Phorbol myristate acetate (PMA) treatment of an EL-4 thymoma cell line (EL-4FARRAR) induced secretion of a factor that inhibited intracellular killing of Leishmania major amastigotes by activated ...macrophages. Analysis of the cytokines produced by EL-4 cells after PMA stimulation identified interleukin-2 (IL-2, 2500 U/ml), IL-4 (1280 U/ml), interferon-gamma (IFN-gamma; 100 U/ml), and granulocyte-macrophage colony-stimulating factor (GM-CSF; 50 U/ml). Neither tumor necrosis factor nor transforming growth factor beta (TGF-beta) was detected. Each of the cytokines present in EL-4 fluids was assessed for capacity to activate macrophages for destruction of parasites or to suppress intracellular killing. IFN-gamma and GM-CSF both activated macrophages to kill Leishmania; IL-2 and IL-4 had no activity for induction of this antimicrobial effector function. IL-2 and IL-4 were tested for their capacity to inhibit lymphokine- or IFN-gamma-induced destruction of L. major by macrophages: IL-4 was ineffective, but IL-2 markedly suppressed the activation of macrophages for intracellular killing. Addition of > or = 10 U/ml of IL-2 at the time of infection, or up to 4 h before, blocked up to 100% of the capacity of activated macrophages to kill intracellular amastigotes. Immunoaffinity treatment of EL-4 fluids with anti-IL-2 antibody resulted in > 80% reduction in suppression of intracellular killing. The suppressive effects of IL-2 were not direct, but mediated by TGF-beta. IL-2 induced resident peritoneal macrophages to secrete > 5000 pg/ml TGF-beta 1, a quantity that is > 500-fold higher than constitutive background levels (20-40 pg/ml) and is sufficient to block intracellular killing activities. This increase in secretion of TGF-beta was not dependent increases in TGF-beta 1 mRNA. Treatment of cultures with EL-4 fluids or recombinant IL-2 in the presence of antibody to TGF-beta 1 blocked the suppressive activity of both. Thus, IL-2 was the major suppressor factor in EL-4 fluids, and it acted indirectly through the induction and autocrine action of TGF-beta.
NRP-154 is a tumorigenic epithelial cell line derived from the preneoplastic dorsal-lateral prostate of rats. These cells are exquisitely sensitive to TGF-β induced apoptosis. In contrast, we find ...that NRP-154 cells can sustain overexpression of exogenous Bax protein, which is different from non-tumor cells where Bax functions as a ubiquitous stimulator of apoptosis. NRP-154 cells stably overexpressing Bax show increased sensitivity to TGF-β induced apoptosis. The degree of TGF-β induced apoptosis displays high correlation with cleavage of Bax at the amino-terminus. Our data indicate that prostate cancer cells can host high levels of latent Bax which can be activated through post-translational modification.
We have developed a nontumorigenic epithelial cell line, DP-153, from the dorsal prostate of a Lobund/Wistar rat treated with N-methyl-N-nitrosourea and testosterone propionate. DP-153 cells express ...cytokeratins 5 and 14, but not cytokeratin 18, consistent with a basal epithelial cell phenotype. Similar to the nontumorigenic NRP-152 prostatic cell line, DP-153 cells do not form tumors in athymic mice and retain many of the properties of normal prostatic cells. They express prostatic acid phosphatase and androgen receptors and require several mitogens (epidermal growth factor, insulin, dexamethasone, and cholera toxin) for sustained growth in culture under serum-containing conditions. DP-153 cells are also growth-stimulated by keratinocyte growth factor and basic fibroblast growth factor and growth-inhibited by all-trans-retinoic acid, 1,25-dihydroxyvitamin D(3), and transforming growth factor (TGF)-beta1. We demonstrate that expression of dominant-negative TGF-beta receptor type II by retroviral transduction of DP-153 cells leads to complete loss of TGF-beta1-induced growth inhibition. When transplanted s.c. in athymic mice, DP-153 cells expressing dominant-negative TGF-beta receptor type II form tumors as early as 4 weeks, in contrast to the vector control and parental cell line, which do not form tumors even 8 months after transplantation, supporting the observation that TGF-beta functions as a tumor suppressor in these cells. Our data further support that DP-153 is a suitable cell line for analysis of normal prostatic growth and carcinogenesis.
