Inhibition of transforming growth factor beta signaling in MCF-7 cells results in resistance to tumor necrosis factor alpha: a role for Bcl-2.

Transforming growth factor beta (TGF-beta) is a multifunctional cytokine capable of regulating diverse cellular processes. In this study we investigated the effect of autocrine TGF-beta signaling on tumor necrosis factor (TNF) alpha-induced cell death. We abrogated the TGF-beta autocrine loop by overexpression of a truncated TGF-beta type II receptor in MCF-7 breast carcinoma cells and found that this generated resistance to TNF-alpha-induced cytotoxicity. To elucidate the molecular basis of the influence of TGF-beta on TNF-alpha-induced cytotoxicity, we evaluated the expression levels or activities of proteins involved in TNF-alpha signal transduction or the regulation of apoptosis in general in TGF-beta-responsive and TGF-beta-nonresponsive MCF-7 cells. We observed no significant difference in the expression of TNF-alpha receptors or the TNF receptor-associated death domain protein. In addition, downstream activation of nuclear factor kappaB by TNF-alpha was not altered in cells that had lost TGF-beta responsiveness. Analysis of members of the Bcl-2 family of apoptosis-regulatory proteins revealed that Bcl-X(L) and Bax expression levels were not changed by disruption of TGF-beta signaling. In contrast, the TGF-beta-nonresponsive cells expressed much higher levels of Bcl-2 protein and mRNA than did cells with an intact TGF-beta autocrine loop. Furthermore, restoration of a TGF-beta signal to MCF-7 cells that had spontaneously acquired resistance to TGF-beta caused a reduction in Bcl-2 protein expression. Taken together, our data indicate that loss of autocrine TGF-beta signaling results in enhanced resistance to TNF-alpha-mediated cell death and that this is likely to be mediated by derepression of Bcl-2 expression.     

Modulation of transforming growth factor beta signalling pathway genes by transforming growth factor beta in human osteoarthritic chondrocytes: involvement of Sp1 in both early and late response cells to transforming growth factor beta

Introduction  

Transforming growth factor beta (TGFβ) plays a central role in morphogenesis, growth, and cell differentiation. This cytokine is particularly important in cartilage where it regulates cell proliferation and extracellular matrix synthesis. While the action of TGFβ on chondrocyte metabolism has been extensively catalogued, the modulation of specific genes that function as mediators of TGFβ signalling is poorly defined. In the current study, elements of the Smad component of the TGFβ intracellular signalling system and TGFβ receptors were characterised in human chondrocytes upon TGFβ1 treatment.     

Nicotine regulates basic fibroblastic growth factor and transforming growth factor beta1 production in endothelial cells

Nicotine, a constituent of cigarette smoking, may induce atherosclerosis through the production of growth factors. The pattern of bFGF and TGF beta1 production and release by bovine aortic endothelial cells (EC) stimulated with nicotine (from 6 x 10(-4) to 6 x 10(-8) M) was studied. EC viability and count were assessed. The presence of bFGF and TGF beta1 in serum-free conditioned media was determined by the inhibition antibody-binding assay and Western blot analysis. Mitogenic activity of nicotine on EC was also determined. Polymerase chain reaction (PCR) was used to study the expression of bFGF and TGF beta1. The bFGF release after nicotine stimulation was greater than controls, whereas TGF beta1 release was lower. At a nicotine concentration of 6 x 10(-6) M we noted the greatest mitogenic activity. The addition of monoclonal antibody anti-bFGF decreased the tritiated thymidine uptake of EC exposed to nicotine but the addition of monoclonal antibody anti-TGF beta1 had no significant effect. bFGF mRNA expression was significantly higher in EC exposed to nicotine than in controls, whereas TGF beta1 mRNA expression was not modified. From these data we concluded that nicotine regulates bFGF production and release and TGF beta1 release and may have a key role in the development and progression of atherosclerosis.     

Autocrine role of transforming growth factor beta1 on rat granulosa cell proliferation

We evaluated the effects of transforming growth factor beta1 (TGFbeta1), alone or in combination with FSH and estradiol, on DNA synthesis in primary cultures of immature rat granulosa cells. 3H-Thymidine incorporation was significantly stimulated by TGFbeta1 (5.6-fold). This effect was enhanced by FSH (20 ng/ml, 27.7-fold) or estradiol (100 ng/ml, 13.4-fold) or by a combination of both hormones (59.2-fold). Measurement of TGFbeta bioactivity showed the presence of significant amounts of TGFbeta in conditioned medium from granulosa cell cultures, and most of the activity was present in the latent form. FSH alone or in combination with estradiol produced a marked suppression of the production of latent and active TGFbeta. Activated conditioned medium from control cultures of granulosa cell elicited a 1.4-fold increase in thymidine incorporation. This effect was markedly amplified by FSH (3-fold) and estradiol (4.3-fold) and by a combination of both (8.7-fold). The peptide containing the cell-binding domain of fibronectin (RGDSPC) partially inhibited thymidine incorporation stimulated by TGFbeta1. Fibronectin did not synergize with FSH, and the interaction between TGFbeta1 and FSH was even observed in the presence of this protein. The conclusions reached were as follows: 1) TGFbeta1 is an autocrine stimulator of rat granulosa cell DNA synthesis, 2) FSH and estradiol produce a suppression of latent and active TGFbeta production but markedly amplify TGFbeta action, presumably at a postreceptor level, and 3) the stimulatory effects of TGFbeta1 may be only partly mediated by the increased fibronectin secretion.     

Modulation of adipocyte differentiation by tumor necrosis factor and transforming growth factor beta

Cultured TA1 adipocytes treated with tumor necrosis factor alpha (TNF) lose intracytoplasmic lipid and, over a period of days, come to resemble their predifferentiated progenitors (preadipocytes). To examine the extent to which this phenotypic reversion represents a return to a less differentiated cell, we examined three major characteristics that distinguish preadipocytes from adipocytes: (a) pattern of gene expression; (b) hormonal requirement for accelerated adipogenesis; and (c) pattern of protein synthesis. We found that within hours of TNF addition to adipocytes, mRNAs for genes whose expression is augmented during adipogenesis decreased to predifferentiated levels; in addition, like preadipocytes, TNF-treated adipocytes required exposure to hormones to accelerate adipogenesis. Further, the pattern of protein synthesis seen on polyacrylamide gels reverted to that seen before differentiation. Transforming growth factor-beta (TGF-beta) also caused a rapid decrease in expression of adipose genes when added to fully differentiated cells, an effect that was achieved by treatment with either TGF-beta 1 or TGF-beta 2. These effects were seen in the absence of a demonstrable proliferative response to either TNF or TGF-beta. Thus characteristics that define the "terminally" differentiated state in adipocytes are subject to modulation by environmental influences.     

Renal transforming growth factor beta 1 production in neonate rats

Abstract. The newborn rat kidney is not fully developed until approximately 12 days after birth. In order to evaluate the possible role of Transforming Growth Factor beta 1 (TGF beta 1) in renal development we analyzed the mRNA level for TGF beta 1 in sixty Wistar rats aged 1,15 and 30 days. We also performed immunohislochemical studies to visualize the distribution of this peptide in the kidney of these rats using a TGF beta 1 antibody. The results show that the mRNA levels for TGF beta 1 are higher in the kidneys of the 1-day-old rats than in the 15 (1.4 fold) and 30-day-old rats (1.7 fold). The immunohistochemical reaction revealed the presence of TGF beta in the kidneys of the rats. The staining intensity was higher in the renal cortex than in the medulla. The data suggests that TGF beta may be important during kidney development.     

