Parathyroid Hormone-related Peptide Treatment Research Articles

Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide.

Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide.

Suppression of Hypertrophy During in vitro Chondrogenesis of Cocultures of Human Mesenchymal Stem Cells and Nasal Chondrocytes Correlates With Lack of in vivo Calcification and Vascular Invasion.

ObjectiveHuman nasal septal chondrocytes (NC) are a promising minimally invasive derivable chondrogenic cell source for cartilage repair. However, the quality of NC-derived cartilage is variable between donors. Coculture of NC with mesenchymal stem cells (MSCs) mitigates the variability but with undesirable markers of chondrocyte hypertrophy, such as type X collagen, and the formation of unstable calcifying cartilage at ectopic sites. In contrast, monoculture NC forms non-calcifying stable cartilage. Formation of a stable NC-MSC coculture cartilage is crucial for clinical application. The aim of this study was to explore the utility of parathyroid hormone-related peptide (PTHrP) hormone to suppress chondrocyte hypertrophy in NC-MSC cocultures and form stable non-calcifying cartilage at ectopic sites.MethodsHuman NC and bone marrow MSCs, and cocultures of NC and MSC (1:3 ratio) were aggregated in pellet form and subjected to in vitro chondrogenesis for 3 weeks in chondrogenic medium in the presence and absence of PTHrP. Following in vitro chondrogenesis, the resulting pellets were implanted in immunodeficient athymic nude mice for 3 weeks.ResultsCoculture of NC and MSC resulted in synergistic cartilage matrix production. PTHrP suppressed the expression of hypertrophy marker, type X collagen (COL10A1), in a dose-dependent fashion without affecting the synergism in cartilage matrix synthesis, and in vivo calcification was eradicated with PTHrP. In contrast, cocultured control (CC) pellets without PTHrP treatment expressed COL10A1, calcified, and became vascularized in vivo.

PTHrP treatment of colon cancer cells promotes tumor associated-angiogenesis by the effect of VEGF

We showed that Parathyroid Hormone-related Peptide (PTHrP) induces proliferation, migration, survival and chemoresistance via MAPKs and PI3K/AKT pathways in colorectal cancer (CRC) cells. The objective of this study was to investigate if PTHrP is also involved in tumor angiogenesis. PTHrP increased VEGF expression and the number of structures with characteristics of neoformed vessels in xenografts tumor. Also, PTHrP increased mRNA levels of VEGF, HIF-1α and MMP-9 via ERK1/2 and PI3K/Akt pathways in Caco-2 and HCT116 cells. Tumor conditioned media (TCMs) from both cell lines treated with PTHrP increases the number of cells, the migration and the tube formation in the endothelial HMEC-1 cells, whereas the neutralizing antibody against VEGF diminished this response. In contrast, PTHrP by direct treatment only increased ERK1/2 phosphorylation and the HMEC-1 cells number. These results provide the first evidence related to the mode of action of PTHrP that leads to its proangiogenic effects in the CRC.

RSK activation via ERK modulates human colon cancer cells response to PTHrP.

Parathyroid hormone-related peptide (PTHrP) is associated with several human cancers such as colon carcinoma. This disease is a complex multistep process that involves enhanced cell cycle progression and migration. Recently we obtained evidence that in the human colorectal adenocarcinoma Caco2 cells, exogenous PTHrP increases the proliferation and positively modulates cell cycle progression via ERK1/2, p38 MAPK and PI3K. The purpose of this study was to explore if the serine/threonine kinase RSK, which is involved in the progress of many cancers and it is emerging as a potential therapeutic target, mediates PTHrP effects on cancer colon cells. Western blot analysis revealed that PTHrP increases RSK phosphorylation via ERK1/2 signaling pathway but not through p38 MAPK. By performing subcellular fractionation, we found that the peptide also induces the nuclear localization of activated RSK, where many of its substrates are located. RSK participates in cell proliferation, in the upregulation of cyclin D1 and CDK6 and in the downregulation of p53 induced by PTHrP. Wound healing and transwell filter assays revealed that cell migration increased after PTHrP treatment. In addition, the hormone increases the protein expression of the focal adhesion kinase FAK, a regulator of cell motility. We observed that PTHrP induces cell migration and modulates FAK protein expression through ERK/RSK signaling pathway but not via p38 MAPK pathway. Finally, in vivo studies revealed that the hormone activates RSK in xenografts tumor. Taken together, our findings provide new insights into the deregulated cell cycle and migration that is characteristic of tumor intestinal cells.

