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1.  Parathyroid Hormone–related Peptide (PTHrP)-dependent and -independent Effects of Transforming Growth Factor β (TGF-β) on Endochondral Bone Formation  
The Journal of Cell Biology  1999;145(4):783-794.
Previously, we showed that expression of a dominant-negative form of the transforming growth factor β (TGF-β) type II receptor in skeletal tissue resulted in increased hypertrophic differentiation in growth plate and articular chondrocytes, suggesting a role for TGF-β in limiting terminal differentiation in vivo. Parathyroid hormone–related peptide (PTHrP) has also been demonstrated to regulate chondrocyte differentiation in vivo. Mice with targeted deletion of the PTHrP gene demonstrate increased endochondral bone formation, and misexpression of PTHrP in cartilage results in delayed bone formation due to slowed conversion of proliferative chondrocytes into hypertrophic chondrocytes. Since the development of skeletal elements requires the coordination of signals from several sources, this report tests the hypothesis that TGF-β and PTHrP act in a common signal cascade to regulate endochondral bone formation. Mouse embryonic metatarsal bone rudiments grown in organ culture were used to demonstrate that TGF-β inhibits several stages of endochondral bone formation, including chondrocyte proliferation, hypertrophic differentiation, and matrix mineralization. Treatment with TGF-β1 also stimulated the expression of PTHrP mRNA. PTHrP added to cultures inhibited hypertrophic differentiation and matrix mineralization but did not affect cell proliferation. Furthermore, terminal differentiation was not inhibited by TGF-β in metatarsal rudiments from PTHrP-null embryos; however, growth and matrix mineralization were still inhibited. The data support the model that TGF-β acts upstream of PTHrP to regulate the rate of hypertrophic differentiation and suggest that TGF-β has both PTHrP-dependent and PTHrP-independent effects on endochondral bone formation.
PMCID: PMC2133184  PMID: 10330406
chondrocyte differentiation; skeletal development; perichondrium; organ culture; transforming growth factor β receptors
2.  Evidence for a causal role of parathyroid hormone-related protein in the pathogenesis of human breast cancer-mediated osteolysis. 
Journal of Clinical Investigation  1996;98(7):1544-1549.
Breast cancer almost invariably metastasizes to bone in patients with advanced disease and causes local osteolysis. Much of the morbidity of advanced breast cancer is a consequence of this process. Despite the importance of the problem, little is known of the pathophysiology of local osteolysis in the skeleton or its prevention and treatment. Observations in patients with bone metastases suggest that breast cancer cells in bone express parathyroid hormone-related protein (PTHrP) more frequently than in soft tissue sites of metastasis or in the primary tumor. Thus, the role of PTHrP in the causation of breast cancer metastases in bone was examined using human breast cancer cell lines. Four of eight established human breast cancer cell lines expressed PTHrP and one of these cell lines, MDA-MB-231, was studied in detail using an in vivo model of osteolytic metastases. Mice inoculated with MDA-MB-231 cells developed osteolytic bone metastasis without hypercalcemia or increased plasma PTHrP concentrations. PTHrP concentrations in bone marrow plasma from femurs affected with osteolytic lesions were increased 2.5-fold over corresponding plasma PTHrP concentrations. In a separate experiment, mice were treated with either a monoclonal antibody directed against PTHrP(1-34), control IgG, or nothing before tumor inoculation with MDA-MB-231 and twice per week for 26 d. Total area of osteolytic lesions was significantly lower in mice treated with PTHrP antibodies compared with mice receiving control IgG or no treatment. Histomorphometric analysis of bone revealed decreased osteoclast number per millimeter of tumor/bone interface and increased bone area, as well as decreased tumor area, in tumor-bearing animals treated with PTHrP antibodies compared with respective controls. These results indicate that tumor-produced PTHrP can cause local bone destruction in breast cancer metastatic to bone, even in the absence of hypercalcemia or increased circulating plasma concentrations of PTHrP. Thus, PTHrP may have an important pathogenetic role in the establishment of osteolytic bone lesions in breast cancer. Neutralizing antibodies to PTHrP may reduce the development of destructive bone lesions as well as the growth of tumor cells in bone.
PMCID: PMC507586  PMID: 8833902
3.  Endotoxin increases parathyroid hormone-related protein mRNA levels in mouse spleen. Mediation by tumor necrosis factor. 
Journal of Clinical Investigation  1993;92(5):2546-2552.
Parathyroid hormone-related protein (PTHrP) causes hypercalcemia in malignancy. However, the role and regulation of PTHrP in normal physiology is just beginning to be explored. PTHrP is found in the spleen and has several other features common to cytokines. Since endotoxin (LPS) causes many of its effects indirectly by inducing cytokines, studies were undertaken to determine whether LPS might also induce splenic PTHrP expression. LPS (100 ng/mouse) increased splenic PTHrP mRNA levels 3.6-fold in C3H/OuJ mice. This effect was maximal at 2 h and returned to baseline by 4 h. PTHrP peptide levels also increased 3.3-fold in splenic extracts in response to LPS (1 microgram/mouse). Murine TNF-alpha and human IL-1 beta, cytokines that mediate many of the effects of LPS, also increased splenic PTHrP mRNA levels. LPS-resistant C3H/HeJ mice, which produce minimal amounts of TNF and IL-1 in response to LPS, were resistant to LPS induction of splenic PTHrP mRNA, while TNF-alpha and IL-1 beta readily increased PTHrP mRNA levels in C3H/HeJ mice. Anti-TNF antibody blocked LPS induction of splenic PTHrP mRNA in C3H/OuJ mice by 68%, indicating that TNF is a mediator of the LPS induction of PTHrP levels. In contrast, an IL-1 receptor antagonist (IL-1ra) was ineffective. The increase in PTHrP in the spleen during the immune response suggests that PTHrP may play an important role in immune modulation, perhaps by mediating changes in lymphocyte proliferation and/or function.
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PMCID: PMC288441  PMID: 8227368
4.  Nuclear localization of parathyroid hormone-related peptide confers resistance to anoikis in prostate cancer cells 
Endocrine-related cancer  2012;19(3):243-254.
