Parathyroid Hormone-Related Protein: A Developmental Regulatory Molecule Necessary for Mammary Gland Development

Parathyroid hormone-related protein (PTHrP) wasoriginally identified as the tumor factor responsiblefor a clinical syndrome known as humoral hypercalcemiaof malignancy. It is now appreciated that PTHrP3 is a developmental regulatory moleculeexpressed during the formation of a wide variety oforgans. Recently, our laboratory has demonstrated thatPTHrP is necessary for mammary gland development. Ourstudies have suggested that this molecule participatesin the regulation of epithelial-mesenchymal interactionsduring embryonic mammary development and perhaps alsoduring adolescent ductal morphogenesis. In addition, it has been suggested that PTHrP plays acritical role in the establishment of bone metastases inbreast cancer. In this article, we will discuss thecurrent knowledge of the mechanisms underlying PTHrPs actions during normal mammary development andin breast cancer.     

Parathyroid Hormone and Parathyroid Hormone-Related Protein Analogs as Therapies for Osteoporosis

Osteoporotic fractures result in significant morbidity and mortality. Anabolic agents reverse the negative skeletal balance that characterizes osteoporosis by stimulating osteoblast-dependent bone formation to a greater degree than osteoclast-dependent bone resorption. Parathyroid hormone (PTH) and parathyroid hormone- related protein (PTHrP) are peptide hormones, which have anabolic actions when administered intermittently. The only FDA-approved anabolic bone agent for the treatment of osteoporosis in the United States is PTH 1-34, or teriparatide, administered by daily subcutaneous injections. However, PTH 1-84 is also available in Europe. Synthetic human PTHrP 1-36 and a PTHrP 1-34 analog, BA058, have also been shown to increase lumbar spine bone density. These agents and several other PTH and PTHrP analogs, including some which are not administered as injections, continue to be investigated as potential anabolic therapies for osteoporosis.     

Parathyroid Hormone-Related Protein and its Receptor in Human Glial Tumors

Summary  Objective. Parathyroid hormone-related protein (PTHrP) and its mRNA have been found to be expressed in a variety of human tumors including breast, prostate, colon, lung, renal and ovarian cancers. The purpose of this study is to evaluate the expression of PTH/PTHrP receptor and ligand in human glial tumors.  Methods. We examined the coexpression of PTH/PTHrP receptor and ligand in 73 glial tumors of different histological grades and 4 nonneoplastic human brain specimens and three glioblastoma cell lines, by using Western Blot analysis and immunohistochemical analysis.  Results. PTHrP and PTH/PTHrP receptors were shown in the neurons, reactive astrocytes and the endothelial cells of normal brain tissue as well as tumor cells, reactive astrocytes and vasculature of nonneoplastic tissue. They were expressed at higher levels in pure astrocytic tumors as compared to tumors with oligodendroglial components.  Conclusion. PTH/PTHrP receptor and PTHrP ligand are co-expressed in human glial tumors. There increased expression suggests an autocrine and/or paracrine loop may exist.     

p21 and Parathyroid Hormone-Related Peptide in the Growth Plate

It is essential for terminal chondrocytes to die before the conversion of calcified cartilage to bone. We have previously demonstrated that apoptosis occurred in the terminal hypertrophic chondrocyte of the growth plate. However, the essential mechanism by which the differentiation of chondrocytes is regulated has not yet been characterized. The purpose of this study was to investigate the mechanism for regulating chondrocyte differentiation. We focused on PTHrP and p21 which regulated the differentiation of chondrocytes and investigated how these factors interacted with each other in chondrocyte differentiation in the growth plate. PTHrP was strongly positive on immunostaining at the interface between the proliferating and the upper zone of the hypertrophic chondrocytes, whereas p21 was negative. On the other hand, p21 was positive in the lower zone of hypertrophic chondrocytes. Furthermore, PTHrP up-regulated the cell proliferation and down-regulated the expression of the p21 messengers in SW-1353 chondrosarcoma cells. These findings indicated that PTHrP might be a negative regulator for p21 in the differentiation of chondrocytes. Received: 1 March 2000 / Accepted: 25 May 2000 / Online publication: 2 November 2000     

Parathyroid Hormone-Related Protein (1–37) Induces cAMP Response in Human Osteoblast-Like Cells

During recent years parathyroid hormone-related protein (PTHrP) research has been focused on the physiological functions of different fragments of the PTHrP molecule. Here we demonstrate that PTHrP (1–37) induced cyclic adenosine monophosphate (cAMP) response in primary human osteoblast-like cells, which were well characterized by the presence of alkaline phosphatase activity and osteocalcin production after stimulation with 1,25-dihydroxyvitamin D₃. However, there was no cAMP response to PTHrP (58–77). Furthermore, the response to PTHrP (1–37) was dose dependent, with a significant increase at 1 nM. The presence of PTHrP (1–37)-induced cAMP response in human osteoblast-like cells implies that aminoterminal PTHrP fragments may exert important functions in the bone. Received: 27 January 1997 / Accepted: 26 June 1997     

Prostate Specific Antigen Cleaves Parathyroid Hormone-Related Protein in the PTH-like Domain: Inactivation of PTHrP-Stimulated cAMP Accumulation in Mouse Osteoblasts

Purpose

To determine whether parathyroid hormone-related protein (PTHrP) is a substrate of prostate-specific antigen (PSA) and how the biological activity of PTHrP may be altered by cleavage with PSA.

Materials and Methods

Prostate-specific antigen cleavage of recombinant human PTHrP 1-141 was conducted in vitro at 37C and analyzed by SDS-PAGE. Five rounds of automated amino-terminal amino acid sequence analysis were performed on blotted PSA-cleaved PTHrP peptide fragments to determine the PSA cleavage sites. The mouse osteoblast cell line MC3T3-E1 was used to test whether PSA cleavage of PTHrP 1-141 altered its ability to stimulate cAMP production.

Results

Prostate-specific antigen was found to specifically cleave PTHrP 1-141 in a time- and dose-dependent manner. Cleavage of PTHrP 1-141 by PSA generated fragments on Coomassiestained acrylamide gels that migrated with mobilities that corresponded to 19.5, 17, 15 and less than 7 kd. The preferred PSA cleavage site of PTHrP 1-141 was determined to be at the carboxyl-terminus of phenylalanine 23, consistent with chymotryptic-like enzymatic activity of PSA. Cleavage of PTHrP by PSA completely abolished the ability of PTHrP to stimulate cAMP production.

Conclusions

Cleavage of PTHrP 1-141 by PSA carboxyl-terminal to phenylalanine 23 represents a unique pattern of PTHrP processing that may be specific to the prostate. Prostate-specific antigen inactivation of the cAMP-inducing activity of PTHrP 1-141 demonstrates that PSA cleavage regulates the biological activity of PTHrP. These results have implications for the role of PTHrP in prostate cancer metastasis to bone and its subsequent regulation of bone remodeling Study of the biological activities of the PSA-generated PTHrP peptides identified in this study merits further investigation.     

Wnt-Signalling in the Embryonic Mammary Gland

The first member of the Wnt-family ligands was identified 30 years ago as a factor in mouse mammary tumours whose expression was deregulated due to the promoter activity emanating from the proximal integration of the Mouse Mammary Tumour Virus genome (Nusse and Varmus, Embo J 31:2670–84, 2012). The Wnt-ligands invoke a number of molecular-genetic signalling cascades fundamental to the patterning of developing tissues and organs during embryogenesis as well as during postnatal development. The Wnt-signalling cascade that controls the activities of β-catenin and the T-cell Factor (Tcf)/Lympoid enhancer factor (Lef1) plays a fundamental role in control of all stages of embryonic mammary gland development. We provide here a brief overview of the known aspects of Wnt-signalling activities in the embryonic mammary gland and its interactions with other signalling cascades in this developing tissue.     

Neuregulin 3 and Erbb Signalling Networks in Embryonic Mammary Gland Development

We review the role of Neuregulin 3 (Nrg3) and Erbb receptor signalling in embryonic mammary gland development. Neuregulins are growth factors that bind and activate its cognate Erbb receptor tyrosine kinases, which form a signalling network with established roles in breast development and breast cancer. Studies have shown that Nrg3 expression profoundly impacts early stages of embryonic mammary development. Network analysis shows how Nrg/Erbb signals could integrate with other major regulators of embryonic mammary development to elicit the morphogenetic processes and cell fate decisions that occur as the mammary lineage is established.     

Parathyroid Hormone-Related Protein (107-139) Decreases Alkaline Phosphatase in Osteoblastic Osteosarcoma Cells UMR 106 by a Protein Kinase C-Dependent Pathway

The C-terminal (107-111) region of parathyroid hormone-related protein (PTHrP) appears to inhibit osteoclastic bone resorption, and to affect osteoblastic growth and differentiation. We tested the effect of human PTHrP (107-139) on alkaline phosphatase (ALP) activity in osteoblastic osteosarcoma UMR 106 cells. We found that this C-terminal PTHrP peptide, between 10 nM and 10 fM, inhibited ALP activity in these cells during the log phase of growth. Human PTHrP (1-34) amide and human PTHrP (1-141) were as potent as PTHrP (107-139) in growing UMR 106 cells. This inhibitory effect of 10 nM PTHrP (107-139) on ALP activity was also observed in serum-depleted cells, and in the presence of 10 nM dexamethasone, which increased ALP activity by 40% in these cells. In addition, this effect of PTHrP (107-139) was blunted by 25 nM bisindolylmaleimide I, a protein kinase C inhibitor. These results support a role for the C-terminal region of PTHrP as a modulator of bone formation. Received: 7 July 1998 / Accepted: 10 January 1999     

Pleiotropic Functions of Fibroblast Growth Factor Signaling in Embryonic Mammary Gland Development

The mammary gland is an ectodermal appendage and a defining feature of mammals. Consistent with it being a recent evolutionary novelty, many of the molecules essential for the ontogeny and morphogenesis of various vertebrate organs, including those in the fibroblast growth factor (FGF) signaling pathway, are co-opted for induction, maintenance and morphogenesis of the mammary glands. Understanding the mechanism whereby FGF signaling regulates the fundamental cell behavior during normal mammary gland develop may facilitate determination of the consequences of its deregulation during breast cancer progression.     

