Temporal changes in the response of chick limb bud mesodermal cells to transforming growth factor beta-type 1

Transforming growth factor beta (TGF beta) has been found in adult and developing bone in vivo and has varied effects on chondrocytes and osteoblasts in culture. We investigated the effects of TGF beta-type 1 on embryonic chick endochondral bone precursors in culture. Stage 24 chick limb bud mesoderm cells were cultured at high density. Under these conditions a dense mat containing nodules of cartilage surrounded by alkaline phosphatase-positive cells formed. Exposure of mesodermal cells to TGF beta-type 1 over the course of 4-7 days or during Days 3 and 4 caused a reduction in alkaline phosphatase activity and [35S]sulfate incorporation into proteoglycans. When the TGF beta-type 1 was applied during Days 1-2, it caused an increase in both parameters: these increases were observed in the absence of a corresponding change in [3H]thymidine incorporation. The specificity of the effects of TGF beta-type 1 was confirmed by neutralizing antibodies. These studies show that TGF beta-type 1 modulates the phenotype of embryonic endochondral bone precursors. The influence may depend on the window of exposure to the growth factor and, therefore, on the state of differentiation of the cells.     

Involvement of Smad signaling in sphingosine 1-phosphate-mediated biological responses of keratinocytes

The lysophospholipid sphingosine 1-phosphate and the cytokine-transforming growth factor beta are both released from degranulating platelets at wound sites, suggesting a broad spectrum of effects involved in wound healing. Interestingly, both of these molecules have been previously shown to induce chemotaxis but to strongly inhibit the growth of keratinocytes, while stimulating the proliferation of fibroblasts. In contrast to sphingosine 1-phosphate, the signaling cascade of the growth factor has been extensively examined. Specifically, Smad3 has been shown to be an essential mediator of transforming growth factor beta-dependent chemotaxis of keratinocytes and mediates, in part, its growth-inhibitory effect. Here we show that sphingosine 1-phosphate, independently of transforming growth factor beta secretion, induces a rapid phosphorylation of Smad3 on its C-terminal serine motif and induces its partnering with Smad4 and the translocation of the complex into the nucleus. Moreover, sphingosine 1-phosphate fails to induce chemotaxis or inhibit the growth of Smad3-deficient keratinocytes, suggesting that Smad3 plays an unexpected functional role as a new target in sphingosine 1-phosphate signaling. Both sphingosine 1-phosphate receptors and the transforming growth factor beta-type I receptor serine/threonine kinase are essential for activation of Smad3 by this lysophospholipid and the dependent biological responses, indicating a novel cross-talk between serine/threonine kinase receptors and G-protein coupled receptors.     

The tumor cells (FA-6) established from a pancreatic cancer associated with humoral hypercalcemia of malignancy: a simultaneous production of parathyroid hormone-like activity and transforming growth factor activity

Human pancreatic cancer cells (FA-6) producing bone resorbing factor were established in culture. A biopsied lymphnode from a patient with pancreatic cancer associated with humoral hypercalcemia of malignancy (HHM) was transplanted to nude mice, and the cells producing high parathyroid hormone (PTH)-like activity were selected by a limited dilution from outgrowth of the xenografts of the tumor grown in nude mice. The conditioned media contained an activity to stimulate the resorption of mouse calvaria in vitro which was indomethacin-insensitive. The conditioned media had both alpha-type and beta-type transforming growth factor (TGF) activity but no interleukin-1 activity. TGF-alpha activity was co-eluted with PTH-like activity from gel-chromatography at around 15 kDa. The FA-6 cells now established are the first cells of pancreatic cancer associated with HHM producing both PTH-like and TGF-alpha activities along with bone resorbing activity.     

Polyomavirus middle tumor antigen increases responsiveness to growth factors.

