Parathyroid Hormone-related Protein Peptides Research Articles

Brief exposure to full length parathyroid hormone-related protein (PTHrP) causes persistent generation of cyclic AMP through an endocytosis-dependent mechanism

Parathyroid hormone (PTH)-related protein (PTHrP) (gene name Pthlh) was discovered as the factor responsible for the humoral hypercalcemia of malignancy. It shares such sequence similarity with PTH in the amino-terminal region that the two are equally able to act through a single G protein-coupled receptor, PTH1R. A number of biological activities are ascribed to domains of PTHrP beyond the amino-terminal domain. PTH functions as a circulating hormone, but PTHrP is generated locally in many tissues including bone, where it acts as a paracrine factor on osteoblasts and osteocytes. The present study compares how PTH and PTHrP influence cyclic AMP (cAMP) formation through adenylyl cyclase, the first event in cell activation through PTH1R. Brief exposure to full length PTHrP(1–141) in several osteoblastic cell culture systems was followed by sustained adenylyl cyclase activity for more than an hour after ligand washout. This effect was dose-dependent and was not found with shorter PTHrP or PTH peptides even though they were fully able to activate adenylyl cyclase with acute treatment. The persistent activation response to PTHrP(1–141) was seen also with later events in the cAMP/PKA pathway, including persistent activation of CRE-luciferase and sustained regulation of several CREB-responsive mRNAs, up to 24 h after the initial exposure. Pharmacologic blockade of endocytosis prevented the persistent activation of cAMP and gene responses.We conclude that full length PTHrP, the likely local physiological effector in bone, differs in intracellular action to PTH by undergoing endosomal translocation to induce a prolonged adenylyl cyclase activation in its target cells.

Parathyroid hormone-related peptide (1–34) reduces alveolar bone loss in type 1 diabetic rats

ObjectiveTo investigate the role of parathyroid hormone related protein (PTHrP) in diabetic periodontitis. MethodsAfter injected with 55mg/kg streptozotocin, diabetic rats were treated subcutaneously with low-dose (40μg/kg, once daily for 5days per week), middle-dose (80μg/kg) or high-dose (160μg/kg) PTHrP(1–34) peptide. Treatment continued for 12 weeks. Changes in periodontal tissues were confirmed by micro-computerized tomography assay and H&E analysis. We used tartrate resistant acid phosphatase (TRAP) staining to identify osteoclast cells. The expression of TNF-α, IL-1β and IL-6 was assessed by immunohistochemistry and Western blot. ResultsTooth-supporting structure loss was observed in periodontal tissues of diabetic rats. PTHrP (1–34) attenuated alveolar bone loss, especially in the middle-dose and high-dose group. Whereas TNF-α, IL-1β and IL-6 protein levels were increased in the diabetic gingival tissues, PTHrP (1–34) treatment inhibited the increase of IL-1β and IL-6, but had no effect on TNF-α. ConclusionType 1 diabetes increased the susceptibility to periodontal disease. Intermittent administration of PTHrP (1–34) exhibited an inhibitory effect on alveolar bone resorption and the gingival inflammation in periodontal tissues of diabetic rats.

Molecular characterisation and expression of parathyroid hormone-related protein in the caudal neurosecretory system of the euryhaline flounder, Platichthys flesus

