Abstract
The transcription and transformation activity of c-Jun is governed by a 27-amino acid regulatory motif, labeled the delta-domain, which is deleted in v-Jun. We have previously shown that c-Jun is a potent inhibitor of the rat prolactin (rPRL) promoter activity induced by either oncogenic Ras or phorbol esters. Here, we have characterized the structural and cell-specific requirements for this c-Jun inhibitory response, and we show that this c-Jun inhibitory response mapped to the rPRL footprint II repressor site, was pituitary-specific and required the c-Jun delta-domain. Moreover, alteration of any one of these features (e.g., cis-element, trans-factor, or cell-specific background) switched c-Jun to a transcriptional activator of the rPRL promoter. In HeLa nonpituitary cells, c-Jun alone activated the rPRL promoter via the most proximal GHF-1/Pit-1 binding site, footprint I, and synergized with GHF-1. Finally, recombinant GHF-1 interacted directly with c-Jun but not c-Fos proteins. These data provide important fundamental insights into the molecular mechanisms by which the c-Jun delta-domain functions as a modulatory switch and further imply that the functional role of c-Jun is dictated by cell-specific influences and the delta-domain motif.
Highlights
C-Jun typically activates gene transcription, examples have accumulated documenting that it can inhibit gene expression
TPA-responsive element that overlaps a critical retinoic acid response element/vitamin D response element required for osteocalcin promoter activity, and sterically interferes with c-Jun is a member of the BZip family of transcription factors, retinoic acid receptor/vitamin D receptor binding [28]
Significant insights into basal and hormone-activated PRL and growth hormone (GH) gene expression have been provided by GH4 rat pituitary tumor cells, which are a clonal cell line that maintains cell type-specific functions and hormonal responses [33, 34, 37,38,39]. Previous experiments in this system have demonstrated that c-Jun does not function as a downstream target for either oncogenic V12 Ras- or TPA-mediated activation of the rat prolactin (rPRL) promoter, but instead c-Jun inhibits both of these signal transduction pathways [40, 41]. This inhibitory effect of c-Jun on the V12 Ras- and TPA-mediated activation of the rPRL promoter is promoter-specific and not GH4 cell-specific, since we demonstrated that c-Jun enhances V12 Ras stimulation of the AP-1-dependent Ϫ73ColCAT promoter-reporter construct [40]
Summary
Plasmid Constructs—The pituitary promoter-luciferase constructs, pA3PRLluc-425, pA3rGHluc, and pA3h␣luc-1760 have been described [39, 42, 43]. The plasmid pc-fosTKluc contains two copies of the c-fos enhancer spanning Ϫ357 to Ϫ276 fused to the TK promoter Ϫ200 to ϩ70 and has been previously described [48]. Western Blot Analysis—GH4 cells transiently transfected with the rPRL promoter-reporter and various effector plasmids were harvested with phosphate-buffered saline with 3 mM EDTA. HeLa cells transiently transfected with the rPRL promoter reporter and various effector plasmids were harvested with phosphate-buffered saline containing 3 mM EDTA. Bacterial cell pellets were resuspended with 7– 8 ml of TST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 5 g/ml each of the protease inhibitors antipain, chymostatin, leupeptin, and pepstatin A) and lysed by sonication, and the debris was pelleted for 20 min at 15,000 rpm in an SS34 rotor. The samples were boiled and loaded onto an SDS 3% stacking, 10% resolving polyacrylamide gel, the signal was enhanced with “Amplify” (Amersham), and the gel was dried and visualized by autoradiography
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