The middle tumor antigen (mT) of polyomavirus is unable to transform a clone of NIH 3T3 cells to anchorage independence (L. Raptis and J.B. Bolen, J. Virol. 63:753-758, 1989). However, this oncogene increased the responsiveness of these cells to the growth factors (alpha-like and beta-type transforming growth factors) produced by cells possessing the whole transforming region of polyomavirus. This resulted in the growth of NIH 3T3 cells, expressing mT under control of the dexamethasone-regulatable mouse mammary tumor virus promoter, in agar medium supplemented with these growth factors upon addition of the inducer. Therefore, mT, a transforming oncogene, is able to enhance the responsiveness of established cells to growth factors, a property previously attributed primarily to myc and other establishment type oncogenes.     

Down-regulation of cellular platelet-derived growth factor receptors induced by an activated neu receptor tyrosine kinase.

The functional integration of growth factor signaling occurs at several levels in target cells. One of the most proximal mechanisms is receptor transmodulation, by which one activated receptor can regulate the expression of other receptors in the same cells. Well-established transregulatory loops involve platelet-derived growth factor (PDGF) down-regulation of epidermal growth factor (EGF) receptors and beta-type transforming growth factors modulation of PDGF receptors. We have studied the relationship between neu tyrosine kinase activation and the expression of the PDGF receptors in transfected NIH/3T3 cells. Expression of the neu oncogene, but not of the neu proto-oncogene, was associated with a decrease of PDGF alpha- and beta-receptors on the cell surface, as measured by [125-I]PDGF-AA and -BB binding. These results were corroborated by metabolic labeling and immunoprecipitation of the PDGF beta-receptors. PDGF alpha- and beta-receptor mRNAs were strongly decreased in the neu oncogene-transformed cells in comparison with control cells expressing the neu proto-oncogene. Down-regulation of the PDGF receptors and their mRNAs was also observed after EGF treatment of cells expressing a chimeric EGF receptor/neu receptor, where the neu tyrosine kinase is activated by EGF binding. These results show that the neu tyrosine kinase can down-modulate PDGF receptor expression, and the effect is mediated via decreased PDGF receptor mRNA levels.     

Synthesis of a Bacillus subtilis small, acid-soluble spore protein in Escherichia coli causes cell DNA to assume some characteristics of spore DNA.

Small, acid-soluble proteins (SASP) of the alpha/beta-type are associated with DNA in spores of Bacillus subtilis. Induction of synthesis of alpha/beta-type SASP in Escherichia coli resulted in rapid cessation of DNA synthesis, followed by a halt in RNA and then protein accumulation, although significant mRNA and protein synthesis continued. There was a significant loss in viability associated with SASP synthesis in E. coli: recA+ cells became extremely long filaments, whereas recA mutant cells became less filamentous. The nucleoids of cells with alpha/beta-type SASP were extremely condensed, as viewed in both light and electron microscopes, and immunoelectron microscopy showed that the alpha/beta-type SASP were associated with the cell DNA. Induction of alpha/beta-type SASP synthesis in E. coli increased the negative superhelical density of plasmid DNA by approximately 20%; UV irradiation of E. coli with alpha/beta-type SASP gave reduced yields of thymine dimers but significant amounts of the spore photoproduct. These changes in E. coli DNA topology and photochemistry due to alpha/beta-type SASP are similar to the effects of alpha/beta-type SASP on the DNA in Bacillus spores, further suggesting that alpha/beta-type SASP are a major factor determining DNA properties in bacterial spores.     

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 (TGFβ1), 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. TGFβ1, which inhibits basal and bFGF-induced endothelial cell growth, had no effect on arachidonic acid matabolism and inositol phosphate formation and did not prevent bFGF-induced arachidonic acid metabolism. These results suggest that the inhibitory action of TGFβ1 on endothelial cell growth occurs through different mechanisms.     

Increased secretion of type beta transforming growth factor accompanies viral transformation of cells.

Cells transformed by Harvey or Moloney sarcoma virus secrete at least 40 times as much type beta transforming growth factor as their respective untransformed control cells. The transformed cells bind only 20 to 50% as much type beta transforming growth factor as the control cells, suggesting that transformation causes down-regulation of the type beta transforming growth factor receptor.     

