Transforming growth factor beta stimulates fibroblast-collagen matrix contraction by different mechanisms in mechanically loaded and unloaded matrices

Studies were carried out to test the idea that transforming growth factor beta (TGFbeta) stimulated fibroblast contraction of collagen matrices by different mechanisms depending on mechanical loading on the cells and matrix. Under mechanically unloaded conditions (floating matrices), TGFbeta stimulated contraction directly as an agonist and indirectly by preactivating cells to express the myofibroblast phenotype. Increased contraction of floating matrices by preactivated cells appeared to result in part from an autocrine mechanism. Under mechanically loaded conditions (stressed matrices), TGFbeta had no direct agonist effect on contraction. Fibroblasts preactivated to become myofibroblasts showed increased ability to transfer tension to stressed matrices, and tension persisted even after the cells' actin cytoskeleton was disrupted. Our findings are consistent with the idea that fibroblasts activated to become myofibroblasts in vitro have increased contractile activity and indicate that multiple mechanisms that differ depending on mechanical loading on the cells and matrix are involved.     

Transforming growth factors and control of neoplastic cell growth

Transforming growth factors (TGFs) are peptides that affect the growth and phenotype of cultured cells and bring about in nonmalignant fibroblastic cells phenotypic properties that resemble those of malignant cells. Two types of TGFs have been well characterized. One of these, TGF alpha, is related to epidermal growth factor (EGF) and binds to the EGF receptor, whereas the other, TGF beta, is not structurally or functionally related to TGF alpha or EGF and mediates its effects via distinct receptors. TGF beta is produced by a variety of normal and malignant cells. Depending upon the assay system employed, TGF beta has both growth-inhibitory and growth-stimulating properties. Many of the mitogenic effects of TGF beta are probably an indirect result of the activation of certain growth factor genes in the target cell. The ubiquitous nature of the TGF beta receptor and the production of TGF beta in a latent form by most cultured cells suggests that the differing cellular responses to TGF beta are regulated either by events involved in the activation of the factor or by postreceptor mechanisms. The combined effects of TGF beta with other growth factors or inhibitors evidently play a central role in the control of normal and malignant cellular growth as well as in cell differentiation and morphogenesis. Since transforming growth factor as a concept has partially proven misleading and insufficient, there is a need to find a new nomenclature for these regulators of cellular growth and differentiation.     

Transforming growth factor beta and basic fibroblast growth factor synergistically promote early bovine embryo development during the fourth cell cycle.

Developmentally competent bovine blastocysts were produced by adding transforming growth factor beta (TGF beta) and basic fibroblast growth factor (bFGF) to serum-free cultures of in vitro produced, 2-cell bovine embryos. The effects of TGF beta were evaluated because this growth factor signals synthesis and secretion of the extracellular matrix component fibronectin and its receptor. Previous investigations have demonstrated that fibronectin promotes early bovine embryo development in vitro. The effects of TGF beta can be potentiated by bFGF; bFGF itself is an effector of protein synthesis and a potent mitogen. A positive interaction between the 2 growth factors resulted in 38.8% of fertilized oocytes maturing beyond the 16-cell stage; of these, 24.6% formed blastocysts. Transfer of early blastocysts produced using serum-free medium supplemented with growth factors resulted in pregnancy in 3 of 9 recipients. These results support the hypothesis that TGF beta and bFGF act synergistically to promote development of bovine embryos beyond the "8-cell block" observed in vitro.     

Transforming growth factor-beta 1 enhances the suppression of human hematopoiesis by tumor necrosis factor-alpha or recombinant interferon-alpha

The effects of transforming growth factor-beta 1 (TGF-beta 1) on human hematopoiesis were evaluated in combination with two other regulatory cytokines, namely, recombinant human tumor necrosis factor-alpha (TNF-alpha) and recombinant human interferon-alpha (rIFN-alpha). Combinations of TNF-alpha and TGF-beta 1 resulted in a synergistic suppression of colony formation by erythroid progenitor cells (BFU-E) and an additive suppression of granulocyte-macrophage (CFU-GM) and multipotential (CFU-GEMM) progenitor cells. In addition, TGF-beta 1 synergized with rIFN-alpha to suppress CFU-GM formation, while the combined suppressive effects of both cytokines on CFU-GEMM and BFU-E were additive. When TGF-beta 1 was tested with TNF-alpha or IFN-alpha on granulocyte/macrophage colony-stimulating factor (GM-CSF)-stimulated bone marrow cells in a 5-day proliferation assay, the antiproliferative effects of TGF-beta 1 and TNF-alpha were additive, while those with TGF-beta 1 and rIFN-alpha were synergistic. A similar pattern was seen in the suppression of the myeloblastic cell line KG-1 where TGF-beta 1 in combination with TNF-alpha resulted in an additive suppression while inhibition by TGF-beta 1 and IFN-alpha was synergistic. These results demonstrate for the first time the cooperative effects between TGF-beta and TNF-alpha and IFN-alpha in the suppression of hematopoietic cell growth, raising the possibility that TGF-beta might be used in concert with TNF-alpha or IFN-alpha in the treatment of various myeloproliferative disorders.     