Prostate cancer remains a leading cause of cancer-related death in men, largely attributable to distant metastases, most frequently to bones. Despite intensive investigations, molecular mechanisms underlying metastasis are not completely understood. Among prostate cancer-derived factors, parathyroid hormone-related peptide (PTHrP), first discovered as an etiologic factor for malignancy-induced hypercalcemia, regulates many cellular functions critical to tumor growth, angiogenesis, and metastasis. In this study, the role of PTHrP in tumor cell survival from detachment-induced apoptosis (i.e. anoikis) was investigated. Reduction of PTHLH (encoding PTHrP) gene expression in human prostate cancer cells (PC-3) increased the percentage of apoptotic cells when cultured in suspension. Conversely, overexpression of PTHrP protected prostate cancer cells (Ace-1 and LNCaP, both typically expressing low or undetectable basal PTHrP) from anoikis. Overexpression of nuclear localization signal (NLS)-defective PTHrP failed to protect cells from anoikis, suggesting that PTHrP-dependent protection from anoikis is an intracrine event. A PCR-based apoptosis-related gene array showed that detachment increased expression of the TNF gene (encoding the proapoptotic protein tumor necrosis factor-α) fourfold greater in PTHrP-knockdown PC-3 cells than in control PC-3 cells. In parallel, TNF gene expression was significantly reduced in PTHrP-overexpressing LNCaP cells, but not in NLS-defective PTHrP overexpressing LNCaP cells, when compared with control LNCaP cells. Subsequently, in a prostate cancer skeletal metastasis mouse model, PTHrP-knockdown PC-3 cells resulted in significantly fewer metastatic lesions compared to control PC-3 cells, suggesting that PTHrP mediated antianoikis events in the bloodstream. In conclusion, nuclear localization of PTHrP confers prostate cancer cell resistance to anoikis, potentially contributing to prostate cancer metastasis.
doi:10.1530/ERC-11-0278
PMCID: PMC3593272  PMID: 22291434
5.  Parathyroid hormone-related protein is induced during lethal endotoxemia and contributes to endotoxin-induced mortality in rodents. 
Molecular Medicine  1996;2(2):204-210.
BACKGROUND: Parathyroid hormone-related protein (PTHrP) is a ubiquitous and highly conserved vasoactive peptide whose role and regulation in normal physiology remain an enigma. Recently, we demonstrated that low-dose endotoxin (LPS) induces intrasplenic, but not systemic, levels of PTHrP; and that tumor necrosis factor, a pro-inflammatory cytokine, is the major mediator of this effect. We have therefore hypothesized that, with higher, lethal doses of endotoxin, PTHrP could be induced in multiple tissues to such a degree that it could contribute to the lethality of septic shock. MATERIALS AND METHODS: Northern blot analysis was used to measure PTHrP mRNA levels in vital organs of rats after administration of a near lethal dose (5 mg/250 g) of LPS (or vehicle alone). Plasma levels of PTHrP were also measured by immunoradiometric assay. The ability of the immunoglobulin fraction of two different PTHrP(1-34) antisera to protect from LPS-induced lethality was also studied in mice using survival analysis. RESULTS: In response to a near-lethal dose of endotoxin, PTHrP mRNA levels increased acutely in every vital organ examined (spleen, lung, heart, kidney, and liver). Circulating levels of PTHrP also increased, peaking 2 hr after administration of high-dose endotoxin. Passive immunization of mice with anti-PTHrP(1-34) antibody 6 hr prior to administration of a lethal dose of LPS protected mice from endotoxin-induced death (p < 0.00005). CONCLUSIONS: These results suggest that PTHrP belongs to the cascade of pro-inflammatory cytokines induced during lethal endotoxemia that is responsible for the toxic effects of LPS.
PMCID: PMC2230122  PMID: 8726463
6.  The N- and C-terminal domains of parathyroid hormone-related protein affect differently the osteogenic and adipogenic potential of human mesenchymal stem cells 
Parathyroid hormone-related protein (PTHrP) is synthesized by diverse tissues, and its processing produces several fragments, each with apparently distinct autocrine and paracrine bioactivities. In bone, PTHrP appears to modulate bone formation in part through promoting osteoblast differentiation. The putative effect of PTH-like and PTH-unrelated fragments of PTHrP on human mesenchymal stem cell (MSCs) is not well known. Human MSCs were treated with PTHrP (1-36) or PTHrP (107-139) or both (each at 10 nM) in osteogenic or adipogenic medium, from the start or after 6 days of exposure to the corresponding medium, and the expression of several osteoblastogenic and adipogenic markers was analyzed. PTHrP (1-36) inhibited adipogenesis in MSCs and favoured the expression of osteogenic early markers. The opposite was observed with treatment of MSCs with PTHrP (107-139). Moreover, inhibition of the adipogenic differentiation by PTHrP (1-36) prevailed in the presence of PTHrP (107-139). The PTH/PTHrP type 1 receptor (PTH1R) gene expression was maximum in the earlier and later stages of osteogenesis and adipogenesis, respectively. While PTHrP (107-139) did not modify the PTH1R overexpression during adipogenesis, PTHrP (1-36) did inhibit it; an effect which was partially affected by PTHrP (7-34), a PTH1R antagonist, at 1 µM. These findings demonstrate that both PTHrP domains can exert varying effects on human MSCs differentiation. PTHrP (107-139) showed a tendency to favor adipogenesis, while PTHrP (1-36) induced a mild osteogenic effect in these cells, and inhibited their adipocytic commitment. This further supports the potential anabolic action of the latter peptide in humans.
doi:10.3858/emm.2010.42.2.010
PMCID: PMC2827833  PMID: 19946180
adipogenesis; bone; mesenchymal stem cells; osteogenesis; parathyroid hormone-related protein
7.  Abundant expression of parathyroid hormone-related protein in primary rat aortic smooth muscle cells accompanies serum-induced proliferation. 
Journal of Clinical Investigation  1991;88(6):1841-1847.