Identification and characterization of polypeptide growth factors secreted by murine embryonal carcinoma cells

Undifferentiated P19 and PC13 murine embryonal carcinoma (EC) cells have been analyzed for their ability to secrete polypeptide growth factors. This has been carried out by a combination of specific bioassays and the use of biochemical and immunological detection methods. Both P19 and PC13 EC cells secrete a platelet-derived growth factor (PDGF)-like growth factor, a type beta transforming growth factor, and insulin-like growth factors. In addition, PC13 EC cells secrete a heparin-binding growth factor functionally related to fibroblast growth factor, while P19 EC cells secrete transforming growth factor-alpha. This is the first demonstration for secretion of transforming growth factor-alpha by an equivalent of early embryonic cells. The possible paracrine growth stimulating effects of these growth factors have been tested on differentiated derivatives of P19 EC cells, corresponding to all three germ layers. The differences in growth factor production by various embryonal carcinoma cells are discussed in relation to the developmental origin of these cell lines.     

Embryonal carcinoma cell growth and differentiation. Production of and response to molecules with transforming growth factor activity

Transforming growth factors are known to induce anchorage-independent growth of non-transformed cells, and are released by a variety of cells, including MSV-transformed cells. This study demonstrates that the differentiated cells derived from F9 and PC-13 embryonal carcinoma cells, but not the parental cells themselves, respond by increased growth to several factors released by MSV-transformed cells, including partially purified sarcoma growth factor. The chemical properties of the growth-promoting activity are shown to match the chemical properties of the transforming growth factors released by MSV-transformed cells. Furthermore, F9 and PC-13 embryonal carcinoma cells, which do not respond to factors released by MSV-transformed cells, are shown to release factors with transforming growth factor activity. Based on the close relationship between mouse embryonal carcinoma cells and cells of early mouse embryos, it is suggested that molecules with transforming growth factor activity may play a role during the early stages of mammalian development.     

Surface proteome analysis identifies platelet derived growth factor receptor-alpha as a critical mediator of transforming growth factor-beta-induced collagen secretion

Fibroblasts are extracellular matrix-producing cells in the lung. Fibroblast activation by transforming growth factor-beta leads to myofibroblast-differentiation and increased extracellular matrix deposition, a hallmark of pulmonary fibrosis. While fibroblast function with respect to migration, invasion, and extracellular matrix deposition has been well-explored, little is known about the surface proteome of lung fibroblasts in general and its specific response to fibrogenic growth factors, in particular transforming growth factor-beta.We thus performed a cell-surface proteome analysis of primary human lung fibroblasts in presence/absence of transforming growth factor-beta, followed by characterization of our findings using FACS analysis, Western blot, and siRNA-mediated knockdown experiments.We identified 213 surface proteins significantly regulated by transforming growth factor-beta, platelet derived growth factor receptor-alpha being one of the top down-regulated proteins. Transforming growth factor beta-induced downregulation of platelet derived growth factor receptor-alpha induced upregulation of platelet derived growth factor receptor-beta expression and phosphorylation of Akt, a downstream target of platelet derived growth factor signaling. Importantly, collagen type V expression and secretion was strongly increased after forced knockdown of platelet derived growth factor receptor-alpha, an effect that was potentiated by transforming growth factor-beta. We therefore show previously underappreciated cross-talk of transforming growth factor-beta and platelet derived growth factor signaling in human lung fibroblasts, resulting in increased extracellular matrix deposition in a platelet derived growth factor receptor-alpha dependent manner. These findings are of particular importance for the treatment of lung fibrosis patients with high pulmonary transforming growth factor-beta activity.     

