The transforming growth factor-betas (TGF-betas) are multifunctional regulators of cellular growth and function and potent inhibitors of epithelial cell proliferation. The widespread expression of TGF-beta indicates a pivotal role in epithelial homeostasis. These features make the TGF-betas attractive candidates for new therapeutic intervention approaches to the prevention and treatment of cancer. TGF-beta plays a major role in adult physiology, as well as in the control of differentiation and morphogenesis in embryonic development. The tissue distribution pattern of the TGF-betas, which include TGF-betas 1, 2, and 3 in mammals, has possible significance for signaling roles in epithelial-mesenchymal interactions during embryogenesis, as well as in cancer and carcinogenesis. TGF-beta is secreted by a variety of normal and malignant cells. The TGF-betas function through a set of cell surface protein receptors that includes TGF?beta type I (RI) and type II (RII). TGF-beta RII can bind TGF-beta directly to form a complex, which then is able to bind TGF-beta RI, and TGF-beta RII is then able to phosphorylate TGF-beta RI, which is necessary for signal transduction. The TGF-beta signaling system has been implicated as a tumor suppressor pathway in several organ systems. Loss of functional TGF-beta RI or RII contributes to loss of TGF-beta responsiveness, resulting in tumor progression. Defects in responsiveness to TGF-beta have been implicated in the pathogenesis of several human epithelial cancers, suggesting that TGF-beta has tumor suppressor properties. But, many advanced tumors show increased expression of TGF-beta, and parallel poor prognosis, suggesting that TGF-beta also has properties of a tumor suppressor. The complex mechanisms of action of TGF-beta in its capacity as a tumor suppressor and a tumor promoter and the target genes that are regulated by TGF-beta in these different capacities must be clearly defined to be able to exploit this gene for clinical therapeutic intervention purposes. Our broad goal is to determine how TGF-beta signaling regulates the development and malignant transformation of epithelial cells, now with a primary emphasis on lung epithelial cells. Our approach is based on the hypotheses that (1) Signaling pathways for TGF-beta occur that are separate from the growth inhibitory pathway and these pathways may be operational in lung cancer cells that are resistant to growth inhibition by TGF-beta; (2) The integrity of the TGF-beta signaling pathways is important for the normal regulation of downstream target molecules of TGF-beta and dysregulation of the activity of these signaling pathways during progressive stages of lung tumorigenesis impacts on the regulation of these target genes; (3) The tumor suppressor and tumor promoter activities of TGF-beta differentially regulate target genes that contribute to these activities. Our research efforts are focused around the proposal that there is a delicate balance between the tumor suppressor and tumor promoter roles of TGF-beta in epithelial tissues. Our premise is that the ability of TGF-beta to act as a tumor suppressor is primary to that of a tumor promoter in normal epithelial cells, and that the ability of TGF-beta to act as a tumor promoter takes on added significance as cells that are sensitive to TGF-beta become transformed and eventually resistant to TGF-beta, expand clonally, and ultimately progress to malignancy. Awareness of the timing of the phenotypic switch from TGF-beta sensitivity to TGF-beta resistance that occurs during tumorigenesis is likely to be important in designing and applying strategies for tumor prevention and treatment. The most recent efforts have examined surfactant proteins in mice bearing tumors. Surfactant protein-D (SP-D) is expressed in ethyl carbamate-induced lung tumors. Serum levels of SP-D are increased in patients with interstitial lung disease and acute respiratory distress syndrome and in rats with acute lung injury, but have not been measured in mice. The serum SP-D concentration was 5.0 +/- 0.2 ng/ml in normal C57BL/6 mice, essentially absent in SP-D null mice, and 63.6 +/- 9.0 ng/ml in SP-D overexpressing mice. SP-D in serum was verified by immunoblotting. Serum SP-D was increased in mice bearing tumors induced by three protocols, and the SP-D level correlated with tumor volume. However, in mice with a single adenoma or a few adenomas, SP-D levels were usually within the normal range. SP-D was expressed by the tumor cells, and there was also a field effect whereby type II cells near the tumor expressed more SP-D than type II cells in the remainder of the lung. Serum SP-D was also increased by lung inflammation. In airway inflammation induced by aerosolized ovalbuman in sensitized BALB/c mice, the serum levels were elevated after challenge. In conclusion, serum SP-D concentration is increased in mice bearing lung tumors, and generally reflects the tumor burden but is also elevated during lung inflammation. To complement our animal model systems, the TGF-beta 1 sensitive epithelial non-small cell lung cancer (NSCLC) cell line NCI-H727, whose growth can be inhibited by TGF-beta 1, was used as a model system to identify potential genes involved in TGF-beta 1 growth inhibition. Comparative cDNA expression patterns between NCI-H727 cells treated with TGF-beta 1 or vehicle alone were generated by differential mRNA display. Among the several cDNA fragments that represent genes that are potentially differentially regulated by TGF-beta 1 that were recovered, one 496-bp cDNA fragment that hybridized to a 2.7-kb mRNA species and whose expression was differentially increased 3-fold by TGF-beta 1 in NCI-H727 cells by northern blot analysis, revealed no significant match to any known gene sequence. The mRNA transcript of this novel gene, which we named Differentially Expressed Nucleolar TGF-beta 1 Target (DENTT), is induced by TGF-beta 1 in NSCLC cells that are sensitive to TGF-beta 1. A human DENTT cDNA probe was used to isolate mouse DENTT cDNA clones. Mouse DENTT mRNA contains a 2031-bp open reading frame that encodes a predicted polypeptide of 677-amino acids with a relative molecular mass of 77,671-daltons. The mouse and human DENTT sequences show 77% and 78% homology at the nucleotide and amino acid level, respectively. Mouse DENTT is predicted to be a nuclear protein with two nuclear localization signals (NLS), two coiled-coil regions, and a domain that shows significant identity to a region that defines the TTSN superfamily. Enhanced green fluorescent protein (EGFP)-tagged full-length mouse DENTT transfected into COS-7 cells showed localization to the nucleolus. Like human DENTT, the bipartite NLS is necessary, but requires the monopartite NLS for nucleolar localization. RT-PCR amplification, northern hybridization, and western blot analyses showed expression of mouse DENTT mRNA and protein throughout mouse embryogenesis. Immunohistochemical staining analysis showed that DENTT is expressed in multiple tissues in a defined spatio-temporal pattern during mouse embryogenesis. The heart and primitive brain were the first organs of the embryo that showed immunoreactivity for the DENTT antibody by day 8 of development (E8). In the developing mouse brain, the choroid plexus was intensely stained for DENTT in all stages of development. The spinal cord and dorsal root ganglia were also positive for DENTT staining beginning in the 11-day-old embryo (E11), where homogeneous immunostaining was observed throughout the developing neurons. Interestingly, by day 16 of development (E16), only a small subset of the neuronal population in the spinal cord and dorsal root ganglia was positively stained.

Agency
National Institute of Health (NIH)
Institute
Division of Clinical Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01SC000166-12
Application #
6946050
Study Section
(CCBB)
Project Start
Project End
Budget Start
Budget End
Support Year
12
Fiscal Year
2003
Total Cost
Indirect Cost
Name
Clinical Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Ozbun, Laurent L; Martinez, Alfredo; Jakowlew, Sonia B (2005) Differentially expressed nucleolar TGF-beta1 target (DENTT) shows tissue-specific nuclear and cytoplasmic localization and increases TGF-beta1-responsive transcription in primates. Biochim Biophys Acta 1728:163-80
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