Transforming growth factor beta s (TGF-beta s), a highly homologous family of 25 kDa dimers, function as both paracrine and autocrine growth modulators which relay a broad spectrum of critical ...biological functions. Although sensitive bioassays for TGF-beta s have been described, their interference by other growth modulators have limited their application. We have previously described a highly sensitive (20-50 pg/ml) and specific sandwich enzyme-linked immunosorbent assay (SELISA) for TGF-beta 1, based on use of rabbit anti-TGF-beta 1 as the capture antibody. Widespread use of this assay was limited by the availability of this rather low-titer antibody. This report describes improved TGF-beta 1 SELISAs with comparable sensitivities to the above assay based on available sources of commercial monoclonal and polyclonal antibodies. These newly developed SELISAs are the most sensitive, specific, precise and convenient methods for the quantitation of TGF-beta 1 in complex biological fluids.
Androgen receptor-associated protein 55 (ARA55/Hic-5) belongs to the LIM protein superfamily and is featured by three or four N-terminal LD motifs and four C-terminal zinc finger-like LIM domains. ...Both LD motifs and LIM domains can serve as protein-protein interaction interfaces. Recently, we found that enforced expression of ARA55 inhibits transforming growth factor-β-mediated up-regulation of Smad binding element-luciferase reporter activity in NRP-154 and NRP-152 rat prostate and LNCaP human prostate cell lines. Moreover, ARA55 also inhibits the induction of Smad-binding element 4-luciferase and 3TP-luciferase (a plasminogen activator inhibitor-1 (PAI-1) promoter construct) reporters by constitutively active (CA)-Smad3 in these cell lines. Co-immunoprecipitation studies suggest an interaction between ARA55 and either CA-Smad3 or wild-type Smad3 in HEK293 cells that occurs through the MH2 domain of Smad3 and the C terminus of ARA55 with wild-type Smad3 having stronger affinity than CA-Smad3 to ARA55. Glutathione S-transferase pull-down assays demonstrate that this interaction can occur in a cell-free system. These results are consistent with the luciferase data showing that the C terminus of ARA55 is critical for suppression of Smad3 activity. Furthermore, using a mammalian two-hybrid system, we confirmed that ARA55 interacts with the MH2 domain of Smad3 and suppresses CA-Smad3-induced transcriptional responses. In conclusion, these results support that ARA55 selectively intercepts transforming growth factor-β signaling through an interaction of the LIM domain of ARA55 with the MH2 domain of Smad3.
The clinical use of anti-oestrogens in breast cancer therapy has traditionally been restricted to tumours that contain measurable oestrogen receptor protein. However, it is now widely recognised that ...the clinical response to adjuvant anti-oestrogen therapy appears to be independent of the oestrogen receptor content of the primary tumour. The study reported here was designed to investigate the possibility that human stromal cells can respond to anti-oestrogens by an increased synthesis of the inhibitory growth factor, transforming growth factor beta (TGF-beta). Two established human fetal fibroblast strains were used as models for the breast cancer stromal fibroblasts. These cells were found to respond to the addition of anti-oestrogens by a large increase in their synthesis of biologically active TGF-beta. Despite the application of ligand binding, immunoassay and Northern analysis, no oestrogen receptor or oestrogen receptor mRNA was detected in either of the human fetal fibroblasts strains. These observations may provide a mechanism of action of anti-oestrogens that is independent of the presence of oestrogen receptor in the tumour epithelial cells, and thus provide an explantation for the counter-intuitive results of adjuvant anti-oestrogen action.