Beta-type transforming growth factor specifies organizational behavior in vascular smooth muscle cell cultures

In culture, vascular smooth muscle cells (SMC) grow in a "hill-and-valley" (multilayered) pattern of organization. We have studied the growth, behavioral organization, and biosynthetic phenotype of rat aortic SMC exposed to purified platelet-derived growth regulatory molecules. We show that multilayered growth is not a constitutive feature of cultured SMC, and that beta-type transforming growth factor (TGF-beta) is the primary determinant of multilayered growth and the hill-and-valley pattern of organization diagnostic for SMC in culture. TGF-beta inhibited, in a dose-dependent manner, the serum- or platelet-derived growth factor-mediated proliferation of these cells in two-dimensional culture, but only when cells were plated at subconfluent densities. The ability of TGF-beta to inhibit SMC growth was inversely correlated to plating cell density. When SMC were plated at monolayer density (5 X 10(4) cells/cm2) to allow maximal cell-to-cell contact, TGF-beta potentiated cell growth. This differential response of SMC to TGF-beta may contribute to the hill-and-valley pattern of organization. Unlike its effect on other cell types, TGF-beta did not enhance the synthesis of fibronectin or its incorporation into the extracellular matrix. However, the synthesis of a number of other secreted proteins was altered by TGF-beta treatment. SMC treated with TGF-beta for 4 or 8 h secreted markedly enhanced amounts of an Mr 38,000-D protein doublet whose synthesis is known to be increased by heparin (another inhibitor of SMC growth), suggesting metabolic similarities between heparin- and TGF-beta-mediated SMC growth inhibition. The data suggest that TGF-beta may play an important and complex regulatory role in SMC proliferation and organization during development and after vascular injury.     

Epithelial-mesenchymal interactions and lung branching morphogenesis. Role of polyamines and transforming growth factor beta1

Lung branching morphogenesis is a result of epithelial-mesenchymal interactions, which are in turn dependent on extracellular matrix composition and cytokine regulation. Polyamines have recently been demonstrated as able to modify chick embryo skin differentiation. In this work we have examined the effects of putrescine and spermidine during chick embryo lung morphogenesis in organotypic cultures by morphological, histochemical and biochemical examination. To verify the role of polyamines, we used specific inhibitors, such as bis-cyclohexylammonium sulphate and alfa-difluoromethylornithine, and transforming growth factor beta1, an ornithine decarboxylase and polyamine stimulator. Our data show that lung morphogenesis is significantly altered following the induced mesenchymal glycosaminoglycan changes. The increase of mesenchymal glycosaminoglycans is correlated with a stimulation of lung development in the presence of polyamines, and with its inhibition when transforming growth factor beta1 is added to the culture medium. The morphometric data show a uniform increase of both the mesenchyme and epithelial branching with spermidine and putrescine stimulus, whereas the mesenchymal substance alone is significantly increased in apical-median lung sections with transforming growth factor beta1 and transforming growth factor beta1 + spermidine lung cultures. Transforming growth factor beta1 and transforming growth factor beta1 + spermidine confirm the blocking of epithelial branching formations and fibroblast activation, and show that polyamines are unable to prevent the blocking of epithelial cells due to the inhibitory effect of transforming growth factor beta1.     

A mouse tumor-derived osteolytic factor stimulates bone resorption by a mechanism involving local prostaglandins production in bone

Culture medium which was conditioned by tissue of a CE mouse breast tumor in vitro contained dose-dependent osteolytic activity. The osteolytic activity was not soluble in dichloromethane and ethylacetate, indicating that it was not attributable to vitamin D metabolites or prostaglandins. However, breast tumor-conditioned medium stimulated production and release of prostaglandin E2 from mouse calvaria in vitro, and the stimulation of bone resorption in vitro by breast tumor-conditioned medium was blocked by a dose of indomethacin that prevented stimulation of mouse calvarial prostaglandin E2 production and release. The resorptive activity of parathyroid hormone (PTH) was not affected by the same dose of indomethacin, suggesting that the osteolytic factor was not PTH. This was further supported by observation that mouse kidney cell cAMP production was stimulated by PTH, but not by the aqueous phase of ethylacetate-extracted breast tumor-conditioned medium. In addition to osteolytic activity, breast tumor-conditioned medium contained a dose-dependent bone cell mitogenic activity, demonstrated by the stimulation of [3H]thymidine incorporation into trichloroacetic acid-insoluble macromolecules and a corresponding increase in bone cell number in monolayer cultures of bone cells. Breast tumor-conditioned medium also contained a dose-dependent transforming growth factor-(TGF-) like activity as defined by its ability to transform anchorage-dependent growth of nontransformed cells to anchorage-independent growth. The TGF in breast tumor-conditioned medium did not compete with epidermal growth factor (EGF) for EGF receptor binding, but its transforming activity was greatly enhanced by EGF, indicating that it was a beta-type TGF. Both the osteolytic and mitogenic activities were nondialyzable, sensitive to reducing agent, and not removable by dichloromethane and ethylacetate extractions. Furthermore, the TGF activity was not removed by ethylacetate extraction. Thus, the possibility that these activities in breast tumor-conditioned medium might be mediated by the same molecule must be considered. In summary, our data suggest that the CE mouse mammary carcinoma cells produce and secrete into the culture medium an osteolytic factor which is neither PTH nor prostaglandin and which stimulates local synthesis in bone of prostaglandin E2 which in turn increases bone resorption in vitro.     