Requirement of Ras/MAPK pathway activation by transforming growth factor beta for transforming growth factor beta 1 production in a Smad-dependent pathway

Our previous results have shown that transforming growth factor beta (TGFbeta) rapidly activates Ras, as well as both ERKs and SAPKs. In order to address the biological significance of the activation of these pathways by TGFbeta, here we examined the role of the Ras/MAPK pathways and the Smads in TGFbeta(3) induction of TGFbeta(1) expression in untransformed lung and intestinal epithelial cells. Expression of either a dominant-negative mutant of Ras (RasN17) or a dominant-negative mutant of MKK4 (DN MKK4), or addition of the MEK1 inhibitor PD98059, inhibited the ability of TGFbeta(3) to induce AP-1 complex formation at the TGFbeta(1) promoter, and the subsequent induction of TGFbeta(1) mRNA. The primary components present in this TGFbeta(3)-inducible AP-1 complex at the TGFbeta(1) promoter were JunD and Fra-2, although c-Jun and FosB were also involved. Furthermore, deletion of the AP-1 site in the TGFbeta(1) promoter or addition of PD98059 inhibited the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Collectively, our data demonstrate that TGFbeta(3) induction of TGFbeta(1) is mediated through a signaling cascade consisting of Ras, the MAPKKs MKK4 and MEK1, the MAPKs SAPKs and ERKs, and the specific AP-1 proteins Fra-2 and JunD. Although Smad3 and Smad4 were not detectable in TGFbeta(3)-inducible AP-1 complexes at the TGFbeta(1) promoter, stable expression of dominant-negative Smad3 could significantly inhibit the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Transient expression of dominant-negative Smad4 also inhibited the ability of TGFbeta(3) to transactivate the TGFbeta(1) promoter. Thus, although the Ras/MAPK pathways are essential for TGFbeta(3) induction of TGFbeta(1), Smads may only contribute to this biological response in an indirect manner.     

Raf and RhoA cooperate to transform intestinal epithelial cells and induce growth resistance to transforming growth factor beta

Although unregulated activation of the Ras/Raf/mitogen-activated protein kinase kinase/Erk signaling pathway is believed to be a central mechanism by which many cell types undergo oncogenic transformation, recent studies indicate that activation of Raf kinase by oncogenic Ras is not sufficient to cause tumorigenic transformation in intestinal epithelial cells. Thus, identification of signaling proteins and pathways that interact with Raf to transform intestinal epithelial cells may be critical for understanding aberrant growth control in the intestinal epithelium. Functional interactions between Raf and the small GTPase RhoA were studied in RIE-1 cells overexpressing both activated Raf(22W) and activated RhoA(63L). Double transfectants were morphologically transformed, formed colonies in soft agar, grew in nude mice, overexpressed cyclin D1 and cyclooxygenase-2 (COX-2), and were resistant to growth inhibition by transforming growth factor (TGF) beta. RIE-Raf and RIE-RhoA single transfectants showed none of these characteristics. Expression of a dominant-negative RhoA(N19) construct in RIE-Ras(12V) cells was associated with markedly reduced COX-2 mRNA, COX-2 protein, and prostaglandin E2 levels when compared with RIE-Ras(12V) cells transfected with vector alone. However, no change in transformed morphology, growth in soft agar, cyclin D1 expression, TGFalpha expression, or TGFbeta sensitivity was observed. In summary, coexpression of activated Raf and RhoA induces transformation and TGFbeta resistance in intestinal epithelial cells. Although blockade of RhoA signaling reverses certain well-described characteristics of RIE-Ras cells, it is insufficient to reverse the transformed phenotype and restore TGFbeta sensitivity. Blockade of additional Rho family members or alternate Ras effector pathways may be necessary to fully reverse the Ras phenotype.     