Parathyroid hormone-related protein (PTHrP), which is responsible for producing hypercalcemia in patients with humoral hypercalcemia of malignancy, has recently been identified in several normal tissues. Because PTHrP, like parathyroid hormone (PTH), is known to exhibit vasodilatory properties, we investigated the expression and regulation of PTHrP mRNA in cultured rat aortic smooth muscle cells (SMC). We report here that PTHrP mRNA is expressed in SMC and is markedly induced by serum in a time- and concentration-dependent fashion. Addition of 10% fetal calf serum to serum-deprived, confluent cells, resulted in a marked induction of PTHrP mRNA by 2 h with a peak at 4-6 h. PTHrP was detected in SMC by immunocytochemistry and radioimmunoassay of conditioned medium, and was shown to be up-regulated within 24 h after the addition of serum. The serum induction of PTHrP mRNA was blocked by actinomycin D and by cycloheximide indicating the need for protein synthesis to evoke the serum effect on PTHrP gene transcription. In addition, treatment with dexamethasone, which has been previously shown to reduce the constitutive expression of PTHrP in human cancer cells, also blunted the serum induction of PTHrP mRNA in SMC. Treatment of quiescent cells with the serum mitogens platelet-derived growth factor or insulin-like growth factor-I had no effect on PTHrP, whereas the vasoactive peptides endothelin, norepinephrine and thrombin stimulated PTHrP expression. Exogenous addition of recombinant PTHrP-(1-141) had no significant effect on SMC DNA synthesis as measured by [3H]thymidine incorporation. In summary, the abundance of PTHrP mRNA and the characteristics of its regulation in SMC suggest a major role for PTHrP as a local modulator in vascular smooth muscle.
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PMCID: PMC295751  PMID: 1752945
8.  Regulation of Articular Chondrocyte Proliferation and Differentiation by Indian Hedgehog and Parathyroid Hormone-related Protein 
Arthritis and rheumatism  2008;58(12):3788-3797.
Objective
The chondrocytes of the epiphyseal growth zone are regulated by the Indian hedgehog (Ihh)-parathyroid hormone-related protein (PTHrP) axis. In weight-bearing joints, this growth zone comes to be subdivided by the secondary ossification center into distinct articular and growth cartilage structures. Here, we explored the cells of origin, localization, regulation of expression, and putative functions of Ihh and PTHrP in articular cartilage in the mouse.
Methods
We assessed Ihh and PTHrP expression in an allelic PTHrP-lacZ knockin mouse and several versions of PTHrP-null mice. Selected joints were unloaded surgically to examine load-induction of PTHrP and Ihh.
Results
The embryonic growth zone appears to serve as the source of PTHrP-expressing proliferative chondrocytes that populate both the forming articular cartilage and growth plate structures. In articular cartilage, these cells take the form of articular chondrocytes in the mid-zone. In PTHrP-knockout mice, mineralizing chondrocytes encroach upon developing articular cartilage but appear to be prevented from mineralizing the joint space by Ihh-driven surface chondrocyte proliferation. In growing and adult mice, PTHrP expression in articular chondrocytes is load-induced, and unloading is associated with rapid changes in PTHrP expression and articular chondrocyte differentiation.
Conclusion
We conclude that the PTHrP-Ihh axis participates in the maintenance of articular cartilage. Dysregulation of this system might contribute to the pathogenesis of arthritis.
doi:10.1002/art.23985
PMCID: PMC2599803  PMID: 19035497
9.  The cyclin-dependent kinase inhibitor p57Kip2 mediates proliferative actions of PTHrP in chondrocytes 
Journal of Clinical Investigation  2004;113(9):1334-1343.
Parathyroid hormone–related peptide (PTHrP) is a positive regulator of chondrocyte proliferation during bone development. In embryonic mice lacking PTHrP, chondrocytes stop proliferating prematurely, with accelerated differentiation. Because the bone phenotype of mice lacking the cyclin-dependent kinase inhibitor p57Kip2 is the opposite of the PTHrP-null phenotype, we hypothesized that PTHrP’s proliferative actions in chondrocytes might be mediated by opposing p57. We generated p57/PTHrP-null embryos, which showed partial rescue of the PTHrP-null phenotype. There was reversal of the loss of proliferative chondrocytes in most bones, with reversal of the accelerated differentiation that occurs in the PTHrP-null phenotype. p57 mRNA and protein were upregulated in proliferative chondrocytes in the absence of PTHrP. Metatarsal culture studies confirmed the action of PTHrP to decrease p57 mRNA and protein levels in a model in which parathyroid hormone (PTH), used as an analog of PTHrP, increased chondrocyte proliferation rate and the length of the proliferative domain. PTH treatment of p57-null metatarsals had no effect on proliferation rate in round proliferative chondrocytes but still stimulated proliferation in columnar chondrocytes. These studies suggest that the effects of PTHrP on both the rate and extent of chondrocyte proliferation are mediated, at least in part, through suppression of p57 expression.
doi:10.1172/JCI200421252
PMCID: PMC398433  PMID: 15124025
10.  Tumor expressed PTHrP facilitates prostate cancer-induced osteoblastic lesions 
Expression of parathyroid hormone-related protein (PTHrP) correlates with prostate cancer skeletal progression; however, the impact of prostate cancer-derived PTHrP on the microenvironment and osteoblastic lesions in skeletal metastasis has not been completely elucidated. In this study, PTHrP overexpressing prostate cancer clones were stably established by transfection of full length rat PTHrP cDNA. Expression and secretion of PTHrP were verified by western blotting and IRMA assay. PTHrP overexpressing prostate cancer cells had higher growth rates in vitro, and generated larger tumors when inoculated subcutaneously into athymic mice. The impact of tumor-derived PTHrP on bone was investigated using a vossicle co-implant model. Histology revealed increased bone mass adjacent to PTHrP overexpressing tumor foci, with increased osteoblastogenesis, osteoclastogenesis and angiogenesis. In vitro analysis demonstrated pro-osteoclastic and pro-osteoblastic effects of PTHrP. PTHrP enhanced proliferation of bone marrow stromal cells and early osteoblast differentiation. PTHrP exerted a pro-angiogenic effect indirectly, as it increased angiogenesis but only in the presence of bone marrow stromal cells. These data suggest PTHrP plays a role in tumorigenesis in prostate cancer, and that PTHrP is a key mediator for communication and interactions between prostate cancer and the bone microenvironment. Prostate cancer-derived PTHrP is actively involved in osteoblastic skeletal progression.