Insulin-like growth factor-I (IGF-I) and transforming growth factor-β (TGF-β) have been shown to be oncogenic and tumor suppressive, respectively, on prostate epithelial cells. Here we show that ...IGF-I inhibits the ability of TGF-β to regulate expression of several genes in the non-tumorigenic rat prostatic epithelial line, NRP-152. In these cells, IGF-I also inhibits TGF-β-induced transcriptional responses, as shown by several promoter reporter constructs, suggesting that IGF-I intercepts an early step in TGF-β signaling. We show that IGF-I does not down-regulate TGF-β receptor levels, as determined by both receptor cross-linking and Western blot analyses. However, Western blot analysis reveals that IGF-I selectively inhibits the TGF-β-triggered activation Smad3 but not Smad2, while not altering expression of total Smads 2, 3, or 4. The phosphatidylinositol 3-kinase (PI3K) inhibitor, LY29004 reverses the ability of IGF-I to inhibit TGF-β-induced transcriptional responses and the activation of Smad3, suggesting that the suppression of TGF-β signaling by IGF-I is mediated through activation of PI3K. Moreover, we show that enforced expression of dominant-negative PI3K (DN-p85α) or phosphatidylinositol 3-phosphate-phosphatase, PTEN, also reverse the suppressive effect of IGF-I on TGF-β-induced 3TP-luciferase reporter activity, whereas constitutively active PI3K (p110αCAAX) completely blocks TGF-β-induced 3TP-luciferase reporter activity. Further transfection experiments including expression of constitutively active and dominant-negative Akt and rapamycin treatment suggest that suppression of TGF-β signaling/Smad3 activation by IGF-I occurs downstream of Akt and through mammalian target of rapamycin activation. In summary, our data suggest that IGF-I inhibits TGF-β transcriptional responses through selective suppression of Smad3 activation via a PI3K/Akt-dependent pathway.
Expression of transforming growth factor-beta s (TGF-beta s) 1-3 was studied in normal liver and during liver regeneration after partial hepatectomy in the rat to determine whether each of these ...isoforms might be involved in hepatocyte growth in vivo. Expression of the mRNAs for all three TGF-beta isoforms increases in the regenerating liver. In addition, the levels of expression of the mRNAs for several extracellular matrix proteins, including fibronectin, vitronectin, laminin, and collagen, also increase in the regenerating liver. Immunohistochemical staining analysis shows a similar distribution of all three TGF-beta s in normal and regenerating liver; however, in both tissues, the level of expression of TGF-beta 1 is 8- to 10-fold higher than that of TGF-beta 2 as determined by sandwich enzyme-linked immunosorbent assay. Expression of all three TGF-beta mRNAs is restricted to liver nonparenchymal cells. Although hepatocytes from normal and regenerating livers do not synthesize TGF-beta, they are sensitive to inhibition of growth by all three TGF-beta isoforms. Hepatocytes from regenerating livers are capable of activating latent TGF-beta 1 complexes in vitro, whereas normal hepatocytes are not. The different TGF-beta isoforms may function in an inhibitory paracrine mechanism that is activated during liver regeneration and may also regulate the synthesis of extracellular matrix components in the regenerating liver.
The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we performed a yeast two-hybrid screen. The results of ...the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type β transforming growth factor (TGF-β ) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-β . DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-β -induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski repressed a TGF-β -inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-β -induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-β -mediated growth inhibition in a prostate-derived epithelial cell line.
In the prostate, androgens negatively regulate the expression of transforming growth factor-beta (TGF-beta) ligands and receptors and Smad activation through unknown mechanisms. We show that ...androgens (dihydrotestosterone and R1881) down-regulate TGF-beta1-induced expression of TGF-beta1, c-Fos, and Egr-1 in the human prostate adenocarcinoma cell line, LNCaP. Moreover, 5alpha-dihydrotestosterone (DHT) inhibits TGF-beta1 activation of three TGF-beta1-responsive promoter constructs, 3TP-luciferase, AP-1-luciferase, and SBE4(BV)-luciferase, in LNCaP cells either with or without enforced expression of TGF-beta receptors (TbetaRI and TbetaRII). Similarly, DHT inhibits the activation of Smad-binding element (SBE)4(BV)-luciferase by either constitutively activated TbetaRI (T204D) or constitutively activated Smad3 (S3*). Activation of SBE4(BV)-luciferase by S3* in the NRP-154 prostatic cell line, which is androgen receptor (AR)-negative but highly responsive to TGF-beta1, is blocked by co-transfection with either full-length AR or AR missing the DNA binding domain. Immunoprecipitation and GST pull-down assays show that AR directly associates with Smad3 but not Smad2 or Smad4. Electrophoretic mobility shift assays indicate that the AR ligand binding domain directly inhibits the association of Smad3 to the Smad-binding element. In conclusion, our data demonstrate for the first time that ligand-bound AR inhibits TGF-beta transcriptional responses through selectively repressing the binding of Smad3 to SBE.
