Effects of transforming growth factors and regulation of their mRNA levels in two human endometrial adenocarcinoma cell lines.

The effects of the transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF) on the growth of cells from 2 endometrial cancer lines, Ishikawa and HEC-50 were evaluated by measuring rates of DNA synthesis and changes in cell numbers during culture. EGF at 17 and 1.7 nM concentrations consistently enhanced HEC-50 cell proliferation. TGF-beta 1 inhibited Ishikawa cell proliferation but, unexpectedly for epithelium-derived cells, stimulated HEC-50 cell growth. This effect is of interest as it indicates that endometrial cells can acquire an altered responsiveness to a growth inhibitor during the process of malignant transformation. Northern blot analyses showed expression of TGF-alpha, TGF-beta 1 and EGF receptors mRNA in both cell lines. Neither estradiol (E2) nor 4-hydroxytamoxifen (OHTam) affected mRNA levels for either TGF-alpha or TGF-beta in HEC-50 cells, a line unresponsive to E2 for proliferation. In Ishikawa cells, previously shown to respond to both E2 and OHTam by increasing proliferation rates, E2 increased TGF-alpha mRNA and reduced TGF-beta mRNA levels. OHTam lowered the levels of both mRNA species, although the effect was greater on TGF-beta than TGF-alpha mRNA. These data are consistent with, but do not prove, the existence of a possible autocrine regulation by TGF-alpha and TGF-beta of human cancer cell proliferation, which might be under E2 influence in Ishikawa cells.     

A model system for tumor angiogenesis: involvement of transforming growth factor-alpha in tube formation of human microvascular endothelial cells induced by esophageal cancer cells.

Tumor growth is dependent on angiogenesis, which is thought to be mediated through growth factors, such as transforming growth factor-alpha (TGF-alpha) and -beta (TGF-beta), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), produced by tumor cells. We have developed a model system for tumor angiogenesis in vitro: tube formation of human omentum microvascular endothelial (HOME) cells in type I collagen gels when these cells are co-cultured with tumor cells. Exogenously added TGF-alpha induced tube formation of HOME cells in collagen gel. In contrast, TGF-beta inhibited the TGF-alpha-induced tube formation of endothelial cells. We investigated whether tube formation could be induced in HOME cells in collagen gel when the HOME cells were co-cultured with three esophageal cancer cell lines, TE1, TE2, and TE5. TE1 and TE2 cells expressed both TGF-alpha and TGF-beta mRNA, but the level of TGF-alpha mRNA in TE2 was found to be much lower than in TE1 cells. TE5 did not express either TGF-alpha or TGF-beta. The tube formation of HOME cell was induced when they were co-cultured with TE1 cells, while both TE2 and TE5 cell lines induced tube formation at much lower rates than TE1. TE1-induced tube formation of HOME cells was specifically blocked by co-administration of anti-TGF-alpha-antibody, but not by anti-bFGF-antibody. The present study suggests that, in our model system, esophageal tumor angiogenesis is partly controlled by TGF-alpha, possibly through a paracrine pathway.     

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.     

Retinoic acid-induced alterations in the expression of growth factors in embryonic mouse palatal shelves