doi:10.1002/ijc.23602
PMCID: PMC3979464  PMID: 18729185
parathyroid hormone-related protein; PTHrP; skeletal metastasis; prostate carcinoma; angiogenesis
11.  Functional characterization and evolution of PTH/PTHrP receptors: insights from the chicken 
Background
The parathyroid hormone (PTH)-family consists of a group of structurally related factors that regulate calcium and bone homeostasis and are also involved in development of organs such as the heart, mammary gland and immune system. They interact with specific members of family 2 B1 G-protein coupled receptors (GPCRs), which have been characterised in teleosts and mammals. Two PTH/PTHrP receptors, PTH1R and PTH2R exist in mammals and in teleost fish a further receptor PTH3R has also been identified. Recently in chicken, PTH-family members involved in calcium transport were characterized and specific PTHRs are suggested to exist although they have not yet been isolated or functionally characterized. The aim of this study is to further explore the evolution and function of the vertebrate PTH/PTHrP system through the isolation, phylogenetic analysis and functional characterization of the chicken receptors.
Results
Two PTHRs were isolated in chicken and sequence comparison and phylogenetic analysis indicate that the chicken receptors correspond to PTH1R and PTH3R, which emerged prior to the teleost/tetrapod divergence since they are present in cartilaginous fish. The vertebrate PTH2R receptor and its ligand TIP39 have been lost from bird genomes. Chicken PTH1R and PTH3R have a divergent and widespread tissue expression and are also evident in very early embryonic stages of development. Receptor stimulation studies using HEK293 cells stably expressing the chicken PTH1R and PTH3R and monitoring cAMP production revealed they are activated by chicken 1–34 N-terminal PTH-family peptides in a dose dependent manner. PTH-L and PTHrP were the most effective peptides in activating PTH1R (EC50 = 7.7 nM and EC50 = 22.7 nM, respectively). In contrast, PTH-L (100 nM) produced a small cAMP accumulation on activation of PTH3R but PTHrP and PTH (EC50 = 2.5 nM and EC50 = 22.1 nM, respectively) readily activated the receptor. PTHrP also stimulated intracellular Ca2+ accumulation on activation of PTH1R but not PTH3R.
Conclusion
Two PTHR homologues of the vertebrate PTH1R and PTH3R were isolated and functionally characterized in chicken. Their distinct pattern of expression during embryo development and in adult tissues, together with their ligand preference, suggests that they have acquired specific functions, which have contributed to their maintenance in the genome. PTH2R and its activating ligand, TIP39, are absent from bird genomes. Nonetheless identification of putative PTH2R and TIP39 in the genome of an ancient agnathan, lamprey, suggests the PTH/PTHrP ligand and receptor family was already present in an early basal paraphyletic group of vertebrates and during the vertebrate radiation diverged via gene/genome duplication and deletion events. Knowledge of the role PTH/PTHrP system in early vertebrates will help to establish evolution of function.
doi:10.1186/1471-2148-12-110
PMCID: PMC3483286  PMID: 22768871
12.  P38 Mitogen-Activated Protein Kinase Inhibitor, FR167653, Inhibits Parathyroid Hormone Related Protein-Induced Osteoclastogenesis and Bone Resorption 
PLoS ONE  2011;6(8):e23199.
p38 mitogen-activated protein kinase (MAPK) acts downstream in the signaling pathway that includes receptor activator of NF-κB (RANK), a powerful inducer of osteoclast formation and activation. We investigated the role of p38 MAPK in parathyroid hormone related protein (PTHrP)-induced osteoclastogenesis in vitro and PTHrP-induced bone resorption in vivo. The ability of FR167653 to inhibit osteoclast formation was evaluated by counting the number of tartrate-resistant acid phosphatase positive multinucleated cells (TRAP-positive MNCs) in in vitro osteoclastgenesis assays. Its mechanisms were evaluated by detecting the expression level of c-Fos and nuclear factor of activated T cells c1 (NFATc1) in bone marrow macrophages(BMMs) stimulated with sRANKL and M-CSF, and by detecting the expression level of osteoprotegerin (OPG) and RANKL in bone marrow stromal cells stimulated with PTHrP in the presence of FR167653. The function of FR167653 on bone resorption was assessed by measuring the bone resorption area radiographically and by counting osteoclast number per unit bone tissue area in calvaria in a mouse model of bone resorption by injecting PTHrP subcutaneously onto calvaria. Whole blood ionized calcium levels were also recorded. FR167653 inhibited PTHrP-induced osteoclast formation and PTHrP-induced c-Fos and NFATc1 expression in bone marrow macrophages, but not the expression levels of RANKL and OPG in primary bone marrow stromal cells treated by PTHrP. Furthermore, bone resorption area and osteoclast number in vivo were significantly decreased by the treatment of FR167653. Systemic hypercalcemia was also partially inhibited. Inhibition of p38 MAPK by FR167653 blocks PTHrP-induced osteoclastogenesis in vitro and PTHrP-induced bone resorption in vivo, suggesting that the p38 MAPK signaling pathway plays a fundamental role in PTHrP-induced osteoclastic bone resorption.
doi:10.1371/journal.pone.0023199
PMCID: PMC3160289  PMID: 21886782
13.  Altered expression of inflammatory cytokines in primary osteoarthritis by human T lymphotropic virus type I retrovirus infection: a cross-sectional study 
Arthritis Research & Therapy  2004;6(4):R347-R354.