Parathyroid hormone-related protein (PTHrP) is a hypercalcemic factor in fish, but the source of circulating PTHrP remains unclear. In this study investigation of the caudal neurosecretory system (CNSS), considered one of major sources of PTHrP in fish, provided valuable insights into this regulatory system. We report pthrpa and pthrpb gene cloning, characterization, expression, and responses to low salinity and hypocalcemia challenge in flounder. The pthrpa and pthrpb precursors, isolated from a European flounder CNSS library, consist of 166 and 192 amino acid residues, respectively, with an overall homology of approximately 59.2%. Both precursors contain a signal peptide and a mature peptide with cleavage and amidation sites. The flounder PTHrPA and PTHrPB peptides share only 41% sequence identity with human PTHrPA. Quantitative PCR analysis demonstrated that the bone and bladder, are respectively major sites of pthrpa and pthrpb expression in flounder. Urophysectomy confirmed the CNSS as a likely contributor to circulating PTHrP peptides. There were no significant differences in CNSS pthrpa and pthrpb mRNA expression or plasma PTHrP levels between seawater (SW) and freshwater (FW)-adapted fish, though plasma total calcium concentrations were higher in FW animals. The intraperitonial administration of EGTA rapidly induced hypocalcemia and concomitant elevation in plasma PTHrP accompanied by increases in both pthrpa and pthrpb expression in the CNSS. Together, these findings support an evolutionary conserved role for PTHrP in the endocrine regulation of calcium.

PTHrP-Derived Peptides Restore Bone Mass and Strength in Diabetic Mice: Additive Effect of Mechanical Loading.

There is an unmet need to understand the mechanisms underlying skeletal deterioration in diabetes mellitus (DM) and to develop therapeutic approaches to treat bone fragility in diabetic patients. We demonstrate herein that mice with type 1 DM induced by streptozotocin exhibited low bone mass, inferior mechanical and material properties, increased bone resorption, decreased bone formation, increased apoptosis of osteocytes, and increased expression of the osteocyte-derived bone formation inhibitor Sost/sclerostin. Further, short treatment of diabetic mice with parathyroid hormone related protein (PTHrP)-derived peptides corrected these changes to levels undistinguishable from non-diabetic mice. In addition, diabetic mice exhibited reduced bone formation in response to mechanical stimulation, which was corrected by treatment with the PTHrP peptides, and higher prevalence of apoptotic osteocytes, which was reduced by loading or by the PTHrP peptides alone and reversed by a combination of loading and PTHrP peptide treatment. In vitro experiments demonstrated that the PTHrP peptides or mechanical stimulation by fluid flow activated the survival kinases ERKs and induced nuclear translocation of the canonical Wnt signaling mediator β-catenin, and prevented the increase in osteocytic cell apoptosis induced by high glucose. Thus, PTHrP-derived peptides cross-talk with mechanical signaling pathways to reverse skeletal deterioration induced by DM in mice. These findings suggest a crucial role of osteocytes in the harmful effects of diabetes on bone and raise the possibility of targeting these cells as a novel approach to treat skeletal deterioration in diabetes. Moreover, our study suggests the potential therapeutic efficacy of combined pharmacological and mechanical stimuli to promote bone accrual and maintenance in diabetic subjects. © 2016 American Society for Bone and Mineral Research.

Anti-senescence and Anti-inflammatory Effects of the C-terminal Moiety of PTHrP Peptides in OA Osteoblasts.

Osteoarthritis (OA) is characterized by degenerative changes in the whole joint leading to physical disability in the elderly population. This condition is associated with altered bone metabolism in subchondral areas suggesting that therapeutic strategies aimed at modifying bone cell metabolism may be of interest. We have investigated the effects of several parathyroid hormone-related protein (PTHrP)-derived peptides (1-37): (N-terminal), (107-111) and (107-139) (C-terminal) on senescence features induced by inflammatory stress in human OA osteoblasts. Incubation of these primary cells with interleukin(IL)-1β led to an increased expression of senescence markers senescence-associated-β-galactosidase activity, γH2AX foci, p16, p21, p53, and caveolin-1. PTHrP (107-111) and PTHrP (107-139) significantly reduced all these parameters. Both peptides decreased the production of IL-6 and prostaglandin E2 which was the consequence of cyclo-oxygenase-2 downregulation. PTHrP (107-139) also reduced tumor necrosis factor-α release. These anti-inflammatory effects would be related to the reduction of nuclear factor-κB activation by both peptides and activator protein-1 by PTHrP (107-139). The three PTHrP peptides favored osteoblastic function although the C-terminal domain of PTHrP was more efficient than its N-terminal domain. Our data support an anti-senescence and anti-inflammatory role for the C-terminal moiety of PTHrP with potential applications in chronic inflammatory conditions such as OA.