Retinoic acid (RA) is teratogenic in many species, producing multiple malformations, including cleft palate. The effects of RA which lead to cleft palate vary depending on the stage of development exposed. After exposure of embryonic mice to RA on gestation day (GD) 10, abnormally small palatal shelves form. After exposure on GD 12 shelves of normal size form, but fail to fuse, as the medial cells proliferate and differentiate into a nasal-like epithelium. Growth factors and their receptors play an important role in regulating development, and the expression of EGF receptors, EGF, TGF-alpha, TFG-beta 1, and TGF-beta 2 has been reported in the mouse embryo. In a variety of cell types in culture, these growth factors are capable of regulating proliferation, differentiation, expression of matrix proteins, and other cellular events including epithelial-mesenchymal transformations. The present study examines immunohistochemically the expression of EGF, TGF-alpha, TGF-beta 1, and TGF-beta 2 in the control embryonic palatal shelves from GD 12 to 15 and the effects of RA treatment on GD 10 or 12 on their expression on GD 14 and 16. These growth factors were shown to have specific temporal and spatial expression in the palatal shelf. With advancing development the levels of TGF-alpha decreased while the expression of EGF increased. TGF-beta 2 localization became regional by GD 14-15, with higher levels found in epithelial cells and chondrogenic mesenchyme. TGF-beta 1 occurred in epithelial and mesenchymal cells and distribution did not change substantially with advancing development. RA exposure altered the expression of TFG-alpha, TGF-beta 1, and TGF-beta 2, but significant effects on EGF were not found. The effects on TGF-alpha and TGF-beta 1 expression were dependent on the gestational age exposed. Levels of TGF-alpha on GD 14 decreased after RA exposure on GD 10, but increased after GD 12 exposure. TGF-beta 1 expression in the mesenchyme was increased after exposure on GD 12, but was unaffected by RA on GD 10. After exposure on either day, the levels of TGF-beta 2 increased in GD 14 nasal epithelial cells. Acting in concert, growth factors could regulate events critical to formation of the secondary palate, including cessation of medial epithelial cell proliferation, synthesis of extracellular matrix proteins in the mesenchyme, programmed cell death of medial epithelial peridermal cells, and transformation of basal epithelial medial cells to mesenchymal cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transforming growth factor-beta modulates the high-affinity receptors for epidermal growth factor and transforming growth factor-alpha

The epidermal growth factor (EGF) receptor mediates the induction of a transformed phenotype in normal rat kidney (NRK) cells by transforming growth factors (TGFs). The ability of EGF and its analogue TGF-alpha to induce the transformed phenotype in NRK cells is greatly potentiated by TGF-beta, a polypeptide that does not interact directly with binding sites for EGF or TGF-alpha. Our evidence indicates that TGF-beta purified from retrovirally transformed rat embryo cells and human platelets induces a rapid (t 1/2 = 0.3 h) decrease in the binding of EGF and TGF-alpha to high-affinity cell surface receptors in NRK cells. No change due to TGF-beta was observed in the binding of EGF or TGF-alpha to lower affinity sites also present in NRK cells. The effect of TGF-beta on EGF/TGF-alpha receptors was observed at concentrations (0.5-20 pM) similar to those at which TGF-beta is active in promoting proliferation of NRK cells in monolayer culture and semisolid medium. Affinity labeling of NRK cells and membranes by cross-linking with receptor-bound 125I-TGF-alpha and 125I-EGF indicated that both factors interact with a common 170-kD receptor structure. Treatment of cells with TGF-beta decreased the intensity of affinity-labeling of this receptor structure. These data suggest that the 170 kD high-affinity receptors for EGF and TGF-alpha in NRK cells are a target for rapid modulation by TGF-beta.     

TGF-beta cooperates with TGF-alpha to induce the self-renewal of normal erythrocytic progenitors: evidence for an autocrine mechanism.

Simultaneous addition of both TGF-alpha and TGF-beta induces the sustained, long-term outgrowth of chicken erythrocytic progenitor cells, referred to as T2ECs from both chick bone marrow and 2-day-old chicken embryos. By analysis for differentiation antigens and gene expression, these cells were shown to represent very immature haematopoietic progenitors committed to the erythrocytic lineage. T2ECs differentiate into almost pure populations of fully mature erythrocytes within 6 days, when TGF-alpha and TGF-beta are withdrawn and the cells exposed to anaemic chicken serum plus insulin. Outgrowth of these cells from various sources invariably required both TGF-alpha and TGF-beta, as well as glucocorticoids. Proliferating, established T2ECs still require TGF-alpha, but are independent of exogenous TGF-beta. Using a TGF-beta-neutralizing antibody or expressing a dominant-negative TGF-beta receptor II, we demonstrate that T2ECs generate an autocrine loop involving TGF-beta during their establishment, which is required for sustained proliferation. Using specific inhibitors, we also show that signalling via Mek-1 is specifically required for induction and maintenance of cell proliferation driven by cooperation between the TGF-alpha and -beta receptors. These results establish a novel mechanism by which self-renewal of erythrocytic progenitors is induced and establish avian T2ECs as a new, quasi-optimal model system to study erythrocytic progenitors.     