Human T cell leukaemia virus type I (HTLV-I) is known to be involved in late-onset chronic polyarthritis as HTLV-I-associated arthropathy. However, it is unclear whether HTLV-I infection could modify the pathophysiology of osteoarthritis (OA). In this study we compared several inflammatory cytokines, such as C-terminal parathyroid hormone-related peptide (C-PTHrP), soluble interleukin-2 receptor (sIL-2R) and interleukin (IL)-6, and an osteo-destruction marker, deoxypyridinoline, in synovial fluid (SF) samples obtained from 22 HTLV-I carriers and 58 control non-carrier patients with OA. These patients were diagnosed clinically and radiographically with primary OA affecting one or both knee joints, and were similar with regard to age, sex and clinical symptoms. We also performed histopathological examination as well as immunohistochemistry of HTLV-I-derived Tax protein in eight synovial tissues taken from carrier patients. C-PTHrP in SF was significantly higher in HTLV-I carriers (287 ± 280 pM) than in non-carriers (69 ± 34 pM), and the concentration in 13 carriers was above the upper range of OA. In HTLV-I carriers, the concentrations of sIL-2R (741 ± 530 IU/ml), IL-6 (55 ± 86 ng/ml) and deoxypyridinoline (3.1 ± 1.8 nM) were higher than in non-carriers (299 ± 303, 2.5 ± 4.0, 0.96 ± 1.0, respectively), and correlated positively with C-PTHrP. C-PTHrP, sIL-2R and IL-6 concentrations in SF positive for IgM antibody against HTLV-I antigen, a marker of persistent viral replication, were higher than of IgM-negative SF. Histologically, five and two synovia showed mild and moderate synovial proliferation with or without some degree of inflammatory reaction, respectively, and could not be distinguished from OA. Tax-positive synoviocytes were observed sparsely in all samples, and often appeared frequently in actively proliferating regions. Our results suggest that although HTLV-I infection does not necessarily worsen the clinical outcome and local synovitis, the virus can potentially modify the pathophysiology of OA by increasing the inflammatory activity in a subset of carrier patients, especially those with IgM antibody. Longitudinal studies are required to assess the association between HTLV-I infection and OA.
doi:10.1186/ar1193
PMCID: PMC464878  PMID: 15225370
Human T cell leukaemia virus type I; osteoarthritis; parathyroid hormone-related peptide; synovial fluid; Tax
14.  Amphiregulin-EGFR signaling regulates PTHrP gene expression in breast cancer cells 
Parathyroid hormone-related protein (PTHrP) is an autocrine/paracrine factor produced by breast cancer cells that is speculated to play a major role in permitting breast cancer cells to grow into the bone microenvironment by stimulating the bone resorption axis. It has been previously shown that EGFR signaling induces the production of PTHrP in several primary and transformed epithelial cell types. Therefore, we investigated the relationship between EGFR and PTHrP gene expression in human breast cancer cells. Of a panel of 7 breast epithelial and cancer cell lines, the osteolytic, EGFR- positive lines (MDA-MB-231 and NS2T2A1) exhibited higher levels of PTHrP transcript expression. Amphiregulin mRNA levels in all lines were approximately 2 orders of magnitude higher than those of TGFα or HBEGF. In the EGFR bearing lines, the receptor was phosphorylated at tyrosine 992 under basal conditions, and the addition of 100 nM amphiregulin did not lead to the phosphorylation of other tyrosine residues typically phosphorylated by the prototypical ligand EGF. Treatment of the EGFR positive lines with the EGFR inhibitor PD153035 and amphiregulin-neutralizing antibodies reduced PTHrP mRNA levels by 50–70%. Stable EGFR expression in the MCF7 line failed to increase basal PTHrP mRNA levels; however, treatment of this cell line with exogenous EGF or amphiregulin increased PTHrP transcription 3-fold. Transient transfection analysis suggests that the MAPK pathway and ETS transcription factors mediate EGFR coupling to PTHrP gene expression. Taken together, it appears that autocrine stimulation of EGFR signaling by amphiregulin is coupled to PTHrP gene expression via EGFR Tyr992 and MAPK, and that this pathway may contribute to PTHrP expression by breast tumor cells.
doi:10.1007/s10549-007-9748-8
PMCID: PMC2730887  PMID: 17882547
Amphiregulin; Breast cancer; Epidermal growth factor receptor (EGFR); Parathyroid hormone-related peptide (PTHrP)
15.  A-Raf and B-Raf Are Dispensable for Normal Endochondral Bone Development, and Parathyroid Hormone-Related Peptide Suppresses Extracellular Signal-Regulated Kinase Activation in Hypertrophic Chondrocytes▿  
Molecular and Cellular Biology  2007;28(1):344-357.
Parathyroid hormone-related peptide (PTHrP) and the parathyroid hormone-PTHrP receptor increase chondrocyte proliferation and delay chondrocyte maturation in endochondral bone development at least partly through cyclic AMP (cAMP)-dependent signaling pathways. Because data suggest that the ability of cAMP to stimulate cell proliferation involves the mitogen-activated protein kinase kinase kinase B-Raf, we hypothesized that B-Raf might mediate the proliferative action of PTHrP in chondrocytes. Though B-Raf is expressed in proliferative chondrocytes, its conditional removal from cartilage did not affect chondrocyte proliferation and maturation or PTHrP-induced chondrocyte proliferation and PTHrP-delayed maturation. Similar results were obtained by conditionally removing B-Raf from osteoblasts. Because A-raf and B-raf are expressed similarly in cartilage, we speculated that they may fulfill redundant functions in this tissue. Surprisingly, mice with chondrocytes deficient in both A-Raf and B-Raf exhibited normal endochondral bone development. Activated extracellular signal-regulated kinase (ERK) was detected primarily in hypertrophic chondrocytes, where C-raf is expressed, and the suppression of ERK activation in these cells by PTHrP or a MEK inhibitor coincided with a delay in chondrocyte maturation. Taken together, these results demonstrate that B-Raf and A-Raf are dispensable for endochondral bone development and they indicate that the main role of ERK in cartilage is to stimulate not cell proliferation, but rather chondrocyte maturation.