Parathyroid hormone-related peptide protects cardiomyocytes from oxidative stress-induced cell death: First evidence of a novel endocrine–cardiovascular interaction

Although there is a growing interest in the molecular cross-talk between the endocrine and cardiovascular systems, the cardiac effects of calcium-regulating hormones (i.e., parathyroid hormone-related peptide (PTHrP)) have not been explored. In this study, we examined the effect of PTHrP on the viability of isolated adult mouse cardiomyocytes subjected to oxidative stress. Myocytes from 19 to 22 week old male 129J/C57BL6 mice were exposed to oxidative insult in the form of H2O2 which led to more than 70% loss of cell viability. Herein we demonstrate, for the first time, that pretreatment with 100 nM PTHrP prior to 100 μM H2O2 incubation prevents H2O2 -induced cell death by more than 50%. Immunoblot analysis revealed H2O2 induction of MKP-1 protein expression while PTHrP decreased MKP-1 expression. Moreover, myocytes derived from MKP1 KO mice were resistant to oxidative injury. No added benefit of PTHrP treatment was noted in MKP-1 null cardiomyocytes. Using specific pharmacological inhibitors we demonstrated that P-p38, P-ERK and P-AKT mediated PTHrP's cardioprotective action. These data provide novel evidence that: i) down-regulation of MKP1 affords profound protection against oxidative stress; and ii) PTHrP is cardioprotective, possibly via down-regulation of MKP-1 and activation of MAPK and PI3K/AKT signaling.

Induction of thermal and mechanical hypersensitivity by parathyroid hormone-related peptide through upregulation of TRPV1 function and trafficking.

The neurobiological mechanisms underlying chronic pain associated with cancers are not well understood. It has been hypothesized that factors specifically elevated in the tumor microenvironment sensitize adjacent nociceptive afferents. We show that parathyroid hormone-related peptide (PTHrP), which is found at elevated levels in the tumor microenvironment of advanced breast and prostate cancers, is a critical modulator of sensory neurons. Intraplantar injection of PTHrP led to the development of thermal and mechanical hypersensitivity in both male and female mice, which were absent in mice lacking functional transient receptor potential vanilloid-1 (TRPV1). The PTHrP treatment of cultured mouse sensory neurons enhanced action potential firing, and increased TRPV1 activation, which was dependent on protein kinase C (PKC) activity. Parathyroid hormone-related peptide induced robust potentiation of TRPV1 activation and enhancement of neuronal firing at mild acidic pH that is relevant to acidic tumor microenvironment. We also observed an increase in plasma membrane TRPV1 protein levels after exposure to PTHrP, leading to upregulation in the proportion of TRPV1-responsive neurons, which was dependent on the activity of PKC and Src kinases. Furthermore, co-injection of PKC or Src inhibitors attenuated PTHrP-induced thermal but not mechanical hypersensitivity. Altogether, our results suggest that PTHrP and mild acidic conditions could induce constitutive pathological activation of sensory neurons through upregulation of TRPV1 function and trafficking, which could serve as a mechanism for peripheral sensitization of nociceptive afferents in the tumor microenvironment.

Abstract 15371: Parathyroid Hormone-Related Peptide Protects Cardiomyocytes from Oxidative Stress: First Evidence of a Novel Endocrine-Cardiovascular Interaction

There is growing interest in the molecular cross-talk between the endocrine and cardiovascular systems, including the effects of hormones on cardiomyocte viability in models of ischemic and oxidative stress. However, current attention has focused on reproductive and metabolic hormones; the cardiac effects of calcium-regulating hormones (i.e., parathyroid hormone-related peptide (PTHrP)) have not been explored. To address this, we interrogated the effect of PTHrP on the viability of isolated adult mouse cardiomyocytes subjected to H 2 O 2 -induced oxidative stress. In Aim 1 , myocytes from wild type (WT) C57 mice were incubated for 16 hrs with 100 nM PTHrP or vehicle and exposed to 100 μM H 2 O 2 for 15 min. Myocyte viability was significantly improved in PTHrP-treated cells vs vehicle control (70±12% vs 20±15%; mean ± SD, p<0.01; Figure). Calcium sensing receptor and PTHrP signaling is purportedly associated with Mitogen-Activated Protein Kinases (MAPKs) and MAPK phosphatase-1 (MKP1). Accordingly, to achieve preliminary mechanistic insight into PTHrP-induced protection ( Aim 2 ), we: (i) incubated WT cardiomyocytes with PTHrP (100 nM) and probed for expression of MKP1; and (ii) repeated the viability assay as described above using myocytes from adult MKP1 knockout (KO) mice. Incubation of WT myocytes with PTHrP evoked a 55% decrease in MKP1 expression. Moreover, myocytes from MKP1 KO mice were resistant to oxidative injury: viability was maintained at 78±12% in vehicle controls (mean ± SD, p<0.01 vs the value of 20±15% in vehicle-treated WT cells), with no added benefit of PTHrP treatment (Figure). Finally ( Aim 3 ), in both models (PTHrP treatment; MKP1 KO), the enhanced cell viability was accompanied by ~2-fold increases in Akt phosphorylation. These data provide novel evidence that: i) down-regulation of MKP1 affords profound protection against oxidative stress; and ii) PTHrP is cardioprotective, possibly via down-regulation of MKP1 and activation of Akt signaling.

Involvement of ERK1/2, p38 MAPK, and PI3K/Akt signaling pathways in the regulation of cell cycle progression by PTHrP in colon adenocarcinoma cells.

Parathyroid hormone-related peptide (PTHrP) is distributed in most fetal and adult tissues, and its expression correlates with the severity of colon carcinoma. Recently we obtained evidence that in Caco-2 cells, a cell line from human colorectal adenocarcinoma, exogenous PTHrP increases the number of live cells, via ERK1/2, p38 MAPK, and PI3-kinase and induces the expression of cyclin D1, a cell cycle regulatory protein. In this study, we further investigated the role of PTHrP in the regulation of the cell cycle progression in these intestinal cells. Flow cytometry analysis revealed that PTHrP treatment diminishes the number of cells in the G0/G1 phase and increases the number in both S and G2/M phases. The hormone increases the expression of CDK6 and diminishes the amount of negative cell cycle regulators p27Kip1, p15INK4B, and p53. However, PTHrP does not modify the expression of cyclin D3, CDK4, and p16INK4A. In addition, inhibitors of ERK1/2 (PD98059), p38 MAPK (SB203580), and PI3Kinase (LY294002) reversed PTHrP response in Caco-2 cells. Taken together, our results suggest that PTHrP positively modulates cell cycle progression and changes the expression of proteins involved in cell cycle regulation via ERK1/2, p38 MAPK, and PI3K signaling pathways in Caco-2 cells.

Involvement of PI3K/Akt pathway in rat condylar chondrocytes regulated by PTHrP treatment

ObjectiveThe aim of this study was to explore the mutual communication of the parathyroid hormone-related peptide (PTHrP) and phosphatidylinositol 3-kinase/threonine protein kinase (PI3K/Akt) pathway on the proliferation and differentiation of condylar chondrocytes from Sprague-Dawley (SD) rats. MethodsCondylar chondrocytes from the condylar cartilage were cultured and an organ culture system of mandibular condyles was employed. The distribution of PI3K, phospho-Akt (p-Akt), and PTHrP in condylar cartilage was detected by either immunohistochemistry or immunofluorescence. The second passage chondrocytes and condyle specimens in the organ culture system were treated with PTHrP, LY294002, PTHrP and LY294002 in combination, or dimethyl sulfoxide (DMSO), separately. The mRNA and protein levels of type II (Col II) and type X collagen (Col X) were investigated by real-time polymerase chain reaction (PCR) and Western blot analysis. The condyle growth in organ culture system was analysed by haematoxylin–eosin staining. ResultsPTHrP, PI3K, and p-Akt were mainly located in the proliferative and hypertrophic zones. PTHrP promoted the proliferation of condylar chondrocytes, while LY294002 limited this effect. The mRNA and protein levels of Col II and Col X in these cells were reduced by PTHrP and enhanced by LY294002. Organ culture showed a significant enhancement of condyle elongation with PTHrP treatment or a combination of PTHrP and LY294002 treatment. After treatment with LY294002, the length of condyles was reduced compared with the samples treated with DMSO. ConclusionsWe conclude that the PI3K/Akt pathway plays an essential role in the proliferation and differentiation of condylar chondrocytes and is a potential target for PTHrP in regulating chondrocyte differentiation at condylar cartilage.