Transforming growth factor beta1 (TGF-beta1) promotes endothelial cell survival during in vitro angiogenesis via an autocrine mechanism implicating TGF-alpha signaling

Mouse capillary endothelial cells (1G11 cell line) embedded in type I collagen gels undergo in vitro angiogenesis. Cells rapidly reorganize and form capillary-like structures when stimulated with serum. Transforming growth factor beta1 (TGF-beta1) alone can substitute for serum and induce cell survival and tubular network formation. This TGF-beta1-mediated angiogenic activity depends on phosphatidylinositol 3-kinase (PI3K) and p42/p44 mitogen-activated protein kinase (MAPK) signaling. We showed that specific inhibitors of either pathway (wortmannin, LY-294002, and PD-98059) all suppressed TGF-beta1-induced angiogenesis mainly by compromising cell survival. We established that TGF-beta1 stimulated the expression of TGF-alpha mRNA and protein, the tyrosine phosphorylation of a 170-kDa membrane protein representing the epidermal growth factor (EGF) receptor, and the delayed activation of PI3K/Akt and p42/p44 MAPK. Moreover, we showed that all these TGF-beta1-mediated signaling events, including tubular network formation, were suppressed by incubating TGF-beta1-stimulated endothelial cells with a soluble form of an EGF receptor (ErbB-1) or tyrphostin AG1478, a specific blocker of EGF receptor tyrosine kinase. Finally, addition of TGF-alpha alone poorly stimulated angiogenesis; however, by reducing cell death, it strongly potentiated the action of TGF-beta1. We therefore propose that TGF-beta1 promotes angiogenesis at least in part via the autocrine secretion of TGF-alpha, a cell survival growth factor, activating PI3K/Akt and p42/p44 MAPK.     

p21(cip1) mRNA is controlled by endogenous transforming growth factor-beta1 in quiescent human hematopoietic stem/progenitor cells

Transforming growth factor-beta1 (TGF-beta1) has been described as an efficient growth inhibitor that maintains the CD34(+) hematopoietic progenitor cells in quiescence. The concept of high proliferative potential-quiescent cells or HPP-Q cells has been introduced as a working model to study the effect of TGF-beta1 in maintaining the reversible quiescence of the more primitive hematopoietic stem cell compartment. HPP-Q cells are primitive quiescent stem/progenitor cells on which TGF-beta1 has downmodulated the cytokine receptors. These cells can be released from quiescence by neutralization of autocrine or endogenous TGF-beta1 with a TGF-beta1 blocking antibody or a TGF-beta1 antisense oligonucleotide. In nonhematopoietic systems, TGF-beta1 cooperates with the cyclin-dependent kinase inhibitor, p21(cip1), to induce cell cycle arrest. We therefore analyzed whether endogenous TGF-beta1 controls the expression of the p21(cip1) in the CD34(+) undifferentiated cells using a sensitive in situ hybridization method. We observed that addition of anti-TGF-beta1 is followed by a rapid decrease in the level of p21(cip1) mRNA whereas TGF-beta1 enhances p21(cip1) mRNA expression concurrently with an inhibitory effect on progenitor cell proliferation. These results suggest the involvement of p21(cip1) in the cell cycle control of early human hematopoietic quiescent stem/progenitors and not only in the differentiation of more mature myeloid cells as previously described. The modulation of p21(cip1) observed in response to TGF-beta1 allows us to further precise the working model of high proliferative potential-quiescent cells.     

