Differential regulation of the parathyroid hormone-related protein gene P1 and P3 promoters by cAMP.
The role of calcitonin, and other agonists which activate the cAMP pathway, in regulating transcription of the human parathyroid hormone-related protein (PTHrP) gene was investigated in a human lung cancer cell line (BEN). Both calcitonin and forskolin caused a 5-6-fold increase in transcription initiated from both the P1 and P3 promoters, but with no observed effect on the P2 promoter. Maximal 6-fold activation of the P1 promoter occurred at 16 h post-stimulation and effects of calcitonin were observed within the pM range. The PKC agonist, phorbol 12-myristate 13-acetate diester (PMA), did not modulate transcription initiated from the P1 promoter. The ionophore ionomycin had a small effect on transcription of the P1 promoter, and transcriptional control may involve an interaction between the cAMP and intracellular calcium second messenger pathways. Deletion mapping studies indicated that increases in transcription of the human PTHrP gene is being mediated via a CRE element situated at -3313 to -3306 upstream of the P1 promoter. Mutational analysis of this CRE element confirmed a role for this sequence in mediating the increase in transcription effected by cAMP. Consistent with these transfection studies, RT-PCR of PTHrP mRNA also indicated a significant increase in transcripts generated from the P1 promoter. Gel retardation assays utilising a fragment of the P1 promoter region, encompassing the putative CRE, determined that nuclear proteins were binding to this region. Competition binding studies with labelled probe and cold competitors determined that the binding was specific for this sequence. A wild-type CRE consensus oligonucleotide also competed for binding with this sequence.
Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Victoria, Australia.
P J
PJ
Chilco
V
V
Leopold
J D
JD
Zajac
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008175:Lung Neoplasms; D016297:Mutagenesis, Site-Directed; D044162:Parathyroid Hormone-Related Protein; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011506:Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D014407:Tumor Cells, Cultured
C470121:PTHLH protein, human; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D011993:Recombinant Fusion Proteins; D005576:Colforsin; D000242:Cyclic AMP; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase