Nuclear and nucleolar localization of parathyroid hormone-related protein

Parathyroid hormone-related protein (PTHrP) was first discovered as the factor causing hypercalcaemia produced by solid tumours frequently associated with the head and neck, breast, lung and kidney. The homology of its amino-terminus to parathyroid hormone (PTH; eight of the first 13 residues are identical), enables it to share the same receptor and perform similar biological functions to PTH. The sequences of PTHrP C-terminal to its PTH-like region confer functions such as transplacental calcium transport, renal bicarbonate excretion and in vitro osteoclast inhibition. Recent findings have shown that PTHrP is a nuclear/nucleolar protein in certain tissues and that this localization is cell cycle-regulated, mediated by the middle portion of the molecule, and involves the nuclear import receptor importin beta1. The present review discusses what is known about the pathway by which PTHrP localizes to the nucleus/nucleolus and the putative roles it may have there.     

Sequences of the cDNAs encoding canine parathyroid hormone-related protein and parathyroid hormone

The cDNA clones encoding canine parathyroid-hormone-related protein (cPTHrP) and parathyroid hormone (cPTH) have been isolated and sequenced. The predicted amino-acid sequences of the mature canine homologs have a high degree of homology to human PTHrP (hPTHrP) and PTH (hPTH), especially in the biologically active regions. The cPTHrP cDNA is unique, since it has homology to exon lA of hPTHrP which suggests that dogs utilize a promoter similar to PI of hPTHrP which has not been demonstrated in other species.     

Purification and characterization of recombinant human parathyroid hormone-related protein

Full-length human parathyroid hormone-related protein (PTHrP-(1-141] as well as a carboxyl-terminal shortened form (PTHrP-(1-108] have been expressed from recombinant DNA-derived clones. These proteins were expressed in Escherichia coli as fusion proteins so that cyanogen bromide cleavage yields the desired product. Both proteins were purified and then characterized by sodium dodecyl sulfate gel electrophoresis, amino-terminal amino acid sequencing, peptide mapping, and mass spectral analysis. Recombinant PTHrP-(1-141), PTHrP-(1-108), synthetic PTHrP-(1-34), and naturally derived PTHrP are all equipotent in the stimulation of cyclic AMP levels in the osteoblast-like cell line UMR 106-01. However, PTHrP-(1-141) and -(1-108) are two to four times more active than PTHrP-(1-34) in the stimulation of plasminogen activator activity from this cell line. PTHrP-(1-141) reacts equipotently with PTHrP-(1-34) in a radioimmunoassay using an antiserum prepared against PTHrP-(1-34). PTHrP-(1-141), -(1-108), and -(1-84) were used as PTHrP-specific mobility standards on sodium dodecyl sulfate gel electrophoresis to determine the approximate length of two forms of naturally derived PTHrP. The data show that PTHrP purified from the lung tumor cell line BEN contains a major form of about 108 amino acids and another form of about 141 amino acids.     

In vitro and in vivo antagonists against parathyroid hormone-related protein

Four analogues of parathyroid hormone-related protein (PTHrP), PTHrP(7-34)NH2, (10-34)NH2, (15-34)NH2 and (20-34)NH2, were synthesized and their antagonistic activity against PTHrP(1-34) was examined in vitro and in vivo. In vitro studies revealed that all four analogues antagonized PTHrP-stimulated cyclic AMP production in rat osteosarcoma cells (ROS 17/2.8), and that PTHrP(7-34)NH2 and PTHrP(10-34)NH2 had potent antagonistic activity. In vivo experiments in nude mice also revealed that PTHrP(7-34)NH2 completely inhibited hypercalcemia induced by PTHrP(1-34), indicating that these analogues antagonize the effects of PTHrP(1-34) in vitro and in vivo.     

Immunochemical characterisation of parathyroid hormone-related protein from tumor and non-tumor cells

The molecular forms of parathyroid hormone-related protein (PTHRP) in conditioned media from the BEN human lung cancer cell line, rat parathyroid cells (PT-r) and human keratinocytes were studied by gel-filtraton chromatography with assay of PTHRP by immunoassays and bioassay. Immunoreactivity (1–86 and 1–34) and bioactivity (1–34) in conditioned media eluted as a coincident major peak (approx. molecular mass 19–22 kDa) and there was evidence of amino-terminal species in the molecular mass range 10–16 kDa in BEN and keratinocyte media. Western blotting of PTHRP affinity purified by monoclonal antibodies directed at regions 1–34 or 37–67, identified a major species in all cell cytosols and media with an apparent molecular mass of 24–25 kDa, consistently slighty larger than recombinant PTHRP(1–141) (mobility of 21 kDa) which may represent an intact or native form of PTHRP. Additional amino-terminal species were identified in medium from keratinocytes (16 and 7 kDa), BEN cells (18 and 14 kDa) and PT-R cells (17 kDa), suggesting that processing occurs at the C-terminus and within the mid-region to form a range of amino-terminal fragments.     