Growth factors specifically alter hair follicle cell proliferation and collagenolytic activity alone or in combination

A three-dimensional culture model for isolated murine pelage hair follicles in a type I collagen gel has been utilized to study the effects of selected growth factors on follicle cell proliferation and release of collagenolytic factors. Cultured follicle organoids differentially express cytokeratins 6 and 14 in a pattern suggesting they contain cells of the outer root sheath, inner root sheath and follicle matrix. Using incorporation of [3H]thymidine as a measure of proliferation, follicle organoids show a peak of DNA synthesis between day 1 and 5 of culture, depending on plating density, and then have a low rate of DNA synthesis. Thymidine incorporation is stimulated by transforming growth factor-alpha (TGF-alpha) in a dose-dependent response. Only peripheral cells presumably of the outer root sheath, incorporate thymidine in basal or stimulated conditions. TGF-beta 1 and TGF-beta 2 inhibit constitutive cell proliferation and oppose growth stimulation by TGF-alpha. Hair follicles lyse the collagen gel matrix when exposed to certain cytokines. Epidermal growth factor (EGF) and TGF-alpha stimulate gel lysis, but TGF-beta 1, TGF-beta 2 and cholera toxin do not. Other skin-derived cells, such as interfollicular epidermal cells, dermal fibroblasts, or combinations thereof, do not lyse gels in this culture model even when exposed to growth factors. Combinations of EGF or TGF-alpha with TGF-beta 1 or TGF-beta 2 are synergistic for collagenase release. These cytokines stimulate release of multiple species of matrix metalloproteinases, but the 92-kDa and 72 kDa type IV procollagenases and their activated derivatives predominate on zymograms. In cytokine-stimulated follicles, both peripheral and centrally located cells in the organoids express the 72-kDa type IV collagenase and a similar immunostaining pattern is present in developing follicles in vivo. Thus growth factors appear to work in concert for certain hair follicle responses and in opposition for others. These combined actions may play a role in different phases of hair follicle development that require cell replication and invasion into the deeper dermis.     

Cell-mediated co-action of transforming growth factors: incubation of type beta with normal rat kidney cells produces a soluble activity that prolongs the ruffling response to type alpha

Intense, continuous ruffling is a characteristic of many transformed cells, but untransformed cells ruffle intensely only briefly after exposure to growth factors. We reported previously that cells of a normal rat kidney (NRK) cell line transformed by Kirsten murine sarcoma virus secrete their own ruffle-inducing agent(s) that cause sustained ruffling in either themselves or untransformed NRK cells. In the present study, we examined the roles of the transforming growth factors TGF-alpha and TGF-beta in the induction and maintenance of ruffling in untransformed NRK cells and observed the following: TGF-alpha caused a transient epidermal growth factor (EGF)-like response, which could be blocked by prior exposure of cells to EGF or by antiserum directed against the COOH-terminus of TGF-alpha. TGF-beta caused no ruffling and did not itself prolong TGF-alpha ruffling. A new, buffer-soluble (transferable) mediator activity produced by incubation of TGF-beta with NRK cells for 6-h extended the duration of maximal TGF-alpha-induced ruffling by several-fold. This study demonstrates that TGF-alpha alone causes an EGF-like, transient ruffling response, but neither TGF-alpha or TGF-beta alone, nor the two together, cause transformation-associated sustained ruffling. Rather, TGF-alpha acts in concert with a new, TGF-beta-dependent activity. This new activity appears to inhibit normal cellular off-regulation of TGF-alpha-induced ruffling. Inhibition of the cellular off-regulation of a growth factor response could play a key role in the unregulated growth associated with malignancy.     

Mammary epithelial cells treated concurrently with TGF-alpha and TGF-beta exhibit enhanced proliferation and death

