Identification and characterization of a GC-rich promoter of the human parathyroid hormone-related peptide gene.

The human PTH-related peptide (PTHrP) gene comprises eight exons spanning more than 15 kilobases of genomic DNA.The gene has a highly complex controlling region, which contains four alternatively spliced, noncoding exons and at le;A two putative promoters, one 5' of exon 1A (up-stream TATA element) and the other 5' of exon 2 (down-stream TATA element).To define important cis regulatory sequences of this gene, a functional dissection of PTHrP 5'-flanking DNA was initiated, using chimeric PTHrP-chloramphenicol acetyltransferase (CAT) constructs.This analysis was carried out in PTHrPnegative human renal carcinoma cells, so that RNA derived from transfected DNA could be studied without interference from endogenous PTHrP sequences.Of the initial series of constructs prepared, the most active was a l.l-kilobase BarnHI-Hindlll PTHrP-CAT plasmid containing 350 basepairs of DNA 5' of exon 1C and extending into exon 3. Analysis of transfection products by RNase protection and primer extension revealed that this region contains a previously unrecognized promoter of the gene.This element is located immediately 5' of exon lC, is active in transfected cells when cloned in isolation up-stream of the CAT gene, and appears to be functional in a number of cell lines and tissues on the basis of primer extension analysis.Unlike the other two PTHrP gene promoters, this element is GC rich and does not possess canonical TATA or CAAT sequences.The human PTHrP gene is one of a handful of genes that appear to use both TATA and GC-rich promoter elements.(Molecular Endocrinology 7: 273-282,1gg3) the syndrome of humoral hypercalcemia of malignancy (1-8).The deduced PTHrP sequence bears similarity to the PTH sequence only in the first 13 N-terminal amino acids, after which the two sequences diverge completely (1, 3-8).It is now known that the PTH and PTHrP genes arose by duplication from a common precursor (7).Whereas the human PTH gene has a simple three-exon structure and appears to be expressed primarily in the parathyroid chief cell (9, lo), the PTHrP gene has evolved into a highly complex transcriptional unit (6-8, 11-l 5) which is expressed in a remarkable variety of normal tissues and cell types, including sites as diverse as the epidermis, pancreatic islets, central nervous system, lactating breast, and preterm myometrium (16)(17)(18)(19)(20).All available data suggest that the peptide acts in an autocrine and/or paracrine fashion in these sites, possibly playing a role in calcium translocation, signalling, or both (18-21).Thus, it seems likely that the syndrome of humoral hypercalcemia of malignancy may represent the only circumstance in which PTHrP enters the systemic circulation in sufficient quantity to exert a conventional endocrine effect, in this case by cross-reacting with classical PTH receptors in bone and kidney.