Andrew C. Karaplis

PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth

The PTH/PTHrP receptor binds to two ligands with distinct functions: the calcium-regulating hormone, parathyroid hormone (PTH), and the paracrine factor, PTH-related protein (PTHrP). Each ligand, in turn, is likely to activate more than one receptor. The functions of the PTH/PTHrP receptor were investigated by deletion of the murine gene by homologous recombination. Most PTH/PTHrP receptor (−/−) mutant mice died in mid-gestation, a phenotype not observed in PTHrP (−/−) mice, perhaps because of the effects of maternal PTHrP. Mice that survived exhibited accelerated differentiation of chondrocytes in bone, and their bones, grown in explant culture, were resistant to the effects of PTHrP and Sonic hedgehog. These results suggest that the PTH/PTHrP receptor mediates the effects of Indian Hedgehog and PTHrP on chondrocyte differentiation.

Nucleolar localization of parathyroid hormone-related peptide enhances survival of chondrocytes under conditions that promote apoptotic cell death.

Parathyroid hormone-related peptide (PTHrP) is a mediator of cellular growth and differentiation as well as a cause of malignancy-induced hypercalcemia. Most of the actions of PTHrP have been attributed to its interaction with a specific cell surface receptor that binds the N-terminal domain of the protein. Here we present evidence that PTHrP promotes some of its cellular effects by translocating to the nucleolus. Localization of transiently expressed PTHrP to the nucleolus was dependent on the presence of a highly basic region at the carboxyl terminus of the molecule that bears homology to nucleolar targeting sequences identified within human retroviral (human immunodeficiency virus type 1 and human T-cell leukemia virus type 1) regulatory proteins. Endogenous PTHrP also localized to the nucleolus in osseous cells in vitro and in vivo. Moreover, expression of PTHrP in chondrocytic cells (CFK2) delayed apoptosis induced by serum deprivation, and this effect depended on the presence of an intact nucleolar targeting signal. The present findings demonstrate a unique intracellular mode of PTHrP action and a novel mechanism by which this peptide growth factor may modulate programmed cell death.

Osteoblast-derived PTHrP is a potent endogenous bone anabolic agent that modifies the therapeutic efficacy of administered PTH 1–34

Mice heterozygous for targeted disruption of Pthrp exhibit, by 3 months of age, diminished bone volume and skeletal microarchitectural changes indicative of advanced osteoporosis. Impaired bone formation arising from decreased BM precursor cell recruitment and increased apoptotic death of osteoblastic cells was identified as the underlying mechanism for low bone mass. The osteoporotic phenotype was recapitulated in mice with osteoblast-specific targeted disruption of Pthrp, generated using Cre-LoxP technology, and defective bone formation was reaffirmed as the underlying etiology. Daily administration of the 1-34 amino-terminal fragment of parathyroid hormone (PTH 1-34) to Pthrp+/- mice resulted in profound improvement in all parameters of skeletal microarchitecture, surpassing the improvement observed in treated WT littermates. These findings establish a pivotal role for osteoblast-derived PTH-related protein (PTHrP) as a potent endogenous bone anabolic factor that potentiates bone formation by altering osteoblast recruitment and survival and whose level of expression in the bone microenvironment influences the therapeutic efficacy of exogenous PTH 1-34.

Parathyroid hormone is essential for normal fetal bone formation

Parathyroid hormone (PTH) is a potent pharmacologic inducer of new bone formation, but no physiologic anabolic effect of PTH on adult bone has been described. We investigated the role of PTH in fetal skeletal development by comparing newborn mice lacking either PTH, PTH-related peptide (PTHrP), or both peptides. PTH-deficient mice were dysmorphic but viable, whereas mice lacking PTHrP died at birth with dyschondroplasia. PTH-deficient mice uniquely demonstrated diminished cartilage matrix mineralization, decreased neovascularization with reduced expression of angiopoietin-1, and reduced metaphyseal osteoblasts and trabecular bone. Compound mutants displayed the combined cartilaginous and osseous defects of both single mutants. These results indicate that coordinated action of both PTH and PTHrP are required to achieve normal fetal skeletal morphogenesis, and they demonstrate an essential function for PTH at the cartilage-bone interface. The effect of PTH on fetal osteoblasts may be relevant to its postnatal anabolic effects on trabecular bone.

