Abstract
We analyzed the effect of rapamycin on autocrine mast cell tumor lines with abnormally stable interleukin-3 (IL-3) transcripts due to a defect in mRNA degradation. Rapamycin inhibited IL-3 mRNA expression specifically, while transcripts of IL-4 and IL-6 were not affected. As indicated by the use of the transcriptional inhibitor actinomycin D or by reporter constructs, inhibition was posttranscriptional and resulted from destabilization of the mRNA. Transcripts from transgenes lacking the AU-rich 3' untranslated region were refractory to drug-induced degradation, suggesting that these 3' sequences contain the target of the rapamycin effect. Rapamycin did not promote IL-3 mRNA degradation in cells of a tumor variant lacking expression of FKBP12, the binding protein of rapamycin. Experiments with wortmannin indicated that rapamycin does not act via p70S6 kinase. FK-506, another ligand of FKBP12 affecting the phosphatase calcineurin, did not antagonize but shared the effect of rapamycin. Our data fit a model whereby both FKBP12 and calcineurin target an unknown regulator of IL-3 mRNA turnover.
Highlights
Lymphokines and their related signal transduction pathways play an important role in the control of cell activation, proliferation, and oncogenesis
Transfection experiments with IL-3 transgenes revealed that deletion of AU-rich element (ARE)-containing 3Ј untranslated region (3ЈUTR) sequences rendered the transcripts drug resistant, indicating that sequences at or near the ARE are a target of the effects of CsA or FK-506 [36]
While both RAPA and FK-506 were inhibitory in the absence of exogenous IL-3 (Fig. 1a), RAPA inhibited growth even in its presence (Fig. 1b)
Summary
Lymphokines and their related signal transduction pathways play an important role in the control of cell activation, proliferation, and oncogenesis These processes can be conveniently studied in murine PB-3c mast cells, which are interleukin-3 (IL-3) dependent but progress to autocrine IL-3-producing tumors after transformation by the v-H-ras oncogene [35]. Evidence for a similar oncogenic mechanism has been previously provided by Schuler and Cole [51] with an autocrine tumor line of monocyte origin In these cells, granulocyte-macrophage colony-stimulating factor mRNA decayed with very slow kinetics, and transfection experiments revealed that the defect occurred in trans. We have recently reported that the abnormally stable IL-3 transcripts expressed in mast cell tumor lines with a decay defect became destabilized upon treatment with the immunosuppressive drugs cyclosporine (CsA) and FK-506. It inhibits serum-induced phosphorylation of 4E-BP1, a binding protein of initiation factor eIF-4E, which blocks initiation of translation [3, 23, 30, 56]
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