We identified transforming growth factor-beta (TGF-beta)-binding proteins which are distinct from previously described TGF-beta
receptors or TGF-beta-binding proteins. These TGF-beta-binding proteins ...migrate as 150- and 180-kDa 125I-TGF-beta 1 affinity-labeled
complexes which are consistently co-expressed in A549, Mv1Lu, MG-63, and BS-C-1 cells. They differ from the types I, II, and
III TGF-beta receptors in their electrophoretic mobilities, their lack of binding to TGF-beta 2, and their failure to undergo
the marked down-regulation seen with types I, II, and III receptors following a 16-h incubation with TGF-beta 1. The 150-
and 180-kDa TGF-beta-binding proteins also are distinct from the recently described disulfide-linked TGF-beta 1-binding proteins
which are present in rat glomeruli. In contrast to the glomerular TGF-beta 1-binding proteins, the electrophoretic mobilities
of the 150- and 180-kDa binding proteins are unchanged following reduction. In addition, the 150- and 180-kDa TGF-beta-binding
proteins are present in the detergent-rich phase during Triton X-114 phase separation, whereas the glomerular TGF-beta-binding
proteins partition exclusively into the detergent-poor phase.
Immunohistochemical staining of skin sections with two polyclonal antibodies (anti-CC 1-30 and anti-LC 1-30), specific for transforming growth factor-β1, revealed increased extracellular and ...decreased intracellular expression of transforming growth factor-β1in retinoic acid-treated, compared to vehicle-treated, skin. Transforming growth factor-β1staining, with both antibodies, was most marked in the upper layers of the epidermis, although dermal staining was also evident. The modulation of transforming growth factor-β1expression by retinoic acid occurred in the absence of any change in its mRNA level. Transforming growth factor-β1protein, as detected by rabbit polyclonal antibody (anti-LC 50-75) and mRNA, were only minimally detected in either retinoic acid- or vehicle-treated skin. Similar changes in TGF-β1and TGF-β2 immunoreactivity and mRNA levels, as observed in retinoic acid-treated skin, were observed in skin following topical application of the irritant sodium lauryl sulfate, indicating that the alterations induced by retinoic acid were not specific. In contrast, mucin depositions which is induced by transforming growth factor-β, was elevated in retinoic acid-treated but not sodium lauryl sulfate-treated skin. Cultured adult human keratinocytes also expressed predominantly transforming growth factor-β1 protein, as measured by ELISA, and mRNA. Treatment of keratinocytes with retinoic acid resulted in a 50% induction of transforming growth factor-β1 protein, without any detectable change in transforming growth factor-β2. These data demonstrate disassociation of modulation of transforming growth factor-β1 expression and mucin deposition by retinoic acid and sodium lauryl sulfate in human skin in vivo. Whereas alterations in transforming growth factor expression were observed in both retinoic acid–and sodium lauryl sulfate-treated skin, accumulation of mucin was specific to retinoic acid–treated skin.
We have identified two distinct classes of transforming growth factor-beta (TGF-beta)-binding proteins by affinity labeling
rat glomeruli with 125I-TGF-beta 1 and 125I-TGF-beta 2. The first type ...consists of a group of proteins that bind TGF-beta
1 but do not bind TGF-beta 2. When 125I-TGF-beta 1 affinity-labeled glomeruli were separated under nonreducing conditions,
four prominent bands with Mr values of 320,000, 260,000, 170,000, and 90,000 were observed. Following reduction, the 320,000
and 170,000 bands yielded only a 100,000 band, the 260,000 complex yielded bands of 200,000, 100,000, and 85,000, and the
90,000 band migrated with an Mr of 85,000. Binding of 125I-TGF-beta 1 to these proteins was unaffected by the addition of
as much as a 1,000-fold excess of TGF-beta 2. The second type of glomerular TGF-beta-binding protein consists of Mr 160,000-200,000
and 280,000 proteins that bind both TGF-beta 1 and beta 2. Digestion of these affinity-labeled proteins with heparitinase
and chondroitinase resulted in a decrease of approximately 40,000 in their apparent molecular weights. Glomerular TGF-beta
1-binding proteins are distinct from previously described TGF-beta-binding proteins in their specificity for TGF-beta 1 and
their formation of disulfide-linked multimers. The TGF-beta 1/beta 2-binding proteins share some properties of the previously
described type III TGF-beta receptor.