The calcium-sensing receptor is required for normal calcium homeostasis independent of parathyroid hormone

The extracellular calcium-sensing receptor (CaR; alternate gene names, CaR or Casr) is a membrane-spanning G protein-coupled receptor. CaR is highly expressed in the parathyroid gland, and is activated by extracellular calcium (Ca(2+)(o)). Mice homozygous for null mutations in the CaR gene (CaR(-/-)) die shortly after birth because of the effects of severe hyperparathyroidism and hypercalcemia. A wide variety of functions have been attributed to CaR. However, the lethal CaR-deficient phenotype has made it difficult to dissect the direct effect of CaR deficiency from the secondary effects of hyperparathyroidism and hypercalcemia. We therefore generated parathyroid hormone-deficient (PTH-deficient) CaR(-/-) mice (Pth(-/-)CaR(-/-)) by intercrossing mice heterozygous for the null CaR allele with mice heterozygous for a null Pth allele. We show that genetic ablation of PTH is sufficient to rescue the lethal CaR(-/-) phenotype. Pth(-/-)CaR(-/-) mice survive to adulthood with no obvious difference in size or appearance relative to control Pth(-/-) littermates. Histologic examination of most organs did not reveal abnormalities. These Pth(-/-)CaR(-/-) mice exhibit a much wider range of values for serum calcium and renal excretion of calcium than we observe in control littermates, despite the absence of any circulating PTH. Thus, CaR is necessary for the fine regulation of serum calcium levels and renal calcium excretion independent of its effect on PTH secretion.

Parathyroid hormone‐related protein and its receptors: nuclear functions and roles in the renal and cardiovascular systems, the placental trophoblasts and the pancreatic islets

The cloning of the so‐called ‘parathyroid hormone‐related protein’ (PTHrP) in 1987 was the result of a long quest for the factor which, by mimicking the actions of PTH in bone and kidney, is responsible for the hypercalcemic paraneoplastic syndrome, humoral calcemia of malignancy. PTHrP is distinct from PTH in a number of ways. First, PTHrP is the product of a separate gene. Second, with the exception of a short N‐terminal region, the structure of PTHrP is not closely related to that of PTH. Third, in contrast to PTH, PTHrP is a paracrine factor expressed throughout the body. Finally, most of the functions of PTHrP have nothing in common with those of PTH. PTHrP is a poly‐hormone which comprises a family of distinct peptide hormones arising from post‐translational endoproteolytic cleavage of the initial PTHrP translation products. Mature N‐terminal, mid‐region and C‐terminal secretory forms of PTHrP are thus generated, each of them having their own physiologic functions and probably their own receptors. The type 1 PTHrP receptor, binding both PTH(1‐34) and PTHrP(1‐36), is the only cloned receptor so far. PTHrP is a PTH‐like calciotropic hormone, a myorelaxant, a growth factor and a developmental regulatory molecule. The present review reports recent aspects of PTHrP pharmacology and physiology, including: (a) the identification of new peptides and receptors of the PTH/PTHrP system; (b) the recently discovered nuclear functions of PTHrP and the role of PTHrP as an intracrine regulator of cell growth and cell death; (c) the physiological and developmental actions of PTHrP in the cardiovascular and the renal glomerulo‐vascular systems; (d) the role of PTHrP as a regulator of pancreatic beta cell growth and functions, and, (e) the interactions of PTHrP and calcium‐sensing receptors for the control of the growth of placental trophoblasts. These new advances have contributed to a better understanding of the pathophysiological role of PTHrP, and will help to identify its therapeutic potential in a number of diseases.

Cloning of Human PEX cDNA EXPRESSION, SUBCELLULAR LOCALIZATION, AND ENDOPEPTIDASE ACTIVITY

Mutations in the PEX gene are responsible for X-linked hypophosphatemic rickets. To gain insight into the role of PEX in normal physiology we have cloned the human full-length cDNA and studied its tissue expression, subcellular localization, and peptidase activity. We show that the cDNA encodes a 749-amino acid protein structurally related to a family of neutral endopeptidases that include neprilysin as prototype. By Northern blot analysis, the size of the full-length PEXtranscript is 6.5 kilobases. PEX expression, as determined by semi-quantitative polymerase chain reaction, is high in bone and in tumor tissue associated with the paraneoplastic syndrome of renal phosphate wasting. PEX is glycosylated in the presence of canine microsomal membranes and partitions exclusively in the detergent phase from Triton X-114 extractions of transiently transfected COS cells. Immunofluorescence studies in A293 cells expressing PEX tagged with a c-myc epitope show a predominant cell-surface location for the protein with its COOH-terminal domain in the extracellular compartment, substantiating the assumption that PEX, like other members of the neutral endopeptidase family, is a type II integral membrane glycoprotein. Cell membranes from cultured COS cells transiently expressing PEX efficiently degrade exogenously added parathyroid hormone-derived peptides, demonstrating for the first time that recombinant PEX can function as an endopeptidase. PEX peptidase activity may provide a convenient target for pharmacological intervention in states of altered phosphate homeostasis and in metabolic bone diseases.