Differential activation of ErbB receptors in the rat olfactory mucosa by transforming growth factor-α and epidermal growth factor in vivo

Transforming growth factor-α (TGF-α) and epidermal growth factor (EGF) are members of the EGF family of growth factors. They have a common receptor, the EGF receptor. This belongs to the tyrosine kinase group of receptors called the ErbB receptor family. Other members are ErbB-2, ErbB-3, and ErbB-4. Binding of either ligand to the receptor elicits an increase in tyrosine kinase activity, resulting in the autophosphorylation of the receptor followed by a phosphorylation cascade of other tyrosine kinase substrates including mitogen-activated protein kinase (MAPK). TGF-α and EGF have been shown to stimulate cell division in the olfactory epithelium in vitro and may regulate cell division in vivo. To investigate whether exogenous TGF-α or EGF has a functional effect on the olfactory mucosa in vivo, 12.5–50 μg of each growth factor was administered to rats via the carotid artery. After 2 min, olfactory mucosa and liver samples were collected, homogenized, and immunoprecipitated with antibodies to the ErbB receptors. The immunoprecipitates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western immunoblotting. Using phosphotyrosine antibody, the receptors were probed for phosphorylation. Activation of MAPK was also investigated using MAPK antibody. Exogenous TGF-α activated EGFR, ErbB-2 and MAPK, whereas EGF activated only the EGFR. TGF-α was a more potent activator of EGFR than EGF. Neither ligand had an effect on ErbB-3 and ErbB-4 receptors. These effects were absent in the control animals which received the same solution without the growth factor. These results are consistent with the notion that binding of TGF-α to EGFR may play a role in olfactory cell division in vivo. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 199–210, 1998     

Transforming growth factors (TGFs): Properties and possible mechanisms of action

Transforming growth factors (TGFs) are growth-promoting polypeptides that cause phenotypic transformation and anchorage-independent growth of normal cells. They have been isolated from several human and animal carcinoma and sarcoma cells. One TGF is sarcoma growth factor (SGF) which is released hy murine sarcoma virus-transformed cells. The TGFs interact with epidermal growth factor (EGF) cell membrane receptors. TGFs are not detectable in culture fluids from cells which contain high numbers of free EGF cell membrane receptors. SGF acts as a tumor promoter in cell culture systems and its effect on the transformed phenotype is blocked by retinoids (vitamin A and synthetic analogs). The production of TGFs by transformed cells and the responses of normal cells to the addition of TGFs to the culture medium raise the possibility that cells “autostimulate” their own growth by releasing factors that rebind at the cell surface. The term “autocrine secretion” has been proposed for this type of situation where a cell secretes a hormone-like substance for which it has external cell membrane receptors. The autocrine concept may provide a partial explanation for some aspects of tumor cell progression.     

Cytokine regulation of adult human osteoblast-like cell prostaglandin biosynthesis

Prostaglandin (PG) biosynthesis by cytokine stimulated normal adult human osteoblast-like (hOB) cells was evaluated by thin layer chromatography, high performance liquid chromatography, and specific immunoassays. PGE₂ was the predominant PG formed under all incubation conditions tested. Control samples produced measurable amounts of PGE₂, and the measured level of this metabolite increased by 22-fold (from 7 to 152 ng/ml) following a 20 h treatment with the combination of TGFβ and tumor necrosis factor-α(TNF). The production of 6-keto-PGF_1α (the stable metabolite of prostacyclin) and of PGF_2α were each increased by about five-fold (from about 0.5 to 2.5 ng/ml) in samples treated with the cytokines. Thus, TGFβ and TNF exerted a regulation of hOB cell PG biosynthesis that was principally directed towards an increased PGE₂ biosynthesis, with lesser effects on the production of other PG metabolites. COX-2 mRNA levels were increased within 2 h of cytokine stimulation, reached a maximum at 6–12 h, and levels had appreciably diminished by 24 h after treatment. Both TGFβ and TNF could independently increase COX-2 mRNA levels and PG biosynthesis. However, the increased production of PGE₂ resulting from TNF stimulation was blocked by the addition of an interleukin-1β (IL-1β) neutralizing antibody, suggesting that TNF regulation of hOB cell PG synthesis was secondary to its capacity to increase hOB cell IL-1β production. TGFβ regulation of PG production was not affected by the addition of the neutralizing antibody. These studies support the proposition that PGs can be important autocrine/paracrine mediators of bone biology, whose production by hOB cells is responsively regulated by osteotropic cytokines. J. Cell. Biochem. 64:618–631. © 1997 Wiley-Liss, Inc.     