doi:10.1128/MCB.00617-07
PMCID: PMC2223319  PMID: 17967876
16.  A Comparison of Parathyroid Hormone-related Protein (1–36) and Parathyroid Hormone (1–34) on Markers of Bone Turnover and Bone Density in Postmenopausal Women: The PrOP Study 
Parathyroid hormone-related protein (PTHrP)(1–36) increases lumbar spine (LS) bone mineral density (BMD), acting as an anabolic agent when injected intermittently, but has not been directly compared to parathyroid hormone (PTH)(1–34). We performed a three month, randomized, prospective study in 105 postmenopausal women with low bone density or osteoporosis comparing daily subcutaneous injections of PTHrP(1–36) to PTH(1–34). Thirty-five women were randomized to each of three groups: PTHrP(1–36) 400 μg/d; PTHrP(1–36) 600 μg/d; and PTH(1–34) 20 μg/d. The primary outcomes measures were changes in amino-terminal telopeptides of procollagen 1 (PINP) and carboxy-terminal telopeptides of collagen 1 (CTX). Secondary measures included safety parameters, 1,25(OH)2vitamin D and BMD. The increase in bone resorption (CTX) by PTH(1–34) (92%) (p<0.005) was greater than for PTHrP(1–36) (30%) (p<0.05). PTH(1–34) also increased bone formation (PINP) (171%) (p<0.0005) more than either dose of PTHrP(1–36) (46 & 87%). The increase in PINP was earlier (day 15) and greater than the increase in CTX for all three groups. LS BMD increased equivalently in each group (p<0.05 for all). Total hip (TH) and femoral neck (FN) BMD increased equivalently in each group but were only significant for the two doses of PTHrP(1–36) (p<0.05) at the TH, and for PTHrP(1–36) 400 (p<0.05) at the FN. PTHrP(1–36) 400 induced mild, transient (day 15) hypercalcemia. PTHrP(1–36) 600 required a dose reduction for hypercalcemia in three subjects. PTH(1–34) was not associated with hypercalcemia. Each peptide induced a marked biphasic increase in 1,25(OH)2D. Adverse events (AE) were similar among the three groups. This study demonstrates that PTHrP(1–36) and PTH(1–34) cause similar increases in LS BMD. PTHrP(1–36) also increased hip BMD. PTH(1–34) induced greater changes in bone turnover than PTHrP(1–36). PTHrP(1–36) was associated with mild transient hypercalcemia. Longer term studies using lower doses of PTHrP(1–36) are needed to define both the optimal dose and full clinical benefits of PTHrP.
doi:10.1002/jbmr.1978
PMCID: PMC3789852  PMID: 23661240
Osteoporosis; PTH; PTHrP; Anabolic; Bone Turnover; DXA
17.  Gene structure, transcripts and calciotropic effects of the PTH family of peptides in Xenopus and chicken 
Background
Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken.
Results
The PTH-L gene is present throughout the vertebrates with the exception of placental mammals. Gene structure of PTH and PTH-L seems to be conserved in vertebrates while PTHrP gene structure is divergent and has acquired new exons and alternative promoters. Splice variants of PTHrP and PTH-L are common in Xenopus and chicken and transcripts of the former have a widespread tissue distribution, although PTH-L is more restricted. PTH is widely expressed in fish tissue but from Xenopus to mammals becomes largely restricted to the parathyroid gland. The N-terminal (1-34) region of PTH, PTHrP and PTH-L in Xenopus and chicken share high sequence conservation and the capacity to modify calcium fluxes across epithelia suggesting a conserved role in calcium metabolism possibly via similar receptors.
Conclusions
The parathyroid hormone family contains 3 principal members, PTH, PTHrP and the recently identified PTH-L. In teleosts there are 5 genes which encode PTHrP (2), PTH (2) and PTH-L and in tetrapods there are 3 genes (PTHrP, PTH and PTH-L), the exception is placental mammals which have 2 genes and lack PTH-L. It is hypothesized that genes of the PTH family appeared at approximately the same time during the vertebrate radiation and evolved via gene duplication/deletion events. PTH-L was lost from the genome of eutherian mammals and PTH, which has a paracrine distribution in lower vertebrates, became the product of a specific endocrine tissue in Amphibia, the parathyroid gland. The PTHrP gene organisation diverged and became more complex in vertebrates and retained its widespread tissue distribution which is congruent with its paracrine nature.
doi:10.1186/1471-2148-10-373
PMCID: PMC3009671  PMID: 21122104
18.  Parathyroid hormone-related peptide as an endogenous inducer of parietal endoderm differentiation 
The Journal of Cell Biology  1993;120(1):235-243.
Parathyroid hormone related peptide (PTHrP), first identified in tumors from patients with the syndrome of "Humoral Hypercalcemia of Malignancy," can replace parathyroid hormone (PTH) in activating the PTH-receptor in responsive cells. Although PTHrP expression is widespread in various adult and fetal tissues, its normal biological function is as yet unknown. We have examined the possible role of PTHrP and the PTH/PTHrP-receptor in early mouse embryo development. Using F9 embryonal carcinoma (EC) cells and ES-5 embryonic stem (ES) cells as in vitro models, we demonstrate that during the differentiation of these cells towards primitive and parietal endoderm-like phenotypes, PTH/PTHrP-receptor mRNA is induced. This phenomenon is correlated with the appearance of functional adenylate cyclase coupled PTH/PTHrP- receptors. These receptors are the mouse homologues of the recently cloned rat bone and opossum kidney PTH/PTHrP-receptors. Addition of exogenous PTH or PTHrP to RA-treated EC or ES cells is an efficient replacement for dBcAMP in inducing full parietal endoderm differentiation. Endogenous PTHrP is detectable at very low levels in undifferentiated EC and ES cells, and is upregulated in their primitive and parietal endoderm-like derivatives as assessed by immunofluorescence. Using confocal laser scanning microscopy on preimplantation mouse embryos, PTHrP is detected from the late morula stage onwards in developing trophectoderm cells, but not in inner cell mass cells. In blastocyst stages PTHrP is in addition found in the first endoderm derivatives of the inner cell mass. Together these results indicate that the PTH/PTHrP-receptor signalling system serves as a para- or autocrine mechanism for parietal endoderm differentiation in the early mouse embryo, thus constituting the earliest hormone receptor system involved in embryogenesis defined to date.