Transforming growth factor-alpha (TGF-alpha) stimulates while TGF-beta inhibits mammary epithelial cell growth, suggesting that when cells are treated concurrently with the growth factors their combined effects would result in no net growth. However, combined treatments stimulate proliferation and cellular transformation in several cell lines. The objective of this paper was to describe the effect of long-term (6 days) concurrent TGF-alpha and TGF-beta treatment on normal mammary epithelial cell growth pattern, morphology, and gene expression. Growth curve analysis showed that TGF-alpha enhanced while TGF-beta suppressed growth rate until Day 4, when cells entered lag phase. However, cells treated concurrently with both growth factors exhibited a dichotomous pattern of growth marked by growth and death phases (with no intermittent lag phase). These changes in growth patterns were due to a marked induction of cell death from Day 2 (16.5%) to Day 4 (89.5%), resulting in the transition from growth to death phases, even though the combined treated cultures had significantly more (P < 0.05) cells in S phase on Day 4. TGF-beta stimulated epithelial to mesenchyme transdifferentiation (EMT) in the presence of TGF-alpha, as characterized by increased expression of fibronectin and changes in TGF-beta receptor binding. Expression patterns of genes that regulate the cell cycle showed significant interaction between treatment and days, with TGF-beta overriding TGF-alpha-stimulated effects on gene expression. Overall, the combined treatments were marked by enhanced rates of cellular proliferation, death, and trans-differentiation, behaviors reminiscent of breast tumors, and thus this system may serve as a good model to study breast tumorigenesis.     

Induction of vasculogenesis and hematopoiesis in vitro.

Despite a large number of investigations of embryonic vascular development, in particular in avian embryos, the conditions under which the endothelial and hematopoietic cell lineages emerge remain unknown. As we demonstrate here, both endothelial and hematopoietic cells can be induced by treatment of dissociated quail epiblast with fibroblast growth factors in vitro. These cells aggregate in characteristic blood islands. In long-term culture, the induced endothelial cells gave rise to vascular structures in vitro, i.e. vasculogenesis. No induction was observed in the absence of fibroblast growth factors, and other growth factors like TGF-beta, TGF-alpha and EGF were not capable of inducing blood island formation. Thus, the dissociated quail epiblast provides a remarkably simple test system to investigate cell lineage diversification in higher vertebrates.     

Phorbol ester or epidermal growth factor (EGF) stimulates the concurrent accumulation of mRNA for the EGF receptor and its ligand transforming growth factor-alpha in a breast cancer cell line

We have previously reported that both 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) can stimulate the synthesis rate of EGF receptors. We now show that the MDA468 breast cancer cells express the mRNA for the EGF-like molecule, transforming growth factor-alpha (TGF-alpha), and demonstrate that TPA or EGF cause an accumulation of both EGF receptor and TGF-alpha mRNA. The levels of EGF receptor mRNA paralleled our earlier protein data, with peak accumulations of 2-3-fold with 10(-9) M EGF and 3-5-fold with 100 ng/ml TPA seen between 6 and 8 h. A 7-fold accumulation of TGF-alpha mRNA was seen following 4 h of treatment with TPA, and a 2-fold accumulation was seen after 8 h with EGF. These changes in EGF receptor and TGF-alpha mRNAs were observed in the absence of any change in the mRNA level of the alpha-subunit of hexosaminidase A (a lysosomal enzyme), demonstrating some degree of specificity. Detectable quantities of immunoreactive TGF-alpha accumulated in the cell culture medium of MDA468 cell treated with the blocking anti-EGF receptor monoclonal antibody B1D8 while no immunoreactive TGF-alpha was detected in the medium of cells with unblocked receptors. The concentration of B1D8 used was sufficient to block the binding of exogenously added 125I-EGF to undetectable levels but had only minor effects on cell growth and no effect on the expression of the TGF-alpha and EGF receptor mRNA.     

A phorbol ester-regulated ribonuclease system controlling transforming growth factor beta 1 gene expression in hematopoietic cells.

12-Tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of U937 promonocytes leads to a 30-fold increase in transforming growth factor beta 1 (TGF-beta 1) gene expression, and this effect results from a stabilized mRNA. Similar up-regulation was detected in TPA-treated K562 erythroblasts but was absent from cell lines that do not differentiate in response to TPA. Related studies in vitro showed that postnuclear extracts of U937 promonocytes contain a ribonuclease system that degrades TGF-beta 1 mRNA selectively and that this system is completely blocked by prior treatment of the cells with TPA. These data identify a new mechanism for regulating TGF-beta 1 mRNA levels and allow us to establish the overall basis for control of TGF-beta 1 gene expression by activation of protein kinase C. Our results also provide a new basis for understanding the long-term up-regulation of TGF-beta 1 gene expression that can accompany hematopoietic cell differentiation.