Expression of a particular beta-N-acetylglucosaminidase isoenzyme in human haematopoietic leukemic cell-lines

N-acetyl-beta-D-glucosaminidase (NAG) activity and isoenzyme profiles were studied in myeloid, histiocytic, B-lymphoid, T-lymphoid and lymphoblastoid continuous cell lines in order to determine if N-acetyl-beta-D-glucosaminidase isoenzyme expression may help to distinguish among various types of leukemic proliferation. Total NAG activity in myeloid, histiocytic, erythroleukemic cell lines were higher than Burkitt's lymphoma derived cell lines (B-lymphoid), T- or lymphoblastoid cell lines. On chromatofocusing by PBE 94 coupled with an automated enzyme assay an intermediate (I) beta-N-acetyl-glucosaminidase form, eluting between forms B and A, was found in all leukemic and in Epstein-Barr virus infected lymphoblastoid cell lines analysed. The different profiles recorded, the expression of the I form and the different I/B ratios may be useful as markers of tumour proliferation.     

Alterations of expression and regulation of transforming growth factor beta in human cancer prostate cell lines

TGFβ can promote and/or suppress prostate tumor growth through multiple and opposing actions. Alterations of its expression, secretion, regulation or of the sensitivity of target cells can lead to a favorable environment for tumor development. To gain a better insight in TGFβ function during cancer progression, we have used different cultured human prostate cells: preneoplastic PNT2 cells, the androgen-dependent LNCaP and the androgen-independent PC3 and DU145 prostate cancer cell lines. We have studied by specific ELISA assays in conditioned media (CM), the secretion of TGFβ1 and TGFβ2 in basal conditions and after hormonal treatment (DHT or E2) and the expression of TGFβ1 mRNA by Northern blot. We have also compared the effect of fibroblast CM on TGFβ secretion by the different cell types. Compared to PNT2 cells, cancer cell lines secrete lower levels of active TGFβ which are not increased in the presence of fibroblast CM. LNCaP cells respond to androgen or estrogen treatment by a 10-fold increase of active TGFβ secretion while PC3 and DU145 are unresponsive. In conclusion, prostate cancer cell lines have lost part of their ability to secrete and activate TGFβ, and to regulate this secretion through stromal–epithelial interactions. Androgen-sensitive cancer cells may compensate this loss by hormonal regulation.     

Transforming growth factor-alpha attenuates the acquisition of aromatase activity by cultured rat granulosa cells

The effect of transforming growth factor-alpha (TGF alpha) on granulosa cell differentiation, as assessed by the acquisition of aromatase activity, was evaluated in vitro by using a primary culture of rat granulosa cells. Harvested from immature, diethylstilbestrol-treated rats, granulosa cells were cultured under serum-free conditions for 72 hr in the presence of saturating concentrations (10(-7)M) of aromatase substrate androstenedione with or without the specific experimental agents. Basal aromatase activity, as assessed by the generation of radioimmunoassayable estrogen was negligible, remaining unaffected by treatment with TGF alpha (10 ng/ml) by itself. Whereas treatment with follicle-stimulating hormone (FSH) resulted in a substantial increase in the extent of aromatization, concurrent treatment with TGF alpha (10 ng/ml) resulted in significant (P less than 0.05), yet reversible inhibition (78 +/- 5.6%) of FSH action. Significantly, this effect of TGF alpha could not be accounted for by a decrease in cellular viability or plating efficiency nor by a decrease in the number of cells or their DNA content. Although independent of the FSH dose employed, the TGF alpha effect proved dose- and time-dependent, with an apparent median inhibitory dose (EC50) of 0.33 +/- 0.04 ng/ml, and a minimal time requirement of 48 hr. Capable of substantial inhibition of the forskolin-stimulated accumulation of extracellular adenosine 3', 5' cyclic monophosphate (cAMP) and estrogen, TGF alpha had a measurable albeit limited effect on N6, 2-'O-Dibutyryladenosine 3':5'-cyclic monophosphate-supported estrogen production. Relative potency comparison revealed epidermal growth factor (EGF; EC50 = 0.24 +/- 0.03 ng/ml) and TGF alpha to be virtually equipotent as regards the attenuation of FSH-stimulated estrogen biosynthesis. Taken together, our findings indicate that TGF alpha, like EGF, acting at subnanomolar concentrations, is capable of attenuating the FSH-stimulated (but not basal) accumulation of estrogen. This effect of TGF alpha proved time- and dose-dependent, involving virtually complete neutralization of FSH action at site(s) both proximal and distal to cAMP generation. As such, these findings provide yet another example of the remarkable qualitative and quantitative similarities between EGF and TGF alpha, thereby reaffirming the prospect that ligands of the EGF/TGF alpha receptor may play a modulatory role in the course of granulosa cell ontogeny.     

