Nuclear PTHrP targeting regulates PTHrP secretion and enhances LoVo cell growth and survival

Parathyroid hormone-related protein (PTHrP) is expressed by human colon cancer tissue and cell lines; expression correlates with colon carcinoma severity. PTHrP is synthesized as a prepro isoform and contains two targeting sequences — a signal sequence and a nuclear localization signal (NLS). The signal peptide (SP) directs PTHrP to the secretory pathway, where it exerts autocrine/paracrine effects. The NLS directs PTHrP to the nucleus/nucleolus, where it exerts intracrine effects. In this study, we used the human colon cancer cell line LoVo as a model system to study the effects of autocrine/paracrine and intracrine PTHrP action on cell growth and survival, hallmarks of malignant tumor cells. We report that PTHrP increases cell growth and survival, protects cells from serum-starvation-induced apoptosis, and promotes anchorage-independent cell growth via an intracrine pathway. Conversely, autocrine/paracrine PTHrP action decreases cell growth and survival. We also show an inverse relationship between secreted and nuclear PTHrP levels, in that cells overexpressing NLS-deleted PTHrP secrete higher PTHrP levels than those overexpressing the wild-type isoform. Conversely, SP deletion results in higher nuclear PTHrP levels. These observations provide evidence of a link between intracrine PTHrP action and cell growth and survival. Targeting PTHrP production in colon cancer may thus prove therapeutically beneficial.     

PTHrP, PTH, and the PTH/PTHrP receptor in endochondral bone development

Endochondral bone development is a fascinating story of proliferation, maturation, and death. An understanding of this process at the molecular level is emerging. In particular, significant advances have been made in understanding the role of parathyroid-hormone-related peptide (PTHrP), parathyroid hormone (PTH), and the PTH/PTHrP receptor in endochondral bone development. Mutations of the PTH/PTHrP receptor have been identified in Jansen metaphyseal chondrodysplasia, Blomstrand's lethal chondrodysplasia, and enchondromatosis. Furthermore, genetic manipulations of the PTHrP, PTH, and the PTH/PTHrP receptor genes, respectively, have demonstrated the critical role of these proteins in regulating both the switch between proliferation and differentiation of chondrocytes, and their replacement by bone cells. A future area of investigation will be the identification of downstream effectors of PTH, PTHrP, and PTH/PTHrP receptor activities. Furthermore, it will be of critical importance to study how these proteins cooperate and integrate with other molecules that are essential for growth plate development.     

Expression of PTHrP and the PTH/PTHrP receptor in growing red deer antler

Antler growth is highly co-ordinated, so that trabecular bone and antler skin (velvet) develop together, at a rapid rate and in a manner reminiscent of their development in the fetus. Parathyroid hormone-related peptide (PTHrP) is expressed in both bone and skin, and is therefore a candidate to effect co-ordination between these tissues. The aim of this study was to localize the expression of PTHrP and its principal receptor, the parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrPR), in antler ("spiker") of one-year-old red deer. Using immunohistochemistry and in situ hybridization, intense and overlapping expression of PTHrP and its receptor was seen in developing osseocartilaginous structures and in the underlying layers of velvet epidermis. PTHrP was located on both the cell surface and within the nuclei. Our results strongly suggest that PTHrP, acting via the PTH/PTHrPR and possibly other intracrine mechanisms, plays a central role in the co-ordinated regulation of cell division and differentiation of developing antler bone and skin.     

PTH and PTHrP signaling in osteoblasts

The striking clinical benefit of PTH in osteoporosis began a new era of skeletal anabolic agents. Several studies have been performed, new studies are emerging out and yet controversies remain on PTH anabolic action in bone. This review focuses on the molecular aspects of PTH and PTHrP signaling in light of old players and recent advances in understanding the control of osteoblast proliferation, differentiation and function.     

PTHrP and cytokeratins in human epidermis

We have demonstrated the presence of parathyroid hormone-related peptide (PTHrP) in cells of human epidermis, employing immunocytochemical techniques. Cells of human epidermal layers demonstrated variable intensity of the reaction. The least pronounced reaction was detected in cells of the basal and the most pronounced reaction in cells of the granular layer. Ultrastructural studies demonstrated that gold particles labeled bundles of keratin filaments. Therefore, at the subsequent stage of the studies we examined the type of filaments to which PTHrP was bound, using immunocytochemical reactions with antibodies against cytokeratins 10, 14, 16 and 19. Positive reaction was obtained for cytokeratins 10, 14 and 16. The reaction pattern obtained for cytokeratins 10 and 16 most closely resembled that of PTHrP. Double labeling with colloidal gold was performed at the ultrastructural level. The results obtained in this way demonstrated that PTHrP most probably binds to filaments built of cytokeratin 16. By binding to the cytokeratin, PTHrP may possibly affect growth and differentiation of keratinocytes.     

