Little is known about the cellular mechanisms by which transforming growth factor beta (TGF-beta) is able to regulate the activities of nontransformed and neoplastic cells. Elucidation of the intracellular pathways of signal transduction should account for the wide variety of biological responses elicited by TGF-beta including both stimulatory actions such as the induction of anchorage-independent growth and neoplastic-like behavior in some types of cells and the inhibition of growth in other cell types. Studies from this laboratory have recently established that TGF-beta, when added in serum-free medium to quiescent Rat-1 cells, will induce a large increase in inositol phosphate generation several hours later, and will dramatically sensitize the cells to the IP3-generating effects of EGF, which normally causes only a feeble increase in inositol phosphate levels. Concomitant with the increase in IP3 levels produced by EGF in TGF-beta-sensitized cells is an increase in the levels of free intracellular calcium, as determined by fura-2 microspectrafluorometry. In a similar time frame as phosphoinositide induction, TGF-beta will induce the transcriptional repression of transin, a transformation-associated metalloprotease whose transcription is activated by the action of several oncogenes and by EGF. In this application we propose to characterize the changes in phosphoinositide metabolism in nontransformed and transformed rat and mouse cell lines. We will investigate the ability of EGF and TGF-beta to induce the production of diacylglycerols and inositol phosphates and the mobilization of intracellular Ca2+. Potential TGF-beta-modulated pathways will be investigated, such as altered phospholipase C, DAG kinase and lipase activities. In an effort to understand the differentiatial responsiveness to TGF-beta of nontransformed and transformed cells, the intracellular responses of some oncogen-transformed and nontransformed cells to TGF-beta will be investigated. To correlate the alteration in 2nd messenger production with transcriptional repression induced by TGF-beta, alterations in transin transcription will be measured using nuclear run-on methods. Finally, in an effort to understand the regulation of transin transcription by both EGF and TGF-beta, transient expression assays will be performed using identified, and the nucleotide sequences responsible for mediating EGF- and TGF- beta-responsiveness of that gene will be identified. Thus, these studies should be able to relate the alterations in 2nd messenger production indeed by TGF-beta to the transcriptional responsiveness of a gene whose transcription in repressed by TGF- beta.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA047404-03
Application #
3191044
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1988-04-05
Project End
1992-03-31
Budget Start
1990-04-01
Budget End
1992-03-31
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
Type
Schools of Medicine
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
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Rodland, K D; Lenormand, P; Muldoon, L L et al. (1992) Regulation of transin/stromelysin and VL30 gene expression by intracellular calcium. J Invest Dermatol 98:12S-16S
Pribnow, D; Muldoon, L L; Fajardo, M et al. (1992) Endothelin induces transcription of fos/jun family genes: a prominent role for calcium ion. Mol Endocrinol 6:1003-12
Foley, J F; Moertel, C G (1991) Improving accrual into cancer clinical trials. J Cancer Educ 6:165-73
Rodland, K D; Muldoon, L L; Magun, B E (1991) Regulation of intracellular Ca2+ and gene expression by endothelin-1. J Cardiovasc Pharmacol 17 Suppl 7:S89-95
Muldoon, L L; Enslen, H; Rodland, K D et al. (1991) Stimulation of Ca2+ influx by endothelin-1 is subject to negative feedback by elevated intracellular Ca2+. Am J Physiol 260:C1273-81
Rodland, K D; Muldoon, L L; Magun, B E (1990) Cellular mechanisms of TGF-beta action. J Invest Dermatol 94:33S-40S
Lenormand, P; Muldoon, L L; Enslen, H et al. (1990) Two tumor promoters, 12-O-tetradecanoylphorbol-13-acetate and thapsigargin, act synergistically via distinct signaling pathways to stimulate gene expression. Cell Growth Differ 1:627-35
Rodland, K D; Muldoon, L L; Lenormand, P et al. (1990) Modulation of RNA expression by intracellular calcium. Existence of a threshold calcium concentration for induction of VL30 RNA by epidermal growth factor, endothelin, and protein kinase C. J Biol Chem 265:11000-7
Muldoon, L L; Pribnow, D; Rodland, K D et al. (1990) Endothelin-1 stimulates DNA synthesis and anchorage-independent growth of Rat-1 fibroblasts through a protein kinase C-dependent mechanism. Cell Regul 1:379-90

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