Local delivery of parathyroid hormone-related protein-derived peptides coated onto a hydroxyapatite-based implant enhances bone regeneration in old and diabetic rats.

Diabetes mellitus (DM) and aging are associated with bone fragility and increased fracture risk. Both (1-37) N- and (107-111) C-terminal parathyroid hormone-related protein (PTHrP) exhibit osteogenic properties. We here aimed to evaluate and compare the efficacy of either PTHrP (1-37) or PTHrP (107-111) loaded into gelatin-glutaraldehyde-coated hydroxyapatite (HA-Gel) foams to improve bone repair of a transcortical tibial defect in aging rats with or without DM, induced by streptozotocin injection at birth. Diabetic old rats showed bone structural deterioration compared to their age-matched controls. Histological and μ-computerized tomography studies showed incomplete bone repair at 4 weeks after implantation of unloaded Ha-Gel foams in the transcortical tibial defects, mainly in old rats with DM. However, enhanced defect healing, as shown by an increase of bone volume/tissue volume and trabecular and cortical thickness and decreased trabecular separation, occurred in the presence of either PTHrP peptide in the implants in old rats with or without DM. This was accompanied by newly formed bone tissue around the osteointegrated HA-Gel implant and increased gene expression of osteocalcin and vascular endothelial growth factor (bone formation and angiogenic markers, respectively), and decreased expression of Sost gene, a negative regulator of bone formation, in the healing bone area. Our findings suggest that local delivery of PTHrP (1-37) or PTHrP (107-111) from a degradable implant is an attractive strategy to improve bone regeneration in aged and diabetic subjects. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2060-2070, 2016.

Abstract 4877: MMP-3 generates a novel PTHrP peptide that impacts osteoblast behavior and contributes to metastatic tumor growth in bone

Abstract Prostate cancer commonly metastasizes to bone, generating incurable mixed lesions that are both osteolytic and osteogenic. To identify new therapeutic targets we performed gene expression analysis and found that matrix metalloproteinase-3 (MMP-3), also known as stromelysin-1 was highly expressed in the prostate tumor-bone microenvironment. In keeping with the literature, immunohistochemical analysis of human bone metastases revealed MMP-3 was largely localized to the stromal compartment. To test whether stromal/host derived MMP-3 contributed to prostate cancer progression in bone, we generated immunocompromized MMP-3 null mice. Using an in vivo intratibial model of prostate to bone metastases (PaIII), we found that tumor growth, as measured by luminescence, and tumor-induced bone remodeling (μCT, histomorphometry) were significantly mitigated (p<0.05) in MMP-3 null mice compared to wild type controls. Using a candidate approach to examine potential mechanisms, we focused on parathyroid hormone-related protein (PTHrP), a powerful regulator of osteoblast behavior in the vicious cycle. We identified that MMP-3 processes mature PTHrP1-36 to yield unique PTHrP1-17, PTHrP18-26, and PTHrP27-36 fragments in vitro. To test the biological significance of the fragments we focused on testing their impact on stromal cell behavior. Using Boyden chamber migration assays, we observed a significant increase in migration of murine MSCs and MC3T3-E1 pre-osteoblasts in response to 10nM PTHrP1-17 compared to that of PTHrP1-36. Further analysis via confocal microscopy revealed a reduction in actin stress fibers as well as variations in lamellipodia and vinculin organization of pre-osteoblasts and primary osteoblasts treated with PTHrP1-17, and live cell microscopy indicated that these effects were transient with the cells reverting to the usual phenotype in a 24-hour period. Reduced phosphorylation of Rho-associated protein kinase (ROCK) substrates such as the myosin light chain suggest that PTHrP1-17 may mediate pre-osteoblast migration by modulating ROCK activity. Our data demonstrate that host MMP-3 contributes to prostate tumor growth and tumor induced changes in bone remodeling in vivo. Further, we have identified that mature PTHrP1-36 is subject to cleavage by MMP-3 resulting in PTHrP1-17, PTHrP18-26, and PTHrP27-36 fragments of which PTHrP1-17 significantly stimulates migration of murine MSCs and MC3T3-E1 pre-osteoblasts. Collectively, these data indicate that specific inhibition of MMP-3 and/or targeting MMP generated neo-epitopes would be efficacious for the treatment of prostate to bone metastases. Citation Format: Jeremy Steven Frieling, Lizzie Atomi Pamen, Shengyu Yang, Conor C. Lynch. MMP-3 generates a novel PTHrP peptide that impacts osteoblast behavior and contributes to metastatic tumor growth in bone. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4877. doi:10.1158/1538-7445.AM2014-4877