Role of transforming growth factor beta in cancer

Transforming growth factor beta (TGF-beta) is an effective and ubiquitous mediator of cell growth. The significance of this cytokine in cancer susceptibility, cancer development and progression has become apparent over the past few years. TGF-beta plays various roles in the process of malignant progression. It is a potent inhibitor of normal stromal, hematopoietic, and epithelial cell growth. However, at some point during cancer development the majority of transformed cells become either partly or completely resistant to TGF-beta growth inhibition. There is growing evidence that in the later stages of cancer development TGF-beta is actively secreted by tumor cells and not merely acts as a bystander but rather contributes to cell growth, invasion, and metastasis and decreases host-tumor immune responses. Subtle alteration of TGF-beta signaling may also contribute to the development of cancer. These various effects are tissue and tumor dependent. Identifying and understanding TGF-beta signaling pathway abnormalities in various malignancies is a promising avenue of study that may yield new modalities to both prevent and treat cancer. The nature, prevalence, and significance of TGF-beta signaling pathway alterations in various forms of human cancer as well as potential preventive and therapeutic interventions are discussed in this review.     

Regulation of extracellular matrix proteins by transforming growth factor beta1 in cultured pulmonary endothelial cells

Transforming growth factor beta-1 (TGF-beta1), which is present in lung tissue, has been suggested to play a role in modulating vascular cell function in vivo. The action of TGF-beta1 in vivo, especially at the local site of application to connective tissue, is anabolic and leads to pulmonary fibrosis and angiogenesis, strongly indicating that TGF-beta may have practical applications in repair of tissue injury caused by burns, trauma, or surgery. In the present study, we have used cultured bovine pulmonary artery endothelial (BPAE) cells as a model system. Expression of various proteins, including SPARC (secreted protein acidic and rich in cysteines), type IV procollagen and fibronectin (FN) was examined by radiolabeling the cells with [3H]proline, immunoprecipitation with specific antibodies, and Northern blot analyses by using specific cDNA probes. Cultured cells were labeled with [3H]proline for 24 h in either the absence or in the presence of TGF-beta1 (0-20 ng/ml). Incorporation of radioactivity was observed in a concentration-dependent manner, maximal at 5 ng/ml. Northern blot hybridization demonstrated that TGF-beta1 (5 ng/ml) treatment of BPAE cells caused an increase in steady-state levels     

Growth modulatory effects of a liver-derived growth inhibitor, transforming growth factor beta 1, and recombinant tumor necrosis factor alpha, in normal and neoplastic cells

The growth modulatory effects of a rat liver-derived growth inhibitor (LDGI), transforming growth factor beta 1 (TGF-beta 1), and recombinant tumor necrosis factor (rTNF-alpha) were examined in a variety of liver-derived and nonliver-derived normal and neoplastic cell culture systems. Normal rat liver epithelial (RLE) cells were highly sensitive to the growth inhibitory effects of LDGI (ID50 = 0.2 ng/ml) and TGF-beta 1 (ID50 = 0.25 ng/ml) but were less sensitive to rTNF-alpha (ID40 = 5000 Units/ml). Aflatoxin B1-transformed RLE cells showed sensitivity to the cytostatic effects of LDGI (ID50 = 1.5 ng/ml); however, these cells were completely resistant to the antiproliferative effects of TGF-beta 1 and rTNF-alpha. Clones isolated from these transformed cells, exhibited a wide range of sensitivities to LDGI but all of the clones were resistant to the growth inhibitory effects of both TGF-beta 1 and rTNF-alpha. Rat hepatoma Reuber cells were extremely sensitive to the antiproliferative effects of rTNF-alpha (ID50 = 10 Units/ml) but exhibited sensitivity to LDGI only at concentrations above 1.5 ng/ml and were resistant to the antiproliferative effects of TGF-beta 1. Rat hepatoma UVM 7777 cells and human hepatoma HepG2 cells, however, were insensitive to the growth inhibitory effects of all three factors. Among the nonliver-derived cells, human breast carcinoma (MCF-7) cells were extremely sensitive to rTNF-alpha (ID50 = 20 Units/ml, exhibited some sensitivity to LDGI (ID50 = 1 ng/ml), and were resistant to the antiproliferative effects of TGF-beta 1. In contrast, the rate of DNA synthesis is rat kidney fibroblasts and human foreskin fibroblasts was significantly stimulated in response to TGF-beta 1, LDGI, and rTNF-alpha. These data demonstrate that LDGI, TGF-beta 1, and rTNF-alpha exert positive and negative modulations of growth in different cell systems and that the growth regulatory effects of LDGI differ from those of TGF-beta 1 and rTNF-alpha in some cell types.     

