Differential regulation of the parathyroid hormone-related protein gene P1 and P3 promoters by cAMP

Abstract

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.

Introduction

Parathyroid hormone-related protein (PTHrP), originally identified as the causative agent of humoral hypercalcemia of malignancy (Moseley et al., 1987, Suva et al., 1987), has been identified in a variety of normal tissues. The hypercalcemic properties of PTHrP have been well documented, with its parathyroid hormone-like (PTH-like) calcium-mobilising activity being attributed to the limited amino terminal homology with PTH. PTHrP has also been reported to demonstrate both growth stimulating and inhibiting properties (Holick et al., 1994). The human PTHrP gene is complex, comprising nine exons and three promoter elements (Mangin et al., 1989, Suva et al., 1989). Several groups have confirmed transcriptional start sites at exon I and IV (Mangin et al., 1989, Suva et al., 1989) and also a promoter at exon 3 (Vasavada et al., 1993). The PTHrP gene promoters P1 and P3, 5′ to exons I and IV respectively, are classical TATA promoters, whereas the P2 promoter, 5′ to exon III, is a GC-rich promoter region. Differential splicing of these exons can result in up to 12 species of mRNA and three peptides, of 139, 141, and 173 amino acids in length. Reverse transcriptase-polymerase chain reaction (RT-PCR) has been utilised by several groups to elucidate the promoter usage and differential splicing of transcripts in different tissues (Campos et al., 1992, Southby et al., 1995), demonstrating tissue specific differences in promoter regulation. Transcription of the human PTHrP gene has recently been demonstrated to be activated via a cAMP-dependent pathway by prostaglandin E₁ in HTLV-I infected cells (Ikeda et al., 1993a). We have previously demonstrated that calcitonin, a small peptide secreted normally by the C cells of the thyroid gland, acting via a cAMP-dependent pathway, increases transcription of the human PTHrP gene (Chilco et al., 1993). Several groups have reported an increase in secretion of PTHrP in response to calcitonin (Deftos et al., 1989, Rizzoli et al., 1994), and we have previously identified a cAMP response element (CRE) which was important for transcriptional regulation of PTHrP P3 promoter by cAMP and calcitonin (Chilco et al., 1993). While calcitonin has potent biological effects on multiple tissues, particularly bone and kidney, the exact physiological role of this peptide in humans remains unclear. Calcitonin has been reported to be secreted by many tumours, particularly medullary thyroid carcinoma and carcinoma of the lung (Luster et al., 1982). Calcitonin has recently been shown to stimulate growth of human prostate cells via increases in cAMP and Ca²⁺ transients (Shah et al., 1994). PTHrP is also secreted by lung tumours (Danks et al., 1989), primarily squamous cell carcinomas, but also small cell tumours, as well as being detected in multiple normal tissues (Danks et al., 1989, Martin et al., 1991). PTHrP expression and production has been demonstrated to be regulated by an array of factors including cycloheximide (Ikeda et al., 1990), glucocorticoids (Ikeda et al., 1989), 1,25-dihydroxyvitamin D₃ (Ikeda et al., 1989), oestradiol (Thiede et al., 1991), phorbol esters (Rodan et al., 1989), angiotensin II (Pirola et al., 1993), EGF (Kremer et al., 1991), TGF β (Casey et al., 1992), IL-2 (Ikeda et al., 1993b), and calcium (Hellman et al., 1992). Thus interactions between calcitonin and PTHrP may be of significance.

Most published reports of regulation of PTHrP gene expression have focused on regulation of the P3 promoter, which appears in many cases to initiate a large proportion of the transcripts produced in response to various agonists (Chilco et al., 1993, Dittmer et al., 1993, Dittmer et al., 1994). There have been limited reports of transcriptional regulation of the P1 promoter (Heath et al., 1995). We present evidence here for the transcriptional regulation of the P1 PTHrP promoter by calcitonin via a cAMP-dependent pathway. Using RT-PCR we have demonstrated a transcriptional effect of both calcitonin and other cAMP agonists on the human PTHrP gene. The studies on transcription of the PTHrP P1 promoter indicate that this promoter is regulated independently of the P3 promoter. Utilising constructs incorporating different reporter genes we have investigated the differential regulation of PTHrP promoters by calcitonin and cAMP agonists in a human lung cancer cell line (BEN).