Inhibition of the canonical Wnt pathway by high glucose can be reversed by parathyroid hormone‐related protein in osteoblastic cells

Recent in vivo findings suggest that the bone sparing effect of parathyroid hormone-related protein (PTHrP) in diabetic mice might occur at least in part through targeting a suppressed Wnt/β-catenin pathway in osteoblasts. We here aimed to examine the inhibitory action of a high glucose environment on specific components of the canonical Wnt pathway, and the putative compensatory effects of PTHrP, in osteoblastic cell cultures. Mouse osteoblastic MC3T3-E1 cells and primary cultures of fetal mouse calvaria were exposed to normal (5.5 mM) or high (25 mM) D-glucose (HG), with or without PTHrP (1-36) or PTHrP (107-139) for different times. In some experiments, MC3T3-E1 cells were incubated with the Wnt pathway activators Wnt3a and LiCl, or were transfected with plasmids encoding either a mutated β-catenin that cannot be targeted for degradation or a human PTHrP (-36/+139) cDNA, or the corresponding empty plasmid, in the presence or absence of HG. The gene expression of Wnt3a and low density receptor-like proteins (LRP)-5 and 6, as well as β-catenin protein stabilization and β-catenin-dependent transcription activity were evaluated. Oxidative stress status under HG condition was also assessed. The present data demonstrate that HG can target different components of the canonical Wnt pathway, while β-catenin degradation appears to be a key event leading to inhibition of Wnt/β-catenin signaling in mouse osteoblastic cells. Both PTHrP peptides tested were able to counteract this deleterious action of HG. These in vitro findings also provide new clues to understand the underlying mechanisms whereby PTHrP can increase bone formation.

Src kinases mediate VEGFR2 transactivation by the osteostatin domain of PTHrP to modulate osteoblastic function

Parathyroid hormone-related protein (PTHrP) stimulates osteoblastic function through its N- and C-terminal domains. Since the osteogenic action of the latter domain appears to depend at least in part on its interaction with the vascular endothelial growth factor (VEGF) system, we aimed to explore the putative mechanism underlying this interaction in osteoblasts. Using native conditions for protein extraction and immunoblotting, we found that both PTHrP (107-139) and the shorter PTHrP (107-111) peptide (known as osteostatin), at 100 nM, promoted the appearance of a VEGF receptor (VEGFR) 2 protein band of apparent Mr. wt. 230 kDa, which likely represents its activation by dimer formation, in mouse osteoblastic MC3T3-E1 cells. Moreover, osteostatin (100 nM) maximally increased VEGFR2 phosphorylation at Tyr-1059 within 5-10 min in both MC3T3-E1 and rat osteoblastic osteosarcoma UMR-106 cells. This phosphorylation elicited by osteostatin appears to be VEGF-independent, but prevented by the VEGFR2 activation inhibitor SU1498 and also by the Src kinase inhibitors SU6656 and PP1. Furthermore, osteostatin induced phosphorylation of Src, extracellular signal-regulated kinase (ERK) and Akt with a similar time course to that observed for VEGFR2 activation in these osteoblastic cells. This osteostatin-dependent induction of ERK and Akt activation was abrogated by SU6656. Up-regulation of VEGF and osteoprotegerin gene expression as well as the pro-survival effect induced by osteostatin treatment were all prevented by both SU1498 and SU6656 in these osteoblastic cells. Collectively, these findings demonstrate that the osteostatin domain of C-terminal PTHrP phosphorylates VEGFR2 through Src activation, which represents a mechanism for modulating osteoblastic function.