NMR restraint analysis of transforming growth factor alpha: a key component for NMR structure refinement.

Structures of the protein, transforming growth factor alpha (TGF-alpha), have been derived from NMR data using distance geometry and subsequent energy refinement. Analysis of the sequential NOE distance bounds using a template algorithm provides a check for consistency in the calculation of bounds, stereospecific assignment of prochiral centers, and secondary structure assignment. Application of the template algorithm to the long range NOEs found within the NMR data sets collected at pH 6.3 and pH 3.4 is used to assess the confidence levels for the accuracy of the structures obtained from modeling. The method also provides critical insight in differentiating regions of the structure that are well defined from those that are not. Use of the restraint analysis protocol is shown to be a powerful adjunct to currently used methods for the assignment of protein structures from NMR data.     

Transforming growth factor β2 is negatively regulated by endogenous retinoic acid during early heart morphogenesis

Vitamin A‐deficient (VAD) quail embryos lack the vitamin A‐active form, retinoic acid (RA) and are characterized by a phenotype that includes a grossly abnormal cardiovascular system that can be rescued by RA. Here we report that the transforming growth factor, TGFβ2 is involved in RA‐regulated cardiovascular development. In VAD embryos TGFβ2 mRNA and protein expression are greatly elevated. The expression of TGFβ receptor II is also elevated in VAD embryos but is normalized by treatment with TGFβ2‐specific antisense oligonucleotides (AS). Administration of this AS or an antibody specific for TGFβ2 to VAD embryos normalizes posterior heart development and vascularization, while the administration of exogenous active TGFβ2 protein to normal quail embryos mimics the excessive TGFβ2 status of VAD embryos and induces VAD cardiovascular phenotype. In VAD embryos pSmad2/3 and pErk1 are not activated, while pErk2 and pcRaf are elevated and pSmad1/5/8 is diminished. We conclude that in the early avian embryo TGFβ2 has a major role in the retinoic acid‐regulated posterior heart morphogenesis for which it does not use Smad2/3 pathways, but may use other signaling pathways. Importantly, we conclude that retinoic acid is a critical negative physiological regulator of the magnitude of TGFβ2 signals during vertebrate heart formation.     

The antineoplasic agent estramustine and the derivative estramustine-phosphate inhibit secretion of interleukin-3 in leukemic cells. Possible roles of MAPs

The antineoplasic drug estramustine is an adduct of estradiol and nor-nitrogen mustard. It has been shown that this drug interferes with microtubule assembly, an effect mediated by estramustine interaction with microtubule-associated proteins (MAPS). In the present report we demonstrate that estramustine and the phosphorylated derivative of the drug, estramustine-phosphate, inhibit the secretion of interleukin-3 by WEHI-3B cells. These studies also show that the estramustine derivative specifically interacts with a MAPs component found in these cells, which exhibited characteristics ressembling those of tau protein isoforms. Western blots using a unique monoclonal antibody MTB6.22 that recognizes microtubule-binding domains on MAPs, indicated that this WEHI protein factor contained the antigenic determinant that are functionally significant for microtubule assembly. ELISA assays using this antibody, also showed a decrease in the levels of the immunoreactive protein in WEHI cells after treatment with EMP. Interestingly, it has been recently described that the action of estramustine-phosphate is mediated by a direct interaction with MAP-binding sites on the microtubule surface, which are recognized by the site-specific monoclonal antibody. These findings together with immuno-precipitation experiments using anti-interleukin-3 antibodies and the inhibitory effect of the estramustine derivative on WEHI secretion process suggest that this anti-mitotic agent may block IL-3 secretion by a mechanism involving its interaction with a tau-like MAPs component present in these cells.