Mammary-specific deletion of parathyroid hormone-related protein preserves bone mass during lactation

Large amounts of calcium are transferred to offspring by milk. This demand results in negative calcium balance in lactating mothers and is associated with rapid bone loss. The mechanisms of bone loss during lactation are only partly understood. Several studies have suggested that parathyroid hormone-related protein (PTHrP) might be secreted into the circulation by the lactating mammary gland and regulate bone turnover during lactation. Because mammary development fails in the absence of PTHrP, conventional PTHrP knockout mice cannot be used to address this possibility. To examine this hypothesis, we therefore used mice carrying a beta-lactoglobulin promoter-driven Cre transgene, one null PTHrP allele, and one floxed PTHrP allele. Expression of Cre specifically in mammary epithelial cells during late pregnancy and lactation resulted in efficient deletion of the PTHrP gene; mammary gland PTHrP mRNA and milk PTHrP protein were almost completely absent. Removal of PTHrP from the lactating mammary glands resulted in reductions in levels of circulating PTHrP and 1,25-dihydroxy vitamin D and urinary cAMP. In addition, bone turnover was reduced and bone loss during lactation was attenuated. We conclude that during lactation mammary epithelial cells are a source of circulating PTHrP that promotes bone loss by increasing rates of bone resorption.

Severe growth retardation and early lethality in mice lacking the nuclear localization sequence and C-terminus of PTH-related protein

Parathyroid hormone (PTH) plays a central role in the regulation of serum calcium and phosphorus homeostasis, while parathyroid hormone-related protein (PTHrP) has important developmental roles. Both peptides signal through the same G protein-coupled receptor, the PTH/PTHrP or PTH type 1 receptor (PTH1R). PTHrP, normally a secreted protein, also contains a nuclear localization signal (NLS) that in vitro imparts functionality to the protein at the level of the nucleus. We investigated this functionality in vivo by introducing a premature termination codon in Pthrp in ES cells and generating mice that express PTHrP (1–84), a truncated form of the protein that is missing the NLS and the C-terminal region of the protein but can still signal through its cell surface receptor. Mice homozygous for the knock-in mutation (Pthrp KI) displayed retarded growth, early senescence, and malnutrition leading postnatally to their rapid demise. Decreased cellular proliferative capacity and increased apoptosis in multiple tissues including bone and bone marrow cells were associated with altered expression and subcellular distribution of the senescence-associated tumor suppressor proteins p16INK4a and p21 and the oncogenes Cyclin D, pRb, and Bmi-1. These findings provide in vivo experimental proof that substantiates the biologic relevance of the NLS and C-terminal portion of PTHrP, a polypeptide ligand that signals mainly via a cell surface G protein-coupled receptor.

PTHrP drives breast tumor initiation, progression, and metastasis in mice and is a potential therapy target

Parathyroid hormone-related protein (PTHrP) is a secreted factor expressed in almost all normal fetal and adult tissues. It is involved in a wide range of developmental and physiological processes, including serum calcium regulation. PTHrP is also associated with the progression of skeletal metastases, and its dysregulated expression in advanced cancers causes malignancy-associated hypercalcemia. Although PTHrP is frequently expressed by breast tumors and other solid cancers, its effects on tumor progression are unclear. Here, we demonstrate in mice pleiotropic involvement of PTHrP in key steps of breast cancer - it influences the initiation and progression of primary tumors and metastases. Pthrp ablation in the mammary epithelium of the PyMT-MMTV breast cancer mouse model caused a delay in primary tumor initiation, inhibited tumor progression, and reduced metastasis to distal sites. Mechanistically, it reduced expression of molecular markers of cell proliferation (Ki67) and angiogenesis (factor VIII), antiapoptotic factor Bcl-2, cell-cycle progression regulator cyclin D1, and survival factor AKT1. PTHrP also influenced expression of the adhesion factor CXCR4, and coexpression of PTHrP and CXCR4 was crucial for metastatic spread. Importantly, PTHrP-specific neutralizing antibodies slowed the progression and metastasis of human breast cancer xenografts. Our data identify what we believe to be new functions for PTHrP in several key steps of breast cancer and suggest that PTHrP may constitute a novel target for therapeutic intervention.