Transcriptomic and proteomic analyses in bone tumor cells: Deciphering parathyroid hormone-related protein regulation of the cell cycle and apoptosis

Giant cell tumor of bone (GCT) is an aggressive skeletal tumor characterized by local bone destruction, high recurrence rates, and metastatic potential. Previous works in our laboratory, including functional assays, have shown that neutralization of parathyroid hormone-related protein (PTHrP) in the cell environment inhibits cell proliferation and induces cell death in GCT stromal cells, indicating a role for PTHrP in cell propagation and survival. The objective of this study was to investigate the global gene and protein expression patterns of GCT cells in order to identify the underlying pathways and mechanisms of neoplastic proliferation provided by PTHrP in the bone microenvironment. Primary stromal cell cultures from 10 patients with GCT were used in this study. Cells were exposed to optimized concentrations of either PTHrP peptide or anti-PTHrP neutralizing antiserum and were analyzed with both cDNA microarray and proteomic microarray assays in triplicate. Hierarchical clustering and principal component analyses confirmed that counteraction of PTHrP in GCT stromal cells results in a clear-cut gene expression pattern distinct from all other treatment groups and the control cell line human fetal osteoblast (hFOB). Multiple bioinformatics tools were used to analyze changes in gene/protein expression and identify important gene ontologies and pathways common to this anti-PTHrP-induced regulatory gene network. PTHrP neutralization interferes with multiple cell survival and apoptosis signaling pathways by triggering both death receptors and cell cycle-mediated apoptosis, particularly via the caspase pathway, TRAIL pathway, JAK-STAT signaling pathway, and cyclin E/CDK2-associated G1/S cell cycle progression. These findings indicate that PTHrP neutralization exhibits anticancer potential by regulating cell-cycle progression and apoptosis in bone tumor cells, with the corollary being that PTHrP is a pro-neoplastic factor that can be targeted in the treatment of bone tumors.

P4-16-01: Proteomic Analysis of Patient Plasma Identifies Parathyroid Hormone Related Protein (PTHrP12-48) as a Potential Biomarker of Breast Cancer Bone Metastasis.

Abstract Background: Bone metastasis is a devastating and often incurable phase of breast cancer progression that significantly compromises patient morbidity and mortality. Despite the well-characterized issues associated with tumor progression, there is currently no reliable method to detect or predict which patients have or are at increased risk for developing bone metastasis. In this study a validated plasma-based proteomic profile for the diagnosis of breast cancer bone metastasis was developed and a highly discriminatory protein component of the profile identified as a novel fragment of parathyroid hormone related protein (PTHrP). Materials and Methods: Plasma samples were collected from a total of 110 breast cancer patients. The training set consisted of 38 breast cancer patients with clinical evidence of bone metastasis and 38 with no evidence of a bone metastasis from time of diagnosis to clinical outcome. The independent validation cohort consisted of 34 breast cancer patients with an unknown bone metastasis classification. The training set was analyzed using surface enhanced laser desorption time-of-flight mass spectrometry (SELDI-TOF MS) and 13 discriminatory protein peaks that contributed to a homogeneous partitioning of the training set (n = 76) in multiple statistical, bioinformatics, and machine learning modeling scenarios were identified and used to construct a RandomForest classifier for the detection of breast cancer bone metastasis (sensitivity: 100%, specificity: 100%). Results: The diagnostic profile identified was then independently confirmed in a blinded fashion using the validation cohort (sensitivity: 97%; specificity: 82%). Importantly, the most discriminatory protein peak (m/z 4260.92 Da) in the plasma of bone metastasis patients was subsequently identified using specific immunodepletion, tryptic peptide mapping and peptide sequencing as a unique proteolytic PTHrP fragment, PTHrP(12-48). Ongoing studies are determining the biogenesis and biological activity of this unique PTHrP peptide. Discussion: Our discovery of PTHrP(12-48) as a novel plasma biomarker of breast cancer bone metastasis validates our plasma proteomic approach and provides the first direct evidence for the existence of a circulating PTHrP biomarker in breast cancer patient plasma that is associated with bone metastasis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-16-01.