Negative regulation of parathyroid hormone-related protein expression by steroid hormones

Elevated parathyroid hormone-related protein (PTHrP) is responsible for humoral hypercalcemia of malignancy (HHM), which is of clinical significance in treatment of terminal patients with malignancies. Steroid hormones were known to cause suppression of PTHrP expression. However, detailed studies linking multiple steroid hormones to PTHrP expression are lacking. Here we studied PTHrP expression in response to steroid hormones in four cell lines with excessive PTHrP production. Our study established that steroid hormones negatively regulate PTHrP expression. Vitamin D receptor, estrogen receptor α, glucocorticoid receptor, and progesterone receptor, were required for repression of PTHrP expression by the cognate ligands. A notable exception was the androgen receptor, which was dispensable for suppression of PTHrP expression in androgen-treated cells. We propose a pathway(s) involving nuclear receptors to suppress PTHrP expression.     

Expression of parathyroid hormone-related protein (PTHrP) in parathyroid tissue under normal and pathological conditions

Parathyroid hormone-related protein (PTHrP), a factor responsible for malignancy associated hypercalcemia, plays a physiological roles such as bone development and placental calcium transport. The expression of PTHrP in adult human parathyroid tissues under normal and pathological conditions was analyzed. By immunohistochemistry, PTHrP expression was detected in 86% of normal parathyroid (12/14 cases), 74% of adenomas (14/19) and 89% of hyperplasia secondary to chronic renal failure (16/18). PTHrP protein was observed mainly in the cytoplasm of oxyphil cells, consistent with the localization of its mRNA demonstrated by in situ hybridization. The rate of PTHrP-positive cells was higher in areas consisting of oxyphil cells than in those of non-oxyphil cells, regardless of whether the parathyroid was normal or pathological. In the normal parathyroid, an age-related increase in PTHrP expression was observed with a relative increase in oxyphil cells, reflecting aging and deterioration of parathyroid tissue. In adenoma, cases with a predominance of oxyphil cells expressed PTHrP, whereas clear cell adenoma did not. In secondary hyperplasia, the rate of PTHrP-expressing cells was higher than in normal parathyroid or adenoma, with varying levels of expression among nodules. We speculate that PTHrP could act through the paracrine/autocrine mechanism to regulate proliferation and differentiation of normal and neoplastic parathyroid cells.     

Characterization of parathyroid hormone-related protein in the human term placenta.

To characterize parathyroid hormone-related protein (PTHrP) in the human placenta, we measured PTHrP-like immunoreactivity (PRP-LI) in the term placenta and studied the elution profiles of placental tissue extracts on Sephadex G-75 chromatography with a specific RIA. We also examined the gene expression of PTHrP mRNA by Northern blot analysis and the localization of PRP-LI in the placenta by immunohistochemistry. The amount of PRP-LI in placental extracts (n = 7) was 20.9 +/- 2.2 pg/g wet tissue (mean +/- SE). Dilution curves of placental tissue ran parallel to those of synthetic PTHrP (1-34) standards. Sephadex G-75 gel chromatography demonstrated two major PRP-LI peaks; the first peak was eluted around the molecular size between 10 kilodaltons (Kda) and 20 Kda and the other around 5 Kda. Northern blot analysis of PTHrP mRNA extracted from placental tissues showed a major hybridization signal around 18S. PTHrP immunohistochemistry showed PRP-LI staining in the cytoplasm of syncytiotrophoblasts and stroma cells (Hofbauer cells) in the term placenta. These results suggest that syncytiotrophoblasts and stroma cells in the term placenta synthesize PTHrP in two major molecular forms, 10 Kda-20 Kda and around 5 Kda.     

Calcitonin-suppressed expression of parathyroid hormone-related protein in breast cancer cells.

