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Inhibition of mink lung epithelial cell proliferation by transforming growth factor-beta is coupled through a pertussis-toxin-sensitive substrate.

ABSTRACT: Transforming growth factor beta 1 (TGF beta 1) inhibits the proliferative response of mink lung epithelial cells (CCL64) to serum and to epidermal growth factor (EGF). This response to TGF beta 1 can be inhibited by prior exposure of the cells to nanogram concentrations of pertussis toxin (PT), suggesting the involvement of a guanine-nucleotide-binding regulatory protein (G-protein) in mediating TGF beta 1-induced growth inhibition. To characterize further this G-protein dependence, we have isolated, by chemical mutagenesis, a CCL64 variant (CCL64-D1) that is resistant to TGF beta 1. Whereas in the parental CCL64 cells TGF beta 1 stimulates both GTP[35S] (guanosine 5'-[gamma-[35S]thio]triphosphate) binding and GTPase activity, in the CCL64-D1 variants TGF beta 1 is without effect. Quantitative immunoblotting with antisera for G-protein alpha- and beta-subunits, as well as PT-catalysed ADP-ribosylation analyses, revealed no appreciable changes in the level of G-protein expression in the CCL64-D1 variants compared with parental cells. In contrast with another TGF beta-resistant clone, MLE-M, which we show lacks detectable type I receptor protein, the CCL64-D1 cells retain all three TGF beta cell-surface binding proteins. On the basis of these studies, we propose that a necessary component of TGF beta 1-mediated growth inhibition in CCL64 epithelial cells is the coupling of TGF beta 1 receptor binding to G-protein activation.

SUBMITTER: Howe PH

PROVIDER: S-EPMC1131165 | biostudies-other | 1990 Mar

REPOSITORIES: biostudies-other

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Project description:In the present study, we determined the agonist specificity and the signalling mechanisms of a putative sphingosine 1-phosphate (S1P) receptor, AGR16. In CHO cells transiently transfected with an AGR16 expression vector, but not in cells transfected with an empty vector, the addition of a low concentration of S1P (1 nM) caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i) by mobilization of Ca2+ from both intra- and extra-cellular pools. To determine the spectrum of agonists for AGR16, we employed K562 cells, which in the naive state do not respond at all to either S1P or structurally related lipids with an increase in [Ca2+]i. In K562 cells stably expressing AGR16, S1P and sphingosylphosphorylcholine (SPC) dose-dependently increased [Ca2+]i with half-maximal values of 3 nM and 100 nM respectively. In CHO cells stably expressing AGR16 (CHO-AGR16), but not in parental CHO cells, we observed specific binding of [32P]S1P, which was displaced by unlabelled S1P and SPC. In CHO-AGR16 cells, but not in parental CHO cells, S1P stimulated the production of inositol phosphates and Ca2+ mobilization which was only 30% inhibited by pertussis toxin (PTX), different from the case of the recently identified S1P receptor EDG1. Also in CHO-AGR16 cells, but not in CHO cells, S1P at higher concentrations activated mitogen-activated protein kinase (MAPK) in a PTX-sensitive and Ras-dependent manner. S1P also induced the activation of two stress-activated MAPKs, c-Jun N-terminal kinase and p38, in a manner that was totally insensitive to PTX. In CHO-AGR16 cells, S1P induced stress-fibre formation, with an increase in myosin light chain phosphorylation, in a PTX-insensitive and Rho-dependent manner. S1P also induced an increase in the cellular cAMP content in CHO-AGR16 cells, which contrasts sharply with the case of EDG1. These results establish that the S1P receptor AGR16 is coupled via both PTX-sensitive and -insensitive G-proteins to multiple effector pathways.

Project description:Transforming growth factor-beta 1 (TGF beta 1) initiates a series of signalling events resulting in diverse cellular responses including stimulation of extracellular matrix protein production. In this study we have investigated the role of pertussis toxin-sensitive G-proteins in mediating the effects of TGF beta 1 on fibroblast procollagen metabolism. TGF beta 1 stimulated human fetal lung fibroblast procollagen synthesis and production in a dose-dependent manner which was maximal at 0.5 ng/ml. TGF beta 1 also decreased the proportion of newly synthesized procollagen degraded intracellularly. Pertussis toxin, a G-protein inhibitor, further stimulated TGF beta 1-induced procollagen synthesis and production, but alone it had no effect on fibroblast procollagen metabolism. Addition of indomethacin also potentiated the TGF beta 1-induced increase in procollagen synthesis and production. The effects of pertussis toxin and indomethacin were not additive. Pertussis toxin and indomethacin did not affect the proportion of newly synthesized procollagen degraded intracellularly, either alone or in combination, by control cells. The TGF beta 1-induced decrease in intracellular procollagen degradation was maintained but not further affected by pertussis toxin or indomethacin. TGF beta 1 increased prostaglandin E2 (PGE2) compared with PGE2 production by control cells. Addition of pertussis toxin or indomethacin blocked the TGF beta 1-induced increase in PGE2 production. The TGF beta 1-induced increase in PGE2 preceded the increase in procollagen production. These results demonstrate that TGF beta 1-induced procollagen synthesis by lung fibroblasts is modulated by production of PGE2. Pertussis toxin and indomethacin block the production of PGE2 and enhance the effect of TGF beta 1 on procollagen synthesis. From these data we conclude that the effects of TGF beta 1 on PGE2 production but not procollagen synthesis are mediated via a receptor linked to a pertussis toxin-sensitive G-protein.

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Inhibition of mink lung epithelial cell proliferation by transforming growth factor-beta is coupled through a pertussis-toxin-sensitive substrate.

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