Systemic and acute administration of parathyroid hormone-related peptide(1–36) stimulates endogenous beta cell proliferation while preserving function in adult mice

A major focus in the treatment of diabetes is to identify factors that stimulate endogenous beta cell growth while preserving function. The first 36 amino acids of parathyroid hormone-related protein (PTHrP) are sufficient to enhance proliferation and function in rodent and human beta cells in vitro. This study examined whether acute and systemic administration of the amino-terminal PTHrP(1-36) peptide can achieve similar effects in rodent beta cells in vivo. Adult male mice were injected with 40, 80 or 160 μg of PTHrP(1-36) per kg body weight or with vehicle for 25 days. Glucose and beta cell homeostasis, as well as expression of differentiation markers and cell cycle genes were analysed. All three doses of PTHrP(1-36) significantly enhanced beta cell proliferation in vivo at day 25, with 160 μg/kg PTHrP(1-36) increasing proliferation as early as day 5. Importantly, the two higher doses of PTHrP(1-36) caused a significant 30% expansion of beta cell mass, with a short-term improvement in glucose tolerance. PTHrP(1-36) did not cause hypercalcaemia, or change islet number, beta cell size, beta cell death or expression of differentiation markers. Analysis of islet G1/S cell cycle proteins revealed that chronic overabundance of PTHrP(1-139) in the beta cell significantly increased the cell cycle activator cyclin D2 and decreased levels of cyclin-dependent kinase 4 inhibitor (p16( Ink4a ) [Ink4a also known as Cdkn2a]), but acute treatment with PTHrP(1-36) did not. Acute and systemic administration of PTHrP(1-36) increases rodent beta cell proliferation and mass without negatively affecting function or survival. These findings highlight the future potential therapeutic effectiveness of this peptide under diabetes-related pathophysiological conditions.

Parathyroid Hormone–Related Protein Enhances Human β-Cell Proliferation and Function With Associated Induction of Cyclin-Dependent Kinase 2 and Cyclin E Expression

OBJECTIVEInducing human β-cell growth while enhancing function is a major goal in the treatment of diabetes. Parathyroid hormone–related protein (PTHrP) enhances rodent β-cell growth and function through the parathyroid hormone-1 receptor (PTH1R). Based on this, we hypothesized that PTH1R is expressed in human β-cells and that PTHrP has the potential to enhance human β-cell proliferation and/or function.RESEARCH DESIGN AND METHODSPTH1R expression, β-cell proliferation, glucose-stimulated insulin secretion (GSIS), and expression of differentiation and cell-cycle genes were analyzed in human islets transduced with adenoviral PTHrP constructs or treated with PTHrP peptides. The effect of overexpression of late G1/S cell cycle molecules was also assessed on human β-cell proliferation.RESULTSWe found that human β-cells express PTH1R. More importantly, overexpression of PTHrP causes a significant approximately threefold increase in human β-cell proliferation. Furthermore, the amino terminus PTHrP(1-36) peptide is sufficient to increase replication as well as expression of the late G1/S cell-cycle proteins cyclin E and cyclin-dependent kinase 2 (cdk2) in human islets. Notably, PTHrP(1-36) also enhances GSIS. Finally, overexpression of cyclin E alone, but not cdk2, augments human β-cell proliferation, and when both molecules are expressed simultaneously there is a further marked synergistic increase in replication.CONCLUSIONSPTHrP(1-36) peptide enhances human β-cell proliferation as well as function, with associated upregulation of two specific cell-cycle activators that together can induce human β-cell proliferation several fold. The future therapeutic potential of PTHrP(1-36) for the treatment of diabetes is especially relevant given the complementary therapeutic efficacy of PTHrP(1-36) in postmenopausal osteoporosis.