Tumor-host interactions in the gallbladder suppress distal angiogenesis and tumor growth: involvement of transforming growth factor beta1.

Angiogenesis inhibitors produced by a primary tumor can create a systemic anti-angiogenic environment and maintain metastatic tumor cells in a state of dormancy. We show here that the gallbladder microenvironment modulates the production of transforming growth factor (TGF)-beta1, a multifunctional cytokine that functions as an endogenous anti-angiogenic and anti-tumor factor in a cranial window preparation. We found that a wide variety of human gallbladder tumors express TGF-beta1 irrespective of histologic type. We implanted a gel impregnated with basic fibroblast growth factor or Mz-ChA-2 tumor in the cranial windows of mice without tumors or mice with subcutaneous or gallbladder tumors to study angiogenesis and tumor growth at a secondary site. Angiogenesis, leukocyte-endothelial interaction in vessels and tumor growth in the cranial window were substantially inhibited in mice with gallbladder tumors. The concentration of TGF-beta1 in the plasma of mice with gallbladder tumors was 300% higher than that in the plasma of mice without tumors or with subcutaneous tumors. In contrast, there was no difference in the plasma levels of other anti- and pro-angiogenic factors. Treatment with neutralizing antibody against TGF-beta1 reversed both angiogenesis suppression and inhibition of leukocyte rolling induced by gallbladder tumors. TGF-beta1 also inhibited Mz-ChA-2 tumor cell proliferation. Our results indicate that the production of anti-angiogenesis/proliferation factors is regulated by tumor-host interactions.     

Proteomic profiling of bone marrow mesenchymal stem cells upon transforming growth factor beta1 stimulation

Bone marrow mesenchymal stem cells (MSCs) can differentiate into different types of cells and have tremendous potential for cell therapy and tissue engineering. Transforming growth factor beta1 (TGF-beta) plays an important role in cell differentiation and vascular remodeling. We showed that TGF-beta induced cell morphology change and an increase in actin fibers in MSCs. To determine the global effects of TGF-beta on MSCs, we employed a proteomic strategy to analyze the effect of TGF-beta on the human MSC proteome. By using two-dimensional gel electrophoresis and electrospray ionization coupled to quadrupole/time-of-flight tandem mass spectrometers, we have generated a proteome reference map of MSCs, and we identified approximately 30 proteins with an increase or decrease in expression or phosphorylation in response to TGF-beta. The proteins regulated by TGF-beta included cytoskeletal proteins, matrix synthesis proteins, membrane proteins, metabolic enzymes, etc. TGF-beta increased the expression of smooth muscle alpha-actin and decreased the expression of gelsolin. Overexpression of gelsolin inhibited TGF-beta-induced assembly of smooth muscle alpha-actin; on the other hand, knocking down gelsolin expression enhanced the assembly of alpha-actin and actin filaments without significantly affecting alpha-actin expression. These results suggest that TGF-beta coordinates the increase of alpha-actin and the decrease of gelsolin to promote MSC differentiation. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.     

Skeletal tissue and transforming growth factor beta

Normal skeletal growth results from a balance between the processes of bone matrix synthesis and resorption. These activities are regulated by both systemic and local factors. Bone turnover is dynamic, and skeletal growth must be maintained throughout life. Although many growth promoters are associated with bone matrix, it is enriched particularly with transforming growth factor beta (TGF-beta) activity. Experimental evidence indicates that TGF-beta regulates replication and differentiation of mesenchymal precursor cells, chondrocytes, osteoblasts, and osteoclasts. Recent studies further suggest that TGF-beta activity in skeletal tissue may be controlled at multiple levels by other local and systemic agents. Consequently, the intricate mechanisms by which TGF-beta regulates bone formation are likely to be fundamental to understanding the processes of skeletal growth during development, maintenance of bone mass in adult life, and healing subsequent to bone fracture.