Cited (CBP/p300-interacting transactivators with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain) 2, which is a CBP/p300-binding transcription co-activator without typical DNA-binding ...domains, has been implicated in control of cell growth and malignant transformation in Rat1 cells. In this report, we provide evidence that Cited2 is an important regulator of transforming growth factor (TGF)-beta signaling. Overexpression of Cited2 enhanced TGF-beta-mediated transcription of a Smad-Binding Element-containing luciferase reporter construct, SBE4-Luc. This may occur through a direct physical association of Cited2 with Smads 2 and 3, as supported by co-immunoprecipitation, mammalian two-hybrid and glutathione S-transferase-pull down assays. The transcription factor p300, which binds to Smad3, was shown to further enhance the interaction between Cited2 and Smad3, and the transcriptional responses of Smad3 by Cited2 in reporter assays. Cited2 enhances TGF-beta-mediated upregulation of matrix metalloproteinase 9 (MMP9) in Cited2 inducible mouse embryo fibroblasts. Overexpression of Cited2 enhanced TGF-beta-mediated MMP9 promoter reporter activity. Moreover, knockdown of Cited2 in MDA-MB-231 cells attenuated TGF-beta-mediated upregulation of MMP9 and TGF-beta-mediated cell invasion. Chromatin immunoprecipitation showed that Cited2 and Smad3 were recruited to MMP9 promoter upon TGF-beta stimulation. This is the first demonstration that Cited2 functions as a Smad3/p300-interacting transcriptional co-activator in modulating the expression of MMP9, which could affect tumor cell invasion mediated by TGF-beta.
Cited (CBP/p300-interacting transactivators with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain) 2, which is a CBP/p300-binding transcription co-activator without typical DNA-binding ...domains, has been implicated in control of cell growth and malignant transformation in Rat1 cells. In this report, we provide evidence that Cited2 is an important regulator of transforming growth factor (TGF)-b signaling. Overexpression of Cited2 enhanced TGF-b-mediated transcription of a Smad-Binding Element-containing luciferase reporter construct, SBE4-Luc. This may occur through a direct physical association of Cited2 with Smads 2 and 3, as supported by co-immunoprecipitation, mammalian two-hybrid and glutathione S-transferase-pull down assays. The transcription factor p300, which binds to Smad3, was shown to further enhance the interaction between Cited2 and Smad3, and the transcriptional responses of Smad3 by Cited2 in reporter assays. Cited2 enhances TGF-b-mediated upregulation of matrix metalloproteinase 9 (MMP9) in Cited2 inducible mouse embryo fibroblasts. Overexpression of Cited2 enhanced TGF-b-mediated MMP9 promoter reporter activity. Moreover, knockdown of Cited2 in MDA-MB-231 cells attenuated TGF-b-mediated upregulation of MMP9 and TGF-b-mediated cell invasion. Chromatin immunoprecipitation showed that Cited2 and Smad3 were recruited to MMP9 promoter upon TGF-b stimulation. This is the first demonstration that Cited2 functions as a Smad3/p300-interacting transcriptional co-activator in modulating the expression of MMP9, which could affect tumor cell invasion mediated by TGF-b.Oncogene (2006) 25, 5547-5560. doi:10.1038/sj.onc.1209552; published online 17 April 2006
Transforming growth factor (TGF)-betas are multifunctional growth factors, the properties of which include the potent inhibition of epithelial cell growth. Expression patterns of TGF-betas and ...TGF-beta receptors in the normal prostate indicate that these growth regulators play key roles in prostatic development and proliferative homeostasis. Importantly, TGF-beta receptor levels are frequently diminished in malignant human prostate tissue. To test the hypothesis that loss of TGF-beta responsiveness is causally involved in the tumorigenic process, we have used retroviral transduction to introduce a dominant-negative mutant type II TGF-beta receptor (DNR) into the premalignant rat prostatic epithelial cell line, NRP-152. High-level expression of the DNR abolished the ability of TGF-beta to inhibit cell growth, to promote cell differentiation, and to induce apoptosis, and it partially blocked the induction of extracellular matrix gene expression. When injected into nude mice, NRP-152-DNR cells formed carcinomas at 13 of 34 sites, compared with 0 of 30 sites for parental and control cells (P = 0.0001). We conclude that the type II TGF-beta receptor is an important tumor suppressor in the prostate, and furthermore, that loss of TGF-beta responsiveness can contribute early in the tumorigenic process by causing the malignant transformation of preneoplastic cells.
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