Parathyroid hormone-related protein (PTHrP) is a key factor behind humoral hypercalcemia of malignancy (HHM). It is produced in most breast tumors and may be an important local mediator of skeletal metastases due to breast cancer. PTHrP may mediate local bone destruction in the absence of increased circulating PTHrP. Calcitonin (CT) is used for treatment of HHM, but there are data showing that CT can increase PTHrP expression and secretion in vitro. We have therefore studied the effect of CT on PTHrP gene expression and secretion in MCF-7 breast cancer cells. PTHrP mRNA decreased significantly after 4, 8, and 16 h incubation with 10 nM salmon calcitonin (sCT) when compared with the respective controls. PTHrP mRNA also decreased significantly and dose-dependently after incubation with sCT at 0.1 to 10 nM for 16 h. The PTHrP levels in the conditioned medium also decreased in a similar dose-dependent manner. The adenylate cyclase agonist forskolin lowered the PTHrP mRNA dose-dependently. In cells exposed to varying concentrations of sCT for 15 min, the cAMP levels increased dose-dependently. In conclusion, sCT can suppress PTHrP gene expression in MCF-7 breast cancer cells. The suppressive effect is probably exerted mainly via the cAMP-protein kinase A pathways.     

Regulation of human organic anion transporter 4 by parathyroid hormone-related protein and protein kinase A

Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters that play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, antihypertensives, and anti-inflammatories. hOAT4 is abundantly expressed in the kidney and placenta. In the current study, we examined the regulation of hOAT4 by parathyroid hormone-related protein (PTHrP) and protein kinase A (PKA) in kidney COS-7 cells. PTHrP induced a time- and concentration-dependent stimulation of hOAT4 transport activity. The stimulation of hOAT4 activity by PTHrP mainly resulted from an increased cell surface expression without a change in total cell expression of the transporter. Activation of PKA by Bt2-cAMP also resulted in a stimulation of hOAT4 activity through an increased cell surface expression of the transporter. However, PTHrP-induced stimulation of hOAT4 activity could not be prevented by treating hOAT4-expressing cells with the PKA inhibitor H89. We concluded that both PTHrP and activation of PKA stimulate hOAT4 activity through redistribution of the transporter from intracellular compartments to the cell surface. However, PTHrP regulates hOAT4 activity by mechanisms independent of PKA pathway.     

Phosphorylation at the cyclin-dependent kinases site (Thr85) of parathyroid hormone-related protein negatively regulates its nuclear localization.

Parathyroid hormone-related protein (PTHrP) is expressed by a wide variety of cells and is considered to act as a secreted factor; however, evidence is accumulating for it to act in an intracrine manner. We have determined that PTHrP localizes to the nucleus at the G1 phase of the cell cycle and is transported to the cytoplasm when cells divide. PTHrP contains a putative nuclear localization sequence (NLS) (residues 61-94) similar to that of SV40 T-antigen, which may be implicated in the nuclear import of the molecule. We identified that Thr85 immediately prior to the NLS of PTHrP was phosphorylated by CDC2-CDK2 and phosphorylation was cell cycle-dependent. Mutation of Thr85 to Ala85 resulted in nuclear accumulation of PTHrP, while mutation to Glu85 to mimic a phosphorylated residue resulted in localization of PTHrP to the cytoplasm. Combined, the data demonstrate that the intracellular localization of PTHrP is phosphorylation- and cell cycle-dependent, and such control further supports a potential intracellular role (10,34,35) for PTHrP.     

Parathyroid hormone-related protein regulates tumor-relevant genes in breast cancer cells

The effect of endogenous parathyroid hormone-related protein (PTHrP) on gene expression in breast cancer cells was studied. We suppressed PTHrP expression in MDA-MB-231 cells by RNA interference and analyzed changes in gene expression by microarray analysis. More than 200 genes showed altered expression in response to a PTHrP-specific small interfering (si) RNA (siPTHrP). Cell cycle-regulating gene CDC2 and genes (CDC25B and Tome-1) that control CDC2 activity showed increased expression in the presence of siPTHrP. CDC2 activity was also found to be higher in siPTHrP-treated cells. Studies with PTHrP peptides 1-34 and 67-86, forskolin, and a PTH1 receptor (PTH1R)-specific siRNA showed that PTHrP regulates CDC2 and CDC25B, at least in part, via PTH1R in a cAMP-independent manner. Other siPTHrP-responsive genes included integrin alpha6 (ITGA6), KISS-1, and PAI-1. When combined, siRNAs against ITGA6, PAI-1, and KISS-1 could mimic the negative effect of siPTHrP on migration, whereas siKISS-1 and siPTHrP similarly reduced the proliferative activity of the cells. Comparative expression analyses with 50 primary breast carcinomas revealed that the RNA level of ITGA6 correlates with that of PTHrP, and higher CDC2 and CDC25B values are found at low PTHrP expression. Our data suggest that PTHrP has a profound effect on gene expression in breast cancer cells and, as a consequence, contributes to the regulation of important cellular activities, such as migration and proliferation.     