Parathyroid hormone-related peptide (PTHrP)

PTHrP, which causes humoral malignant hypercalcaemia in man and animals, acts on bone and kidney in a way similar to that of parathyroid hormone. PTHrP released by fetal parathyroid glands stimulates placental calcium transport in pregnant ewes and maintains the calcium gradient from the dam to its foetus. PTHrP, which is also present in the mammary gland, colostrum and milk, might play an important physiological role in regulating calcium secretion through milk and calcium metabolism in newborn animals.     

Estrogen stimulates PTHrP but not PTH/PTHrP receptor gene expression in the kidney of ovariectomized rat

The aim of the present study was to test the hypothesis that the decreased renal tubular reabsorption of calcium observed in estrogen deficiency is associated with a local regulation of either PTHrP or PTH/PTHrP receptor genes in the kidney. Rats were randomly sham-operated (S) or ovariectomized receiving either vehicule (OVX) or 4 μg E2/kg/day (OVX+E4) or 40 μg E2/kg/d (OVX+E40) during 14 days using alzet minipumps. Plasma PTH and calcium levels were lower in untreated OVX animals than in all other groups (P < 0.01). Plasma PTH was higher in OVX+E40 than in OVX+E4 (P < 0.05). PTHrP mRNA expression in the kidney was unaffected by ovariectomy but was increased in OVX+E40 (0.984 ± 0.452 for PTHrP/GAPDH mRNAs expression vs. 0.213 ± 0.078 in sham, P < 0.01). PTH/PTHrP receptor mRNA expression and the cAMP response of renal membranes to PTH were unaffected by ovariectomy and estrogen substitution. In conclusion, renal PTHrP and PTH/PTHrP receptor mRNAs are not modified by ovariectomy. However, 17β-estradiol increases renal expression of PTHrP mRNA without evident changes in its receptor expression and function. This may help to explain the pharmacological action of estrogen in the kidney, especially how it prevents the renal leak of calcium in postmenopausal women. J. Cell. Biochem. 70:84–93, 1998. © 1998 Wiley-Liss, Inc.     

Coexpression of PTHrP and PTH/PTHrP receptor in a myoepithelial cell line derived from normal human breast

Parathyroid hormone-related peptide (PTHrP) is the cause of humoral hipercalcaemia of malignancy syndrome (HHM). It is known that the peptide as well as its receptors are widely distributed in many normal organs and tissues, where it influences an array of diverse functions which are realized through paracrine or autocrine pathway. PTHrP is present in large amounts in lactating mammary gland but its function is not fully elucidated. In this study, production of parathyroid hormone-related peptide (PTHrP) by the Hs578Bst cell line corresponding to mammary myoepithelial cells was examined by immunocytochemistry. Using RNA extracted from these cells we analyzed expression of mRNA for PTHrP and for the PTH/PTHrP receptor by RT-PCR. The obtained results demonstrated that Hs578Bst cells produced PTHrP and synthesized mRNA for PTHrP and PTH/PTHrP type I receptor. It provides evidence that myoepithelial cells are target cells for PTHrP. The data support that PTHrP may be an important autocrine/paracrine factor, involved in the regulation of myoepithelial cell function as well as in growth and differentiation of the mammary gland.     

Deletion of the Nuclear Localization Sequences and C-Terminus of PTHrP Impairs Embryonic Mammary Development but also Inhibits PTHrP Production

Parathyroid hormone-related protein (PTHrP) can be secreted from cells and interact with its receptor, the Type 1 PTH/PTHrP Receptor (PTHR1) in an autocrine, paracrine or endocrine fashion. PTHrP can also remain inside cells and be transported into the nucleus, where its functions are unclear, although recent experiments suggest that it may broadly regulate cell survival and senescence. Disruption of either the PTHrP or PTHR1 gene results in many abnormalities including a failure of embryonic mammary gland development in mice and in humans. In order to examine the potential functions of nuclear PTHrP in the breast, we examined mammary gland development in PTHrP (1–84) knock-in mice, which express a mutant form of PTHrP that lacks the C-terminus and nuclear localization signals and which can be secreted but cannot enter the nucleus. Interestingly, we found that PTHrP (1–84) knock-in mice had defects in mammary mesenchyme differentiation and mammary duct outgrowth that were nearly identical to those previously described in PTHrP^−/− and PTHR1^−/− mice. However, the mammary buds in PTHrP (1–84) knock-in mice had severe reductions in mutant PTHrP mRNA levels, suggesting that the developmental defects were due to insufficient production of PTHrP by mammary epithelial cells and not loss of PTHrP nuclear function. Examination of the effects of nuclear PTHrP in the mammary gland in vivo will require the development of alternative animal models.     

Parathyroid hormone-related peptide (PTHrP) induces parietal endoderm formation exclusively via the type I PTH/PTHrP receptor.

