ABSTRACT
Glucocorticoids have been shown to inhibit the activity of the human prolactin (hPRL) promoter. Using transient expression experiments in rat pituitary cells, we located the sequence conferring glucocorticoid inhibition to a region which contains Pit-1 binding sites, responsible for pituitary-specific expression, but does not seem to contain a glucocorticoid receptor (GR) binding site. Co-transfection experiments in non-pituitary cell lines, using expression vectors for Pit-1 and different mutants of the human GR show that inhibition of the hPRL gene is seen only in the presence of Pit-1 and GR, and that the DNA binding function of the receptor is not required. Immunoprecipitation studies show that either anti-GR or anti-Pit-1 antibodies are able to co-precipitate GR and Pit-1, suggesting an interaction between these factors. We conclude that the activated GR functionally interferes with the pituitary specific factor Pit-1, thereby leading to the observed transcriptional repression.
Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation , Prolactin/antagonists & inhibitors , Prolactin/genetics , Receptors, Glucocorticoid/physiology , Transcription Factors/metabolism , Animals , COS Cells , Cyclic AMP/metabolism , DNA/metabolism , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/physiology , Gene Expression Regulation/drug effects , HeLa Cells , Humans , Promoter Regions, Genetic/drug effects , Protein Binding/genetics , Rats , Transcription Factor Pit-1 , Transcription Factors/drug effects , Transcription Factors/physiology , Tumor Cells, CulturedABSTRACT
Pituitary GH3 cells were transfected with different deletion mutants of the human prolactin (hPRL) promoter fused to the CAT reporter gene. The proximal region (-250 to -42) was sufficient to confer stimulation by both thyrotropin-releasing hormone (TRH) and epidermal growth factor (EGF). Further deletion analyses demonstrated the importance of the three proximal Pit-1 binding sites in this response. However, Pit-1 binding oligonucleotides confer neither TRH nor EGF induction to a linked neutral promoter, suggesting that other elements might be involved. We have previously shown that sequence A (-115 to -85) is needed together with Pit-1 binding sites for full cyclic AMP response of hPRL-CAT. Mutation of this sequence strongly affects TRH and EGF induction. On the other hand, three copies of sequence A confer both TRH and EGF response to a linked neutral promoter. In conclusion, although TRH and EGF activate mostly different intracellular pathways, they mediate transcriptional induction of the hPRL promoter via identical cis elements.
Subject(s)
DNA-Binding Proteins , Epidermal Growth Factor/pharmacology , Prolactin/genetics , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , Thyrotropin-Releasing Hormone/pharmacology , Animals , Binding Sites , Cyclic AMP/physiology , Humans , Pituitary Neoplasms , Prolactin/biosynthesis , Rats , Recombinant Fusion Proteins/biosynthesis , Thymidine Kinase/genetics , Transcription Factor Pit-1 , Transcription Factors/metabolism , Transfection , Tumor Cells, CulturedABSTRACT
cAMP strongly stimulates the activity of the human prolactin (hPRL) promoter. We have previously shown that two types of cis-element are required for this cAMP regulation; binding sites for the pituitary-specific factor Pit-1, and the sequence spanning nucleotides -115 to -85 (named sequence A). Sequence A contains the TGACG motif found in the consensus sequence of the cAMP-responsive element (CRE). In this study, we show that a mutation in the TGACG motif of sequence A strongly reduces not only the cAMP regulation but also the Ca2+ regulation and basal activity of the hPRL promoter. Furthermore, gel-shift assays indicate that the mutation prevents binding of a ubiquitous factor which is not the CRE-binding protein. Southwestern experiments suggest that this ubiquitous factor's molecular mass is approximately 100 kDa. We conclude that binding of a 100-kDa ubiquitous factor to sequence A is required for full basal and hormonal regulation of hPRL-promoter activity.