Upregulation of HER2/ErbB2/Neu occurs in 15-30% of human breast cancers and correlates with poor prognosis. Identification of ErbB2/Neu transcriptional targets should facilitate development of novel ...therapeutic approaches. Development of breast cancer is a multistep process; thus, to identify the transcriptomes associated with different stages of progression of tumorigenesis, we compared expression profiles of mammary tumors and preneoplastic mammary tissue from MMTV-Neu transgenic mice to expression profiles of wild-type mammary glands using Affymetrix microarrays. We identified 324 candidate genes that were unique to ErbB2/Neu-induced tumors relative to normal mammary gland tissue from wild-type controls. Expression of a subset of these genes (82) was also changed in the preneoplastic mammary glands compared to wild-type controls, indicating that they may play a pivotal role during early events of ErbB2/Neu-initiated mammary tumorigenesis. Further analysis of the microarray data revealed that expression of several known transforming growth factor (TGF)-beta target genes was altered, suggesting that the TGF-beta signaling cascade is downregulated in ErbB2/Neu-induced tumors. Western blot analysis for TGF-beta-Receptor-I/ALK5 and immunohistochemistry for TGF-beta-Receptor-I/ALK5 and phosphorylated/activated Smad2 confirmed that the Smad-dependent TGF-beta signaling cascade was inactive in these tumors. Although absent in most of the tumor, phosphorylated Smad2 was present in the periphery of tumors. Interestingly, presence of phosphorylated/activated Smad2 correlated with expression of Activin-Receptor-IB/ALK4, suggesting that although Smad-dependent TGF-beta signaling is absent in ErbB2/Neu-induced tumors, Activin signaling may be active at the leading edge of these tumors. Cumulatively, these data indicate that the TGF-beta pathway is intrinsically suppressed in ErbB2/Neu tumors via a mechanism involving loss of TGF-beta-Receptor-I/ALK5.
Transforming growth factor beta (TGF-beta) signals through TGF-beta receptor serine/threonine kinases (TbetaRI and TbetaRII) and Smads, regulating cell growth and apoptosis. Although loss of TGF-beta ...receptor levels is strongly selected for during the progression of most cancers, tumor cells frequently escape from complete loss of TGF-beta receptors through unknown mechanisms. Here, we provide the first evidence that epidermal growth factor (EGF) signaling, which is generally enhanced in cancer, is permissive for regulation of gene expression and growth suppression by TGF-beta in LNCaP prostate adenocarcinoma cells. Our results support that these permissive effects occur through enhanced stability of TbetaRII mRNA and reversal of TGF-beta-mediated TbetaRII mRNA loss. Changes in stability of TbetaRII mRNA occur soon after EGF or TGF-beta1 addition (optimal within 3 h) and are independent of de novo protein synthesis or transcription. Remarkably, such loss of TbetaRII by TGF-beta can be mediated by a kinase-dead TbetaRII (K277R), as well as by other forms of this receptor harboring mutations at prominent autophosphorylation sites. Moreover, Smad3 small interfering RNA, which blocks TGF-beta-induced AP-1 promoter activity, does not block changes in the expression of TbetaRII by EGF or TGF-beta. We have also shown that changes in TbetaRII levels by EGF are EGF receptor-kinase-dependent and are controlled by signals downstream of MEK1/2. Our findings provide invaluable insights on the role of the EGF receptor-kinase in enhancing TGF-beta responses during prostate carcinogenesis.
Here we describe a protein product of the human septin H5/PNUTL2/CDCrel2b gene, which we call ARTS (for apoptosis-related protein in the TGF-beta signalling pathway). ARTS is expressed in many cells ...and acts to enhance cell death induced by TGF-beta or, to a lesser extent, by other apoptotic agents. Unlike related septin gene products, ARTS is localized to mitochondria and translocates to the nucleus when apoptosis occurs. Mutation of the P-loop of ARTS abrogates its competence to activate caspase 3 and to induce apoptosis. Taken together, these observations expand the functional attributes of septins previously described as having roles in cytokinesis and cellular morphogenesis.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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