The effect of transforming growth factor beta on the intensity of cellular DNA synthesis in relation to the type and conditions of cultivation

A study was made of the influence of transforming growth factor beta (0.05-50.00 ng/ml) on the intensity of 3H-thymidine incorporation in the DNAs of pseudonormal mouse fibroblasts of NIH 3T3 line, of cells of NRK-49F line from normal rat kidney, and of cells of A-549 line from human lung adenocarcinoma. The experiments were carried out in the absence and in the presence of epidermal growth factor (5 ng/ml), and(or) insulin (1 micrograms/ml), as well as in the presence of different concentrations of fetal calf serum, and while using different time of incubation of cells with the above mentioned growth factors. It was shown that depending on the culture conditions the transforming growth factor beta exerted stimulatory, inhibitory or no action on the intensity of DNA synthesis in the cells of the same type. An attempt was made to analyse the reasons, which may significantly determine the direction of regulatory influence of the transforming growth factor beta on DNA replication in the cells.     

DNA in dormant spores of Bacillus species is in an A-like conformation

The DNA in dormant spores of Bacillus species is associated with alpha/beta-type small, acid-soluble proteins (SASP), which are double-stranded DNA-binding proteins whose amino acid sequence has been highly conserved in evolution. In vitro these proteins bind most strongly to DNA which readily adopts an A-like conformation, as binding of alpha/beta-type SASP causes DNA to assume an A-like conformation. As predicted by this conformational change in DNA, binding of alpha/beta-type SASP to relaxed but covalently closed plasmid DNA results in the introduction of a large number of negative supercoils. Associated with the conformational change in DNA brought about by alpha/beta-type SASP binding is a change in its photochemistry such that ultraviolet irradiation does not generate pyrimidine dimers, but rather a thyminyl-thymine adduct termed spore photoproduct (SP). The latter two properties of DNA complexed with alpha/beta-type SASP in vitro are similar to those of DNA in dormant spores of Bacillus species in which: (i) plasmid DNA has a much higher number of negative supercoils than plasmid in growing cells; and (ii) ultraviolet irradiation produces SP and no pyrimidine dimers, while only pyrimidine dimers are formed in growing cells. During sporulation these changes in the properties of spore DNA take place in parallel with synthesis of alpha/beta-type SASP, and the magnitude of the changes is greatly reduced in mutants that make low amounts of these proteins. A straightforward interpretation of these data is that DNA in dormant spores of Bacillus species is in an A-like conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cadmium produces a delayed mitogenic response and modulates the EGF response in quiescent NRK cells