Investigating Molecular Mechanisms of Embryonic Mammary Gland Development by Bead-Implantation in Embryonic Flank Explant Cultures – A Protocol

The involvement of molecular mechanisms in a particular process such as embryonic mammary gland development, can be revealed by modulation of one or several factors that purportedly act in that process. If those factors or their inhibitors are soluble, their function can be tested by loading them onto small inert beads, which are then implanted in cultured explants of the tissue of interest, in this case embryonic flanks. We here describe a protocol for such experiments.     

A Crucial Role for Fibroblast Growth Factor Signaling in Embryonic Mammary Gland Development

The fibroblast growth factors (Fgfs) represent a large group of intercellular signaling molecules that mediate their effects by binding to a class of cell surface enzymes belonging to the receptor tyrosine kinase family (FgfRs). In vitro, Fgf signaling can induce potent mitogenic, motogenic, and angiogenic cellular responses, and has been associated with a multitude of biological processes. The development of gene targeting and transgenic strategies has provided unequivocal evidence for the key involvement of Fgf signaling in mammalian developmental processes. In this review we highlight recent findings that demonstrate a critical requirement for Fgf signaling in the induction and development of the embryonic mammary gland. Furthermore, we briefly discuss the potential of Fgfs to act as oncogenic factors in mammary neoplasia.     

Nodal and Cripto-1: Embryonic Pattern Formation Genes Involved in Mammary Gland Development and Tumorigenesis

Members of the TGFbeta superfamily and EGF-CFC family, such as Nodal and Cripto, are important mediators of anterior-posterior and left-right axis specification during embryogenesis. In this paper, we review the role of Nodal and Cripto as critical morphogen-like molecules, with an emphasis on Nodal and EGF-CFC signaling during embryonic pattern formation. New evidence from gene expression and transgenic mouse studies have shown that both Nodal and Cripto-1 are expressed within the mammary duct and that modulation of these genes can disrupt normal branching morphogenesis resulting in epithelial disorganization and defective ductal architecture. We describe these new findings and propose that Cripto and Nodal are candidate mammary morphogens. Finally, the data linking overexpression of Cripto and perturbations of Cripto signaling to cell transformation and tumor formation are discussed. The fact that Cripto can modulate multiple pathways suggests it may act to deregulate growth inhibitors/homeostasis factors early in the cell transformation process and then activate prosurvival pathways dependent on MAPK and PI3K/Akt later in fully transformed phenotypes.     

Comparison of the Abilities of Human Parathyroid Hormone(1-31)NH2 and Human Parathyroid Hormone-Related Protein(1-31)NH2 to Stimulate Femoral Trabecular Bone Growth in Ovariectomized Rats

N-terminal fragments of PTH and PTHrP, such as hPTH-(1-34) and hPTHrP-(1-34), are sufficiently similar with respect to amino acid sequence, location of functional domains, and higher order configuration to activate the same PTH/PTHrP receptor and the same two signal enzymes, adenylyl cyclase and phospholipase-Cβ. Therefore, it was expected that hPTHrP-(1-31)NH₂ would stimulate bone growth in ovariectomized rats as strongly as hPTH-(1-31)NH₂. Like hPTH-(1-31)NH₂, hPTHrP-(1-31)NH₂ stimulated adenyly cyclase in ROS 17/2 osteosarcoma cells as strongly as the standard hPTH-(1-34) and like hPTH-(1-31)NH₂, triggered a large drop in mean blood pressure when injected intravenously. Unlike hPTH-(1-31)NH₂, however, hPTHrP-(1-31)NH₂ could not stimulate trabecular growth in the distal femurs of young, sexually mature, ovariectomized rats. Received: 12 December 1996 / Accepted: 23 April 1997     

A Whole-mount Immunofluorescence Protocol for Three-dimensional Imaging of the Embryonic Mammary Primordium

Whole-mount immunofluorescent staining facilitates the profiling of protein expression patterns within diverse and complex tissues. Thanks to the application of antibodies on whole mounted instead of sectioned specimens, this technique has many advantages with respect to the preservation of biological and pathological features of specimens when compared to conventional immunohistological methods. Here, we describe a protocol and optimal conditions of whole-mount immunofluorescence for studying the formation of mammary primordia. We also show an example three-dimensional reconstruction of a mammary primordium based on z-stacked images of a whole-mount stained specimen using confocal microscopy and image analysis software.