PMCID: PMC2119495  PMID: 8380175
19.  Alternative splicing of parathyroid hormone-related protein mRNA: expression and stability 
Parathyroid hormone-related protein (PTHrP) is a multifunctional protein that is often dysregulated in cancer. The human PTHrP gene is alternatively spliced into three isoforms, each with a unique 3′-untranslated region (3′-UTR), encoding 139, 173 and 141 amino acid proteins. The regulation of PTHrP mRNA isoform expression has not been completely elucidated, but it may be affected by transforming growth factor-β1 (TGF-β1). In this study, we examined differences in the PTHrP mRNA isoform expression in two squamous carcinoma cell lines (SCC2/88 and HARA), an immortalized keratinocyte cell line (HaCaT), and spontaneous human lung cancer with adjacent normal tissue. In addition, the effect of TGF-β1 on PTHrP mRNA isoform expression and stability was examined. Cell-type specific expression of PTHrP mRNA isoforms occurred between the various cell lines, normal human lung, and immortalized human keratinocytes (HaCaT). PTHrP isoform expression pattern was significantly altered between normal lung tissue and the adjacent lung cancer. In vitro studies revealed that TGF-β1 differentially altered the mRNA steady-state levels and mRNA stability of the PTHrP isoforms. Protein–RNA binding studies identified different proteins binding to the 3′-UTR of the PTHrP isoforms (139) and (141), which may be important in the differential mRNA stability and response to cytokines between the PTHrP isoforms. The data demonstrate that there is cell-type specific expression of PTHrP mRNA isoforms, and disruption of the normal regulation during cancer progression may in part be associated with TGF-β1-induced changes in PTHrP mRNA isoform expression and stability.
PMCID: PMC3049244  PMID: 15291755
20.  Increased Cell Survival, Migration, Invasion, and Akt Expression in PTHrP-Overexpressing LoVo Colon Cancer Cell Lines 
Regulatory peptides  2007;141(1-3):61-72.
Parathyroid hormone-related protein (PTHrP) has been localized in human colon cancer tissue and cell lines. We have previously shown that PTHrP increases colon cancer cell proliferation, extracellular matrix adhesion, and cell-surface integrin α6β4 expression. Since cancer cell migration, invasion, and survival are crucial components of metastasis, and colon cancer has a high metastatic potential, in this study we used the human colon cancer cell line LoVo as a model system to study the effects of PTHrP on these parameters. PTHrP expression was modulated by stable transfection with a construct expressing PTHrP (−36 to +139). We report that PTHrP increases cell migration, invasion, and survival. PTHrP altered cell morphology, with PTHrP-overexpressing cells exhibiting increased spreading and several long protrusions. PTHrP also increased the steady-state mRNA levels of the integrin α6 and β4 subunits, indicating a direct and/or indirect effect of PTHrP on the transcriptional and/or posttranscriptional regulation of integrin α6 and β4 expression. Integrin α6β4 activates the phosphoinositol 3-kinase (PI3-K)/Akt pathway, leading to glycogen synthase kinase-3 (GSK-3) deactivation. PTHrP overexpression also led to an increase in active Akt and inactive GSK-3 levels, indicating that the PTHrP-mediated upregulation of integrin α6β4 expression may activate the PI3-K pathway, resulting in increased cell survival, migration and invasion.
doi:10.1016/j.regpep.2006.12.017
PMCID: PMC1892531  PMID: 17276526
Parathyroid hormone-related protein; integrin α6β4; apoptosis; glycogen synthase kinase-3; laminin
21.  Parathyroid Hormone-related Protein Regulates Cell Survival Pathways via Integrin α6β4-mediated Activation of PI3-K/Akt Signaling 
Molecular cancer research : MCR  2009;7(7):1119-1131.
Parathyroid hormone-related protein (PTHrP) is expressed by human prostatic tissues and cancer cell lines. PTHrP enhances tumor cell growth and metastasis in vivo and upregulates pro-invasive integrin α6β4 expression in vitro. Hallmarks of malignant tumor cells include resistance to apoptosis and anchorage-independent cell growth. In this study, we used the human prostate cancer cell lines C4-2 and PC-3 as model systems to study the effects of PTHrP on these processes. We report that PTHrP protects these cells from doxorubicin-induced apoptosis and promotes anchorage-independent cell growth via an intracrine pathway. Conversely, autocrine/paracrine PTHrP action increases apoptosis in C4-2 cells and has no effect on apoptosis in PC-3 cells. The intracrine effects of PTHrP on apoptosis are mediated via activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. PTHrP also affects the phosphorylation state of Akt substrates implicated in apoptosis suppression, including glycogen synthase kinase-3 and Bad. The pro-survival effects of PTHrP are accompanied by increases in the ratio of anti- to pro-apoptosis members of the Bcl-2 family and in levels of c-myc. PTHrP also increases NF-κB activity via a PI3-K-dependent pathway. Integrin α6β4 is known to activate PI3-K. Here we also show that knockdown of integrin α6β4 negates the PTHrP-mediated activation of the PI3-K/Akt pathway. Taken together, these observations provide evidence of a link between PTHrP and the PI3-K/Akt signaling pathway through integrin α6β4, resulting in the activation of survival pathways. Targeting PTHrP production in prostate cancer may thus prove therapeutically beneficial.
