogen-responsive normal and transformed cells. The effect of this polypeptide growth factor on the estrogen-responsive pituitary lactotropes has not been determined. To evaluate the role of TGF-beta 1 in the control of lactotropic growth, the action and production of TGF-beta 1 in the anterior pituitary was studied in rats. The growth factor suppressed basal PRL release from the primary culture of enriched rat lactotropes in a concentration-dependent manner in the range of 2 pg/ml-20 ng/ml. The growth factor did not affect the secretion of other pituitary hormones in the cultures. The inhibitory action of TGF-beta 1 on PRL release was time dependent. The minimum time required to produce a significant effect was 4 h. The growth factor also suppressed estradiol-induced PRL release as well as it inhibited estradiol-induced proliferation of lactotropes. TGF-beta 1 immunoreactivity was detected in the cellular extracts of cultured anterior pituitary cells and in the extracts of anterior pituitary tissue. In addition, the primary culture of enriched rat lactotropes secreted TGF-beta 1. Using Northern blot techniques, a 2.4-kilobase transcript of pro-TGF-beta 1 mRNA was detected both in the anterior pituitary tissue and in the primary culture of anterior pituitary cells. These data suggest that TGF-beta 1 is produced in the pituitary gland and inhibits the secretion of PRL and growth of lactotropes in an autocrine and/or paracrine fashion.

Signal transduction by bFGF, but not TGF beta 1, involves arachidonic acid metabolism in endothelial cells.

We investigated the stimulation of early cellular events resulting from the interaction of the growth factor basic FGF (bFGF) and of the growth inhibitor transforming growth factor beta-type 1 (TGF beta 1), with their specific receptors on bovine endothelial cells. At mitogenic concentrations, bFGF stimulated the rapid release of arachidonic acid and its metabolites from (3H)-arachidonic acid labeled cells. When arachidonic acid metabolism was stimulated by addition of the calcium ionophore A23187, the effect of bFGF was amplified. Nordihydroguaïaretic acid, an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism, decreased the mitogenic effect of bFGF, whereas indomethacin, an inhibitor of the cyclooxygenase pathway, was ineffective. These findings suggest that metabolism of arachidonic acid to lipoxygenase products may be necessary for the mitogenic effect of bFGF. Basic FGF did not stimulate the production of inositol phosphates from cells labelled with myo-(2-3H)-inositol nor did it induce calcium mobilization, as measured by fura-2 fluorescence, indicating that bFGF does not activate phosphoinositide-specific phospholipase C in endothelial cells, but rather, that bFGF-induced arachidonic acid metabolism is mediated by another phospholipase. TGF beta 1, which inhibits basal and bFGF-induced endothelial cell growth, had no effect on arachidonic acid metabolism and inositol phosphate formation and did not prevent bFGF-induced arachidonic acid metabolism. These results suggest that the inhibitory action of TGF beta 1 on endothelial cell growth occurs through different mechanisms.

Transforming growth factor beta is a potent inhibitor of interleukin 1 (IL-1) receptor expression: proposed mechanism of inhibition of IL-1 action.

Transforming growth factor beta (TGF-beta) acts as a potent inhibitor of the growth and functions of lymphoid and hemopoietic progenitor cells. Cell proliferation depends not only on the presence of growth factors, but also on the development of specific receptor-signal transducing complexes. We therefore investigated whether the inhibitory actions of TGF-beta could be mediated by inhibition of growth factor receptors. TGF-beta inhibited the constitutive level of interleukin 1 receptor (IL-1R) expression on several murine lymphoid and myeloid progenitor cell lines, as well as IL-1R expression induced by interleukin 3 (IL-3) on normal murine and human bone marrow cells. Furthermore, treatment of bone marrow progenitor cells with TGF-beta concomitantly inhibited the ability of IL-1 to promote high proliferative potential (HPP) colony formation as well as blocked IL-1-induced IL-2 production by EL-4 6.1 cells. These findings provide the first evidence that the inhibitory action of TGF-beta on the growth and functional activities of hematopoietic and T cells is associated with a reduction in the cell surface receptor expression for IL-1.

Transforming growth factor alpha may be a physiological regulator of liver regeneration by means of an autocrine mechanism.

We investigated whether transforming growth factor alpha (TGF-alpha) is involved in hepatocyte growth responses both in vivo and in culture. During liver regeneration after partial hepatectomy in rats, TGF-alpha mRNA increased; it reached a maximum (approximately 9-fold higher than normal) at the peak of DNA synthesis. The message and the peptide were localized in hepatocytes and found in higher amounts in hepatocytes obtained from regenerating liver. TGF-alpha caused a 13-fold elevation of DNA synthesis in hepatocytes in primary culture and was slightly more effective than epidermal growth factor. TGF-beta blocked TGF-alpha stimulation when added either simultaneously with TGF-alpha or a day later. TGF-alpha message increased in hepatocytes stimulated to undergo DNA synthesis by TGF-alpha or epidermal growth factor, and the peptide was detected in the culture medium by RIA. In the regenerating liver, the increase in TGF-alpha mRNA during the first day after partial hepatectomy coincided with an increase in epidermal growth factor/TGF-alpha receptor mRNA and a decrease (already reported) in the number of these receptors. We conclude that TGF-alpha may function as a physiological inducer of hepatocyte DNA synthesis during liver regeneration by means of an autocrine mechanism and that its stimulatory effects in this growth process are balanced by the inhibitory action of TGF-beta 1.

TGF-beta inhibits growth factor-induced DNA synthesis in hamster fibroblasts without affecting the early mitogenic events.

Transforming growth factor beta (TGF-beta) was found to inhibit (IC50 = 0.1 ng/ml) alpha-thrombin or FGF-induced mitogenicity in G0-arrested Chinese hamster lung fibroblasts. Growth factor-stimulated cells became rapidly insensitive to TGF-beta addition during their progression through G0/G1 suggesting that an early step of the mitogenic response was the target of TGF-beta action. Surprisingly, none of the well characterized early mitogenic events commonly triggered by growth factors was found to be affected by TGF-beta addition. These responses included: phosphoinositide breakdown, activation of protein kinase C as determined by EGF receptor down-modulation, subsequent rises in pHi, c-fos, and c-myc mRNA levels, ribosomal protein S6 phosphorylation, the increase in RNA and protein synthesis, induction of ornithine decarboxylase. Only the induction of thymidine kinase, a marker of entry in the S phase, was found to be repressed by TGF-beta, with maximal inhibition when TGF-beta was added early in G1. These results indicate that the inhibitory action of TGF-beta does not affect the growth factors signalling pathways but touches an early event different from those so far analyzed.