The osteoinductive properties of mesoporous silicate coated with osteostatin in a rabbit femur cavity defect model

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone formation and remodeling. Our recent findings demonstrate that PTHrP (107–111) (osteostatin) loaded onto silica-based ordered mesoporous SBA15 materials exhibit osteogenic features in osteoblastic cell cultures. We aimed here to elucidate whether these peptide-coated materials might be suitable for promoting bone repair following a cavitary defect in the rabbit femur. Histological examination revealed the absence of significant inflammation or bone resorption within the time of study (4 and 8 weeks) after implantation. At 8 weeks, the peptide-unloaded materials were still separated from the bone marrow by a fibrous cap, which was greatly diminished by the presence of the PTHrP peptide. By using μCT analysis, new bone formation was evident at different distances from the implants, mainly for the latter peptide-loaded biomaterials. This was confirmed by performing immunostaining for different osteoblast markers. Our findings demonstrate that these PTHrP (107–111)-loaded bioceramics significantly improve local bone induction, as compared to that observed with the unloaded material.

Role of the N- and C-terminal Fragments of Parathyroid-Hormone-Related Protein as Putative Therapies to Improve Bone Regeneration Under High Glucocorticoid Treatment

The parathyroid-hormone-related protein (PTHrP) is an important modulator of bone formation and bone remodeling. High and/or prolonged glucocorticoid (GC) treatments inhibit PTHrP expression in osteoblastic cells and bone formation and repair. We assessed the ability of the N- and C-terminal PTHrP fragments to restore the GC-altered bone regeneration after bone marrow ablation in mice. Animals were administered 3-methylprednisolone or vehicle and PTHrP (1-36) or PTHrP (107-139) every other day, beginning 4 days before marrow ablation in the tibia, and euthanized 12 days later. GC-treated mice showed in the ablated tibia a decrease in bone formation and in osteoblast and sclerostin-positive osteocyte numbers, reduced expression of osteoblastic factors, decreased osteogenesis of bone-marrow-derived cells, an increase in the numbers of multinucleated osteoclasts and adipocytes, and decreased cortical vascularization, as well as altered bone structure (measured by microcomputerized tomography) in the intact femur. These effects were reversed at least in part by either PTHrP peptide. The present novel findings support the use of both PTHrP peptides tested as putative bone regenerative therapies in GC-related bone diseases.

Potentiation of ATP- and Bradykinin-Induced [Ca2+]c Responses by PTHrP Peptides in the HaCaT Cell Line

In the epidermis, local and systemic factors including extracellular nucleotides and parathyroid hormone-related protein (PTHrP) regulate keratinocyte proliferation and differentiation. Extracellular nucleotides increase proliferation via activation of P2 receptors and induction of calcium transients, while endoproteases cleave PTHrP, resulting in fragments with different cellular functions. We investigated the effects of adenosine 5'-triphosphate (ATP) alone and in combination with synthetic PTHrP peptides on calcium transients in HaCaT cells. ATP induced calcium transients, while PTHrP peptides did not. C-terminal and mid-molecule PTHrP peptides (1-100 pM) potentiated ATP-induced calcium transients independently of calcium influx. 3-Isobutyl-1-methylxanthine potentiated ATP-induced calcium transients, suggesting that a cyclic monophosphate is responsible. Cyclic AMP is not involved, but cyclic GMP is a likely candidate since the protein kinase G inhibitor, KT5823, inhibited potentiation. Co-stimulation with ATP and either PTHrP (43-52) or PTHrP (70-77) increased proliferation, suggesting that this is important in the regulation of cell turnover and wound healing and may be a mechanism for hyperproliferation in skin disorders such as psoriasis. Finally, PTHrP fragments potentiated bradykinin-induced calcium transients, suggesting a role in inflammation in the skin. Since PTHrP is found in many normal and malignant cells, potentiation is likely to have a wider role in modulating signal transduction events.