doi:10.1158/1541-7786.MCR-08-0568
PMCID: PMC2755566  PMID: 19584267
22.  EBP50 Inhibits the Anti-Mitogenic Action of the Parathyroid Hormone Type 1 Receptor in Vascular Smooth Muscle Cells 
Parathyroid hormone-related protein (PTHrP) and the parathyroid hormone type 1 receptor (PTH1R) are important regulators of vascular remodeling. PTHrP expression is associated to increased proliferation of vascular smooth muscle cells (VSMC). In contrast, signaling via the PTH1R inhibits cell growth. The mechanisms regulating the dual effect of PTHrP and PTH1R on VSMC proliferation are only partially understood. In this study we examined the role of the adaptor protein ezrin-radixin-moesin-binding phosphoprotein (EBP50) on PTH1R expression, trafficking, signaling and control of A10 cells proliferation. In normal rat vascular tissues, EBP50 was restricted to the endothelium with little expression in VSMC. EBP50 expression significantly increased in VSMC following angioplasty in parallel with PTHrP. Interestingly, PTHrP was able to induce EBP50 expression. In the clonal rat aortic smooth muscle cell line A10, EBP50 increased the recruitment of PTH1R to the cells membrane and delayed its internalization in response to PTHrP(1-36). This effect required an intact C-terminal motif in the PTH1R. In naive A10 cells, PTHrP(1-36) stimulated cAMP production but not intracellular calcium release. In contrast, PTHrP(1-36) induced both cAMP and calcium signaling in A10 cells over-expressing EBP50. Finally, EBP50 attenuated the induction of p27 kip1 and the antiproliferative effect of PTHrP(1-36). In summary, this study demonstrates the dynamic expression of EBP50 in vessels following injury and the effects of EBP50 on PTH1R function in VSMC. These finding highlight one of the mechanisms leading to increased VSMC proliferation and have important implication in the understanding of the molecular events leading to restenosis.
doi:10.1016/j.yjmcc.2010.08.025
PMCID: PMC2975869  PMID: 20843475
Vascular smooth muscle cell; Parathyroid hormone-related protein; Parathyroid hormone type 1 receptor; Ezrin-radixin-moesin-binding phosphoprotein EBP50; Proliferation; Cyclic AMP; Calcium; Restenosis; Neointima
23.  Absence of functional receptors for parathyroid hormone and parathyroid hormone-related peptide in Blomstrand chondrodysplasia. 
We report the absence of functional parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptors (PTH/PTHrP receptor) in Blomstrand chondrodysplasia, a genetic disorder characterized by advanced endochondral bone maturation. Analysis of PTH/PTHrP receptor genomic DNA from a patient with Blomstrand chondrodysplasia demonstrated that the patient was heterozygous for a point mutation (G--> A substitution at nucleotide 1176) inherited from the mother. Analysis of PTH/PTHrP receptor cDNA demonstrated that: (a) this point mutation caused the deletion of the first 11 amino acids of exon M5 (encoding the fifth transmembrane domain of the receptor), resulting from the use of a novel splice site created by the base substitution; (b) the mutant receptor was well expressed in COS-7 cells, but did not bind PTH or PTHrP, and failed to induce detectable stimulation of either cAMP or inositol phosphate production in response to these ligands; and (c) the paternal allele was not expressed. Thus, only the abnormal and nonfunctional PTH/PTHrP receptors encoded by the maternal allele were expressed by chondrocytes from this patient. In view of the known role played by the PTH/PTHrP receptor in bone and cartilage development, these results strongly support the conclusion that the absence of functional PTH/ PTHrP receptors is responsible for the skeletal abnormalities seen in Blomstrand chondrodysplasia, abnormalities that are the mirror image of those observed in Jansen's chondrodysplasia. These findings emphasize the importance of signaling through this receptor in human fetal skeletal development.
PMCID: PMC509062  PMID: 9649554
24.  The Paracrine Feedback Loop Between Vitamin D3 (1,25(OH)2D3) and PTHrP in Prehypertrophic Chondrocytes 
Journal of Cellular Physiology  2014;229(12):1999-2014.
The endocrine feedback loop between vitamin D3 (1,25(OH)2D3) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH-related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)2D3 and PTH. This was investigated in ATDC5 cells treated with 10−8 M 1,25(OH)2D3 or PTHrP, Col2-pd2EGFP transgenic mice, and primary Col2-pd2EGFP growth plate chondrocytes isolated by FACS, using RT-qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)2D3 receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)2D3 decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α- and 24-hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)2D3 treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate. 1,25(OH)2D3 decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)2D3 by increasing VDR production. In light of the role of 1,25(OH)2D3 and PTHrP in modulating chondrocyte differentiation, 1,25(OH)2D3 in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration.
doi:10.1002/jcp.24658
PMCID: PMC4298802  PMID: 24777663
25.  Evaluating the Role of PTH in Promotion of Chondrosarcoma Cell Proliferation and Invasion by Inhibiting Primary Cilia Expression 
Chondrosarcoma is characterized by secretion of a cartilage-like matrix, with high proliferation ability and metastatic potential. Previous studies have shown that parathyroid hormone-related protein (PTHrP) has a close relationship with various tumor types. The objectives of this study were to research the function played by PTHrP in human chondrosarcoma, especially targeting cell proliferation and invasion, and to search for the potential interaction between PTHrP and primary cilia in tumorigenesis. Surgical resection tissues and the human chondrosarcoma cell line SW1353 were used in the scientific research. Cells were stimulated with an optimum concentration of recombinant PTH (1-84), and siRNA was used to interfere with internal PTHrP. Cell proliferation and invasion assays were applied, including MTS-8 cell proliferation assay, Western blot, RT-PCR, Transwell invasion assay, and immunohistochemistry and immunofluorescence assays. A high level of PTHrP expression was found in human chondrosarcoma tissues, and recombinant PTH exhibited positive promotion in tumor cell proliferation and invasion. In the meantime, PTHrP could inhibit the assembly of primary cilia and regulate downstream gene expression. These findings indicate that PTHrP can regulate tumor cell proliferation and invasion ability, possibly through suppression of primary cilia assembly. Thus, restricting PTHrP over-expression is a feasible potential therapeutic method for chondrosarcoma.
doi:10.3390/ijms151119816
PMCID: PMC4264140  PMID: 25365173
chondrosarcoma; primary cilia expression; parathyroid hormone-related protein; PTHrP

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