Leptin mediates the parathyroid hormone-related protein paracrine stimulation of fetal lung maturation

Developing rat lung lipofibroblasts express leptin beginning on embryonic day (E) 17, increasing 7- to 10-fold by E20. Leptin and its receptor are expressed mutually exclusively by fetal lung fibroblasts and type II cells, suggesting a paracrine signaling "loop." This hypothesized mechanism is supported by the following experimental data: 1) leptin stimulates the de novo synthesis of surfactant phospholipid by both fetal rat type II cells (400% x 100 ng(-1) x ml(-1) x 24 h(-1)) and adult human airway epithelial cells (85% x 100 ng(-1) x 24 h(-1)); 2) leptin is secreted by lipofibroblasts in amounts that stimulate type II cell surfactant phospholipid synthesis in vitro; 3) epithelial cell secretions such as parathyroid hormone-related protein (PTHrP), PGE(2), and dexamethasone stimulate leptin expression by fetal rat lung fibroblasts; 4) PTHrP or leptin stimulate the de novo synthesis of surfactant phospholipid (2- to 2.5-fold/24 h) and the expression of surfactant protein B (SP-B; >25-fold/24 h) by fetal rat lung explants, an effect that is blocked by a leptin antibody; and 5) a PTHrP receptor antagonist inhibits the expression of leptin mRNA by explants but does not inhibit leptin stimulation of surfactant phospholipid or SP-B expression, indicating that PTHrP paracrine stimulation of type II cell maturation requires leptin expression by lipofibroblasts. This is the first demonstration of a paracrine loop that functionally cooperates to induce alveolar acinar lung development.     

The parathyroid hormone-related protein associated with malignancy is secreted by neuroendocrine tumors

We have demonstrated the production of the PTH-related protein (PTHrP) associated with hypercalcemia of malignancy by human neuroendocrine cell lines that also produce calcitonin gene products and chromogranin A. PTHrP was demonstrable in the cells by immunocytochemistry and immunoassay and Northern analysis of the cells revealed the presence of multiple mRNAs for PTHrP. The cell lines also secreted PTHrP in a regulated fashion, with the most potent secretagogue being phorbol. Thus, PTHrP is secreted by neuroendocrine cells and it may have neuroectodermal lineage. The coexpression of calcitonin gene products and chromogranin A, also neuroendocrine, with PTHRP may influence its secretion and ultimate biological effects in vivo.     

Parathyroid hormone-related protein regulates apoptosis in lung cancer cells through protein kinase A

Parathyroid hormone-related protein (PTHrP)-(1–34) and PTHrP-(140–173) protect lung cancer cells from apoptosis after ultraviolet (UV) irradiation. This study evaluated upstream signaling in PTHrP-mediated alteration of lung cancer cell sensitivity to apoptosis. The two peptides increased cAMP levels in BEN lung cancer cells by 15–35% in a dose-dependent fashion, suggesting signaling through protein kinase A (PKA). In line with this view, the PKA inhibitor H89 abrogated the protective effects of PTHrP-(1–34) and PTHrP-(140–173) against caspase activation and DNA loss. PKA activation by forskolin, 3-isobutyl-1-methylxanthine (IBMX), or 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate attenuated and H89 augmented apoptosis after UV exposure as indicated by caspase-3 activation, cell DNA loss, and morphological criteria. Studies with IBMX and varying doses of forskolin indicated that small increases in cAMP, on the order of those generated by IBMX alone and the PTHrP peptides, were sufficient to protect lung cancer cells from apoptosis. In summary, PTHrP-(1–34) and PTHrP-(140–173) stimulate PKA in lung carcinoma cells and protect cells against UV-induced caspase-3 activation and DNA fragmentation. PKA activation by other means also induces resistance to apoptosis, and the protective effect of the PTHrP peptide is blocked by PKA inhibition. Thus PKA appears to have a role in the regulatory effects of PTHrP on lung cancer cell survival. caspases; cell surface receptors; growth substances; signal transduction