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.

Nuclear targeting of a midregion PTHrP fragment is necessary for stimulating growth in breast cancer cells

Parathyroid-hormone related protein (PTHrP) is the primary factor in humoral hypercalcemia of malignancy and is highly secreted by breast cancers. The pro-hormone undergoes post-translational processing and cleavage to give rise to mature secretory peptides, one of which is midregion PTHrP (38-94/95/101) containing a nuclear localisation sequence (NLS) in amino acids (87-106). The current study investigates whether the NLS in midregion PTHrP is important in breast cancer growth. PTHrP-(67-101), a midregion PTHrP fragment containing NLS-(87-101) significantly increased growth of MCF-7 and MDA-MB231 cells (126.3 and 121.3% of control respectively in serum conditions), independent of PTHR1 whereas PTHrP-(67-86), which lacks the NLS did not. Fluorescent-labelled PTHrP-(67-101) translocated to the nucleus, whereas PTHrP-(67-86) remained cytosolic and a scrambled(+NLS) peptide was not internalised. In comparison, no growth influence or uptake was seen in non-tumour breast cells (Hs578Bst). Increases in intracellular calcium mobilisation were observed in breast cancer cells stimulated with both PTHrP-(67-101) and PTHrP-(67-86) (EC(50) of 3.2 pM and 2.2 pM respectively for MCF-7 cells), whereas inositide turnover was not detected. Both nuclear uptake and calcium signalling were attenuated in the presence of EGTA, but not with U73122 or N-terminal PTHrP peptides. Our studies indicate that the NLS-containing midregion PTHrP peptide is dependent on both internalisation and nuclear translocation to induce growth in breast cancer cells. These findings highlight the importance of midregion PTHrP and its receptor in breast cancer growth and may provide potential targets for future therapeutic intervention.

Parathyroid hormone-related protein as a common target molecule in specific immunotherapy for a wide variety of tumor types

Parathyroid hormone-related protein (PTH-rP) has been considered to be responsible for malignancy-associated hypercalcemia and is thought to participate in pathological changes in bone metastases of cancer. In this study, we determined whether or not PTH-rP could be a common target molecule in specific immunotherapy for patients with a wide variety of tumor types. Various types of tumor cell lines were examined for PTH-rP expression at the mRNA and protein levels by RT-PCR, flow cytometry, and immunocytochemistry. We also determined whether or not cancer-reactive cytotoxic T lymphocytes (CTLs) could be induced from the peripheral blood mononuclear cells (PBMCs) of HLA-A24+ patients with gastric, colon, renal, or cervical cancer by in vitro stimulation with two PTH-rP peptides. As a result, PTH-rP mRNA was expressed in the majority of gastric, breast, lung, colon, cervical, and renal cancer cell lines. Expression of the protein was confirmed by both flow cytometry and immunocytochemistry. Furthermore, PTH-rP peptide-specific and cancer-reactive CTLs were successfully generated from the PBMCs of HLA-A24+ patients with different tumor types using in vitro stimulation with either the PTH-rP(102-111) or PTH-rP(110-119) peptide. These findings indicate that PTH-rP could be a common target molecule in specific immunotherapy for patients with a wide variety of tumor types, particularly bone metastases.

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.