A number of studies suggest a role for PTHrP and the classical PTH/PTHrP receptor (type I) in one of the first differentiation processes in mouse embryogenesis, i.e. the formation of parietal endoderm (PE). We previously reported that although in type I receptor (-/-) embryos PE formation seemed normal, the embryos were smaller from at least day 9.5 p.c. and 60% had died before day 12.5 p.c. Here we show that the observed growth defect commences even earlier, at day 8.5 p.c. Using two novel antibodies, we show that the expression of the type I receptor protein at this stage is confined to extraembryonic endoderm only. In addition, we show that large amounts of PTHrP protein are present in the adjacent trophoblast giant cells, suggesting a paracrine interaction of PTHrP and the type I PTH/PTHrP receptor in PE formation. The involvement in PE differentiation of other recently described receptors for PTHrP would explain a possible redundancy for the type I receptor in PE formation. However, deletion of the type I PTH/PTHrP receptor in ES cells by homologous recombination completely prevents PTHrP-induced PE differentiation. Based upon these observations, we propose that PTHrP and the type I PTH/PTHrP receptor, although not required for the initial formation of PE, are required for its proper differentiation and/or functioning.     

Altered selectivity of parathyroid hormone (PTH) and PTH-related protein (PTHrP) for distinct conformations of the PTH/PTHrP receptor

PTH and PTHrP use the same G protein-coupled receptor, the PTH/PTHrP receptor (PTHR), to mediate their distinct biological actions. The extent to which the mechanisms by which the two ligands bind to the PTHR differ is unclear. We examined this question using several pharmacological and biophysical approaches. Kinetic dissociation and equilibrium binding assays revealed that the binding of [(125)I]PTHrP(1-36) to the PTHR was more sensitive to GTPgammaS (added to functionally uncouple PTHR-G protein complexes) than was the binding of [(125)I]PTH(1-34) ( approximately 75% maximal inhibition vs. approximately 20%). Fluorescence resonance energy transfer-based kinetic analyses revealed that PTHrP(1-36) bound to the PTHR more slowly and dissociated from it more rapidly than did PTH(1-34). The cAMP signaling response capacity of PTHrP(1-36) in cells decayed more rapidly than did that of PTH(1-34) (t(1/2) = approximately 1 vs. approximately 2 h). Divergent residue 5 in the ligand, Ile in PTH and His in PTHrP, was identified as a key determinant of the altered receptor-interaction responses exhibited by the two peptides. We conclude that whereas PTH and PTHrP bind similarly to the G protein-coupled PTHR conformation (RG), PTH has a greater capacity to bind to the G protein-uncoupled conformation (R(0)) and, hence, can produce cumulatively greater signaling responses (via R(0)-->RG isomerization) than can PTHrP. Such conformational selectivity may relate to the distinct modes by which PTH and PTHrP act biologically, endocrine vs. paracrine, and may help explain reported differences in the effects that the ligands have on calcium and bone metabolism when administered to humans.     

Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy

Chondrocyte hypertrophy is an essential process required for endochondral bone formation. Proper regulation of chondrocyte hypertrophy is also required in postnatal cartilage homeostasis. Indian hedgehog (Ihh) and PTHrP signaling play crucial roles in regulating the onset of chondrocyte hypertrophy by forming a negative feedback loop, in which Ihh signaling regulates chondrocyte hypertrophy by controlling PTHrP expression. To understand whether there is a PTHrP-independent role of Ihh signaling in regulating chondrocyte hypertrophy, we have both activated and inactivated Ihh signaling in the absence of PTHrP during endochondral skeletal development. We found that upregulating Ihh signaling in the developing cartilage by treating PTHrP(-/-) limb explants with sonic hedgehog (Shh) protein in vitro, or overexpressing Ihh in the cartilage of PTHrP(-/-) embryos or inactivating patched 1 (Ptch1), a negative regulator of hedgehog (Hh) signaling, accelerated chondrocyte hypertrophy in the PTHrP(-/-) embryos. Conversely, when Hh signaling was blocked by cyclopamine or by removing Smoothened (Smo), a positive regulator of Hh signaling, chondrocyte hypertrophy was delayed in the PTHrP(-/-) embryo. Furthermore, we show that upregulated Hh signaling in the postnatal cartilage led to accelerated chondrocyte hypertrophy during secondary ossification, which in turn caused reduction of joint cartilage. Our results revealed a novel role of Ihh signaling in promoting chondrocyte hypertrophy independently of PTHrP, which is particularly important in postnatal cartilage development and homeostasis. In addition, we found that bone morphogenetic protein (Bmp) and Wnt/beta-catenin signaling in the cartilage may both mediate the effect of upregulated Ihh signaling in promoting chondrocyte hypertrophy.     

Immunolocalization of PTHrP in the sublingual glands of three mammals

The present study deals with immunohistochemical localization of PTHrP in sublingual glands of white mouse, bank vole, and common vole. PTHrP immunoreactivity was observed in epithelial cells of striated, interlobular and main excretory ducts of the salivary glands in all the three animal species tested. However, we found no positive reaction for PTHrP in epithelial cells of the intercalated ducts. In striated duct cells, the reaction intensity was species-dependent. In bank vole and common vole, the reaction was very strong, while in white mouse very weak. In the remaining segments of excretory ducts (interlobular and main excretory duct) we found no species-related differences in the reaction intensity or character. Myoepithelial cells surrounding ducts and mucous tubules with serous demilunes in sublingual glands were also PTHrP-negative in all the three animal species tested.