Recent studies have shown that cadmium, at subtoxic levels, may induce a response characteristic of that elicited by a type of growth factor that supports the anchorage independent growth of cells that are not fully transformed. That is, Cd⁺⁺ was found to replace transforming growth factor beta in supporting soft agar growth of NRK-49F cells. To tes the extent to which Cd⁺⁺ further mimics transforming growth factor beta in its effects and to establish response patterns that suggest possible molecular mechnisms of action, we have determined the effects of Cd⁺⁺ and/or epidermal growth factor (EGF) on DNA synthesis in quiescent NRK-49F cells. We found that subtoxic doses of Cd⁺⁺ modulate EGF-induced DNA synthesis in a dose-dependent fashion. Although Cd⁺⁺ effects on early (16–24 hr) EGF-induced DNA synthesis are primarily inhibitory, later effects involve stimulation as well. Subtoxic doses of Cd⁺⁺ did not stimulate DNA synthesis in quiescent cells within 24 hr of addition. At later times (40 or 64 hr), however, an increase in DNA synthesis of up to threefold was induced by 0.25 M Cd⁺⁺. This pattern of mitogenic response, involving inhibition of early growth-factor induced DNA synthesis and stimulation of late DNA synthesis, is consistent with that reported to be effected in some instances by transforming growth factor beta. Because a defined pattern of gene expression also is associated with the mitogenic responses to transforming growth factor beta, future studies at the molecular level can definitively test the degree to which Cd⁺⁺ and transforming growth factor beta effects are common.Abbreviations CFE colony forming efficiency - EGF epidermal growth factor - MT metallothionein - PGDF paltelet derived growth factor - TGF transforming growth factor     

The role of transforming growth factor beta in atherosclerosis: novel insights and future perspectives

PURPOSE OF REVIEW: Atherosclerosis is a disease of the arterial wall that seems to be tightly modulated by the local inflammatory balance. Transforming growth factors beta 1, 2 and 3 are cytokines/growth factors with broad activities on cells and tissues in the cardiovascular system, and have been suggested to play a role in the pathogenesis of atherosclerosis. RECENT FINDINGS: In the present review, we discuss recent developments in the role of transforming growth factor beta in the regulation of the immuno-inflammatory balance that modulates atherosclerosis. Such studies strongly suggest that the inhibition of endogenous transforming growth factor beta signalling favours the development of atherosclerotic lesions with an increased inflammatory component (T cells and macrophages) and decreased collagen content, features that are characteristic of unstable atherosclerotic plaques. SUMMARY: Transforming growth factor beta is identified as a critical modulator of the immuno-inflammatory balance in atherosclerosis, and a crucial plaque-stabilizing factor. Future studies should aim at defining the precise molecular mechanisms responsible for this protective effect, and developing immunomodulatory strategies based on the promotion of transforming growth factor beta activity (T regulatory T helper type 3 cells) to limit disease complications.     

The elastogenic effect of recombinant transforming growth factor-beta on porcine aortic smooth muscle cells

Transforming growth factor-beta, a peptide growth factor, is known to be a multifunctional regulator of cellular activity. The effect of this growth factor on extracellular matrix formation is well established, but its effects on elastin, a critical component of lung, skin, and blood vessels are unknown. In the present study, by use of an Enzyme-Linked Immunoassay method, we found that transforming growth factor-beta strongly increased elastin production in cultured porcine aortic smooth muscle cells. In a dosage-dependent study, 1.0-10.0 ng/ml transforming growth factor-beta promoted elastin production 2-3 fold. In a time-dependent study, at least an 8 h pre-treatment with 10.0 ng/ml transforming growth factor-beta was required for sustained increases in elastin production. The effects of transforming growth factor-beta on cultured aortic smooth muscle cells suggest that this cytokine may be an important mediator of elastin formation during atherosclerosis and hypertension.     

The effect of transforming growth factor-beta on the alkaline phosphatase activity in rabbit renal cortical tubular cell cultures

The effect of various growth regulators including epidermal growth factor and transforming growth factor-beta on the alkaline phosphatase activity of rabbit renal cortical tubular cells has been investigated in a serum-free culture. As a result, it was found that transforming growth factor-beta, known to be a growth inhibitor of renal tubular cells, increased the alkaline phosphatase activity of the tubular cells dose-dependently and that cycloheximide blocked any increase in the activity of this factor. In contrast, epidermal growth factor decreased the alkaline phosphatase activity in the tubular cells.