PTHrP promotes chondrogenesis and suppresses hypertrophy from both bone marrow-derived and adipose tissue-derived MSCs

During chondrogenesis from mesenchymal stem cells (MSCs), inadequate differentiation and hypertrophic differentiation are two important limitations. The purpose of this study was to test the hypothesis that chondrogenesis is enhanced and unwanted hypertrophic changes are suppressed by treating bone marrow-derived (BMMSCs) and adipose tissue-derived mesenchymal stem cells (ATMSCs) with parathyroid hormone-related peptide (PTHrP). To induce chondrogenesis, in vitro pellet cultures were carried out using 2.5 × 10 5 MSCs at passage 3 in chondrogenic medium containing 5 ng/ml of TGF-β 2 for BMMSCs, and 5 ng/ml of TGF-β 2 and 100 ng/ml of BMP-7 for ATMSCs. From the 14th day of culture, subsets of pellets were treated with PTHrP [0, 10, 100 ng/ml], and after two more weeks of in vitro culture, pellets were harvested for analysis. The addition of PTHrP dose-dependently increased DNA contents in both BMMSCs and ATMSCs. GAG contents also increased after PTHrP treatment. The gene expression of COL1A1 decreased by three-fourths, while the decrease was not evident in ATMSCs after PTHrP treatment. SOX-9 mRNA increased up to four fold in both BMMSCs and ATMSCs, and COL2A1 gene expression sharply increased to sevenfold in BMMSCs and to 4 fold in ATMSCs. COL10A1 gene expression decreased by a third in both cell types, and Runx-2 expression dropped sharply in both cell types after PTHrP treatment. Safranin-O and immunohistochemistry for type I, II, X collagen and Runx-2 generally paralleled qRT-PCR findings with minor variations. In conclusion, PTHrP was found to promote chondrogenesis and suppress hypertrophy during in vitro chondrogenesis from both BMMSCs and ATMSCs, which supports its use for cartilage tissue engineering.

Novel Late Response Genes of PTHrP in Chondrocytes

To gain more insight into the downstream effectors of parathyroid hormone (PTH) related peptide (PTHrP) signaling in chondrocytes, we performed microarray analysis to identify late PTHrP response genes using the chondrogenic ATDC5 cell line and studied their response in the osteoblastic KS483 cell line and explanted metatarsals. At day 8 of micromass culture, ATDC5 cells have pre-hypertrophic-like characteristics and at this time point the cells were stimulated with PTHrP for 24 and 72 h and RNA was isolated. PTHrP treatment inhibited outgrowth of cartilage matrix and decreased the expression of Col10a1 mRNA, which is in line with the inhibitory effects of PTHrP on chondrocyte differentiation. Using cDNA microarray analysis, a list of 9 genes (p< 10^–3) was generated, including 3 upregulated (IGFBP4, Csrp2, and Ecm1) and 6 downregulated (Col9a1, Col2a1, Agc, Hmgn2, Calm1, and Mxd4) response genes. Four out of 9 genes are novel PTHrP response genes and 2 out of 9 have not yet been identified in cartilage. Four out of 9 genes are components of the extra-cellular matrix and the remaining genes are involved in signal transduction and transcription regulation. The response to PTHrP was validated by quantitative PCR, using the same RNA samples as labeled in the microarray experiments and RNA samples isolated from a new experiment. In addition, we examined whether these genes also reacted to PTHrP in other PTHrP responsive models, like KS483 osteoblasts and explanted metatarsals. The expression of late PTHrP response genes varied between ATDC5 chondrocytes, KS483 osteoblasts and metatarsals, suggesting that the expression of late response genes is dependent on the cellular context of the PTHrP responsive cells.

Runx2 expression and action in chondrocytes are regulated by retinoid signaling and parathyroid hormone-related peptide (PTHrP)

Objective Runx2 (also known as Cbfa1) is a transcription factor required for chondrocyte maturation and osteoblast differentiation. While there is information on the regulation of its expression during osteogenesis, much less is known about it during cartilage maturation. Here we asked whether Runx2 expression and function are affected by retinoic acid (RA) and parathyroid hormone-related peptide (PTHrP), which represent an important stimulator and inhibitor of chondrocyte maturation, respectively.Design We first cloned and characterized Runx2 expressed by chick chondrocytes (cRunx2). We then constructed expression vectors of cRunx2 and a dominant-negative form (DN-cRunx2) and determined their effects on chondrocyte maturation in culture before and during retinoid and PTHrP treatment.Results cRunx2 showed similar transactivation activity to that of its mammalian counterparts although it has a very short QA domain and lacks a small portion of the PST domain. cRunx2 over-expression stimulated chondrocyte maturation, as indicated by increases in alkaline phosphatase activity (APase), mineralization, and type X collagen and MMP-13 expression, and by maintenance of Indian hedgehog (Ihh) expression. RA treatment stimulated cRunx2 gene expression and boosted its pro-maturation effects. PTHrP treatment blocked Runx2 expression and its pro-maturation effects. Over-expression of DN-cRunx2 inhibited maturation and even prevented RA from exerting its pro-maturation role.Conclusions As previously indicated by mammalian studies, cRunx2 has chondrocyte pro-maturation activity. Its expression and roles are favorably modulated by retinoid signaling but are completely inhibited by PTHrP. A model integrating cRunx2 with PTHrP, Ihh and retinoid signaling and operating during skeletogenesis is proposed.Copyright 2003 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.

PTHrP Modulates Chondrocyte Differentiation through AP-1 and CREB Signaling

During the process of differentiation, chondrocytes integrate a complex array of signals from local or systemic factors like parathyroid hormone-related peptide (PTHrP), Indian hedgehog, bone morphogenetic proteins and transforming growth factor beta. While PTHrP is known to be a critical regulator of chondrocyte proliferation and differentiation, the signaling pathways through which this factor acts remain to be elucidated. Here we show that both cAMP response element-binding protein (CREB) and AP-1 activation are critical to PTHrP signaling in chondrocytes. PTHrP treatment leads to rapid CREB phosphorylation and activation, while CREB DNA binding activity is constitutive. In contrast, PTHrP induces AP-1 DNA binding activity through induction of c-Fos protein expression. PTHrP activates CRE and TRE reporter constructs primarily through PKA-mediated signaling events. Both signaling pathways were found to be important mediators of PTHrP effects on chondrocyte phenotype. Alone, PTHrP suppresses maturation and stimulates proliferation of the chondrocyte cultures. However, in the presence of dominant negative inhibitors of CREB and c-Fos, these PTHrP effects were suppressed, and chondrocyte maturation was accelerated. Moreover, in combination, the effects of dominant negative c-Fos and CREB are synergistic, suggesting interaction between these signaling pathways during chondrocyte differentiation.

Macrophage colony-stimulating factor release and receptor expression in bone cells.

Colony-stimulating factors (CSF) may play a role in bone resorption. To examine whether osteoblasts secrete colony-stimulating activity (CSA) in response to parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), conditioned medium (CM) from ROS 17/2.8 cells and primary rat osteoblasts were examined for induction of clonal growth of cultured rat bone marrow cells. Untreated cells constitutively secreted CSA, which increased with PTH and PTHrP treatment. The colonies formed were principally comprised of macrophages, and preincubation of CM with antiserum to murine macrophage colony-stimulating factor (M-CSF) neutralized most of the CSA, suggesting that the osteoblast-derived CSA was predominantly due to M-CSF. PTHrP treatment upregulated steady-state M-CSF mRNA levels. To investigate a paracrine role for M-CSF in bone we examined bone tissue and cells for the M-CSF receptor c-fms using immunohistochemical techniques and demonstrated staining of mature osteoclasts both in situ and after isolation. We conclude that M-CSF is responsible for the majority of the CSA released by PTH- and PTHrP-treated rat osteoblasts. In addition we identified CSF-1 receptor expression in mature osteoclasts. These data suggest that M-CSF is a mediator of osteoblast-osteoclast interaction in PTH- and PTHrP-induced bone resorption.